c-Fos expression in ouabain-treated vascular smooth muscle cells from rat aorta: evidence for an intracellular-sodium-mediated, calcium-independent mechanism.
In this study, we examined the effect of Na(+)-K(+) pump inhibition on the expression of early response genes in vascular smooth muscle cells (VSMC) as possible intermediates of the massive RNA synthesis and protection against apoptosis seen in ouabain-treated VSMC in our previous experiments. Incubation of VSMC with ouabain resulted in rapid induction of c-Fos protein expression with an approximately sixfold elevation after 2 h of incubation. c-Jun expression was increased by approximately fourfold after 12 h, whereas expression of activating transcription factor 2, cAMP/Ca(2+) response element binding protein (CREB)-1 and c-Myc was not altered. Markedly augmented c-Fos expression was also observed under Na(+)-K(+) pump inhibition in potassium-depleted medium. Na(+)-K(+) pump inhibition triggered c-Fos expression via elevation of the [Na(+)](i)/[K(+)](i) ratio. This conclusion follows from experiments showing the lack of effect of ouabain on c-Fos expression in high-potassium-low-sodium medium and from the comparison of dose responses of Na(+)-K(+) pump activity, [Na(+)](i) and [K(+)](i) content and c-Fos expression to ouabain. A fourfold increment of c-Fos mRNA was revealed 30 min following addition of ouabain to the incubation medium. At this time point, treatment with ouabain resulted in an approximately fourfold elevation of [Na(+)](i) but did not affect [K(+)](i). Augmented c-Fos expression was also observed under VSMC depolarization in high-potassium medium. Increments in both c-Fos expression and (45)Ca uptake in depolarized VSMC were abolished under inhibition of L-type Ca(2+) channels with 0.1 microM nicardipine. Ouabain did not affect the free [Ca(2+)](i) or the content of exchangeable [Ca(2+)](i). Ouabain-induced c-Fos expression was also insensitive to the presence of nicardipine and [Ca(2+)](o), as well as chelators of [Ca(2+)](o) (EGTA) and [Ca(2+)](i) (BAPTA). The effect of ouabain and serum on c-Fos expression was additive. In contrast to serum, however, ouabain failed to activate the Elk-1, serum response factor, CREB and activator protein-1 transcription factors identified within the c-Fos promoter. These results suggest that Na(+)-K(+) pump inhibition triggers c-Fos expression via [Na(+)](i)-sensitive [Ca(2+)](i)-independent transcription factor(s) distinct from factors interacting with known response elements of this gene promoter.
Taurin
Sebastien
S
Centre de Recherche, Centre Hospitalier de l'Université de Montréal - Hôtel-Dieu, PQ, Canada.
Dulin
Nickolai O
NO
Pchejetski
Dimitri
D
Grygorczyk
Ryszard
R
Tremblay
Johanne
J
Hamet
Pavel
P
Orlov
Sergei N
SN
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D001798:Blood Proteins / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015870:Gene Expression / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology*; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics*; D051381:Rats; D011914:Rats, Inbred BN; D020218:Response Elements; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
D001798:Blood Proteins; D004791:Enzyme Inhibitors; D016760:Proto-Oncogene Proteins c-fos; D014157:Transcription Factors; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium; D002118:Calcium