The Phosphatidylinositol 3-Kinase/Akt Pathway Enhances Smad3-stimulated Mesangial Cell Collagen I Expression in Response to Transforming Growth Factor-β1
2004; Elsevier BV; Volume: 279; Issue: 4 Linguagem: Inglês
10.1074/jbc.m310412200
ISSN1083-351X
AutoresConstance E. Runyan, H. William Schnaper, Anne-Christine Poncelet,
Tópico(s)Pancreatic and Hepatic Oncology Research
ResumoTransforming 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 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. 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 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. Transforming growth factor (TGF) 1The abbreviations used are: TGF, transforming growth factor; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PDGF, platelet-derived growth factor; FBS, fetal bovine serum; PI3K, phosphatidylinositol 3-kinase; BMP, bone morphogenetic protein.-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.Sharma K. Ziyadeh F.N. Diabetes. 1995; 44: 1139-1146Crossref PubMed Scopus (555) Google Scholar, 2.Border W.A. Noble N.A. Kidney Int. 1997; 51: 1388-1396Abstract Full Text PDF PubMed Scopus (377) Google Scholar). 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.Border W.A. Noble N.A. Kidney Int. 1997; 51: 1388-1396Abstract Full Text PDF PubMed Scopus (377) Google Scholar). 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 down-stream from the TGF-β family receptors to transduce signal to the nucleus (3.Piek E. Heldin C.-H. ten Dijke P. FASEB J. 1999; 13: 2105-2124Crossref PubMed Scopus (747) Google Scholar, 4.Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (479) Google Scholar, 5.Massagué J. Wotton D. EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar). Upon ligand binding, the type II receptor recruits and phosphorylates type I receptor (TβRI). The receptor-regulated or pathway-restricted Smads (R-Smads) contain an 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.Piek E. Heldin C.-H. ten Dijke P. FASEB J. 1999; 13: 2105-2124Crossref PubMed Scopus (747) Google Scholar, 4.Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (479) Google Scholar, 5.Massagué J. Wotton D. EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar). 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 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 kinase (class Ia) or heteromeric G-protein-coupled (class Ib) receptors (6.Vanhaesebroeck B. Waterfield M.D. Exp. Cell Res. 1999; 253: 239-254Crossref PubMed Scopus (763) Google Scholar, 7.Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1399) Google Scholar, 8.Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4657) Google Scholar). 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.Toker A. Mol. Pharmacol. 2000; 57: 652-658Crossref PubMed Scopus (285) Google Scholar). A few studies have suggested that the PI3K signaling pathway could be modulated by members of the TGF-β family (10.Higaki M. Shimokado K. Arteriosler. Thromb. Vasc. Biol. 1999; 19: 2127-2132Crossref PubMed Scopus (61) Google Scholar, 11.Bakin A.V. Tomlinson A.K. Bhowmick N.A. Moses H.L. Arteaga C.L. J. Biol. Chem. 2000; 275: 36803-36810Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 12.Ghosh-Choudhury N. Abboud S.L. Nishimura R. Celeste A. Mahimainathan L. Ghosh Choudhury G. J. Biol. Chem. 2002; 277: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 13.Dupont J. McNeilly J. Vaiman A. Canepa S. Combarnous Y. Taragnat C. Biol. Reprod. 2003; 68: 1877-1887Crossref PubMed Scopus (76) Google Scholar). 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.Krymskaya V.P. Hoffman R. Eszterhas A. Ciocca V. Panettieri R.A.J. Am. J. Physiol. Lung Cell. Mol. Physiol. 1997; 273: L1220-L1227Crossref PubMed Google Scholar). More recently, Bakin et al. (11.Bakin A.V. Tomlinson A.K. Bhowmick N.A. Moses H.L. Arteaga C.L. J. Biol. Chem. 2000; 275: 36803-36810Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar) 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. We previously showed that TGF-β1-induced collagen I gene expression is Smad3-dependent in human mesangial cells (15.Poncelet A.-C. de Caestecker M.P. Schnaper H.W. Kidney Int. 1999; 56: 1354-1365Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 16.Poncelet A.-C. Schnaper H.W. J. Biol. Chem. 2001; 276: 6983-6992Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). 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. 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 (Ser-473), 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-horse-radish peroxidase (HRP), from Santa Cruz Biotechnology (Santa Cruz, CA); anti-rabbit IgG-HRP, luciferase and β-galactosidase assay systems from Promega (Madison, WI); rabbit anti-phosphoserine antibody and protein G-Sepharose from Zymed Laboratories Inc. (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 Me2SO. 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 Dulbecco's modified Eagle's medium/Ham's F12 medium supplemented with 20% heat-inactivated fetal bovine serum (FBS), glutamine, penicillin/streptomycin, sodium pyruvate, Hepes buffer, and 8 μg/ml insulin (Invitrogen Life Technologies, Carlsbad, CA) as previously described (17.Poncelet A.-C. Schnaper H.W. Am. J. Physiol. Renal Physiol. 1998; 275: F458-F466Crossref PubMed Google Scholar) 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 radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 7.5; 150 mm NaCl; 1% Nonidet P-40; 0.1% SDS) containing protease inhibitors (Sigma). After clarification by centrifugation, the protein content was determined by Bradford protein assay (BioRad). Cell lysates were incubated with a SGK1 peptide substrate (Upstate, Waltham, MA) in the presence of [γ-32P]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 Biotechnologies) 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 Me2SO as vehicle control for 1 h prior to TGF-β1. For the immunoprecipitation experiments, 500 μg of lysates were immunoprecipitated with 1 μg of anti-Smad2/3 antibody (N-19) followed by precipitation with 40 μl of protein G-Sepharose as previously described (16.Poncelet A.-C. Schnaper H.W. J. Biol. Chem. 2001; 276: 6983-6992Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). The resulting immunoprecipitates or whole cell lysates were analyzed by immunoblotting with anti-phosphoserine 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). 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 of 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-100 soluble fraction, representing the 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 × 104 cells per well. Eighteen hours later, cells were switched to 1% FBS medium and transfected with the indicated constructs along with CMV-SPORT-β-galactosidase as a control of transfection efficiency. Transfection was performed with the FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN) as previously described (15.Poncelet A.-C. de Caestecker M.P. Schnaper H.W. Kidney Int. 1999; 56: 1354-1365Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). After 3 h, 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. 18–24 h later, the cells were harvested in 300 μl of reporter lysis buffer (Promega). Luciferase and β-galactosidase activities were measured as previously described (15.Poncelet A.-C. de Caestecker M.P. Schnaper H.W. Kidney Int. 1999; 56: 1354-1365Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Luciferase assay results were normalized for β-galactosidase 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) reporter gene was previously described (15.Poncelet A.-C. de Caestecker M.P. Schnaper H.W. Kidney Int. 1999; 56: 1354-1365Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The SBE-LUC (18.Zawel L. Le Dai J. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar) reporter construct was kindly provided by Dr. B. Vogelstein. The vectors expressing the indicated Smad3 variants (19.Liu X. Sun Y. Constantinescu S.N. Karam E. Weinberg R.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10669-10674Crossref PubMed Scopus (332) Google Scholar) were kindly provided by Drs. H. F. Lodish and X. Liu. The Gal4-Smad constructs (20.de Caestecker M.P. Yahata T. Wang D. Parks W.T. Huang S. Hill C.S. Shioda T. Roberts A.B. Lechleider R.J. J. Biol. Chem. 2000; 275: 2115-2122Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) were kindly provided by Dr. M. P. de Caestecker. The constitutively active p110 PI3K construct (p110αK227E) (21.Rodriguez-Viviana P. Warne P.H. Vanhaesebroeck B. Waterfield M.D. Downward J. EMBO J. 1996; 15: 2442-2451Crossref PubMed Scopus (500) Google Scholar) was kindly provided by Dr. J. Downward. The CMV-SPORT-β-galactosidase was purchased from Invitrogen, the pFR-LUC from Stratagene (La Jolla, CA) and the Myc-Akt and Akt dominant-negative constructs from Upstate. 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.Poncelet A.-C. Schnaper H.W. Am. J. Physiol. Renal Physiol. 1998; 275: F458-F466Crossref PubMed Google Scholar). The same blots were successively rehybridized with additional probes after complete stripping. cDNAs for human α1(I) (clone Hf677, Ref. 22.Chu M.-L. Myers J.C. Bernard M.P. Ding J.-F. Ramirez F. Nucleic Acids Res. 1982; 10: 5925-5934Crossref PubMed Scopus (381) Google Scholar) and α2(I) collagen (clone Hf1131, Ref. 23.Bernard M.P. Myers J.C. Chu M.-L. Ramirez F. Eikenberry E.F. Prockop D.J. Biochemistry. 1983; 22: 1139-1145Crossref PubMed Scopus (190) Google Scholar) 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. The difference between two comparative groups was further analyzed by unpaired Student's t test. 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. Fig. 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. Because 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 (Fig. 2, A 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 (Fig. 2, C and D). LY294002, a PI3K inhibitor, blocks Akt membrane translocation (Fig. 2E) and phosphorylation (Fig. 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.Poncelet A.-C. de Caestecker M.P. Schnaper H.W. Kidney Int. 1999; 56: 1354-1365Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). 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.Poncelet A.-C. de Caestecker M.P. Schnaper H.W. Kidney Int. 1999; 56: 1354-1365Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) 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 (Fig. 3). Similarly, co-transfection of a vector expressing a kinase-deficient (K179M) Akt (shown to act as a dominant-negative Ref. 12.Ghosh-Choudhury N. Abboud S.L. Nishimura R. Celeste A. Mahimainathan L. Ghosh Choudhury G. J. Biol. Chem. 2002; 277: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) 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. 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.Rodriguez-Viviana P. Warne P.H. Vanhaesebroeck B. Waterfield M.D. Downward J. EMBO J. 1996; 15: 2442-2451Crossref PubMed Scopus (500) Google Scholar) or the corresponding empty vector. As shown in Fig. 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 with cells transfected with empty vector (data not shown). PDGF has been shown to activate PI3K in various cell types (24.Kazlauskas A. Cooper J.A. EMBO J. 1990; 9: 3279-3286Crossref PubMed Scopus (158) Google Scholar, 25.Conway A.M. Rakhit S. Pyne S. Pyne N.J. Biochem. J. 1999; 337: 171-177Crossref PubMed Scopus (116) Google Scholar, 26.Reif S. Lang A. Lindquist J.N. Yata Y. Gäbele E. Scanga A. Brenner D.A. Rippe R.A. J. Biol. Chem. 2003; 278: 8083-8090Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). 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. (Fig. 4B). Thus, PDGF is able to activate the PI3K pathway in mesangial cells, in agreement with Ghosh Choudhury et al. (27.Ghosh Choudhury G. Karamitsos C. Hernandez J. Gentilini A. Bardgette J. Abboud H.E. Am. J. Physiol. Renal Physiol. 1997; 273: F931-F938Crossref PubMed Google Scholar) 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 (Fig. 4A). We also measured collagen I mRNA expression after treatment with PDGF or TGF-β1. As expected, PDGF is not able to increase α1(I) and α2(I) collagen mRNA levels (Fig. 4C). In contrast, TGF-β1 stimulates collagen I mRNA expression as previously reported (17.Poncelet A.-C. Schnaper H.W. Am. J. Physiol. Renal Physiol. 1998; 275: F458-F466Crossref PubMed Google Scholar), and the induction is blocked by LY294002 (Fig. 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.Poncelet A.-C. de Caestecker M.P. Schnaper H.W. Kidney Int. 1999; 56: 1354-1365Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 16.Poncelet A.-C. Schnaper H.W. J. Biol. Chem. 2001; 276: 6983-6992Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). 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.Poncelet A.-C. de Caestecker M.P. Schnaper H.W. Kidney Int. 1999; 56: 1354-1365Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), while PDGF does not activate Smad3 (Fig. 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 reduces both ligand-dependent and ligand-independent Smad3-mediated COL1A2 promoter activity (Fig. 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 transactivation 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 (Fig. 7A). Similarly, co-transfection of the dominant-negative Akt construct reduces Smad3 activity (Fig. 7B). These data suggest that the PI3K pathway modulates Smad3 transactivation 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 GTCTAGAC sequence that has been shown to bind recombinant Smad3 and Smad4 (18.Zawel L. Le Dai J. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar). 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% (Fig. 8, pEXL histograms) as well as transcriptional activity of over-expressed Smad3 (Fig. 8, Smad3 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 demonstrated (19.Liu X. Sun Y. Constantinescu S.N. Karam E. Weinberg R.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10669-10674Crossref PubMed Scopus (332) Google Scholar, 28.Runyan C.E. Schnaper H.W. Poncelet A.-C. Am. J. Physiol. Renal Physiol. 2003
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