Our previous study using MEFs derived from CasΔex2/Δex2 mice showed that Cas Δex2 possesses reduced function in FN-mediated signaling.32 Thus, to examine the Cas requirement for SEC function, we first attempted to knock down endogenous Cas in NP31 cells by RNA interference. However, we found that NP31 cells rapidly lost fenestra formation when they were exposed to transfection reagents with nonspecific small interfering RNA Saracatinib ic50 or even no RNA (data not shown). We thus used an alternative Cas mutant overexpression approach. We used Cas ΔSH3 because the SH3 domain represents virtually the functional domain of exon 2 (Fig.
1) and other motifs in exon 2, YLVP and YQxPs, have been demonstrated to Ipatasertib clinical trial be redundant or dispensable for Cas-mediated signaling.34 In fact, Cas ΔSH3–expressing NP31 cells exhibited biochemical properties similar to those of
CasΔex2/Δex2 MEFs, such as impaired Cas tyrosine phosphorylation and reduced interaction of Cas with CrkII32 (Fig. 4). Thus, Cas ΔSH3 is biochemically equivalent to and functionally recapitulates Cas exon 2 deletion. In agreement with these biochemical alterations, we demonstrated that Cas ΔSH3 abolished reorganization of the actin cytoskeleton and critically inhibited the formation of fenestrae (Fig. 5). These findings strongly indicate that the Cas exon 2 deficiency affected actin cytoskeleton reorganization and SEC fenestration in CasΔex2/Δex2 embryos, and the impaired SEC fenestration subsequently induced massive hepatocyte apoptosis during liver development. Previous in vitro studies in SECs showed that
treatment of the cells with anti-actin agents and artificial modulation of Rho small GTPases impaired SEC fenestration35-40; in addition, SEC fenestration was required for hepatocyte survival.5, 6 These findings are consistent with the notion described previously. Monoiodotyrosine The current study highlights the importance of Cas in liver development. It also unveils an unexpectedly intimate interaction between SEC cytoskeletal turnover and hepatocyte development by illustrating the indirect influence of SEC fenestration on hepatocyte survival. It has previously been reported that the numbers and diameters of fenestrae are sensitive to growth factors such as vascular endothelial growth factor,41-43 endothelin-1,44-46 and transforming growth factor β.43 Intriguingly, these factors are known to tyrosine-phosphorylate Cas,47-49 and this strongly suggests that Cas tyrosine phosphorylation is involved in the induced changes of fenestrae. Defenestration has been reported in various liver diseases such as alcoholic liver damage,50, 51 hepatitis and liver cirrhosis,52, 53 and liver cancer54, 55 and causes portal hypertension and liver dysfunction.