EPAS1 trans-Activation during Hypoxia Requires p42/p44 MAPK

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-␣/HLF/ HRF). This protein is 48% homologous to hypoxia-inducible factor 1-␣ (HIF1-␣). To date, virtually nothing is known about the signaling pathways that lead to either EPAS1 or HIF1-␣ 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 hypoxiainduced trans-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 the trans-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.Regulation of gene expression is a primary response by which cells adapt to changes in the environment. The mechanisms involved in regulation of gene expression in response to hypoxia are beginning to be understood. Transcription factors that are activated by hypoxia include the hypoxia-inducible factor (HIF1-␣), 1 c-fos, and CREB (1-4). HIF1-␣ has been shown to be critical for hypoxia-induced regulation of a number of genes, including glycolytic enzymes, vascular endothelial growth factor, and erythropoieitin (5-7). Recently, endothelial PAS-domain protein 1 (EPAS1, also known as HIF2-␣, HLF, and HRF) was identified as a hypoxia-inducible transcription factor (8 -10). EPAS1 is a basic helix-loop-helix transcription factor, which shares 48% sequence identity with HIF1-␣ (8). EPAS1 protein levels, like HIF1-␣ levels, are relatively low under basal conditions and accumulate upon exposure of cells to hypoxia (11). These factors then translocate to the nucleus and trans-activate target genes containing the sequence 5Ј-GCCCTACGTGCTGTCTCA-3Ј, which is commonly referred to as the hypoxia response element (HRE) (8, 12). EPAS1 is expressed in many tissues and is particularly abundant in the type I oxygen-sensing cells of the carotid body (13). Type I cells act as the primary O 2 sensors in mammals and are responsible for matching changes in art...

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Hypoxia Induces the Activation of the Phosphatidylinositol 3-Kinase/Akt Cell Survival Pathway in PC12 Cells

“…Hypoxia-inducible factors are the best characterized; others include cAMP response element-binding protein (4), NF-B (36), and c-Fos (2). Although we cannot formally rule out that hypoxia-inducible transcription factors are involved in Akt activation, indirect evidence suggests that it is not the case, because both calmodulin antagonists and the MEK inhibitor PD98059 prevent EPAS activity in PC12 cells (25) without affecting hypoxia-induced Akt activation.…”

“…2 Finally, we tested whether other signaling pathways contributed to Akt phosphorylation. The calmodulin-regulated and mitogen-activated protein kinase (MAPK) cascades are activated by hypoxia and mediate some of its effects in PC12 cells (25)(26)(27). Inhibition of MAPK/extracellular signal-regulated kinase kinase (MEK), one of the upstream kinases in the MAPK cascade, with PD59098 or treatment with antagonists of calmodulin signaling (W12 and W13 2 ; calmidazolium) had no effect on Akt phosphorylation induced by hypoxia (Fig.…”

A variant of nuclear localization signal of bipartite-type is required for the nuclear translocation of hypoxia inducible factors (1α, 2α and 3α)

“…Thus, the HIF complex formation and transcription-activating function mainly depend on the regulation of the protein stability and nuclear entry of HIFa subunits. Progress has been made in understanding the protein stability and the transcriptional activation of the HIFa subunits during hypoxia, but the mechanism underlying its nuclear import is poorly understood (Conrad et al, 1999;Ema et al, 1999).…”