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

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.

Figure 1. Effect of Na⁺,K⁺-ATPase inhibition on the intracellular content of monovalent ions.

RASMC were incubated in control and K⁺-free medium or in the presence of 3 mM ouabain for 6 hr. Means ± S.E. from 3 independent experiments performed in quadruplicate are shown. *p<0.05 compared to controls.

Figure 2. 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.

Figure 3. Effect of Na⁺,K⁺-ATPase inhibition on the RASMC transcriptome.

Cells were incubated for 6 hr in control DMEM, K⁺-free DMEM or DMEM containing 3 mM ouabain. All experiments are repeated 3 times. A. PCA of transcriptomic changes. Ellipsoids highlight portioning of samples based on type of treatment. The principal components in 3-dimensional graphs (PC#1, PC#2 and PC#3) represent the variability of gene expression level within datasets. B. Comparative analysis of the impact of Na⁺,K⁺-ATPase inhibition by ouabain and K⁺-free medium on the RASMC transcriptome. The total number of genes whose expression is altered by ouabain and K⁺-free medium by more than 1.2-fold with p<0.05 is indicated; the number of genes affected by both stimuli appears in bold.

Figure 4. Correlation analysis of transcripts whose expression is altered by ouabain and K⁺-free medium in RVSMC by more than by 1.2-fold with p<0.05.

The total number of transcripts subjected to analysis is shown in Figure 2B. Transcript expression in control cells was taken as 1.00. The fold change was determined as log transformed treatment/control expression ratio.

Figure 5. Distribution of up- and down- regulated [Na⁺]_i/[K⁺]_i-sensitive genes listed in Tables 2 and 3 among major functional categories.

Figure 6

A. Representative Western blots of HIF-1α and GAPDH in RASMC subjected to 24-hr incubation under control conditions (normoxia), hypoxia/glucose deprivation, 3 mM ouabain or hypoxia/glucose deprivation in cells transfected with Hif-1α siRNA. B. Effect of hypoxia/glucose deprivation and ouabain on relative content of HIF-1α protein in RASMC. The HIF-1α/GAPDH ratio in control conditions was taken as 1.00. Data obtained in 3 independent experiments are reported as means ± S.E.

Figure 7. Effect of hypoxia and ouabain on gene expression in RASMC.

Cells were exposed to normoxia, hypoxia/glucose deprivation or 3 mM ouabain for 24 hr in control high-Na⁺, low-K⁺ medium (A, C), or high-K⁺, low-Na⁺ medium (B). In some experiments, RASMC were transfected with Hif-1α siRNA (C). mRNA content in normoxia was taken as 1.00 and shown by broken lines. For more details, see figure 4 legend.

Figure 8. Position of (A/G)CGTG consensus within 10,000 bp 5′-UTR of genes listed in Table 5.

Figure 9. Position of (A/G)CGTG consensus within 1,500 bp 5′-UTR of genes listed in Table 5.

Figure 10. Possible mechanisms of the involvement of elevated [Na⁺]_i/[K⁺]_i ratio in the transcriptomic changes evoked by hypoxia: a working hypothesis.

1– Na⁺,K⁺-ATPase; 2– Na⁺/Ca²⁺ exchanger; CaM – calmodulin; CRE – Ca²⁺-response elements. For other abbreviations, see text.