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Activation of the Pro-survival Phosphatidylinositol 3-Kinase/AKT Pathway by Transforming Growth Factor-β1 in Mesenchymal Cells Is Mediated by p38 MAPK-dependent Induction of an Autocrine Growth Factor

2004; Elsevier BV; Volume: 279; Issue: 2 Linguagem: Inglês

10.1074/jbc.m306248200

ISSN

1083-351X

Autores

Jeffrey C. Horowitz, Daniel Lee, Meghna Waghray, Venkateshwar G. Keshamouni, Peedikayil E. Thomas, Hengmin Zhang, Zongbin Cui, Victor J. Thannickal,

Tópico(s)

Neonatal Respiratory Health Research

Resumo

Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine involved in differentiation, growth, and survival of mesenchymal cells while inhibiting growth/survival of most other cell types. The mechanism(s) of pro-survival signaling by TGF-β1 in mesenchymal cells is unclear. In this report, we demonstrate that TGF-β1 protects against serum deprivation-induced apoptosis of mesenchymal cells isolated from patients with acute lung injury and of normal human fetal lung fibroblasts (IMR-90). TGF-β receptor(s)-activated signaling in these cells involves rapid activation of the Smad and p38 MAPK pathways within minutes of TGF-β1 treatment followed by a more delayed activation of the pro-survival phosphatidylinositol 3-kinase-protein kinase B (PKB)/Akt pathway. Pharmacological inhibition of p38 MAPK with SB203580 or expression of a p38 kinase-deficient mutant protein inhibits TGF-β1-induced PKB/Akt phosphorylation. Conditioned medium from TGF-β1-treated cells rapidly induces PKB/Akt activation in an SB203580- and suramin-sensitive manner, suggesting p38 MAPK-dependent production of a secreted growth factor that activates this pro-survival pathway by an autocrine/paracrine mechanism. Inhibition of the phosphatidylinositol 3-kinase-PKB/Akt pathway blocks TGF-β1-induced resistance to apoptosis. These results demonstrate the activation of a novel TGF-β1-activated pro-survival/anti-apoptotic signaling pathway in mesenchymal cells/fibroblasts that may explain cell-specific actions of TGF-β1 and provide mechanistic insights into its pro-fibrotic and tumor-promoting effects. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine involved in differentiation, growth, and survival of mesenchymal cells while inhibiting growth/survival of most other cell types. The mechanism(s) of pro-survival signaling by TGF-β1 in mesenchymal cells is unclear. In this report, we demonstrate that TGF-β1 protects against serum deprivation-induced apoptosis of mesenchymal cells isolated from patients with acute lung injury and of normal human fetal lung fibroblasts (IMR-90). TGF-β receptor(s)-activated signaling in these cells involves rapid activation of the Smad and p38 MAPK pathways within minutes of TGF-β1 treatment followed by a more delayed activation of the pro-survival phosphatidylinositol 3-kinase-protein kinase B (PKB)/Akt pathway. Pharmacological inhibition of p38 MAPK with SB203580 or expression of a p38 kinase-deficient mutant protein inhibits TGF-β1-induced PKB/Akt phosphorylation. Conditioned medium from TGF-β1-treated cells rapidly induces PKB/Akt activation in an SB203580- and suramin-sensitive manner, suggesting p38 MAPK-dependent production of a secreted growth factor that activates this pro-survival pathway by an autocrine/paracrine mechanism. Inhibition of the phosphatidylinositol 3-kinase-PKB/Akt pathway blocks TGF-β1-induced resistance to apoptosis. These results demonstrate the activation of a novel TGF-β1-activated pro-survival/anti-apoptotic signaling pathway in mesenchymal cells/fibroblasts that may explain cell-specific actions of TGF-β1 and provide mechanistic insights into its pro-fibrotic and tumor-promoting effects. Transforming growth factor-β1 (TGF-β1) 1The abbreviations used are: TGF-β, transforming growth factor-β1; MAP, mitogen-activated protein; MAPK, MAP kinase; PI3K, phosphatidylinositol 3-kinase; EMT, epithelial-mesenchymal transition; DAPI, 4,6-diamidino-2-phenylindole; PIPES, 1,4-piperazinediethanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; ssDNA, single-stranded DNA; ELISA, enzyme-linked immunosorbent assay; PKB, protein kinase B; ALI, acute lung injury; MC, mesenchymal cells; KM, kinase mutant. is a multifunctional cytokine that regulates a number of biological responses including chemotaxis, cell cycle progression, differentiation, and apoptosis of target cells in a context- and cell-specific manner (1.Massague J. Nat. Rev. Mol. Cell. Biol. 2000; 1: 169-178Crossref PubMed Scopus (1653) Google Scholar, 2.Roberts, A. B., and Derynck, R. (2001) Science's STKE http:/www.stke.org/cgi/content/full/OC_sigtrans;2001/PE43Google Scholar). TGF-β1 is critically involved in tissue injury and repair processes (3.Border W.A. Ruoslahti E. J. Clin. Investig. 1992; 90: 1-7Crossref PubMed Scopus (1046) Google Scholar, 4.Grande J.P. Proc. Soc. Exp. Biol. Med. 1997; 214: 27-40Crossref PubMed Google Scholar). Rapid release of TGF-β1 at sites of tissue injury is chemotactic for both inflammatory cells (5.Wahl S.M. Hunt D.A. Wakefield L.M. McCartney-Francis N. Wahl L.M. Roberts A.B. Sporn M.B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5788-5792Crossref PubMed Scopus (1101) Google Scholar) and fibroblasts (6.Postlethwaite A.E. Keski-Oja J. Moses H.L. Kang A.H. J. Exp. Med. 1987; 165: 251-256Crossref PubMed Scopus (653) Google Scholar). Pro-angiogenic effects are likely to be important in formation of granulation tissue in the “proliferative” phase of wound healing (7.Roberts A.B. Sporn M.B. Assoian R.K. Smith J.M. Roche N.S. Wakefield L.M. Heine U.I. Liotta L.A. Falanga V. Kehrl J.H. Fauci A.S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4167-4171Crossref PubMed Scopus (2415) Google Scholar). TGF-β1 exerts multiple effects in the later “maturation” phase of wound repair by inducing extracellular matrix production/remodeling (8.Ignotz R.A. Massague J. J. Biol. Chem. 1986; 261: 4337-4345Abstract Full Text PDF PubMed Google Scholar, 9.Tomasek J.J. Gabbiani G. Hinz B. Chaponnier C. Brown R.A. Nat. Rev. Mol. Cell. Biol. 2002; 3: 349-363Crossref PubMed Scopus (3159) Google Scholar) and myofibroblast differentiation (10.Desmouliere A. Geinoz A. Gabbiani F. Gabbiani G. J. Cell Biol. 1993; 122: 103-111Crossref PubMed Scopus (1880) Google Scholar, 11.Thannickal V.J. Lee D.Y. White E.S. Cui Z. Larios J.M. Chacon R. Horowitz J.C. Day R.M. Thomas P.E. J. Biol. Chem. 2003; 278: 12384-12389Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). Apoptosis of fibroblasts/myofibroblasts is essential for the normal resolution of repair responses and the prevention of scarring/fibrosis (12.Desmouliere A. Redard M. Darby I. Gabbiani G. Am. J. Pathol. 1995; 146: 56-66PubMed Google Scholar, 13.Grinnell F. Zhu M. Carlson M.A. Abrams J.M. Exp. Cell Res. 1999; 248: 608-619Crossref PubMed Scopus (239) Google Scholar). The persistence of mesenchymal cells and the up-regulated expression/activation of TGF-β1 at sites of tissue injury and repair are associated with progressive fibrosis with subsequent organ dysfunction in diverse systems including the kidney, liver, and lung (14.Blobe G.C. Schiemann W.P. Lodish H.F. N. Engl. J. Med. 2000; 342: 1350-1358Crossref PubMed Scopus (2189) Google Scholar, 15.Border W.A. Noble N.A. N. Engl. J. Med. 1994; 331: 1286-1292Crossref PubMed Scopus (3014) Google Scholar). The mechanisms by which TGF-β1 regulates apoptosis/survival signals in mesenchymal cells are not well understood. There is better understanding of the growth-inhibitory/pro-apoptotic effects of TGF-β1 on immune cells (16.Brown T.L. Patil S. Cianci C.D. Morrow J.S. Howe P.H. J. Biol. Chem. 1999; 274: 23256-23262Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and epithelial cells (17.Dai C. Yang J. Liu Y. J. Biol. Chem. 2003; 278: 12537-12545Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), consistent with its anti-inflammatory and tumor-suppressive functions. In contrast to these “suppressive” functions, TGF-β1 generally promotes growth and survival of mesenchymal cells. Growth-promoting effects of TGF-β1 appear to be primarily mediated by indirect effects on the induction of mitogenic growth factor synthesis (18.Leof E.B. Proper J.A. Goustin A.S. Shipley G.D. DiCorleto P.E. Moses H.L. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2453-2457Crossref PubMed Scopus (407) Google Scholar, 19.Finlay G.A. Thannickal V.J. Fanburg B.L. Paulson K.E. J. Biol. Chem. 2000; 275: 27650-27656Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) and/or their receptor(s) up-regulation (20.Thannickal V.J. Aldweib K.D. Rajan T. Fanburg B.L. Biochem. Biophys. Res. Commun. 1998; 251: 437-441Crossref PubMed Scopus (50) Google Scholar). Relatively few studies have examined direct effects of TGF-β1 on mesenchymal cell/fibroblast apoptosis. Anti-apoptotic effects of TGF-β1 on fibroblasts and myofibroblasts have been reported previously (21.Jelaska A. Korn J.H. Arthritis Rheum. 2000; 43: 2230-2239Crossref PubMed Scopus (144) Google Scholar, 22.Zhang H.Y. Phan S.H. Am. J. Respir. Cell Mol. Biol. 1999; 21: 658-665Crossref PubMed Scopus (301) Google Scholar, 23.Kim G. Jun J.B. Elkon K.B. Arthritis Rheum. 2002; 46: 1504-1511Crossref PubMed Scopus (64) Google Scholar), although mechanisms are not well understood. The phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/Akt) pathway regulates a number of cellular processes including cell cycle progression, glucose metabolism, angiogenesis, cell motility, and apoptosis (24.Brazil D.P. Park J. Hemmings B.A. Cell. 2002; 111: 293-303Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar). Multiple targets of PKB/Akt mediate pro-survival/anti-apoptotic effects (reviewed in Ref. 25.Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3721) Google Scholar). Activation of the PI3K/Akt pathway in response to TGF-β1 has been demonstrated in epithelial cells where it mediates epithelial-mesenchymal transition (EMT) (26.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 (830) Google Scholar, 27.Nicolas F.J. Lehmann K. Warne P.H. Hill C.S. Downward J. J. Biol. Chem. 2003; 278: 3251-3256Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Moreover, activation of the PKB/Akt pathway has been demonstrated to “rescue” Hep3B cells from TGF-β1-induced apoptosis (28.Chen R.H. Su Y.H. Chuang R.L. Chang T.Y. Oncogene. 1998; 17: 1959-1968Crossref PubMed Scopus (177) Google Scholar). There is limited information, however, on the role and regulation of this pathway by TGF-β1 in mesenchymal cells. TGF-β family members signal via heteromeric transmembrane complexes of type II and type I serine-threonine receptor kinases (1.Massague J. Nat. Rev. Mol. Cell. Biol. 2000; 1: 169-178Crossref PubMed Scopus (1653) Google Scholar, 2.Roberts, A. B., and Derynck, R. (2001) Science's STKE http:/www.stke.org/cgi/content/full/OC_sigtrans;2001/PE43Google Scholar). The best known direct effectors of TGF-β receptor(s) signaling are the Smad proteins that, when activated, function as transcriptional regulators (1.Massague J. Nat. Rev. Mol. Cell. Biol. 2000; 1: 169-178Crossref PubMed Scopus (1653) Google Scholar). More recently, early post-receptor signaling via Smad-independent pathways have been increasingly recognized (2.Roberts, A. B., and Derynck, R. (2001) Science's STKE http:/www.stke.org/cgi/content/full/OC_sigtrans;2001/PE43Google Scholar). The p38 mitogen-activated protein kinase (MAPK) appears to be an important transducer of such responses (29.Hanafusa H. Ninomiya-Tsuji J. Masuyama N. Nishita M. Fujisawa J. Shibuya H. Matsumoto K. Nishida E. J. Biol. Chem. 1999; 274: 27161-27167Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 30.Yu L. Hebert M.C. Zhang Y.E. EMBO J. 2002; 21: 3749-3759Crossref PubMed Scopus (588) Google Scholar, 31.Edlund S. Bu S. Schuster N. Aspenstrom P. Heuchel R. Heldin N.E. Ten Dijke P. Heldin C.H. Landstrom M. Mol. Biol. Cell. 2003; 14: 529-544Crossref PubMed Scopus (187) Google Scholar). A direct link between activation of the p38 MAPK pathway and TGF-β receptor(s) activation appears to be through binding of X chromosome-linked inhibitor of apoptosis protein (32.Yamaguchi K. Nagai S. Ninomiya-Tsuji J. Nishita M. Tamai K. Irie K. Ueno N. Nishida E. Shibuya H. Matsumoto K. EMBO J. 1999; 18: 179-187Crossref PubMed Scopus (325) Google Scholar, 33.Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (520) Google Scholar). Activation of p38 MAPK by TGF-β1 has been shown to induce either EMT (34.Bhowmick N.A. Zent R. Ghiassi M. McDonnell M. Moses H.L. J. Biol. Chem. 2001; 276: 46707-46713Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar) or pro-apoptotic effects in epithelial cells (17.Dai C. Yang J. Liu Y. J. Biol. Chem. 2003; 278: 12537-12545Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 30.Yu L. Hebert M.C. Zhang Y.E. EMBO J. 2002; 21: 3749-3759Crossref PubMed Scopus (588) Google Scholar, 31.Edlund S. Bu S. Schuster N. Aspenstrom P. Heuchel R. Heldin N.E. Ten Dijke P. Heldin C.H. Landstrom M. Mol. Biol. Cell. 2003; 14: 529-544Crossref PubMed Scopus (187) Google Scholar). The role of the p38 MAPK pathway in the regulation of apoptosis in mesenchymal cells is not well understood. We hypothesized that activation of PI3K/Akt by TGF-β1 may induce an anti-apoptotic phenotype in mesenchymal cells/fibroblasts and that upstream activation of p38 MAPK may regulate this activity. This study was undertaken to determine effects of TGF-β1 on apoptotic susceptibility of primary cultures of untransformed human lung fibroblasts and the potential role of p38 MAPK-dependent PI3K-Akt activation in the expression of this phenotype. Research protocols involving human subjects received prior approval by the Institutional Review Board at the University of Michigan. Lung mesenchymal cells were isolated by bronchoalveolar lavage from adult patients with respiratory failure due to acute lung injury (ALI) (35.Ware L.B. Matthay M.A. N. Engl. J. Med. 2000; 342: 1334-1349Crossref PubMed Scopus (4495) Google Scholar). Cells were cultured in medium consisting of Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Sigma), 100 units/ml penicillin/streptomycin (Sigma), and fungizone (Invitrogen); medium was changed every 2 days. Studies were performed on passage 3-5 of a morphologically homogeneous population of spindle-shaped cells that uniformly stained positive for the fibroblast marker, prolyl 4-hydroxylase (36.Janin A. Konttinen Y.T. Gronblad M. Karhunen P. Gosset D. Malmstrom M. Clin. Exp. Rheumatol. 1990; 8: 237-242PubMed Google Scholar). Normal human fetal lung fibroblasts (IMR-90; Institute for Medical Research, Camden, NJ) were cultured under similar conditions, and studies were performed at passage 5-9. Cells were plated on 60-mm cell culture dishes at a density of 5 × 105 cells/dish or on 96-well ELISA cell culture plates at a density of 15,000 cells per well and incubated in 5% CO2, 95% air. When cells reached 80% confluence, they were growth-arrested for 48 h in DMEM with 0.01% FBS prior to treatment. Porcine-derived TGF-β1 was obtained from R & D Systems, Minneapolis, MN. SB203580, SP600125, wortmannin, and Y-27632 were from Calbiochem. PD98059 and LY294002 were obtained from Cell Signaling Technology, Beverly, MA. Rabbit polyclonal antibodies to phospho-Akt (Ser-473), phospho-ATF-2 (Thr-71), total Akt, and total p38 MAP kinase were from Cell Signaling Technology. Mouse monoclonal antibody to phospho-p38 MAPK (Thr-180/Tyr-182) was from Cell Signaling Technology. Rabbit polyclonal antibody to phospho-Smad2 (Ser-465/467) was from Upstate Biotechnology, Inc., Lake Placid, NY. Goat polyclonal antibody to total Smad2 was from Santa Cruz Biotechnology, Santa Cruz, CA. Mouse monoclonal antibodies to α-smooth muscle actin and β-actin were obtained from Sigma. Mouse monoclonal antibody to single-stranded DNA (ssDNA) and rabbit polyclonal antibody against activated caspase 3 were from Chemicon International, Temecula, CA. Secondary horseradish peroxidase-conjugated anti-goat, anti-mouse, and anti-rabbit antibodies were obtained from Pierce. All other reagents including suramin were from Sigma. The mammalian expression plasmids, pcDNA3-HA-p38 (wild type), pcDNA3-p38-KM (kinase mutant), and pcDNA3-MKK3, were provided by Dr. Kun Liang Guan, Department of Biological Chemistry, University of Michigan, Ann Arbor. Plasmid transfections of IMR-90 cells using the cationic lipid reagent LipofectAMINE (Invitrogen) were performed according the manufacturer's instructions. The optimal ratio of DNA (μg) to LipofectAMINE (μl) was determined to be ∼1:5 for IMR-90 cells. Cells were incubated with DNA-lipid complexes in serum-free Opti-MEM I medium (Invitrogen) for 4-5 h prior to introducing 10% FBS for 16-20 h. The next day, transfection medium was replaced by DMEM supplemented with 10% FBS and geneticin (Invitrogen). Geneticin concentrations were 400 μg/ml for selection of stable transfectants and 200 μg/ml as a maintenance dose in cell cultures prior to performing experiments. Cell lysates were prepared in RIPA buffer, subjected to SDS-PAGE, and Western blot analyses performed as described previously (37.Thannickal V.J. Aldweib K.D. Fanburg B.L. J. Biol. Chem. 1998; 273: 23611-23615Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In vitro p38 MAP kinase activity was measured using a nonradioactive assay obtained from Cell Signaling Technology, Beverly, MA. Immobilized antibody to phospho-p38 MAPK was added to 200 μg of whole cell lysates in RIPA buffer and incubated overnight at 4 °C. This was then centrifuged, and the supernatant was removed and the pellet washed three times with lysis buffer and kinase buffer as per the manufacturer's protocol. The pellet was then suspended in kinase buffer supplemented with ATP and an ATF-2 fusion protein for 30 min at 30 °C. The reaction was terminated with 6× SDS sample buffer, and the samples were boiled, vortexed, and centrifuged. Samples were then subjected to SDS-PAGE followed by immunoblotting with an antibody to phospho-ATF-2. ELISA for ssDNA—Apoptosis was quantitated with the use of an ELISA-based assay for ssDNA (Apoptosis ELISA Kit, Chemicon International, Temecula, CA) according to the manufacturer's instructions, with minor modifications (38.Frankfurt O.S. Krishan A. J. Immunol. Methods. 2001; 253: 133-144Crossref PubMed Scopus (80) Google Scholar). Cells were seeded directly into 96-well cell culture plates, grown to 80% confluence, growth-arrested for 48 h, and then treated with or without TGF-β1 in the presence of 10% FBS or under serum-deprived conditions for another 120 h. Prior to the assay, the 96-well plate was centrifuged for 5 min. The medium was removed, and cells were fixed to the plate with 80% methanol. The methanol was then removed, and the plate was dried in a 37 °C water bath for 20 min, after which 50 μl of formamide was added to each well for 10 min at room temperature followed by 10 min in a 75 °C water bath. After cooling, the formamide was removed, and 200 μl of 1% bovine serum albumin was added to each well for 1 h to block nonspecific binding. The blocking solution was then removed, and 50 μl of mouse monoclonal anti-ssDNA antibody (1:100) was added to each well for 30 min. After washing, 200 μl of horseradish peroxidase-conjugated anti-mouse secondary antibody was added for 30 min. After repeat washing, 100 μl of 2,2′-azino-bis-(3-benzthiazoline-6-sulfonic) acid solution was added to each well for 30 min followed by “stop solution” supplied by the manufacturer. Absorbance was read with an ELISA plate reader at 405 nm. The “background” absorbance of cells receiving no primary antibody was subtracted, and a relative apoptosis index calculated by dividing the corrected absorbance by cell counts (measured by Coulter counter) was obtained prior to fixing the cells. Activated Caspase 3/Immunofluorescence Staining—Activated caspase 3 was detected by immunofluorescence staining of fixed cells. Briefly, IMR-90 cells plated on 35 mm of tissue culture were grown to 50% confluence and growth-arrested in medium containing 0.01% serum for 48 h prior to treatments for defined times. Cells were then fixed in 5% formaldehyde and washed three times with cold phosphate-buffered saline prior to permeabilization and after each subsequent step. Permeabilization was performed in buffer consisting of 0.1% Triton in 50 mm PIPES (pH 7.0), 90 mm HEPES (pH 7.0), 0.5 mm MgCl2, 0.5 mm EGTA, and 75 mm KCl. Nonspecific binding sites were blocked with 1% bovine serum albumin for 15 min prior to the addition of antibody to activated caspase 3 (1:25 dilution) for 1 h followed by fluorescein isothiocyanate-conjugated secondary antibody (1:40 dilution) for 1 h. Counterstaining was with DAPI for nuclear staining, and cells were visualized and photographed using a Zeiss fluorescence microscope. Statistical analysis was performed using Student's t test and one-way analysis of variance with Bonferonni post test using GraphPad Prism version 3.0 for Windows, GraphPad Software, San Diego (www.graphpad.com). Densitometric analysis of Western blots was performed using the public domain NIH Image program available on the internet at rsb.info.nih.gov/nih-image. TGF-β1 Protects Human Lung Mesenchymal Cells/Fibroblasts from Serum Deprivation-induced Apoptosis—Adult mesenchymal cells, particularly when isolated from injured tissues, often represent a variably heterogeneous population of cells; moreover, there may be differences in cellular phenotypes and responsiveness between adult and fetal fibroblasts/mesenchymal cells. Therefore, we examined the effects of TGF-β1 on mesenchymal cells isolated both from adult patients with acute lung injury (ALI-MC) and on normal human fetal lung fibroblasts (IMR-90). Spontaneous rates of apoptosis in the presence of serum were slightly higher in ALI-MC than in IMR-90 cells (Fig. 1). Serum deprivation for 5 days increased apoptotic rates in both groups. Treatment of serum-deprived cells with TGF-β1 (2 ng/ml) reduced apoptotic rates in ALI-MC and IMR-90 cells by 50 and 48%, respectively (Fig. 1). Interestingly, TGF-β1 also protected against spontaneous apoptosis (in the presence of serum) in ALI-MC cells (Fig. 1A). These results suggest that TGF-β1 is able to induce cellular resistance against both spontaneous and serum deprivation-induced apoptosis in different fibroblasts/mesenchymal cells, including heterogeneous adult mesenchymal cells (ALI-MC) and more homogeneous cultures of untransformed, human fetal lung fibroblasts (IMR-90). TGF-β1 Induces Early Activation of the Smad and p38 MAPK Followed by Delayed Activation of the PI3K-Akt Pathway in Lung Fibroblasts—The PI3K-Akt pathway is known to transduce signals critical for cell survival (24.Brazil D.P. Park J. Hemmings B.A. Cell. 2002; 111: 293-303Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 25.Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3721) Google Scholar). To determine whether the pro-survival/anti-apoptotic effects of TGF-β1 on fibroblasts may, at least in part, be attributed to this pathway, we examined the effects of TGF-β1 on PKB/Akt activation. In both ALI-MC and IMR-90 cells, phosphorylation of PKB/Akt was observed as early as 3-6 h following TGF-β1 (2 ng/ml) treatment with peak effects at 12 h (Fig. 2). Peak effects at 12 h were completely inhibited by treatment with LY294002 (10 μm) or wortmannin (1 μm) for 2 h prior to cell lysis (data not shown). TGF-β1-induced PKB/Akt phosphorylation was sustained for 24 h and decreased significantly by 48 h following treatment (Fig. 2). The delayed responses in PKB/Akt phosphorylation suggested that other more proximal TGF-β1 signals mediate this effect. Rapid signaling from TGF-β receptor(s) activation is best recognized to occur by the Smad proteins (1.Massague J. Nat. Rev. Mol. Cell. Biol. 2000; 1: 169-178Crossref PubMed Scopus (1653) Google Scholar). Recent studies suggest the involvement of Smad-independent pathways involving p38 MAPK (2.Roberts, A. B., and Derynck, R. (2001) Science's STKE http:/www.stke.org/cgi/content/full/OC_sigtrans;2001/PE43Google Scholar, 30.Yu L. Hebert M.C. Zhang Y.E. EMBO J. 2002; 21: 3749-3759Crossref PubMed Scopus (588) Google Scholar). Moreover, p38 MAPK appears to mediate PKB/Akt activation in mouse epithelial cells exposed to UV light (39.Nomura M. Kaji A. He Z. Ma W.Y. Miyamoto K. Yang C.S. Dong Z. J. Biol. Chem. 2001; 276: 46624-46631Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). To determine whether p38 MAPK is rapidly activated in response to TGF-β1 in lung fibroblasts, we assessed the phosphorylated (activated) state of p38 MAPK and compared it with that of Smad2 phosphorylation. Fig. 3 demonstrates that both p38 MAPK and Smad2 are activated within 10 min of TGF-β1 (2 ng/ml) stimulation. Peak activation of these responses to TGF-β1 is observed at 1 h. These results demonstrate that the p38 MAPK pathway is activated rapidly in response to TGF-β1, with similar kinetics to that of Smad2 phosphorylation, and suggested that PKB/Akt phosphorylation might be mediated via p38 MAPK. Pharmacological Inhibition of p38 MAPK Blocks TGF-β1-induced PKB/Akt Phosphorylation—To determine whether early p38 MAPK activation is necessary for the more delayed activation of PKB/Akt, we examined the effect of various protein kinase inhibitors on TGF-β1-mediated PKB/Akt phosphorylation. In both ALI-MC and IMR-90 cells, blockade of the ERK-1/2 MAPK pathway with the MEK1 inhibitor, PD98059 (20 μm), had little effect on PKB/Akt phosphorylation (Fig. 4). In contrast, the p38 MAPK inhibitor, SB203580 (6 μm), completely inhibited TGF-β1-induced PKB/Akt phosphorylation to below base-line levels in both ALI-MC and IMR-90 cells (Fig. 4). Similar results were obtained when the effects of TGF-β1 were examined separately at 12 and 24 h after treatment (data not shown). An inhibitor of c-Jun N-terminal kinase (SP600125, 100 nm) more modestly attenuated the induction of PKB/Akt phosphorylation by 48% in ALI-MC and 27% in IMR-90 cells (Fig. 4). In the presence of the PI3K inhibitor, LY294002 (10 μm), PKB/Akt phosphorylation was virtually undetectable in IMR-90 cells (Fig. 4, C and D). Similarly, wortmannin (1 μm) reduced PKB/Akt phosphorylation to about 50% of control levels in ALI-MC (Fig. 4, A and B). Interestingly, the Rho kinase inhibitor, Y-27632 (15 nm), inhibited PKB/Akt phosphorylation to a greater degree in ALI-MC than in IMR-90 cells. Overall, these results suggest that pharmacological inhibition of the p38 MAPK pathway reliably and consistently blocks TGF-β1-induced PKB/Akt phosphorylation; more variable and apparently cell-specific responses are observed with an inhibitor of Rho kinase. Protein kinase inhibitors may exert non-specific effects, particularly at higher concentrations (40.Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3945) Google Scholar). SB203580 may directly inhibit PKB/Akt, although the IC50 values are 100-500-fold higher than for p38 MAPK (40.Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3945) Google Scholar). In vitro assays indicate that at a concentration of 10 μm, SB203580 almost completely inhibits p38 MAPK activity (98%), whereas PKB/Akt is inhibited by about 38% (40.Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3945) Google Scholar). To define better effective concentrations at which SB203580 inhibits TGF-β1-induced PKB/Akt phosphorylation, we performed a dose-response study by co-treating cells with increasing doses (0.6, 3, 6, and 18 μm) of SB203580 and TGF-β1 (2 ng/ml for 12 h). Fig. 5 demonstrates dose-dependent inhibition of TGF-β1-induced PKB/Akt phosphorylation. An ∼50% inhibition was noted at 3 μm, and complete inhibition was achieved at 6 μm; at 18 μm, there was additional inhibition of the base-line levels of PKB/Akt phosphorylation (Fig. 5). Moreover, 6 μm SB203580 alone (in the absence of TGF-β1) had no effect on base-line PKB/Akt phosphorylation in these cells (data not shown). These findings suggest that the observed blockade of TGF-β1 stimulated PKB/Akt phosphorylation is more likely related to inhibitory effects of SB203580 on upstream p38 MAPK activation than to more direct effects of this compound on PKB/Akt. PKB/Akt Phosphorylation by TGF-β1 Is Dependent on Functional p38 MAPK Activation—To address further the role of p38 MAPK activation in TGF-β1-induced PKB/Akt phosphorylation, we generated stable cell lines of IMR-90 cells expressing a kinase-deficient/mutant p38 MAPK (p38-KM), a constitutively active MKK3 (MKK3), and control (pcDNA, empty vector) plasmid constructs. TGF-β1 (2 ng/ml for 1 h) induced phosphorylation of p38 MAPK in all cell lines (Fig. 6A, top panel). However, p38 MAPK activity measured by in vitro phosphorylation of ATF-2 was not induced in p38-KM cells at the same time point (Fig. 6A, upper middle panel), consistent with functionally inactive p38 MAPK in these cells. Overexpression of MKK3 did not appear to augment further the ATF-2 phosphorylation in response to TGF-β1, and basal levels appeared to be lower than control pcDNA cells. The pattern of ATF-2 phosphorylation/p38 activity closely correlated with PKB/Akt phosphorylation induced by TGF-β1 in all cell lines. Importantly, TGF-β1-induced PKB/Akt phosphorylation was completely inhibited in p38-KM cells (Fig. 6A, lower middle panel, and B). Moreover, the activation of this pathway appears to be less important for TGF-β1-induced α-smooth muscle cell actin, a marker of myofibroblast differentiation, because this response is preserved in p38-KM cells (Fig. 6A, lower panel). These results indicate that activation of p38 MAPK by TGF-β1 is required for the induction of PKB/Akt and suggest that activation of this pathway may be relatively more specific for pro-survival signaling than for myofibroblast differentiation-inducing effects of TGF-β1. The morphology of these stable cell l

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