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

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. Invest. 1997; 100: 589-596Crossref PubMed Scopus (172) Google Scholar, 9Stocker R. Free Radical Res. Commun. 1990; 9: 101-112Crossref PubMed Scopus (306) Google Scholar). The induction of HO-1 is thought to be an adaptive response that offers cytoprotection to cells/tissue against oxidant stress (9Stocker R. Free Radical Res. Commun. 1990; 9: 101-112Crossref PubMed Scopus (306) Google Scholar). A previous study reported an increase of HO-1 mRNA and protein expression by TGF-β1 in human retinal pigment epithelial cells and suggested that induction of HO-1 attenuates the adverse effects of elevated TGF-β1 (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). The initial purpose of this study was to evaluate the molecular mechanism of TGF-β1-mediated induction of the human HO-1 gene in renal epithelial cells as well as to elucidate the signaling pathways involved in HO-1 gene induction. 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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PubMed Scopus Google and The was on a to a and with a human HO-1 was by mRNA and with for The and with a human to for and The also with a TβR-I to Following was by for and Smad7 as well as constitutively active TβR-I by and and have been described T. M. A. M. Nature. 1997; PubMed Scopus Google Scholar). Smad6, or Smad7 expression in cells by and by and The expression for TβR-I a constitutively active mutant of TβR-I, was into the of cells at of with in the glycine- and serine-rich in constitutive activation of TβR-I such that growth and extracellular matrix occur in the of or TβR-II R. Wrana J.L. Massague J. J. 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The induction of HO-1 by TGF-β1 may serve as a protective response to the effects of In this at the Smad7 pathway in expression of its cytoprotective We speculate that the beneficial effects of TGF-β1 may be mediated via the induction of HO-1 and that the effects of TGF-β1 are by HO-1 induction. are potential mechanisms of the beneficial effects of HO-1 induction in response to in increased cellular which is from heme proteins, such as and The heme is and G. U. Invest. Google Scholar, J. G. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar, S. Kidney Int. 1998; Full Text PDF PubMed Scopus Google resulting in to and components of the nucleus S. Kidney Int. 1998; Full Text PDF PubMed Scopus Google Scholar). HO-1 heme injury. HO-1 is for a into is converted to an which is of and R. Y. Science. PubMed Scopus Google Scholar, 1994; PubMed Scopus Google Scholar). an for is with of the iron the of heme G. J. M. F. J. Biol. Full Text PDF PubMed Google Scholar). has as a signaling similar to has effects mediated via and also and J. M. M. 6: PubMed Scopus Google Scholar). induction of HO-1 may be for of the wound properties with TGF-β1 as well as a cytoprotective response to an of In we have demonstrated that TGF-β1 HO-1 gene expression in a and in renal proximal tubule A against TGF-β1 the of and effects of that may be in HO-1 induction by TGF-β1 is mediated via mechanisms and requires de novo protein synthesis. In that TGF-β1-mediated HO-1 induction via the Smad Smad7 mRNA is induced by TGF-β1 as as h of of a constitutively active TβR-I mutant receptor HO-1 in cells with or Smad6, the BMP signaling Smad, while cells with Smad7, the for TGF-β signaling, of TGF-β1-mediated HO-1 gene expression. that the Smad pathway is involved in TGF-β1-mediated HO-1 induction and that Smad7 expression of an endogenous We are to and of The of the for for the expression for Smad6, Smad7, and TβR-I