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Regulation of the Expression and Function of Exchange Protein Directly Activated by cAMP (EPAC) by Hypoxia
Author(s) -
Olah Mark,
Justus Lillian,
Kiefer Erika,
Shelley Jessica
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.1060.8
Subject(s) - downregulation and upregulation , forskolin , medicine , hypoxia (environmental) , endocrinology , rap1 , adenylyl cyclase , microbiology and biotechnology , protein kinase b , chemistry , signal transduction , rhoa , guanine nucleotide exchange factor , biology , receptor , biochemistry , oxygen , organic chemistry , gene
Exchange protein directly activated by cAMP (EPAC) is a signaling protein implicated in several physiologic and pathophysiologic processes. Following binding of cAMP, EPAC activates small GTPases such as Rap1 to induce downstream effects. In vascular endothelial cells, EPAC may have a role in the regulation of permeability, vascular tone and angiogenesis. As low oxygen tension initiates many vascular events including those modulated by EPAC, we hypothesized that hypoxia regulates EPAC expression and function in human microvascular endothelial cells (HMVEC). HMVEC maintained in 2.0% oxygen for 48 hours demonstrated a significant decrease in expression of EPAC protein relative to normoxic cells or HMVEC exposed to hypoxia for shorter periods. At 48 hours of hypoxia, EPAC expression was 53.9 ± 7.4% of that in normoxic cells. To begin to delineate the mechanism underlying this downregulation of EPAC, we examined the role of the transcription factor hypoxia‐inducible factor‐1α (HIF‐1α). Deferoxamine, an iron chelator, and IOX‐2, an inhibitor of prolyl hydroxylase, both increased expression of HIF‐1α at short time periods as did exposure to 2.0% oxygen. At 48 hours of treatment, deferoxamine and IOX‐2 mimicked the effects of hypoxia by decreasing EPAC expression to levels 40.5 ± 12.1% and 43.0 ± 12.7% of control, respectively. To assess the functional significance of hypoxia‐induced downregulation of EPAC, we examined activation of the downstream effector Akt by determining levels of phospho‐Akt (Ser473) by Western blotting. The A2B adenosine receptor agonist 5′‐N‐ethylcarboxamidoadenosine (NECA) and the adenylyl cyclase activator forskolin, both of which increase intracellular cAMP levels, as well as the EPAC specific activator 8‐(4‐chlorophenylthio)‐2′‐O‐methyladenosine 3′,5′‐cyclic monophosphate (8‐CPT) induced a decreased Akt stimulation in hypoxic HMVEC relative to normoxic cells. In conclusion, hypoxia downregulates protein expression of EPAC in HMVEC and this may be mediated by HIF‐1α. This decrease may result in reduced EPAC‐mediated Akt activation. Studies are currently further exploring the mechanism of the hypoxia‐induced downregulation of EPAC and examining effects on Akt‐mediated functional responses such as cell survival. Support or Funding Information Supported by funds from The Raabe College of Pharmacy, Ohio Northern University