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TGR5 suppresses high glucose-induced upregulation of fibronectin and transforming growth factor-β1 in rat glomerular mesangial cells by inhibiting RhoA/ROCK signaling

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Abstract

RhoA/ROCK can cause renal inflammation and fibrosis in the context of diabetes by activating nuclear factor-κB (NF-κB). TGR5 is known for its role in maintaining metabolic homeostasis and anti-inflammation, which is closely related to NF-κB inhibition. Given that TGR5 is highly enriched in kidney, we aim to investigate the regulatory role of TGR5 on fibronectin (FN) and transforming growth factor-β1 (TGF-β1) in high glucose (HG)-treated rat glomerular mesangial cells (GMCs). Both the factors are closely related to renal inflammations and mediated by NF-κB. Moreover, our study determines whether such regulation is achieved by the inhibition of RhoA/ROCK and the subsequent NF-κB suppression. Polymerase chain reaction was taken to test the mRNA level of TGR5. Western blot was used to measure the protein expressions of TGR5, FN, TGF-β1, p65, IκBα, phospho-MYPT1 (Thr853), and MYPT1. Glutathione S-transferase-pull down and immunofluorescence were conducted to test the activation of RhoA, the distribution of TGR5, and p65, respectively. Electrophoretic mobility shift assay was adopted to measure the DNA binding activity of NF-κB. In GMCs, TGR5 activation or overexpression significantly suppressed FN and TGF-β1 protein expressions, NF-κB, and RhoA/ROCK activation induced by HG or transfection of constitutively active RhoA. By contrast, TGR5 RNA interference caused enhancement of FN, TGF-β1 protein expressions, increase of RhoA/ROCK activation. However, TGR5 cannot suppress RhoA/ROCK activation when a selective Protein kinase A (PKA) inhibitor was used. This study suggests that in HG-treated GMCs, TGR5 significantly suppresses the NF-κB-mediated upregulation of FN and TGF-β1, which are hallmarks of diabetic nephropathy. These functions are closely related to the suppression of RhoA/ROCK via PKA.

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References

  1. Y.S. Kanwar, L. Sun, P. Xie, F.Y. Liu, S. Chen, A glimpse of various pathogenetic mechanisms of diabetic nephropathy. Annu. Rev. Pathol. 6, 395–423 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. C. Ponchiardi, M. Mauer, B. Najafian, Temporal profile of diabetic nephropathy pathologic changes. Curr. Diab. Rep. 13(4), 592–599 (2013)

    Article  CAS  PubMed  Google Scholar 

  3. A. Cove-Smith, H. BM, The regulation of mesangial cell proliferation. Nephron. Exp. Nephrol. 108(4), e74–e79 (2008)

    Article  PubMed  Google Scholar 

  4. H.E. Abboud, Mesangial cell biology. Exp. Cell Res. 318(9), 979–985 (2012)

    Article  CAS  PubMed  Google Scholar 

  5. J.F. Navarro-Gonzalez, C. Mora-Fernandez, M. Muros de Fuentes, J. Garcia-Perez, Inflammatory molecules and pathways in the pathogenesis of diabetic nephropathy. Nat. Rev. Nephrol. 7(6), 327–340 (2011)

    Article  CAS  PubMed  Google Scholar 

  6. K.I. Woroniecka, A.S. Park, D. Mohtat, D.B. Thomas, J.M. Pullman, K. Susztak, Transcriptome analysis of human diabetic kidney disease. Diabetes 60(9), 2354–2369 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. I.A. Bondar’, V.V. Klimontov, A.P. Nadeev, Urinary excretion of proinflammatory cytokines and transforming growth factor beta at early stages of diabetic nephropathy. Ter. Arkh. 80(1), 52–56 (2008)

    PubMed  Google Scholar 

  8. A.P. Sanchez, K. Sharma, Transcription factors in the pathogenesis of diabetic nephropathy. Expert Rev. Mol. Med. 11, e13 (2009)

    Article  PubMed  Google Scholar 

  9. H. Schmid, A. Boucherot, Y. Yasuda, A. Henger, B. Brunner, F. Eichinger, A. Nitsche, E. Kiss, M. Bleich, H.J. Grone, P.J. Nelson, D. Schlondorff, C.D. Cohen, M. Kretzler; European Renal c, D.N.A.B.C., Modular activation of nuclear factor-kappaB transcriptional programs in human diabetic nephropathy. Diabetes 55(11), 2993–3003 (2006)

    Article  CAS  PubMed  Google Scholar 

  10. S. Mezzano, C. Aros, A. Droguett, M.E. Burgos, L. Ardiles, C. Flores, H. Schneider, M. Ruiz-Ortega, J. Egido, NF-kappaB activation and overexpression of regulated genes in human diabetic nephropathy. Nephrol. Dial. Transplant. 19(10), 2505–2512 (2004)

    Article  CAS  PubMed  Google Scholar 

  11. Y. Kawamata, R. Fujii, M. Hosoya, M. Harada, H. Yoshida, M. Miwa, S. Fukusumi, Y. Habata, T. Itoh, Y. Shintani, S. Hinuma, Y. Fujisawa, M. Fujino, A G protein-coupled receptor responsive to bile acids. J. Biol. Chem. 278(11), 9435–9440 (2003)

    Article  CAS  PubMed  Google Scholar 

  12. T. Maruyama, Y. Miyamoto, T. Nakamura, Y. Tamai, H. Okada, E. Sugiyama, T. Nakamura, H. Itadani, K. Tanaka, Identification of membrane-type receptor for bile acids (M-BAR). Biochem. Biophys. Res. Commun. 298(5), 714–719 (2002)

    Article  CAS  PubMed  Google Scholar 

  13. T.W. Pols, L.G. Noriega, M. Nomura, J. Auwerx, K. Schoonjans, The bile acid membrane receptor TGR5 as an emerging target in metabolism and inflammation. J. Hepatol. 54(6), 1263–1272 (2011)

    Article  CAS  PubMed  Google Scholar 

  14. K.L. Pierce, R.T. Premont, R.J. Lefkowitz, Seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol. 3(9), 639–650 (2002)

    Article  CAS  PubMed  Google Scholar 

  15. C. Thomas, A. Gioiello, L. Noriega, A. Strehle, J. Oury, G. Rizzo, A. Macchiarulo, H. Yamamoto, C. Mataki, M. Pruzanski, R. Pellicciari, J. Auwerx, K. Schoonjans, TGR5-mediated bile acid sensing controls glucose homeostasis. Cell Metab. 10(3), 167–177 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. M. Watanabe, S.M. Houten, C. Mataki, M.A. Christoffolete, B.W. Kim, H. Sato, N. Messaddeq, J.W. Harney, O. Ezaki, T. Kodama, K. Schoonjans, A.C. Bianco, J. Auwerx, Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation. Nature 439(7075), 484–489 (2006)

    Article  CAS  PubMed  Google Scholar 

  17. T.W. Pols, M. Nomura, T. Harach, G. Lo Sasso, M.H. Oosterveer, C. Thomas, G. Rizzo, A. Gioiello, L. Adorini, R. Pellicciari, J. Auwerx, K. Schoonjans, TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation and lipid loading. Cell Metab. 14(6), 747–757 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Y.D. Wang, W.D. Chen, D. Yu, B.M. Forman, W. Huang, The G-protein-coupled bile acid receptor, Gpbar1 (TGR5), negatively regulates hepatic inflammatory response through antagonizing nuclear factor kappa light-chain enhancer of activated B cells (NF-kappaB) in mice. Hepatology 54(4), 1421–1432 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. C. Guo, H. Qi, Y. Yu, Q. Zhang, J. Su, D. Yu, W. Huang, W.D. Chen, Y.D. Wang, The G-protein-coupled bile acid receptor Gpbar1 (TGR5) inhibits gastric inflammation through antagonizing NF-kappaB signaling pathway. Front. Pharmacol. 6, 287 (2015)

    Article  PubMed  PubMed Central  Google Scholar 

  20. D. Bar-Sagi, A. Hall, Ras and Rho GTPases: a family reunion. Cell 103(2), 227–238 (2000)

    Article  CAS  PubMed  Google Scholar 

  21. T. Matsui, M. Amano, T. Yamamoto, K. Chihara, M. Nakafuku, M. Ito, T. Nakano, K. Okawa, A. Iwamatsu, K. Kaibuchi, Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J. 15(9), 2208–2216 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. H. Zhou, Y. Li, Long-term diabetic complications may be ameliorated by targeting Rho kinase. Diabetes Metab. Res. Rev. 27(4), 318–330 (2011)

    Article  CAS  PubMed  Google Scholar 

  23. E. Amin, B.N. Dubey, S.C. Zhang, L. Gremer, R. Dvorsky, J.M. Moll, M.S. Taha, L. Nagel-Steger, R.P. Piekorz, A.V. Somlyo, M.R. Ahmadian, Rho-kinase: regulation, (dys)function, and inhibition. Biol. Chem. 394(11), 1399–1410 (2013)

    Article  CAS  PubMed  Google Scholar 

  24. F. Peng, D. Wu, B. Gao, A.J. Ingram, B. Zhang, K. Chorneyko, R. McKenzie, J.C. Krepinsky, RhoA/Rho-kinase contribute to the pathogenesis of diabetic renal disease. Diabetes 57(6), 1683–1692 (2008)

    Article  CAS  PubMed  Google Scholar 

  25. V. Kolavennu, L. Zeng, H. Peng, Y. Wang, F.R. Danesh, Targeting of RhoA/ROCK signaling ameliorates progression of diabetic nephropathy independent of glucose control. Diabetes 57(3), 714–723 (2008)

    Article  CAS  PubMed  Google Scholar 

  26. X. Xie, J. Peng, X. Chang, K. Huang, J. Huang, S. Wang, X. Shen, P. Liu, H. Huang, Activation of RhoA/ROCK regulates NF-kappaB signaling pathway in experimental diabetic nephropathy. Mol. Cell. Endocrinol. 369(1–2), 86–97 (2013)

    Article  CAS  PubMed  Google Scholar 

  27. A. Gojo, K. Utsunomiya, K. Taniguchi, T. Yokota, S. Ishizawa, Y. Kanazawa, H. Kurata, N. Tajima, The Rho-kinase inhibitor, fasudil, attenuates diabetic nephropathy in streptozotocin-induced diabetic rats. Eur. J. Pharmacol. 568(1–3), 242–247 (2007)

    Article  CAS  PubMed  Google Scholar 

  28. R. Komers, T.T. Oyama, D.R. Beard, C. Tikellis, B. Xu, D.F. Lotspeich, S. Anderson, Rho kinase inhibition protects kidneys from diabetic nephropathy without reducing blood pressure. Kidney Int. 79(4), 432–442 (2011)

    Article  CAS  PubMed  Google Scholar 

  29. R. Samarakoon, S.P. Higgins, C.E. Higgins, P.J. Higgins, TGF-beta1-induced plasminogen activator inhibitor-1 expression in vascular smooth muscle cells requires pp60(c-src)/EGFR(Y845) and Rho/ROCK signaling. J. Mol. Cell. Cardiol. 44(3), 527–538 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. H. Shimada, L.E. Rajagopalan, Rho kinase-2 activation in human endothelial cells drives lysophosphatidic acid-mediated expression of cell adhesion molecules via NF-kappaB p65. J. Biol. Chem. 285(17), 12536–12542 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. H. Iwasaki, R. Okamoto, S. Kato, K. Konishi, H. Mizutani, N. Yamada, N. Isaka, T. Nakano, M. Ito, High glucose induces plasminogen activator inhibitor-1 expression through Rho/Rho-kinase-mediated NF-kappaB activation in bovine aortic endothelial cells. Atherosclerosis 196(1), 22–28 (2008)

    Article  CAS  PubMed  Google Scholar 

  32. K. Geoffroy, N.M. Wiernsperger, B.S. El, Bimodal effect of advanced glycation end products on mesangial cell proliferation is mediated by neutral ceramidase regulation and endogenous sphingolipids. J. Biol. Chem. 279(33), 34343–34352 (2004)

    Article  CAS  PubMed  Google Scholar 

  33. M.C. Subauste, M. Von Herrath, V. Benard, C.E. Chamberlain, T.H. Chuang, K. Chu, G.M. Bokoch, K.M. Hahn, Rho family proteins modulate rapid apoptosis induced by cytotoxic T lymphocytes and Fas. J. Biol. Chem. 275(13), 9725–9733 (2000)

    Article  CAS  PubMed  Google Scholar 

  34. Q. Jiang, P. Liu, X. Wu, W. Liu, X. Shen, T. Lan, S. Xu, J. Peng, X. Xie, H. Huang, Berberine attenuates lipopolysaccharide-induced extracelluar matrix accumulation and inflammation in rat mesangial cells: involvement of NF-kappaB signaling pathway. Mol. Cell. Endocrinol. 331(1), 34–40 (2011)

    Article  CAS  PubMed  Google Scholar 

  35. Y. Rikitake, J.K. Liao, Rho-kinase mediates hyperglycemia-induced plasminogen activator inhibitor-1 expression in vascular endothelial cells. Circulation 111(24), 3261–3268 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. T. Lan, X. Shen, P. Liu, W. Liu, S. Xu, X. Xie, Q. Jiang, W. Li, H. Huang, Berberine ameliorates renal injury in diabetic C57BL/6 mice: Involvement of suppression of SphK-S1P signaling pathway. Arch. Biochem. Biophys. 502(2), 112–120 (2010)

    Article  CAS  PubMed  Google Scholar 

  37. Y.X. Tao, Constitutive activation of G protein-coupled receptors and diseases: insights into mechanisms of activation and therapeutics. Pharmacol. Ther. 120(2), 129–148 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. R. Seifert, K. Wenzel-Seifert, Constitutive activity of G-protein-coupled receptors: cause of disease and common property of wild-type receptors. Naunyn Schmiedebergs Arch. Pharmacol. 366(5), 381–416 (2002)

    Article  CAS  PubMed  Google Scholar 

  39. A.B. Sanz, M.D. Sanchez-Nino, A.M. Ramos, J.A. Moreno, B. Santamaria, M. Ruiz-Ortega, J. Egido, A. Ortiz, NF-kappaB in renal inflammation. J. Am. Soc. Nephrol. 21(8), 1254–1262 (2010)

    Article  CAS  PubMed  Google Scholar 

  40. T. Ishizaki, M. Uehata, I. Tamechika, J. Keel, K. Nonomura, M. Maekawa, S. Narumiya, Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol. Pharmacol. 57(5), 976–983 (2000)

    CAS  PubMed  Google Scholar 

  41. T. Chijiwa, A. Mishima, M. Hagiwara, M. Sano, K. Hayashi, T. Inoue, K. Naito, T. Toshioka, H. Hidaka, Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J. Biol. Chem. 265(9), 5267–5272 (1990)

    CAS  PubMed  Google Scholar 

  42. C. Eboh, T.A. Chowdhury, Management of diabetic renal disease. Ann. Transl. Med 3(11), 154 (2015)

    PubMed  PubMed Central  Google Scholar 

  43. K. Reidy, H.M. Kang, T. Hostetter, K. Susztak, Molecular mechanisms of diabetic kidney disease. J. Clin. Invest. 124(6), 2333–2340 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. F.G. Schaap, M. Trauner, P.L. Jansen, Bile acid receptors as targets for drug development. Nat. Rev. Gastrol. Hepatol. 11(1), 55–67 (2014)

    Article  CAS  Google Scholar 

  45. V. Stepanov, K. Stankov, M. Mikov, The bile acid membrane receptor TGR5: a novel pharmacological target in metabolic, inflammatory and neoplastic disorders. J. Recept. Signal Transduct. Res. 33(4), 213–223 (2013)

    Article  CAS  PubMed  Google Scholar 

  46. R. Pellicciari, A. Gioiello, A. Macchiarulo, C. Thomas, E. Rosatelli, B. Natalini, R. Sardella, M. Pruzanski, A. Roda, E. Pastorini, K. Schoonjans, J. Auwerx, Discovery of 6alpha-ethyl-23(S)-methylcholic acid (S-EMCA, INT-777) as a potent and selective agonist for the TGR5 receptor, a novel target for diabesity. J. Med. Chem. 52(24), 7958–7961 (2009)

    Article  CAS  PubMed  Google Scholar 

  47. X. Chen, G. Lou, Z. Meng, W. Huang, TGR5: a novel target for weight maintenance and glucose metabolism. Exp. Diabetes Res. 2011, 853501 (2011)

    PubMed  PubMed Central  Google Scholar 

  48. X.X. Wang, M.H. Edelstein, U. Gafter, L. Qiu, Y. Luo, E. Dobrinskikh, S. Lucia, L. Adorini, V.D. D'Agati, J. Levi, A. Rosenberg, J.B. Kopp, D.R. Gius, M.A. Saleem, M. Levi, protein-coupled bile acid receptor TGR5 activation inhibits kidney disease in obesity and diabetes. J. Am. Soc. Nephrol. (2015).

  49. P. Lang, F. Gesbert, M. Delespine-Carmagnat, R. Stancou, M. Pouchelet, J. Bertoglio, Protein kinase A phosphorylation of RhoA mediates the morphological and functional effects of cyclic AMP in cytotoxic lymphocytes. EMBO J. 15(3), 510–519 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  50. S. Rajagopal, D.P. Kumar, S. Mahavadi, S. Bhattacharya, R. Zhou, C.U. Corvera, N.W. Bunnett, J.R. Grider, K.S. Murthy, Activation of G protein-coupled bile acid receptor, TGR5, induces smooth muscle relaxation via both Epac- and PKA-mediated inhibition of RhoA/Rho kinase pathway. Am. J. Physiol. Gastrointest. Liver Physiol. 304(5), G527–G535 (2013)

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This study was supported by research grants from the National Natural Science Foundation of China (No. 81373457, No. 81573477), the National Science and Technology Major Project (No. 2014ZX09301307-008), Doctoral Fund Project of the Ministry of Education of China (No. 20130171110097), the Science and Technology Program of Guangdong province, PR China (No. 2012B050300017, No. 2014A020210007), the Natural Science Foundation of Guangdong Province (No. S2013010015765).

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    1. Laboratory of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China

      Fengxiao Xiong, Zhiying Yang, Yu Wang, Wenyan Gong, Junying Huang, Cheng Chen, Peiqing Liu & Heqing Huang

    2. National and Local United Engineering Lab of Druggability and New Drugs Evaluation, Guangzhou, 510006, China

      Fengxiao Xiong, Peiqing Liu & Heqing Huang

    3. Guangzhou Key Laboratory of Druggability Assessment for Biologically Active Compounds, Guangzhou, 510006, China

      Fengxiao Xiong, Peiqing Liu & Heqing Huang

    4. Dept of Pharmacy, Shenzhen Children’s Hospital, Shenzhen, Guangdong, 518026, China

      Xuejuan Li

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    Xiong, F., Li, X., Yang, Z. et al. TGR5 suppresses high glucose-induced upregulation of fibronectin and transforming growth factor-β1 in rat glomerular mesangial cells by inhibiting RhoA/ROCK signaling. Endocrine 54, 657–670 (2016). https://doi.org/10.1007/s12020-016-1032-4

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