EPAS1 trans-Activation during Hypoxia Requires p42/p44 MAPK*
- From the University of Cincinnati, College of Medicine, Department of Molecular and Cellular Physiology, Cincinnati, Ohio 45267-0576
Abstract
Hypoxia is a common environmental stress that regulates gene expression and cell function. A number of hypoxia-regulated transcription factors have been identified and have been shown to play critical roles in mediating cellular responses to hypoxia. One of these is the endothelial PAS-domain protein 1 (EPAS1/HIF2-α/HLF/HRF). This protein is 48% homologous to hypoxia-inducible factor 1-α (HIF1-α). To date, virtually nothing is known about the signaling pathways that lead to either EPAS1 or HIF1-α activation. Here we show that EPAS1 is phosphorylated when PC12 cells are exposed to hypoxia and that p42/p44 MAPK is a critical mediator of EPAS1 activation. Pretreatment of PC12 cells with the MEK inhibitor, PD98059, completely blocked hypoxia-inducedtrans-activation of a hypoxia response element (HRE) reporter gene by transfected EPAS1. Likewise, expression of a constitutively active MEK1 mimicked the effects of hypoxia on HRE reporter gene expression. However, pretreatment with PD98059 had no effect on EPAS1 phosphorylation during hypoxia, suggesting that MAPK targets other proteins that are critical for thetrans-activation of EPAS1. We further show that hypoxia-induced trans-activation of EPAS1 is independent of Ras. Finally, pretreatment with calmodulin antagonists nearly completely blocked both the hypoxia-induced phosphorylation of MAPK and the EPAS1 trans-activation of HRE-Luc. These results demonstrate that the MAPK pathway is a critical mediator of EPAS1 activation and that activation of MAPK and EPAS1 occurs through a calmodulin-sensitive pathway and not through the GTPase, Ras. These results are the first to identify a specific signaling pathway involved in EPAS1 activation.
Footnotes
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↵* This work was supported by National Institutes of Health Grants R37HL33831 and RO1HL59945 (to D. E. M.), United States Army Grant DAMD179919544 (to D. E. M.), Grant 9806242 from the American Heart Association (Ohio Valley Affiliate) and a grant from the Parker B. Francis Foundation (to D. B. J.), and a National Institutes of Health Training Grant HL07571 (to P. W. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. of Molecular and Cellular Physiology, University of Cincinnati, College of Medicine, P. O. Box 67-0576, Cincinnati, Ohio 45267–0576. Tel.: 513-558-5636; Fax: 513-558-5738; E-mail: david.millhorn@uc.edu.
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↵2 D. Beitner-Johnson and D. E. Millhorn, unpublished observations.
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↵3 T. L. Freeman, unpublished observation.
- Abbreviations:
- HIF1-α
-
hypoxia-inducible factor
- CREB
-
cyclic-AMP response element-binding protein
- EPAS1
-
endothelial PAS-domain protein
- HLF
-
HIF-like factor
- HRF
-
HIF-related factor
- HRE
-
hypoxia response element
- PC12
-
pheochromocytoma
- MAPK
-
mitogen-activated protein kinase
- SAPK
-
stress-activated protein kinase
- DMEM
-
Dulbecco's modified Eagle's medium
- NGF
-
nerve growth factor
- CMZ
-
calmidazolium chloride
- CBP
-
CREB-binding protein
- VHL
-
von Hippel Lindau
- PAGE
-
polyacrylamide gel electrophoresis
-
- Received June 18, 1999.
- Revision received August 20, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
