HIF-1alpha, HIF-2alpha and VHL mRNA expression in different cell lines during hypoxia

Hypoxia inducible factor-1alpha (HIF-1alpha) mRNA expression is significantly decreased under hypoxia in different cell lines exposed directly to hypoxia or treated with dimethyloxalylglycine which mimics hypoxic effects under normoxic conditions. However, the decreased expression of HIF-1alpha mRNA is accompanied by an increase of HIF-1alpha protein (pHIF-1alpha) level as well as by overexpression of known HIF-dependent genes (VEGF, Glut1, PFKFB-3 and PFKFB-4) under hypoxic conditions or with the use of dimethyloxalylglycine. Expression of HIF-1alpha mRNA also depends on iron because desferrioxamine and cobalt chloride produce similar to hypoxia effects on the levels of this mRNA. It was shown that HIF-1alpha mRNA expression did not change significantly in some cell lines (SKBR3, MDA-MB468 and BT549) under hypoxia. However, in these cell lines hypoxia decreases expression of HIF-2alpha mRNA, another member of HIF-alpha gene family, as a result of cell specific regulation of HIF-alpha genes under hypoxia. Moreover, hypoxia slightly induces expression of PFKFB-4 mRNA in SKBR3, MDA-MB468 and BT549 as compared to other cell lines where this effect of hypoxia was much stronger and adaptation to hypoxia is controlled by HIF-1alpha. Hypoxia slightly reduces expression of tumor suppressor VHL which targets HIF-1alpha for ubiquitination. Thus, our results clearly demonstrated down regulation of HIF-1alpha or HIF-2alpha in different cell lines by hypoxia.     

Induction of hypoxia-inducible-factor 1 by nitric oxide is mediated via the PI 3K pathway

Adaptation to hypoxic stress provokes activation of the hypoxia-inducible-factor-1 (HIF-1) which mediates gene expression of, e.g., erythropoietin or vascular endothelial growth factor. Detailed information on signaling pathways that stabilize HIF-1 is missing, but reactive oxygen species degrade the HIF-1 alpha subunit, whereas phosphorylation causes its stabilization. It was believed that hypoxia resembles the only HIF-1 inducer but recent evidence characterized other activators of HIF-1 such as nitric oxide (NO). Herein, we concentrated on NO-evoked HIF-1 induction as a heretofore unappreciated inflammatory response in association with massive NO formation. We demonstrated that S-nitrosoglutathione induces HIF-1 alpha accumulation and concomitant DNA binding. The response was attenuated by the kinase inhibitor genistein and blockers of phosphatidylinositol 3-kinase such as Ly 294002 or wortmannin. Whereas mitogen-activated protein kinases were not involved, we noticed phosphorylation/activation of Akt in correlation with HIF-1 alpha stabilization. NO appears to regulate HIF-1 alpha via the PI 3K/Akt pathway under normoxic conditions.     

Insulin-like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression,which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells

Stimulation of human colon cancer cells with insulin-like growth factor 1 (IGF-1) induces expression of the VEGF gene, encoding vascular endothelial growth factor. In this article we demonstrate that exposure of HCT116 human colon carcinoma cells to IGF-1 induces the expression of HIF-1 alpha, the regulated subunit of hypoxia-inducible factor 1, a known transactivator of the VEGF gene. In contrast to hypoxia, which induces HIF-1 alpha expression by inhibiting its ubiquitination and degradation, IGF-1 did not inhibit these processes, indicating an effect on HIF-1 alpha protein synthesis. IGF-1 stimulation of HIF-1 alpha protein and VEGF mRNA expression was inhibited by treating cells with inhibitors of phosphatidylinositol 3-kinase and MAP kinase signaling pathways. These inhibitors also blocked the IGF-1-induced phosphorylation of the translational regulatory proteins 4E-BP1, p70 S6 kinase, and eIF-4E, thus providing a mechanism for the modulation of HIF-1 alpha protein synthesis. Forced expression of a constitutively active form of the MAP kinase kinase, MEK2, was sufficient to induce HIF-1 alpha protein and VEGF mRNA expression. Involvement of the MAP kinase pathway represents a novel mechanism for the induction of HIF-1 alpha protein expression in human cancer cells.     

Hypoxia-inducible Factor-1-mediated Regulation of Semaphorin 4D Affects Tumor Growth and Vascularity

Tumor progression and metastasis depend on the ability of cancer cells to initiate angiogenesis to ensure delivery of oxygen, nutrients, and growth factors to tumor cells and provide access to the systemic circulation. Hypoxia-inducible factor-1 (HIF-1) can activate expression of a broad range of genes that mediate many of the adaptive responses to decreased oxygen concentration, such as enhanced glucose uptake and formation of new blood vessels. Acting through Plexin-B1 on endothelial cells, Semaphorin 4D (Sema4D) has been shown to promote angiogenesis and enhance invasive growth and proliferation in some tumors. Here we show that the gene for Sema4D, the product of which is elevated in head and neck squamous cell carcinoma (HNSCC) cells, contains upstream hypoxia response elements (HRE) and is strongly induced in hypoxia in a HIF-1-dependent manner. Knocking down Sema4D expression with short hairpin (sh) RNA reduces in vitro endothelial cell migration and growth and vascularity of HNSCC xenografts expressing a degradation resistant HIF-1α subunit. We also demonstrate a correlation between HIF-1 activity and Sema4D expression in HNSCC specimens. These findings indicate that Sema4D is induced by hypoxia in a HIF-1-dependent manner and influences endothelial cell migration and tumor vascularity. Expression of Sema4D may be a strategy by which carcinomas promote angiogenesis and therefore could represent a therapeutic target for these malignancies.