SOX9 Protein Induces a Chondrogenic Phenotype of Mesangial Cells and Contributes to Advanced Diabetic Nephropathy

Abstract

Diabetic nephropathy (DN) is the most important chronic kidney disease. We previously reported that Smad1 transcriptionally regulates the expression of extracellular matrix in DN. Phenotypic change in mesangial cells (MCs) is a key pathologic event in the progression of DN. The aim of this study is to investigate a novel mechanism underlying chondrogenic phenotypic change in MCs that results in the development of DN. MCs showed chondrogenic potential in a micromass culture, and BMP4 induced the expression of chondrocyte markers (SRY-related HMG Box 9 (SOX9) and type II collagen (COL2)). Advanced glycation end products induced the expression of chondrocyte marker proteins downstream from the BMP4-Smad1 signaling pathway in MCs. In addition, hypoxia also induced the expression of BMP4, hypoxia-inducible factor-1α (HIF-1α), and chondrocyte markers. Overexpression of SOX9 caused ectopic expression of proteoglycans and COL2 in MCs. Furthermore, forced expression of Smad1 induced chondrocyte markers as well. Dorsomorphin inhibited these inductions. Glomerular expressions of HIF-1α, BMP4, and chondrocyte markers were observed in diabetic nephropathy mice. These positive stainings were observed in mesangial sclerotic lesions. SOX9 was partially colocalized with HIF-1α and BMP4 in diabetic glomeruli. BMP4 knock-in transgenic mice showed not only similar pathological lesions to DN, but also the induction of chondrocyte markers in the sclerotic lesions. Here we demonstrate that HIF-1α and BMP4 induce SOX9 expression and subsequent chondrogenic phenotype change in DN. The results suggested that the transdifferentiation of MCs into chondrocyte-like cells in chronic hypoxic stress may result in irreversible structural change in DN. Diabetic nephropathy (DN) is the most important chronic kidney disease. We previously reported that Smad1 transcriptionally regulates the expression of extracellular matrix in DN. Phenotypic change in mesangial cells (MCs) is a key pathologic event in the progression of DN. The aim of this study is to investigate a novel mechanism underlying chondrogenic phenotypic change in MCs that results in the development of DN. MCs showed chondrogenic potential in a micromass culture, and BMP4 induced the expression of chondrocyte markers (SRY-related HMG Box 9 (SOX9) and type II collagen (COL2)). Advanced glycation end products induced the expression of chondrocyte marker proteins downstream from the BMP4-Smad1 signaling pathway in MCs. In addition, hypoxia also induced the expression of BMP4, hypoxia-inducible factor-1α (HIF-1α), and chondrocyte markers. Overexpression of SOX9 caused ectopic expression of proteoglycans and COL2 in MCs. Furthermore, forced expression of Smad1 induced chondrocyte markers as well. Dorsomorphin inhibited these inductions. Glomerular expressions of HIF-1α, BMP4, and chondrocyte markers were observed in diabetic nephropathy mice. These positive stainings were observed in mesangial sclerotic lesions. SOX9 was partially colocalized with HIF-1α and BMP4 in diabetic glomeruli. BMP4 knock-in transgenic mice showed not only similar pathological lesions to DN, but also the induction of chondrocyte markers in the sclerotic lesions. Here we demonstrate that HIF-1α and BMP4 induce SOX9 expression and subsequent chondrogenic phenotype change in DN. The results suggested that the transdifferentiation of MCs into chondrocyte-like cells in chronic hypoxic stress may result in irreversible structural change in DN. IntroductionDiabetic nephropathy (DN) 2The abbreviations used are: DNdiabetic nephropathyBMP4bone morphogenetic protein 4SOX9SRY-related HMG Box 9pSOX9phosphorylated SOX9COL2type II collagenAGEsadvanced glycation end-productsHIF-1αhypoxia-inducible factor-1αESKDend-stage kidney diseaseMCsmesangial cellsiNOSinducible nitric-oxide synthaseTgmtransgenic mice. is the most common and important chronic kidney disease and is the leading cause of end-stage kidney disease (ESKD) worldwide. The characteristic glomerular changes resulting from DN are thickening of the glomerular basement membrane (GBM) and expansion of the mesangial area caused by increased mesangial extracellular matrix (ECM). Mesangial cells (MCs) are the major source of ECM synthesis in diabetic glomeruli (1Lenz O. Striker L.J. Jacot T.A. Elliot S.J. Killen P.D. Striker G.E. J. Am. Soc. Nephrol. 1998; 9: 2040-2047PubMed Google Scholar, 2Lupia E. Elliot S.J. Lenz O. Zheng F. Hattori M. Striker G.E. Striker L.J. Diabetes. 1999; 48: 1638-1644Crossref PubMed Scopus (87) Google Scholar, 3Xia L. Wang H. Munk S. Kwan J. Goldberg H.J. Fantus I.G. Whiteside C.I. Am. J. Physiol. Renal Physiol. 2008; 295: F1705-F1714Crossref PubMed Scopus (62) Google Scholar). Hyperglycemia, advanced glycation end products (AGEs), and transforming growth factor-β (TGF-β) are implicated in the progression of DN by stimulating ECM production in MCs (4Abe H. Matsubara T. Iehara N. Nagai K. Takahashi T. Arai H. Kita T. Doi T. J. Biol. Chem. 2004; 279: 14201-14206Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 5Fornoni A. Striker L.J. Zheng F. Striker G.E. Diabetes. 2002; 51: 499-505Crossref PubMed Scopus (31) Google Scholar, 6Simonson M.S. Kidney Int. 2007; 71: 846-854Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). In addition, the phenotypic modulation and dedifferentiation of MCs, which are derived from mesenchymal cells, are key pathological events (7Makino H. Kashihara N. Sugiyama H. Kanao K. Sekikawa T. Shikata K. Nagai R. Ota Z. J. Diabetes Complications. 1995; 9: 282-284Crossref PubMed Scopus (24) Google Scholar, 8Johnson R.J. Kidney Int. 1994; 45: 1769-1782Abstract Full Text PDF PubMed Scopus (220) Google Scholar). MCs acquired phenotypes not only of activated smooth muscle cells but also of fibroblasts, and subsequently secrete excess ECM proteins.We previously reported that Smad1 is a direct transcriptional regulator of type IV collagen (COL4), a major component of increased mesangial ECM in DN, as well as other ECM proteins such as type I collagen (COL1), osteopontin, and α-smooth muscle actin (α-SMA) (4Abe H. Matsubara T. Iehara N. Nagai K. Takahashi T. Arai H. Kita T. Doi T. J. Biol. Chem. 2004; 279: 14201-14206Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 9Matsubara T. Abe H. Arai H. Nagai K. Mima A. Kanamori H. Sumi E. Takahashi T. Matsuura M. Iehara N. Fukatsu A. Kita T. Doi T. Lab. Invest. 2006; 86: 357-368Crossref PubMed Scopus (59) Google Scholar). The most critical feature of glomerulosclerosis is mesangial expansion, which is strongly correlated with decreased glomerular filtration rate (GFR) (10Mauer S.M. Steffes M.W. Ellis E.N. Sutherland D.E. Brown D.M. Goetz F.C. J. Clin. Invest. 1984; 74: 1143-1155Crossref PubMed Scopus (1056) Google Scholar). Most importantly, we found that the glomerular expression levels of Smad1 are clearly correlated with the severities of mesangial matrix expansion in rodent DN (9Matsubara T. Abe H. Arai H. Nagai K. Mima A. Kanamori H. Sumi E. Takahashi T. Matsuura M. Iehara N. Fukatsu A. Kita T. Doi T. Lab. Invest. 2006; 86: 357-368Crossref PubMed Scopus (59) Google Scholar, 11Mima A. Arai H. Matsubara T. Abe H. Nagai K. Tamura Y. Torikoshi K. Araki M. Kanamori H. Takahashi T. Tominaga T. Matsuura M. Iehara N. Fukatsu A. Kita T. Doi T. Diabetes. 2008; 57: 1712-1722Crossref PubMed Scopus (44) Google Scholar). Therefore, we have concluded that Smad1 plays a central role in the development of DN. However, we still lack mechanistic insight into how Smad1 is involved in phenotypic alteration of MCs and in the irreversible progression of DN. It is generally acknowledged that Smad1 transduces TGF-β signals and also transduces BMP signals through its receptors (12Chen W. Fu X. Sheng Z. Chin. Med. J. 2002; 115: 446-450PubMed Google Scholar, 13Massagué J. Blain S.W. Lo R.S. Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2052) Google Scholar). Although impairment of vascular wall integrity caused by mesangial matrix expansion leads to irreversible glomerulosclerosis, the molecular mechanisms underlying this irreversible change in the phenotypes of MCs in advanced DN are still unknown. We also demonstrated previously that a transcription factor, SRY-related HMG Box 9 (SOX9), is involved in the modulation of transcriptional enhancement of COL4 (14Sumi E. Iehara N. Akiyama H. Matsubara T. Mima A. Kanamori H. Fukatsu A. Salant D.J. Kita T. Arai H. Doi T. Am. J. Pathol. 2007; 170: 1854-1864Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), but we still do not know how this influences the phenotypic alteration of MCs.It is well recognized that SOX9 is a cartilage-specific transcription factor; that is, the expression of SOX9 is required for the commitment and differentiation of mesenchymal cells toward the chondrogenic lineage in all developing skeletal elements (15Akiyama H. Chaboissier M.C. Martin J.F. Schedl A. de Crombrugghe B. Genes Dev. 2002; 16: 2813-2828Crossref PubMed Scopus (1344) Google Scholar). Moreover, SOX9 is responsible for the deposition of ECM, which is composed predominantly of type II collagen (COL2) in chondrocytes (16Bi W. Deng J.M. Zhang Z. Behringer R.R. de Crombrugghe B. Nat. Genet. 1999; 22: 85-89Crossref PubMed Scopus (1376) Google Scholar). Precise regulation of both induction and silencing of SOX9 is required for the normal development of chondrocytes. Conversely, inappropriate or reactivated expression of SOX9 was reported to impair cell phenotype and promote ECM deposition in vascular diseases and fibrosis (17Farrington-Rock C. Crofts N.J. Doherty M.J. Ashton B.A. Griffin-Jones C. Canfield A.E. Circulation. 2004; 110: 2226-2232Crossref PubMed Scopus (371) Google Scholar, 18Hanley K.P. Oakley F. Sugden S. Wilson D.I. Mann D.A. Hanley N.A. J. Biol. Chem. 2008; 283: 14063-14071Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 19Tintut Y. Alfonso Z. Saini T. Radcliff K. Watson K. Boström K. Demer L.L. Circulation. 2003; 108: 2505-2510Crossref PubMed Scopus (307) Google Scholar, 20Chen J.H. Yip C.Y. Sone E.D. Simmons C.A. Am. J. Pathol. 2009; 174: 1109-1119Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Among the many signaling molecules involved in chondrogenesis, the biological significance of bone morphogenetic proteins (BMPs) has been well established by utilizing pluripotent C3H10T1/2 cells as a model for the initial events of mesenchymal chondrogenesis (21Ahrens M. Ankenbauer T. Schröder D. Hollnagel A. Mayer H. Gross G. DNA Cell Biol. 1993; 12: 871-880Crossref PubMed Scopus (309) Google Scholar).We hypothesized that BMP4 and SOX9 would be closely linked to the phenotypic alteration of MCs and to the irreversible progression of diabetic glomerulosclerosis. However, the regulatory mechanism in the interaction between BMP4-Smad1 signaling and SOX9 expression in MCs during the process of diabetic glomerular injuries has not been fully elucidated.Though hypoxia has been considered a pivotal contributor to tubular atrophy and interstitial fibrosis in the progression of kidney disease (22Manotham K. Tanaka T. Matsumoto M. Ohse T. Miyata T. Inagi R. Kurokawa K. Fujita T. M. J. Am. Soc. Nephrol. 2004; PubMed Scopus Google Scholar), that hypoxia or stress also MCs in kidney diseases H. Inagi R. H. M. D. Fujita T. M. J. Am. Soc. Nephrol. 2008; PubMed Scopus Google Scholar, G. C. B. M. S. K. A. M. Am. J. Pathol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, S. K. M. H.J. D. Am. J. Pathol. Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, in to hypoxia or stress and expression also be important the advanced of diabetic glomerulosclerosis. key in to hypoxic is hypoxia-inducible a transcription of a and that also HIF-1α expression in MCs L. Wang H. Munk S. H. Goldberg H.J. Fantus I.G. Whiteside C.I. Am. J. Physiol. 2007; PubMed Scopus Google Scholar). Therefore, HIF-1α also be important in the development of glomerulosclerosis. The chondrocyte is a cell a of and hypoxia and HIF-1α matrix D. T. E. R.S. J. Cell 2003; PubMed Scopus Google Scholar, T. E. R.S. B. D. 2004; 12: Full Text Full Text PDF PubMed Scopus Google and are in chondrocyte differentiation Y. M. Am. J. Physiol. Cell Physiol. 2006; PubMed Scopus Google Scholar, Y. M. E. 2007; PubMed Scopus Google Scholar). Furthermore, matrix such as and are not direct of but are by hypoxia through cartilage-specific transcription SOX9 R. A. R.S. E. 2007; PubMed Scopus Google these we hypothesized that MCs a chondrocyte-like phenotype that SOX9 during glomerulosclerosis We a novel mechanism of chondrogenic phenotype change of MCs in the developing a derived structural to the by ECM that the mesangial matrix D. J. PubMed Scopus Google Scholar). or cells have differentiation development the and In addition, phenotypic change in MCs is important pathological change in glomerular and is the of glomerulosclerosis. Although many have demonstrated that the of MCs to activated cells is a characteristic in this study we showed that MCs have chondrogenic potential the micromass that MCs have chondrogenic potential in DN. novel potential of is by the induction of a transcriptional of of have implicated SOX9 expression in diseases such as the of E. S. Kidney Int. 2007; Full Text Full Text PDF PubMed Scopus (87) Google Scholar), in lesions of M. H. M. Tanaka T. H. S. K. Genes PubMed Scopus Google Scholar), and in fibrosis K.P. Oakley F. Sugden S. Wilson D.I. Mann D.A. Hanley N.A. J. Biol. Chem. 2008; 283: 14063-14071Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), irreversible changes with ECM Therefore, the of the phenotype in MCs may be involved in the irreversible changes of diabetic to a of MCs are generally to into a of mesenchymal cells that of extracellular matrix proteins type I and type that are not in the mesangial matrix and the expression of proteins (4Abe H. Matsubara T. Iehara N. Nagai K. Takahashi T. Arai H. Kita T. Doi T. J. Biol. Chem. 2004; 279: 14201-14206Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, A. A. F. G. J. Cell Biol. 1993; PubMed Scopus Google Scholar, R.J. H. M.W. S.M. K. J. Clin. Invest. PubMed Scopus Google Scholar). We previously demonstrated that Smad1 transcriptionally regulates the expression of and Moreover, we have that SOX9 influences the transcriptional enhancement of COL4 (14Sumi E. Iehara N. Akiyama H. Matsubara T. Mima A. Kanamori H. Fukatsu A. Salant D.J. Kita T. Arai H. Doi T. Am. J. Pathol. 2007; 170: 1854-1864Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), but we still lack mechanistic insight into how SOX9 expression is in DN. Here we have demonstrated that the expression of SOX9 be induced by BMP4 in MCs. also induced SOX9 expression as well as BMP4 in MCs, that the signaling pathway is activated in MCs in chronic hypoxia of do not chondrocyte as these cells to have mechanisms to to this R. Z. J. Cell. Physiol. PubMed Scopus Google Scholar). is a that is required for the regulation of chondrocyte Although hypoxia has been considered a pivotal to tubular atrophy and interstitial fibrosis in the progression of kidney disease H. Inagi R. H. M. D. Fujita T. M. J. Am. Soc. Nephrol. 2008; PubMed Scopus Google Scholar), the role of hypoxia the progression of irreversible changes in diabetic glomeruli has to be In the we have that hypoxic stress as well as phenotypic alteration of MCs. In to hypoxia is to be important in the progression of diabetic kidney into a chondrogenic phenotype to to chronic hypoxia may be a critical event during the development of glomerulosclerosis. Although is for in the or muscle J.M. J. Clin. Invest. 2000; PubMed Scopus Google Scholar), similar do not in advanced diabetic glomerulosclerosis to the of glomerular in In matrix has a the cell lineage S. D.E. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), and the chondrogenic phenotypic change in MCs and subsequent chondrogenic ECM which are of be implicated in irreversible glomerular structural changes leading to of the production of of the DN is mesangial which is by of ECM in the mesangial leading to of the vascular and subsequent in its advanced Therefore, we mesangial expansion and lesions advanced of DN, the other diabetic model glomerular We the from a and of the mesangial and These changes are similar to DN. The responsible of this established model to be these glomerular lesions were by the signaling These is model for advanced of DN Y. Doi T. H. S. M. T. S. S. S. H. H. J. Clin. Invest. 108: PubMed Scopus Google Scholar, S. Abe H. Takahashi T. Y. M. Arai H. K. Kita T. H. H. Doi T. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Doi T. A. H. T. H. M. K. N. M. S. H. H. Diabetes. 2006; PubMed Scopus Google the glomerulosclerosis and showed ectopic induction of BMP4, and In BMP4 is a regulator in the process of chondrogenesis that the of and the expression of chondrogenic SOX9 K. Takahashi Takahashi K. R. L. Dev. 2000; PubMed Scopus Google Scholar, C. H. J. 1999; PubMed Scopus Google Scholar). Furthermore, we that BMP4 plays a central role in the development of DN. BMP4 showed pathological changes with the of diabetic mesangial matrix and also showed ectopic induction of SOX9 and the BMP pathway and SOX9 are required for the of chondrogenesis, that BMP4 and SOX9 are of phenotypic change of in the advanced of study that MCs have chondrogenic a mechanism of differentiation the induction of ectopic are the only cells found in and hypoxic mesenchymal S. 2003; PubMed Scopus Google Scholar). Therefore, that MCs the chondrogenic phenotype by ectopic SOX9 to a mechanism for chronic pathological hypoxic stress in the BMP4 and HIF-1α, which are to be molecules of have important influences the phenotypic change of into a chondrocyte-like cell in DN. differentiation mechanism of MCs be a key event in the development and progression of the irreversible of DN. IntroductionDiabetic nephropathy (DN) 2The abbreviations used are: DNdiabetic nephropathyBMP4bone morphogenetic protein 4SOX9SRY-related HMG Box 9pSOX9phosphorylated SOX9COL2type II collagenAGEsadvanced glycation end-productsHIF-1αhypoxia-inducible factor-1αESKDend-stage kidney diseaseMCsmesangial cellsiNOSinducible nitric-oxide synthaseTgmtransgenic mice. is the most common and important chronic kidney disease and is the leading cause of end-stage kidney disease (ESKD) worldwide. The characteristic glomerular changes resulting from DN are thickening of the glomerular basement membrane (GBM) and expansion of the mesangial area caused by increased mesangial extracellular matrix (ECM). Mesangial cells (MCs) are the major source of ECM synthesis in diabetic glomeruli (1Lenz O. Striker L.J. Jacot T.A. Elliot S.J. Killen P.D. Striker G.E. J. Am. Soc. Nephrol. 1998; 9: 2040-2047PubMed Google Scholar, 2Lupia E. Elliot S.J. Lenz O. Zheng F. Hattori M. Striker G.E. Striker L.J. Diabetes. 1999; 48: 1638-1644Crossref PubMed Scopus (87) Google Scholar, 3Xia L. Wang H. Munk S. Kwan J. Goldberg H.J. Fantus I.G. Whiteside C.I. Am. J. Physiol. Renal Physiol. 2008; 295: F1705-F1714Crossref PubMed Scopus (62) Google Scholar). Hyperglycemia, advanced glycation end products (AGEs), and transforming growth factor-β (TGF-β) are implicated in the progression of DN by stimulating ECM production in MCs (4Abe H. Matsubara T. Iehara N. Nagai K. Takahashi T. Arai H. Kita T. Doi T. J. Biol. Chem. 2004; 279: 14201-14206Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 5Fornoni A. Striker L.J. Zheng F. Striker G.E. Diabetes. 2002; 51: 499-505Crossref PubMed Scopus (31) Google Scholar, 6Simonson M.S. Kidney Int. 2007; 71: 846-854Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). In addition, the phenotypic modulation and dedifferentiation of MCs, which are derived from mesenchymal cells, are key pathological events (7Makino H. Kashihara N. Sugiyama H. Kanao K. Sekikawa T. Shikata K. Nagai R. Ota Z. J. Diabetes Complications. 1995; 9: 282-284Crossref PubMed Scopus (24) Google Scholar, 8Johnson R.J. Kidney Int. 1994; 45: 1769-1782Abstract Full Text PDF PubMed Scopus (220) Google Scholar). MCs acquired phenotypes not only of activated smooth muscle cells but also of fibroblasts, and subsequently secrete excess ECM proteins.We previously reported that Smad1 is a direct transcriptional regulator of type IV collagen (COL4), a major component of increased mesangial ECM in DN, as well as other ECM proteins such as type I collagen (COL1), osteopontin, and α-smooth muscle actin (α-SMA) (4Abe H. Matsubara T. Iehara N. Nagai K. Takahashi T. Arai H. Kita T. Doi T. J. Biol. Chem. 2004; 279: 14201-14206Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 9Matsubara T. Abe H. Arai H. Nagai K. Mima A. Kanamori H. Sumi E. Takahashi T. Matsuura M. Iehara N. Fukatsu A. Kita T. Doi T. Lab. Invest. 2006; 86: 357-368Crossref PubMed Scopus (59) Google Scholar). The most critical feature of glomerulosclerosis is mesangial expansion, which is strongly correlated with decreased glomerular filtration rate (GFR) (10Mauer S.M. Steffes M.W. Ellis E.N. Sutherland D.E. Brown D.M. Goetz F.C. J. Clin. Invest. 1984; 74: 1143-1155Crossref PubMed Scopus (1056) Google Scholar). Most importantly, we found that the glomerular expression levels of Smad1 are clearly correlated with the severities of mesangial matrix expansion in rodent DN (9Matsubara T. Abe H. Arai H. Nagai K. Mima A. Kanamori H. Sumi E. Takahashi T. Matsuura M. Iehara N. Fukatsu A. Kita T. Doi T. Lab. Invest. 2006; 86: 357-368Crossref PubMed Scopus (59) Google Scholar, 11Mima A. Arai H. Matsubara T. Abe H. Nagai K. Tamura Y. Torikoshi K. Araki M. Kanamori H. Takahashi T. Tominaga T. Matsuura M. Iehara N. Fukatsu A. Kita T. Doi T. Diabetes. 2008; 57: 1712-1722Crossref PubMed Scopus (44) Google Scholar). Therefore, we have concluded that Smad1 plays a central role in the development of DN. However, we still lack mechanistic insight into how Smad1 is involved in phenotypic alteration of MCs and in the irreversible progression of DN. It is generally acknowledged that Smad1 transduces TGF-β signals and also transduces BMP signals through its receptors (12Chen W. Fu X. Sheng Z. Chin. Med. J. 2002; 115: 446-450PubMed Google Scholar, 13Massagué J. Blain S.W. Lo R.S. Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2052) Google Scholar). Although impairment of vascular wall integrity caused by mesangial matrix expansion leads to irreversible glomerulosclerosis, the molecular mechanisms underlying this irreversible change in the phenotypes of MCs in advanced DN are still unknown. We also demonstrated previously that a transcription factor, SRY-related HMG Box 9 (SOX9), is involved in the modulation of transcriptional enhancement of COL4 (14Sumi E. Iehara N. Akiyama H. Matsubara T. Mima A. Kanamori H. Fukatsu A. Salant D.J. Kita T. Arai H. Doi T. Am. J. Pathol. 2007; 170: 1854-1864Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), but we still do not know how this influences the phenotypic alteration of MCs.It is well recognized that SOX9 is a cartilage-specific transcription factor; that is, the expression of SOX9 is required for the commitment and differentiation of mesenchymal cells toward the chondrogenic lineage in all developing skeletal elements (15Akiyama H. Chaboissier M.C. Martin J.F. Schedl A. de Crombrugghe B. Genes Dev. 2002; 16: 2813-2828Crossref PubMed Scopus (1344) Google Scholar). Moreover, SOX9 is responsible for the deposition of ECM, which is composed predominantly of type II collagen (COL2) in chondrocytes (16Bi W. Deng J.M. Zhang Z. Behringer R.R. de Crombrugghe B. Nat. Genet. 1999; 22: 85-89Crossref PubMed Scopus (1376) Google Scholar). Precise regulation of both induction and silencing of SOX9 is required for the normal development of chondrocytes. Conversely, inappropriate or reactivated expression of SOX9 was reported to impair cell phenotype and promote ECM deposition in vascular diseases and fibrosis (17Farrington-Rock C. Crofts N.J. Doherty M.J. Ashton B.A. Griffin-Jones C. Canfield A.E. Circulation. 2004; 110: 2226-2232Crossref PubMed Scopus (371) Google Scholar, 18Hanley K.P. Oakley F. Sugden S. Wilson D.I. Mann D.A. Hanley N.A. J. Biol. Chem. 2008; 283: 14063-14071Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 19Tintut Y. Alfonso Z. Saini T. Radcliff K. Watson K. Boström K. Demer L.L. Circulation. 2003; 108: 2505-2510Crossref PubMed Scopus (307) Google Scholar, 20Chen J.H. Yip C.Y. Sone E.D. Simmons C.A. Am. J. Pathol. 2009; 174: 1109-1119Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Among the many signaling molecules involved in chondrogenesis, the biological significance of bone morphogenetic proteins (BMPs) has been well established by utilizing pluripotent C3H10T1/2 cells as a model for the initial events of mesenchymal chondrogenesis (21Ahrens M. Ankenbauer T. Schröder D. Hollnagel A. Mayer H. Gross G. DNA Cell Biol. 1993; 12: 871-880Crossref PubMed Scopus (309) Google Scholar).We hypothesized that BMP4 and SOX9 would be closely linked to the phenotypic alteration of MCs and to the irreversible progression of diabetic glomerulosclerosis. However, the regulatory mechanism in the interaction between BMP4-Smad1 signaling and SOX9 expression in MCs during the process of diabetic glomerular injuries has not been fully elucidated.Though hypoxia has been considered a pivotal contributor to tubular atrophy and interstitial fibrosis in the progression of kidney disease (22Manotham K. Tanaka T. Matsumoto M. Ohse T. Miyata T. Inagi R. Kurokawa K. Fujita T. M. J. Am. Soc. Nephrol. 2004; PubMed Scopus Google Scholar), that hypoxia or stress also MCs in kidney diseases H. Inagi R. H. M. D. Fujita T. M. J. Am. Soc. Nephrol. 2008; PubMed Scopus Google Scholar, G. C. B. M. S. K. A. M. Am. J. Pathol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, S. K. M. H.J. D. Am. J. Pathol. Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, in to hypoxia or stress and expression also be important the advanced of diabetic glomerulosclerosis. key in to hypoxic is hypoxia-inducible a transcription of a and that also HIF-1α expression in MCs L. Wang H. Munk S. H. Goldberg H.J. Fantus I.G. Whiteside C.I. Am. J. Physiol. 2007; PubMed Scopus Google Scholar). Therefore, HIF-1α also be important in the development of glomerulosclerosis. The chondrocyte is a cell a of and hypoxia and HIF-1α matrix D. T. E. R.S. J. Cell 2003; PubMed Scopus Google Scholar, T. E. R.S. B. D. 2004; 12: Full Text Full Text PDF PubMed Scopus Google and are in chondrocyte differentiation Y. M. Am. J. Physiol. Cell Physiol. 2006; PubMed Scopus Google Scholar, Y. M. E. 2007; PubMed Scopus Google Scholar). Furthermore, matrix such as and are not direct of but are by hypoxia through cartilage-specific transcription SOX9 R. A. R.S. E. 2007; PubMed Scopus Google these we hypothesized that MCs a chondrocyte-like phenotype that SOX9 during glomerulosclerosis We a novel mechanism of chondrogenic phenotype change of MCs in the developing DN.