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Cells in the body are subjected to mechanical stresses such as tension, compression, and shear stress. These mechanical stresses play important roles in both physiological and pathological processes; however, mechanisms transducing mechanical stresses into biochemical signals remain elusive. Here, we demonstrated that equibiaxial stretch inhibited lamellipodia formation through deactivation of Rac. Nearly maximal effects on Rac activity were obtained with 10% strain. GAP-resistant, constitutively active V12Rac reversed this inhibition, supporting a critical role for Rac inhibition in the response to stretch. In contrast, activation of endogenous Rac with a constitutively active nucleotide exchange factor did not, suggesting that regulation of GAP activity most likely mediates the inhibition. Uniaxial stretch suppressed lamellipodia along the sides lengthened by stretch and increased it at the adjacent ends. A fluorescence assay for localized Rac showed comparable changes in activity along the sides versus the ends after uniaxial stretch. Blocking polarization of Rac activity by expressing V12Rac prevented subsequent alignment of actin stress fibers. Treatment with Y-27632 or ML-7 that inhibits myosin phosphorylation and contractility increased lamellipodia through Rac activation and decreased cell polarization. We hypothesize that regulation of Rac activity by tension may be important for motility, polarization, and directionality of cell movement.

Increased expression of vascular endothelial growth factor (VEGF) contributes to the growth of many tumors by increasing angiogenesis. Although hypoxia is a potent inducer of VEGF, we previously showed that epidermal growth factor receptor amplification and loss of PTEN, both of which can increase phosphatidylinositol-3-kinase (PI3K) activity, increase VEGF expression. Using both adenoviral vectors and a cell line permanently expressing constitutively active myristoylated Akt (myrAkt), we show that activation of Akt, which is downstream of PI3K, increases VEGF expression in vitro and increases angiogenesis in a Matrigel plug assay. Transient transfection experiments using reporter constructs containing the VEGF promoter showed that up-regulation of VEGF by Akt is mediated through Sp1 binding sites located in the proximal promoter. Small interfering RNA directed against Sp1 prevented the induction of VEGF mRNA in response to myrAkt but not to hypoxia. Expression of myrAkt is associated with increased phosphorylation of Sp1 and its increased binding to a probe corresponding to the ؊88/؊66 promoter region. In conclusion, our results indicate that Sp1 is required for transactivation of the VEGF by Akt. Others have proposed that the PI3K/Akt pathway can increase VEGF expression via the hypoxia-inducible factor 1 (HIF-1); however, our results suggest an alternative mechanism can also operate. INTRODUCTIONVascular endothelial growth factor (VEGF), which is an important mediator of angiogenesis in a variety of settings (Ferrara, 2002), is often overexpressed in cancers and may be important for their continued growth beyond a certain size. This is underscored by the fact that strategies to inhibit VEGF expression and function, including antibodies, kinase inhibitors, and soluble VEGF receptors, efficiently inhibit tumor growth in animal models of gliomas and other tumors (Manley et al., 2002;Bogler and Mikkelsen, 2003). Therefore, defining the mechanisms that regulate VEGF expression in cancer cells may have important implications for understanding tumor biology.VEGF is strongly induced by hypoxia, and undoubtedly this is an important mechanism of induction in tumors (Shweiki et al., 1992). However, when grown in standard tissue culture conditions under ambient oxygen conditions, many cell lines express high levels of VEGF expression, indicating that other factors can also play a role in regulating VEGF (Feldkamp et al., 1999). Our previous work suggested that genetic changes found in many cancers, specifically activation of the epidermal growth factor receptor (EGFR) and mutation of the tumor suppressor gene PTEN, may lead to up-regulation of VEGF (Maity et al., 2000;Pore et al., 2003).PTEN (phosphatase and tensin homolog deleted on chromosome ten), also known as MMAC-1 or TEP-1, functions primarily as a lipid phosphatase to dephosphorylate the D-3 position of phosphoinositide phosphates such as PI(3,4,5)P 3 to convert them to PI(4,5)P 2 (Vivanco and Sawyers, 2002). The PTEN tumor suppressor gene product therefore ac...

Stability of the vascular endothelial growth factor (VEGF) mRNA is tightly regulated through its 3-untranslated region (3-UTR). Here, we demonstrate that VEGF mRNA levels are increased by anisomycin, a strong activator of stress-activated protein kinases. Hence, VEGF mRNA induction is inhibited by SB202190, an inhibitor of JNK and p38/HOG kinase. Furthermore, VEGF mRNA expression is increased in cells that overexpress JNK and p38/HOG by an increase in its stability. We show by two different approaches that anisomycin exerts its effect on the VEGF mRNA 3-UTR. First, by using an in vitro mRNA degradation assay, the half-life of the VEGF mRNA 3-UTR region transcript was found to be increased when incubated with extracts from anisomycin-treated cells; and second, the 3-UTR was also sufficient to confer mRNA instability to the Nhe3 (Na ؉ /H ؉ exchanger 3) heterologous reporter gene, and anisomycin treatment stabilized the chimeric mRNA (Nhe3 fused to the VEGF mRNA 3-UTR). This chimeric mRNA is also more stable in cells overexpressing p38/ HOG and JNK that have been stimulated by anisomycin. We show that such regulation is mediated through an AU-rich region of the 3-UTR contained within a stable hairpin structure. By RNA electrophoretic mobility shift assays, we show that this region binds proteins specifically induced by anisomycin treatment. These findings clearly demonstrate a major role of stress-activated protein kinases in the post-transcriptional regulation of VEGF.