TGF-β1 induces the dissolution of tight junctions in human renal proximal tubular cells: Role of the RhoA/ROCK signaling pathway

Zhang,Ke; Zhang,Hao; Xiang,Hui; Liu,Jishi; Liu,Yan; Zhang,Xianming; Wang,Jianwen; Tang,Youzhou

doi:10.3892/ijmm.2013.1396

TGF-β1 induces the dissolution of tight junctions in human renal proximal tubular cells: Role of the RhoA/ROCK signaling pathway

Authors:
- Ke Zhang
- Hao Zhang
- Hui Xiang
- Jishi Liu
- Yan Liu
- Xianming Zhang
- Jianwen Wang
- Youzhou Tang
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Affiliations: Department of Nephrology, The Third Xiangya Hospital, Central South University, Changsha, Hunan 410013, P.R. China
Published online on: May 29, 2013 https://doi.org/10.3892/ijmm.2013.1396
Pages: 464-468

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Abstract

The RhoA/ROCK signaling pathway plays a significant role in transforming growth factor (TGF)-β1‑mediated epithelial-mesenchymal transition (EMT). It remains unclear, however, whether the RhoA/ROCK signaling pathway mediates TGF-β1-induced EMT by promoting the dissolution of tight junctions (TJs) in renal proximal tubular epithelial cells. In this study, we aimed to investigate the association between TGF-β1-mediated Rho/ROCK signaling and TJs in a cell line derived from human renal proximal tubular cells (HK-2 cells). HK-2 cells were treated with 5 ng/ml TGF-β1 for 0, 12, 24 and 48 h. Zona occludens protein 1 (also known as tight junction protein 1; ZO-1) and occludin mRNA and protein levels were determined by real-time PCR and western blot analysis, respectively. The HK-2 cells were then divided into three groups: a control group (serum-free culture medium for 24 h); a TGF-β1 group (treated with 5 ng/ml TGF-β1 for 24 h); and a TGF-β1 + Y-27632 (a specific ROCK inhibitor) group (pre-treated with 10 µM Y-27632 for 2 h and subsequently treated with 5 ng/ml TGF-β1 for 24 h). The levels of ZO-1 and occludin were detected by real-time PCR, western blot analysis and immunofluorescence. As shown by our results, the mRNA and protein levels of ZO-1 and occludin were decreased in the HK-2 cells following treatment with TGF-β1 in a time-dependent manner; in addition, ZO-1 and occludin levels in the TGF-β1 + Y-27632 group were significantly increased compared with those of the TGF-β1 group (P<0.05), with no significant changes compared with the control group. Our results indicate that the Rho/ROCK signaling pathway mediated by TGF-β1 plays a role in the dissolution of TJs during EMT.

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1	Rastaldi MP: Epithelial-mesenchymal transition and its implications for the development of renal tubulointerstitial fibrosis. J Nephrol. 19:407–412. 2006.PubMed/NCBI
2	Li MX and Liu BC: Epithelial to mesenchymal transition in the progression of tubulointerstitial fibrosis. Chinese Med J. 120:1925–1930. 2007.PubMed/NCBI
3	Liu Y: Cellular and molecular mechanisms of renal fibrosis. Nat Rev. 7:684–696. 2011.PubMed/NCBI
4	Blanco D, Vicent S, Elizegi E, Pino I, Fraga MF, Esteller M, Saffiotti U, Lecanda F and Montuenga LM: Altered expression of adhesion molecules and epithelial-mesenchymal transition in silica-induced rat lung carcinogenesis. Lab Invest. 84:999–1012. 2004. View Article : Google Scholar : PubMed/NCBI
5	Ozdamar B, Bose R, Barrios-Rodiles M, Wang HR, Zhang Y and Wrana JL: Regulation of the polarity protein Par6 by TGFbeta receptors controls epithelial cell plasticity. Science. 307:1603–1609. 2005. View Article : Google Scholar : PubMed/NCBI
6	Kolosova I, Nethery D and Kern JA: Role of Smad2/3 and p38 MAP kinase in TGF-beta1-induced epithelial-mesenchymal transition of pulmonary epithelial cells. J Cell Physiol. 226:1248–1254. 2011. View Article : Google Scholar : PubMed/NCBI
7	Lampropoulos P, Zizi-Sermpetzoglou A, Rizos S, Kostakis A, Nikiteas N and Papavassiliou AG: TGF-beta signalling in colon carcinogenesis. Cancer Lett. 314:1–7. 2011. View Article : Google Scholar
8	Lv ZM, Wang Q, Wan Q, Lin JG, Hu MS, Liu YX and Wang R: The role of the p38 MAPK signaling pathway in high glucose-induced epithelial-mesenchymal transition of cultured human renal tubular epithelial cells. PloS One. 6:e228062011. View Article : Google Scholar : PubMed/NCBI
9	Matsuzaki K: Smad phosphoisoform signaling specificity: the right place at the right time. Carcinogenesis. 32:1578–1588. 2011. View Article : Google Scholar : PubMed/NCBI
10	Schneider D and Janshoff A: Inhibition of actin dynamics during epithelial-to-mesenchymal transition. Biochem Biophys Res Commun. 419:221–225. 2012. View Article : Google Scholar : PubMed/NCBI
11	Tian YC, Fraser D, Attisano L and Phillips AO: TGF-beta1-mediated alterations of renal proximal tubular epithelial cell phenotype. Am J Physiol. 285:F130–F142. 2003.PubMed/NCBI
12	Zhang H, Liu X, Liu Y, Yi B and Yu X: Epithelial-mesenchymal transition of rat peritoneal mesothelial cells via Rhoa/Rock pathway. In Vitro Cell Dev Biol. 47:165–172. 2010. View Article : Google Scholar : PubMed/NCBI
13	Zhang H, Liu FY and Liu YH: Effect of TGF-beta1 stimulation on the smad signal transduction pathway of human peritoneal mesothelial cells. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 29:142–147. 2004.(In Chinese).
14	Ryan MJ, Johnson G, Kirk J, Fuerstenberg SM, Zager RA and Torok-Storb B: HK-2: an immortalized proximal tubule epithelial cell line from normal adult human kidney. Kidney Int. 45:48–57. 1994. View Article : Google Scholar : PubMed/NCBI
15	Black D, Lyman S, Qian T, Lemasters JJ, Rippe RA, Nitta T, Kim JS and Behrns KE: Transforming growth factor beta mediates hepatocyte apoptosis through Smad3 generation of reactive oxygen species. Biochimie. 89:1464–1473. 2007. View Article : Google Scholar : PubMed/NCBI
16	Sun L, Xiao L, Nie J, Liu FY, Ling GH, Zhu XJ, Tang WB, Chen WC, Xia YC, Zhan M, Ma MM, Peng YM, Liu H, Liu YH and Kanwar YS: p66Shc mediates high-glucose and angiotensin II-induced oxidative stress renal tubular injury via mitochondrial-dependent apoptotic pathway. Am J Physiol. 299:F1014–F1025. 2010.PubMed/NCBI
17	Ronaldson PT, Demarco KM, Sanchez-Covarrubias L, Solinsky CM and Davis TP: Transforming growth factor-beta signaling alters substrate permeability and tight junction protein expression at the blood-brain barrier during inflammatory pain. J Cereb Blood Flow Metab. 29:1084–1098. 2009. View Article : Google Scholar
18	Inumaru J, Nagano O, Takahashi E, Ishimoto T, Nakamura S, Suzuki Y, Niwa S, Umezawa K, Tanihara H and Saya H: Molecular mechanisms regulating dissociation of cell-cell junction of epithelial cells by oxidative stress. Genes Cells. 14:703–716. 2009. View Article : Google Scholar : PubMed/NCBI
19	Deroanne CF, Hamelryckx D, Ho TT, Lambert CA, Catroux P, Lapiere CM and Nusgens BV: Cdc42 downregulates MMP-1 expression by inhibiting the ERK1/2 pathway. J Cell Sci. 118:1173–1183. 2005. View Article : Google Scholar : PubMed/NCBI
20	Dovas A, Choi Y, Yoneda A, Multhaupt HA, Kwon SH, Kang D, Oh ES and Couchman JR: Serine 34 phosphorylation of rho guanine dissociation inhibitor (RhoGDIalpha) links signaling from conventional protein kinase C to RhoGTPase in cell adhesion. J Biol Chem. 285:23296–23308. 2010. View Article : Google Scholar : PubMed/NCBI
21	Leung TH, Ching YP, Yam JW, Wong CM, Yau TO, Jin DY and Ng IO: Deleted in liver cancer 2 (DLC2) suppresses cell transformation by means of inhibition of RhoA activity. Proc Natl Acad Sci USA. 102:15207–15212. 2005. View Article : Google Scholar : PubMed/NCBI
22	Edlund S, Landstrom M, Heldin CH and Aspenstrom P: Transforming growth factor-beta-induced mobilization of actin cytoskeleton requires signaling by small GTPases Cdc42 and RhoA. Mol Biol Cell. 13:902–914. 2002. View Article : Google Scholar : PubMed/NCBI
23	Lee HT and Kay EP: FGF-2 induced reorganization and disruption of actin cytoskeleton through PI 3-kinase, Rho, and Cdc42 in corneal endothelial cells. Mol Vision. 9:624–634. 2003.PubMed/NCBI
24	Patel S, Takagi KI, Suzuki J, Imaizumi A, Kimura T, Mason RM, Kamimura T and Zhang Z: RhoGTPase activation is a key step in renal epithelial mesenchymal transdifferentiation. J Am Soc Nephrol. 16:1977–1984. 2005. View Article : Google Scholar : PubMed/NCBI
25	Ruiz-Loredo AY, Lopez E and Lopez-Colome AM: Thrombin promotes actin stress fiber formation in RPE through Rho/ROCK-mediated MLC phosphorylation. J Cell Physiol. 226:414–423. 2011. View Article : Google Scholar : PubMed/NCBI
26	Lapointe TK, O’Connor PM, Jones NL, Menard D and Buret AG: Interleukin-1 receptor phosphorylation activates Rho kinase to disrupt human gastric tight junctional claudin-4 during Helicobacter pylori infection. Cell Microbiol. 12:692–703. 2010. View Article : Google Scholar : PubMed/NCBI
27	Yin J and Yu FS: Rho kinases regulate corneal epithelial wound healing. Am J Physiol. 295:C378–C387. 2008. View Article : Google Scholar : PubMed/NCBI
28	Bruewer M, Hopkins AM, Hobert ME, Nusrat A and Madara JL: RhoA, Rac1, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin. Am J Physiol. 287:C327–C335. 2004. View Article : Google Scholar : PubMed/NCBI