Abstract
Abstract: :
Purpose: In vertebrates during postnatal lens growth, cell proliferation is restricted to the germinative zone with the more anterior epithelium remaining relatively quiescent. In vivo and in vitro studies have shown PDGF to be a mitogen for epithelial cells ( Reneker & Overbeek,1996; Kok et al, 2002). Recent studies in our laboratory have implicated a role for both ERK1/2 and PI-3 kinase during PDGF-induced lens epithelial cell proliferation (ARVO, 2002). Other kinases such as the family of non-receptor Src tyrosine kinases have also been implicated in playing a pivotal role in PDGF signalling. To determine if Src tyrosine kinases can influence PDGF-induced ERK1/2 signalling and proliferation in lens epithelial cells, we employed the potent and selective Src tyrosine kinase inhibitor, PP2. Methods: Rat lens epithelial explants treated with PDGF-AA (10ng/ml) were cultured for either 10 mins or 48 hrs. Cell proliferation was assayed using immunolabelling for incorporation of BrdU. Activation of ERK1/2 was assayed using western blotting and immunofluorescence with specific antibodies. Blocking experiments using PP2 (1mM) were also used to assess the role for Src signalling in PDGF-induced lens cell proliferation. Results: PP2 was shown to markedly inhibit phosphorylation of PDGF-induced ERK1/2 at 10 mins; however by 48 hrs, PP2 had little or no effect on PDGF-induced ERK activation. These results were consistent with our immunofluorescence data. In addition, presence of the Src inhibitor in lens epithelial cultures had minimal effect on proliferation. Surprisingly, control lens epithelial cells treated with PP2 demonstrated an increase in phospho-ERK1/2 by 10 mins which was significantly enhanced by 48 hrs. Conclusions: Src tyrosine kinases may act as pivotal signalling molecules during the very early events leading to activation of PDGF-induced ERK activation. Src kinases may also play a putative role in negatively regulating quiescent cells thus demonstrating the diverse roles of Src tyrosine kinases in governing cell function.
Keywords: growth factors/growth factor receptors • signal transduction • proliferation