Inducible Expression of Endothelial PAS Domain Protein-1 by Hypoxia in Human Lung Adenocarcinoma A549 Cells

Abstract

Hypoxia is a potent inducer of tumor angiogenesis, the process of which is mostly mediated by induction of vascular endothelial growth factor (VEGF). In this study, we investigated the effect of hypoxia on the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and endothelial PAS domain protein-1 (EPAS1). These two similar but distinct basic helix-loop-helix-PAS proteins have been postulated to activate VEGF expression in response to hypoxia. We showed that EPAS1, but not HIF-1alpha, is abundantly expressed in human lung adenocarcinoma A549 cells. Exposure of cultured A549 cells to hypoxia increased EPAS1 mRNA and protein levels. A specific inhibitor for Src family kinases, PP1, abolished the hypoxia-induced expression of EPAS1. Transient transfection assays revealed that forced expression of EPAS1 increased the reporter gene activity driven by EPAS1 promoter as well as by VEGF promoter. Finally, overexpression of EPAS1 by infection of adenoviral vector expressing EPAS1 cDNA evidently induced the endogenous EPAS1 gene expression. Together, these data demonstrate Src family kinases mediate the hypoxia-mediated EPAS1 gene expression, which in turn positively autoregulates its own expression. Given an EPAS1 as a potent activator of the VEGF gene, these findings will provide a novel insight into the mechanisms underlying the enhancement of growth property of EPAS1-expressing tumor cells under the hypoxic environment.

Hypoxia upregulates hypoxia inducible factor (HIF)-3α expression in lung epithelial cells: characterization and comparison with HIF-1α

“…This study demonstrates that when cells were exposed to 1% oxygen for 2 h, a major increase in HIF-3α and HIF-1α protein was observed, whereas HIF-1α protein levels decreased from 8 to 16 h. In addition to the decrease in HIF-1α protein, hypoxia also induced a decrease in HIF-1α mRNA. These observations in agreement with some previous reports showing that HIF-1α protein and mRNA levels decreased during sustained hypoxia stimulation in alveolar epithelial cells and many other cell lines [20,21,23,35]. On the other hand, the protein levels HIF-3α exposed to hypoxia appear to have peaked over 8-16 h, while the mRNA levels peaked from 4 h to 16 h. One possible explanation for such a discrepancy between mRNA and protein levels is that an inducible expression of HIF-3α protein by hypoxia is regulated at the protein level as well as at the mRNA levels.…”

“…The presence of HIF-1α and HIF-2α in lung alveolar epithelial cells has been reported in separate studies, suggesting that the two isoforms are co-expressed [20,21]. The hypoxic induction of HIF-2α has been demonstrated in vivo and in vitro in different organs under various conditions [22,23].…”

Gene regulation in the adaptive process to hypoxia in lung epithelial cells

“…Interestingly, the temporal characteristics and responses of HIF-1␣ expression to varying O 2 concentrations were remarkably similar in vivo and in vitro. The presence of HIF-2␣ and of HIF-3␣, in alveolar epithelial cells, has been evidenced more recently (28,48,58). HIF-2␣ was detected in primary and A549 alveolar epithelial cells (58), whereas at this time HIF-3␣ was reported in A549 cells (28).…”

“…Interestingly, the three isoforms were reported to be differentially regulated during prolonged hypoxia. When A549 cells were exposed to more than 6 h of severe hypoxia, HIF-1␣ protein stimulation disappeared, whereas HIF-2␣ protein level remained high and stable (48,58). Also, a recent study showed that HIF-3␣ remained stable during sustained hypoxia in A549 cells in contrast to HIF-1␣ (28).…”

Rho Guanine Nucleotide Exchange Factor 5 Increases Lung Cancer Cell Tumorigenesis via MMP-2 and Cyclin D1 Upregulation

“…Src may stimulate tumorigenesis in NSCLC in a variety of ways, for example, affecting the STAT-3 and focal adhesion kinase-related pathways, both of which are involved in tumor survival (25,26). Indeed, Src activates the VEGF pathway via STAT-3 (27) in response to hypoxia in human lung adenocarcinoma cells, thus increasing blood supply to the oxygenstarved tumors (28).…”