The PI3K-Akt pathway enhances Smad3-stimulated mesangial cell

collagen I expression in response to TGF-β1

Constance E. Runyan, H. William Schnaper and Anne-Christine Poncelet*

Northwestern University, Pediatrics, Chicago, IL, 60611 USA

Running title: Smad3 and PI3K regulate COL1A2 expression

*corresponding author

Northwestern University

Department of Pediatrics, W-140

Feinberg School of Medicine

303 E. Chicago Ave.

Chicago, IL 60611-3008, USA

Phone: (312)503-0089

Fax: (312)503-1181

Email: anne-c@northwestern.edu

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SUMMARY

Transforming growth factor (TGF)-β has been associated with renal glomerular matrix

accumulation. We previously showed that Smad3 promotes COL1A2 gene activation by TGF-

β1 in human glomerular mesangial cells. Here, we report that the PI3K/Akt pathway also plays a

role in TGF-β1-increased collagen I expression. TGF-β1 stimulates the activity of

phosphoinositide-dependent kinase (PDK)-1, a downstream target of PI3K, starting at 1 min.

Akt, a kinase downstream of PDK-1, is phosphorylated and concentrates in the membrane

fraction within 5 min of TGF-β1 treatment. The PI3K inhibitor LY294002 decreases TGF-β1-

stimulated α1(I) and α2(I) collagen mRNA expression. Similarly, LY294002 or an Akt

dominant negative construct blocks TGF-β1 induction of COL1A2 promoter activity. However,

PI3K stimulation alone is not sufficient to increase collagen I expression, since neither a

constitutively active p110 PI3K construct nor PDGF, which induces Akt phosphorylation, is able

to stimulate COL1A2 promoter activity or mRNA expression, respectively. LY294002 inhibits

stimulation of COL1A2 promoter activity by Smad3. In a Gal4-LUC assay system, blockade of

the PI3K pathway significantly decreases TGF-β1-induced transcriptional activity of Gal4-

Smad3. Activity of SBE-LUC, a Smad3/4-responsive construct, is stimulated by over-

expression of Smad3 or Smad3D, in which the three C-terminal serine phospho-acceptor

residues are mutated. This induction is blocked by LY294002, suggesting that inhibition of the

PI3K pathway decreases Smad3 transcriptional activity independently of C-terminal serine

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phosphorylation. However, TGF-β1-induced total serine phosphorylation of Smad3 is

decreased by LY294002, suggesting that Smad3 is phosphorylated by the PI3K pathway at serine

residues other than the direct TGF-β receptor I target site. Thus, although the PI3K-PDK1-

Akt pathway alone is insufficient to stimulate COL1A2 gene transcription, its activation by

TGF-β1 enhances Smad3 transcriptional activity leading to increased collagen I expression in

human mesangial cells. This cross-talk between the Smad and PI3K pathways likely contributes

to TGF-β1 induction of glomerular scarring.

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INTRODUCTION

Transforming growth factor (TGF-)β is a pleiotropic cytokine involved in activities such as

differentiation, growth, apoptosis, inflammation, tissue remodeling and wound healing. A

number of studies indicate that TGF-β plays a critical role in renal matrix accumulation. TGF-β

has been linked to fibrogenesis in experimental models of glomerulonephritis and diabetic

nephropathy (1, 2). Its expression is elevated in human glomeruli under conditions associated

with increased extracellular matrix (ECM) deposition such as diabetic nephropathy and focal

segmental glomerulosclerosis (2).

Members of the TGF-β superfamily signal via heteromeric complexes of transmembrane

serine/threonine kinases, the type I and type II receptors. The Smad proteins function

downstream from the TGF-β family receptors to transduce signal to the nucleus (3-5). Upon

ligand binding, the type II receptor recruits and phosphorylates type I receptor (TβRI). The

receptor-regulated or pathway-restricted Smads (R-Smads) contain a SSXS phosphorylation site

at their C-terminal end that is a direct target of TβRI. Once phosphorylated, the R-Smads

associate with the common-partner Smad, Smad4. The resulting heteromultimer translocates to

the nucleus where it regulates expression of TGF-β target genes by direct binding to DNA

and/or interaction with other transcription factors (3-5).

Phosphoinositide 3-kinases (PI3Ks) phosphorylate inositol-containing lipids at the D-3

position of the inositol ring. They are divided into three classes in mammalian cells. Class III

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PI3Ks produce phosphatidylinositol (PtdIns)-3-P which is constitutively present in all cells. In

vitro, class I and class II PI3Ks can utilize PtdIns, PtdIns-4-P and PtdIns-4,5-P2. However, in

the cells, class I PI3Ks preferentially convert PtdIns-4,5-P2 into PtdIns-3,4,5-P3 (PIP3)

following stimulation by tyrosine kinases (class Ia) or heteromeric G-protein-coupled (class Ib)

receptors (6-8). Class I PI3Ks are heterodimers of a 110-kDa catalytic subunit (p110α, p110β,

p110δ and p110γ) and an adaptator/regulator subunit (p85α, p85β, p55 and p101). Following

PI3K activation, PIP3 recruits the phosphoinositide-dependent kinase (PDK)-1 and Akt/PKB,

bringing these proteins into proximity at the plasma membrane where Akt is phosphorylated on

threonine 308 by PDK-1. This is followed by phosphorylation at serine 473 by a yet-to-be

identified mechanism. Once activated, Akt leaves the plasma membrane to phosphorylate

intracellular substrates. Akt also has been shown to translocate to the nucleus where it can

phosphorylate transcription factors (9).

A few studies have suggested that the PI3K signaling pathway could be modulated by

members of the TGF-β family (10-13). In human airway smooth muscle cells, the p85

regulatory subunit of PI3K associates with type I and type II TGF-β receptors and TGF-β1

enhances activation of PI3K by epidermal growth factor (14). More recently, Bakin et al. (11)

showed that LY294002, an inhibitor of PI3K, blocks TGF-β1-induced Smad2 phosphorylation

in breast cancer cells, suggesting that Smad proteins are potential targets of the PI3K pathway.

However, the role of PI3K in fibrosis in response to TGF-β1 has not been investigated.

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We previously showed that TGF-β1-induced collagen I gene expression is Smad3-

dependent in human mesangial cells (15, 16). Here, we demonstrate that TGF-β1 activates

PDK-1 and Akt, two downstream targets of PI3K, in these cells. Inhibition of the PI3K pathway

with LY294002 or an Akt dominant-negative construct abrogates TGF-β1-stimulated COL1A2

gene transcription. Furthermore, blockade of the PI3K pathway decreases Smad3 activity in

transcriptional assays. These data suggest that cross-talk between Smads and the PI3K pathway

regulates collagen I expression in response to TGF-β1.

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EXPERIMENTAL PROCEDURES

Materials

Reagents were purchased from the following vendors: active, human recombinant TGF-β1

and platelet-derived growth factor (PDGF)-BB from R&D Systems (Minneapolis, MN); rabbit

anti-phospho-Akt (Ser473), rabbit anti-Akt antibody, LY294002 from Cell Signaling (Beverly,

MA); rabbit anti-phospho-Smad2 (Ser 465/467) from Upstate (Lake Placid, NY); mouse anti-

cMyc, mouse anti-Smad4 IgG (B-8), mouse anti-Smad1/2/3 IgG (H-2), rabbit anti-phospho-

Smad2/3 IgG (Ser433/435), goat anti-Smad2/3 IgG (N19), anti-goat IgG-Horseradish

Peroxidase (HRP), from Santa Cruz Biotechnology (Santa Cruz, CA); anti-rabbit IgG-HRP,

Luciferase and βgalactosidase assay systems from Promega (Madison, WI); rabbit anti-

phospho-serine antibody and protein G-Sepharose from Zymed (South San Francisco, CA).

Stock solutions were made as follows: TGF-β1 in 4 mM HCl containing 1 mg/ml bovine serum

albumin; PDGF in water; LY294002 in DMSO.

Cell culture

Human mesangial cells were isolated from glomeruli by differential sieving of minced

normal human renal cortex obtained from anonymous surgery or autopsy specimens. The cells

were grown in DMEM/Ham’s F12 medium supplemented with 20% heat-inactivated fetal

bovine serum (FBS), glutamine, penicillin/streptomycin, sodium pyruvate, Hepes buffer, and 8

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µg/ml insulin (Invitrogen life technologies, Carlsbad, CA) as previously described (17) and were

used between passages 5 and 8.

PDK-1 activity assay

Cells in medium containing 1% FBS were treated with 1 ng/ml TGF-β1 for various time

periods leading up to simultaneous harvest in RIPA buffer (50 mM Tris/HCl, pH 7.5; 150 mM

NaCl; 1% Nonidet P-40; 0.1% SDS) containing protease inhibitors (Sigma, St-Louis, MO).

After clarification by centrifugation, the protein content was determined by Bradford protein

assay (BioRad, Hercules, CA). Cell lysates were incubated with a SGK1 peptide substrate

(Upstate, Waltham, MA) in the presence of [γ32P] ATP for 10 min at 30 °C. The reactions were

spotted on phosphocellulose and washed four times with 1% phosphoric acid followed by an

acetone rinse. The amount of radioactivity incorporated into the substrate was determined by

scintillation counting. Active PDK-1 (Upstate) was used as a positive control.

Preparation of cell lysates, immunoprecipitation and western blot analysis

Cells were switched to medium containing 1% FBS and treated with 1 ng/ml TGF-β1 or 5

ng/ml PDGF for various time periods leading to simultaneous harvest. In some experiments, the

cells were preincubated with 10 µM LY294002 or DMSO as vehicle control for 1 h prior to

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TGF-β1. For the immunoprecipitation experiments, 500 µg of lysates were immunoprecipitated

with 1 µg anti-Smad2/3 antibody (N-19) followed by precipitation with 40 µl protein G-

Sepharose as previously described (16). The resulting immunoprecipitates or whole cell lysates

were analyzed by immunoblotting with anti-phospho-serine antibody (1 µg/ml); anti-Smad2/3,

anti-Smad1/2/3 or anti-Smad4 antibody (0.2 µg/ml); anti-phospho-Smad2 antibody (1 µg/ml);

anti-phospho-Smad3 antibody (2 µg/ml); anti-phospho-Akt or anti-Akt antibody (1:1,000).

The blots were developed with chemiluminescence reagents according to the manufacturer’s

protocol (Santa Cruz Biotechnology, Santa Cruz, CA). Autoradiograms were scanned with an

Arcus II Scanner (AGFA) in transparency mode and densitometric analysis was performed using

the NIH Image 1.61 program for Macintosh.

Cell fractionation

Cells were scraped into a detergent-free buffer (20 mM Tris/HCl, pH 7.5; 0.5 mM EDTA;

0.5 mM EGTA; 10 mM β-mercaptoethanol) containing protease and phosphatase inhibitors.

The cells were then disrupted by 15 strokes of a Dounce homogenizer. After 45 min

centrifugation at 100,000 g, the supernatant was removed and saved as the S-100 fraction,

representing the cytosolic fraction. The pellet, representing the particulate fraction, was

resuspended in buffer containing 0.5% Triton-X 100 and syringe sheared. After 30 min

incubation on ice, the insoluble material was removed by 10 min centrifugation at 18,000 g, and

the resulting supernatant was saved as the Triton-X soluble fraction, representing the

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membrane-enriched fraction. After determination of the protein content, each fraction was

analyzed by immunoblotting with anti-Akt antibody as described above.

Transient transfection and luciferase assay

The day before the transfection, cells were seeded on 6-well plates at 6.5 to 8 x 104 cells per

well. Eighteen hours later, cells were switched to 1% FBS medium and transfected with the

indicated constructs along with CMV-SPORT-βgalactosidase as a control of transfection

efficiency. Transfection was performed with the Fugene6 transfection reagent (Roche Applied

Science, Indianapolis, IN) as previously described (15). After three hours, 1 ng/ml TGF-β1 or

control vehicle was added to the cells. In some experiments, the transfected cells were pretreated

for 1 h with LY294002 before adding TGF-β1. Eighteen to twenty-four hours later, the cells

were harvested in 300 µl reporter lysis buffer (Promega, Madison, WI). Luciferase and

βgalactosidase activities were measured as previously described (15). Luciferase assay results

were normalized for βgalactosidase activity. Experimental points were performed in triplicate in

several independent experiments.

Plasmid constructs

The 376COL1A2-LUC construct containing the sequence 376 bp of the α2(I) collagen

(COL1A2) promoter and 58 bp of the transcribed sequence fused to the luciferase (LUC)

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reporter gene was previously described (15). The SBE-LUC (18) reporter construct was kindly

provided by Dr. B. Vogelstein. The vectors expressing the indicated Smad3 variants (19) were

kindly provided by Drs H. F. Lodish and X. Liu. The Gal4-Smad constructs (20) were kindly

provided by Dr. M. P de Caestecker. The constitutively active p110 PI3K construct

(p110αK227E) (21) was kindly provided by Dr. J. Downward. The CMV-SPORT-βGalactosidase was

purchased from Gibco BRL, the pFR-LUC from Stratagene (La Jolla, CA) and the Myc-Akt

and Akt dominant-negative constructs from Upstate (Chalotteville, VA).

RNA isolation and Northern blot

Cells were switched to medium containing 1% FBS. They were preincubated with 10 µM

LY294002 for 1 h before addition of 1 ng/ml TGF-β1, 5 ng/ml PDGF or control vehicle for 24

h. Total RNA was harvested using Trizol (Invitrogen life technologies) and analyzed by

Northern blot as previously described (17). The same blots were successively rehybridized with

additional probes after complete stripping. cDNAs for human α1(I) (clone Hf677 (22)) and

α2(I) collagen (clone Hf1131 (23)) chains were obtained from Dr. Y. Yamada.

Statistical analysis

Statistical differences between experimental groups were determined by analysis of variance

using StatView 4.02 software program for Macintosh. Values of P<0.05 by Fisher’s PLSD were

considered significant. Difference between two comparative groups was further analyzed by

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unpaired student t test.

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RESULTS

Activation of the PI3K pathway by TGF-β1.

Only a limited number of studies have examined the role of PI3K in TGF-β signaling. None

have investigated the potential role of the PI3K pathway in fibrosis induced by TGF-β1. We

used human glomerular mesangial cells treated with TGF-β1 as our model to study the

mechanism of abnormal matrix accumulation by kidney cells. First, we investigated whether the

PI3K pathway is activated in response to TGF-β1 in these cells. We examined whether the

activity of PDK-1 (a downstream target of PI3K) is increased by TGF-β1 treatment. Human

mesangial cells were treated with 1 ng/ml TGF-β1 for different time periods leading up to

simultaneous harvest. Lysates were used for an in vitro kinase assay with a SGK1 peptide

substrate. Figure 1 shows that TGF-β1 stimulates PDK-1 activity beginning at 1 min. The

increased activity is sustained for up to 24 h. These data suggest the PI3K pathway is activated

by TGF-β1 in human mesangial cells.

Since one major target of PDK-1 is Akt, we sought to determine whether PDK-1 activation

by TGF-β1 leads to increased Akt activity as measured by membrane translocation and

phosphorylation. Cells, treated with TGF-β1 for various durations, were fractionated into

cytosol and membrane fractions for analysis by immunoblot with an anti-Akt antibody. Akt

translocates to the membrane-enriched fraction in a biphasic manner following TGF-β1

treatment. An initial peak of membrane-associated Akt is observed 5 min after adding TGF-β1

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(Figure 2A and B). A second peak is detected at 30 min and membrane association remains

elevated at up to 24 h of treatment. Increased membrane association correlates with increased

phosphorylation (Figure 2C and D). LY294002, a PI3K inhibitor, blocks Akt membrane

translocation (Figure 2E) and phosphorylation (Figure 2F). These results indicate that activation

of Akt follows TGF-β1-stimulated PDK-1 activity.

TGF-β1-induced PI3K modulates collagen I gene activity.

We previously have shown that TGF-β1 stimulates α2(I) collagen (COL1A2) gene

expression in mesangial cells (15). Since the PI3K pathway is activated by TGF-β1, we

investigated whether this pathway modulates COL1A2 promoter activity in response to TGF-β1.

We performed transient transfection experiments with the 376COL1A2-LUC construct (15) and

determined the effect of inhibiting the PI3K pathway on its activity. The transfected cells were

pretreated for 1 h with LY294002, a specific inhibitor of PI3K. TGF-β1 was then added for 24

h and luciferase activity was determined. Inhibition of PI3K almost completely blocks TGF-

β1-induced COL1A2 promoter activity (Figure 3). Similarly, co-transfection of a vector

expressing a kinase deficient (K179M) Akt (shown to act as a dominant-negative (12)) prevents

the response to TGF-β1. Of note, basal promoter activity is also decreased by the dominant-

negative construct. These results suggest that the PI3K pathway is necessary for the

transcriptional activation of COL1A2 by TGF-β1.

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Activation of PI3K/Akt is not sufficient for COL1A2 stimulation

We next sought to determine whether increased PI3K activity is sufficient to stimulate

collagen I gene expression. Cells were transfected with the 376COL1A2-LUC construct and a

vector expressing a constitutively active p110 (p110*) PI3K subunit (21) or the corresponding

empty vector. As shown in Figure 4A, the constitutively active p110 alone does not increase

COL1A2 promoter activity. To confirm that the constitutively active p110 construct was active

in mesangial cells, lysates from cells co-transfected with Myc-Akt and either p110* or empty

vector were immunoprecipitated with an anti-cMyc antibody and the immunoprecipitated

complexes were analyzed by immunoblotting with anti-phospho-Akt. Akt phosphorylation

levels are increased by ∼2 fold in cells transfected with the constitutively active p110 compared

to cells transfected with empty vector (data not shown).

Platelet-derived growth factor (PDGF) has been shown to activate PI3K in various cell types

(24-26). We examined whether the PI3K/Akt pathway was activated by PDGF in human

mesangial cells. As expected, PDGF stimulates phosphorylation of Akt at serine 473 in a time

dependent manner. (Figure 4B). Thus, PDGF is able to activate the PI3K pathway in mesangial

cells, in agreement with Ghosh Choudhury and colleagues (27) who have demonstrated that

PDGF stimulates PI3K activity, as determined by increased PIP production.

Next, we compared the effects of PDGF and TGF-β1 on collagen I transcription. In

mesangial cells that were transfected with the 376COL1A2-LUC reporter construct, the collagen

I promoter is not stimulated by PDGF but is responsive to TGF-β1 (Figure 4A). We also

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measured collagen I mRNA expression after treatment with PDGF or TGF-β1. As expected,

PDGF is not able to increase α(1)I and α(2)I collagen mRNA levels (Figure 4C). In contrast,

TGF-β1 stimulates collagen I mRNA expression as previously reported (17) and the induction is

blocked by LY294002 (Figure 4C). These data further support a role for PI3K in TGF-β1-

increased collagen I transcription while PDGF, another activator of the PI3K/Akt pathway, is not

able to stimulate COL1A2 gene expression.

PI3K modulation of collagen I gene expression is through modulation of Smad3 activity

We previously have shown that Smad3 stimulates COL1A2 gene transcription (15, 16). We

investigated whether the reason why PDGF could not activate collagen I expression was due to

an inability to stimulate Smad3 activity. Cells were treated with TGF-β1 or PDGF for different

durations and Smad3 phosphorylation levels were examined. TGF-β1 induces increased Smad3

phosphorylation in a time-dependent manner, as previously shown (15), while PDGF does not

activate Smad3 (Figure 5). Thus, although both PDGF and TGF-β1 are able to stimulate the

PI3K pathway, only TGF-β1 leads to phosphorylation of Smad3 that correlates with increased

COL1A2 promoter activity.

Next, we determined whether inhibition of the PI3K pathway would block Smad3-mediated

COL1A2 activation. Mesangial cells were co-transfected with the 376COL1A2-LUC construct

and the expression vector for Smad3 or the corresponding empty vector. One hour after adding

LY294002 or control vehicle, the cells were treated with TGF-β1 for 24 h. Inhibition of PI3K

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reduces both ligand-dependent and ligand-independent Smad3-mediated COL1A2 promoter

activity (Figure 6). Together, these data suggest that TGF-β1-stimulated PI3K pathway activity

contributes to Smad3 induction of COL1A2 gene transcription.

To investigate whether TGF-β1-stimulated PI3K activation modulates Smad3 trans-

activation activity. Cells were co-transfected with a reporter construct containing five Gal4

binding sites in front of the luciferase gene and a construct expressing the Gal4 DNA binding

domain fused to full-length Smad3 (Gal4-Smad3). The cells were pretreated for 1 h with

vehicle or LY294002 and then incubated with TGF-β1 for 24 h. The PI3K inhibitor

significantly decreases TGF-β1-stimulated Smad3 transcriptional activity (Figure 7A).

Similarly, co-transfection of the dominant-negative Akt construct reduces Smad3 activity

(Figure 7B). These data suggest that the PI3K pathway modulates Smad3 trans-activation

activity, at least in part, independently of Smad3 DNA binding activity. We also evaluated the

effect of PI3K inhibition on the activity of the SBE-LUC reporter construct containing four

copies of the CTCTAGAC sequence that has been shown to bind recombinant Smad3 and

Smad4 (18). Cells were co-transfected with SBE-LUC and a construct expressing wild-type

Smad3, or the empty vector (pEXL). Pretreatment for 1 h with LY294002 decreases TGF-β1-

induced activity of endogenous Smad3 by ~60% (Figure 8, pEXL histograms) as well as

transcriptional activity of over-expressed Smad3 (Figure 8, Sd3 histograms). Smad3D, a

construct in which the three C-terminal serine residues of Smad3 are replaced by three aspartic

acid residues, can function as a transcriptional activator in the absence of TGF-β1 as previously

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demonstrated (19, 28). In mesangial cells, LY294003 inhibits Smad3D activity (Figure 8).

These results indicate that modulation of Smad3 transcriptional activity by the PI3K pathway is

probably independent of phosphorylation at the C-terminal TβRI target site.

In addition to the directly receptor-regulated C-terminal phosphorylation motif, several

reports have demonstrated that other sites in R-Smads can be regulated by phosphorylation in

response to signaling pathways such as extracellular signal regulated kinase (ERK), c-Jun N-

terminal kinase (JNK), protein kinase C (PKC) and Ca2+-calmodulin-dependent kinase II

(CaMKII) (29-34). Thus we investigated whether inhibition of the PI3K pathway affects total

serine phosphorylation of Smad3 as determined by Western blot of immunoprecipitated Smad2/3

complexes using an anti-phospho-serine antibody. In the presence of LY294002, total serine

phosphorylation of both Smad2 and Smad3 is significantly decreased (Figure 9A and B). In

contrast, phosphorylation at the C-terminal site is not inhibited by LY294002 pretreament as

determined by immunoblotting with a phospho-Smad2 or phospho-Smad2/3 antibody

specifically recognizing phosphorylation at Ser465/467 or Ser433/435, respectively (Figure 9C

and D). Following TGF-β1 stimulation, activated R-Smads form complexes with Smad4 (35-

37). Paralleling its effect on Smad2/3 phosphorylation, inhibition of the PI3K pathway also

decreases association with Smad4 without affecting the expression levels of Smad2, Smad3 or

Smad4 (Figure 9A and B). These data suggest that TGF-β1-induced PI3K activation leads to

phosphorylation of Smad2/3 outside the C-terminal phospho-acceptor site and facilitates for

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Smad4 association with R-Smads.

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DISCUSSION

In the present paper, we demonstrate that TGF-β1 activates the PI3K pathway in human

mesangial cells and that modulation of Smad3 transcriptional activity by PI3K, possibly through

an effect on phosphorylation, contributes to increased collagen I expression in response to TGF-

β1. The PI3K downstream targets, PDK-1 and Akt, are rapidly activated by TGF-β1 in human

mesangial cells. Increased PDK-1 activity is detected within one minute of TGF-β1 treatment

and is followed by increased membrane translocation and phosphorylation of Akt. In the

NMuMG mammary epithelial cell line, epithelial-to-mesenchymal transition induced by TGF-

β1 is preceded by increased Akt phosphorylation (11). In Swiss 3T3 cells, TGF-β stimulates PI3K

activity and Akt phosphorylation (10). Ghosh-Choudhury et al. (12) have demonstrated

activation of the PI3K pathway by bone morphogenetic protein (BMP)-2, another member of the

TGF-β family. In contrast, Krymskaya and colleagues (14) found that, in human airway smooth

muscle cells, TGF-β1 alone does not alter PI3K activation but enhances the stimulatory effect of

epidermal growth factor. Other reports describe inhibition by TGF-β1 of cytokine-induced Akt

phosphorylation in lymphocyte cell lines (38, 39). Thus, activation or inhibition of PI3K

signaling by TGF-β1 is likely to be cell type-specific. Here, we show stimulation of this

pathway by TGF-β1 in kidney mesangial cells. This activation is likely to be direct considering

the timing of increased PDK-1 activity and Akt phosphorylation in response to TGF-β1.

Moreover, coimmunoprecipitation between the p85 subunit of PI3K and both TβRI and TβRII

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have been demonstrated in airway smooth muscle cell (14).

The reports suggesting activation of the PI3K pathway by TGF-β1 have not examined

whether TGF-β1-induced PI3K plays a role in extracellular matrix accumulation. Here we

show that inhibition of PI3K with LY294002 decreases TGF-β1-stimulated α1(I) and α2(I)

collagen mRNA expression. Similarly, LY294002 or a construct expressing an Akt dominant

negative mutant blocks COL1A2 promoter activation by TGF-β1. Although we could not

exclude an effect on mRNA stability, our transfection experiments clearly indicate that, in human

mesangial cells, the PI3K pathway contributes to TGF-β1-stimulated COL1A2 expression at the

transcriptional level.

Ricupero and colleagues reported that, in the absence of any stimulus, incubation with

LY294002 inhibited basal α1(I) collagen mRNA expression in human lung fibroblasts within 2

to 5 h (40). In contrast, in our experiments, LY294002 had a minimal effect on basal α1(I) and

α2(I) collagen I mRNA levels. Thus involvement of the PI3K pathway in basal expression of type

I collagen could depend on the cell type. Alternatively, PI3K activation could result from

autocrine stimulation or continuous activation by the culture conditions. Another possibility is

that the timing of inhibition by LY294002 is cell-specific and may reflect a difference in the

stability of collagen I mRNA in different cell types. Indeed, Reif et al. (26) have demonstrated

that, in hepatic stellate cells, blockade of PI3K with LY294002 leads to a decrease in basal α1(I)

collagen mRNA levels at a much slower rate (48 to 72 h after treatment) than in lung fibroblasts.

In our experiments, LY294002 substantially decreased the response to TGF-β1-stimulated

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COL1A2 mRNA expression and promoter activity, indicating that the PI3K pathway mediates

the increase in collagen I expression in response to TGF-β1, at least in part at the transcriptional

level. However, activation of the PI3K pathway alone is not sufficient to increase COL1A2 gene

expression since neither a constitutively active p110 PI3K construct nor PDGF is unable to

stimulate collagen I promoter activity or mRNA expression, respectively. Smad3 is

phosphorylated in response to TGF-β1 in a time-dependent manner and mediates COL1A2

transcription (this report and references 15, 16). Although PDGF is able to increase PI3K

activity (27) and Akt phosphorylation (this report) in mesangial cells, it does not stimulate

Smad3 phosphorylation. Together these results strongly suggest that PI3K can modulate

collagen I expression only when the Smad proteins also are activated.

Our data indicate that the PI3K pathway positively modulates Smad3 transcriptional activity.

Stimulation of COL1A2 by Smad3 is partially blocked by LY294002. Using the Gal4-LUC

assay system or the SBE-LUC reporter construct, we further demonstrate inhibition of Smad3

trans-activation activity either by LY294002 or an Akt dominant-negative construct. Bakin et

al. (11) showed that Smad2 C-terminal phosphorylation was decreased by LY294002 in

NMuMG cells, suggesting that PI3K is involved in TGF-β1-mediated phosphorylation of

Smad2. In contrast, we do not detect any change in Smad2 phosphorylation at the C-terminal

site in the presence of LY294002 in mesangial cells, while total serine phosphorylation of Smad2

is decreased. Similarly, C-terminal phosphorylation of Smad3 in response to TGF-β1 is not

affected by inhibition of the PI3K pathway, whereas total serine phosphorylation is partially

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inhibited. Moreover, the transcriptional activity of Smad3 mutated at its C-terminal serines is

still inhibited by LY294002. This further supports the hypothesis that activation of Smad3 by

the PI3K pathway is independent of Smad3 phosphorylation at its TβRI phosphorylation target

site. Inhibition of PI3K not only decreases total serine phosphorylation of Smad2/3 but it also

decreases association between Smad2/3 and Smad4, suggesting that PI3K activity contributes to

Smad2/3 phosphorylation-dependent association with Smad4. Our data are consistent with work

from others who have identified additional sites in the R-Smads whose phosphorylation could be

mediated by various pathways such as ERK (29-31), JNK (32), PKC (33) and CaMKII (34).

However, Smad2 and Smad3 have not been described as a target for phosphorylation by the

PI3K/Akt pathway, and whether Smad2/3 can be directly phosphorylated by Akt or other

downstream kinases of PI3K remains to be determined.

Examples of kinases that could act downstream of PI3K to modulate Smad3 activity include

the MEK/ERK pathway and PKC. We previously have shown that ERK1/2 is activated by

TGF-β1 in human mesangial cells (41). Similar to PI3K inhibition, blockade of the MEK/ERK

pathway with PD98059 or U0126 leads to decreased TGF-β1-induced total serine

phosphorylation of Smad2/3 without affecting phosphorylation of the C-terminal phospho-

acceptor site (31). Since PI3K has been shown to activate ERK in some cells lines (42, 43),

phosphorylation of Smad2/3 by PI3K in response to TGF-β1 could be mediated by ERK. On

the other hand, it has been shown that PI3K associates with PKCδ in the erythroleukemia cell

line TF-1 upon stimulation by cytokines (44) and that phosphorylation of PKCδ in response to

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serum in the human embryonic kidney 293 cells is PI3K-and PDK1-dependent (45).

Previously, we have demonstrated that TGF-β1-stimulated PKCδ activity in human mesangial

cells positively regulates Smad3 trans-activation activity (28). Moreover, both ERK and PKC

have been shown to phosphorylate R-Smads outside the C-terminal phosphorylation site,

enhancing or decreasing Smad activity depending upon cell type and/or stimulus (29-31, 33).

Therefore, these kinases could mediate some of the effects of PI3K on Smad activity. However,

the role of ERK or PKCδ in mediating phosphorylation of Smads after direct activation of the

PI3K pathway by TGF-β1 remains to be investigated.

Recently, it was demonstrated that another member of the TGF-β family, BMP-2, stimulates

the PI3K pathway during osteoblast differentiation and that a dominant negative Akt construct

inhibited nuclear translocation of Smad1/5, the specific BMP R-Smads (12). In contrast,

inhibition of the PI3K pathway with LY294002 does not seem to affect Smad2/3 translocation in

human mesangial cells1, suggesting that modulation of Smads by the PI3K pathway might

depend on cell type or the initiating stimulus and the R-Smads that are involved.

In summary, we show that, in human mesangial cells, TGF-β1 stimulates PDK-1 activity as

well as Akt phophorylation and membrane translocation. The PI3K pathway positively regulates

Smad3 transcriptional activity and is required for increased collagen I production, suggesting that

activation and interaction of multiple signaling pathways contribute to the pathogenesis of

glomerular matrix accumulation.

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ACKNOWLEDGEMENTS

Supported by a Young Investigator Research Grant the National Kidney Foundation of

Illinois to ACP; Grant DK49362 from the National Institute of Diabetes, Digestive and Kidney

Disease; and the Children’s Memorial Institute for Education and Research. We thank Drs M. P

de Caestecker, H. F. Lodish, X. Liu, J. Downward and Y. Yamada for the constructs described

under Materials and Methods. We express our gratitude to the members of the Schnaper lab for

their helpful discussion.

FOOTNOTES

1 Runyan and Poncelet, unpublished data.

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FIGURE LEGENDS

Figure 1: Activity of PDK-1 is increased by TGF-β1. Human mesangial cells in medium

containing 1% FBS were treated with 1 ng/ml TGF-β1 for the indicated time periods. Lysates

were used in an in vitro kinase assay with a SGK1 peptide substrate. Results are presented as the

mean fold-induction over untreated cells in at least 3 independent experiments.

Figure 2: TGF-β1 stimulates Akt activation in a biphasic manner. Cells in medium containing

1% FBS were treated with 1 ng/ml TGF-β1 for the indicated time periods. In some experiments

10 µM LY294002 or DMSO as vehicle control (-) was added 1 h prior to treatment with TGF-

β1 for 30 min. A, The cells were separated into cytosol and membrane fractions before analysis by

immunoblotting with the anti-Akt antibody. B, Densitometric analysis of at least 3 independent

experiments showing Akt levels in the membrane-enriched fraction. C, The whole-cell lysates

were harvested and analyzed by western blotting with anti-phospho-Akt (PAkt) or anti-Akt

antibody. D, Densitometric analysis of several independent experiments. The results are

presented as fold-induction of Akt phosphorylation over cells at time 0 after correction for total

Akt levels. E, Densitometric analysis of 3 independent experiments showing the effect of

LY294002 on the ratio between membrane-associated Akt in TGF-β1-treated (T) cells to that

in control (C) cells (* P<0.005). F, Effect of LY294002 on TGF-β1-stimulated Akt

phosphorylation as determined by immunoblotting.

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Figure 3: Inhibition of the PI3K pathway blocks TGF-β1-induced COL1A2 promoter activity.

Mesangial cells were transfected with 0.5 µg of 376COL1A2-LUC construct and 0.5 µg of a

construct expressing the dominant negative Akt mutant (DNAkt) or the control empty vector

pcDNA3 (E). The cells were co-transfected with 0.5 µg of CMV-βgalactosidase as a control for

transfection efficiency. After 3 h, the cells were pretreated with 10 µM LY294002 (LY) or

DMSO as vehicle control (-). One hour later, TGF-β1 was added at a final concentration of 1

ng/ml. The cells were harvested after 24 h, and luciferase and βgalactosidase activities were

measured. Luciferase activity was normalized to βgalactosidase activity. The results are shown

as means ± SE of at least 3 independent experiments. *P<0.02, ns: not significant.

Figure 4: Activation of the PI3K pathway alone is not sufficient to stimulate collagen I

expression. A, PI3K stimulation alone does not increase COL1A2 promoter activity. Mesangial

cells were transfected with 0.5 µg of 376COL1A2-LUC construct, 0.5 µg of a construct

expressing the constitutively active p110 PI3K (p110*) or the control empty vector pSG5 (E),

along with 0.5 µg of CMV-βgalactosidase as a control for transfection efficiency. After 4 h, the

cells were treated with 1 ng/ml TGF-β1, 5 ng/ml PDGF or left untreated. The cells were

harvested after 24 h, and luciferase and βgalactosidase activities were measured. Luciferase

activity was normalized to βgalactosidase activity. Values are given as means ± SE of triplicate

wells from a representative experiment. #P<0.003 compared to control cells. B, PDGF

stimulates Akt phosphorylation. Cells incubated in medium containing 1% FBS with 5 ng/ml

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PDGF for different durations were harvested and the whole-cell lysates were analyzed by

western blotting with anti-phospho-Akt or anti-Akt antibody. C, TGF-β1-increased α1(I) and

α2(I) collagen mRNA expression is blunted by LY294002 pretreament. Mesangial cells were

incubated for 1 h with 10 µM LY294002 (LY) or DMSO as vehicle control (-) before addition of

1 ng/ml TGF-β1 (T), 5 ng/ml PDGF (P) or left untreated (C). Total RNA was collected after 24

h of treatment and COL1A1 and COL1A2 mRNA levels were assessed by Northern blotting.

18s rRNA ethidium bromide staining demonstrates equal loading.

Figure 5: TGF-β1, but not PDGF, stimulates Smad2/3 phosphorylation. Cells were treated with

(A) 1 ng/ml TGF-β1 or (B) 5 ng/ml PDGF for the indicated time periods and then

simultaneously harvested. Lysates were immunoprecipitated with an anti-Smad2/3 antibody

before electrophoresesis and immunoblotting. Top panels were developed with anti-phospho-

serine antibody (PSer). Bottom panels show the total Smad2/3 levels. Normal goat IgG does not

precipitate the Smad2/3 bands in A. Thirty-minute incubation with TGF-β1 (T) was used as a

positive control in B.

Figure 6: LY294002 abolishes ligand-dependent and -independent activation of the COL1A2

promoter by Smad3. Cells were co-transfected with 0.5 µg of the 376COL1A2-LUC reporter

construct, and either 0.5 µg of the vector encoding wild-type Smad3 or the empty vector pEXL;

along with 0.5 µg of CMV-βgalactosidase vector. The transfected cells were incubated for 3 h

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with 10 µM LY294002 and then treated with 1 ng/ml TGF-β1 for 18 to 24 h. Luciferase activity

was normalized to βgalactosidase activity. Values are means ± SE of triplicate wells from a

representative experiment out of 4 independent experiments. *P<0.005; **P=0.0001 compared

to control cells transfected with the same expression vector, in the absence of LY294002;

#P<0.04 compared to TGF-β1-treated cells transfected with the same expression vector, in the

absence of LY294002; ns: not significant

Figure 7: Inhibition of the PI3K pathway significantly decreases Smad3 trans-activation activity.

Cells were transfected with 0.5 µg of the pFR-LUC reporter construct (containing five Gal4

binding sites in front of the luciferase gene) and 0.5 µg of CMV-βgalactosidase as a control for

transfection efficiency. The cells were co-transfected with (A) 0.5 µg of Smad3 fused to the

Gal4 DNA binding domain (Gal4-Smad3); or (B) 0.25 µg Gal4-Smad3, and 0.25 µg DNAkt or

the control empty vector (E). After 3 h, the cells were incubated with 10 µM LY294002 or

DMSO as vehicle control for 1 h before adding 1 ng/ml TGF-β1. The results are shown as

means ± SE of luciferase activity corrected for βgalactosidase activity. A, *P<0.003; #P<0.04

compared to TGF-β1-treated cells in the absence of LY294002 (n=6 from independent

experiments). B, **P<0.03; ##P<0.02 compared to TGF-β1-treated cells transfected with

empty vector in the absence of LY294002 (n=3 from triplicate wells from a representative

experiment out of 3 independent experiments).

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Figure 8: LY294002 decreases Smad3 activity independently of phosphorylation at the C-

terminal site. Cells were co-transfected with 0.5 µg of the SBE-LUC reporter construct

(containing four DNA binding sites for Smad3/Smad4), and either 0.5 µg of the vector encoding

wild-type Smad3, mutated Smad3 (Smad3D) or the empty expression vector pEXL, along with

0.5 µg of CMV-βgalactosidase vector. The transfected cells were incubated with 10 µM

LY294002 and then treated with 1 ng/ml TGF-β1 for 18-24 h. Luciferase activity was

normalized to βgalactosidase activity. Values are means ± SE of triplicate wells from a

representative experiments out of 3 independent experiments. #P<0.004 compared to control

cells transfected with the same expression vector, in the absence of LY294002; *P<0.03

compared to TGF-β1-treated cells transfected with the same expression vector, in the absence

of LY294002.

Figure 9: Blockade of PI3K decreases total serine phosphorylation of Smad2/3 and association

with Smad4. Cells were pretreated with 10 µM LY294002 for 1 h. After 30 min incubation with

1 ng/ml TGF-β1, the cell lysates were harvested and immunoprecipitated with an anti-Smad2/3

antibody. Immunoprecipitated complexes (IP) and cells lysates were electrophoresed and

immunoblotted with the indicated antibodies. A, A representative experiment showing partial

inhibition of Smad2/3 total serine phosphorylation and Smad4 association after PI3K blockade.

B, The graph shows the ratio between total serine phosphorylation and R-Smad for Smad2 and

Smad3; and the ratio between associated Smad4 and total Smad2/3. The ratios in the absence of

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inhibitor are arbitrarily set up as 1. *P<0.05 (n=3). C, A representative experiment showing no

change of C-terminal phosphorylation levels. D, The graph shows ratio between C-terminal

serine phosphorylation and R-Smad, in the absence (open bars) or presence (shaded bars) of

LY294002, after TGF-β1 treatment.

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Constance E. Runyan, H. William Schnaper and Anne-Christine Poncelet1βexpression in response to TGF-

The PI3K-Akt pathway enhances Smad3-stimulated mesangial cell collagen I

published online November 10, 2003J. Biol. Chem. 

  10.1074/jbc.M310412200Access the most updated version of this article at doi:

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