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Vol. 64 No. 3: Issue 3

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TGFβ1-mediated expression and alternative splicing of Fibronectin Extra Domain A in human podocyte culture

https://doi.org/10.14715/cmb/2018.64.3.4
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

Alternative splicing is a fundamental phenomenon to build protein diversity in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin protein present in the extra cellular matrix (ECM) in renal fibrosis. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. TGFβ1 is a strong stimulator of ECM proteins in renal injury. In this study, we have investigated alternative splicing of EDA+ Fn in human podocytes in response to TGFβ1. We have performed western blotting and immunofluorescence to characterise the expression of the EDA+Fn protein, real-time PCR for RNA expression and RT-PCR to look for alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We used TGFβ1 as a stimulator and SB431542 and SRPIN340 for inhibitory studies. In this work, for the first time we have demonstrated in human podocytes culture EDA+Fn is expressed in the basal condition and TGFβ1 2.5ng/ml induced the Fn mRNA and EDA+Fn protein expression demonstrated by real-time PCR, western blotting and immunofluorescence. TGFβ1 2.5ng/ml induced the alternative splicing of EDA+Fn shown by conventional RT-PCR. Studies with ALK5 inhibitor SB431542 and SRPIN340 show that TGFβ1 induced alternative splicing of EDA+Fn was by the ALK5 receptor and the SR proteins.  In human podocytes culture, alternative splicing of EDA+Fn occurs at basal conditions and TGFβ1 further induced the alternative splicing of EDA+Fn via ALK5 receptor activation and SR proteins. This is the first evidence of basal and TGFβ1 mediated alternative splicing of EDA+Fn in human podocytes culture.

Keywords

Extra Domain A Fibronectin Podocyte TGFβ1 Alternative splicing.
Madne, T. H., & Dockrell, M. E. C. (2018). TGFβ1-mediated expression and alternative splicing of Fibronectin Extra Domain A in human podocyte culture. Cellular and Molecular Biology, 64(3), 17–24. https://doi.org/10.14715/cmb/2018.64.3.4
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Author Biography

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
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