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Transcriptomic changes triggered by hypoxia: evidence for HIF-1α-independent, [Na+]i/[K+]i-mediated, excitation-transcription coupling.

Koltsova SV, Shilov B, Birulina JG, Akimova OA, Haloui M, Kapilevich LV, Gusakova SV, Tremblay J, Hamet P, Orlov SN - PLoS ONE (2014)

Bottom Line: Incubation of RASMC in ischemic conditions resulted in ∼3-fold elevation of [Na+]i and 2-fold reduction of [K+]i.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Moscow State University, Moscow, Russia; Department of Medicine, Centre de recherche, Centre hospitalier de l'Université de Montréal, Montreal, Quebec, Canada.

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.

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Effect of ouabain and hypoxia on intracellular Na+, K+ and ATP concentrations.RASMC were incubated for 24 hr under normal oxygen partial pressure (5% CO2/air - control) ±3 µM ouabain or exposure to hypoxia (5% CO2/95% N2)/glucose deprivation in normal high-Na+, low-K+ ([Na+]o/[K+]o = 140/5) or in low-Na+, high-K+ DMEM-like medium ([Na+]o/[K+]o = 131/115). Means ± S.E. from 3 independent experiments performed in quadruplicate are shown. *p<0.05 compared to the controls.
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pone-0110597-g002: Effect of ouabain and hypoxia on intracellular Na+, K+ and ATP concentrations.RASMC were incubated for 24 hr under normal oxygen partial pressure (5% CO2/air - control) ±3 µM ouabain or exposure to hypoxia (5% CO2/95% N2)/glucose deprivation in normal high-Na+, low-K+ ([Na+]o/[K+]o = 140/5) or in low-Na+, high-K+ DMEM-like medium ([Na+]o/[K+]o = 131/115). Means ± S.E. from 3 independent experiments performed in quadruplicate are shown. *p<0.05 compared to the controls.

Mentions: Figure 2 shows that 24-hr incubation of RASMC in hypoxia and glucose starvation decreased intracellular ATP content by ∼3-fold whereas ouabain attenuated this parameter by less than 20%. The actions of hypoxia and ouabain on ATP content were preserved in low-Na+, high-K+ medium. Treatment with ouabain resulted in almost 10-fold gain of [Na+]i and virtually similar loss of [K+]i. In hypoxic conditions, [Na+]i and [K+]i were increased and decreased by 3- and 2-fold, respectively. As predicted, dissipation of the transmembrane gradients of monovalent cations in low-Na+, high-K+ medium almost completely abolished the actions of ouabain and hypoxia on the [Na+]i/]K+]i ratio (Fig. 2). Viewed collectively, 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]. Data considered below support this hypothesis.


Transcriptomic changes triggered by hypoxia: evidence for HIF-1α-independent, [Na+]i/[K+]i-mediated, excitation-transcription coupling.

Koltsova SV, Shilov B, Birulina JG, Akimova OA, Haloui M, Kapilevich LV, Gusakova SV, Tremblay J, Hamet P, Orlov SN - PLoS ONE (2014)

Effect of ouabain and hypoxia on intracellular Na+, K+ and ATP concentrations.RASMC were incubated for 24 hr under normal oxygen partial pressure (5% CO2/air - control) ±3 µM ouabain or exposure to hypoxia (5% CO2/95% N2)/glucose deprivation in normal high-Na+, low-K+ ([Na+]o/[K+]o = 140/5) or in low-Na+, high-K+ DMEM-like medium ([Na+]o/[K+]o = 131/115). Means ± S.E. from 3 independent experiments performed in quadruplicate are shown. *p<0.05 compared to the controls.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222758&req=5

pone-0110597-g002: Effect of ouabain and hypoxia on intracellular Na+, K+ and ATP concentrations.RASMC were incubated for 24 hr under normal oxygen partial pressure (5% CO2/air - control) ±3 µM ouabain or exposure to hypoxia (5% CO2/95% N2)/glucose deprivation in normal high-Na+, low-K+ ([Na+]o/[K+]o = 140/5) or in low-Na+, high-K+ DMEM-like medium ([Na+]o/[K+]o = 131/115). Means ± S.E. from 3 independent experiments performed in quadruplicate are shown. *p<0.05 compared to the controls.
Mentions: Figure 2 shows that 24-hr incubation of RASMC in hypoxia and glucose starvation decreased intracellular ATP content by ∼3-fold whereas ouabain attenuated this parameter by less than 20%. The actions of hypoxia and ouabain on ATP content were preserved in low-Na+, high-K+ medium. Treatment with ouabain resulted in almost 10-fold gain of [Na+]i and virtually similar loss of [K+]i. In hypoxic conditions, [Na+]i and [K+]i were increased and decreased by 3- and 2-fold, respectively. As predicted, dissipation of the transmembrane gradients of monovalent cations in low-Na+, high-K+ medium almost completely abolished the actions of ouabain and hypoxia on the [Na+]i/]K+]i ratio (Fig. 2). Viewed collectively, 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]. Data considered below support this hypothesis.

Bottom Line: Incubation of RASMC in ischemic conditions resulted in ∼3-fold elevation of [Na+]i and 2-fold reduction of [K+]i.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Moscow State University, Moscow, Russia; Department of Medicine, Centre de recherche, Centre hospitalier de l'Université de Montréal, Montreal, Quebec, Canada.

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.

Show MeSH
Related in: MedlinePlus