Abstract
This study examines the relative impact of canonical hypoxia-inducible factor-1alpha- (HIF-1α and Na+ i/K+ i-mediated signaling on transcriptomic changes evoked by hypoxia and glucose deprivation. Incubation of RASMC in ischemic conditions resulted in ∼3-fold elevation of [Na+]i and 2-fold reduction of [K+]i. Using global gene expression profiling we found that Na+,K+-ATPase inhibition by ouabain or K+-free medium in rat aortic vascular smooth muscle cells (RASMC) led to the differential expression of dozens of genes whose altered expression was previously detected in cells subjected to hypoxia and ischemia/reperfusion. For further investigations, we selected Cyp1a1, Fos, Atf3, Klf10, Ptgs2, Nr4a1, Per2 and Hes1, i.e. genes possessing the highest increments of expression under sustained Na+,K+-ATPase inhibition and whose implication in the pathogenesis of hypoxia was proved in previous studies. In ouabain-treated RASMC, low-Na+, high-K+ medium abolished amplification of the [Na+]i/[K+]i ratio as well as the increased expression of all tested genes. In cells subjected to hypoxia and glucose deprivation, dissipation of the transmembrane gradient of Na+ and K+ completely eliminated increment of Fos, Atf3, Ptgs2 and Per2 mRNAs and sharply diminished augmentation expression of Klf10, Edn1, Nr4a1 and Hes1. In contrast to low-Na+, high-K+ medium, RASMC transfection with Hif-1a siRNA attenuated increments of Vegfa, Edn1, Klf10 and Nr4a1 mRNAs triggered by hypoxia but did not impact Fos, Atf3, Ptgs2 and Per2 expression. Thus, our investigation demonstrates, for the first time, that Na+ i/K+ i-mediated, Hif-1α- -independent excitation-transcription coupling contributes to transcriptomic changes evoked in RASMC by hypoxia and glucose deprivation.
Highlights
Hypoxia is characteristic of numerous pathologies, including inflammation [1], cancer [2], obesity [3], systemic and pulmonary hypertension [4;5], atherosclerosis [6] and kidney disease [7]
These results allowed us to hypothesize that transcriptomics changes triggered by hypoxia are at least partially caused by Na+i/K+i-mediated excitation-transcription coupling discovered in our recent studies [26]
Do these transcription factors contribute to [Na+]i/[K+]i-mediated transcriptomic changes evoked by hypoxia? We found a negligible impact of Na+,K+-ATPase inhibition on Hif-1b expression and 2fold elevation of mRNA encoding aryl hydrocarbon receptors for dioxins, benzopyrenes and other environmental pollutions (AhR) (Table 3)
Summary
Hypoxia is characteristic of numerous pathologies, including inflammation [1], cancer [2], obesity [3], systemic and pulmonary hypertension [4;5], atherosclerosis [6] and kidney disease [7]. The protective action of brief ischemia was documented in other tissues, including blood vessels [9]. The prophylactic influence of ischemic preconditioning was at least partially blocked by inhibitors of RNA synthesis [10;11], suggesting a key role of profound transcriptomic changes documented in global gene expression profiling studies of ischemic tissues [12,13,14,15,16,17,18]. Hypoxia-inducible factor 1alpha (HIF-1a), considered to be a major oxygen sensor, regulates gene expression in ischemic tissues via interaction of HIF-1a/HIF-1b heterodimer with hypoxia response elements (HREs) in promoter/enhancer regions of the target gene’s DNA. The list of HIF-1-sensitive genes comprises Hif-1a per se, and others related to vasomotor control (nitric oxide synthase-2, adrenomedulin, endothelin-1), angiogenesis (vascular endothelial growth factor (Vegf) and its receptor Flt1), erythropoiesis and iron metabolism (erythropoietin, transferrin, transferrin receptor, ceruloplasmin), cell proliferation (Igf, Igfbp, Tgfb), energy metabolism (glucose transporters Glut1-Glut, phosphoenolpyruvate carboxylase, lactate dehydrogenase A, aldose, phosphoglucokinase-1, -L and -C, endolase, tyrosine hydroxylase and plasminogen activator inhibitor-1)
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