Focal adhesion kinase and Src phosphorylations in HGF-induced proliferation and invasion of human cholangiocarcinoma cell line, HuCCA-1

AIM: To study the role of focal adhesion kinase (FAK) and its association with Src in hepatocyte growth factor (HGF)-induced cell signaling in cholangiocarcinoma progression.METHODS: Previously isolated HuCCA-1 cells were re-characterized by immunofluorescent staining and reverse transcriptase-polymerase chain reaction assay for the expression of cytokeratin 19, HGF and c-Met mRNA. Cultured HuCCA-1 cells were treated with HGF and determined for cell proliferation and invasion effects by MTT and invasion assays. Western blotting, immunoprecipitation, and co-immunoprecipitation were also performed to study the phosphorylation and interaction of FAK and Src. A novel Src inhibitor (AZM555130) was applied in cultures to investigate the effects on FAK phosphorylation inhibition and on cell proliferation and invasion.RESULTS: HGF enhanced HuCCA-1 cell proliferation and invasion by mediating FAK and Src phosphorylations.FAK-Src interaction occurred in a time-dependent manner that Src was proved to be an upstream signaling molecule to FAK. The inhibitor to Src decreased FAK phosphorylation level in correlation with the reduction of cell proliferation and invasion.CONCLUSION: FAK plays a significant role in signaling pathway of HGF-responsive cell line derived from cholangiocarcinoma. Autophosphorylated Src, induced by HGF, mediates Src kinase activation, which subsequently phosphorylates its substrate, FAK, and signals to cell proliferation and invasion.

Distinct mechanisms of FAK mechanoactivation by different extracellular matrix proteins

Introduction Cells can sense and respond to the mechanical microenvironment by converting forces into biochemical signals inside the cells,i.e.mechanotransduction[1-3].Focal adhesions are the major sites of interaction between a cell and its extracellular matrix（ECM）microenvironment,thus outside mechanical signals can be sensed at focal adhesions through transmembrane receptor integrins.In particular,it has been shown that matrix elasticity can control the cell fate[4]by modulating the interactions between ECM proteins and their receptor integrins[5,6].For example,different rigidity of polyacrylamide（PA）gels can lead to different density of ECM ancho-

Inactivation of Cdc42 in embryonic brain results in hydrocephalus with ependymal cell defects in mice

The establishment of a polarized cellular morphology is essential for a variety of processes including neural tube morphogenesis and the development of the brain.Cdc42 is a Ras-related GTPase that plays an essential role in controlling cell polarity through the regulation of the actin and microtubule cytoskeleton architecture.Previous studies have shown that Cdc42 plays an indispensable role in telencephalon development in earlier embryo developmental stage(before E12.5).However,the functions of Cdc42 in other parts of brain in later embryo developmental stage or in adult brain remain unclear.Thus,in order to address the role of Cdc42 in the whole brain in later embryo developmental stage or in adulthood,we used Cre/loxP technology to generate two lines of tissue-specific Cdc42-knock-out mice.Inactivation of Cdc42 was achieved in neuroepithelial cells by crossing Cdc42/flox mice with Nestin-Cre mice and resulted in hydrocephalus,causing death to occur within the postnatal stage.Histological analyses of the brains from these mice showed that ependymal cell differentiation was disrupted,resulting in aqueductal stenosis.Deletion of Cdc42 in the cerebral cortex also induced obvious defects in interki-netic nuclear migration and hypoplasia.To further explore the role of Cdc42 in adult mice brain,we examined the effects of knocking-out Cdc42 in radial glial cells by crossing Cdc42/flox mice with human glial fi brillary acidic protein(GFAP)-Cre mice.Inactivation of Cdc42 in radial glial cells resulted in hydrocephalus and ependymal cell denudation.Taken together,these results highlight the importance of Cdc42 for ependymal cell differentiation and maintaining,and suggest that these functions likely contribute to the essential roles played by Cdc42 in the development of the brain.