| Περίληψη: | Angiogenesis is a major aspect of tumor biology. Expanding tumors soon become hypoxic and demand new blood supply in order to support their rapid growth and to promote metastasis. Hypoxia and HIFs are the main factors involved in the expression of genes that turn on the 'angiogenic switch'. Among these, VEGF-A and its receptors, VEGFR1 and VEGFR2 are the most well known regulators of angiogenesis in both normoxic and hypoxic conditions. Our group has previously shown that VEGF165 binds to the receptor RPTPβ/δ, leading to phosphorylation of the β3 subunit of integrin αvβ3 at Tyr773 and cell membrane localization of the multifunctional protein NCL. Cell surface NCL is glycosylated and mediates the stimulatory effect of several angiogenic factors, including VEGF and PTN. PTN is a heparin-binding growth factor that was initially found to promote neurite outgrowth in the rat brain during late development. PTN has been found to be over-expressed by many human malignancies and is involved in tumor angiogenesis. Despite its important biological actions, little is known on the regulation of PTN expression. Our group has previously shown that FGF-2, ROS and NO induce PTN expression and secretion through transcriptional activation of ptn promoter by AP-1. RPTPβ/δ is the main receptor involved in PTN-induced migration of endothelial and cancer cells. Under some circumstances, PTN may also act as an angiostatic factor: PTN is found to directly interact with VEGF165 and inhibit its effect on endothelial cell proliferation and tube formation. In addition, PTN antisense expression increases angiogenesis both in vitro and in vivo. In the present study, we examined the effect of hypoxia and VEGF on PTN expression and function by human endothelial and cancer cells. We showed by using Western blot analyses, quantitative Real Time PCR and luciferase assays that acute and chemical hypoxia increase PTN expression and secretion through transcriptional activation of the corresponding gene. Using decoy oligonucleotide technology, we showed that in hypoxia, this effect is mediated by HIFs and partially through AP-1. The inductive effect of hypoxia on PTN secretion was not VEGF-mediated since VEGF decreased PTN secretion both in hypoxia and normoxia. Boyden assays in PTN knock-down cancer cells showed that PTN down-regulation enhances hypoxia-induced cell migration and RPTPβ/δ is not involved in this effect. By using Western blot analyses, quantitative Real Time PCR and luciferase assays it was shown that VEGF decreased PTN expression and secretion through transcriptional inhibition of the corresponding gene. Treatment of cells with the monoclonal antibody bevacizumab showed that the receptor-binding domain of VEGF is not involved in its inhibitory effect on PTN expression and moreover, bevacizumab by itself may increase PTN secretion. HB-19 peptide that targets cell surface NCL inhibited VEGF effect on PTN secretion in human endothelial cells. However, HB-19 inhibited by itself both PTN secretion and ptn transcription and VEGF did not cause any further decrease in glioblastoma cells. VEGF-induced inhibition of PTN secretion depended on the presence or phosphorylation of the β3 subunit of integrin αvβ3 at Tyr773. The functional antibody LM-609 which does not inhibit cell surface localization of NCL failed to abolish the effect of VEGF on PTN secretion. Down-regulation of RPTPβ/δ by siRNA which inhibits Tyr773 phosphorylation abolished the inhibitory effect of VEGF on PTN secretion. Collectively, these data suggest that RPTPβ/δ-mediated αλβ3-induced cell surface NCL localization is involved in the inhibitory effect of VEGF on PTN secretion. In order to find the possible functional significance of these data, we tested the effect of PTN in VEGF-induced endothelial cell migration and found that it decreased migration to the levels of its own effect, while PTN112-136 completely abolished the stimulatory effect on cell migration. These data are in line with data from immunoprecipitation/Western blot analyses showing that VEGF binding to RPTPβ/δ could be abolished by either PTN or PTN112-136. The migratory effect of VEGF could be also inhibited by HB-19 and CS-E, which could be explained by the fact that they also inhibit VEGF-NCL interaction, as is already known for HB-19 and shown in the present work by proximity ligation assays. Collectively, these data suggest that hypoxia increases whereas VEGF decreases PTN expression and secretion. In both cases, PTN seems to play an important role in limiting hypoxia and/or VEGF induced angiogenic activities, acting as an endogenous break of VEGF-induced aberrant angiogenesis. Mechanistically, the effect of hypoxia is both HIF- and AP-1-dependent and the effect of VEGF seems to be mediated by cell surface nucleolin and to require RPTPβ/δ-mediated αλβ3 Tyr775 phosphorylation. PTN112-136, HB-19 and CS-E inhibit interaction of VEGF with RPTPβ/δ or NCL and completely inhibit the migratory effect of VEGF on endothelial cells, suggesting that they could be further exploited as potential anti-angiogenic agents.
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