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

Svetlana V Koltsova,Johanne Tremblay,Mounsif Haloui,Sergei N Orlov,Pavel Hamet,Yulia Trushina,Olga A Akimova,Yoshiaki Tsuji

doi:10.1371/journal.pone.0038032

Abstract

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.

Highlights

Gene expression is regulated by diverse stimuli to achieve tissuespecific functional responses via coordinate synthesis of the cell’s macromolecular components [1]
More recent studies demonstrated that side-by-side with the above-listed ‘‘classic’’ Na+i,K+i-dependent cellular processes, sustained elevation of the [Na+]i/[K+]i ratio in vascular smooth muscle cells, cardiomyocytes, hepatocytes, renal epithelial and neuronal cells causes differential expression of c-Fos and other immediate response genes (IRG), as well as cell type-specific late response genes, such as tumour growth factor-b, the a1- and b1-subunits of Na+,K+-ATPase, myosin light chain, skeletal muscle actin, atrial natriuretic factor and mortalin
In RVSMC the action of K+-free medium on the [Na+]i/[K+]i ratio was similar, whereas in human umbilical vein endothelial cells (HUVEC) and HeLa cells the gain of Na+i and the loss of K+i triggered by K+-free medium were higher compared to ouabain by

Summary

Introduction

Gene expression is regulated by diverse stimuli to achieve tissuespecific functional responses via coordinate synthesis of the cell’s macromolecular components [1]. Electrochemical gradients of monovalent cations across the plasma membrane (high intracellular potassium, [K+]i vs low intracellular sodium, [Na+]i) are created by the Na+,K+-pump and determine a large variety of physiologically important processes. These processes include maintenance of resting and action electrical membrane potentials, regulation of cell volume, secondary transport of mono- and divalent ions (such as chloride, calcium and phosphate), and accumulation of nutrients (glucose, amino acids, nucleotides) and other relevant molecules [2]. Sensitive pathways – a phenomenon termed excitation-transcription coupling [6,7,8] It is well-documented that elevation of the [Na+]i/[K+]i ratio typically leads to increases in [Ca2+]i via activation of the Na+/Ca2+ exchanger [9] and/or voltage-gated

Methods

Results

Conclusion