Fig. 6.

FAK, but not PYK2, regulates LIF and IL-6 expression in human and mouse brain endothelial cells. Human CMEC cells were passaged and resuspended in serum-free medium and maintained in suspension for 1 h. Cells were then plated onto plastic (PBS; Ctrl), or VTN-, laminin- (LN) or fibronectin (FN1)-treated plates for 1 h. (A) VTN caused a much greater increase in pFAK Y397 and STAT3 Y705 over laminin and fibronectin. CMEC cells were pre-incubated with integrin β3- or β5-blocking antibodies before plating onto VTN. At 4 h after plating, induction of LIF (B) and IL-6 (C) by VTN were reduced when CMEC cells were pre-incubated with P11, β3- or β5-blocking antibodies (mean±s.e.m.; n=4–6, ANOVA with Fisher LSD test). To determine the role and specifity of FAK in regulating baseline LIF and IL-6 mRNA expression, we performed knockdown of FAK (siFAK) or PYK2 (siPYK2) using siRNAs in human CMEC cells over 6 days. siFAK reduced LIF (A) and IL-6 (B), while increasing CNTF (C) gene expression relative to non-targeting control siRNA. siPYK2 had no effect. In bEnd5 cells, siFAK also diminished both LIF (D) and IL-6 (E) expression while increasing CNTF (F) (mean±s.e.m.; n=3). Pharmacological FAK inhibition for 4 h with PF573228 (PF228) also suppressed LIF (G) and IL-6 (H) while upregulating CNTF (I) mRNA expression relative to vehicle-treated controls (Ctrl), as measured by RT-qPCR (n=4). Data are means±s.e.m. from n=4 independent experiments each. *P<0.05, **P<0.01, ***P<0.001; NS, not significant.