Involvement of ERK1/2 pathway in TGF-beta1-induced VEGF secretion in normal human cytotrophoblast cells

Transforming growth factor-beta1 (TGF-beta1) plays a pivotal role in the angiogenesis during the development of placenta, but the intracellular signaling mechanism by which TGF-beta1 stimulates this process remains poorly understood. In this article, we demonstrated that exposure of normal human cytotrophoblast cells to TGF-beta1 stimulated the secretion of the VEGF gene encoding vascular endothelial growth factor, which is a key factor in angiogenesis. Meanwhile, treatment of normal human cytotrophoblast cells with TGF-beta1-induced expression of HIF-1a, the regulated subunit of hypoxia-inducible factor 1, a known transactivator of the VEGF gene. Our data indicated that TGF-beta1 induced extracellular signal- regulated kinase (ERK) 1/2 phosphorylation in normal human cytotrophoblast cells. Moreover, treating cells with PD98059, an inhibitor of ERK1/2 signaling, inhibited TGF-beta1 stimulation of VEGF secretion and HIF-1a protein expression. These data indicated that in normal human cytotrophoblast cells, TGF-beta1 induced HIF-1a-mediated VEGF secretion, and TGF-beta1-stimulated-ERK1/2 activation may be involved in this process.     

p38 Signaling-mediated hypoxia-inducible factor 1alpha and vascular endothelial growth factor induction by Cr(VI) in DU145 human prostate carcinoma cells

Chromium(VI) (Cr(VI)) is widely used in industry and is a potent inducer of tumors in animals. The present study demonstrates that Cr(VI) induces hypoxia-inducible factor 1 (HIF-1) activity through the specific expression of HIF-1alpha but not HIF-1beta subunit and increases the level of vascular endothelial growth factor (VEGF) expression in DU145 human prostate carcinoma cells. To dissect the signaling pathways involved in Cr(VI)-induced HIF-1 expression, we found that p38 mitogen-activated protein kinase signaling was required for HIF-1alpha expression induced by Cr(VI). Neither phosphatidylinositol 3-kinase nor extracellular signal-regulated kinase activity was required for Cr(VI)-induced HIF-1 expression. Cr(VI) induced expression of HIF-1 and VEGF through the production of reactive oxygen species in DU145 cells. The major species of reactive oxygen species responsible for the induction of HIF-1 and VEGF expression is H(2)O(2). These results suggest that the expression of HIF-1 and VEGF induced by Cr(VI) may be an important signaling pathway in the Cr(VI)-induced carcinogenesis.     

Hypoxia-induced HIF-1 alpha accumulation is augmented in a co-culture of keloid fibroblasts and human mast cells: involvement of ERK1/2 and PI-3K/Akt

Keloids represent a prolonged inflammatory fibrotic state with areas that display distinctive histological features characterized by an abundant extracellular matrix stroma, a local infiltration of inflammatory cells including mast cells, and a milieu of enriched cytokines. Previous studies from our laboratory demonstrated an intrinsic higher level of HIF-1alpha and VEGF protein expression in keloid tissues compared with their adjacent unremarkable skins. To further investigate the mechanisms underlying the elevated expression of HIF-1alpha and VEGF in keloids, we exposed a co-culture of keloid fibroblasts and mast cells (HMC-1) to hypoxic conditions and studied the expression of HIF-1alpha and its target gene, VEGF. Our results showed that hypoxia-dependent HIF-1alpha protein accumulation and VEGF expression is augmented in keloid fibroblasts when co-cultured with HMC-1 cells under the condition where direct cell-cell contact is allowed. But such augmentation is not observed in the transwell co-culture system whereas fibroblasts and HMC-1 cells were separated by a porous membrane. Our results also indicated that the enhancement of hypoxia-mediated activation of ERK1/2 and Akt requires direct cell-cell interaction between mast cells and keloid fibroblasts, and activation of both ERK1/2 and Akt is involved in the hypoxia-dependent HIF-1alpha protein accumulation and VEGF expression in the co-culture system. These findings suggest that under hypoxic conditions mast cells may contribute, at least in part, to an elevated expression of HIF-1alpha and VEGF protein in keloids via direct cell-cell interaction with fibroblasts.     

Involvement of PI3K and MAPK signaling in bcl-2-induced vascular endothelial growth factor expression in melanoma cells

We have previously demonstrated that bcl-2 overexpression in tumor cells exposed to hypoxia increases the expression of vascular endothelial growth factor (VEGF) gene through the hypoxia-inducible factor-1 (HIF-1). In this article, we demonstrate that exposure of bcl-2 overexpressing melanoma cells to hypoxia induced phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2 proteins. On the contrary, no modulation of these pathways by bcl-2 was observed under normoxic conditions. When HIF-1alpha expression was reduced by RNA interference, AKT and ERK1/2 phosphorylation were still induced by bcl-2. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways reduced the induction of VEGF and HIF-1 in response to bcl-2 overexpression in hypoxia. No differences were observed between control and bcl-2-overexpressing cells in normoxia, in terms of VEGF protein secretion and in response to PI3K and MAPK inhibitors. We also demonstrated that RNA interference-mediated down-regulation of bcl-2 expression resulted in a decrease in the ERK1/2 phosphorylation and VEGF secretion only in bcl-2-overexpressing cell exposed to hypoxia but not in control cells. In conclusion, our results indicate, for the first time, that bcl-2 synergizes with hypoxia to promote expression of angiogenesis factors in melanoma cells through both PI3K- and MAPK-dependent pathways.     

BRCA1 plays a role in the hypoxic response by regulating HIF-1alpha stability and by modulating vascular endothelial growth factor expression

A recent study of breast cancer patients with and without BRCA1 gene mutations found significantly lower levels of VEGF in serum from patients with BRCA1 mutations (Tarnowski, B., Chudecka-Glaz, A., Gorski, B., and Rzepka-Gorska, I. (2004) Breast Cancer Res. Treat. 88, 287-288). Here, we describe a possible mechanistic explanation for this correlation. Because hypoxia in tumors stimulates VEGF expression and secretion we hypothesized that altered BRCA1 protein levels in breast tumors could affect hypoxia-stimulated VEGF promoter activity. This possibility was tested in cells transfected with various combinations of expression plasmids for BRCA1, BRCA1 specific inhibitory RNAs (BRCA1-siRNAs), HIF-1alpha, and a VEGF promoter-reporter and then incubated in normoxia (21%, O2) or hypoxia (0.1%, O2). As predicted, increased BRCA1 levels enhanced both hypoxia-stimulated VEGF promoter activity and the amounts of VEGF mRNA, as determined by semiquantitative RT-PCR and quantitative real time PCR. Using the ChIP assay, we discovered that BRCA1 could be recruited to the endogenous human VEGF promoter along with HIF-1alpha following hypoxia. An interaction between BRCA1 and HIF-1alpha was found in human breast cancer cells. We also found that hypoxia-stimulated VEGF promoter activity and secretion was reduced in cells containing reduced amounts of endogenous BRCA1 protein (obtained by transfecting with BRCA1 siRNAs). A mechanistic explanation for these effects is provided by our finding a reduced half-life and reduced accumulation of HIF-1alpha in hypoxic cells transfected with BRCA1-siRNAs and that proteasome inhibitors blocked these effects of BRCA1-siRNAs. Thus, our results suggest that normal amounts of BRCA1 function in hypoxia to regulate HIF-1alpha stability, probably by interacting with HIF-1alpha.     

Evidence for a role of p38 kinase in hypoxia-inducible factor 1-independent induction of vascular endothelial growth factor expression by sodium arsenite

Recently we have demonstrated that sodium arsenite induces the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) protein and vascular endothelial growth factor (VEGF) in OVCAR-3 human ovarian cancer cells. We now show that arsenic trioxide, an experimental anticancer drug, exerts the same effects. The involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) pathways in the effects of sodium arsenite was investigated. By using kinase inhibitors in OVCAR-3 cells, both effects of sodium arsenite were found to be independent of phosphatidylinositol 3-kinase and p44/p42 MAPKS but were attenuated by inhibition of p38 MAPK. A role for p38 in the regulation of HIF-1alpha and VEGF expression was supported further by analysis of activation kinetics. Experiments in mouse fibroblast cell lines, lacking expression of c-Jun N-terminal kinases 1 and 2, suggested that these kinases are not required for induction of HIF-1alpha protein and VEGF mRNA. Unexpectedly, sodium arsenite did not activate a HIF-1-dependent reporter gene in OVCAR-3 cells, indicating that functional HIF-1 was not induced. In agreement with this hypothesis, up-regulation of VEGF mRNA was not reduced in HIF-1alpha(-/-) mouse fibroblast cell lines. Altogether, these data suggest that not HIF-1, but rather p38, mediates induction of VEGF mRNA expression by sodium arsenite.