The FAK Src signaling pathway mediates caveolin 1 expression We have shown previously that the integrin FAK Src axis triggered hepatocyte dedifferentiation. Hence, we subsequent investigated whether or not this pathway also contributed to caveolin 1 induction. Inhibition of FAK was achieved employing the small chemical inhibitor PF573228 which led to abroga tion of FAK tyrosine 397 and to a reduction of Src tyrosine 527 phosphorylation. The blockage of FAK subsequently attenuated caveolin 1 upregulation. Addition ally, downstream signaling of FAK, when it comes to pSrc, pAKT and pERK was affected. To additional elucidate that Src members of the family are crucial in mediating activa tion of AKT and ERK, two Src inhibitors have been applied and downstream signaling was evaluated. Inhibition of Src with SU6656 and PP2 yielded in reduced activity of ERK and AKT pathways.
Application of different inhibitors of Src family members is adequate in our experimental set ting as a result of their variant substrate specificity and thus distinct downstream effects. Similarly, as observed using the FAK inhibitor, Src block age considerably prevented upregulation of caveolin 1 in hepatocytes on protein Entinostat price and mRNA levels. Attenuated hepatocyte dedifferentiation was demonstrated by decreased expression of N Cadherin and Collagen 11 mRNA, too as the full details sustained E Cadherin ex pression when cells have been cultured in presence of Src family inhibitors. AKT and ERK contribute to caveolin 1 upregulation As a result of a recent report regarding the relevance of MAPK ERK and AKT signaling pathways in modulating hepato cyte plasticity in monolayer culture, and the observation of impacted ERK and AKT phosphorylation upon FAK Src inhibition, we intended to define the relevance of those pathways on caveolin 1 expres sion.
Blocking culture dependent AKT activation with Ly294002 inhibitor sig nificantly decreased caveolin 1 pro tein and mRNA levels. As also Collagen 11 induction was significantly blunted, in hibition of AKT affected hepatocyte dedifferentiation and caveolin 1 induction. Subsequent, U0126 was applied to interfere with ERK1 two activity for the duration of hepatocyte culture on collagen monolayer. This led to reduced caveolin 1 and Collagen 11 expression, similar to the result obtained upon AKT pathway blunting. In summary, both ERK1 two and AKT pathways influenced hepatocyte dedifferentiation and caveolin 1 expression. Snai1 is not involved in hepatocyte dedifferentiation and caveolin 1 induction Considering the exclusive role of Snail transcription factor family members in driving EMT, we tested whether Snai1 and Slug were involved in the culture dependent dedifferentiation method of hepatocytes. Intriguingly, during culture, Snai1 mRNA levels only slightly improved. As a potent mediator of EMT, TGF B was previously shown to induce Snai1 expression.