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Vol. 64 No. 5: Issue 5

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TGFβ1-mediated PI3K/Akt and p38 MAP kinase dependent alternative splicing of fibronectin extra domain A in human podocyte culture

https://doi.org/10.14715/cmb/2018.64.5.22
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. 5: Issue 5

Abstract

Alternative splicing is an important gene regulation process to distribute proteins in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin (Fn) protein, present in the extra cellular matrix (ECM) and a recognised marker of various pathologies. TGFβ1 has been shown to induce alternative splicing of EDA+Fn in many cell types. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. In our previous study we have demonstrated expression and alternative splicing of EDA+Fn in basal condition in human podocytes culture. TGFβ1 further induced the basal expression and alternative splicing of EDA+Fn through Alk5 receptor and SR proteins. In this study, we have investigated TGFβ1 mediated signalling involved in alternative splicing of EDA+Fn in human podocytes. We have performed western blotting to characterise the expression of the EDA+Fn protein and other signalling proteins and RT-PCR to look for signalling pathways involved in regulation of alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We have used TGFβ1 as a stimulator and SB431542, SB202190 and LY294002 for inhibitory studies. In this work, we have demonstrated in human podocytes culture TGFβ1 2.5ng/ml induced phosphorylation of Smad1/5/8, Smad2 and Smad3 via the ALK5 receptor. TGFβ1 significantly induced the PI3K/Akt pathway and the PI3K/Akt pathway inhibitor LY294002 significantly downregulated basal as well as TGFβ1 induced alternative splicing of EDA+Fn in human podocytes. In addition to this, TGFβ1 significantly induced the p38 MAP kinase signalling pathway and p38 MAP kinase signalling pathway inhibitor SB202190 downregulated the TGFβ1-mediated alternative splicing of EDA+Fn in human podocytes. The results with PI3K and p38 MAP kinase signalling pathway suggest that inhibiting PI3K signalling pathway downregulated the basal alternative splicing of EDA+Fn in human podocytes and its the inhibition of p38 Map Kinase signalling pathway which had specifically downregulated the TGFβ1 mediated alternative splicing of EDA+Fn in human podocytes culture. Activation of TGFβ1-mediated Smad1/5/8 via Alk5 receptor suggests that TGFβ1 signalling pathway involved Alk5/Alk1 receptor axis signalling in human podocytes.

Keywords

Extra Domain A Fibronectin Alternative splicing Podocyte TGFβ1 PI3k/Akt pathway p38 MAP Kinase pathway.
Madne, T. H., & Dockrell, M. E. C. (2018). TGFβ1-mediated PI3K/Akt and p38 MAP kinase dependent alternative splicing of fibronectin extra domain A in human podocyte culture. Cellular and Molecular Biology, 64(5), 127–135. https://doi.org/10.14715/cmb/2018.64.5.22
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