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Vol. 64 No. 3: Issue 3

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Human podocytes responses to alternatively spliced Extra domain A Fibronectin in culture

https://doi.org/10.14715/cmb/2018.64.3.8
Tarunkumar Hemraj Madne
Southwest Thames Institute for Renal Research, Renal Unit, St Helier Hospital, Wrythe Lane, Carshalton, Surrey, SM5 1AA, St Georges, University of London. London, United Kingdom
Mark Edward Carl Dockrell
Southwest Thames Institute for Renal Research, Renal Unit, St Helier Hospital, Wrythe Lane, Carshalton, Surrey, SM5 1AA, St Georges, University of London. London, United Kingdom

Corresponding Author(s) : Tarunkumar Hemraj Madne

tarun2madne@yahoo.co.in

Cellular and Molecular Biology, Vol. 64 No. 3: Issue 3

Abstract

The interactions of the extracellular matrix (ECM) proteins with cells strongly regulate cell behaviour. The glomerular basement membrane (GBM) is a dynamic structure made up of protein secreted by endothelial cells and podocyte. These proteins could regulate the behaviour of these cells in health and diseases. Extra Domain A + Fn (EDA+Fn) is an alternatively spliced form of Fibronectin (Fn) recently identified in GBM and a recognised marker of various pathologies. In this study for the first time, we have investigated the responses of human podocytes cultured on different composition of GBM proteins which are cellular Fn (EDA+), plasma Fn (EDA-) and collagen IV. Conditionally immortalised human podocyte were grown on the dishes coated with different matrices; collagen IV (Col IV), cellular fibronectin (CFn) containing the EDA Exon, plasma fibronectin (PFn), which lacks the EDA Exon (EDA-Fn). We have performed western blotting to characterise the expression of the different proteins, real time PCR and RT-PCR to look for gene expression and alternative splicing of EDA+Fn. We have used TGFβ1 as a stimulator. We have used HEK-Blue-hTLR4 cells to determine the biological activity of cellular Fn. Conditionally immortalised human podocyte show marked differences in their morphology grown on the dishes coated with different matrices; Col IV, CFn, and PFn. CFn was biologically active as it activated the TLR4 signalling in HEK-Blue-hTLR4 cells. Different matrices effects basal as well as TGFβ1 mediated alternative splicing of EDA+Fn. TGFβ1 was active on different matrices as it induced phosphorylation of pSmad3 however it did not affect phosphorylation of pAkt and p38. Interestingly, different cellular matrices affected basal phosphorylation of pAkt. CFn downregulated gene expression of synaptopodin and increased gene expression of collagen I and Fn. CFn increased cell death in detached human podocytes. Alteration of the constituents of the GBM is likely to significantly alter podocyte cellular responses to growth factors involved in podocytopathies, such as TGFβ.

Keywords

Extra Domain A Fibronectin Podocyte TGFβ1 Alternative splicing Glomerular basement membrane Extra cellular matrix.
Madne, T. H., & Dockrell, M. E. C. (2018). Human podocytes responses to alternatively spliced Extra domain A Fibronectin in culture. Cellular and Molecular Biology, 64(3), 45–52. https://doi.org/10.14715/cmb/2018.64.3.8
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References
  1. Byron A, Randles MJ, Humphries JD, Mironov A, Hamidi H, Harris S, et al. Glomerular cell cross-talk influences composition and assembly of extracellular matrix. J Am Soc Nephrol. 2014 May [cited 2014 Dec 30];25(5):953–66.
  3. Pelisch F, Gerez J, Druker J, Schor IE, Muñoz MJ, Risso G, et al. The serine/arginine-rich protein SF2/ASF regulates protein sumoylation. Proc Natl Acad Sci U S A. 2010 Sep 14 [cited 2014 Dec 18];107(37):16119–24.
  5. Miner JH. The glomerular basement membrane. Exp Cell Res. 2012 May 15 [cited 2014 Dec 30];318(9):973–8.
  6. Pavenstädt H, Kriz W, Kretzler M. Cell biology of the glomerular podocyte. Physiol Rev. 2003 Jan;83(1):253–307.
  8. Satoi S, Kitade H, Hiramatsu Y, Kwon AH, Takahashi H, Sekiguchi K, et al. Increased extra domain-A containing fibronectin and hepatic dysfunction during septic response: an in vivo and in vitro study. Shock. 2000 Jun [cited 2014 Dec 26];13(6):492–6.
  10. Has C, Spartí G, Kiritsi D, Weibel L, Moeller A, Vega-Warner V, et al. Integrin α3 mutations with kidney, lung, and skin disease. N Engl J Med. 2012 Apr 19 [cited 2014 Oct 29];366(16):1508–14.
  11. Margadant C, Sonnenberg A. Integrin-TGF-beta crosstalk in fibrosis, cancer and wound healing. EMBO Rep. 2010 Feb [cited 2014 Dec 3];11(2):97–105.
  12. Qu H, Tu Y, Shi X, Larjava H, Saleem M a, Shattil SJ, et al. Kindlin-2 regulates podocyte adhesion and fibronectin matrix deposition through interactions with phosphoinositides and integrins. J Cell Sci. 2011 Mar 15 [cited 2012 Sep 4];124(Pt 6):879–91.
  14. Chen C-A, Hwang J, Guh J, Tsai J, Chen H. TGF-beta1 and integrin synergistically facilitate the differentiation of rat podocytes by increasing alpha-smooth muscle actin expression. Transl Res. 2006 Sep [cited 2014 Dec 30];148(3):134–41.
  15. Dai C, Stolz DB, Bastacky SI, St-Arnaud R, Wu C, Dedhar S, et al. Essential role of integrin-linked kinase in podocyte biology: Bridging the integrin and slit diaphragm signaling. J Am Soc Nephrol. 2006 Aug [cited 2014 Oct 29];17(8):2164–75.
  17. Henderson NC, Sheppard D. Integrin-mediated regulation of TGFβ in fibrosis. Biochim Biophys Acta. 2013 Jul [cited 2014 Dec 23];1832(7):891–6.
  19. Pankov R. Fibronectin at a glance. J Cell Sci. 2002 Oct 15 [cited 2012 Jul 13];115(20):3861–3.
  21. Pozzi A, Jarad G, Moeckel GW, Coffa S, Zhang X, Gewin L, et al. Beta1 integrin expression by podocytes is required to maintain glomerular structural integrity. Dev Biol. 2008 Apr 15 [cited 2014 Oct 29];316(2):288–301.
  23. Baelde HJ, Eikmans M, van Vliet AI, Bergijk EC, de Heer E, Bruijn JA. Alternatively spliced isoforms of fibronectin in immune-mediated glomerulosclerosis: the role of TGFbeta and IL-4. J Pathol. 2004 Nov [cited 2014 Dec 30];204(3):248–57.
  24. Muro AF, Moretti F a, Moore BB, Yan M, Atrasz RG, Wilke C a, et al. An essential role for fibronectin extra type III domain A in pulmonary fibrosis. Am J Respir Crit Care Med. 2008 Mar 15 [cited 2012 Sep 7];177(6):638–45.
  25. Olsen AL, Sackey BK, Marcinkiewicz C, Boettiger D, Wells RG. Fibronectin extra domain-A promotes hepatic stellate cell motility but not differentiation into myofibroblasts. Gastroenterology. 2012 Apr [cited 2014 Dec 30];142(4):928–937.e3.
  26. Rybak J, Roesli C, Kaspar M, Villa A, Neri D. The extra-domain A of fibronectin is a vascular marker of solid tumors and metastases. Cancer Res. 2007 Dec 15 [cited 2014 Dec 31];67(22):10948–57.
  27. Courtoy PJ, Kanwar YS, Hynes RO, Farquhar MG. Fibronectin localization in the rat glomerulus. J Cell Biol. 1980 Dec [cited 2014 Dec 30];87(3 Pt 1):691–6.
  28. Kohan M, Muro AF, White ES, Berkman N. EDA-containing cellular fibronectin induces fibroblast differentiation through binding to alpha4beta7 integrin receptor and MAPK/Erk 1/2-dependent signaling. FASEB J. 2010 Nov [cited 2014 Oct 10];24(11):4503–12.
  30. Ou J, Li J, Pan F, Xie G, Zhou Q, Huang H, et al. Endostatin suppresses colorectal tumor-induced lymphangiogenesis by inhibiting expression of fibronectin extra domain A and integrin α9. J Cell Biochem. 2011 Aug [cited 2014 Oct 29];112(8):2106–14.
  31. Shinde A V, Bystroff C, Wang C, Vogelezang MG, Vincent P a, Hynes RO, et al. Identification of the peptide sequences within the EIIIA (EDA) segment of fibronectin that mediate integrin alpha9beta1-dependent cellular activities. J Biol Chem. 2008 Feb 1 [cited 2014 Oct 15];283(5):2858–70.
  32. Gunwar S, Ballester F, Noelken ME, Sado Y, Ninomiya Y, Hudson BG. Glomerular Basement Membrane: identification of a novel disulfide-cross-linked network of 3, 4, and 5 chains of type iv collagen and its implications for the pathogenesis of alport syndrome. J Biol Chem. 1998 Apr 10 [cited 2014 Dec 30];273(15):8767–75.
  34. Greka A, Mundel P. Cell biology and pathology of podocytes. Annu Rev Physiol. 2012 Jan [cited 2012 Sep 22];74:299–323.
  35. To WS, Midwood KS. Plasma and cellular fibronectin: distinct and independent functions during tissue repair. Fibrogenesis Tissue Repair. 2011 Jan [cited 2012 Nov 20];4(1):21.
  36. Okamura Y, Watari M, Jerud ES, Young DW, Ishizaka ST, Rose J, et al. The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem. 2001 Mar 30 [cited 2014 Nov 27];276(13):10229–33.
  37. Yaswen P. Cell Shape-dependent Regulation of Protein 4.1Alternative Pre-mRNA Splicing in Mammary Epithelial Cells. J Biol Chem. 1997 Apr 11 [cited 2014 Dec 31];272(15):10254–9.
  38. Srebrow A, Blaustein M, Kornblihtt AR. Regulation of fibronectin alternative splicing by a basement membrane-like extracellular matrix. FEBS Lett. 2002 Mar [cited 2014 Oct 29];514(2–3):285–9.
  39. Wei C, Möller CC, Altintas MM, Li J, Schwarz K, Zacchigna S, et al. Modification of kidney barrier function by the urokinase receptor. Nat Med. 2008 Jan [cited 2014 Dec 31];14(1):55–63.
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