Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation

Abstract

Angiogenesis, the formation of new microvasculature by capillary sprouting, is crucial for tumour development. Hypoxic regions of solid tumours produce the powerful and directly acting angiogenic protein VEGF/VPF (vascular endothelial growth factor/vascular permeability factor). We now investigate the signal transduction pathway involved in hypoxic induction of VEGF expression. Hypoxia is known to induce a tyrosine kinase cascade that results in the activation of nitrogen-fixation genes in Rhizobium meliloti, and activation of tyrosine kinases is critical in signalling triggered by growth factors and ultraviolet light. We show here that genistein, an inhibitor of protein tyrosine kinase, blocks VEGF induction. Hypoxia increases the kinase activity of pp60c-src and its phosphorylation on tyrosine 416 but does not activate Fyn or Yes. Expression of either a dominant-negative mutant form of c-Src or of Raf-1 markedly reduces VEGF induction. VEGF induction by hypoxia in c-src(-) cells is impaired, although there is a compensatory activation of Fyn. Our results provide an insight into hypoxia-triggered intracellular signalling, define VEGF as a new downstream target for c-SRC, and suggest a role for c-SRc in promoting angiogenesis.

Development and malignant progression of astrocytomas in GFAP-v-src transgenic mice

“…In situ hybridization analysis revealed expression of VEGF in single astrocytes in the brains of transgenic mice already at early stages (Figure 6f). This in vivo result extends earlier observations showing transactivation of the VEGF promoter by v-Src in U87 and 293 cells (Mukhopadhyay et al, 1995a) in vitro. However, in preneoplastic lesions and tumors, expression of VEGF was widespread and most prominent in pseudopalisading cells surrounding necrotic areas, suggesting additional upregulation by hypoxia (Figure 6c).…”

Sp1 recognition sites in the proximal promoter of the human vascular endothelial growth factor gene are essential for platelet-derived growth factor-induced gene expression

“…Enhanced VEGF expression has been reported upon activation of protein kinase C by tumor promoters, and in cells harbouring activated oncogenes, such as ras, raf, and src (Grugel et al, 1995;Rak et al, 1995;Mukhopadhyay et al, 1995). In addition, hypoxia, as well as inactivation of p53 and of von Hippel-Lindau tumor suppressor genes, respectively, result in elevated VEGF mRNA levels (Schweiki et al, 1992;Finkenzeller et al, 1995;Kieser et al, 1994;Siemeister et al, 1996).…”

Modeling malignant melanoma in mice: pathogenesis and maintenance

“…Speci®cally, although a decline in VEGF mRNA and protein was associated with a decrease in RAS activity following doxycycline withdrawal in cell culture, an initial increase in VEGF was observed in vivo following (Chin et al, 1999). These rising levels of VEGF are coincident with the initial stages of regression and tumor collapse and thus point to RASindependent mechanisms of VEGF stimulation, such as hypoxia-induced VEGF upregulation (Schweiki et al, 1992;Goldberg and Schneider, 1994;Mukhopadhyay et al, 1995;Mazure et al, 1996). Furthermore, to determine whether sustained VEGF expression can rescue the tumor regression phenotype, we have generated retrovirally transduced-VEGF expressing doxycycline-responsive melanoma cell line.…”