[Na + ] i ‐induced c‐Fos expression is not mediated by activation of the 5′‐promoter containing known transcriptional elements

In vascular smooth muscle cells and several other cell types, inhibition of Na + /K + ‐ATPase leads to the expression of early response genes, including c‐Fos . We designed this study to examine whether or not a putative Na + i /K + i ‐sensitive element is located within the c‐Fos 5′‐UTR from − 650 to + 103 containing all known response elements activated by ‘classic’ stimuli, such as growth factors and Ca 2+ i ‐raising compounds. In HeLa cells, the highest increment of c‐Fos mRNA content was noted after 6 h of Na + /K + ‐ATPase inhibition with ouabain that was abolished by actinomycin D, an inhibitor of RNA synthesis. c‐Fos protein accumulation in ouabain‐treated cells correlated with a gain of Na + i and loss of K + i . Augmented c‐Fos expression was also observed under inhibition of Na + /K + ‐ATPase in K + ‐free medium and in the presence of the Na + ionophore monensin. The effect of ouabain on c‐Fos expression was sharply attenuated under dissipation of the transmembrane Na + gradient, but was preserved in the presence of Ca 2+ chelators and the extracellular regulated kinase inhibitor PD98059, thus indicating an Na + i ‐mediated, Ca 2+ i ‐ and extracellular regulated kinase‐independent mechanism of gene expression. In contrast to massive c‐Fos expression, we failed to detect any effect of ouabain on accumulation of luciferase driven by the c‐Fos 5′‐UTR. Negative results were also obtained in ouabain‐treated vascular smooth muscle cells and C11 Madin–Darby canine kidney cells possessing augmented c‐Fos expression. Our results reveal that Na + i ‐induced c‐Fos expression is not mediated by the 5′‐UTR containing transcriptional elements activated by growth factors and other ‘classic stimuli’.