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藥學博士學位論文

A Novel Function of Myc-nick, a Cleavage Product

of c-Myc, in Resolution of Inflammation

염증해소 반응에 있어서 Myc-nick 의 새로운 기능

2018 年 8 月

서울大學校 大學院

藥學科 醫藥生命科學 專攻

钟 宪 财

A Novel Function of Myc-nick, a Cleavage Product

of c-Myc, in Resolution of Inflammation

염증해소 반응에 있어서 Myc-nick 의 새로운 기능

指导教授 徐 荣 俊

이論文을 藥學博士 學位論文으로 提出함

2018 年 5 月

서울大學校 大學院

藥學科 醫藥生命科學 專攻

钟宪财

钟宪财의 藥學博士 學位論文을 認準함

2018 年 8 月

委 員 長 印

副委員長 印

委 員 印

委 員 印

委 員 印

A Novel Function of Myc-nick, a Cleavage Product

of c-Myc, in Resolution of Inflammation

by

Xiancai Zhong

A thesis submitted in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

(Pharmacy: Pharmaceutical biosciences major)

Under the supervision of Professor Young-Joon Surh

at the

College of Pharmacy

Seoul National University

May 2018

i

ABSTRACT

A Novel Function of Myc-nick, a Cleavage Product

of c-Myc, in Resolution of Inflammation

Xiancai Zhong

Under the supervision of Professor Young-Joon Surh

at the college of Pharmacy, Seoul Nation University

Acute inflammatory response is characterized by sequential events including an

immediate recruitment of polymorphonuclear leukocytes (PMNs), a subsequent

clearance of leukocytes and an eventual resolution. Eventually, restoration of body

homeostasis is accomplished when inflammation is resolved successfully, which is

underpinned by adaptive immunity at the post-resolution phase. An impaired

resolution causes chronic inflammatory response, leading to persistent tissue damage.

In the past many years, researchers have focused on developing drugs such as non-

steroidal anti-inflammatory drugs to treat patients with inflammatory diseases by

shutting off inflammatory response. However, this is not effective in augmenting

ii

cellular adaptive response to acute inflammation through potentiation of innate

immunity as they often interrupt the consecutive host-protective process. Recent

work has been hence shifted to discovery of drugs that actively facilitate resolution

of inflammation, achieving a termination of inflammatory response utilizing

intrinsic mechanism and a simultaneous recovery of body homeostasis. To achieve

the active resolution, two physiologic processes are employed. These include

recruitment of leukocytes (typically neutrophils) that eventually die, mostly via

apoptosis, after fighting infectious agents and subsequent clearance of dead cells and

debris. Macrophages, especially pro-resolving macrophages, are typical cells that are

effective on clearance of apoptotic cells by phagocytic engulfment (efferocytosis).

These pro-resolving macrophages carries M2 (alternatively activated) phenotypic

characteristics.

Carcinogenesis is closely associated with chronic inflammation. Chronic

inflammation can cause activation and accumulation of immunosuppressive cells,

which favors initial tumor formation and later survival of tumor cells. In recent years,

resolution of cancer-associated inflammation is becoming a new approach for cancer

therapy. Pro-resolving treatment not only eliminates excessive inflammatory signal,

but mediates a post-resolution reaction that generates adaptive immune response.

The influx of adaptive immune cells possibly potentiate therapy as these cells have

been demonstrated to be effective in immunotherapy. Therefore, it is of great

interest to apply pro-resolving strategies for the treatment of inflammatory disorders.

iii

The transcription factor, c-Myc has been known to play a role in manifestation or

maintenance of phenotypic characteristics of alternatively activated macrophages.

Contradictory to this notion, I found that peritoneal macrophages from zymosan A-

treated mice, in the M2-like state, exhibited a markedly reduced level of c-Myc. This

was accompanied by accumulation of Myc-nick, a truncated protein generated by a

Calpain-mediated proteolytic cleavage of full-length c-Myc. Further, I noticed that

the generation of Myc-nick promoted the M2 polarization of macrophages,

enhanced the efferocytic capability of these cells and accelerated resolution of

inflammation. Another study showed that c-Myc cleavage was not obviously

affected by treatment of pro-resolving lipid mediator, resolvin D1 (RvD1), to HCT

116 cancer cells. This is probably due to a lack of Calpain activation during a pro-

resolving treatment. It is notable that RvD1 exerts anti-cancer effect by facilitating

c-Myc degradation in HCT 116 cells. My studies indicate that Myc-nick might be an

innovative therapeutic target in macrophages but not cancer cells. Since tumor-

associated macrophages (TAM) are largely present in tumor microenvironment, the

role of Myc-nick in the function of TAM merits further investigation in the context

of development of novel anticancer strategies.

Keywords: Resolution of inflammation, Efferocytosis, M2 polarization, Myc-nick,

Student Number: 2011-31329

iv

TABLE OF CONTENTS

ABSTRACT ........................................................................................................... i

TABLE OF CONTENTS..................................................................................... iv

List of Figures .................................................................................................... viii

List of Abbreviations .............................................................................................x

Chapter I ................................................................................................................1

1.1. Introduction.....................................................................................................2

1.2. Resolution of inflammation.............................................................................3

1.3. Efferocytosis ....................................................................................................8

1.4. Role of cytoskeleton during efferocytosis .....................................................11

1.4.1. Actin cytoskeleton for efferocytosis........................................................12

1.4.2. Microtubule cytoskeleton for efferocytosis ............................................14

1.5. LC3-associated phagocytosis ........................................................................15

1.6. Phenotypic characteristics of macrophage during resolution .....................16

1.7. Role of c-Myc.................................................................................................18

1.8. Myc-nick........................................................................................................19

1.9. Perspectives ...................................................................................................22

Chapter II.............................................................................................................27

2.1. Abstract .........................................................................................................28

2.2. Introdutcion...................................................................................................29

v

2.3. Materials and methods..................................................................................32

2.3.1. Materials .................................................................................................32

2.3.2. Methods...................................................................................................33

2.3.2.1. Cell culture .......................................................................................33

2.3.2.2. Zymosan- and thioglycollate-induced peritonitis ............................33

2.3.2.3. Preparation of bone marrow derived macrophages (BMDMs)......34

2.3.2.4. Isolation of mouse thymocytes and generation of apoptotic cells ...35

2.3.2.5. Efferocytosis assay............................................................................35

2.3.2.6. Flow cytometry assay .......................................................................36

2.3.2.7. Immunofluorescent staining and imaging .......................................37

2.3.2.8. Western blotting and PCR analysis .................................................38

2.3.2.9. Bioinformatic Studies.......................................................................39

2.3.2.10. Statistical analysis ..........................................................................40

2.4. Results............................................................................................................41

2.4.1. Murine macrophages have diverse characteristics at different stages..41

2.4.2. During the resolution of zymosan A-induced murine peritonitis, pro-

inflammatory macrophages switch to pro-resolving macrophages with an

M2-like phenotype............................................................................................44

2.4.3. Full length c-Myc is cleaved into Myc-nick during the resolution of

zymosan-induced peritonitis. ...........................................................................47

2.4.4. Myc-nick localizes in the cytoplasm. ......................................................49

vi

2.4.5. Myc-nick production is associated with efferocytosis............................49

2.4.6. Myc-nick enhances efferocytic activity of macrophages. ......................54

2.4.7. Myc-nick promotes M2 polarization of macrophages during

efferocytosis. .....................................................................................................57

2.4.8. Myc-nick potentiates efferocytosis through α-tubulin acetylation........60

2.4.9. Myc-nick induces LC3-associated phagocytosis (LAP).........................64

2.5. Discussion ......................................................................................................68

Chapter III ...........................................................................................................73

3.1. Abstract .........................................................................................................74

3.2. Introduction...................................................................................................75

3.3. Materials and methods..................................................................................78

3.3.1. Materials .................................................................................................78

3.3.2. Methods...................................................................................................78

3.3.2.1. Animal experiment ...........................................................................78

3.3.2.2. Cell culture .......................................................................................79

3.3.2.3. Fractionation of cytosolic and nuclear extracts...............................79

3.3.2.4. Western blot and reverse transcription-polymerase chain reaction

(RT-PCR) analysis ........................................................................................80

3.3.2.5. Electrophoresis mobility shift assay (EMSA)..................................81

3.3.2.6. Colony formation assay....................................................................82

3.3.2.7. Integrative network and correlation analysis..................................82

vii

3.3.2.8. Statistical analysis ............................................................................83

3.4. Results............................................................................................................84

3.4.1. c-Myc expression is elevated in colitis-associated experimental

carcinogenesis and human cancer cells ...........................................................84

3.4.2. RvD1 inhibits TNFα-induced overexpression of c-Myc in normal colon

cells....................................................................................................................87

3.4.3. RvD1 reduces the stability of c-Myc in colon cancer cells.....................91

3.4.4. RvD1 inhibits c-Myc through interaction with the G-protein coupled

formyl peptide receptor 2 (ALX/FPR2)...........................................................91

3.5. Discussion ......................................................................................................97

Conclusion and Perspectives .............................................................................100

Reference............................................................................................................106

Korean Abstract.................................................................................................127

Biographical Data ..............................................................................................131

viii

List of Figures

Chapter I

Figure 1-1. A process of inflammation and its resolution.....................................5

Figure 1-2. Strategies for inflammation termination..........................................24

Chapter II

Figure 2-1. Constitution of peritoneal leukocytes upon zymosan-induced

peritonitis. ............................................................................................................42

Figure 2-2. Gene expression of M1 and M2 markers in peritoneal macrophages.

..............................................................................................................................45

Figure 2-3. Cleavage of c-Myc into Myc-nick during the resolution of peritonitis.

..............................................................................................................................48

Figure 2-4. Localization of Myc-nick. .................................................................50

Figure 2-5. Myc-nick production by efferocytosis. .............................................52

Figure 2-6. Effects of Myc-nick overexpression and inhibition of its production

on efferocytosis.....................................................................................................55

Figure 2-7. M2 polarization of macrophages promoted by ectopically expressed

Myc-nick during efferocytosis. ............................................................................58

Figure 2-8. Involvement of acetylated α-tubulin in efferocytosis and subsequent

M2 polarization....................................................................................................61

ix

Figure 2-9. Involvement of acetylated α-tubulin in the expression profile of M1

and M2 markers...................................................................................................63

Figure 2-10. LC3-associated phagocytosis is involved in Myc-nick-induced

efferocytosis. .........................................................................................................65

Chapter III

Figure 3-1. Expression profile of c-Myc in colon cancer. ...................................85

Figure 3-2. Inhibition of TNFα-induced overexpression of c-Myc by RvD1 in

normal intestinal cells. .........................................................................................89

Figure 3-3. Effect of RvD1 on stability of c-Myc hyper-expressed in HCT

116 cells.................................................................................................................92

Figure 3-4. Inhibition of ERK activity by RvD1 via interaction with ALX/RPR2

receptor. ...............................................................................................................94

Conclusion and Perspectives

Figure 4-1. Role of Myc-nick in the resolution of inflammation. .....................101

x

List of Abbreviations

Adgrb1/Bai1: adhesion G protein-coupled receptor B1;

Arg1: arginase 1;

BMC: bone marrow cell;

CAC: colitis-associated cancer;

C/EBPβ: CCAAT/enhancer-binding protein β

Cdc42: cell division cycle 42;

CTPH2: cyclopentylidene-[4-(4’-chlorophenyl)thiazol-2-yl]hydrazine;

F4/80int: F4/80intermediate;

Gas6: growth arrest specific 6;

Hdac6: histone deacetylase 6; Kat2a/Gcn5, lysine (K) acetyltransferase 2a;

IRF4: interferon regulatory factor 4

LC3: microtubule-associated protein 1 light chain 3;

LAP: LC3-associated phagocytosis;

Mertk: c-mer proto-oncogene tyrosine kinase;

Mfge8: milk fat globule-EGF factor 8 protein;

MT: microtubule; p38, mitogen-activated protein kinase 14;

pHrodo-SE: pHrodo-succinimidyl ester;

PM: peritoneal macrophage;

PMN: polymorphonuclear neutrophil

Pparγ: peroxisome proliferator activated receptor γ;

xi

PS: phosphatidylserine;

Rab7: RAB7, member RAS oncogene family;

Rac1: RAS-related C3 botulinum substrate 1;

ROS: Reactive oxygen species;

RSV: Rous sarcoma virus

RvD1: resolvin D1

SPM: specialized pro-resolving mediators

STAT6: signal transducer and activator of transcription 6

Stab2: stabilin 2;

TAM: tumor-associated macrophage;

Timd4/Tim4, T cell immunoglobulin and mucin domain containing 4;

Treg: regulatory T cell.

1

Chapter I

Prospect of Resolution and Cancer Therapy

2

1.1. Introduction

Inflammation is an essential process in the host, which enables the elimination of

a noxious substance in response to microbial infection or tissue injury. It promotes

tissue repair and in case of infection, establishes a memory so that the host mounts a

faster and more precise response to a future encounter1. Inflammation can be

classified as either acute inflammation, which occurs over short term, or chronic

inflammation, which occurs over a longer period of time. In acute conditions, a large

variety of stimuli are sensed by pattern recognition receptors, leading to signal

cascades that induce the release of inflammatory mediators, such as eicosanoids and

cytokines. These mediators recruit neutrophils, monocytes and macrophages to the

site of inflammation to eliminate or mitigate the pro-inflammatory insults. Once the

insults are eliminated, neutrophils undergo constitutive apoptosis and are cleared by

macrophages, subsequently leading to resolution of inflammation2.

The clearance of dying/dead neutrophils delivers an imperative signal to switch

inflammatory macrophages into pro-resolving macrophages. The mediators

produced by pro-resolving macrophages counteract inflammatory signals and foster

tissue repair. Therefore, acute inflammation is considered a physiological response

that can be successfully terminated in time to prevent continuous damage to the

tissue. In contrast, chronic inflammation leads to the pathogenesis and progression

of various diseases. It occurs if the host is unable to clear the initiating stimulus or

3

fails to generate a resolution response, which is needed in order to override the self-

sustaining inflammation program3. All the factors of the inflammatory response are

found in the tumor tissues as well, and these factors have been demonstrated to be

associated with development and progression of cancer. Therefore, strategies

targeting resolution of inflammation not only promises novel approaches for the

prevention and treatment of chronic inflammatory diseases4, but discloses innovative

methods for cancer therapy5.

1.2. Resolution of inflammation

Resolution is known as an active process that not only shuts off pro-

inflammatory signaling, but also normalizes to clear apoptotic polymorphonuclear

neutrophils (PMNs) at the inflamed tissue site6. This process comprises an

elimination of harmful stimuli, stopping pro-inflammatory signals, promoting

apoptosis of PMNs, clearing apoptotic cells and cellular debris by macrophages, and

draining these myeloid cells by lymph nodes or incorporating these cells into the

resident populations (Fig. 1-1). It is properly programmed by a large range of

mediators such as chemokines, cytokines and chemicals. Improper resolution has

been implicated in chronic inflammation, persistent tissue damage, abnormal tissue

healing and development of autoimmunity7. Hence, it is of great interest to identify

and decode mediators involved in the resolution of inflammation.

4

5

Figure 1-1. A process of inflammation and its resolution.

During an acute inflammation, inflammatory stimuli such as microorganisms are

sensed by patrolling monocytes and tissue resident macrophages. These cells release

various cytokines and chemokines to recruit neutrophils to the inflamed site, leading

to a clearance of those pathogens. At a later time, monocyte-derived macrophages

are recruited and activated as inflammatory ones to further promote pathogen

clearance. Once pathogens completely eliminated, neutrophils undergo apoptosis

followed by engulfing and degradation by macrophages, a process termed

efferocytosis. At the end of resolution, macrophages leave the site by lymphatic

6

drainage and the resolution is expected to be completed. Adopted from Headland

and Norling. (2015).

7

Generally, mediators switch from pro-inflammatory to pro-resolving ones once

resolution is initiated. Although the latter molecules act in the resolution phase, they

are primed by early events occurring in acute inflammation-the process that can be

defined “the beginning programs the end”8. One of the early events involved in the

process of resolution is an activation of self-termination signaling in the initial phase

of inflammation, thereby converting eicosanoids from ‘alpha’ form to an ‘omega’

one9. Besides, the apoptosis of inflammatory neutrophils acts as a prominent

checkpoint for subsequent resolution10, including release of intracellular taurine and

thereby affecting phagocytic activity of macrophages11. The pro-resolving cells are

mainly composed of T regulatory cells, macrophages and myeloid-derived

suppressive cells. Among them, macrophages dominate the clearance of apoptotic

neutrophils and cellular debris by phagocytic engulfment. There is a class of lipid

mediators, collectively termed ‘specialized pro-resolving mediators’ (SPMs)

involved in the accelerating macrophage uptake of apoptotic neutrophils and cell

debris.

These pro-resolving cells and mediators not merely switch off inflammation but

also facilitate the restoration of homeostasis. However, an ideal pharmacologic

resolution should be applied to the inflammation peak since the onset of

inflammation is required for clearing microorganism or other noxious substances4.

Also it should be harnessed by targeting endogenous signaling molecules that are

involved in resolution only, or by modifying an inflammation steady state, in a way

8

that the cellular antimicrobial function is maintained. Researchers have identified

numerous endogenous agonists of resolution such as SPMs12 that actively promote

resolution and tissue repair without compromising host defense6.

1.3. Efferocytosis

Efferocytosis (Figure 1-1), defined as phagocytic removal of apoptotic cells, is a

carefully orchestrated process for clearing these dead cells in a non-phlogistic

manner. It is mediated by phagocytes such as macrophages, mast cells and dendritic

cells. Macrophages have been known as professional phagocytes due to their high

capacity for engulfing apoptotic cells. To initiate an engulfment, these cells are

firstly activated and attracted by ‘find-me’ signals sent by dying cell13. The

phagocytes then distinguish the apoptotic cell from healthy living cells via specific

engulfment receptors, which recognize ‘eat-me’ signals on the dying cell14. Next, the

phagocyte undergoes extensive cytoskeletal rearrangement to internalize cellular

‘corpses’, leading to the final ingestion of apoptotic bodies and subsequent release

of anti-inflammatory mediators that help terminate the immune response15.

The critical first step is a release of ‘find-me’ signals by apoptotic cells in many

tissues, as these signals recruit a sufficient amount of phagocytes to the site with

dying cells. This process is particularly important for the developing thymus on

account of that thymocytes lack the ability to engulf apoptotic neighbor cells.

9

Therefore, distant phagocytes must be recruited to the proximity of apoptotic

thymocytes for clearance of these dead cells. This can be accomplished through the

release of mediators from the dying cell, including chemokines (CX3CL1)16,

nucleotides (ATP and UTP)17, and the lipids lysophosphatidylcholine and

sphingosine 1-phosphate18, 19. It is possible that ‘find-me’ signals might influence

and/or enhance the engulfing capacity of the phagocytes. For example, CX3CL1

stimulates the expression of MFG-E8 (‘milk fat globule–epidermal growth factor 8’)

by phagocytes, which bridge apoptotic cells to the phagocytes, thereby facilitating

engulfment20.

The following step is the tethering of apoptotic cells by engulfment receptors on

the phagocytes through recognition of specific ‘eat-me’ signals on apoptotic cells.

The most largely studied ‘eat-me’ signal is the exposure of lipid phosphatidylserine

on apoptotic cells. Phosphatidylserine is actively restrained to the inner plasma

membrane in living cells, but the restriction is uncontrollable in apoptotic cells due

to loss of energy21. In addition to phosphatidylserine, other moieties that are variably

exposed on apoptotic cells include a modified form of the intracellular adhesion

molecule ICAM-3, oxidized low-density lipoprotein, calreticulin, annexin I, cell

surface-bound thrombospondin and complement C1q; other alterations on the

surface include changes in the surface protein charge and glycosylation status22.

Engulfment receptors show high specificity to these ‘eat-me’ signal molecules23.

These receptors include phosphatidylserine receptors (e.g. TIM4, BAI1, and integrin

10

αvβ3/αvβ5), scavenger receptors (e.g. CD68 and CD36) that recognize sites

resembling oxidized low-density lipoprotein particles (oxLDL) and receptors that

identify other ‘eat-me’ signals.

The engulfment of apoptotic cells is involved in cytoskeleton rearrangement to

mediate membrane protrusion, phagocytic cup formation, and membrane closure.

This is regulated by the signal pathways that are triggered by the binding of

engulfment receptors to the apoptotic cells. There are primaryly two types of signal

pathways for regulating cytoskeleton rearrangement. One is mediated by the

recruitment of the Elmo-Dock (‘engulfment and cell motility downstream of Crk’)

complex, which functions as a guanine-exchange factor for the small GTPase Rac

(e.g. BAI1)24. Activation of Rac promotes remodeling of the actin cytoskeleton

required for engulfment of the apoptotic cellular ‘corpse’. The other one is mediated

by the activation of kinase FAK subsequent activation of Rac via integrins αVβ3 or

αVβ5 and the TAM receptors (MerTK, Tyro3 and Axl)25. TAM receptors comprise

a unique family of tyrosine kinases that also activate cell-signaling pathways

involving the kinases Src and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)

and the phospholipase C (PLC)26. TIM-4, as a phosphatidylserine receptor, only

directs a tethering of macrophage to apoptotic cell due to the lack of intracellular

domain that transduces signals for activating downstream molecules of

phosphatidylserine recognition27. In addition, it utilizes and functions cooperatively

with other co-receptors (integrins) to transduce signals for cytoskeleton

11

rearrangement28.

Phagosome maturation is viewed as the end of the phagocytic process, during

which internalized apoptotic cells are efficiently degraded. The phagosome

sequentially acquire various proteins (e.g. motor proteins and GTPases) during

maturation and fuse with acidic lysosome eventually. A deficiency in degradation of

apoptotic cell can result in autoimmune disease29. Therefore, phagosome maturation

is of great importance to homeostasis of the immune system. The nascent

phagosome undergoes a series of sequential membrane fusion and fission

interactions with early endosomes, late endosomes and lysosomes thereby forming

matured phagosome, also referred to as the phagolysosome30. Motor proteins

dominate the transportation of phagosome to the center area where the lysosomes

are more acidic31. The transportation is a coordination of centripetal and centrifugal

forces from these motor proteins32. Rab GTPases mediate the conversion of

phagosomes in part. Rab5 activity is required for the transition from an early to a

late phagosome33. Rab5 facilitates fusion of the nascent phagosome with early

endosomes and subsequently recruits and activates a number of proteins involved in

phagosome maturation, including Rab7. As maturation proceeds, Rab5 is lost, and

Rab7 mediates the fusion of the phagosome with late endosomes and ultimately,

lysosomes34.

1.4. Role of cytoskeleton during efferocytosis

12

The cytoskeleton is a structure that maintains the shape and internal organization

of cells, and it also enables cells to execute essential functions like movement and

division. There are several different components that work together to form the

networks of cytoskeleton, including microfilaments, microtubules and intermediate

filaments35. Microfilaments, composed of F-actin (filamentous), mainly govern

membrane plasticity (including cytoskeleton-propelled deformation and protrusion)

and cell motility by a high rate of actin polymerization or de-polymerization. By

generating mechanical force through contraction and relaxation, they also control

cell morphology and cell movement36. A microtubule is a hollow cylindrical

structure consisting of 13 parallel proto-filaments, each built from alternating α- and

β-tubulin molecules. It forms large structures on the outside of the cells, mainly

serving a transportation and cell movement, and supports cell division37.

Intermediate filaments consist of a variety of proteins in different types of cells,

which is distinct from that actin filaments and microtubules are composed of single

types of components. They are highly stable and involved in structural support, but

not show polarized growth and support motor-based cargo transport38.

1.4.1. Actin cytoskeleton for efferocytosis

The primary scaffold structure in eukaryotic cells is actin cytoskeleton which is

larger than any intracellular organelle. The monomeric subunit of this cytoskeleton,

13

globular actin, polymerizes to form filamentous actin with a diameter of

approximately 7 nm and a length up to several micrometers39. Actin filaments are

highly concentrated underneath the plasma membrane, the periphery of cells, where

they constitute a network that provides dominant mechanical support for

maintaining cell morphology39. The movement of cells also requires the

participation of actin cytoskeleton via a rearrangement of actin proteins. In engulfing

macrophages, the uptake of apoptotic cell is mediated by the reorganization of actin

cytoskeleton and subsequent movement of membranes, thereby forming a

phagocytic cup at the initiating stage and an eventual phagosome once the apoptotic

cell is totally internalized40. An accumulation of myosin and actin filaments at the

site of advancing cup provides forward force for the extension of the membrane over

the surface of apoptotic cell. And the following contractile activities drive the close

of the extended membrane for accomplishing internalization 41.

An initial contact between phagocytes and apoptotic cells was proposed to be

driven by random events, such as Brownian collisions and blood flow42. It has been

elucidated recently that macrophages actively detect apoptotic cells by dynamic

membrane extensions42. Actin polymerization and the recruitment of other

regulatory proteins have been implicated in the membrane extension occurred in the

whole process of engulfing apoptotic cells40. The movements of the cell membrane

are mediated by a combination of localized actin polymerization and

depolymerization, together with the organized gelation, solation and contraction of

14

actin-filament networks. The primary polymerization of actin is governed by Arp2/3

complex43. Other proteins that affect actin polymerization during phagocytosis

include WASP, WAVE2, amphiphysin-2 and coronin40. The enzymes that control

the actin cytoskeleton are controlled by small GTPases and the guanine nucleotide

exchange factors (GEFs) and other GTPase-activating proteins. These GTPases,

including Rac1, Cdc42 and RhoA, regulate actin dynamics once they are activated

by GEFs such as Elmo/Dock complex44 and other kinases (e.g. FAK)45.

1.4.2. Microtubule cytoskeleton for efferocytosis

A microtubule is a hollow cylindrical structure consisting of 13 parallel proto-

filaments, each built from alternating α- and β-tubulin molecules46. It has a distinct

structural polarity with two distinct ends: a plus end that points to the periphery area

of the cell, and a minus end that is often anchored to the microtubule organization

center (MTOC), mostly centrosome47. The plus end, where both growth and

shrinkage are fast, is more dynamic and can undergo rapid polymerization and

depolymerization47. Transport of proteins or organelle cargos along microtubules is

accomplished by coordination of centrifugal and centripetal forces from kinesin and

dynein motor proteins respectively, which are powered by ATP hydrolysis,

permitting them to move in a stepwise fashion48. In this way, transport can be

directed either towards the periphery or center area of the cell.

The engulfment of apoptotic neutrophils accompanies cytoskeleton

15

reorganization and membrane ruffling in macrophages to facilitate phagocytic cup

formation and final internalization of apoptotic cells24, 27, 28, 49, 50. Microtubules

(MTs), a family of the main cytoskeleton filaments, function in numerous cellular

process, such as intracellular transport, organelle positioning, cell shape and cell

motility51. MTs undergo various covalent modifications. One of the most prevalent

MT modifications is tubulin acetylation51. Although there is a growing body of

evidence suggesting that tubulin acetylation might be involved in M2 polarization of

macrophages52, the underlying molecular mechanisms are poorly understood.

1.5. LC3-associated phagocytosis

A large body of evidence exhibits that autophagy plays essential roles in various

processes of immune reponses53. In macrophages, the autophagy-related proteins

can modulate the cell survival, regulate cytokine secretion and pathogen clearance54.

Recently, one of the autophagy-related proteins, microtubule-associated protein

1A/1B-light chain 3 (LC3), has been found that it is recruited to phagosome

membrane thereby facilitating degradation of apoptotic cells and pathogens55, 56. The

recruitment of LC3 to phagosome is mediated by the cooperation between a motor

protein, dynein, and a scaffold protein, JIP157. This process also requires

transporting tracks, microtubules, for a direction to phagosome. Hence, these

16

cytoskeletons are not only essential for initiating engulfment but also the eventual

degradation of apoptotic cells.

1.6. Phenotypic characteristics of macrophage during

resolution

Macrophages play central roles in the maintenance of tissue integrity during

development and its restoration after injury, as well as the initiation and resolution

of innate and adaptive immunity58. These cells carry out rapid engulfment and

clearance of apoptotic PMNs, a process called ‘efferocytosis’22, thereby preventing

exposure of intracellular content to cells at inflamed sites. Impaired efferocytosis

causes accumulation of dead cells, which consequently hampers timely resolution,

provoking pathologic conditions including atherosclerosis, rheumatoid arthritis,

systemic lupus erythematosus, etc59. Hence, the efforts to develop strategies based

on facilitating efferocytosis are a new area of resolution pharmacology. An early

efferocytosis, initiated by the pro-inflammatory (M1 type) macrophages, leads them

to become anti-inflammatory ones (M2 type) with enhanced efferocytic capability60.

Pro-resolving macrophages have been shown to be the M2 type61 which is

characterized by their property to release anti-inflammatory cytokines, such as

interleukin (IL)-10 and transforming growth factor β62. The activation of M2

macrophages is transcriptionally controlled by various proteins including signal

17

transducer and activator of transcription 6 (STAT6), interferon regulatory factor 4

(IRF4), CCAAT/enhancer-binding protein β (C/EBPβ) and peroxisome proliferator-

activated receptor γ (PPARγ)63. The efferocytic process is initiated by recognition of

the “eat me” signals transmitted from cells undergoing apoptosis64. One of the

molecular signatures in apoptotic cells is the exposure of phosphatidylserine on the

outer surface of their plasma membrane which is recognized by bridge molecules

(e.g., Gas6 and MFGE8)65, 66 and phosphatidylserine receptors (e.g., Bai1, Tim4 and

Stab2)24, 49, 67 expressed on the surface of M2 macrophages.

However, macrophage or monocyte populations might possess diverse

phenotypes that are neither M1 nor M268, despite experiencing the same

inflammatory cues. Macrophages together with dendritic cells (DCs) and myeloid-

derived suppressor cells (MDSCs) with the phenotype of Ly6Chigh character amplify

the ensuing adaptive immune response and maintained immune tolerance by

generating inducible regulatory T cells68. These macrophages are a hybrid of the

conventional M1- and M2-like cell phenotypes. The precise nature of macrophage

phenotypes involved in each disease will depend on which disease mechanism

predominates in a given organ. It indicates that there are diverse routes to resolution;

thus, it is unlikely that one single therapeutic intervention will be a panacea for all

chronic inflammatory diseases. Instead, it will be increasingly imperative to focus

on a specific pathology, appreciating both the heterogeneity within classical clinical

diagnoses and the factors that influence the inflammatory phenotype, to successfully

18

administer targeted pro-resolution therapy.

1.7. Role of c-Myc

Myc proteins, a family of proto-oncogenic transcription factors, composed of

three members, c-Myc, N-Myc and L-Myc, and regulate expression of extensive

genes thereby mediating diverse cellular events. The discovery of Myc is initiated

from P. Rous’ finding that entity from avian sarcoma filtrates could generate

tumors69. And an in-depth study identified an oncogenic gene which is named as v-

myc in myelocytomatosis from avian retrovirus MC29 in 1970s. Then researchers

found an analogous gene in cells (c-myc)70, 71. During cellular and tissue homeostasis,

c-Myc is regulated both at the transcriptional and post-transcriptional levels, and

constitutes a direct target and effector of growth-regulatory pathways, influencing

basic cellular processes such as cell proliferation, growth, apoptosis, energy

metabolism and various biosynthetic pathways72. c-Myc has been shown to be an

attractive target for treating diverse types of cancer. Direct evidence showed that

Myc inhibition triggers rapid regression of tumors by inducing apoptosis to tumor

cells but only cell cycle arrest of normal regeneration tissues which are well

tolerated and rapidly and completely reversible73.

In the last few years, c-Myc has been noted that it plays a role in alternative

activation of human macrophages and is proposed as one of the M2 macrophage

19

markers74, 75. It transcriptionally regulates some of M2-related genes in cultured

macrophages upon stimulation with external stimuli (e.g. IL-4), thereby facilitating

M2 polarization75. On the basis of this notion, enhancing c-Myc expression is likely

to be a potential strategy for seeking efficient resolution of inflammation. However,

whether c-Myc indeed favors the resolution of inflammation in vivo remained

overlooked. In contrary to the increase of c-Myc in vitro, I observed a decrease in

the pro-resolving macrophages, M2-like cells76. There is not any prerequisite

condition for facilitating gene expression of M2 genes by c-Myc. And macrophages

tend to terminate further synthesis of c-Myc at the beginning of inflammation and

eliminate protein at a later phase. Given the fact that macrophages engulf and

degrade apoptotic neutrophils during acute inflammation, the presence of high level

of c-Myc probably hampers the clearance of apoptotic cells, and hence, c-Myc

should be eliminated by the host.

1.8. Myc-nick

Myc-nick is a smaller form of c-Myc with an approximate molecular weight of

42 kDa. This truncated protein possesses amino acids 1-298 at the N terminal but

lacks C terminal domain including a nuclear localization sequence and DNA binding

domain. It is distinct from other variants of c-Myc on account of that its generation

is not directed by a translation of mRNA but a cleavage of an already exited protein.

20

This indicates that c-Myc has transcription-independent roles in cells. The

generation of Myc-nick is derived from a proteolytic cleavage at residue Lys 298,

catalyzed by a non-lysosomal cysteine protease, Calpain77. Calpains constitute a

superfamily since there are 15 genes identified in human and one or more transcripts

for each gene78. Activity of Calpains is directly associated with a binding of

calcium molecule to the catalytic core79, illustrating a requirement of calcium flux

for the c-Myc cleavage.

Myc-nick was firstly identified in differentiating cells and tissues such as

fibroblast cells and skeletal muscle77. In these cells or tissues, there is a great deal of

storage of calcium ion in mitochondrial and endoplasmic reticulum. The wave of

calcium has been characterized in neuron and muscle cells80 as well as epithelial

cells81. Yet it has not been well elucidated how calcium signal is stimulated for c-

Myc cleavage. The influx of calcium has been shown to dominate macrophages

activity during phagocytosis82, 83. This is consistent with my finding of Myc-nick in

engulfing macrophages. As Myc-nick was reported to be involved in α-tubulin

acetylation77, a modification that amplifies IL-10 secretion52, this prompted me to

investigate whether Myc-nick has a potential role in the resolution of inflammation.

A recent report for an over-expression of L-type calcium channels deciphers the

mechanism of filopodia formation in epithelial cancer cells84. A similar calcium flux

is likely to occur in stressed condition that accounts for the generation of Myc-nick85.

Subsequently the Myc-nick favors cancer cell survival and migration as well85, 86.

21

22

1.9. Perspectives

A spontaneous resolution is not only the course of inflammation termination but

also a process to assist tissue repair and restoration of homeostasis. The ideal pro-

resolving treatment should be started at the beginning of the resolution phase but not

the phase of inflammation initiation since the preceded inflammation is required for

elimination of toxic insults or ending continuous tissue damage (Fig. 1-2A).

However, traditional anti-inflammatory treatment neglect the importance of the

initial onset of inflammation. An immediate consequence is that this treatment

lessens the degree of inflammatory response (Fig. 1-2B). The treatment is

acceptable in case of that the host can achieve a similar resolution (①). Under the

circumstance of an inflammation related to tissue damage or microbial infection, it

may cause a compromise to aforementioned pathologic conditions. Therefore, the

resolution process will be prolonged (②) or even turn into unattainable (③). Many

researchers use pharmacological inhibition of inflammatory factors present during

the early inflammatory phase, or generate mice carry knockout/knock-in of genes

that involved in inflammation or resolution for this study. These conditions

correspond to an attenuated onset of inflammation as well. It makes an incomparable

condition to the control one. In the case of a pre-existed over-expression of pro-

resolving gene, mice with may be observed with higher inflammatory state than

23

those with normal expression if the resolution is delayed (②, ③). It is indiscernible

to the role of that

24

Figure 1-2. Strategies for inflammation termination.

(A) An ideal pro-resolving treatment is carried out at the time point when the host

gets the maximum inflammation. (B) Traditional anti-inflammatory treatment or

knockout of pro-inflammatory genes hamper inflammation initiation and reduces the

intensity of inflammation. Adopted from Fullerton and Gilroy. (2016).

25

gene. Hence, it is important design a comparable experimental condition for

studying resolution of inflammation4.

Resolution of acute inflammation is an effective approach against chronic

inflammation. Whether seeking resolution is equal for cancer therapy is an

interesting and urgent issue to be addressed. It has been extensively stated that

carcinogenesis is promoted by inflammation and pro-inflammatory mediators.

Although pro-resolving strategies have been considered for cancer therapy in recent

years5, the mechanisms remain largely unknown. Cancer cannot be cured if a

resolution merely achieves an elimination of inflammation or pro-inflammatory

mediators. On one hand, inflammatory cells involved in adaptive immunity, have a

substantial effect on cancer treatment. Cytokine-induced killer cell87, tumor-

infiltrating lymphocytes88, Natural killer cell89 and γδ T cells90 are being applied to

clinical investigation for anticancer therapy. On the other hand, tumor itself creates

an immunosuppressive environment which is similar to the condition for pro-

resolving events. Anti-inflammatory cells such as regulatory T cells (Tregs) and

tumor-associated macrophages (TAMs) jeopardize CD8+ T cell activation by

suppressing co-stimulatory ligands. Cytotoxic T cells do not regularly kill tumor

cells since they generally express checkpoint molecules91. Converting tumor to a

“hot” one is therefore considered to be a key factor for immunotherapy91. According

to these points, resolution conflicts with cancer therapy to some extent. Practically,

pro-resolving resolvins have been applied for cancer treatment, and these mediators

26

exert substantial effect92. It indicates that resolution may have more unknown effects

that have not been uncovered yet. A discovery of post-resolution, an adaptive

immune response after resolution, may provide one clue for this issue.

27

Chapter II

c-Myc Cleavage Mediates an Effective Efferocytosis And

a Consequent Resolution of Inflammation

28

2.1. Abstract

A key event required for effective resolution of inflammation is efferocytosis,

which is defined as phagocytic removal of apoptotic cells mostly by macrophages

acquiring an alternatively activated phenotype (M2). c-Myc has been reported to

play a role in alternative activation of human macrophages and is proposed as one of

the M2macrophagemarkers. I found that M2-like peritoneal macrophages from

zymosan A–treated mice exhibited marked accumulation of Myc-nick, a truncated

protein generated by a Calpain-mediated proteolytic cleavage of full-length c-Myc.

Further, ectopic expression of Myc-nick in murine bone marrow-derived

macrophages promoted the M2 polarization and, consequently, enhanced their

efferocytic capability. Notably, Myc-nick-induced efferocytosis was found to be

tightly associated witha-tubulin acetylation by K acetyltransferase 2a (Kat2a/Gcn5)

activity. These findings suggest Myc-nick as a novel pro-resolving mediator that has

a fundamental function in maintaining homeostasis under inflammatory conditions.

29

2.2. Introdutcion

Resolution of inflammation is an active process that not only halts pro-

inflammatory signaling, but also normalizes a chemokine gradient to clear apoptotic

polymorphonuclear neutrophils (PMNs) at the inflamed tissue site6. Phagocytes,

particularly macrophages, mediate rapid engulfment and clearance of apoptotic cells

in a process called efferocytosis22. An early efferocytosis, initiated by the classically

activated M1 type macrophages, leads them to become alternatively activated ones

(M2 type) with enhanced efferocytic capability60. The M2 polarized macrophages

are characterized by their ability to release anti-inflammatory cytokines, such as IL-

10 and TGF-β62. Impaired efferocytosis causes accumulation of dead cells, which

consequently hampers timely resolution, provoking pathologic conditions including

atherosclerosis, rheumatoid arthritis, systemic lupus erythematosus, etc.59. The

efferocytic process is initiated by recognition of the “eat me” signals transmitted

from cells undergoing apoptosis64. One of the molecular signatures in apoptotic cells

is the exposure of phosphatidylserine on the outer surface of their plasma membrane

which is recognized by bridge molecules [e.g., Gas6 (growth arrest–specific 6) and

MFGE8 (milk fat globule–EGF factor 8 protein)]65, 66 and phosphatidylserine

receptors [e.g., Bai1 (brain-specific angiogenesis inhibitor 1)24, Tim4 (T-cell

30

immunoglobulin and mucin domain containing 4)67, and Stab2 (stabilin 2)49]

expressed on the surface of M2 macrophages.

c-Myc, a transcription factor, has been proposed as an authentic marker of M2

macrophages74, 75. It transcriptionally regulates some of the M2-related genes in

cultured human macrophages stimulated with IL-475. However, whether this

transcription factor indeed favors resolution of inflammation in vivo remained

overlooked. This prompted us to assess possible roles of c-Myc in efferocytosis and

eventual resolution of experimentally induced acute inflammation. Notably, there

was accumulation of Myc-nick, a truncated protein generated by a Calpain-mediated

proteolytic cleavage of fulllength c-Myc. Compared with full-length c-Myc, little

has been known about physiologic functions of Myc-nick.

The engulfment of apoptotic neutrophils accompanies cytoskeleton

reorganization and membrane ruffling in macrophages to facilitate phagocytic cup

formation and final internalization of apoptotic cells22. Microtubules (MTs), a major

family of cytoskeleton filaments, function in many cellular processes, such as

intracellular transport, organelle positioning, cell shape, and cell motility51. A

protein called tubulin is the basic structural unit of MTs. MTs undergo various

covalent modifications. One of the most prevalent MT modifications is tubulin

acetylation. Although there is growing body of evidence suggesting that tubulin

acetylation might be involved in M2 polarization of macrophages52, the underlying

molecular mechanisms are poorly understood. As Myc-nick was reported to be

31

involved in a-tubulin acetylation77, I investigated the potential role of Myc-nick in

the resolution of inflammation.

32

2.3. Materials and methods

2.3.1. Materials

Dulbecco’s modified Eagle’s medium (DMEM) medium, penicillin,

streptomycin and fetal bovine serum were obtained from Gibco-BRL (Grand Island,

NY, USA). Antibody against c-Myc-N-terminal was bought from Abcam plc.

(Cambridge, UK). Zymosan, dexamethasone and antibodies against actin and

acetylated tubulin were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

Calpain inhibitor VI, Kat2a/Gcn5 inhibitor cyclopentylidene-[4-(4'-

chlorophenyl)thiazol-2-yl)hydrazine (CPTH2) and antibodies against c-Myc (N262)

and Pparγ were provided by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Recombinant murine IL-4 was a product from PEPROTECH (Rocky Hill, NJ, USA).

Recombinant mouse M-CSF and carboxylfluorescein succinimidyl ester (CFSE)

were products from BioLegend Inc. (San Diego, CA, USA). pHrodo red,

succinimidyl ester (SE) was provided by Thermo Fisher Scientific Inc. (Waltham,

MA, USA). Red blood cell lysis buffer was a product from iNtRON biotechnology.

Antibodies against acetylated-lysine and LC3B were supplied by Cell Signaling

Technology, Inc. (Danvers, MA, USA). The anti-rabbit and anti-mouse horseradish

peroxidase- conjugated secondary antibodies were obtained from Zymed

Laboratories (San Francisco, CA, USA). Polyvinylidene difluoride (PVDF)

membrane was purchased from Gelman Laboratory (Ann, Arbor, MI, USA). The

33

enhanced Chemiluminescent (ECL) detection kit was obtained from Amersham

Pharmacia Biotech (Buckinghamshire, UK). pIRESneo-EGFP-α tubulin was a gift

from Patricia Wadsworth (Addgene plasmid #12298). pIRESneo-EGFP-α tubulin

K40R vector was generated by site-direct mutagenesis from the wild type vector.

Myc-nick vectors were created by cloning mouse cDNA sequence expressing a

truncated c-Myc including 1-298 amino acid residues into pTurbo-RFP and PCS2-

3Flag vectors respectively.

2.3.2. Methods

2.3.2.1. Cell culture

Murine NIH/3T3 fibroblasts were obtained from American Type Culture

Collection (ATCC, Manassas, VA, USA). The primary peritoneal macrophages

were elicited by using thioglycollate and isolated from mouse peritoneum. NIH/3T3

cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) and peritoneal

macrophages were cultured in DMEM Nutrient Mixture F-12 (DMEM/F-12). Both

media were supplemented with 10% v/v FBS and 100 units/mL penicillin and 100

μg/ml streptomycin at 37oC in an incubator with humidified atmosphere of 95%

O2/5% CO2.

2.3.2.2. Zymosan- and thioglycollate-induced peritonitis

To induce peritonitis, 6-8-week-old C57BL/6 mice obtained from ORIENT Bio,

34

Inc. (Seoul, South Korea) were intraperitoneally injected with zymosan (5 mg/kg) or

1 ml thioglycollate broth (3%). For isolation of PMs, total leukocytes were collected

from peritoneal lavage fluid followed by an incubation with red blood cell (RBC)

lysis buffer (iNtRON Biotechnology) to eliminate RBCs. The cells were then

allowed to attach to culture plates by culturing in DMEM/F12 media for 2 h. After

removing non-adherent cells, PMs isolated at different time points (12 h, 18h, 24 h,

day 2, day 3 and day 6) were used for further assessment of cell characteristics. For

evaluation of in vivo efferocytosis, after clearing red blood cells, total leukocytes

were stained by an F4/80 antibody to label monocytes/macrophages. Next,

neutrophils were recognized by a Gr-1 antibody after a permeabilization of cell

membrane. Samples were then applied to flow cytometric assay.

2.3.2.3. Preparation of bone marrow derived macrophages (BMDMs)

Isolation of BMDMs was performed as previously described93. In brief, femur

and tibia were isolated from 8 weeks old C57BL/6 mouse. Bone marrow was

flushed out by cold PBS containing 2% heat inactivated fetal bovine serum (FBS)

via 3 ml syringe. Cells were dissociated to be single by passing through 23G syringe

needle for 4-6 times. The cells from bone marrow were then filtered through 70 μm

FALCON nylon cell strainer (Thermo Fisher Scientific Inc) to remove bone, hair,

cell clumps, and other cells/tissues, and then centrifuged at 500 x g for 5 min at 4 oC.

RBCs were then removed by a lysis buffer (iNtRON Biotechnology) by incubating

35

on ice for 1 min. After removing lysis buffer, the remained cells were re-suspended

in DMEM containing 10% heat inactivated FBS and 20 ng/ml M-CSF, seeded in

petri dishes at around 2x106 cells/plate and incubated at 37oC for 7 days in a

humidified incubator containing 5% CO2. Media containing M-CSF was changed

once after 3 days incubation. Bone marrow derived macrophages were detached

from petri dishes and seeded again into new culture plates for additional experiment.

2.3.2.4. Isolation of mouse thymocytes and generation of apoptotic cells

The thymus was collected from C57BL/6 mouse and ground between frosted

ends of two microscope glass slides until completely homogenized without

destroying cells. Collected cells were filtered with 40 μm FALCON nylon cell

strainer (Thermo Fisher Scientific Inc.) to remove remained tissue and clumped cells.

Cells were washed twice with PBS, followed by resuspension in DMEM medium at

107 cells/ml, and then cultured at 37°C in a humidified incubator containing 5% CO2

(Miksa et al., 2009). Apoptosis of cells was triggered by treatment of 0.1 μM

dexamethasone (DEX) to thymocytes for 16 h.

2.3.2.5. Efferocytosis assay

The apoptotic cells were collected and stained with 1 μl of 1 mg/ml pHrodo-SE

in 50 ml cell suspension (final concentration 20 ng/ml and 106 cells) for 30 min at

room temperature. The labelled Cells were collected by centrifugation and washed

36

twice with PBS and re-suspended in Opti-MEM. For 1×106 BMDMs on a 60 mm

plate, 3×106 pHrodo-SE-labelled apoptotic thymocytes were added in 4 ml medium

and incubated at 37°C in a humidified atmosphere containing 5% CO2 for 1 h.

Efferocytosis was verified under the light microscope prior to labeling and detaching

cells. After incubation, the BMDMs were washed to remove free apoptotic cells,

treated with 5 mM EDTA, fixed with 2% formaldehyde and analyzed by flow

cytometry assay or confocal microscopy. To quench fluorescence from non-

phagocytized pHrodo-SE-labelled apoptotic cells, cells were washed and re-

suspended in basic buffer (pH 8.8) before flow cytometry assay 94. For an in vivo

assay, mice were pretreated with Calpain inhibitor VI or vehicle (2.5% DMSO and 2%

methylcellulose in PBS) for 4 hours before i.p. injection of pHrodo-SE-labelled

apoptotic thymocytes. The peritoneal cells were isolated and analyzed at 2 h post-

injection of apoptotic cells by gating out F4/80- cells.

2.3.2.6. Flow cytometry assay

The samples were prepared for flow cytometry assay as described earlier95.

Briefly, cells ready to analyze were detached from plates and prepared by spinning

down 5 min at 300 x g, 4oC. For assessment of intracellular proteins, cells were

fixed by 2% formaldehyde in PBS for 30 min at 4oC. A permeabilization of cells

was performed by 0.2% Tween-20 in PBS for 15 min at 37oC. For staining cell

surface proteins, it is optional to fix cells but necessary to avoid permeabilization.

37

To block non-specific antibody binding, the cells were incubated with an unlabeled

isotype control antibody in staining buffer containing 1% bovine serum albumin

(BSA; fraction V) and 0.1% sodium azide (NaN3) in PBS for 15 min. Following to

fully removing free isotype control antibody, an additional incubation with CD16/32

antibody was performed to block non-specific binding to Fc receptors. Specific

antibodies w/o fluorophore conjugation were added to samples in the presence of

CD16/32 antibody and incubated for 30 min. For use of non-conjugated primary

antibodies, the fluorophore-conjugated secondary antibodies were used to stain for

another 30 min. Samples were analyzed by a FACSCalibur™ flow cytometer (BD,

Franklin Lakes, NJ) and Flowjo software.

2.3.2.7. Immunofluorescent staining and imaging

Immunofluorescent imaging was performed as described previously96. Briefly,

cells were cultured on sterilized coverslips up to 20-50% confluent before

experiment. Following fixation of cells by incubation in PBS with 2% formaldehyde

at room temperature for 10-15 min, cells were permeabilized for 5 min at room

temperature using 0.2% Triton X-100 in PBS. To block nonspecific sites of antibody

adsorption, cells were blocked with 3% bovine serum albumin (BSA) in PBS

containing 0.1% Triton X-100 for 1 h at room temperature. Primary antibody was

diluted in PBS containing 1% BSA and added to samples for incubation for 1 hour

at room temperature or overnight at 4oC. Fluorophore-conjugated secondary

38

antibodies (Alexa fluor® 488 or 568) were incubated for another 1 hour at room

temperature. Samples were imaged at 20x using an Eclipse Ti-U inverted

microscope integrated by an NIS-Elements imaging software (Nikon, Japan). Image

analysis was performed using ImageJ (http://imagej.net) software for correcting

background, splitting channels, measuring fluorescent intensity and analyzing co-

localization.

2.3.2.8. Western blotting and PCR analysis

Cells were collected, and lysed in ice-cold lysis buffer [2% Triton X-100,

10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM NaN3, and 2 mM EDTA]

containing protease inhibitors (Roche Life Sciences). Animal tissue was

homogenized using a homogenizer in cold lysis buffer. Lysates were harvested and

centrifuged to eliminate nuclei. The protein concentration was determined using the

BCA protein assay reagents, and 20 μg of protein was electrophoresed in a SDS-

PAGE under reducing conditions and transferred to PVDF membranes. The

membranes were then blocked with 5% w/v nonfat dry milk in phosphate buffered

saline containing 0.1% Tween 20 (PBST) buffer for 1 h at room temperature.

Afterwards, the blots were incubated with the primary antibody diluted in 3% non-

fat milk PBST buffer overnight at 4 °C. After washing, the blots were incubated

with proper horseradish peroxidase-conjugated secondary antibody. The

immunoblot with protein-antibody complexes were detected by using an enhanced

39

chemiluminescence detection kit (Amersham Pharmacia Biotech, Buckinghamshire,

UK). Cells for PCR analysis were applied to mRNA isolation using TRIzolTM

Reagent (Thermo Fisher Scientific) and cDNA creation by RT-PCR. Amplification

of genes was performed by PCR analysis using SolgTM 2x Taq PCR Smart Mix

(Solgent, Korea) according to instruction from the manufactures. Primer sequences

are indicated in Supplementary Table 1. PCR product were run on 2% agarose gel

followed by a staining of DNA using SYBR® Safe DNA gel stain (Thermo Fisher

Scientific, USA). Digital image analysis of Western blot and PCR were performed

using the ImageQuantTM LAS 4000 luminescent image analyzer (GE Healthcare

Life Sciences, USA).

2.3.2.9. Bioinformatic Studies

For hierarchical cluster analysis, microarray data were obtained from the

ArrayExpress (E-MEXP-3189)97. Expression values were extracted after

normalization of original data using rma method. For profile of single gene

expression, expression values were extracted from expression set directly and

plotted into graph. While differentially expressed genes (DEGs) were selected

according to statistical significance (FDR < 0. 05) in the macrophages isolated from

zymosan-treated mice (day 3) compared to those from naive mice. DEGs were

further selected according to gene ontology terms related to calcium transport using

DAVID 6.7 software (Huang et al., 2009). DEGs mapped into clusters using cluster

40

3.0 (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm) and visualized

and analyzed by Java TreeView program. Statistical computing microarray data was

performed in Bioconductor (R version 3.4.0).

2.3.2.10. Statistical analysis

All data are analyzed by means ± SE and are based on experiments performed at

least in triplicate. Statistical significance was calculated with the Student’s t-test and

Sigmaplot software.

41

2.4. Results

2.4.1. Murine macrophages have diverse characteristics at different

stages.

To identify pro-resolving macrophages, I utilized a self-resolving model of

zymosan A-induced murine peritonitis. Intraperitoneal (i.p.) injection of zymosan A

caused inflammation of peritoneum with a maximal accumulation of PMNs at 12 h.

On account of an almost elimination of neutrophils at 24 h, the resolution phase is

appropriately defined as the period from this point to the complete resolution (day 6).

To characterize PMs, I performed flow cytometry analysis by staining cells for

several markers with a gating strategy shown in Fig. 2-1A. In the zymosan-induced

peritonitis, the tissue-resident macrophages (CD11bhighF4/80highLy6c-) were replaced

by monocyte-derived infiltrating macrophages (CD11bintermediate (int) F4/80lowLy6c+/-)

at the inflammatory phase and hereafter by pro-resolving macrophages (CD11bint-

highF4/80intLy6c-) during resolution (Fig. 2-1B, C). I reasoned that the day 3 when

PMs did not present Ly6c (Fig. 2-1C, top) and regained expression of Tim4 (Fig. 2-

1C, bottom), a marker of tissue-resident macrophages in naïve mice, would be

appropriate for the evaluation of pro-resolving macrophages. Further, tissue resident

macrophages (CD11bhiF4/80hiTim4+) are likely to dominate the initiation but not

later phases of peritonitis since these cells take only a small proportion of

macrophages at later phases.

42

Figure 2-1. Constitution of peritoneal leukocytes upon zymosan-induced

peritonitis.

43

A resolving peritonitis model was established by intraperitoneally injection of

Zymosan (5 mg/kg) for 0 h, 18h, 3 days and 6 days. (A) Gating strategy for

identifying peritoneal macrophages based on surface molecules. (B)

Characterization of peritoneal macrophages by CD11b and F4/80 at 0 h, 18h, day3

and day6. (C) Macrophages were gated by CD11bhighF4/80+ population and

followed by analysis of Ly6c (top panels) and Tim4 (bottom panels). Data shown

are representative of 3 experiments.

44

2.4.2. During the resolution of zymosan A-induced murine

peritonitis, pro-inflammatory macrophages switch to pro-resolving

macrophages with an M2-like phenotype.

To unravel the activation states of the pro-resolving macrophages, I analyzed the

public GEO database (GSE28621). I executed an unbiased analysis of murine

M1/M2 markers and transcription factors that were validated previously. In general,

infiltrating macrophages acquired an inflammatory feature at an early stage (4 h). As

shown in Fig. 2-2A, the canonical M1 markers, such as CD80, CD86, TNFα and IL-

1β, were upregulated in M1 array. Simultaneously, there was a downregulation of

M2 markers such as CD163, Fizz1 (Retnla) and Arg1 in M2 array. A quantitative

PCR (qPCR) indicated that, at a late inflammatory phase (18 h), macrophages

manifested an intermediate feature which was reflected by upregulation of some M2

markers including CD206 (Mrc1), CD163 and Fizz1 (Retnla) and maintained the

high expression of M1 markers, such as GPCR18 (Gpr18), IL-1β, and TNFα. At a

resolution phase, PMs elicited enhanced expression M2 markers completely and

downregulation of M1 markers. A similar change of M1 and M2 genes was also

observed in sorted macrophages from mice treated with zymosan A for indicated

time (Fig. 2-2B, C). I therefore defined late inflammatory stage (18 h) as an

intermediate phase which bridged the gap between inflammation and resolution.

These findings suggest a development of pro-resolving macrophages from

inflammatory cells, which is consistent with a vanishing of tissue-resident

45

macrophages (CD11bhighF4/80high) 4

Figure 2-2. Gene expression of M1 and M2 markers in peritoneal macrophages.

(A) Heatmaps of expression levels of M1 and M2 markers in the zymosan-induced

peritoneal macrophages. The data were extracted from GSE28621. (B, C)

46

Quantitative reverse-transcription polymerase chain reaction assays (qPCR) of IL-10

(Il10), Arg1, Fizz (Retnla) and CD206 (Mrc1) (B), and iNOS (Nos2), IL-6 (Il6) and

TNFα (Tnf) (C) in sorted PMs as indicated in Fig. 1A. Data represent the means ±

s.e.m. (replication number n = 3, *p < 0.05, **p < 0.01).

47

h (data not shown). Therefore, I propose that pro-resolving macrophages undergo an

M2 polarization from M1 macrophages. I noticed that there was an additional influx

of monocyte-derived macrophages (Ly6c+) at the day 6 (Fig. 2-1B). It probably

represents an influx of cells that trigger adaptive immune response.

2.4.3. Full length c-Myc is cleaved into Myc-nick during the

resolution of zymosan-induced peritonitis.

A further analysis of PMs by using two antibodies that recognize N- and C-

terminal c-Myc epitopes revealed a presence of a cell population with positive signal

of N-terminus but negative signal of C-terminus. The proportion of these cells

increased at an intermediate phase (18 h) and throughout the resolution phase (3 d;

Fig. 2-3A). This implies that some of the PMs underwent a cleavage of c-Myc

which was previously observed in fibroblasts. The resulting truncated protein merely

detected by N-terminal antibody, termed as Myc-nick, lacks the C-terminal portion

of c-Myc and an interior nuclear localization sequence while preserving an intact N

terminus comprising Myc box regions. An in vitro efferocytosis was performed to

mimic the in vivo engulfment of apoptotic cells during a resolution. The cleavage

was further verified by a Western blotting using the anti-C-terminal c-Myc antibody

(Fig. 2-3B). The PMs isolated at the day 3 exhibited a dramatic reduction of full-

length c-Myc and a significant accumulation of Myc-nick.

48

Figure 2-3. Cleavage of c-Myc into Myc-nick during the resolution of peritonitis.

(A) Flow cytometric plots shows a cleavage of c-Myc. The cleavage was indicated

by the percentage of cells showing N-terminal (N-term) signal but not C-terminal

(C-term) signal. Data are representative of 3 experiments. (B) Detection of c-Myc

and Myc-nick in PMs from naive and zymosan-treated mice by WB. The isolation of

macrophages were performed at the day 3 after zymosan treatment. Data represent

the means ± s.e.m. (replication number n = 3-5, **p < 0.01, ***p < 0.001.).

49

2.4.4. Myc-nick localizes in the cytoplasm.

To specify the location of Myc-nick, I performed immunofluorescent staining by

using those two antibodies detecting N- and C-terminal domains of c-Myc. I

determined the co-localization as well as the distribution of nuclear vs. cytoplasmic

N- and C-terminal signals. As shown in Fig. 2-4A, naive PMs showed a co-

occurrence of fluorescent signals detectable by both antibodies, suggesting that these

cells maintained full length c-Myc. PMs collected at 18 h and day 3 of zymosan A

administration exhibited spatially separated fluorescence derived from N- and C-

terminal domains of c-Myc, indicative of a generation of Myc-nick. In line with this

notion, there was a reduced co-localization of two signals and a significantly

reduced ratio of nuclear vs. cytoplasmic N-terminal signal. NIH/3T3 cells

transfected with a RFP-tagged Myc-nick also exhibited a cytoplasmic localization

(Fig. 2-4B).

2.4.5. Myc-nick production is associated with efferocytosis.

Considering an important role of macrophages in clearance of apoptotic cells, I

investigated possible association between peritonitis-induced Myc-nick production

in PMs and their efferocytic activity. An in vitro efferocytosis assay was performed

to mimic the in vivo engulfment of apoptotic neutrophils, a key event that occurs at

intermediate phase (Fig. 2-5A). Myc-nick production was assessed by both flow

cytometric and immunofluorescence analyses to measure PMs retaining

50

predominantly the N-terminus of c-Myc. I observed a significant increase of the

Figure 2-4. Localization of Myc-nick.

51

(A) Immunocytometry analysis of c-Myc cleavage and subsequent production of

Myc-nick in sorted PMs (green: N-term, red: C-term, blue: DAPI). The cleavage

was shown by the extent of co-localizaiton of N- and C-terminal signals and the

mean fluorescence intensities (MFIs) ratio (nucleus/cytoplasm) of each signal. (B)

Localization of RFP or RFP-tagged murine Myc-nick in NIH/3T3 murine fibroblast

cells (green: α-tubulin; red: RFP or RFP-Myc-nick; blue: DAPI). Scale bars

represent 5 mm. Data represent the means ± s.e.m. (*p < 0.05, **p < 0.01.).

52

Figure 2-5. Myc-nick production by efferocytosis.

(A) Efferocytosis of apoptotic thymocytes by BMDMs (green: CFSE-labelled

BMDMs; red: pHrodo-SE-labelled apoptotic thymocytes; blue: DAPI). (B) The

assessment of c-Myc cleavage into Myc-nick by flow cytometry at 4 h after

53

efferocytosis. A time course is shown in Supplemental Fig. 3A, B. (C) The

representative of c-Myc cleavage in macrophages after engulfment of apoptotic

thymocytes by immunofluorescent staining of c-Myc (green: N-term; red: C-term;

blue: DAPI). (D) WB and (E) flow cytometry assay of Myc-nick generation in

BMDMs. Scale bars represent 20 mm. Data represent the means ± s.e.m. (replication

number n = 3, ***p < 0.001.).

54

proportion of the PMs producing Myc-nick at 4 h after co-incubating BMDMs with

apoptotic thymocytes (Fig. 2-5B, C). The maximum cleavage was observed at 4 h

(Fig. 2-5D) and the proportion of cleaved cells increased as the incubation time

elongated (Fig. 2-5E), which is consistent with an increased engulfing cells.

2.4.6. Myc-nick enhances efferocytic activity of macrophages.

To determine the role of Myc-nick in efferocytosis, I generated a Myc-nick

vector by cloning cDNA encoding N-terminal amino acids (1-298) of c-Myc.

Compared with control cells transfected with a mock vector, those expressing

ectopic Myc-nick exhibited an enhanced uptake of apoptotic thymocytes (Fig. 2-6A,

B). I extended these findings by blocking cleavage of c-Myc using Calpain inhibitor

VI (Fig. 2-6C). The efferocytic ability of BMDMs was consequently dampened in

the presence of Calpain inhibitor VI (Fig. 2-6D). I next assessed the possible

involvement of endogenously produced Myc-nick in inducing activity of

macrophages in the peritoneum of zymosan-treated mice in vivo. Inhibition of Myc-

nick production by blocking a c-Myc cleavage by administration of Calpain

inhibitor VI led to a substantial reduction in the proportion of macrophages

engulfing apoptotic thymocytes (Fig. 2-6E).

55

Figure 2-6. Effects of Myc-nick overexpression and inhibition of its production

on efferocytosis.

(A, B) Flow cytometric (A) and immunofluorescent (B) analyses of efferocytosis by

BMDMs following transfection with mock or Myc-nick vectors (green: CFSE-

56

labelled BMDMs; red: pHrodo-SE-labelled apoptotic thymocytes; blue: DAPI). (C)

Suppression of Myc-nick production from c-Myc by Calpain inhibitor VI. (D)

Efferocytosis by BMDMs in the absence or presence of Calpain inhibitor VI (10

mM). (E) Effect of Calpain inhibitor VI (i.p., 10 mg/kg) on engulfment of apoptotic

thymocytes in vivo. Data represent the means ± s.e.m. (replication number n = 3 to 5,

*p < 0.05, **p < 0.01.).

57

2.4.7. Myc-nick promotes M2 polarization of macrophages during

efferocytosis.

In another experiment, BMDMs treated with apoptotic cells were assessed for

the activation state after a complete digestion at 24 h. These cells showed an

upregulation of M2 markers including Il10, Arg1, Retnla and Mrc1 (Fig. 2-7A).

Ectopic overexpression of Myc-nick further enhanced the extent of the M2 state of

BMDMs upon digestion of apoptotic cells (Fig. 2-7B). On the contrary, the

blockage of Myc-nick generation by use of Calpain inhibitor VI attenuated the M2

polarization of macrophages (Fig. 2-7C). The resolution state of mice treated with

zymosan was assessed at the day 3 with or without Calpain inhibitor treatment. A

normal resolution upon zymosan administration was indicated by disappearance of

inflammatory macrophages expressing Ly6c (Fig. 2-1C and 2-7D). Mice co-treated

with the Calpain inhibitor VI retained the Ly6c+ inflammatory macrophages to

some extent (Fig. 2-7D). I also observed an increase of F4/80low macrophages in

mice treated with Calpain inhibitor VI, which revealed an influx of inflammatory

macrophages (Fig. 2-6E).

58

Figure 2-7. M2 polarization of macrophages promoted by ectopically expressed

Myc-nick during efferocytosis.

(A-C) Measurement of mRNA expression of IL-10 (Il10), Arg1, Fizz (Retnla) and

59

CD206 (Mrc1) in BMDMs (n = 3-4 each) by qPCR assays in the absence or

presence of apoptotic thymocytes (A), in the presence of apoptotic thymocytes with

or without ectopic expression of Myc-nick (B) and in the absence or presence of

apoptotic thymoctyes and/or Calpain inhibitor VI (C). Analysis of these genes was

carried out at 24 h after incubation with apoptotic thymocytes. The Myc-nick vector

was cloned from cDNA sequence containing 1-298 amino acids of c-Myc. (D)

Evaluation of the resolution of zymosan-induced peritonitis in mice treated with

vehicle or Calpain inibitor VI at day 3 (n = 3/group). All values show means ± s.e.m.

(error bars). Asterisks indicate statistical significance (*p < 0.05, **p < 0.01, ***p <

0.001).

60

2.4.8. Myc-nick potentiates efferocytosis through α-tubulin

acetylation.

In a subsequent experiment, I attempted to elucidate the mechanism by which

Myc-nick induces efferocytosis and M2 polarization. Myc-nick recruits

acetyltransferases and thereby it acetylates the cytoskeletal protein α-tubulin.

Although the physiological significance of α-tubulin acetylation remains largely

unresolved, I found that acetylated α-tubulin is involved in phagocytic cup

formation during engulfment of apoptotic cells by macrophages (Fig. 2-8A). In

accord with this finding, an upregulation of acetylated α-tubulin was detected in

BMDMs expressing ectopic Myc-nick (Fig. 2-8B). Gcn5 is an acetyltransferase that

mediates Myc-nick-induced acetylation of α-tubulin. The accumulation of acetylated

α-tubulin was diminished upon treatment of CPTH2, an inhibitor of Gcn5 (Fig. 2-

8C). Further, I measured the efferocytic ability of PMs in the presence of CPTH2.

As illustrated in Fig. 2-8D, the efferocytic index declined markedly in CPTH2-

treated macrophages compared to vehicle-treated cells. To ensure that the

acetylation of tubulin is directly involved in the process of efferocytosis, I

transfected PMs with the wild-type (WT) and mutated (K40R) α-tubulin. The K40R

mutation of α-tubulin could mimic an acetylation deficiency in Lysine 40.

Compared with macrophages transfected with WT α-tubulin, those harboring the

non-acetylatable mutant form (K40R) of α-tubulin showed significantly lower

capability of engulfing apoptotic thymocytes (Fig. 2-8E). Therefore, α-tubulin

61

acetylation is considered essential for efficient engulfment of

Figure 2-8. Involvement of acetylated α-tubulin in efferocytosis and subsequent

M2 polarization.

(A) Representative of fluorescent microscopic graphs showing different localization

62

pattern of α-tubulin and acetylated α-tubulin in macrophages with or without

engulfment of apoptotic thymocytes (green: α-tubulin; red: Ac-tubulin; blue: DAPI;

arrow: phagocytic cup). Engulfed apoptotic cells are recognized by their nucleuses

(blue) in the peripheral region of macrophages. Scale bars represent 20 mm. (B, C)

Western blot of acetylated α-tubulin in the macrophages transfected with Mock or

Myc-nick vector (B) and the cells with or without treatment of Gcn5 inhibitor

(CPTH2; C). (D, E) Efferoctyosis of apoptotic thymocytes by BMDMs with or

without treatment of CPTH2 (D) and BMDMs transfected with WT or K40R GFP-

α-tubulin (E). (F) qPCR assays for measuring the mRNA transcripts of IL-10 (Il10),

Arg1, Fizz (Retnla) and CD206 (Mrc1) in BMDMs expressing WT or K40R α-

tubulin (n = 3-4 each). The values represent means ± s.e.m. and are representative of

at least three experiments. Asterisks indicate statistical significance (*p < 0.05, **p

< 0.01; ns: no significance).

63

Figure 2-9. Involvement of acetylated α-tubulin in the expression profile of M1

and M2 markers.

(A) Representative Western blots of in the macrophages transfected with GFP-α-

tubulin vectors without or with mutation (K40R). (B) The expression levels of TNFα

(Tnf), IL-6 (Il6) and iNOS (Nos2) in the BMDMs expression WT or K40R GFP-α-

tubulin.

64

apoptotic cells during efferocytosis.

I next investigated whether α-tubulin acetylation could affect the M1 or M2 state.

For this purpose, I utilized acetylation-resistant mutant α-tubulin (K40R). Compared

to the cells harboring WT α-tubulin, those transfected with K40R α-tubulin

exhibited the reduced expression of M2 marker genes, Arg1, Retnla, and Mrc1 (Fig.

2-8F). In contrast, the expression levels of M1 genes including Tnf, and Il6 were

significantly increased in the cells expressing mutated α-tubulin (Fig. 2-9).

2.4.9. Myc-nick induces LC3-associated phagocytosis (LAP).

While investigating the role of Myc-nick in efferocytosis, I came across its co-

localization with LC3, one of the most well-defined autophagy-related proteins (Fig.

2-10A). Since LC3 is associated with efficient degradation of apoptotic bodies upon

engulfment, and its lapidated form (LC3-II) dominates phagosome maturation, I was

tempted to determine whether Myc-nick-induced α-tubulin acetylation is causally

related to the accumulation of LC3-II. The inhibition of tubulin acetylation by the

Gcn5 inhibitor, CPTH2 was accompanied by a decline in the level of LC3-II (Fig. 2-

10B), suggesting that acetylated-tubulin is required for LC3-II accumulation. I

further investigated whether LC3 could be involved in efferocytosis. The ex vivo

efferocytosis experiment revealed an upregulation of LC3-II expression (Fig. 2-10C)

and accumulation of LC3 in the membrane of phagosomes with totally engulfed

apoptotic bodies (Fig. 2-10D, arrow). However, LC3 accumulation was not evident

65

Figure 2-10. LC3-associated phagocytosis is involved in Myc-nick-induced

efferocytosis.

(A) Co-localization of LC3 (green) with Myc-nick (red) in thioglycollate-elicited

PMs transfected with RFP tagged Myc-nick vector (blue: DAPI; dash: outline of

66

macrophages). (B, C) Western blot analysis of LC3-II expression in thioglycollate-

elicited PMs in the absence or presence of CPTH2 (B) and apoptotic thymocytes (C).

(D) Localization of LC3 (green) in PMs with and without engulfment of apoptotic

cells (magenta). Arrow indicates LC3 recruited to phagosomes after efferocytosis,

and asterisk represents a tethered apoptotic cell without accumulation of LC3. (E)

Localization of LC3 (green) and acetylated tubulin (red) in macrophages with and

without engulfment of apoptotic cells (recognized by peripheral nucleuses). Arrow

indicates co-accumulation and co-localization of LC3 and acetylated tubulin to

phagosomes after efferocytosis. (F) Immunofluorescent images of LC3 (green) and

acetylated tubulin (magenta) in macrophages expressing mock or Myc-nick vectors.

Arrows indicate accumulation and co-localization of LC3 and acetylated tubulin.

Scale bars represent 20 mm.

67

in the area adjacent to dead cells tethered by macrophages (Fig. 2-10D, marked with

asterisk), suggesting that LC3 is unlikely to be involved in phagocytic cup formation

but rather mediates later events after internalization. Since acetylated tubulin was

reported to recruit LC3-II for efficient digestion of dead bodies, I assume that the

acetylated tubulin accumulation during engulfment of apoptotic cells might facilitate

the translocation of LC3-II to internalized phagosomes. The fluorescent data showed

co-localization of acetylated tubulin with LC3 in the membrane of phagosomes (Fig.

2-10E, arrow). Likewise, the expression of ectopic Myc-nick in macrophages

resulted in remarkable interaction between LC3 and acetylated tubulin (Fig. 2-10F,

arrow).

68

2.5. Discussion

Because of its prominent role in alternative macrophage polarization, c-Myc has

been suggested as an M2 marker74, 75, 98 in cultured human or murine macrophages.

However, it is unclear whether it has similar functions in the murine peritoneum in

vivo. I initially explored the potential involvement of c-Myc in resolution of

experimentally induced acute inflammation by using the zymosan-induced murine

peritonitis model. Surprisingly, I discovered that pro-resolving macrophages

exhibiting an M2 polarized phenotype have a substantially reduced level of c-Myc.

Moreover, by analyzing databases available in public domain, I found a decrease of

c-Myc mRNA immediately after zymosan administration (4 h; not shown).

Although the c-Myc mRNA expression in pro-resolving macrophages collected

during resolution (72 h) of zymosan-induced peritonitis was restored to some extent,

it did not reach the level in naive macrophages from untreated control mice. I

noticed that a Calpain-mediated cleavage of c-Myc into Myc-nick was attributed to a

rapid reduction of the level of full-length c-Myc observed in pro-resolving

macrophages. Manifestation of the anti-inflammatory/pro-resolving M2 phenotype

is one of the early events in inflammatory stage8. In this context, M2 polarization in

vivo may not be ascribed to the transcriptional regulation of M2-related genes by c-

Myc itself, but is likely to be mediated, at least in part, by its cleaved product Myc-

nick.

Efficient resolution relies on phagocytic engulfment of apoptotic neutrophils by

69

macrophages. Importantly, an early calcium flux in the macrophages is essential for

formation of phagocytic cup, internalization of dead cells, and initiation of anti-

inflammatory signaling83, 99-101. Calcium mobilization is hence required for transition

of pro-inflammatory macrophages to anti-inflammatory/pro-resolving ones102, 103.

However, the mechanisms responsible for such calcium flux-governed events

remain largely unresolved. Calpains that belong to a distinct group of proteases are

directly regulated by calcium104, and they are possibly involved in these calcium-

dependent events. In macrophages, I have noticed that Calpains mimic pro-resolving

effects exerted by Myc-nick formed by a Calpain-mediated proteolytic cleavage of

c-Myc. Thus, blockade of Calpain action, either by pharmacologic inhibition or by

calcium depletion, hampered efferocytosis as well as Myc-nick generation and M2

macrophage polarization which is consistent with the above notion. Compared to

M2 polarized macrophages stimulated with external stimulus, such as IL-4, I

consider that bona fide pro-resolving macrophages from mice subjected to acute

inflammation are appropriate for the study of M2 polarization.

For the effective efferocytosis to occur, the precise cytoskeleton rearrangement

is pivotal for internalization of corpse once the apoptotic cell is captured105.

Reorganization of actin cytoskeleton has been reported to be crucial in controlling

phagocytic cup formation and membrane protrusion and extension. MTs contact and

modify cell membranes, thereby providing a protrusive force for engulfment of

apoptotic neutrophils106 or a retraction force for phagosome transport32 by

70

interacting with microtubule-associated proteins. My study reveals that α-tubulin

acetylation is critical for internalization of apoptotic cells through promotion of

phagocytic cup formation. As acetylation prevalently reduces rigidity of MTs107,

acetylated tubulin may allow a flexible structure for extension of cellular membrane

of macrophages over surface of apoptotic cells. In support of this speculation, non-

opsonized silica particles are internalized by macrophages in a manner

dependent on the acetylated MT cytoskeleton as well108. Myc-nick induces α-

tubulin acetylation by recruiting histone acetyltransferase GCN5 to microtubules. It

is also noticeable that acetylated α-tubulin not only provides physical force, by

means of constituting MTs, but also mediates signaling responsible for expression of

M2 marker genes and those encoding PS receptors. I discovered a spatial

accumulation of acetylated MTs in juxta-nuclear zone (Fig. 2-8A) in addition to its

recruitment to phagocytic cups in macrophages executing efferocytosis. The nuclear

periphery provides an environment for efficient degradation of cell corpses 31.

Therefore, accumulation of acetylated MTs in this area suggests its involvement in

lysosomal degradation. There are various ways by which microtubules promote

phagosome fusion with lysosomes. By interacting with MT cytoskeleton, LC3 plays

an essential role in this process of fusion of two organelles56, 109. In a similar way by

which LC3 works, Rab7 and dynein32 drive transport of phagosomes to lysosomes

and subsequent fusion. My finding that acetylated tubulin formed by Myc-nick

accumulates in this central area supports its possible involvement in the process of

71

autophagosome fusion with lysosome by recruitment of LC3-II to

autophagosomes110. Therefore, accumulation of acetylated MT is likely to

coordinate effective engulfment and efficient degradation of apoptotic cells via the

LAP pathway during efferocytosis.

Following uptake of apoptotic cells by macrophages, phagocytic respiratory

burst generates reactive oxidant species (ROS), which is mediated by activation of

NOX2, the major NADPH oxidase complex present in macrophages111. This triggers

the recruitment of autophagy-related proteins during LAP55. However, excessive

ROS accumulation can be potentially harmful to macrophages as well111. Therefore,

efferocytosis-induced ROS generation should be controlled in a manner that the

resulting oxidative stress is not intense enough to induce apoptosis. Upon

engulfment of apoptotic cells, macrophages tend to maintain ROS at an optimal

level through activation of PPARγ112, 113. Some PPARγ agonists/ligands have been

reported to inhibit endothelial ROS production114 and to ameliorate vascular

oxidative stress and NADPH oxidase expression in diabetic mice115. In these

macrophages Myc-nick is speculated to regulate redox balance by upregulating of

PPARγ expression for maintaining a non-phlogistic state. PPARγ acetylation has

been reported to be associated with its transcriptional activity in adipocytes116. As

Myc-nick produced in response to growth arrest or stress can interact with

acetyltransferase and acetylate cytoplasmic proteins, such as α-tubulin, its possible

involvement in acetylation of PPARγ in macrophages upon oxidative burst in the

72

context of their regulating redox homeostasis merits further investigation.

Based on my observation, full-length c-Myc itself is likely to be dispensable for

M2 polarization. It has been reported that knockout of c-Myc results in deficiency in

M2 polarization in vivo117, 118. However, knocking out of c-Myc diminishes Myc-

nick as well. Even the application of the c-Myc inhibitor, 10058-F475, not only

inhibits c-Myc-Max dimerization119, but also decreases the level of c-Myc protein120,

121, consequently reducing the availability of the substrate for Calpains in Myc-nick

generation. Thus, it is better to generate a conditional knockout of c-Myc without

affecting generation of Myc-nick. This may be achieved by deleting genomic DNA

harboring the sequence encoding C-terminal residue of c-Myc in mouse.

73

Chapter III

Therapeutic Potentials of Resolvin D1 (RvD1) for

Treatment of Inflammation-associated Colon Cancer

74

3.1. Abstract

Inflammatory bowel diseases, including ulcerative colitis and Crohn's disease,

are persistent disorders that lead to development of colitis-associated cancer (CAC).

Facilitated resolution of colitis has been addressed as a novel therapeutic strategy to

control development of CAC. Resolvin D1 (RvD1) is an endogenous lipid mediator

that is generated from docosahexaenoic acid during the resolution of inflammation.

Although the pro-resolving effects of RvDs have been extensively investigated and

well defined, the role for RvD1 in CAC remains largely unknown. In this study, I

found that RvD1 inhibited the expression of c-Myc in normal colon cells stimulated

with tumor necrosis factor-α (TNFα) and also in colon cancer cells. The suppression

of TNFα-induced upregulation of c-Myc in normal cells was mediated through

attenuation of NF-κB signaling. Notably, RvD1 destabilized the constitutively

overexpressed c-Myc protein in HCT 116 human colon cancer cells by stimulating

its ubiquitination and subsequent proteasomal degradation. Further, I revealed that

RvD1 stimulated c-Myc degradation through direct interaction with the ALX/FPR2

receptor. This interaction resulted in inhibition of activation of extracellular signal–

regulated kinase, thereby attenuating phosphorylation-dependent stabilization of c-

Myc.

75

3.2. Introduction

While acute inflammation functions to eliminate the damage from original

pathogen or physical and chemical insults, initiate tissue repair and restore

homeostasis, a loss of controlling to the response leads to a persistent inflammatory

process, namely chronic inflammation, which involves a progressive change in the

cell types at the inflammatory sites. The proposition of that chronic inflammation

triggers tumorigenesis was first raised by Rudolf Virchow in 1863 after the

discovery of a prevalent infiltration of leukocytes in tumors122. Thereafter, numerous

studies provide abundant evidence for that claim123, and it is generally recognized

that a certain portion of human malignancies is related to chronic inflammation.

Chronic inflammation endowed cancer cells with capability of liberation from

exogenous growth factors, resistance to anti-proliferation signals, evading from

apoptosis, acquiring unlimited replicative potential, developing sustained

angiogenesis, enabling cancer invasion and metastasis124. Diverse inflammatory

mediators such as cytokines and reactive oxygen species (ROS) induce a pre-

malignant lesions and silence tumor suppressor genes at initial phase of tumor

development, facilitate survival and growth for malignant cells during tumor

promotive phase, and promote angiogenesis for supplying adequate oxygen and

nutrition to tumor cells125, 126. Hence, the microenvironment is modulated by

inflammatory cells as well as tumor cells themselves, resulting in an escape of

76

cancer cells from immune surveillance127. A pro-resolving strategy is therefore

proposed as a reasonable approach for cancer prevention and even treatment5.

Among cancer-related inflammatory diseases, the inflammatory bowel disease,

including ulcerative colitis and Crohn's disease, has been associated with an

increased risk of developing colorectal cancer (CAC)128. This chronic disorder

provokes a prolonged and persistent inflammation in the intestine, which facilitates

the colorectal carcinogenesis through generation of excessive reactive oxygen

species and subsequent DNA damage and genotoxicity129. The epigenetic

modifications, such as DNA methylation130, 131 and posttranslational modification of

histone proteins, play a critical role in the cancer development as well. Once the

cells acquire the characteristics of cancer, the constitutive expression of chemokines

and cytokines, such as interleukin (IL)-1β, IL-6 and tumor necrosis factor α

(TNFα)132, 133, by immune cells enhances the interaction of cancer cells with

surrounding stromal cells in the local tumor microenvironment and subsequently

promotes tumor progression. Diverse inflammatory cells comprising of innate and

adoptive immune cells constitute the complicated and unhealed environment.

c-Myc is one of the most frequently overexpressed oncoproteins in a wide

variety of human malignancies, and its aberrant amplification leads to tumor

aggression and poor clinical outcome134, 135. The overactivation of c-Myc has been

implicated in aberrant cell cycle progression, genomic instability, malignant

transformation, immortalization and eventual migration and metastasis, via

77

regulation of the numerous target genes72. Although the amplification of c-Myc

could be achieved by transcriptional regulation, the protein undergoes rapid

ubiquitination and subsequent degradation. The principle stabilization of c-Myc

relies on phosphorylation on its Ser62 residue mediated predominantly by hyper-

activated extracellular signal–regulated kinases (ERK)136. Therefore, targeting the

ERK-c-Myc axis may confer an attractive strategy for cancer therapy137.

Some of lipid mediators have been shown to carry out active resolution of

inflammation6. Recent study has demonstrated that resolvins92. Among these,

resolvin D1 (RvD1), derived from docosahexaenoic acid, has been widely

investigated with regards to its prominent role in the body's defense against

microbial infection and other inflammatory insults138-140. Although there is

substantial evidence for the pro-resolving functions of RvD1138, 141, its effect on

cancer development remained largely unraveled. In this study, I examined the effect

of RvD1 on the TNFα-induced c-Myc expression in normal colonic epithelial cells

and also on constitutive c-Myc overexpression in cancer cells.

78

3.3. Materials and methods

3.3.1. Materials

RvD1 was obtained from Cayman Chemical Co. (Ann Arbor, MI, USA).

Recombinant human TNFα was produced by R&D systems (Minneapolis, MN,

USA). Recombinant human Apo-SAA was a product from PEPROTECH (Rocky

Hill, NJ, USA). Antibodies against c-Myc, FPR2/3, phospho-IκB kinase (IKK)α/β,

SAA, p50, IκBα, ERK1/2, phospho-ERK (Tyr 204) and actin were bought from

Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against p65,

IKKα, IKKβ, and phospho-IκBα were obtained from Cell Signaling (Beverly, MA,

USA). Antibody against phospho-c-Myc (Ser62) was obtained from Abcam plc.

(Cambridge, UK). The anti-rabbit and anti-mouse horseradish peroxidase-

conjugated secondary antibodies were purchased from Zymed Laboratories (San

Francisco, CA, USA).

3.3.2. Methods

3.3.2.1. Animal experiment

Male C57BL/6 mice obtained from ORIENT Bio, Inc. (Seoul, South Korea)

were maintained in a pathogen-free environment in animal center of Seoul National

University. All animal experiments were conducted in accordance with the

guidelines of the Institutional Animal Use and Care Committee at Seoul National

79

University. And the experimental protocol was approved by the Animal

Experimental Ethics Committee, Seoul National University (authorization number

SNU-150305-10). For inducing colon carcinogenesis, mice were administered with

a single intraperitoneal (10 mg/kg) injection of azoxymethane followed by treatment

of drinking water containing 2% (w/v) dextransulfate sodium (DSS) for 1 week. All

mice were euthanized at 16 weeks.

3.3.2.2. Cell culture

CCD841CoN and HCT 116 human colon cell lines were obtained from

American Type Culture Collection (ATCC, Manassas, VA, USA). These cell lines

were cultured in Minimal Essential Medium (MEM) and Dulbecco's Modified Eagle

Medium (DMEM), respectively. Both media were supplemented with 10% v/v FBS,

100 units/mL penicillin and 100 μg/ml streptomycin at 37 °C in an incubator with

humidified atmosphere of 95% O2/5% CO2.

3.3.2.3. Fractionation of cytosolic and nuclear extracts

After harvesting cells pellets were treated with hypotonic buffer A [10 mM 4-(2-

hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, pH 7.8), 1.5 mM MgCl2,

10 mM KCl, 1 mM sithiothreitol (DTT), 0.1 mM EDTA and 0.1 mM

phenylmethylsulfonyl fluoride (PMSF)] on ice for 15 min. Cell lysates were

supplemented with 10% Nonidet P-40 (NP-40) and incubated for another 5 min,

80

followed by a centrifugation at 12, 000 rpm for 5 min. The cytosolic extracts

(supernatants) were collected and stored at -70 °C until use. The remained pellets

were washed with buffer A containing 10% NP-40 for 3 times to totally eliminate

cytosolic components. Pellets were then resuspended in buffer C [20 mM HEPES

(pH 7.8), 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM

PMSF and 20% glycerol]. After centrifugation at 12, 000 rpm for 15 min, the

nuclear extracts were collected and stored at -70 °C until use.

3.3.2.4. Western blot and reverse transcription-polymerase chain reaction (RT-

PCR) analysis

A standard Western blotting was carried out as described previously76. Cells for

PCR analysis were applied to mRNA isolation using TRIzol™ reagent (Thermo

Fisher Scientific, Waltham, MA, USA) and cDNA creation by RT-PCR.

Amplification of genes was performed by PCR analysis using Solg™ 2x Taq PCR

Smart Mix (SolGent, Daejeon, South Korea) according to instructions of the

manufactures. PCR products were run on 2% agarose gel followed by a staining of

DNA using SYBR® Safe DNA gel stain (Thermo Fisher Scientific, Waltham, MA,

USA). Digital image analysis of Western blot and PCR were performed using the

ImageQuant™ LAS 4000 luminescent image analyzer (GE Healthcare Life Sciences,

USA).

81

3.3.2.5. Electrophoresis mobility shift assay (EMSA)

The EMSA was previously introduced138. Briefly, customized double-stranded

oligonucleotide corresponding to the NF-κB binding domains (Promega, Madison,

WI, USA) was labeled with [γ-32P]ATP by employing T4 polynucleotide kinase and

purified on a Nick column (Amersham Pharmacia Biotech, Buckinghamshire, UK).

The nuclear extracts (10 μg) were mixed with 4 μl of concentrated incubation buffer

[10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 4% glycerol

and 0.1 mg/ml sonicated salmon sperm DNA], and the hypertonic buffer was added

to make up the final volume 20 μl. After incubation at room temperature for 15 min,

the labeled oligonucleotide (400,000 cpm) was added to the nuclear fraction and

incubation was continued for an additional 50 min at room temperature. To ensure

the specific binding of the labeled oligonucleotide to nuclear protein, a competition

assay was carried out with the excess amounts of unlabeled oligonucleotide. After

the incubation, 0.1% Bromophenol Blue (2 μl) was added, and NF-κB-DNA

complexes were separated from the unbound free probe by electrophoresis on 6%

nondenaturing polyacrylamide gel in 1× TBE buffer [90 mM Tris base, 90 mM boric

acid and 0.5 mM EDTA (pH 8.0)] at 140 V for 3 h. Gels were dried and exposed to

X-ray film.

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3.3.2.6. Colony formation assay

The CCD 841 CoN cells transfected with Mock or c-Myc vector were collected

and diluted to a proper concentration. A total 200 cells were seeded to 60 mm

culture plates. The colonies were fixed by formaldehyde (4% v/v) at room

temperature for 10–15 min and stained with crystal violet (0.5% w/v) after culture

for 10 days. The colony number was counted for further analysis.

3.3.2.7. Integrative network and correlation analysis

For gene interaction analysis, microarray data from Gene Expression Omnibus

(GEO) database (GSE31106) were extracted and analyzed. Differentially expressed

genes (DEGs) with statistical significance (p < 0.05) in the colons of AOM+DSS-

treated mice (8 week) relative to controls were selected. Upregulated genes (fold

change>1.5) were further categorized according to gene ontology terms (DAVID 6.7

software). Gene interaction network was achieved using STRING and visualized by

Cytoscape 3.0.0 software. For the analysis of correlation between TNFα and c-Myc,

gene expression levels of Tnf and Myc (0, 4, 6, 8, 20 week) were extracted and

analyzed.

83

3.3.2.8. Statistical analysis

All data are analyzed by means ± s.e.m. and are based on experiments performed

at least in triplicate. Statistical significance was calculated with the Student's t-test

and Sigmaplot software.

84

3.4. Results

3.4.1. c-Myc expression is elevated in colitis-associated experimental

carcinogenesis and human cancer cells

I first performed systematic analysis of gene expression profiles in an AOM plus

DSS-induced carcinogenesis model by utilizing the GEO database (GSE31106). A

functional enrichment analysis revealed that 509 upregulated genes are significantly

associated with “regulation of cell migration” (Fig. 3-1A, left panel). By a network

analysis, I found 6 (Myc, Igf1, MMP9, Vim, Timp1 and Thbs1) are in the core

network of those genes (Fig. 3-1A, right panel). Notably, the microarray data

showed that the overexpression of c-Myc was more significant in the stage of

dysplasia development (4–8 week) than the stage of eventual adenocarcinoma

formation (20 week), indicative of the importance of c-Myc in the initiation of CAC

(Fig. 3-1B). I also validated the involvement of c-Myc in the AOM and DSS-

induced adenocarcinoma. The mice treated with AOM and DSS developed tumors at

16 weeks. A significantly elevated c-Myc expression was observed in the colon

adenomas of treated mice (Fig. 3-1C). In another study, I compared the levels of c-

Myc in normal and cancerous colon cells. Compared to CCD 841 CoN normal colon

epithelial cells, HCT 116 colon cancer cells exhibited higher expression of c-Myc

mRNA (Fig. 3-1D) and protein (Fig. 3-1E). Moreover, ectopic overexpression of c-

Myc enhanced the

85

Figure 3-1. Expression profile of c-Myc in colon cancer.

(A) Gene interaction enrichment and network analysis of microarray data set

(GSE31106) of colon samples from control and AOM plus DSS-treated mice. Left

panel: top ten enriched pathways of upregulated genes. Right panel: the interaction

86

network for the DEGs involved in the “regulation of cell migration” pathway

achieved using STRING database and visualized by Cytoscape 3.0.0 software. (B) A

time course of the relative gene expression of c-Myc which was extracted from

GSE31106. (C) Elevated expression of c-Myc in the colon tumors of mice treated

with AOM and DSS. Mice were treated with a single intraperitoneal dose of AOM

(10 mg/kg) which was followed by treatment with 2% DSS (w/v) in drinking water

for 1 week. All mice were euthanized at the 16th week. (D, E) Measurement of the

mRNA and protein levels of c-Myc in human CCD 841 CoN (CCD) non-cancerous

human intestinal cells and HCT 116 (HCT) human colon cancer cells. (F) Colony

formation of CCD cells transfected with mock or c-Myc vector. The colonies were

counted after staining with 0.5% crystal violet at the day 10. (G) A representative

blot illustrating the phosphorylation of ERK in CCD and HCT cells. All values

show means ± s.e.m. (*p < .05, **p < .01, ***p < .001; n.s.: no significance).

87

proliferation of CCD 841 CoN cells as assessed by their capability to form colonies

(Fig. 3-1F). c-Myc protein stability has been known to be maintained through its

phosphorylation by ERK. I noticed that ERK phosphorylation was highly elevated in

c-Myc overexpressing HCT 116 colon cancer cells compared with normal CCD 841

CoN normal colon epithelial cells (Fig. 3-1G).

3.4.2. RvD1 inhibits TNFα-induced overexpression of c-Myc in

normal colon cells

Considering an important role of TNFα in the initiation CRC142, I evaluated the

expression of TNFα and c-Myc expression in murine colon samples from GEO

dataset. I identified a significant correlation between TNFα and c-Myc in the process

of colorectal cancer development (Fig. 3-2A). To elucidate the molecular

mechanisms underlying the regulation of c-Myc during cancer-promoting

inflammation, I examined expression of c-Myc in CCD 841 CoN cells treated with

the prototypic pro-inflammatory tumor promotors TNFα and phorbol 12-myristate

13-acetate (PMA). I observed an activation of the NF-κB signaling pathway as

evidenced by an augmented nuclear accumulation of its functionally active subunit,

p65 in the cells upon treatment of TNFα or PMA (Fig. 3-2B). Under these

circumstances, the expression of c-Myc was also upregulated in these cells (Fig. 3-

2B). Further, a silencing of p65 with siRNA abolished the TNFα-induced expression

88

of c-Myc (Fig. 3-2C). This finding suggests that the upregulation of c-Myc by TNFα

is dependent on the NF-κB signaling

89

Figure 3-2. Inhibition of TNFα-induced overexpression of c-Myc by RvD1 in

normal intestinal cells.

(A) Correlation between Tnf and c-Myc expression in the colon of control and AOM

plus DSS-treated mice (n = 15) based on microarray data (GSE31106). Pearson’ r

90

and p were computed with the cor.test function (R-CRAN). (B) Determination of

inflammatory mediators that regulate c-Myc. (C) Effects of TNFα on the c-Myc

expression with and without silencing p65. CCD 841 CoN cells transfected with

either negative siRNA control or si-p65 RNA for 24 h were treated with TNFα

(10 ng/ml) for additional 1 h. (D) Effects of RvD1 on c-Myc mRNA expression in

human CCD 841 CoN cells challenged with TNFα. RvD1 (100 nM) was pre-treated

to the cells for 15 min, which was followed by treatment of TNFα for additional 1 h.

(E, F) Inhibitory effects of RvD1 on NF-κB signaling in the CCD 841 CoN cells.

The cells were treated with RvD1 for 15 min followed by stimulation with TNFα for

30 min. (G) Inhibition of TNFα-induced NF-κB-DNA binding by RvD1 in CCD 841

CoN cells. Cells were preincibated with RvD1 for 15 min followed by TNFα

treatment for additional 30 min. Nuclear fractions were isolated and incubated with

the [γ-32P]-labeled oligonucleotides containing the NF-κB consensus motif.

Electrophoresis was performed to achieve a separation of the NF-κB-DNA complex

from free probe. The values represent means ± s.e.m. and are representative of at

least three experiments. p < .05, **p < .01, ***p < .001.

91

pathway. RvD1 abrogated the TNFα-induced overexpression of c-Myc mRNA (Fig.

3-2D), IKK-mediated phosphorylation and degradation of IκBα, an NF-κB

inhibitory protein (Fig. 3-2E) and nuclear accumulation of NF-κB subunits, p65 and

p50 as well as c-Myc (Fig. 3-2F). Consequently, the DNA binding activity of NF-

κB was attenuated (Fig. 3-2G).

3.4.3. RvD1 reduces the stability of c-Myc in colon cancer cells

I next investigated the effect of RvD1 on constitutively overexpressed c-Myc in

colon cancer cells. When HCT 116 cells were treated with RvD1 for 24 h, there was

a significant reduction of c-Myc in the whole lysates (Fig. 3-3A) and nuclear

extracts (Fig. 3-3B). However, the mRNA level of c-Myc was not affected (Fig. 3-

3C), suggesting that RvD1 may reduce the stability of c-Myc without influencing its

expression at the transcriptional levels. The RvD1-mediated suppression of c-Myc

was abrogated by MG132, a proteasome inhibitor (Fig. 3-3D). Further, I observed

elevated ubiquitination of c-Myc in the cells treated with RvD1 (Fig. 3-3E).

3.4.4. RvD1 inhibits c-Myc through interaction with the G-protein

coupled formyl peptide receptor 2 (ALX/FPR2)

Considering that the stability of c-Myc is regulated via its phosphorylation, I

investigated the activity of ERK1/2 that predominantly phosphorylates the Ser 62

92

residue of c-Myc. As illustrated in Fig. 3-4A, RvD1 inhibited phosphorylation of

both

Figure 3-3. Effect of RvD1 on stability of c-Myc hyper-expressed in HCT

116 cells.

HCT 116 colon cancer cells were treated with vehicle (DMSO) or RvD1 (100 nM)

for 24 h. (A) Effect of RvD1 on the intracellular accumulation of c-Myc protein. (B)

Comparison of the c-Myc nuclear localization in the absence or presence of RvD1.

(C) Effect of RvD1 on expression of c-Myc mRNA. (D) Effect of RvD1 on the c-

Myc degradation in the absence or presence of a proteasome inhibitor, MG132 (2

μM). The cells were co-treated with RvD1 and MG132 for 24 h. (E) Enhancement

93

of the c-Myc ubiquitination by RvD1. Data represent the means ± s.e.m. (*p < .05,

***p < .001; n.s.: no significance).

94

Figure 3-4. Inhibition of ERK activity by RvD1 via interaction with ALX/RPR2

receptor.

(A) Inhibitory effect of RvD1 on ERK-c-Myc signaling axis at different time

intervals. (B, C) Effect of RvD1 (B) and the ERK inhibitor, U0126 (20 μM) (C), on

the degradation of wild type (WT) or S62E mutant c-Myc. After transfection with

GFP-tagged c-Myc WT or S62E vector for 24 h, HCT 116 cells were treated with

RvD1 or U0126 for additional 24 h and 6 h, respectively. The level of ectopic c-Myc

95

was measured by Western blot analysis. (D) Involvement of ALX receptor in the

regulation of c-Myc by RvD1. (E) Suppression of SAA-stimulated ERK

phosphorylation by RvD1. HCT 116 cells were pre-incubated with DMSO or RvD1

for 15 min prior to treatment with SAA (1 μg/ml) for additional 30 min. Data

represent the means ± s.e.m. (**p < .01, ***p < .001; n.s.: no significance).

96

ERK1/2 and c-Myc. To determine whether RvD1 could reduce the stability of

overexpressed c-Myc by inhibiting its phosphorylation at the Ser 62 residue, I

utilized a constitutive phosphorylation-mimetic mutant (S62E). RvD1 failed to

suppress phosphorylation of mutated c-Myc even though it could still attenuate the

phosphorylation of ERK1/2 (Fig. 3-4B). This mutated c-Myc was also insensitive to

the specific ERK inhibitor, U0126 (Fig. 3-4C). Taken together, these results suggest

that RvD1 reduces the c-Myc stability by blocking ERK-directed phosphorylation of

c-Myc.

RvD1 is known to exert its anti-inflammatory and pro-resolving effects through

interaction with ALX/FPR2 143, 144. Because of predominant activation by RvD1 and

its analogues, ALX/FPR2 was proposed as a RvD1 receptor143. siRNA knock down

of ALX/FPR2 upregulated phosphorylation of ERK1/2 which was accompanied by

an enhancement of c-Myc accumulation (Fig.3-4D). In ALX/FPR2 silenced cells,

RvD1 failed to inhibit the phosphorylation of ERK1/2 and c-Myc. (Fig. 3-4D).

Serum amyloid A (SAA), a pro-inflammatory agonist of ALX/FPR2, triggers

phosphorylation of ERK1/2 in the HCT cells (Fig. 3-4E). I found that RvD1

abrogated SAA-induced enhancement of ERK1/2 phosphorylation (Fig. 3-4E).

97

3.5. Discussion

The c-Myc oncoprotein, commonly overexpressed in various human cancers, is

involved in the cancer initiation and maintaining145. An elimination of Myc function

could lead to a rapid and irreversible regression of tumor73. Thus, repression of c-

Myc overexpression or stimulation of its degradation has been considered as a

promising strategy for cancer therapy. In this study, I found that c-Myc was

overexpressed in CAC, especially in the stage of dysplasia. This indicates a

prominent role of c-Myc in the development of colorectal cancer even though it may

also play an important role in the progression of CAC. Notably, there was a

significant correlation between pro-inflammatory TNFα and c-Myc in

experimentally induced colon carcinogenesis, indicative of the possible role for c-

Myc overexpression in inflammatory tumor microenvironment. These two factors

have been reported to be involved in the dedifferentiation of colon epithelial cells

and subsequent occurrence of tumor-initiating cells142. Thus, it is speculated that

stimulation of resolution of inflammation by blocking c-Myc over-expression is

likely to be an encouraging strategy for prevention of CAC.

A family of endogenous pro-resolving lipid mediators have been extensively

investigated with regards to their potential role in timely termination of

inflammation without compromising host defense6. In recent years, some of these

molecules applied in treatment of various cancers exhibited promising effect146-148.

98

Although pro-resolving RvD1 has been widely involved in resolution of various

inflammatory diseases138, 141, its anti-tumor effect remained largely elusive. I

demonstrated that RvD inhibits TNFα-induced overexpression of c-Myc. Further, I

revealed that RvD1 reduces the stability of c-Myc constitutively overexpressed in

colon cancer cells. In accordance with my finding, RvD1 has been shown to inhibit

TGF-β-induced epithelial-mesenchymal transition of lung cancer cells149 and

enhance caspase-3 activity in pancreatic ductal adenocarcinoma cells150. There has

been increasing concern about the potential application of RvD1 for cancer

therapy151. Therefore, it will be of great interest to investigate the chemotherapeutic

as well as chemopreventive role of RvD1 in other carcinogenesis models.

RvD1 exerts anti-inflammatory and pro-resolving effects through interaction with

ALX/FPR2143, 144. However, it remains largely unknown whether this receptor

influences the outcome of cancer development and progression. I demonstrated an

inhibitory effect of ALX/FPR2 on the constitutive activation of ERK, thereby

destabilizing the c-Myc protein in HCT 116 colon cancer cells. This finding

suggests potential anticarcinogenic as well as anti-inflammatory characteristics of

ALX/FPR2 152. In contrast to my notion, Xiang et al. have reported a role of

ALX/FPR2 in manifestation of a malignant phenotype in human colon cells153. Such

differential effects elicited by ALX/FPR2 appear to largely rely on the nature of the

ligands. Thus, activation of ALX/FPR2 by RvD1 has been shown to resolve

cytokine-mediated inflammatory responses in mammalian cells152, while SAA and

99

some peptide agonists convey pro-inflammatory signals by interacting with the same

receptor154, 155.

100

Conclusion and Perspectives

In my studies, I uncovered a novel function for c-Myc in M2 macrophage

polarization in vivo. As summarized in Fig. 4-1, in macrophages challenged with

inflammatory insults, c-Myc undergoes Calpain-mediated proteolytic cleavage to

generate Myc-nick, which accomplishes a task beyond the transcriptional activity of

full-length c-Myc for the successful resolution of inflammation. This cleaved form

of c-Myc drives formation of phagosomes and the subsequent apoptotic cell

degradation, in which spatial aggregation of MT cytoskeleton is involved. Notably,

Myc-nick-mediated acetylation of tubulin facilitates the M2 polarization of

macrophages and engulfment of apoptotic cells. Thus, targeting Myc-nick might be

an innovative therapeutic option for optimal resolution of inflammation in the

management of inflammatory disorders.

The pro-resolving mediators benefit for regaining homeostasis after an acute

inflammation by affecting multiple aspects. The most notable one is the clearance of

cell debris at the inflamed sites. The cleavage of c-Myc occurs during the

engulfment and then facilitates posterior uptake and degradation, suggesting host

has an autonomous system for getting restoration. My study verified that strategies

for promoting c-Myc generation favor effective resolution of inflammation. If this

approach can be implemented equally in cancer-related inflammation, it is

undoubted an exciting method for anticancer therapy. A report on the therapeutic

101

effect of resolvins boosted our confidence on this subject92. However, it has yet to

be elucidated whether c-Myc cleavage can be targeted for cancer therapy. I found

that a

Figure 4-1. Role of Myc-nick in the resolution of inflammation.

Inflammatory macrophages engulf apoptotic neutrophils at early phase of resolution

which mediates an influx of calcium. Calpains are activated by the influx of calcium

thereby triggering cleavage of c-Myc into Myc-nick. A subsequent acetylation of

microtubules is promoted by Myc-nick when Gcn5 is recruited. The acetylated

102

microtubules are involved in further engulfment of apoptotic cells at later phase and

degradation of engulfed cells.

103

similar polarization to M2 type occurred in macrophages engulfed apoptotic cancer

cells. Since the M2-like phenotype of TAM facilitates a formation of

immunosuppressive tumor environment, the acceleration of c-Myc cleavage is likely

linked to an inefficient cancer therapy. I propose that an effective engulfment of

cancer cell debris but with a simultaneous abolishment of c-Myc cleavage is useful

to adjuvant cancer therapy. Since resolvins is applicable to cancer therapy, they

may play other roles in cancer therapy, which counteracts with immunosuppressive

effect after clearance of debris. At least, my study demonstrated the anticancer effect

RvD1156, which substantiates my hypothesis. For a cancer therapy by targeting c-

Myc cleavage, it still largely remained to be investigated.

To uncover the potential of c-Myc cleavage, TAM is the most ideal object for an

examination up to now since a calcium flux is validated in macrophages during

uptake of apoptotic cells. However, the engulfment of dead cancer cells is not

largely observed in a solid tumor without chemotherapy. A great number of

macrophages have been found engulfing apoptotic cancer cells after treatment of

canticancer agents. As mentioned earlier, this cause an immunosuppressive

character of TAM, which probably reduces the efficacy of chemotherapy even

though the treatment can reduce the size of tumor. A current literature demonstrated

that a reprogramming of TAM toward pro-inflammatory state inspires T cell

response against the tumor157. Given the anti-inflammatory role of Myc-nick, a

blocking of c-Myc cleavage is likely to be essential adjuvant treatment to

104

chemotherapy to dampen the expression of anti-inflammatory cytokines. To achieve

it, specific pharmacological inhibitors should be discovered for suppression Calpain

activity to eliminate off target interruption of other intracellular responses. As

Calpains are a superfamily, an identification of cell specific expression of those

proteins is required for further development proper inhibitors.

In recent years, the immunotherapy, such as chimeric antigen receptor (CAR)-T

cell treatment and blocking checkpoint of T cells, becomes a new hot issue in cancer

treatment. There is an inevitable problem is that patients treated with CAR-T are

often accompanied by a cytokine storm even though it is effective158. Moreover, a

high price for CAR-T and vaccines against T cell checkpoint hindered the practical

application to most of the patients. Therefore, the developing Calpain inhibitors

targeting c-Myc cleavage might be an alternative strategy for extensive cancer

therapy.

While c-Myc is cleaved in macrophages during a resolution of inflammation, it

is not observed in cancer cells treated with pro-resolving RvD1. The latter study

revealed that RvD1 suppressed TNFα-induced overexpression of c-Myc through

inhibition of NF-κB signaling in non-cancerous human intestinal epithelial cells.

Further, RvD1 attenuates stability of c-Myc protein constitutively overexpressed in

human colon cancer cells through interaction with the ALX/FPR2 receptor and

subsequent inhibition of phosphorylation of ERK1/2. These findings suggest that

RvD1 has cancer preventive and therapeutic potential in the management of CAC.

105

The calcium influx and subsequent Calpain activation upon engulfing apoptotic

cells contributes to the generation of Myc-nick. A treatment of RvD1 probably

rarely affects Calpain activity of cancer cells, suggesting mechanisms of pro-

resolving treatment targeting inflammatory cells differ from that targeting cancer

cells. This observation also stated another pathway for resolvins-mediated

suppression of tumor growth which is claimed as a consequence of engulfing cancer

debris by macrophages151. To further investigate the potentials of Myc-nick in

cancer therapy, effects of pro-resolving mediators (e.g. RvD1) on c-Myc cleavage in

inflammatory cells (e.g. TAM) merit further investigation.

106

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Korean Abstract

염증해소 반응에 있어서 Myc-nick 의 새로운 기능

급성 염증 반응의 특징은 염증 부위로의 다핵성 호중구의 즉각적인 동원과

이후 백혈구의 제거 및 최종적인 염증 해소를 포함한 순차적인 과정이다. 급성

염증 반응으로 염증이 해소되고 나면 인체는 체내의 항상성을 회복할 수 있다.

이를 염증 해소 후 단계 (post-resolution) 이라고 하며 이 단계를 거치고

나면 획득 면역은 더욱 공고해 진다. 하지만 염증의 해소가 제대로 이루어지지

못하면 급성 염증은 만성 염증으로 발전하여 염증이 발생한 조직에 지속적인

손상을 줄 수 있다. 지난 수년 간 연구자들은 염증성 질병의 치료제로

비스테로이드성 소염진통제와 같은 약의 개발에 초점을 맞추었다. 하지만

이런 약들은 면역체계를 교란하여 체내방어기전을 방해할 수 있기 때문에

우리 몸의 항상성 회복에 효과적이지 못하다. 그래서 최근에는 염증 해소를

촉진시켜 내인성 면역반응을 위주로 염증을 억제함과 동시에 체내 항상성을

회복할 수 있는 할 수 있는 약물을 개발하는 쪽으로 항 염증과 관련된 연구의

동향이 옮겨가고 있다. 적극적인 염증 해소를 위해서 두가지 생리학적인

프로세스가 수반되어야 한다. 여기에는 먼저 백혈구의 사멸(대체적으로

호중구세포의 사멸)을 유발한 다음 사멸한 세포와 세포 잔해를 제거하는

128

과정이 포함되어야 한다. 염증해소에 관여하는 대식세포는 식세포작용을 통해

사멸한 세포들을 효과적으로 정리해 주는 것으로 알려진 세포이다. 이런 염증

해소 촉진 세포는 M2 표현형을 가진 세포로 정의하며, 염증 작용을 억제할

뿐만 아니라 식세포작용을 포함한 특정한 내인성 면역체계를 작동시켜서

염증을 해소하는데 도움을 준다.

암화 과정은 만성 염증과 밀접한 관계를 맺고있다. 만성 염증은 염증부위로의

염증반응을 억제하는 세포들의 활성화와 축적 야기시키며 초기 종양 형성과

암세포의 생존을 용이하게 한다. 최근에 암-연관 염정반응에서 염증 해소

작용은 암의 치료제 개발에 대한 새로운 접근법으로 대두되고 있다. 염증

해소를 촉진하는 치료법은 과도한 염증성 신호전달물질을 제거해 줄 뿐만

아니라 획득 면역 체계를 만듦으로 염증 해소 후 작용에도 도움을 준다. 획득

면역 세포의 작용은 효과적인 염증의 치료법으로 알려져 있으며 염증 해소를

촉진함으로써 이러한 작용을 촉진 시킬 수 있다. 그러므로 염증 해소 작용에

대한 연구는 만성 염증성 질환의 치료에 대한 흥미로운 전략이 될 것으로

사료된다.

c-Myc 은 대식세포의 활성화와 식세포 작용 활성 유지에 중요한 역할을

수행하는 전사인자이다. 하지만 이와는 상반되게 본 연구에서 염증을

유발하는 zymosan-A 를 처리한 마우스의 복강에서 채취한 M2 표현형의

대식세포에서 Myc 단백질 양이 아주 적은 것을 확인하였다. 또 이와 동시에

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잘려진 c-Myc 단백질 (Myc-nick)이 축적되는 것이 관찰되었다. Full-length

c-Myc 에서 잘린 단백질을 Myc-nick 이라 정의하는데, 이 단백질은 단백질

분해효소인 Calpain 에 의해 조절되는 단백질 분해로 형성된다. 더불어 본

연구에서는 Myc-nick 의 증가가 M2 표현형을 가진 대식세포의 분화를

촉진시켜 식세포작용과 염증 해소 작용을 효과적으로 유도함을 확인하였다.

한편 대장암세포인 HCT-116 세포를 이용한 실험에서는 내인성 염증 해소

인자인 Resolvin D1 (RvD1)은 c-Myc 이 Myc-nick 으로 잘리지 않는 것이

관찰되었는데 이는 암세포인 HCT-116 세포에는 염증 해소 작용 에 관여하는

효소인 Calpain 활성화하지 않기 때문인 것으로 사료된다. 하지만 흥미롭게도

RvD1 은 HCT-116 세포의 c-Myc 단백질을 완전히 분해시켜 암세포의

증식을 억제하는 역할을 하여, 항암 작용에 관여한다는 것을 확인하였다.

본 연구 내용을 바탕으로 본 연구에서는 Myc-nick 은 암세포가 아닌

대식세포에서 활성화되어 염증해소 작용을 돕는 역할을 함으로써 만성

염증과 관련된 질환의 혁신적인 치료제가 될 가능성을 제시하였다. 암-연관

대식세포 (tumor associated macrophage; TAM)는 종양미세환경에서

광범위하게 존재하는 면역세포로, Myc-nick 이 종양 환경에서도 TAM 의

작용을 조절할 가능성이 있으며 더불어 새로운 암 치료제의 개발에도 기여할

것으로 사료된다.

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주요어: Myc-nick, 염증 해소, 식세포작용 (Efferocytosis), M2 양극화,

tubulin 의 아세틸화, LC3 관련한 식세포작용

학번: 2011-31329

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Biographical Data

Xiancai Zhong, Ph.D. Candidate

Tumor Microenvironment Global Core Research Center,

College of Pharmacy, Seoul National University

Gwanak-ro 1, Gwanak-gu, Seoul 08826, South Korea

Fax: +82-2-874-9775

E-mail: zhongxiancai@163.com

EDUCATION

Ph.D., Seoul National University Seoul, South Korea

Pharmaceutical Bioscience, 94.60/100 Sep. 2011 _ Aug. 2018

Dissertation: A novel function of Myc-nick, a cleavage product of c-Myc, in

resolution of inflammation.

Advisor: Young-Joon Surh (Professor and Director)

M.S., Seoul National University Seoul, South Korea

Pharmaceutical Bioscience, 90.80/100 Sep. 2008 _ Feb. 2011

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Dissertation: Chemopreventive effects of baicalein on dextran sulfate sodium-

induced mouse colitis.

Advisor: Young-Joon Surh (Professor and Director)

B.S., Jilin University Changchun, China

Biological Sciences, 92.70/100 (top 5) Sep. 2004 _ Jun. 2008

WORK EXPERIENCE

Assistant Researcher

Seoul National University, South Korea Mar. 2011 – Aug.

2011

SCHOLARSHIPS AND AWARDS

Development Fund Scholarship

Seoul National University, South Korea Sep. 2016

SNU Global Scholarship (2 times)

Seoul National University, South Korea Sep. 2011 - Aug. 2012

Outstanding Poster Award

2011 International Conference on Food Factors, Taiwan, China Dec. 2011

Lecture & Research Scholarship

Seoul National University, South Korea Mar. 2011

Superior Academic Performance Scholarship

133

Seoul National University, South Korea Mar. 2010

Third-class Superior Academic Performance Scholarship

Jilin University, China Sep. 2006 _ Aug. 2007

Second-class Superior Academic Performance Scholarship (2 times)

Jilin University, China Sep. 2004 _ Aug.

2006

CONFERENCES

International Cell Death Society Meeting 2018

Seoul, South Korea May 31, 2018 - Jun. 2,

2018

American Association for Cancer Research (AACR) annual meeting

New Orleans, USA Apr. 16, 2016 - Apr. 29,

2016

The 4th International Conference on Nutrition & Physical Activity

in Aging, Obesity and Cancer,

Pattaya, Thailand Aug. 14, 2011 - Aug. 17,

2013

International Conference on Food Factors (ICoFF)

Taipei, China Nov. 20, 2011 - Nov. 23,

2011

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PUBLICATIONS

1. Zhong X., H.N. Lee, S.H. Kim, S.A. Park, W. Kim, Y.N. Cha and Y.J. Surh.

2018. Myc-nick promotes efferocytosis through M2 macrophage polarization during

resolution of inflammation. FASEB J.

2. Yang, J.Y., X. Zhong, S.J. Kim, D.H. Kim, H.S. Kim, J.S. Lee, H.W. Yum, J.

Lee, H.K. Na, and Y.J. Surh. 2018. Comparative effects of curcumin and

tetrahydrocurcumin on dextran sulfate sodium-induced colitis and inflammatory

signaling in mice. J. Cancer Prev. 23:18-24.

3. Zhong, X., H.N. Lee, and Y.J. Surh. 2018. RvD1 inhibits TNFα-induced c-Myc

expression in normal intestinal epithelial cells and destabilizes hyper-expressed c-

Myc in colon cancer cells. Biochem. Biophys. Res. Commun. 496:316-323.

4. Kim, S.H., X. Zhong, W. Kim, K. Kim, Y.G. Suh, C. Kim, Y. Joe, H.T. Chung,

Y.N. Cha, and Y.J. Surh. 2018. Taurine chloramine potentiates phagocytic activity

of peritoneal macrophages through up-regulation of dectin-1 mediated by heme

oxygenase-1-derived carbon monoxide. FASEB J. 32:2246-2257.

5. Dae Park, D., H.W. Yum, X. Zhong, S.H. Kim, S.H. Kim, D.H. Kim, S.J. Kim,

H.K. Na, A. Sato, T. Miura, and Y.J. Surh. 2017. Perilla frutescens extracts protects

against dextran sulfate sodium-induced murine colitis: NF-κB, STAT3, and Nrf2 as

putative targets. Front. Pharmacol. 8:482.

6. Ngo, H.K., H.G. Lee, J.Y. Piao, X. Zhong, H.N. Lee, H.J. Han, W. Kim, D.H.

Kim, Y.N. Cha, H.K. Na, and Y.J. Surh. 2016. Helicobacter pylori induces Snail

135

expression through ROS-mediated activation of Erk and inactivation of GSK-3β in

human gastric cancer cells. Mol. Carcinog. 55:2236-2246.

7. Yum, H.W., X. Zhong, J. Park, H.K. Na, N. Kim, H.S. Lee, and Y.J. Surh. 2013.

Oligonol inhibits dextran sulfate sodium-induced colitis and colonic adenoma

formation in mice. Antioxid. Redox Signal. 19:102-114.

8. Yang, J.Y., X. Zhong, H.W. Yum, H.J. Lee, J.K. Kundu, H.K.Na, and Y.J. Surh.

2013. Curcumin inhibits STAT3 signaling in the colon of dextran sulfate sodium-

treated mice. J. Cancer Prev. 18:186-191.