Ubiquitous [Na+]i/[K+]i-Sensitive Transcriptome in Mammalian Cells: Evidence for Ca2+i-Independent Excitation-Transcription Coupling

Stimulus-dependent elevation of intracellular Ca2+ ([Ca2+]i) affects the expression of numerous genes – a phenomenon known as excitation-transcription coupling. Recently, we found that increases in [Na+]i trigger c-Fos expression via a novel Ca2+i-independent pathway. In the present study, we identified ubiquitous and tissue-specific [Na+]i/[K+]i-sensitive transcriptomes by comparative analysis of differentially expressed genes in vascular smooth muscle cells from rat aorta (RVSMC), the human adenocarcinoma cell line HeLa, and human umbilical vein endothelial cells (HUVEC). To augment [Na+]i and reduce [K+]i, cells were treated for 3 hrs with the Na+,K+-ATPase inhibitor ouabain or placed for the same time in the K+-free medium. Employing Affymetrix-based technology, we detected changes in expression levels of 684, 737 and 1839 transcripts in HeLa, HUVEC and RVSMC, respectively, that were highly correlated between two treatments (p<0.0001; R2>0.62). Among these Na+i/K+i-sensitive genes, 80 transcripts were common for all three types of cells. To establish if changes in gene expression are dependent on increases in [Ca2+]i, we performed identical experiments in Ca2+-free media supplemented with extracellular and intracellular Ca2+ chelators. Surprisingly, this procedure elevated rather than decreased the number of ubiquitous and cell-type specific Na+i/K+i-sensitive genes. Among the ubiquitous Na+i/K+i-sensitive genes whose expression was regulated independently of the presence of Ca2+ chelators by more than 3-fold, we discovered several transcription factors (Fos, Jun, Hes1, Nfkbia), interleukin-6, protein phosphatase 1 regulatory subunit, dual specificity phosphatase (Dusp8), prostaglandin-endoperoxide synthase 2, cyclin L1, whereas expression of metallopeptidase Adamts1, adrenomedulin, Dups1, Dusp10 and Dusp16 was detected exclusively in Ca2+-depleted cells. Overall, our findings indicate that Ca2+i-independent mechanisms of excitation-transcription coupling are involved in transcriptomic alterations triggered by elevation of the [Na+]i/[K+]i ratio. There results likely have profound implications for normal and pathological regulation of mammalian cells, including sustained excitation of neuronal cells, intensive exercise and ischemia-triggered disorders

Counter-regulation by atorvastatin of gene modulations induced by L-NAME hypertension is associated with vascular protection

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Mechanisms underlying transcriptomic alterations in normal and pathophysiological conditions: <i>a working hypothesis</i>.

<p>Activation of Na⁺-permeable channels such as NMDA receptors (<b>1</b>), Na⁺/H⁺ exchanger (<b>2</b>), voltage-gated K⁺ channels (<b>6</b>) and inhibition of Na⁺,K⁺-ATPase (<b>4</b>) lead to elevation of the [Na⁺]_i/[K⁺]_i ratio. In cells abundant with Na⁺/Ca²⁺ exchanger (<b>3</b>), the dissipation of transmembrane gradients of monovalent cations is accompanied by elevation of [Ca²⁺]_i. Elevation of the [Na⁺]_i/[K⁺]_i ratio affects the expression of <b>X_1,2,3…</b> and <b>Z_1,2,3…</b> genes via activation of unknown Na/K sensor(s) and Na/K response elements (<b>Na/KRE</b>). Expression of <b>Z_1,2,3…</b> genes is also subjected to regulation by elevated [Ca²⁺]_i via its interaction with calmodulin (<b>CaM</b>) and other Ca²⁺_i sensors and diverse Ca²⁺-response elements (<b>CaRE</b>), whereas <b>Y_1,2,3…</b> genes lacking Na/KRE are controlled by [Ca²⁺]_i only. The set of Na⁺_i,K⁺_i-sensitive transcription regulators shown as <b>X_n</b>, <b>Y_n</b> and <b>Z_n</b> contributes to overall transcriptomic changes via activation of canonical response elements (<b>RE</b>) within <b>V_1,2,3…</b> genes. Autocrine pathways triggered by the release of interleukin 6 and other [Na⁺]_i/[K⁺]_i-sensitive regulators of gene expression (<b>Z_m</b>) may also contribute to overall transcriptomic changes via activation of their receptors (<b>7</b>).</p

Effect of ouabain and K⁺-free medium on intracellular Na⁺ (a–c) and K⁺ (d–f) content in HeLa (a,d), HUVEC (b,e) and RVSMC (c,f).

<p>Cells were incubated in control or K⁺-free medium during 3 hr and ouabain was added at a final concentration of 3 µM (HeLa and HUVEC) or 3 mM (RVSMC). Mean ± S.E. values of experiments performed in quadruplicate are shown.</p

Ubiquitous Na⁺_i/K⁺_i-sensitive genes whose expression was up- and down-regulated by more than 1.2-fold (p≤0.05) in control (Ca²⁺-containing) medium.

<p>HeLa, HUVEC and RVSMC were treated with ouabain or K+-free medium for 3 hr. Genes whose expression is not affected by Na⁺,K⁺-ATPase inhibition at least in one type of Ca²⁺-depleted cells are shown in <b><i>italics</i></b>. Genes whose expression is not affected by Na⁺,K⁺-ATPase inhibition in all 3 types of cells are shown in <b><i><u>underlined italics</u></i></b>. Functional categories are indicated in the left column as: <b>t</b> – regulators of transcription/translation, RNA processing and degradation; <b>d</b> – regulators of cell adhesion, migration, proliferation, differentiation and death; <b>f</b> - protein folding and ubiquitination; <b>i</b> – inflammation and immune response; <b>o</b> – other functional categories and genes with unknown function.</p

<b><i>RVSMC:</i></b> the list of genes whose expression was changed by more than 4-fold in 3 hr of Na⁺,K⁺-ATPase inhibition in control (Ca²⁺ containing) medium.

<p>Genes whose differential expression is limited to RVSMC are shown in <b>bold</b>. Genes whose expression were also detected in HeLa or HUVEC are shown in <b><u>underlined bold</u></b>. Genes whose expression was not affected by Na⁺,K⁺-ATPase inhibition in Ca²⁺-depleted cells are shown in <i>italics</i>.</p