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miR-200a Prevents renal fibrogenesis through repression of TGF-β2 expression.

Wang B, Koh P, Winbanks C, Coughlan MT, McClelland A, Watson A, Jandeleit-Dahm K, Burns WC, Thomas MC, Cooper ME, Kantharidis P - Diabetes (2010)

Bottom Line: TGF-β1 and TGF-β2 also downregulated expression of miR-200a.The renal expression of miR-141 and miR-200a was also reduced in mouse models representing early and advanced kidney disease. miR-200a and miR-141 significantly impact on the development and progression of TGF-β-dependent EMT and fibrosis in vitro and in vivo.These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-β signaling and as components of feedback regulation, and as such represent important new targets for the prevention of progressive kidney disease in the context of diabetes.

View Article: PubMed Central - PubMed

Affiliation: JDRF Danielle Alberti Memorial Centre for Diabetes Complications, Melbourne, Australia.

ABSTRACT

Objective: Progressive fibrosis in the diabetic kidney is driven and sustained by a diverse range of profibrotic factors. This study examines the critical role of microRNAs (miRNAs) in the regulation of the key fibrotic mediators, TGF-β1 and TGF-β2.

Research design and methods: Rat proximal-tubular epithelial cells (NRK52E) were treated with TGF-β1 and TGF-β2 for 3 days, and expression of markers of epithelial-to-mesenchymal transition (EMT) and fibrogenesis were assessed by RT-PCR and Western blotting. The expression of miR-141 and miR-200a was also assessed, as was their role as translational repressors of TGF-β signaling. Finally, these pathways were explored in two different mouse models, representing early and advanced diabetic nephropathy.

Results: Both TGF-β1 and TGF-β2 induced EMT and fibrogenesis in NRK52E cells. TGF-β1 and TGF-β2 also downregulated expression of miR-200a. The importance of these changes was demonstrated by the finding that ectopic expression miR-200a downregulated smad-3 activity and the expression of matrix proteins and prevented TGF-β-dependent EMT. miR-200a also downregulated the expression of TGF-β2, via direct interaction with the 3' untranslated region of TGF-β2. The renal expression of miR-141 and miR-200a was also reduced in mouse models representing early and advanced kidney disease.

Conclusions: miR-200a and miR-141 significantly impact on the development and progression of TGF-β-dependent EMT and fibrosis in vitro and in vivo. These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-β signaling and as components of feedback regulation, and as such represent important new targets for the prevention of progressive kidney disease in the context of diabetes.

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TGF-β1-induced ECM gene and TGF-β2 expression changes in proximal tubular epithelial cells. A: NRK52E cells (Dulbecco's modified Eagle medium, 25 mmol/l glucose, 2% serum) were treated with TGF-β1 (10 ng/ml, 3 days), and the expression of several genes was assessed by real-time QPCR. Significant changes are indicated (*P < 0.05 compared with control). B: The change in expression of TGF-β1 and TGF-β2 genes was assessed by real-time QPCR, and significant changes are indicated (*P < 0.0005 compared with control). C: TGF-β2 proteins levels were measured by ELISA and expressed as pg/mg (*P < 0.0005 compared with control). D: NRK52E cells were incubated with either control IgG (1 μg/ml) or TGF-β2-specific neutralizing antibody (1 μg/ml) for 1 h, and then TGF-β1 was added (10 ng/ml). Cells were harvested 3 days later and subjected to real-time QPCR analysis. The TGF-β2 antibody prevented the increased expression of αSMA, Collagen I, and fibronectin that is induced by TGF-β1, compared with the IgG control antibody (*P < 0.05 compared with control). E: Cells were then treated with TGF-β1 (10 ng/ml, 3 days) in the presence or absence of SB432542, the TGF-β2 receptor inhibitor. Real-time QPCR analysis confirmed that the gene expression changes induced by TGF-β1 are attenuated by SB432542 (*P < 0.05 and #P < 0.001 compared with control).
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Figure 2: TGF-β1-induced ECM gene and TGF-β2 expression changes in proximal tubular epithelial cells. A: NRK52E cells (Dulbecco's modified Eagle medium, 25 mmol/l glucose, 2% serum) were treated with TGF-β1 (10 ng/ml, 3 days), and the expression of several genes was assessed by real-time QPCR. Significant changes are indicated (*P < 0.05 compared with control). B: The change in expression of TGF-β1 and TGF-β2 genes was assessed by real-time QPCR, and significant changes are indicated (*P < 0.0005 compared with control). C: TGF-β2 proteins levels were measured by ELISA and expressed as pg/mg (*P < 0.0005 compared with control). D: NRK52E cells were incubated with either control IgG (1 μg/ml) or TGF-β2-specific neutralizing antibody (1 μg/ml) for 1 h, and then TGF-β1 was added (10 ng/ml). Cells were harvested 3 days later and subjected to real-time QPCR analysis. The TGF-β2 antibody prevented the increased expression of αSMA, Collagen I, and fibronectin that is induced by TGF-β1, compared with the IgG control antibody (*P < 0.05 compared with control). E: Cells were then treated with TGF-β1 (10 ng/ml, 3 days) in the presence or absence of SB432542, the TGF-β2 receptor inhibitor. Real-time QPCR analysis confirmed that the gene expression changes induced by TGF-β1 are attenuated by SB432542 (*P < 0.05 and #P < 0.001 compared with control).

Mentions: Treatment of the NRK52E cells with TGF-β1 (10 ng/ml, 3 days) also resulted in EMT and associated changes in gene expression (Fig. 2A). TGF-β1 was also able to induce its own expression (Fig. 2B) and that of TGF-β2 at a gene (Fig. 2B) and protein level (Fig. 2C). To explore the functional role of TGF-β2 changes induced by TGF-β1, a neutralizing antibody specific for TGF-β2 was added to NRK52E cells followed by TGF-β1 treatment. This attenuated the induction of EMT and subsequent fibrogenesis associated with TGF-β1 treatment (Fig. 2D). EMT was also blocked by the TGF-β type 1 receptor antagonist SB431542 (Fig. 2E), as was the induction of TGF-β2.


miR-200a Prevents renal fibrogenesis through repression of TGF-β2 expression.

Wang B, Koh P, Winbanks C, Coughlan MT, McClelland A, Watson A, Jandeleit-Dahm K, Burns WC, Thomas MC, Cooper ME, Kantharidis P - Diabetes (2010)

TGF-β1-induced ECM gene and TGF-β2 expression changes in proximal tubular epithelial cells. A: NRK52E cells (Dulbecco's modified Eagle medium, 25 mmol/l glucose, 2% serum) were treated with TGF-β1 (10 ng/ml, 3 days), and the expression of several genes was assessed by real-time QPCR. Significant changes are indicated (*P < 0.05 compared with control). B: The change in expression of TGF-β1 and TGF-β2 genes was assessed by real-time QPCR, and significant changes are indicated (*P < 0.0005 compared with control). C: TGF-β2 proteins levels were measured by ELISA and expressed as pg/mg (*P < 0.0005 compared with control). D: NRK52E cells were incubated with either control IgG (1 μg/ml) or TGF-β2-specific neutralizing antibody (1 μg/ml) for 1 h, and then TGF-β1 was added (10 ng/ml). Cells were harvested 3 days later and subjected to real-time QPCR analysis. The TGF-β2 antibody prevented the increased expression of αSMA, Collagen I, and fibronectin that is induced by TGF-β1, compared with the IgG control antibody (*P < 0.05 compared with control). E: Cells were then treated with TGF-β1 (10 ng/ml, 3 days) in the presence or absence of SB432542, the TGF-β2 receptor inhibitor. Real-time QPCR analysis confirmed that the gene expression changes induced by TGF-β1 are attenuated by SB432542 (*P < 0.05 and #P < 0.001 compared with control).
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Figure 2: TGF-β1-induced ECM gene and TGF-β2 expression changes in proximal tubular epithelial cells. A: NRK52E cells (Dulbecco's modified Eagle medium, 25 mmol/l glucose, 2% serum) were treated with TGF-β1 (10 ng/ml, 3 days), and the expression of several genes was assessed by real-time QPCR. Significant changes are indicated (*P < 0.05 compared with control). B: The change in expression of TGF-β1 and TGF-β2 genes was assessed by real-time QPCR, and significant changes are indicated (*P < 0.0005 compared with control). C: TGF-β2 proteins levels were measured by ELISA and expressed as pg/mg (*P < 0.0005 compared with control). D: NRK52E cells were incubated with either control IgG (1 μg/ml) or TGF-β2-specific neutralizing antibody (1 μg/ml) for 1 h, and then TGF-β1 was added (10 ng/ml). Cells were harvested 3 days later and subjected to real-time QPCR analysis. The TGF-β2 antibody prevented the increased expression of αSMA, Collagen I, and fibronectin that is induced by TGF-β1, compared with the IgG control antibody (*P < 0.05 compared with control). E: Cells were then treated with TGF-β1 (10 ng/ml, 3 days) in the presence or absence of SB432542, the TGF-β2 receptor inhibitor. Real-time QPCR analysis confirmed that the gene expression changes induced by TGF-β1 are attenuated by SB432542 (*P < 0.05 and #P < 0.001 compared with control).
Mentions: Treatment of the NRK52E cells with TGF-β1 (10 ng/ml, 3 days) also resulted in EMT and associated changes in gene expression (Fig. 2A). TGF-β1 was also able to induce its own expression (Fig. 2B) and that of TGF-β2 at a gene (Fig. 2B) and protein level (Fig. 2C). To explore the functional role of TGF-β2 changes induced by TGF-β1, a neutralizing antibody specific for TGF-β2 was added to NRK52E cells followed by TGF-β1 treatment. This attenuated the induction of EMT and subsequent fibrogenesis associated with TGF-β1 treatment (Fig. 2D). EMT was also blocked by the TGF-β type 1 receptor antagonist SB431542 (Fig. 2E), as was the induction of TGF-β2.

Bottom Line: TGF-β1 and TGF-β2 also downregulated expression of miR-200a.The renal expression of miR-141 and miR-200a was also reduced in mouse models representing early and advanced kidney disease. miR-200a and miR-141 significantly impact on the development and progression of TGF-β-dependent EMT and fibrosis in vitro and in vivo.These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-β signaling and as components of feedback regulation, and as such represent important new targets for the prevention of progressive kidney disease in the context of diabetes.

View Article: PubMed Central - PubMed

Affiliation: JDRF Danielle Alberti Memorial Centre for Diabetes Complications, Melbourne, Australia.

ABSTRACT

Objective: Progressive fibrosis in the diabetic kidney is driven and sustained by a diverse range of profibrotic factors. This study examines the critical role of microRNAs (miRNAs) in the regulation of the key fibrotic mediators, TGF-β1 and TGF-β2.

Research design and methods: Rat proximal-tubular epithelial cells (NRK52E) were treated with TGF-β1 and TGF-β2 for 3 days, and expression of markers of epithelial-to-mesenchymal transition (EMT) and fibrogenesis were assessed by RT-PCR and Western blotting. The expression of miR-141 and miR-200a was also assessed, as was their role as translational repressors of TGF-β signaling. Finally, these pathways were explored in two different mouse models, representing early and advanced diabetic nephropathy.

Results: Both TGF-β1 and TGF-β2 induced EMT and fibrogenesis in NRK52E cells. TGF-β1 and TGF-β2 also downregulated expression of miR-200a. The importance of these changes was demonstrated by the finding that ectopic expression miR-200a downregulated smad-3 activity and the expression of matrix proteins and prevented TGF-β-dependent EMT. miR-200a also downregulated the expression of TGF-β2, via direct interaction with the 3' untranslated region of TGF-β2. The renal expression of miR-141 and miR-200a was also reduced in mouse models representing early and advanced kidney disease.

Conclusions: miR-200a and miR-141 significantly impact on the development and progression of TGF-β-dependent EMT and fibrosis in vitro and in vivo. These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-β signaling and as components of feedback regulation, and as such represent important new targets for the prevention of progressive kidney disease in the context of diabetes.

Show MeSH
Related in: MedlinePlus