TY - JOUR
T1 - Smad pathway is activated in the diabetic mouse kidney and smad3 mediates TGF-β-induced fibronectin in mesangial cells
AU - Isono, Motohide
AU - Chen, Sheldon
AU - Won Hong, Soon
AU - Carmen Iglesias-de la Cruz, M.
AU - Ziyadeh, Fuad N.
N1 - Funding Information:
We thank Drs. Harvey F. Lodish and Xuedong Liu for pEXL-Flag-Smad3 and pEXL-Flag-Smad3D, Dr. Joan Massagué for 3TP-Lux, Dr. Bert Vogelstein for SBE4-Luc, Dr. Peter ten Dijke for pFlag-Smad7, and Dr. Byung-Heon Lee for pGL2F1900. We also thank Dr. Jia Guo for her excellent technical assistance. This study was supported in part by the Juvenile Diabetes Foundation International (F.N.Z., M.I., and S.C.) and the National Institutes of Health (Grants DK-44513, DK-45191, and DK-54608 to F.N.Z.; training Grant DK-07006 and Individual National Research Service Award DK-09993 to S.C.). S.W.H. is a visiting scholar at the University of Pennsylvania and is supported by Yonsei University, Seoul, Korea. M.C. Iglesias-de la Cruz is a postdoctoral fellow at the University of Pennsylvania and is supported by the Ministerio de Educación y Cultura of Spain.
PY - 2002
Y1 - 2002
N2 - Activation of the transforming growth factor-β (TGF-β) system has been implicated in the pathological changes of diabetic nephropathy such as renal hypertrophy and accumulation of extracellular matrix. Streptozotocin-induced diabetic mice were used to examine whether the Smad pathway, which transduces the TGF-β signal, is activated in the diabetic kidney, employing Southwestern histochemistry with labeled Smad-binding element (SBE) oligonucleotides and immunoblotting of nuclear protein extracts for Smad3. Mouse mesangial cells were used to study the role of Smads in mediating the effects of high glucose and TGF-β on fibronectin expression, using transient transfections of Smad expression vectors and TGF-β-responsive reporter assays. By Southwestern histochemistry, the binding of nuclear proteins to labeled SBE increased in both glomeruli and tubules at 1, 3, and 6 weeks of diabetes. Likewise, immunoblotting demonstrated that nuclear accumulation of Smad3 was increased in the kidney of diabetic mice. Both increases were prevented by insulin treatment. In mesangial cells, high glucose potentiated the effect of low-dose TGF-β1 (0.2 ng/ml) on the following TGF-β-responsive constructs: 3TP-Lux (containing AP-1 sites and PAI-1 promoter), SBE4-Luc (containing four tandem repeats of SBE sequence), and the fibronectin promoter. Additionally, Smad3 overexpression increased fibronectin promoter activity, an effect that was enhanced by high ambient glucose or treatment with TGF-β1 (2 ng/ml). The TGF-β-stimulated activity of the fibronectin promoter was prevented by transfection with either a dominant-negative Smad3 or the inhibitory Smad7. We conclude that hyperglycemia activates the intrarenal TGF-β/Smad signaling pathway, which then promotes mesangial matrix gene expression in diabetic nephropathy.
AB - Activation of the transforming growth factor-β (TGF-β) system has been implicated in the pathological changes of diabetic nephropathy such as renal hypertrophy and accumulation of extracellular matrix. Streptozotocin-induced diabetic mice were used to examine whether the Smad pathway, which transduces the TGF-β signal, is activated in the diabetic kidney, employing Southwestern histochemistry with labeled Smad-binding element (SBE) oligonucleotides and immunoblotting of nuclear protein extracts for Smad3. Mouse mesangial cells were used to study the role of Smads in mediating the effects of high glucose and TGF-β on fibronectin expression, using transient transfections of Smad expression vectors and TGF-β-responsive reporter assays. By Southwestern histochemistry, the binding of nuclear proteins to labeled SBE increased in both glomeruli and tubules at 1, 3, and 6 weeks of diabetes. Likewise, immunoblotting demonstrated that nuclear accumulation of Smad3 was increased in the kidney of diabetic mice. Both increases were prevented by insulin treatment. In mesangial cells, high glucose potentiated the effect of low-dose TGF-β1 (0.2 ng/ml) on the following TGF-β-responsive constructs: 3TP-Lux (containing AP-1 sites and PAI-1 promoter), SBE4-Luc (containing four tandem repeats of SBE sequence), and the fibronectin promoter. Additionally, Smad3 overexpression increased fibronectin promoter activity, an effect that was enhanced by high ambient glucose or treatment with TGF-β1 (2 ng/ml). The TGF-β-stimulated activity of the fibronectin promoter was prevented by transfection with either a dominant-negative Smad3 or the inhibitory Smad7. We conclude that hyperglycemia activates the intrarenal TGF-β/Smad signaling pathway, which then promotes mesangial matrix gene expression in diabetic nephropathy.
KW - Diabetic nephropathies
KW - Extracellular matrix
KW - Kidney glomerulus
KW - Smad7
KW - Streptozotocin
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U2 - 10.1016/S0006-291X(02)02084-3
DO - 10.1016/S0006-291X(02)02084-3
M3 - Article
C2 - 12207925
AN - SCOPUS:0036387022
SN - 0006-291X
VL - 296
SP - 1356
EP - 1365
JO - Biochemical and biophysical research communications
JF - Biochemical and biophysical research communications
IS - 5
ER -