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Smad7-dependent Regulation of Heme Oxygenase-1 by Transforming Growth Factor-β in Human Renal Epithelial Cells

2000; Elsevier BV; Volume: 275; Issue: 52 Linguagem: Inglês

10.1074/jbc.m006621200

ISSN

1083-351X

Autores

Nathalie Hill‐Kapturczak, Leigh Truong, Vijayalakshmi Thamilselvan, Gary Visner, Harry S. Nick, Anupam Agarwal,

Tópico(s)

Mesenchymal stem cell research

Resumo

Heme oxygenase-1 (HO-1), a 32-kDa microsomal enzyme, is induced as a beneficial and adaptive response in cells/tissues exposed to oxidative stress. Transforming growth factor-β1 (TGF-β1) is a regulatory cytokine that has been implicated in a variety of renal diseases where it promotes extracellular matrix deposition and proinflammatory events. We hypothesize that the release of TGF-β1 via autocrine and/or paracrine pathways may induce HO-1 and serve as a protective response in renal injury. To understand the molecular mechanism of HO-1 induction by TGF-β1, we exposed confluent human renal proximal tubule cells to TGF-β1 and observed a significant induction of HO-1 mRNA at 4 h with a maximal induction at 8 h. This induction was accompanied by increased expression of HO-1 protein. TGF-β1 treatment in conjunction with actinomycin D or cycloheximide demonstrated that induction of HO-1 mRNA requires de novo transcription and, in part, protein synthesis. Exposure to TGF-β1 resulted in marked induction of Smad7 mRNA with no effect on Smad6 expression. Overexpression of Smad7, but not Smad6, inhibited TGF-β1-mediated induction of endogenous HO-1 gene expression. We speculate that the induction of HO-1 in the kidney is an adaptive response to the inflammatory effects of TGF-β1 and manipulations of the Smad pathway to alter HO-1 expression may serve as a potential therapeutic target. Heme oxygenase-1 (HO-1), a 32-kDa microsomal enzyme, is induced as a beneficial and adaptive response in cells/tissues exposed to oxidative stress. Transforming growth factor-β1 (TGF-β1) is a regulatory cytokine that has been implicated in a variety of renal diseases where it promotes extracellular matrix deposition and proinflammatory events. We hypothesize that the release of TGF-β1 via autocrine and/or paracrine pathways may induce HO-1 and serve as a protective response in renal injury. To understand the molecular mechanism of HO-1 induction by TGF-β1, we exposed confluent human renal proximal tubule cells to TGF-β1 and observed a significant induction of HO-1 mRNA at 4 h with a maximal induction at 8 h. This induction was accompanied by increased expression of HO-1 protein. TGF-β1 treatment in conjunction with actinomycin D or cycloheximide demonstrated that induction of HO-1 mRNA requires de novo transcription and, in part, protein synthesis. Exposure to TGF-β1 resulted in marked induction of Smad7 mRNA with no effect on Smad6 expression. Overexpression of Smad7, but not Smad6, inhibited TGF-β1-mediated induction of endogenous HO-1 gene expression. We speculate that the induction of HO-1 in the kidney is an adaptive response to the inflammatory effects of TGF-β1 and manipulations of the Smad pathway to alter HO-1 expression may serve as a potential therapeutic target. transforming growth factor bone morphogenetic protein glyceraldehyde-3-phosphate dehydrogenase human renal proximal tubular cell mitogen-activated protein kinase phenylmethanesulfonyl fluoride peroxidase type I and II receptor for transforming growth factor β, respectively constitutively active mutant type I receptor for transforming growth factor β hemagglutinin Tris-buffered saline phosphate-buffered saline Kidney diseases such as IgA nephropathy, focal and segmental glomerulosclerosis, crescentic glomerulonephritis, lupus nephritis, diabetic nephropathy, and chronic rejection are characterized by the deposition of extracellular matrix and have increased expression of transforming growth factor-β1 (TGF-β1)1 in glomeruli and tubulointerstitium as compared with normal kidneys (1Yamamoto T. Noble N.A. Cohen A.H. Nast C.C. Hishida A. Gold L.I. Border W.A. Kidney Int. 1996; 49: 461-469Abstract Full Text PDF PubMed Scopus (418) Google Scholar, 2Sharma V.K. Bologa R.M. Xu G-P. Li B. Mouradian J. Wang J. Serur D. Rao V. Suthanthiran M. Kidney Int. 1996; 49: 1297-1303Abstract Full Text PDF PubMed Scopus (173) Google Scholar). TGF-β1 is a member of the TGF-β superfamily, which includes the TGF-βs, the activins/inhibins, and the bone morphogenetic proteins (BMPs). TGF-β1 is implicated in a wide range of cellular events such as cell proliferation and migration, wound healing, inflammatory responses, and stimulation of extracellular matrix components (3Kays S.E. Nowak G. Schnellmann R.G. J. Biochem. Toxicol. 1996; 11: 79-84Crossref PubMed Scopus (5) Google Scholar, 4Massague J. Annu. Rev. Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (3001) Google Scholar). In response to injury, TGF-β1 and other growth factors are released via autocrine and/or paracrine mechanisms to maintain cellular homeostasis. While chronic elevation of TGF-β1 plays an important role in the progression of renal diseases, TGF-β1 can also stabilize and attenuate tissue injury through the activation of cytoprotective proteins. We hypothesize that the paradoxical effects of TGF-β1 in response to cellular injury may be in part mediated by the induction of an antioxidant enzyme, heme oxygenase-1 (HO-1). Heme oxygenase is a microsomal enzyme that catalyzes the conversion of heme to biliverdin, releasing equimolar amounts of carbon monoxide (CO) and iron (5Maines M.D. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 517-554Crossref PubMed Scopus (2198) Google Scholar). Subsequently, biliverdin is converted to bilirubin by biliverdin reductase (5Maines M.D. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 517-554Crossref PubMed Scopus (2198) Google Scholar). Three isoforms of heme oxygenase (HO-1, HO-2, and HO-3) have been described (5Maines M.D. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 517-554Crossref PubMed Scopus (2198) Google Scholar, 6McCoubrey W.K. Huang A.T.J. Maines M.D. Eur. J. Biochem. 1997; 247: 725-732Crossref PubMed Scopus (735) Google Scholar). HO-1 is inducible, while HO-2 is refractory to most stimuli and, thus, the constitutive form. The third isoform, HO-3, with properties similar to HO-2, has only recently been described (6McCoubrey W.K. Huang A.T.J. Maines M.D. Eur. J. Biochem. 1997; 247: 725-732Crossref PubMed Scopus (735) Google Scholar). HO-1 is induced by heme products and a wide variety of nonheme stimuli, which include hydrogen peroxide, ultraviolet A radiation, heavy metals, endotoxin, cytokines, and oxidant stress (7Keyse S.M. Tyrrell R.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 99-103Crossref PubMed Scopus (1105) Google Scholar, 8Wagner C.T. Durante W. Christodoulides N. Hellums J.D. Schafer A.I. J. Clin. 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TGF-β initiates signaling through type I (TβR-I) and type II (TβR-II) receptors, binding directly with TβR-II, which then interacts transiently with TβR-I, forming a heteromeric complex (11Chen R.H. Ebner R. Derynck R. Science. 1993; 260: 1335-1338Crossref PubMed Scopus (357) Google Scholar). Ligand binding is followed by TβR-II transphosphorylation of TβR-I, mainly in the conserved glycine- and serine-rich domain, resulting in activation of the TβR-I kinase that initiates downstream signaling events. The molecular mechanism of TGF-β signal transduction from the cell surface to the nucleus has been recently identified to occur through a novel group of structurally related proteins called Smads (12Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 1998; 10: 188-194Crossref PubMed Scopus (177) Google Scholar, 13Derynck R. Zhang Y. Feng X. Cell. 1998; 95: 737-740Abstract Full Text Full Text PDF PubMed Scopus (948) Google Scholar). 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Heldin N. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1549) Google Scholar, 25Hayashi H. Abdollah S. Qui Y. Cai J. Xu Y.-Y. Grinnell B.W. Richardson M.A. Topper J.N. Gimbrone J.M.A. Wrana J.L. Falb D. Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1151) Google Scholar). The molecular mechanism of TGF-β1 signal transduction through Smads has been studied extensively in cancer models. The anti-Smad pathway has been shown to inhibit activation of other genes such as human plasminogen activator inhibitor-1 (PAI-1) and collagen (25Hayashi H. Abdollah S. Qui Y. Cai J. Xu Y.-Y. Grinnell B.W. Richardson M.A. Topper J.N. Gimbrone J.M.A. Wrana J.L. Falb D. Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1151) Google Scholar, 26Atfi A. Djelloul S. Chastre E. Davis R. Gespach C. J. Biol. Chem. 1997; 272: 1429-1432Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 27Zhu H.J. Iaria J. Sizeland A.M. J. Biol. 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Chem. 1999; 274: 32258-32264Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 28Nakao A. Fujii M. Matsumura R. Kumano K. Saito Y. Miyazono K. Iwamoto I. J. Clin. Invest. 1999; 104: 5-11Crossref PubMed Scopus (384) Google Scholar, 29Engel M.E. Datta P.K. Moses H.L. J. Cell. Biochem. Suppl. 1998; 30/31: 111-122Crossref Google Scholar). In this regard, our studies have not only demonstrated that TGF-β1 induces HO-1 and Smad7 in human renal epithelial cells but also represent the first demonstration that overexpression of Smad7 inhibits the induction of the endogenous HO-1 gene. Recombinant human TGF-β1 and anti-TGF-β antibody were obtained from R & D systems (Minneapolis, MN). Another preparation of recombinant human TGF-β1 was obtained from Genzyme (Cambridge, MA). Actinomycin D, anti-FLAG® M2 monoclonal antibody, anti-mouse IgG-agarose, cycloheximide, and hemin were purchased from Sigma. Anti-HA high affinity rat monoclonal antibody, peroxidase-conjugated goat anti-mouse IgG antibody, and streptavidin-peroxidase were purchased from Roche Molecular Biochemicals. Calf serum; fetal bovine serum; G418 (Geneticin); and transfection reagents LipofectAMINETM, LipofectAMINETM Plus, and OptiMEM were obtained from Life Technologies, Inc. FugeneTM transfection reagent was purchased from Roche Molecular Biochemicals. Kinase inhibitors PD98059 and SB203580 were obtained from Calbiochem. Human proximal tubule cells (HPTCs; Clonetics, Walkersville, MD) were grown in renal epithelial basal medium (Clonetics) supplemented with fetal bovine serum (5%), gentamicin (50 μg/ml), amphotericin B (50 μg/ml), insulin (5 μg/ml), transferrin (10 μg/ml), triiodothyronine (6.5 ng/ml), hydrocortisone (0.5 μg/ml), epinephrine (0.5 μg/ml), and human epidermal growth factor (10 ng/ml) at 37 °C in 95% air and 5% carbon dioxide. HPTCs were used over a range of 4–5 passages. Human embryonic kidney cells (HEK 293 cells; ATCC, Manassas, VA) were grown in Dulbecco's minimum essential medium supplemented with calf serum (10%) and HEPES buffer (25 mm) at 37 °C in 90% air and 10% carbon dioxide. Total RNA was extracted from cultured cells grown in 60-mm plates using the method described by Chomczynski and Sacchi (30Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63087) Google Scholar) and purified using RNeasy mini kits (Qiagen). The RNA (10–15 μg) was electrophoresed on a 1% agarose gel containing formaldehyde, electrotransferred to a nylon membrane, and hybridized with a 32P-labeled 1.0-kilobase pair human HO-1 cDNA probe. For Smad6/7 expression, poly(A)+ RNA was obtained by purifying total RNA using Oligotex mRNA mini kits (Qiagen) and probed with cDNAs for Smad6/7. The nylon membranes were stripped and rehybridized with a human GAPDH cDNA probe to control for handling and loading. The blots were also reprobed with a TβR-I (TD) cDNA probe to confirm overexpression. Following autoradiography, quantitation was performed by densitometry using NIH Image. Expression constructs for FLAG-tagged pcDNA3-Smad6 and Smad7 as well as HA-tagged pcDNA3 constitutively active TβR-I (TD) were generously provided by Takeshi Imamura and Kohei Miyazono and have been described previously (23Imamura T. Takase M. Nishihara A. Oeda E. Hanai J.-I. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar). Stable transfectants using either vector, Smad6, or Smad7 expression plasmids, were made in HEK 293 cells by antibiotic selection (G418; 400 μg/ml), and clones were confirmed by northern and immunoblot analyses. Transfections were performed using LipofectAMINETM Plus reagent. The expression plasmid for TβR-I (TD), a constitutively active mutant of TβR-I, was transfected into the above stable clones of HEK 293 cells at 50% confluency using FugeneTM 6. Replacement of Thr204 with aspartic acid in the glycine- and serine-rich domain results in constitutive activation of TβR-I such that TGF-β-specific responses (e.g. growth inhibition and extracellular matrix deposition) occur in the absence of ligand or TβR-II (31Wieser R. Wrana J.L. Massague J. EMBO J. 1995; 14: 2199-2208Crossref PubMed Scopus (595) Google Scholar). Transfection efficiency was 40% based on co-transfections using a β-galactosidase expression plasmid (pcDNA3.1/LacZ). For HO-1 immunoblots, cells were treated with stimulus for the concentrations and times indicated. Cells were then washed twice with ice-cold PBS and lysed in a buffer containing a broad spectrum mixture of protease inhibitors consisting of 10 μg/ml aprotinin, 5 mm EDTA, 1 μg/ml leupeptin, 0.7 μg/ml pepstatin A, 1 mmphenylmethanesulfonyl fluoride, and Triton X-100. Protein concentration of lysates was assessed by the bicinchoninic acid assay (Pierce). Samples were separated in a 10% SDS-polyacrylamide gel and then transferred onto a polyvinylidene difluoride membrane. The membranes were incubated for 1.5 h with the anti-HO-1 antibody (1:500 dilution) followed by incubation with peroxidase-conjugated goat anti-rabbit IgG antibody (1:10,000 dilution) for 1 h. Labeled protein bands were examined by using a chemiluminescence method according to the manufacturer's recommendation. FLAG-tagged Smad6 and Smad7, and HA-tagged TβR-I (TD) were immunoprecipitated prior to immunoblot analysis. For immunoprecipitation, cells were washed twice with ice-cold PBS and lysed in a buffer containing 50 mm Tris, pH 7.5, 150 mm NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate along with a broad spectrum mixture of protease inhibitors (as described above). The supernatants of cell lysates from vector alone, Smad6- or Smad7-stable transfectants were immunoprecipitated with anti-mouse IgG-agarose bound to anti-FLAG® M2 according to the manufacturer's directions. To confirm the presence of TβR-I (TD) protein, the supernatants from cells transiently transfected with TβR-I (TD) were immunoprecipitated with protein G-agarose (Roche Molecular Biochemicals) followed by an anti-HA high affinity rat monoclonal antibody (Roche Molecular Biochemicals) as indicated by the manufacturer. Protein concentration of lysates was assessed by the bicinchoninic acid assay (Pierce). Immunoprecipitated samples were then subjected to immunoblot analysis by separation on a 7.5% SDS-polyacrylamide gel and transferred subsequently onto a polyvinylidene difluoride membrane. For FLAG-tagged Smad6 and Smad7, membranes were incubated with a murine anti-FLAG® M2 monoclonal antibody (10 μg/ml) in 5% nonfat dry milk/TBS-Tween (0.05%) for 1 h at room temperature. Membranes were washed with TBS-Tween (0.05%) and then incubated with anti-mouse IgG peroxidase conjugate (secondary antibody) at the manufacturer's recommended concentration in TBS-Tween (0.05%) for 1 h. For HA-tagged TβR-I (TD), membranes were incubated following manufacturer recommendations with anti-HA high affinity rat monoclonal antibody (200 ng/ml) in 5% nonfat dry milk/TBS for 1 h, washed with TBS-Tween (0.05%), and incubated with peroxidase-conjugated goat anti-mouse IgG antibody (20 ng/ml; Roche Molecular Biochemicals) for 30 min. The HA immunoblots were washed and incubated with streptavidin-peroxidase (15 milliunits/ml; Roche Molecular Biochemicals) for 30 min. All labeled protein bands (FLAG- or HA-tagged) were examined using the chemiluminescence method (Pierce). Increased levels of TGF-β1 play an important role in the pathogenesis of renal injury (1Yamamoto T. Noble N.A. Cohen A.H. Nast C.C. Hishida A. Gold L.I. Border W.A. Kidney Int. 1996; 49: 461-469Abstract Full Text PDF PubMed Scopus (418) Google Scholar, 2Sharma V.K. Bologa R.M. Xu G-P. Li B. Mouradian J. Wang J. Serur D. Rao V. Suthanthiran M. Kidney Int. 1996; 49: 1297-1303Abstract Full Text PDF PubMed Scopus (173) Google Scholar). A previous study by Kutty et al. (10Kutty R.K. Nagineni C.N. Kutty G. Hooks J.J. Chader G.J. Wiggert B. J. Cell. Physiol. 1994; 159: 371-378Crossref PubMed Scopus (102) Google Scholar) reported an increase of HO-1 mRNA and protein by TGF-β1 in retinal pigment epithelial cells. We hypothesized that the induction of cytoprotective enzymes such as HO-1 may explain the paradoxical effects of TGF-β1 in response to renal injury. Steady-state levels of HO-1 mRNA in HPTCs were examined by Northern blot analyses after exposure to TGF-β1. HPTCs treated with TGF-β1 at concentrations of 0.05, 0.5, and 2.0 ng/ml exhibited a significant, dose-dependent induction in HO-1 mRNA of 6-, 12-, and 13-fold, respectively, over control (PBS) at 4 h (Fig. 1 A). Hemin (5 μm) was used as a positive control. Immunoblot analysis demonstrated that induction of HO-1 mRNA by TGF-β1, obtained from two different sources, was accompanied by a 3.5-fold increase in HO-1 protein (Fig. 1 B). Furthermore, HO-1 mRNA levels were induced by TGF-β1 (2.0 ng/ml) in a time-dependent manner with a maximum induction observed at 8 h (Fig. 1 C). Neutralization of TGF-β1 activity with anti-TGF-β antibody (1000 ng/ml) completely blocked HO-1 mRNA induction (Fig. 2 A), demonstrating the specificity of TGF-β1. To further evaluate the mechanism(s) of TGF-β1-mediated HO-1 induction, confluent HPTCs were co-treated with TGF-β1 (1.0 ng/ml) and actinomycin D (4 μm), a transcriptional inhibitor, or cycloheximide (20 μm), an inhibitor of protein synthesis. Actinomycin D completely blocked TGF-β1-mediated HO-1 mRNA induction (Fig. 2 B). Cycloheximide attenuated the up-regulation of HO-1 mRNA steady state levels in response to TGF-β1 by 50% (Fig. 2 B). These data suggest that HO-1 induction by TGF-β1 is dependent onde novo mRNA expression and, in part, on new protein synthesis. Recent studies have reported that TGF-β1 up-regulates its inhibitory proteins, Smad6 and Smad7, in mink lung epithelial cells and other TGF-β-responsive cell lines (24Nakao A. Afrakhte M. Morén A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin C. Heldin N. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1549) Google Scholar, 32Itoh S. Landström M. Hermansson A. Itoh F. Heldin C. Heldin N. ten Dijke P. J. Biol. Chem. 1998; 273: 29195-29201Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 33Afrakhte M. Morén A. Jossan S. Itoh S. Sampath K. Westermark B. Heldin C. Heldin N. ten Dijke P. Biochem. Biophys. Res. Commun. 1998; 249: 505-511Crossref PubMed Scopus (297) Google Scholar). While Smad6 is involved mainly in BMP signaling, Smad7 inhibits both BMP and TGF-β1 signaling (22Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (578) Google Scholar, 23Imamura T. Takase M. Nishihara A. Oeda E. Hanai J.-I. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 24Nakao A. Afrakhte M. Morén A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin C. Heldin N. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1549) Google Scholar, 25Hayashi H. Abdollah S. Qui Y. Cai J. Xu Y.-Y. Grinnell B.W. Richardson M.A. Topper J.N. Gimbrone J.M.A. Wrana J.L. Falb D. Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1151) Google Scholar). Smad7 translocates from the nucleus to the cytoplasm on ligand binding and inhibits TGF-β1-mediated downstream signaling events through either an association with receptor regulated Smads and TβR-I (23Imamura T. Takase M. Nishihara A. Oeda E. Hanai J.-I. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 24Nakao A. Afrakhte M. Morén A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin C. Heldin N. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1549) Google Scholar, 25Hayashi H. Abdollah S. Qui Y. Cai J. Xu Y.-Y. Grinnell B.W. Richardson M.A. Topper J.N. Gimbrone J.M.A. Wrana J.L. Falb D. Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1151) Google Scholar) or competition with Smad4 (22Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (578) Google Scholar). Previous studies have also reported that the anti-Smads inhibit activation of TGF-β1-responsive promoter-reporter systems such as p3TP-lux (25Hayashi H. Abdollah S. Qui Y. Cai J. Xu Y.-Y. Grinnell B.W. Richardson M.A. Topper J.N. Gimbrone J.M.A. Wrana J.L. Falb D. Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1151) Google Scholar, 27Zhu H.J. Iaria J. Sizeland A.M. J. Biol. Chem. 1999; 274: 32258-32264Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 29Engel M.E. Datta P.K. Moses H.L. J. Cell. Biochem. Suppl. 1998; 30/31: 111-122Crossref Google Scholar). To extend these observations to activation of an endogenous gene, we evaluated the role of the anti-Smads in TGF-β1-mediated activation of HO-1. To examine the presence/absence of the inhibitory Smad pathway in HPTCs, the induction of Smad6 and Smad7 following TGF-β1 stimulation was first examined. Steady-state levels of Smad7 mRNA were examined by Northern blot analyses after exposure to TGF-β1. HPTCs treated with TGF-β1 (1.0 ng/ml) for 0, 0.5, 1.0, and 1.5 h exhibited a significant induction (12-fold) in Smad7 mRNA as early as 1 h (Fig. 3). No induction of Smad6 mRNA was observed (data not shown). In order to further investigate the effect of anti-Smads on TGF-β1-mediated HO-1 mRNA induction, mammalian expression vectors harboring the cDNAs for Smad6 and Smad7 were stably transfected into HEK 293 cells. Since HEK 293 cells are only minimally responsive to TGF-β1, the stable Smad6 and Smad7 clones were transfected with TβR-I (TD) to mimic the actions of TGF-β. Transfection of the TβR-I (TD) expression plasmid results in constitutive activation of TβR-I such that TGF-β specific responses occur in the absence of ligand or TβR-II (31Wieser R. Wrana J.L. Massague J. EMBO J. 1995; 14: 2199-2208Crossref PubMed Scopus (595) Google Scholar). Expression of Smad6 and Smad7 was confirmed by immunoprecipitation using a FLAG antibody (Fig. 4 A). Expression of TβR-I (TD) was confirmed by immunoprecipitation using a HA antibody (and/or Northern analysis) (Figs. 4 B and5). 24 and 48 h following transfection of TβR-I (TD) into the stable Smad6 and Smad7 clones, steady-state levels of HO-1 mRNA were examined by Northern blot analyses. TβR-I (TD) increased the steady state levels of HO-1 mRNA in vector alone and in Smad6-transfected cells (Fig. 5). However, the expression of Smad7 inhibited TβR-I (TD)-mediated induction of HO-1 mRNA (Fig. 5). These data indicate that overexpression of Smad7 can inhibit TGF-β1-mediated induction of the endogenous HO-1 gene.Figure 5Effect of TβR-I (TD) on HO-1 mRNA in HEK 293 cells. Vector alone, Smad6- and Smad7-stable transfectants were transiently transfected with TβR-1 (TD). Total RNA was isolated 24 and 48 h after transient transfection of TβR-I (TD), electrophoresed, transferred to a nylon membrane, and hybridized with a 32P-labeled human HO-1 cDNA probe. The membrane was stripped and reprobed with a TβR-I (TD) cDNA probe to control for transfection efficiency. The membrane was again stripped and reprobed with a human GAPDH cDNA probe to control for loading and transfer. Results are representative of three independent experiments.View Large Image Figure ViewerDownload (PPT) The effects of TGF-β1 and other growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor, fibroblast growth factor, TGF-β2, TGF-α, and insulin-like growth factor-1 on HO-1 expression are cell type-specific. For example, in retinal pigment epithelial cells, TGF-β1 induces HO-1, while PDGF, fibroblast growth factor, insulin-like growth factor-1, and TGF-α do not (10Kutty R.K. Nagineni C.N. Kutty G. Hooks J.J. Chader G.J. Wiggert B. J. Cell. Physiol. 1994; 159: 371-378Crossref PubMed Scopus (102) Google Scholar). PDGF has been reported to induce HO-1 in vascular smooth muscle cells (34Durante W. Peyton K.J. Schafer A.I. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2666-2672Crossref PubMed Scopus (56) Google Scholar). TGF-β1 induces HO-1 expression in bovine choroid fibroblasts but not in HeLa, human lung fibroblasts, or bovine corneal fibroblasts (10Kutty R.K. Nagineni C.N. Kutty G. Hooks J.J. Chader G.J. Wiggert B. J. Cell. Physiol. 1994; 159: 371-378Crossref PubMed Scopus (102) Google Scholar). Furthermore, TGF-β1 down-regulates endotoxin-mediated induction of HO-1 in rat vascular smooth muscle cells (35Pellacani A. Wiesel P. Sharma A. Foster L.C. Huggins G.S. Yet S.F. Perrella M.A. Circ. Res. 1998; 83: 396-403Crossref PubMed Scopus (55)

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