VEGF is essential for vasculogenesis, development of the embryonic vasculture and is a potent angiogenic factor in pathological angiogenesis. Here we determined the effect a novel splice variant of VEGF, VEGF165b on VEGF-R2 receptor tyrosine kinase mediated phosphorylation of mitogen activated protein kinase (p42/p44 MAPK).
Chinese Hamster Ovary cells (CHOs) were transfected with either an empty vector (pcDNA3) or VEGF-R2 containing expression vector. Cells were incubated with media or with media containing 1 nM VEGF165, 1 nM VEGF165b, or 1 nM VEGF165 and 1 nM VEGF165b for 20 min at 37°C. Western blot analysis was used to determine whether VEGF165b inhibits VEGF-R2 induced phosphorylation of MAPK by using specific antibodies against phospho-p44/p42 MAP kinases.
A basal level p44/p42 MAP kinase phosphorylation was detected in pcDNA3 transfected cells, regardless of treatment. A similar p44/p42 MAP kinase phosphorylation level was observed in VEGF-R2 transfected cells that were incubated in the absence of a VEGF isoform (control). As expected, VEGF165 increased the intensity of the phosphorylated polypeptide band by mean ± S.E.M. 222 ± 60 % of the untreated cells. In contrast, when VEGF-R2 transfected cells were treated with VEGF165b, only a small increase (122 ± 42 %) in the level of p44/p42 MAPK kinase phosphorylation was detected compared to control. This was significantly lower than that of VEGF165-induced stimulation (paired t test, P < 0.05). Treatment of VEGF-R2 transfected cells with a combination of both VEGF165 and VEGF165b, resulted in an 190 ± 56 % increase in the level of p44/p42 MAP Kinase phosphorylation compared to the control.
These results show that the intensity of VEGF165b-induced phosphorylation of the signalling proteins p44/p42 MAP kinases is significantly less than that detected for VEGF165-induced stimulation. Furthermore, combination of VEGF165 and VEGF165b-induced phosphorylation of p44/p42 MAP kinases was 32 % less than that detected for VEGF165-induced stimulation. VEGF165b therefore appears to be able to inhibit VEGF mediated signalling through VEGF-R2.
This work was supported by Wellcome Trust Grant 69029