P226
SRp40 MEDIATES TGF-INDUCED POST-TRANSCRIPTIONAL SPLICING OF
FIBRONECTIN IN RENAL EPITHELIAL CELLS
Phanish, M, Shirali, S, Dockrell, M
SWT Institute for Renal Research, St Helieer Hospital, Carshalton
More than two thirds of the human protein-coding genes undergo alternative splicing,
facilitating the production of several polypeptides from a single gene. Fibronectin (Fn), the
important exctracellular matrix protein, is a good example of post-transcriptional splicing.
The solubility and activity of fibronectin is highly dependent on the splicing that occurs.
Compelling evidence suggests that the isoform containing Extra Domain A (EDA+) has profibrotic activity and targeting its production may be an anti-fibrotic strategy. Our group has
previously produced evidence for a putative role for the arginine serine rich protein SRp40 in
mediating EDA+ Fn production from human tubule epithelial cells in response to TGF1 and
have a suggested a possible role for PI-3kinase. Cdc2-like kinases (Clks) have been
implicated as one of the key kinases that regulate SR protein activity; and evidence suggests
that Clk may be either up-stream or down-stream of PI-3kinase depending on the cellular
context. Here we investigate the regulation of SRp40 by TGF and assess possible roles for
Clk and PI3 kinase in EDA+ Fn production.
Primary and transformed human PTEC were used. Intracellular SRp40 localisation was
assessed by immunofluorescent microscopy. Cell were treated with TGF1 (2.5ng/ml) for 648 hr, before fixing. To investigate the role of Clk, cells were treated with TGF1 +/- a Clk
inhibitor, Tg-0003, 10 M for 48 h and EDA+ Fn production assessed by Western Blotting.
Direct association of phoshpho-Akt (pAkt) and SRp40 was investigated by treating cells with
TGF1 +/- Ly294002 (5M), the PI-3K inhibitor. Cells were subsequently lysed and the
lysates subjected to immuno-precipitation by an SRp40 antibody and the IP subjected to
immunoblotting.
TGF1 induced a time dependent intracellular relocalisation of SRp40 as determined by
immunofluorescent microscopy. An apparent shift to a more nuclear localisation was
observed 24 h after TGF1 treatment which then returned to basal conditions by 48h. This
intracellular redistribution appeared to be PI3kinase dependent as it was modified by a
submaximal dose of the PI3-kinase inhibitor LY294002. To investigate whether Clk might be
involved in the process cells were treated with TGF +/- Tg003; however Tg003 had no
effect on TGF-induced EDA+ Fn production. We then investigated whether Akt the down
stream target of PI3 kinase may have a direct interaction with SRp40. Immunoprecipitation
experiments demonstrated that TGF1 caused an association of phospho-Akt and SRp40
which could be inhibited by pre-treatment with the PI3-kinase inhibitor LY294002.
Previously our group has demonstrated a requirement of SRp40 expression for TGF1induced EDA+ Fn expression. Here we demonstrate that TGF1 induced an intracellular
redistribution of SRp40 in PI3 kinase dependent manner. We also provided evidence for the
activation of SRp40 being independent of Clk activity but that TGF1 treatment is associated
with a direct interaction of SRp40 pAKT.