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J. Biol. Chem., Vol. 283, Issue 6, 3365-3375, February 8, 2008
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¶||1
From the
||Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29401, the Departments of ¶Medicine and
Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29403, and the
Department of Medical Chemistry, Biochemistry, and Biotechnology, University of Milan, Segrate, Milan 20090, Italy
Sphingosine 1-phosphate (S1P), a sphingolipid metabolite that plays an important role in the regulation of cell survival, growth, migration, and angiogenesis, acts both inside the cells and as an extracellular mediator through binding to five G protein-coupled receptors (S1P1-5). Sphingosine kinase 1 (SK1), the enzyme responsible for S1P production, is overexpressed in many solid tumors, including gliomas. One common feature of these tumors is the presence of "hypoxic regions," characterized by cells expressing high levels of hypoxia-inducible factors HIF-1
and HIF-2
, two transcription regulators that modulate the levels of proteins with crucial roles in tumor progression. So far, nothing is known about the role and the regulation of SK1 during tumor-induced hypoxia or about SK1 regulation and HIFs. Here we investigated the role of HIF-1
and HIF-2
in the regulation of SK1 during hypoxic stress in glioma-derived U87MG cells. We report that hypoxia increases SK1 mRNA levels, protein expression, and enzyme activity, followed by intracellular S1P production and S1P release. Interestingly, knockdown of HIF-2
by small interfering RNA abolished the induction of SK1 and the production of extracellular S1P after CoCl2 treatment, whereas HIF-1
small interfering RNA resulted in an increase of HIF-2
and of SK1 protein levels. Moreover, using chromatin immunoprecipitation analysis, we demonstrate that HIF-2
binds the SK1 promoter. Functionally, we demonstrate that conditioned medium from hypoxia-treated tumor cells results in neoangiogenesis in human umbilical vein endothelial cells in a S1P receptor-dependent manner. These studies provide evidence of a link between S1P production as a potent angiogenic agent and the hypoxic phenotype observed in many tumors.
Received for publication, October 3, 2007 , and in revised form, November 30, 2007.
* This work was supported in part by NCI, National Institutes of Health (NIH), Grant P01 CA097132, NIGMS, NIH, Grant R01 GM062887, and a MERIT Award (to L. M. O.) from the Office of Research and Development, Department of Veterans Affairs, Ralph H. Johnson Veterans Affairs Medical Center (Charleston, SC). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.
1 To whom correspondence should be addressed: Medical University of South Carolina, Dept. of Medicine, Division of General Internal Medicine/Geriatrics, P.O. Box 250779, Charleston, SC 29403. Tel.: 843-876-5169; Fax: 843-876-5172; E-mail: obeid{at}musc.edu.
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