TNF-α increased A549 permeability by repressing VASP expression, through the activation of HIF-1α.

(A) A549 cells were transfected with siRNA-HIF-1α or pEGFP-C1-HIF-1α for 24 hours as indicated. Scrambled siRNA and pEGFP-C1 vector were used as controls. The HIF-1α and VASP protein expression levels were then determined by western blotting. GAPDH served as a loading control. (B) A549 cells were transfected with siRNA-HIF-1α or pEGFP-C1-HIF-1α for 24 hours as indicated. HIF-1α and VASP mRNA expression levels were then determined by RT-PCR. Figure 2A and 2B indicate that VASP expression of both mRNA levels and protein levels were repressed by HIF-1α and that VASP expression was dramatically activated by HIF-1α knockdown in A549 cells. In contrast, HIF-1α overexpression led to the decreased VASP expression. (C) Cells were treated with TNF-α after transfection with siRNA-HIF-1α for 24 h. Scrambled siRNA was used as a control. The HIF-1α and VASP protein expression levels were then determined by western blotting. (D) Cells were treated with TNF-α after transfection with siRNA-HIF-1α for 24 h. Scrambled siRNA was used as a control. The HIF-1α and VASP mRNA expression levels were then determined by RT-PCR. Figure 2C and 2D indicate that in the absence of TNF-α, the expression of both HIF-1α and VASP were maintained at moderate protein levels and the knockdown of HIF-1α led to increased VASP expression in A549 cells. Upon stimulation by TNF-α, the level of HIF-1 α was elevated accompanied by a suppression of VASP expression. However, the suppression of VASP by TNF-α could be relieved through HIF-1α knockdown. (E) A549 cells were transfected with shRNA-VASP or scrambled shRNA and then treated with 10 ng/mL TNF-α or untreated for 24 hours as indicated. The HIF-1α and VASP expression levels were then determined by western blotting. In the absence of TNF-α, VASP expression in the shRNA-VASP group decreased by 15.6% versus the scrambled group (**p0.05; scrambled+TNF-α vs. shRNA-VASP+TNF-α, p>0.05). Conversely, TNF-α stimulation dramatically elevated HIF-1α expression (scrambled vs. scrambled+TNF-α, **p2 or vehicle for 24 hours. The HIF-1α and VASP expression levels were then detected via western blotting. HIF-1α expression increased by 64.9% in the CoCl2 group compared with the vehicle group (**p2 group compared with the vehicle group (*p**p**p**p