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Response gene to complement 32 interacts with Smad3 to promote epithelial-mesenchymal transition of human renal tubular cells.

ABSTRACT: Previous studies demonstrate that response gene to complement 32 (RGC-32) mediates transforming growth factor-?(1)-induced epithelial-mesenchymal transition (EMT) of human renal proximal tubular cells. However, the mechanisms underlying RGC-32 function remain largely unknown. In the present study, we found that RGC-32 function in EMT is associated with Smad3. Coexpression of RGC-32 and Smad3, but not Smad2, induces a higher mesenchymal marker ?-smooth muscle actin (?-SMA) protein expression as compared with RGC-32 or Smad3 alone, while knockdown of Smad3 using short hairpin interfering RNA blocks RGC-32-induced ?-SMA expression. These data suggest that RGC-32 interacts with Smad3, but not Smad2, in the regulation of EMT. In addition to ?-SMA, RGC-32 and Smad3 also synergistically activate the expression of extracellular matrix protein fibronectin and downregulate the epithelial marker E-cadherin. RGC-32 colocalizes with Smad3 in the nuclei of renal proximal tubular cells. Coimmunoprecipitation assays showed that Smad3, but not Smad2, physically interacts with RGC-32 in renal proximal tubular cells. Mechanistically, RGC-32 and Smad3 coordinate the induction of EMT by regulating the EMT regulators Slug and Snail. Taken together, our data demonstrate for the first time that RGC-32 interacts with Smad3 to mediate the EMT of human renal proximal tubular cells.

SUBMITTER: Guo X

PROVIDER: S-EPMC3118632 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Response gene to complement 32 interacts with Smad3 to promote epithelial-mesenchymal transition of human renal tubular cells.

Guo Xia X Jose Pedro A PA Chen Shi-You SY

American journal of physiology. Cell physiology 20110209 6

Previous studies demonstrate that response gene to complement 32 (RGC-32) mediates transforming growth factor-β(1)-induced epithelial-mesenchymal transition (EMT) of human renal proximal tubular cells. However, the mechanisms underlying RGC-32 function remain largely unknown. In the present study, we found that RGC-32 function in EMT is associated with Smad3. Coexpression of RGC-32 and Smad3, but not Smad2, induces a higher mesenchymal marker α-smooth muscle actin (α-SMA) protein expression as c ...[more]

PMID: 21307346

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Project description:Epithelial-mesenchymal transition (EMT) occurs in several disease states, including renal fibrosis and carcinogenesis. Myofibroblasts produced from EMT of renal tubular cells are responsible for the deposition of extracellular matrix components in a large portion of renal interstitial fibrosis. Transforming growth factor-beta (TGF-beta) plays an essential role in the EMT of renal tubular cells, but the molecular mechanism governing this process remains largely unknown. In this study, we found that RGC-32 (response gene to complement 32) is critical for TGF-beta-induced EMT of human renal proximal tubular cells (HPTCs). RGC-32 is not normally expressed in the HPTCs. However, TGF-beta stimulation markedly activates RGC-32 while inducing an EMT, as shown by the induction of smooth muscle alpha-actin (alpha-SMA) and extracellular matrix proteins collagen I and fibronectin, as well as the reduction of epithelial marker E-cadherin. TGF-beta function is mediated by several signaling pathways, but RGC-32 expression in HPTCs appears to be mainly regulated by Smad. Functionally, RGC-32 appears to mediate TGF-beta-induced EMT of HPTCs. Blockage of RGC-32 using short hairpin interfering RNA significantly inhibits TGF-beta induction of myofibroblast marker gene alpha-SMA while repressing the expression of E-cadherin. In contrast, overexpression of RGC-32 induces alpha-SMA expression while restoring E-cadherin. RGC-32 also inhibits the expression of another adherens junction protein, N-cadherin, suggesting that RGC-32 alone induces the phenotypic conversion of renal epithelial cells to myofibroblasts. Additional studies show that RGC-32 stimulates the production of extracellular matrix components fibronectin and collagen I. Mechanistically, RGC-32 induces EMT via the activation of other transcription factors such as Snail and Slug. RGC-32 knockdown inhibits the expression of Snail and Slug during TGF-beta-induced EMT. Taken together, our data demonstrate for the first time that RGC-32 plays a critical role in TGF-beta-induced EMT of renal tubular cells.

Project description:BackgroundOur previous study indicated that Ginkgo biloba leaf extract (EGb) could protect against cisplatin-induced acute kidney injury in rabbits. The present study aimed to determine the effects and potential molecular mechanisms of EGb on chronic renal interstitial fibrosis induced by cisplatin using in vivo and in vitro models.MethodsRats received a single dose of cisplatin on Day 1, and a subset of rats was intraperitoneally injected with EGb daily between Days 22-40. In vitro, HK-2 cells were treated with cisplatin, and a subset of cells was cultivated with EGb or SIS3 (Smad3 inhibitor) for 48 h. Renal function of rats was assessed by detecting the levels of serum creatinine (Scr), blood urea nitrogen (BUN) and urinary N-acetyl-β-D-glucosaminidase (NAG). Hematoxylin and eosin staining and Masson's trichrome staining were used to evaluate the damage and fibrosis of renal tissue. Western blotting, immunohistochemistry and immunofluorescence were used to detect the protein levels of fibrosis-associated proteins and signaling pathway-related proteins. RT-qPCR analysis was used to examine the mRNA levels of related indicators.ResultsEGb significantly decreased the increased levels of Scr, BUN and urinary NAG and attenuated renal damage and the relative area of renal interstitial fibrosis induced by cisplatin. Additionally, EGb decreased the protein levels of α-SMA, Col I, TGF-β1, smad2/3, phosphorylated (p)-smad2/3, p38 MAPK, and p-p38 MAPK; the ratio of p-p38 MAPK/p38 MAPK; and the mRNA level of p38 MAPK in renal tissues induced by cisplatin. In agreement with in vivo studies, EGb significantly reduced the increased protein levels of these indicators. Additionally, EGb significantly reduced the increased protein levels of vimentin, TIMP-1, and CTGF, as well as the mRNA levels of α-SMA, vimentin, and TGF-β1, while it significantly increased the reduced E-cadherin protein level and the MMP-1/TIMP-1 ratio in HK-2 cells induced by cisplatin. It's worth noting that the effects of SIS3 in changing the above indicators were similar to those of EGb.ConclusionOur study demonstrated that EGb improved cisplatin-induced chronic renal interstitial fibrosis, and its mechanisms were associated with inhibiting the epithelial-mesenchymal transition of renal tubular epithelial cells via the Smad3/TGF-β1 and Smad3/p38 MAPK pathways.

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Response gene to complement 32 interacts with Smad3 to promote epithelial-mesenchymal transition of human renal tubular cells.

Publications

Response gene to complement 32 interacts with Smad3 to promote epithelial-mesenchymal transition of human renal tubular cells.

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