저 시-비 리- 경 지 2.0 한민

는 아래 조건 르는 경 에 한하여 게

l 저 물 복제, 포, 전송, 전시, 공연 송할 수 습니다.

다 과 같 조건 라야 합니다:

l 하는, 저 물 나 포 경 , 저 물에 적 된 허락조건 명확하게 나타내어야 합니다.

l 저 터 허가를 면 러한 조건들 적 되지 않습니다.

저 에 른 리는 내 에 하여 향 지 않습니다.

것 허락규약(Legal Code) 해하 쉽게 약한 것 니다.

Disclaimer

저 시. 하는 원저 를 시하여야 합니다.

비 리. 하는 저 물 리 목적 할 수 없습니다.

경 지. 하는 저 물 개 , 형 또는 가공할 수 없습니다.

의학 석사 학위 논문

Roles of transglutaminases

in the regulation

of dendritic cell function

수지상세포 기능의 조절에 있어서

트랜스글루타미네이즈의 역할

2012 년 8월

서울대학교 대학원

의과대학 의과학과

노 지 아

수지상세포 기능의 조절에 있어서

트랜스글루타미네이즈의 역할

지도교수 김 인 규

이 논문을 의학석사 학위논문으로 제출함

2012 년 8월

서울대학교 대학원

의과대학 의과학과 전공

노 지 아

노지아의 의학석사 학위논문을 인준함

2012 년 8월

위 원 장 (인)

부위원장 (인)

위 원 원 (인)

Roles of transglutminases

in the regulation

of dendritic cell function

by

Jiah Noh

A Thesis Submitted in Partial Fulfillment

of the Requirements for the Degree of

Master of Science in Medicine

(Biomedical Sciences)

at the Seoul National University College of Medicine

October 2012

Approved by thesis committee:

Professor Chairman

Professor Vice Chairman

Professor

i

Abstract

Jiah Noh

Major in Biomedical Sciences

Department of Biomedical Sciences

Seoul National University Graduate School

Dendritic cell (DC) is professional antigen presenting cell, regulating

innate immunity and adaptive immunity. It has been reported that

treatment of DC with LPS up-regulates the expression of TGase 2, a

member of calcium-dependent enzyme family that catalyzes the

post-translational modification of protein by forming polyaminated

proteins or cross-linked proteins. In this study, role(s) of LPS-

induced TGase 2 in DC was investigated using TGase 2-deficient DC.

When bone marrow cells isolated from wild-type and TGase 2-

deficient mice were compared, there was no difference between

wild-type and TGase 2-deficient cells in the ability of differentiation

and maturation. Moreover, TGase 2-deficient DC showed no

difference in the level of secreted cytokine, phagocytosis, migration,

and apoptosis compared with those of wild-type DC. However, wild-

type DC showed higher level of ROS than TGase 2-deficient DC. In

addition, Real time RT-PCR revealed that increase of TGase activity

by LPS treatment is due to expression of TGase 4. These results

vi

indicate that TGase activity might be involved in the generation of

ROS and in the process of DC maturation.

Key word : Transglutaminase, Dendritic cell, Differentiation,

Maturation

vii

Contents

Abstracts ------------------------------------------- ⅰ

Contents ------------------------------------------ ⅲ

List of Tables ------------------------------------- ⅳ

List of Figures ------------------------------------ ⅳ

Introduction ---------------------------------------- 1

Purpose ------------------------------------------ 28

Materials and Methods --------------------------- 29

Results ------------------------------------------- 36

Discussion ----------------------------------------- 61

Reference ---------------------------------------- 63

Abstract (in Korea) ------------------------------ 69

viii

List of Tables

Table 1. Summary of characteristics of nine transglutaminases-----6

Table 2. Known inducers of TGase 2 expression---------------14

Table 3. NOX isoenzymes --------------------------------23

Table 4. Primers for TGases ------------------------------45

List of Figures

Figure1. Schematic representation of enzymatic reaction of

transglutaminase ---------------------------------------- 5

Figure 2. The schematic representation of TGase 2 structure ----- 8

Figure 3. Structure of TGase 2 ----------------------------- 9

Figure 4. Correlation between the development of myeloid and

lymphoid dendritic cells (DCs) ---------------------------- 19

Figure 5. Domain structure of regulatory proteins for NOX enzymes --

---------------------------------------------------- 26

Figure 6. Activation of reactive oxygen species (ROS) generation by

assembly of Phox regulatory proteins ---------------------- 27

Figure 7. A process of mDCs differentiation and maturation ------ 31

Figure 8. TGase 2 is not involved in DC differentiation ------- 37

ix

Figure 9. TGase 2 is not involved in LPS-induced DC maturation - 39

Figure 10. Cystamine inhibits DC maturation via TGase 2-

independent pathway ----------------------------------- 41

Figure 11. KCC009 also inhibits DC maturation via TGase 2-

independent pathway ------------------------------------ 42

Figure 12. TGase 4 is a strong candidate that is involved in DC

maturation -------------------------------------------- 46

Figure 13. TGase 4 is increased along with differentiation of DC -- 47

Figure 14. TGase 4 is involved in maturation of DC ------------- 48

Figure 15. TGase 2 is not involved in function of matured DCs ---- 50

Figure 16. TGase 2 is not involved in phagocytosis of DCs ------- 52

Figure 17. TGase 2 is not involved in migration of maturated DC -- 54

Figure 18. TGase 2 is not involved in expression of CCR7 ------- 55

Figure 19. TGase 2 is not involved in apoptosis of maturated DC -- 57

Figure 20. TGase 2 is not involved in ROS release of maturated DC --

---------------------------------------------------- 59

Figure 21. TGase 2 is involved in mRNA level of NOX2 components --

--------------------------------------------------- 60

１

Introduction

1. Transglutaminase

The transglutaminases (TGases) catalyze Ca2+-dependent acyl

transfer reactions between proteins or protein and polyamines

(Lorand and Graham, 2003). TGase-mediated modification of

glutamine residue can be made by three ways: polyamine

incorporation, deamidation, and crosslinking reaction. This reaction

involves a glutamine-containing protein or peptides as acyl acceptor

substrate. TGase forms a covalently linked γ-glutamythioester, the

acylezyme intermediate, at the active site cysteine with release of

ammonia (Iismaa, Mearns et al. 2009). This initial acylation step is

followed by a direct attack by small molecule amine (polyamine

incorporation), water (deamidation), and amino group of protein-

bound lysine residues (crosslinking) (Figure 1).

To date, nine TGase isoforms have been identified in human

(Esposito and Caputo, 2005). TGase 1 (encoded by the gene TGM1

on human chromosome 14q11.2) is expressed in almost all kinds of

epithelial cells and associated with plasma membrane in lung, kidney,

and liver (Hiiragi T and M, 1999). It is also expressed at low level in

proliferating keratinocytes differentiation (Steinert PM, 1996). It

functions mainly in the formation of cornified cell envelope or

terminally differentiated epidermal keratinocyte (Chakravarty R,

２

1989). It has been reported that mutation in the TGase 1 gene were

found in lamellar ichthyosis, autosomal recessive sin disease. This

finding indicated that TGase 1 has esterase enzyme activity in

addition to transamidation activity for lipid and protein barrier

formation.

TGase 2 (encoded by TGM2 on human chromosome 20q11-12), a

74-to 80-kDa protein, is ubiquitously expressed, and found in all

mammalian tissues (Begg, Carrington et al., 2006). It is localized in

cytosol, nuclear, membrane, cell surface, and extracellular. It is not

present in the mitochondria (Krasnikov, Kim et al., 2005). TGase 2

functions both as transamidating enzyme and GTP binding and

hydrolyzing enzyme. The transamidase activity plays an extracellular

role in matrix stabilization, and it essential for angiogenesis, wound

healing, and bone remodeling. And it also plays an intracellular role

in the cross-linking of proteins during apoptosis.

TGase 3 (encoded by TGM3 on human chromosome 20q11-12) is

also expressed in epithelial cells and hair follicle (Kim HC, 1990).

The predicted protein contains 692 amino acids and is 75% identical

with that of mouse. It has been reported that while TGase 3 is of

unique functional importance in hair development, loss of TGase 3 in

the epidermis could be compensated by other TGase family members.

Hair lacking TGase 3 is thinner, and shows major alterations in the

cuticle cells and markedly decreased cross-linking of hair protein

(John, Thiebach et al., 2012).

３

TGase 4 (encoded by TGM4 on human chromosome 3q21-22) is

unique in that it is espressed in the prostate gland (Willian-Ashman,

1972). Recently, it has been shown that TGase 4 may be involved in

prostate cancer cell growth, migration and invasiveness, tumor–

endothelial interaction and epithelial–mesenchymal transition.

Moreover, TGase 4 has a wide-range of interaction with other

molecular complexes, implicating that TGase 4 could be used as a

possible biomarker of aggressive cancer as well as a therapeutic

factor (Jiang and Ablin, 2011).

TGase 5 (encoded by TGM5 on human chromosome 15q15.2) is

very similar to that of TGase 1 in aspect of tissue distribution and

functions. TGase 5 is widely expressed in the epidermis and involved

in protein cross-linking. It is thought to be required for structural

integrity of the outermost epidermal layers (Cassidy, van Steensel et

al., 2005), Recently it is reported that TGM5 mutations were found

in acral peeling skin syndrome (Pigors, Kiritsi et al., 2012).

TGase 6 (encoded by TGM6 on human chromosome 20q11) is

expressed in a human carcinoma cell line with neuronal

characteristics and in mouse brain. Biochemical data show that

TGase 6 is allosterically regulated by Ca2+ and guanine nucleotides

(Thomas, Beck et al., 2011). TGase 7 is encoded by TGM7 on human

chromosome 15q15.2. Enzymatic properties and biological function of

TGase 6 and 7 are not fully characterized yet.

Factor XⅢa (encoded by F13A1 on human chromosome 6p24-25)

is expressed in blood. It is zymogen that is activated by thrombin

４

cleavage. The activated Factor XⅢa crosslinks fibrin monomer to

stabilize the blood clot (McDonagh and McDonagh, 1975). Factor

XⅢa consists of 731-amino acids with a molecular mass of 83kDa

(Takahashi, Takahashi et al., 1986).

Lastly, band4.2 (encoded by EPB42 on human chromosome

15q15.2) is a catalytically inactive member of the TGase family due

to absence of cysteine at active site of enzyme. It contributes to

membrane shape and stability and associates with the Rhesus protein

complex in RBC membranes (Mouro-Chanteloup, Delaunay et al.,

2003). Characteristics of TGases are summarized in Table 1.

５

Figure 1. Schematic representation of enzymatic reaction of

transglutaminase

Transglutaminases catalyze Ca2+-dependent protein-modification of

glutamine residue: polyamine incorporation, deamidation, and

crosslinking reaction.

６

Table 1. Summary of characteristics of nine transglutaminases

(Lorand and Graham, 2003)

７

2. Transglutaminase 2

2.1 Cloning and genomic structure

TGase 2 may be the most interesting member of TGases. It is

first isoenzyme identified among TGase family. TGase 2 was purified

from guinea pig liver homogenate (Sarkar et al., 1957), and then

cDNA clone was isolated from mouse macrophage and human

endothelial cell. By fluorescence in situ hybridization (FISH), TGase

2 maps on chromosome 20q11.2-12 (Genitil V, 1991). Analysis of

genomic structure showed that TGase 2 gene is 32.5 kilobases (Fraij

and Gonzales, 1996). There are two alternatively splied isoform of

TGase 2. One is TGase-H (TGase-homologue) or TGase-S (TGase-

short) that is 1.9 kilobases transcript and coded 584 amino acids

protein. The other is TGase-H2 that is 2.4 kilobases transcript and

coded 349 amino acids protein.

TGase 2 is comprised of a dimer form in complex and has four

distinct domains, N-terminal β-sandwich, catalytic core domain and

two C-terminal β-barrel domains (Figure 2). In the absence of

calcium, TGase 2 shows a compact conformation. When TGase 2 is

activated, there is a remarkably large conformational change from

the compact form to extended form, and then TGase active site is

exposed. The catalytic triad of enzyme consists of a cysteine,

histidine, and aspartate residue, and a conserved tryptophan

stabilizes the transition state. These are all essential for catalytic

activity (Iismaa, Mearns et al., 2009) (Figure 3).

８

Figure 2. The schematic representation of TGase 2 structure

Transglutaminase 2 consists of four domains; β-sandwich, catalytic

core, β-barrel 1, and β-barrel 2 (Fesus L 2002).

９

(A)

(B)

Figure 3. Structure of TGase 2

(A) Compact, inactive TGase 2 conformation (left) and extented

inhibitor-bound TGase 2 conformation (right) (B) TGase 2 is

comprised of four domains and catalytic core domain consists of a

cysteine, histidine, aspartate, and tryptophan residue.

１０

2.2 Functions

2.2.1 The function of TGase 2 in cells

Seeing as the in vivo expression pattern of the enzyme, TGase 2

may have multiple roles. The cell type that expresses TGase 2 can

be divided into two groups. There are cell type such as smooth

muscle and endothelial cells that constitutively express TGase 2 at

high levels. In the other group, TGase 2 is induced by signalling

pathways.

At the membrane, TGase 2 is involved in transmitting signals from

seven-transmembrane helix receptor to phospholipase C (Iismaa, Wu

et al., 2000). TGase 2 also has important roles in diverse cellular

processes like apoptosis, cell adhesion, cell cycle and receptor-

mediated endocytosis. TGase 2 is thought to be responsible for the

formation of intracellular cross-linked protein polymers that

constitute the major component in an apoptotic envelope and it

stabilizes the apoptotic body (Melino G, 1998). TGase 2 activated by

Ca2+ interacts with and modifies key components of the cytoskeleton.

In response to RA treatment, TGase 2-dependant transamidation of

RhoA results in the increased binding of RhoA GTPase to ROCK-2

protein kinase, autophosphorylation of ROCK-2 and phosphorylation

of vimentin (Singh, Kunar et al., 2001) which can lead to the

formation of stress fibers and increased cell adhesion. In the cells

under the apoptosis in vivo, TGase 2 is induced. Recently it is

reported that TGase 2 sensitizes cells during apoptosis by interacting

１１

with mitochondria, and it alters redox status (Mauro Piacentini et al.,

2002).

2.2.2 The function of TGase 2 on the cell surface and in the

extracellular matrix

On the cell surface, integrin-bound TGase 2 offers a binding site

for fibronectin. The TGase 2 binds to the sequences outside the

integrin ligand-binding pocket. On the surface of many different cell

type, some integrins could be complexed with TGase 2 (Akimov SS,

2001). In addition to fibronectin, other proteins were reported to be

substrates for TGase 2. TGase 2 may play an important role in

stabilizing the ECM by making the matrix resistant to mechanical and

proteolytic degradation. Such a stabilization of the ECM has been

proposed to play a role in tumor cell invasiveness as a result of

altered cell-matrix interactions. TGase 2 is also involved in

angiogenesis and wound healing (Haroon, Hettasch et al., 1999).

2.3 Pathogenic role of TGase 2

It has been reported that TGase 2 is involved in many pathogenic

processes such as celiac disease, cataract, bleomycine-induced lung

fibrosis, and neurodegenerative diseases.

In the case of celiac disease, it is a malabsorbtion syndrome

characterized by almost total atrophy of villi in the jejunum on

exposure to dietary glutens. TGase 2 is involved in generating gluten

peptides which stimulates T cell through deamidation of specific

１２

glutamines (Anderson, Degano et al., 2000; Vader, de Ru et al., 2002).

The TGase 2-formed disease-triggering epitopes stimulate a

pathological immune response that destroys the jejunal epithelium.

Cataract is a clouding that develops in the crystalline lens of the

eye or in its envelope, varying in degree from slight to complete

opacity by obstructing the passage of light. Age-related cataracts

are characterized by aggregation of lens proteins with subsequent

lens clouding. TGase 2-mediated cross-linking of the β-crystallins,

αB-crystallins, and the intermediate filament protein vimentin is

likely to be involved in cataracogenesis (Lorand, Hsu et al., 1981;

Mulders, Hoekman et al., 1987; Velasco and Lorand 1987).

TGase 2 is activated by oxidative stress in human lens epithelial

cells. Oxidative stress induces the release of transforming growth

factor (TGF)-2 which in turn activates Smad3 signaling pathway.

Modulation of gene expression by Smad signalling pathway results in

activation of TGase 2, leading to crosslinking and aggregation of lens

proteins (Shin, Jeon et al., 2008).

Huntington disease (HD) is a neurodegenerative genetic disorder

that affects muscle coordination and leads to cognitive decline and

psychiatric problems. HD is caused by the expansion of CAG

trinucleotide repeats in the gene encoding huntingtin (htt).

Accumulation of ubiquitinated htt aggregate in the nucleus, and

progressive loss of neuronal cells are observed. One of the proposed

mechanisms of htt aggregation is based on the action of TGase 2

because expanded polyglutamine repeats are excellent glutaminyl-

１３

donor substrates for TGase2-catalyzed cross-linking (Lesort,

Tucholski et al., 2000). In addition, TGase 2 is highly expressed in

several cancerous tissues including glioblastoma, malignant

melanoma, and pancreatic ductal adenocarcinoma (Siegel and Khosla,

2007). The cancer cells resistant to chemothrapeutic drug or

obtained from metastatic cancer tissue also showed elevated

expression of TGase 2. Elevated TGase 2 induced constitutive

activation of transcription factor of NF-κB (nuclear factor κB),

important regulator in cell proliferation and survival (Mann et al.,

2006), which is probably associated with TGase 2 up-regulation in

drug resistant and metastatic cells. Furthermore, treatment of

irreversible TGase 2 inhibitors results in apoptosis of cancer cells

and sensitization to chemotherapy in glioblastoma (Yuan, Choi et al.,

2005).

１４

Table 2. Known inducers of TGase 2 expression

１５

3. Dendritic cell

Dendritic cells (DCs) are immune cells participating in the

mammalian immune system. Although DCs are scanty, they are

broadly distributed in many tissues (Shortman and Naik, 2007). DCs

are at the center of immune control, linking innate immunity and

adaptive immunity. DCs are one of the professional antigen

presenting cells (APC) derived from hemopoietic cells and

specialized for T cell-mediated immunity and tolerance. Major

histocompatibility complex (MHC) of DC surface interacts with T cell

receptor (TCR). DCs present peptide epitope of antigen to T cells,

and associates with TCR, activating naive T cell to effector T cells.

The interaction between DCs and naive T cells initiate immune

response (Shortman and Naik, 2007).

Peripheral tolerance to self-antigens is maintained by immature

DCs. Activation of DCs is essential to induce immunity. DCs derived

from progenitor cells go through maturation step upon encountering

antigen. Then, DCs migrate to lymphoid tissues, possessing ability to

induce T cells. During last several years, it is identified that DCs

have many distinct subtype. Each subtype can be classified according

to many aspects such as location, phenotype and functions (Shortman

and Liu, 2002). Recently, therapeutic DC vaccine has been proposed

as a novel approach for treatment of cancer and chronic infections

(Freire, Kadaria et al., 2010), suggesting potential roles for DCs

other than immune induction (Chauvin and Josien 2008).

１６

3.1 History of DCs

In the 19th century, Dendritic cells were first explained by Ralph M.

Steinman and Zanvil A. Cohn, described as a large stellate cells with

distinct properties from the former cell types (Steinman RM 2007). In

1998, Jacques Banchereau and Ralph M. Steinman explain that B and

T lymphocytes are the mediators of immunity, but their function is

under the control of dendritic cell. Thus, dendritic cell is thought to

be as a powerful tool for manipulating the immune system. For this

finding, Steinman was awarded the Nobel Prize in Physiology and

Medicine in 2011. Almost 30 years on, dendritic cells are one of the

most well studied cells in immunology.

3.2 Development of DCs

The key advance in DC biology for the past few years have been

facilitated by the ability to propagate large numbers of DCs in vitro

with defined growth factors. One of the most important finding is DCs

are not a single cell type, but a system of cells that arise from

distinct, bone morrow-derived, hematopoietic lineages. The earliest

DC progenitors are normally present in the bone morrow, and DC

progenitors/precursors are released from the bone morrow and

patrol through the blood and lymphoid organs that are ready to

receive differentiation signals.

１７

3.2.1 Lymphoid-derived DCs

Lymphoid DC is a distinct DC subtype that is closely linked to the

lymphocyte lineage. Lymphoid DCs may be differentiated from

progenitors that also have the potential to mature into T and natural

killer (NK) cells (Marquez, Trigueros et al., 1998). Such progenitors

are distributed in the thymus and in the T cell areas of secondary

lymphoid tissue (Grouard, Rissoan et al., 1997). Lymphoid DCs also

may be developed from thymic progenitors by stimulation with

interleukin 3 (IL-3), but not granulocyte-macrophage colony-

stimulating factor (GM-CSF), and from lymphoid precursors in human

tonsil by treatment with CD40 ligand (CD40L).

A various functions of lymphoid DCs have been reported (Mott,

UnderHill et al., 2008). Above all, they promote negative selection in

the thymus and are costimulators of CD4+ and CD8+T cells.

Lymphoid DCs primarily mediate regulatory rather than stimulatory

immune effector functions. In human, early progenitors of the

lymphoid related DC pathway express CD34, CD10 and CD38;

intermediate precursors have been described as CD25+, CD86+, IL-

3R+, CD4+, MHC class Ⅰ, and MHC class Ⅱ positive.

3.2.2 Myeloid-derived DCs

CD34+ progenitor and PB mononuclear cells (MNC) have described

two other DC pathways, both associated with the myeloid lineage

(Cavanagh LL, 1998). One pathway is derived from a CD14+

precursor and is represented by a CD14+CD1a+ intermediate; the

１８

other is derived from a CD14-CD1a+ precursor. Both produce mature

DCs capable of stimulating strong naive T cell responses. Mature

DCs of both path way express high level of MHC class I, MHC class

Ⅱ molecules and CD86. Tumor necrosis factor (TNF), GM-CSF, and

IL-4 induce the maturation of CD14-derived DCs from CD34+

progenitors or CD14+PB monocytes (Rosenzwajg, Camus et al.,

1998). For CD1a-derived DCs, transforming growth factor β (TGF-

β) appears to be a principal differentiation factor. CD14-derived DCs

most likely correspond to interstitial and/or circulating blood DCs,

whereas CD1a-derived DCs are related to epidermal DCs (Strobl et

al., 1998).

１９

Figure 4. Correlation between the development of myeloid and

lymphoid dendritic cells (DCs) (Kumar and Jack 2006)

２０

3.3 Maturation and activation of DCs

Life cycle of DCs can be divided into two stages: immature and

mature stage based on the expression of surface markers. While

peripheral tolerance to self-antigens is maintained by immature DCs,

activation (maturation) of DC is essential to induce immunity.

Immature DCs have capacity to capture and internalize antigens due

to surface expression of receptors that enables the recognition and

endocytosis of potential antigens. These receptors include members

of pattern recognition receptors (PRRs) family such as Toll-like

receptors, C-type lectin receptors (CLRs), intracytoplasmic

nucleotide oligomerization domain (NOD)-like receptors, and so on.

Immature DCs act as immunological sensors that receive stimuli from

environment to alert the immune system. However, immature DCs

are poor stimulators of T cells and must undergo a maturation

process.

Upon antigen capture y immature DCs, a complex process of

maturation is initiated based on the nature of the stimuli. There are

stimuli that control DC maturation such as cytokines (TNF, IL-1,

IFNs, and TSLP), microbial products (LPS, CpG, dsRNA and MDP),

danger signals (Urates, HSPs), NK cells (INFγ), and T helper cells

(CD40L). And it is showed the series of phenotypic changes that

enables DCs to initiate immunity as professional APCs (Villadangos

and Schnorrer, 2007), which include (1) loss of phagocytic receptor;

(2) morphological changes; (3) secretion of cytokines and

chemokines such as IL-1β, IL-10, IL-12, TNF, CCL19, and CCL22;

２１

(4) up-regulation of costimulatory and adhesion molecules such as

CD80, CD86, CD40, CD54 and CD58; (5) translocation of MHC class

Ⅰ,Ⅱ compartents to the cell surface; and (6) migration of mature

DCs into regional/draining lymphoid tissue. As a result, antigen

acquired in peripheral sites is retained, processed and presented to

T cells in lymph nodes.

２２

4. NADPH oxidase

Reactive oxygen species (ROS) are a heterogeneous group of highly

reactive molecules that oxidize targets in a biologic system. ROS are

widely recognized as important mediators of cell growth, adhesion,

differentiation, senescence, and apoptosis. During steady-state

conditions, ROS are constantly produced in the electron-transport

chain during cellular respiration and by various constitutively active

oxidases. ROS production can also be induced by activation of

NADPH oxidase family in a process generally referred to as an

oxidative burst. NADPH oxidase is a superoxide (O2−)-generating

enzyme first identified in phagocytes such as neutrophils that shows

bactericidal activities. After that it has also been reported that O2−

is released in an NADPH dependent manner in non-phagocytes. But

studies on NADPH oxidases have been performed mainly in

phagocytes because mutations in the gene encoding the phagocyte

NADPH oxidase were found in patients with chronic granulomatous

disease (CGD) (Royerpokora, Kunkel et al., 1986)

4.1. NADPH oxidase iosenzymes

NADPH oxidases are composed of five homologs that are

distinguished by catalytic subunit from NOX1 to NOX5, and two

related DUOX1, 2.

２３

Table 3. NOX isoenzymes

２４

4.2 Structure and subunits of NOX2

NOX2/NADPH oxidase is composed of NOX2 (gp91phox), p22phox,

p67phox, p47phox, p40phox, and the small GTP-binding protein Rac.

gp91phox and p22phox are located at the plasma membrane and p22phox

forms a mutually stabilizing complex with gp91phox. The complex of

gp91phox and p22phox is referred to as flavocytochrome b558. p67phox,

p47phox, and p40phox are located at cytosol and referred to as

cytosolic subunits. Under resting conditions, the SH3 (SRC-

homology3) domains of p47phox bind intramolecularly to the

autoinhibitory region (AIR) in the middle of C-terminal, preventing its

binding to p22phox. The phagocyte oxidase (PX) domain in N-terminal

of p47phox is known for role of binding to membrane lipids. Other

cytosolic subunit p40phox binds to PB1 (Phox and Bem1p proteins)

domain of p67phox. Similarly, SH3 domain of p67phox binds to PR

(prolline rich) domain of p47phox. p40phox, p47phox, and p67phox are

assembled into complex in cytosol. A small GTP-binding protein Rac

is interacted with inhibitory protein RhoGDP-dissociation inhibitor

(RhoGDI).

4.3 Activation mechanisms of NOX2

Activation of the NOX2 system can be divided into at least three

signaling triggers; lipid metabolism, phosphorylation, and Guanine-

nucleotide exchange. First, after any stimulation phosphatidylinositol

3-kinase (PI3K) and phospholipase D produce 3-phosphorylated

phosphatidylintositols (PtdInsP) and phosphatidic acid, respectively,

２５

providing lipids to which the p47phox and p40phox PX domains bind.

Second, when it comes to complex of cytosolic subunits (p40phox,

p47phox, and p67phox) they translocate to membrane and assemble with

flavocytochrome b558. To translocate from cytosol to membrane,

protein kinases are required.

Protein kinases like AKT catalyse AIR domain of p47phox. It allows

transformation of p47phox, which makes p47phox to bind to p22phox. It

also relieves inhibition of the PX domain of p47phox, allowing binding

to lipids. Last, activation allows GTP binding. The conformational

change of RAC-GDP reduces interaction RAC-GDP with Rho GDI.

And it makes for RAC-GTP to bind to membrane. p67phox subunit has

activation domain that activates electron transfer. It produces

superoxide.

２６

Figure 5. Domain structure of regulatory proteins for NOX enzymes

２７

Figure 6. Activation of reactive oxygen species (ROS) generation by

assembly of Phox regulatory proteins

２８

Purpose

TGase 2 is involved in some inflammatory disease such as celiac

disease and bleomycine-induced lung fibrosis. Moreover, the role of

DCs in septic shock has been highlighted. It has been reported that

TGase 2 is a possible player in molecular and cellular mechanisms

leading to improper regulation of inflammatory response involved in

sepsis (Falasca L, 2008). Moreover, treatment with KCC009, inhibitor

for TGase 2, did not alter monocyte differentiation to phenotypically

immature DCs, but influence the ability of DCs to mature when

stimulated with LPS (Matic, Sacchi et al., 2010). The aim of this

study is to confirm the role of TGase 2 in differentiation and

maturation of DC using TGase 2 knock-out mice. Interestingly,

results of our research with TGase 2 knock-out mice show that

TGase 2 is not involved in DCs maturation and phenotype.

２９

Material and Method

1. Mice

C57BL/6J mice were obtained from The Jackson Laboratory. TG2-

null mice were backcrossed to C57BL/6J mice for 10 generations

(N10). Mice were kept in essentially specific pathogen-free

environment at the animal facility of Seoul National University

College of Medicine. All animal experiments were carried out with

the approval of the Institutional Animal Care and Use Committee at

Seoul National University.

2. Cell culture

Bone marrow (BM) cells were isolated by flushing femur and tibia.

The cells were centrifuged and then resuspended in culture medium

consisting of Isocove’s Modified Dulbecco’s Media (Gibco, #12240)

supplemented with 10% heat-inactivated fetal bovine serum (Gibco,

#26140), 0.1 % 2-mercaptoethanol (Gibco, #21985-023), 0.1 %

gentamicine (Gibco, #15750-060), 1% Penicillin-Streptomycin

glutamine (Gibco, #10378), gentamicine (Gibco, #15750-060), 3

ng/ml IL-4, and GM-CSF (peprotech, #214-14 and #315-3). BM

cells were cultured at 1ｘ106 cell/ml in 24well plates in culture

medium. The cells were incubated at 37 ℃ in a humidified atosphere

containing 5% CO2 for 6 days as shown in Figure 7. Media has to be

３０

changed per 2 days for 6 days. DCs were maturated for 20 hr by the

addition of 1 μg/ml LPS (sigma, #L6529).

３１

Figure 7. A process of mDCs differentiation and maturation

３２

3. Flow cytometry

Cells were harvested by vigorous pipetting and removal of

nonadherent cells. Before incubatation with mAbs, the cells were

blocked at 4 ℃ for 20 min in mixture FACS buffer (1 % BSA, 1mM

EDTA, and 0.02% NaN3 in PBS), super block 1:1. Cells were incubated

with directly conjugated mAbs at 4 ℃ for 30 min.

The following mAbs were used: PE Rat anti-mouse CD40 (BD Ph

armingenTM), PE/Cy7 anti-mouse, C86 (BioLegend), FITC Mouse

anti-mousel-A[b](BDPharmingenTM), APC conjugated anti-mouse

CD80(B7-1) (eBioscience.com), APC/Cy7 anti-mouse CD11c

(BioLegend), and PE rat anti-mouse CD11b (BDPharmingenTM). After

washing two times with FACs buffer, cells conjugated mAbs were

measured by flow cytometry.

4. Real-time quantitative PCR

Quantitative assessment of mRNA levels of IL-10, IL-12, CCR7, and

NOX2 subunits was performed by real-time quantitative PCR using a

CFX96TM Real-Time system (Bio-Rad). The cDNA templates from

each cell were amplified using Maxime RT PreMix Kit (iNtRON).

KAPA SYBR® FAST qPCR Master Mix (KAPA Biosystems)

isusedtoreal-timePCR.

３３

5. Intracellular TG activity assay

Cells were incubated with 100mM biotinylated pentylamine (BP,

Pierce) for 1 hr at 37 ℃ before harvesting. Cell extracts were

prepared by disrupting the cells in lysis buffer (50 mM Tris-Cl, pH

7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) followed by

centrifugation (12,000 g) for 10 min at 4 ℃ and were subjected to

SDS-PAGE using 10 % gel and then transferred to nitrocellulose

membrane. The proteins that crosslink with BP were visualized by

probing with HRP-conjugated strept-avidin, followed by enhanced

chemiluminescence reagents (Pierce).

6. Western blot analysis

Cells were suspended with 1 % Tritronｘ100 buffer and sonicated (2

sec pulse and 2 sec pulse, 5 times at 14 % amplitude). Cell lysate

were centrifuged at 12000 g for 10min at 4 ℃ and supernatant was

transferred to e-tube. Samples were boiled in sample loading buffer

at 100 ℃ for 10 min, seperated on 10 % SDS-polyacrylamide gel,

and transferred to a nitrocellulose membranes. The membrane was

incubated with 5 % skim milk in TBS-T (50 mM Tris, 200 mM NaCl,

0.05 % Tween 20, PH7.5) for 1 hr to block nonspecific protein

binding. After washing for 5 min with 0.1 % TBS-T, the membranes

were then incubated with anti-TGase 4 antibody (1:1000), anti-

TGase 2 antibody (1:1000), or anti-gp91phox antibody (1:1000) in 5 %

skim milk in TBST. Immunoreative antibodies were further incubated

３４

with appropriate secondary antibodies conjugated to horseradish

peroxidase in 5 % skim milk in TBST for 1 hr. After washing three

times with 0.1 % TBS-T, the membranes were detected by

SuperSignal West Substrate (Pierce, #34080) and exposed to X-ray

film.

7. Phagocytosis assay

BM-drived DCs (105/sample) were suspended with culture medium.

Fluorescein isothiocyanate-dextran particles (sigma, Korea) were

added to each sample at a final concentration of 1 mg/ml and

incubation was continued for 45 min. Cells were rinsed twice with

FACS buffer containing 1 % BSA, 1 mM EDTA, and 0.02 % NaN3 in

PBS and analysed using flow cytometry. First, cells were gated out

according to CD11c+, CD11b+ differentiated DCs population and then

phagocytosing BM-drived DCs were identified on a forward scatter-

side scatter plot and the fluorescence of the cells was measured. Ten

thousands of DCs were counted in each sample.

8. In vitro 3D chemotaxis assays

Mixture of 50ml collagen (1.5 mg/ml) and 50 ml of a cell suspension

containing 5 × 105 BM-drived DCs was placed in 1 μ-Slide Ⅵ flat

ibiTreat microscopy chamber and incubated for 30 min. 30 ml culture

medium containing chemokines (CCL21) was inserted in lower

chamber and 30 ml culture medium in upper chamber. Cells migated

３５

from the lower and upper chamber, and were recorded onto confocal

microscope per 2 min for 4 hr.

9. In vitro survival assay

BM-drived DCs were seeded in 96-well micro plates at a

concentration of 1ｘ105/ml in 100ml of culture medium in the

presence or absence of LPS (1 μg/ml) and/or thapsigargin (50 nM).

For each time cells were added 20 μl/well Cell Titer-Blue Reagent

(Promega, #G8080). And then record fluorescence 560/590 nm.

10. Measurement of ROS production

DCs (3ｘ106 cells) from wild type mice and TGase 2 knock-out mice

were treated with PMA or LPS. ROS generation in cultured medium

was measured by luminal-enhanced chemiluminescence. That is, DCs

were collected and resuspended in 200l of CO2-independent

medium containing luminol (12.5 M) and horseradish peroxidase

(6.25 unit). Cell suspensions were preheated to 37℃ in the

thermostated chamber of the luminometer (Berthold-Biolumat LB937)

and allowed to stabilize for 5min. Then, they were stimulated with

50l medium containing 5 g/ml LPS, 2.5 g/ml PMA or 50 M DPI.

Changes in chemiluminiscence were measured over the indicated

times, every 1 min for 120 min.

３６

Results

TGase 2 is not involved in differentiation of DC

To investigate the role of TGase 2 in differentiation of DCs, we

examined the protein level of TGase 2 and transamidation activity

during BMCs is induced to differentiate into DCs. Streptavidin-HRP

that recognizes proteins incorporated with biotinylated-pentylamine

(BP) was used to measure the transamidation activity. When BMCs

were differentiated into DCs, the protein level of TGase 2 and

transamidation activity were gradually increased. Moreover, the

protein level of TGase 2 and transamidation activity were further

increased with treatment DCs with LPS at day 6. However, when

compared the percentage of DC population by flow cytometry

measuring the expression of CD11b and CD11c, we found that there

is no difference in differentiated DCs population between BMCs from

wild-type and TGase 2-deficient mice (Figure 8). These results

indicate that TGase 2 is not involved in DC differentiation in contrast

to previous reports (Matic et al., 2010).

３７

Figure 8. TGase 2 is not involved in DC differentiation.

We differentiated mouse bone marrow cells (BMCs) into DCs by

treatment of GM-CSF and IL-4 for 6 days. (A) BM-derived DCs are

incubated with 500uM of biotinylated pentylamine for 1hr before it is

collected. After 6 day DCs are treated 1 μg/ml LPS. (B) DCs not

treated LPS are collected and then expression of CD11c, CD11b is

measured by flow cytometry.

３８

TGase 2 is not involved in maturation of DC

There was report that TGase 2 plays an important role in

maturation of DCs by LPS (Matic et al., 2010). However, they didn't

show this effect in DCs from TGase 2-deficient mice. So, we tested

whether DCs obtained from TGase 2-deficient mice are abnormally

maturated by LPS or not. After 6 day DCs differentiated from wild-

type BMCs and TGase 2-deficient BMCs were treated with different

concentration of LPS for 20 hr. And then, CD80+, CD86+, CD40+, and

MHCⅡ+ populations that are markers of maturated DCs were

measured. Unlike previous reports, there was no difference between

DCs obtained from wild type mice and TGase 2-deficient mice

(Figure 9). DCs obtained from TGase 2-deficient mice were normally

maturated by LPS dose dependently. Thus, TGase 2 is not involved

in maturation of DCs.

３９

Figure 9. TGase 2 is not involved in LPS-induced DC maturation.

Cells were treated with different concentration of LPS for 18 hr.

The expression of CD80+CD86+(A), CD40+ MHC2+(B) that indicate

mature DCs is tested by flow cytometry. The expression level is

increased by LPS dose dependant. But there is no difference

between wile type mice and TGase 2 knock-out mice.

４０

Cystamine and KCC009 inhibit maturation of DC via TGase 2-

independent pathway

Although our data showed that TGase 2 is not involved in

maturation of DCs, we decided to test whether DCs maturation is

decreased by TGase 2 inhibitor as previous study showed that TGase

inhibitor suppresses the process of DC maturation. At day 6 of

differentiation, DCs were treated with TGase 2 inhibitor, cystamine,

and LPS. Interestingly, DCs treated with cystamine showed

decreased expression of surface markers in the both DCs obtained

from wild-type and TGase 2-deficient mice. Likewise, TGase

activity was decreased with cystamine treatment in a dose dependent

manner in the both DCs from wild type and TGase 2-deficient mice

(Figure 10). To confirm these results, we treated DC with another

TGase 2 inhibitor, KCC009. The expression of surface markers was

similar with those treated with cystamine (Figure 11). These results

indicate that cystamine and KCC009 inhibit maturation of DCs via

TGase 2-independent pathway, suggesting that other TGase

isoenzyme may play a role in the process of DC maturation.

４１

Figure 10. Cystamine inhibits DC maturation via TGase 2-

independent pathway.

BM-drived DCs were collected after 6 days of culture with GM-CSF

and IL-4 and in the presence of the LPS stimulation and the different

concentrations of cystamine, TGase 2 specific inhibior. (A) After LPS

stimulati0n, CD80, CD86, CD40, and MHC2 molecules of each DC

treated cystamine were analyzed by flow cytometry. (B) Maturated

DCs from wild-type and TGase 2-deficient BMCs were lysed and the

lasates were immunoblatted with horseradish peroxidase conjugated

strep-avidin to visualize intracellular TG activity or antibody for

actin.

４２

Figure 11. KCC009 also inhibits DC maturation via TGase 2-

independent pathway.

Another TGase 2 specific inhibitor, KCC009, also displays the same

tendency with result from cystamine. (A) flow cytometry analysis (B)

Western blot for TGase activity and actin

４３

Expression of TGase 4 is increased during DC maturation

To investigate which TGase isoenzyme(s) is involved in maturation

of DC, we first screened mRNA expression level of from TGase 1 to

FⅩⅢa. DCs were differentiated for 6 day and treated with LPS for 3

hr, 6 hr, and 18 hr. Cells were then collected, and total RNA was

extracted using TRIzol reagent (Invitrogen). Primer pairs used for

RT-PCR were summerized in Table 4. Compared with control DCs,

DCs treated with LPS showed comparable increase of TGase 1,

TGase 2, TGase 3, and TGase 5 mRNA level after 3hr LPS treat, and

decrease of TGase 7 and FⅩⅢa mRNA level. By contrast, the level

of TGase 4 mRNA was increased about 100 folds after 3hr LPS

treatment (Figure 12). To confirm these results in protein level,

BMCs were differentiated to DCs for 1 day, 3 day, and 6 day, and the

treated with LPS for 20 hr. Cells were collected and subjected to

Western blot analysis using TGase 4 specific antibody. Western blot

analysis showed that TGase 4 protein level was increased during

differentiation from BMCs to DCs, and protein level of TGase 4 was

further increased with LPS treatment (Figure 13).

To further confirm the involvement of TGase 4 in DC maturation,

we examined the knock-down effect of TGase 4 on DC maturation.

BMCs were differentiated to DCs for 6 day, and cells were

transfected with siRNA for GFP and TGase 4. GFPi was used as

positive control. Surface markers of maturated DCs were measured

by FACS analysis. DCs transfected with siRNA for TGase 4 showed

significantly decreased expression of CD80, CD86 compared to

４４

transfected with siRNA for GFP (Figure 14). These results indicate

that TGase 4 is involved in the maturation of DC.

４５

Table 4. Primers of TGases

４６

Figure 12. TGase 4 is a strong candidate that is involved in DC

maturation.

After 6 days DCs were treated LPS (1 μg/ml) for 3, 6, and 18 hr.

And then DCs were collected and total RNA was extracted from

cultured DCs using TRIzol reagent (Invitrogen). The RNA was then

employed in the first-strand cDNA synthesis reations using Maxime

RT PreMix Kit (iNtRON). The PCR products were amplfied using

each primer.

４７

Figure 13. TGase 4 is increased along with differentiation of DC.

40 mg of concentrated BM-drived DCs for each time point after

differentiation and in the presence of the LPS stimulation were

loaded and immunoblot was performed with anti-TGase 4 monoclonal

antibody.

４８

Figure 14. TGase 4 is involved in maturation of DC

We transfect siRNA for TGase 4 and GFP after 6 day of DC culture

And then DCs were treated with LPS or not for 20 hr. Maturation markers

were analyzed by FACS.

４９

Functional analysis of TGase 2 deficient DCs: cytokine secretion

Figure 10 and 11 showed significantly higher TGase activity of

wild-type DCs than TGase 2 deficient DCs. To investigate the role of

TGase 2 in DC function, we examined cytokine level secreted by DCs

upon LPS treatment. Under proper stimulation, DCs are able to

secrete IL-10 and IL-12 which play a central role in the regulation of

the immune response (Matic, Sacchi et al., 2010). We measured IL-

10 and IL-12 released from DCs after LPS stimulation. We found no

difference between wild-type and TGase 2-deficient DCs, indicating

that TGase 2 is not involved in cytokine secretion of DC (Figure 15).

５０

Figure 15. TGase 2 is not involved in function of matured DCs.

The quantification of mouse IL-10(A), 12(B) mRNA in DCs from wild

-type BMCs as well as TGase 2-deficient BMCs treated with LPS for

20 hr or not by using real-time quantitative PCR.

５１

Functional analysis of TGase 2 deficient DCs: antigen uptake

Previous reports showed that TGase 2 play a role in phagocytosis

through phagocytosis-associate crosstalk between macrophages and

apoptotic cells. Moreover, TGase 2 also contributes to phagocytotic

ability of neutrophil (Balajthy et al., 2006). To test whether TGase 2

also play a role in phagocytosis of DC, BMCs were differentiated to

DCs for 6 day, and cells were incubated with FITC labeled dextran

for 15 min, 30 min, and 45 min. Flow cytometric analysis revealed

that there is no difference in percentage of FITC positive DC

populations between wild-type and TGase 2-deficient DCs,

indicating that TGase 2 is not involved in phagocytosis of DC (Figure

16).

５２

Figure 16. TGase 2 is not involved in phagocytosis of DCs

DCs differentiated from TGase 2-deficient BMCs and wild-type

BMCs for 6 days were incubated with FITC-labeled E coli at 37 ℃

for each designated times. FITC positive cells out of total BM-drived

DCs were counted by FACS analysis. Each experimental group

included 2 to 6 mice, and each individual experiment was performed

2 or 3 timed in triplicates.

５３

Functional analysis of TGase 2 deficient DCs: migration

In regulating immune response, DCs have a unique ability to migrate

peripheral lymph node and then to stimulate naive T lymphocytes.

We tested whether TGase 2 affects migration ability of DCs. When

DCs are activated, DCs express receptors to their surface that

enable to migrate to peripheral lymph node. One of major receptors

participating migration is CCR7. To investigate migration of DCs we

use specially designed microscopy chamber. It has two sides. DCs

were placed on one side and ligand of CCR7, CCL21, were placed on

the other side. And then DCs moving toward CCL21 were measured

by confocal microscopy every 2 min for 4 hr. Four sets were

measured independently; wild type DC, wild-type DC treated with

LPS, TGase 2 deficient DC and TGase 2 deficient DC treated with

LPS. Data obtained from confocal microscopy were analyzed at the

three factors which are accumulated distance, euclidean distance,

and velocity. In all factors, no difference was observed (Figure 17).

Moreover, when measured mRNA and protein level of CCR7, major

factor affecting this assay, both wild-type and TGase 2-deficient

DCs showed the similar expression level of CCT7 mRNA and protein

(Figure 18), indicating that TGase 2 is not involved in migration of

DCs.

５４

Figure 17. TGase 2 is not involved in migration of maturated DC.

Accumulated distances, Euclidean distances, and Velocities of

chemotaxing BM-derived single DCs (dot;line,mean) towards CCL21

in the presence or absence of LPS stimulation (1 μg/ml) in 3D

collagen matrices.

５５

Figure 18. TGase 2 is not involved in expression of CCR7.

(A) After LPS stimulation, CCR7 positive DCs positive were

analyzed by flow cytometry analysis. (B) The quantification of mouse

CCR7 mRNA in DCs from wild-type BMCs as well as TGase 2-

deficient BMCs treated with LPS for 20 hr or not by using real-time

quantative PCR.

５６

Functional analysis of TGase 2 deficient DCs: apoptosis

It has been considered that apoptosis of DC is critical for immune

suppression. Therefore, defect in apoptosis of DC can induce

autoimmune diseases (Chen, Wang et al., 2006). Moreover, TGase 2

is involved in apoptosis. We investigated whether TGase 2 is

involved in DC apoptosis. DCs differentiated from wild type and

TGase 2-deficient BMCs for 6 days were activated with LPS or LPS

and thapsigargin. The percentages of living cells were measured at

various time points. No difference between wild-type and TGase 2

deficient DC was observed (Figure 19), indicating that TGase 2 is not

involved in apoptosis of DCs.

５７

Figure 19. TGase 2 is not involved in apoptosis of maturated DC.

BM-drived DCs from wild-type mice as well as TGase 2-deficient

mice survival after incubation with LPS (1 μg/ml) or thapsigargin (50

nM). Survival of unstimulated cells kept in culture medium.

５８

Functional analysis of TGase 2 deficient DCs: ROS production

Previous reports showed that gp91phox, one of major components of

NOX2, is down-regulated in TGase 2-deficient neutrophil

granulocyte (Balajthy et al., 2006). In human dendritic cells, NADPH

oxidase is regulated by Toll receptor. Oxygen radicals generated by

NADPH oxidase is not associated with DC differentiation, maturation,

cytokine production, and induction of T cell proliferation (Vulcano et

al., 2004), but is required for DC killing of intracellular Escherichia

coli. Therefore, we tested whether TGase 2 is involved in ROS

generation through modulation of NOX2 expression. When DCs were

treated with LPS, wild-type DC showed significant increase of

p67phox and gp91phox expression, whereas TGase 2-deficient DCs

showed no change in the expression of NOX2 components (Figure

21). Thus, TGase 2 is involved in the regulation NOX2 expression of

DC.

５９

Figure 20. TGase 2 is not involved in ROS release of maturated DC.

Release of ROS from BM-drived DCs from wild-type mice and TGase2

-deficient mice left unstimulated or stimulated with LPS (1 g/ml)

(a,b) and with PMA (0.5 g/ml) or PMA and DPI (10 M)

６０

Figure 21. TGase 2 is involved in mRNA level of NOX2 components

After 6 days DCs were treated LPS (1 μg/ml) for 3, 6, and 18 hr.

And then DCs were collected and total RNA was extracted from

cultured DCs using TRIzol reagent (Invitrogen). The RNA was then

employed in the first-strand cDNA synthesis reactions using Maxime

RT PreMix Kit (iNtRON). The PCR products were amplified using

each primer.

６１

Discussion

Regulating innate immunity and adaptive immunity by DC is one of

the most important immune responses in immune system. It has

capacity to respond directly to an invading pathogen. So, DC is

considered as great therapeutic target. Recently, it has been

reported that treatment of DC with LPS up-regulates the expression

of TGase 2 that catalyzes the post-translational modification of

protein by forming polyaminated proteins or cross-linked proteins.

However, role(s) of TGase 2 in DC has been unclear. In this study,

role(s) of LPS-induced TGase 2 in DC was investigated using TGase

2-deficient DC.

Although DC treated with LPS up-regulates the expression of

TGase 2 as well as transamidation activity, differentiated DC

population and matured DC markers didn’t show difference between

DCs from wild-type and TGase 2-deficient mice. However, like

previous study that showed TGase inhibitor suppresses the process

of DC maturation, our data also showed the same results. That is

other TGase isoenzyme may play a role in the process of DC

maturation. And we found that increase of TGase activity by LPS

treatment is due to expression of TGase 4.

Our data also showed significantly higher TGase activity of wild-

type DCs than TGase 2-deficient DCs and it suggested that TGase 2

may play the important role in DC function. Seeing our data, TGase

６２

2-deficient DC showed no difference in the level of secreted

cytokine, phagocytosis, migration, and apoptosis compared with

those of wild-type DC. However, wild-type DC showed higher level

of ROS than TGase 2-deficient DC.

Taken together, our results indicated that role of TGase 2 in DC

function might be involved in the generation of ROS. To be more

definite, we need to more studies by which mechanism(s) TGase 2 is

involved in the generation of ROS.

６３

References

Akimov SS, B. A. (2001). "Cell-surface transglutaminase promotes

fibronectin assembly via interaction with the gelatin-binding domain

of fibronectin a role in TGFβ-dependent matrix deposition.pdf."

Anderson, R. P., P. Degano, et al. (2000). "In vivo antigen challenge in celiac

disease identifies a single transglutaminase-modified peptide as the

dominant A-gliadin T-cell epitope." Nature Medicine 6(3): 337-342.

Balajthy, Z., K. Csomos, et al. (2006). "Tissue-transglutaminase contributes

to neutrophil granulocyte differentiation and functions." Blood 108(6):

2045-2054.

Begg, G. E., L. Carrington, et al. (2006). "Mechanism of allosteric regulation

of transglutaminase 2 by GTP." Proc Natl Acad Sci U S A 103(52):

19683-19688.

Cassidy, A. J., M. A. van Steensel, et al. ( 2005). "A homozygous missense

mutation in TGM5 abolishes epidermal transglutaminase 5 activity

and causes acral peeling skin syndrome." Am J Hum Genet 77(6):

909-917.

Cavanagh LL, S. R., Grimmett KL, Thomas R. (1998). "<Proliferation in

monocyte derived dendritic cell cultures is caused by progenitors

cells capable of myeloid differentiation.pdf>."

Chakravarty R, R. R. (1989). "Acylation of Keratinocyte Transglutaminase

by Palmitic and Myristic acids in the membrane anchorage

region.pdf."

Chauvin, C. and R. Josien (2008). "Dendritic cells as killers: Mechanistic

aspects and potential roles." Journal of Immunology 181(1): 11-16.

６４

Chen, M., Y. H. Wang, et al. (2006). "Dendritic cell apoptosis in the

maintenance of immune tolerance." Science 311(5764): 1160-1164.

Esposito, C. and I. Caputo (2005). "Mammalian transglutaminases.

Identification of substrates as a key to physiological function and

physiopathological relevance." FEBS J 272(3): 615-631.

Falasca L, F. M., Rinaldi A, Tuosto L, Melino G, Piacentini M. (2008).

"<Transglutamine Type Ⅱ is involved in the pathogenesis of

endotoxic shock.pdf>."

Fesus L, P. M. (2002). "<Transglutaminase 2 an enigmatic enzyme with

diverse functions.pdf>."

Fraij, B. M. and R. A. Gonzales (1996). "A third human tissue

transglutaminase homologue as a result of alternative gene

transcripts." Biochimica Et Biophysica Acta-Gene Structure and

Expression 1306(1): 63-74.

Freire, A. X., D. Kadaria, et al. (2010). "Obstructive Sleep Apnea and

Immunity: Relationship of Lymphocyte Count and Apnea Hypopnea

Index." Southern Medical Journal 103(8): 771-774.

Genitil V, S. M., Chiocca EA, Akande O, Birckbichler PJ, Lee KN, Stin JP,

Davies PJ (1991). "<The human tissue transglutaminase gene maps

on chromosome 20q12 by in situ fluorescence hybridization..pdf>."

Grouard, G., M. C. Rissoan, et al. (1997). "The enigmatic plasmacytoid T

cells develop into dendritic cells with interleukin (IL)-3 and CD40-

ligand." Journal of Experimental Medicine 185(6): 1101-1111.

Haroon, Z. A., J. M. Hettasch, et al. (1999). "Tissue transglutaminase is

expressed, active, and directly involved in rat dermal wound healing

and angiogenesis." Faseb Journal 13(13): 1787-1795.

Hiiragi T, S. H., Nagafuchi A, Sabe H, Shen SC, Matsuki and Y. K. M,

６５

Tsukita S. (1999). "<Transglutaminase Type 1 and Its Cross-linking

Activity Are concentrated at adherens junctions in simple epithelial

cells..pdf>."

Iismaa, S. E., B. M. Mearns, et al. (2009). "Transglutaminases and disease:

lessons from genetically engineered mouse models and inherited

disorders." Physiol Rev 89(3): 991-1023.

Iismaa, S. E., M. J. Wu, et al. (2000). "GTP binding and signaling by

Gh/transglutaminase II involves distinct residues in a unique GTP-

binding pocket." J Biol Chem 275(24): 18259-18265.

Jiang, W. G. and R. J. Ablin (2011). "Prostate transglutaminase: a unique

transglutaminase and its role in prostate cancer." Biomark Med 5(3):

285-291.

John, S., L. Thiebach, et al. (2012). "Epidermal transglutaminase (TGase 3)

is required for proper hair development, but not the forma tion of the

epidermal barrier." PLoS One 7(4): e34252.

Kim HC, L. M., Gorman JJ, Park SC, Girard JE, Folk JE, Chung SI. (1990).

"<protransglutaminas D from guinea pig skin isolation and partial

characterization.pdf>."

Krasnikov, B. F., S. Y. Kim, et al. (2005). "Transglutaminase activity is

present in highly purified nonsynaptosomal mouse brain and liver

mitochondria." Biochemistry 44(21): 7830-7843.

Kumar, S. and R. Jack (2006). "Origin of monocytes and their differentiation

to macrophages and dendritic cells." Journal of Endotoxin Research

12(5): 278-284.

Lesort, M., J. Tucholski, et al. (2000). "Tissue transglutaminase: a possible

role in neurodegenerative diseases." Progress in Neurobiology 61(5):

439-463.

６６

Lorand, L. and R. M. Graham (2003). "Transglutam inases: crosslinking

enzymes with pleiotropic functions." Nat Rev Mol Cell Biol 4(2):

140-156.

Lorand, L., L. K. H. Hsu, et al. (1981). "Lens Transglutaminase and Cataract

Formation." Proceedings of the National Academy of Sciences of the

United States of America-Biological Sciences 78(3): 1356-1360.

Marquez, C., C. Trigueros, et al. (1998). "Identification of a common

developmental pathway for thymic natural killer cells and dendritic

cells." Blood 91(8): 2760-2771.

Matic, I., A. Sacchi, et al. (2010). "Characterization of transglutaminase type

II role in dendritic cell differentiation and function." J Leukoc Biol

88(1): 181-188.

Mauro Piacentini, Maria Grazia Farrace,* Lucia Piredda,* Paola Matarrese,,

L. F. Fabiola Ciccosanti, Carlo Rodolfo,* Anna Maria Giammarioli,,

et al. (2002). "<Transglutaminase overexpression sensitizes

neuronal cell lines to apoptosis by increasing mitochondrial

membrane potential and cellular oxidative stress.pdf>."

McDonagh, J. and R. P. McDonagh (1975). "Alternative pathways fo r the

activation of factor XIII." Br J Haematol 30(4): 465-477.

Melino G, P. M. (1998). "'Tissue' transglutaminase in cell death a

downstream or a multifunctional upstream effector.pdf."

Mott, K. R., D. UnderHill, et al. (2008). "Lymphoid -Related CD11c(+) CD8

alpha(+) Dendritic Cells Are Involved in Enhancing Herpes Simplex

Virus Type 1 Latency." Journal of Virology 82(20): 9870-9879.

Mouro-Chanteloup, I., J. Delaunay, et al. (2003). "Evidence that the red cell

skeleton protein 4.2 interacts with the Rh membrane complex

member CD47." Blood 101(1): 338-344.

６７

Mulders, J. W. M., W. A. Hoekman, et al. (1987). "Beta-B1 Crystallin Is an

Amine-Donor Substrate for Tissue Transglutaminase." Experimental

Cell Research 171(2): 296-305.

Pigors, M., D. Kiritsi, et al. (2012). "TGM5 Mutations Impact Epidermal

Differentiation in Acral Peeling Skin Syndrome." J Invest Dermatol.

Rosenzwajg, M., S. Camus, et al. (1998). "The influence of interleukin (IL) -4,

IL-13, and Flt3 ligand on human dendritic cell d ifferentiation from

cord blood CD34(+) progenitor cells." Experimental Hematology

26(1): 63-72.

Royerpokora, B., L. M. Kunkel, et al. (1986). "Cloning the Gene for an

Inherited Human Disorder - Chronic Granulomatous-Disease - on

the Basis of Its Chromosomal Location." Nature 322(6074): 32-38.

Shin, D. M., J. H. Jeon, et al. (2008). "TGFbeta mediates activation of

transglutaminase 2 in response to oxidative stress that leads to

protein aggregation." Faseb Journal 22(7): 2498-2507.

Shortman, K. and Y. J. Liu (2002). "Mouse and human dendritic cell

subtypes." Nature Reviews Immunology 2(3): 151-161.

Shortman, K. and S. H. Naik (2007). "Steady-state and inflammatory

dendritic -cell development." Nat Rev Immunol 7(1): 19-30.

Siegel, M. and C. Khosla (2007). "Transglutaminase 2 inhibitors and their

therapeutic role in disease states." Pharmacology & Therapeutics

115(2): 232-245.

Singh, U. S., M. T. Kunar, et al. (2001). "Role of transglutaminase II in

retinoic acid-induced activation of RhoA-associated kinase-2."

Embo Journal 20(10): 2413-2423.

Steinert PM, C. S., Kim SY. (1996). "Inactive Zymogen and Highly Active

Proteolytically Processed membrane-bound forms of the

６８

transglutaminase 1 enzyme in human epidermal keratinocytes.pdf."

Steinman RM, C. Z. (2007). "Pillars Article Identification of a novel cell type

in peripheral lymphoid organs of mice.I. Morphology, quantitation,

tissue distribution.pdf."

Strobl, H., E. Riedl, et al. (1998). "Epidermal Langerhans cell development

and differentiation." Immunobiology 198(5): 588-605.

Takahashi, N., Y. Takahashi, et al. (1986). "Primary structure of blood

coagulation factor XIIIa (fibrinoligase, transglutaminase) from human

placenta." Proc Natl Acad Sci U S A 83(21): 8019-8023.

Thomas, H., K. Beck, et al. (2011). "Transglutaminase 6: a protein

associated with central nervous system development and motor

function." Amino Acids.

Vader, L. W., A. de Ru, et al. (2002). "Specificity of tissue transglutaminase

explains cereal toxicity in celiac disease." Journal of Experimental

Medicine 195(5): 643-649.

Velasco, P. T. and L. Lorand (1987). "Acceptor Donor Relationships in the

Transglutaminase-Mediated Cross-Linking of Lens Beta-Crystallin

Subunits." Biochemistry 26(15): 4629-4634.

Villadangos, J. A. and P. Schnorrer (2007). "Intrinsic and cooperative

antigen-presenting functions of dendritic-cell subsets in vivo."

Nature Reviews Immunology 7(7): 543-555.

Vulcano, M., S. Dusi, et al. (2004). "Toll receptor-mediated regulation of

NADPH oxidase in human dendritic cells." Journal of Immunology

173(9): 5749-5756.

Yuan, L., K. Choi, et al. (2005). "Tissue transglutaminase 2 inhibition

promotes cell death and chemosensitivity in glioblastomas." Mol

Cancer Ther 4(9): 1293-1302.

６９

요약 (국문 초록)

수지상세포는 항원을 present하는 세포로서, innate 와 adaptive 면역의

조절에 중요한 역할을 한다. 트랜스글루타미네이즈 2 (TG2)는 단백질의

글루타민 잔기와 라이신잔기 사이의 교결합 형성하거나 또는 단백질에

폴리아민을 결합시키는 칼슘 의존성 효소이다. 따라서 TG2 는 이러한

수식을 촉매함으로서 단백질 기능을 조절하는 역할을 한다. 또한 TG2는

수지상세포에서도 발현되는데, LPS 로 처리하면 발현이 증가한다. 본

연구는 수지상세포에서 LPS 에 의하여 증가된 TG2 의 기능을 규명하기

위하여 시행되었다. 이를 위하여 wild-type 과 TG2-/-mice 골수에서

채취한 세포를 이용하여, 수지상세포 분화와 성숙 marker 의 발현을

비교하였다. 그 결과, TG2 가 없는 수지상세포는 wild-type 과

비교하여 분화와 성숙능력에서 차이가 없었다. 뿐만 아니라, 분화된

수지상세포의 기능인 사이토카인 분비, 세포이동, 포식작용 그리고

세포사멸에서 두 종류의 수지상세포간에 차이가 없었다. 반면, wild-

type 세포는 활성산소를 많이 생산하였다. 또한 LPS 자극에 의한

TG 활성의 증가는 TG4 발현증가에 기인함을 밝혔다. 이 결과는

수지상세포에서 TG2 가 분화나 성숙과정에는 관여하지 않으나,

활성산소 생산에 중요한 역할을 하며, TG4 가 성숙과정에 중요한

역할을 할 가능성을 보여준다.

주요어 : 트랜스글루타미네이즈, 수지상세포, 분화, 성숙

학 번 : 2010-23740