Neural stem cells are capable of differentiating into three major neural cell types, but the underlying molecular mechanisms remain unclear. Here, we investigated the mechanism by which integrin β4 modulates mouse neural stem cell differentiation in vitro. Inhibition of endogenous integrin β4 by RNA interference inhibited the cell differentiation and the expression of fibroblast growth factor receptor 2 but not fibroblast growth factor receptor 1 or fibroblast growth factor receptor 3. Overexpression of integrin β4 in neural stem cells promoted neural stem cell differentiation. Furthermore, integrin β4-induced differentiation of neural stem cells was attenuated by SU5402, the inhibitor of fibroblast growth factor receptors. Finally, we investigated the role of integrin β4 in neural stem cell survival: knockdown of integrin β4 did not affect survival or apoptosis of neural stem cells. These data provide evidence that integrin β4 promotes differentiation of mouse neural stem cells in vitro possibly through fibroblast growth factor receptor 2.

Recently, the specific roles of integrin β4 in the signaling networks that drive pathological angiogenesis and tumor progression have been revealed. Our previous study showed that integrin β4 might be involved in neuron survival signal transduction. To further our study on the role of integrin β4 in the survival and apoptosis of primary cultured mouse neurons, we inhibited the expression of integrin β4 by its specific small interfering RNA. Viability of the cells remarkably declined, and neurons underwent apoptosis with down-regulation of integrin β4. Next, we investigated the effect of siRNA-mediated down-regulation of integrin β4 on the level of intracellular reactive oxygen species and the activities of NADPH oxidase and superoxide dismutase. The level of reactive oxygen species in the neurons was elevated significantly, the activities of manganese-dependent superoxide dismutase and copper/zinc-dependent superoxide dismutase were not altered, but the activity of NADPH oxidase was increased. Furthermore, inhibition of NADPH oxidase by its specific inhibitor dibenziodolium chloride attenuated the neuronal death induced by integrin β4 knockdown. The data suggest that integrin β4 is a key factor in neuron survival and apoptosis and indicate that this integrin subunit might perform its action through regulating NADPH oxidase and the level of reactive oxygen species in neuronal survival and apoptosis.

To understand whether integrin β4 is involved in vascular endothelial cell (VEC) senescence, we examined integrin β4 level changes, as well as P53 and reactive oxygen species (ROS) levels and alterations of phosphatidylcholine-specific phospholipase C (PC-PLC) activity before and after knocking-down integrin β4 by small interfering RNA. We found integrin β4, P53 and ROS levels increased significantly, while Ca2+-independent PC-PLC activity obviously decreased during VEC senescence. On the other hand, integrin β4 down-regulation attenuated the senescence phenotype and reversed Ca2+-independent PC-PLC activity, and P53 and ROS levels. The data suggested that integrin β4 might mediate VEC senescence through depressing Ca2+-independent PC-PLC and elevating the levels of P53 and ROS.

Previously, we found that 3,4-(methylenedioxy)-1-(2′,3′- epoxypropyl)-benzene (safrole oxide) induced a typical apoptosis in A549 human lung cancer cells by activating caspase-3, -8, and -9. In this study, we further investigated which upstream pathways were activated by safrole oxide during the apoptosis. Immunofluorescence assay combined with laser scanning confocal microscopy revealed that both Fas and Fas ligand (FasL) were up-regulated by the small molecule. In addition, Fas protein distribution was altered, showing a clustering distribution instead of a homogeneous one. Subsequently, Western blot analysis confirmed the up-regulations of Fas and its membrane-binding form of FasL (m-FasL), as well as P53 protein. Conversely, safrole oxide hardly affected integrin β4 subunit expression or distribution, which was reflected from the data obtained by immunofluorescence assay combined with laser scanning confocal microscopy. The results suggested that Fas/FasL pathway might be involved in safrole oxide-induced apoptosis of A549 cells, while integrin β4 might be irrelevant to the apoptosis. Nevertheless, we first found the strong expression of integrin β4 in A549 cells. The study first suggested that safrole oxide might be used as a small molecular promoter of Fas/FasL pathway to elicit apoptosis in A549 cells, which would lay the foundation for us to insight into the new strategies for lung cancer therapy.