c-Fos expression in ouabain-treated vascular smooth muscle cells from rat aorta: evidence for an intracellular-sodium-mediated, calcium-independent mechanism

Abstract

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.

Figure 3. Effect of ouabain on the Na⁺–K⁺ pump, [Na⁺]_i and [K⁺]_i and expression of c-Fos protein in quiescent VSMC

A, dose dependency of the effect of ouabain on Na⁺–K⁺ pump activity (curve 1, ▪) and intracellular content of exchangeable Na⁺ and K⁺ (curves 2, •, and 3, ▴, respectively). Means ± s.e.m. from experiments performed in quadruplicate are shown. B, representative Northern blot illustrating c-Fos expression after incubation of VSMC with different concentrations of ouabain 6 h. C, dose dependency of the ouabain effect on c-Fos expression. c-Fos content in the absence of ouabain was taken as 1.0. Means ± s.e.m. from three experiments are shown.

Figure 1. Effect of ouabain on early response gene (ERG) expression in quiescent vascular smooth muscle cells (VSMC)

A, representative blot showing expression of c-Fos, c-Jun, activating transcription factor (ATF)-2 and cAMP-Ca²⁺ response element binding protein (CREB)-1 proteins after incubation of VSMC for up to 24 h with 1 mm ouabain. B, kinetics of c-Fos (•) and c-Jun (○) expression in ouabain-treated quiescent VSMC. ERG content in the absence of ouabain was taken as 1.0. Means ± s.e.m. from three experiments are shown.

Figure 2. Effect of ouabain and potassium-depleted medium on the expression of c-Fos protein in quiescent VSMC

VSMC were incubated for 6 h in control medium with or without 1 mm ouabain or in potassium-depleted medium. Concentrations of Na⁺ and K⁺ in the media are shown at the top of the figure. The control media contained (mm): NaCl 121, KCl 5, CaCl₂ 1.8, MgSO₄ 0.8, NaHCO₃ 12, NaH₂PO₄ 0.9, Hepes 4.2 (pH 7.2), glucose 5, and vitamins and amino acids at concentrations indicated for Dulbecco's modified Eagle's medium (DMEM) recipes. In potassium-depleted medium, KCl was substituted with NaCl.

Figure 4. Ouabain-induced modulation of [Na⁺]_i, [K⁺]_i and the c-Fos/S18 ratio

A, kinetics of the modulation by ouabain of [Na⁺]_i (1, •) and [K⁺]_i (2, ○) content and the c-Fos mRNA/ribosomal S18 RNA ratio (3, ▴). To measure [Na⁺]_i and [K⁺]_i, quiescent VSMC were preincubated for 6 h in DMEM with 0.5 μCi ml⁻¹⁸⁶RbCl or 2 μCi ml⁻¹²²NaCl, and then aliquots of DMEM with the same radioactivity and 1 mm ouabain were added for the next 0.25, 0.5, 1, 2 and 5 h. The content of exchangeable Na⁺, K⁺ and the c-Fos/S18 RNA ratio in the absence of ouabain was taken as 100 %. Means ± s.e.m. from three (intracellular ions) and five (c-Fos mRNA) experiments are given. B, representative blot showing the kinetics of c-Fos mRNA expression in ouabain-treated VSMC.

Figure 5. Role of cell volume in c-Fos expression

A, kinetics of cell volume modulation by ouabain. Quiescent VSMC were preincubated for 6 h in DMEM containing 2 μCi ml⁻¹[¹⁴C]urea, and then ouabain was added at a final concentration of 1 mm. Means ± s.e.m. from experiments performed in triplicate are given. B, effect of hyperosmotic medium on VSMC volume. The osmolality of the medium was increased by the addition of either 100 or 300 mm mannitol to DMEM. Means ± s.e.m. from experiments performed in quadruplicate are given. C, effect of hyperosmotic medium on c-Fos protein expression. Quiescent VSMC were incubated in DMEM containing 100 or 300 mm mannitol for 6 h. c-Fos expression in the absence of mannitol was taken as 100 %. Means ± s.e.m. from experiments performed in triplicate are given.

Figure 6. Effect of nicardipine (0.1 μM) on c-Fos protein expression after incubation of VSMC for 6 h in control and high-potassium media

For composition of the control medium, see Fig. 2. In high-potassium medium, NaCl was substituted with KCl. Ouabain was added at final concentration of 1 mm.

Figure 7. [Ca²⁺]_i in control and ouabain-treated cells

A, representative records of the effect of ouabain and thapsigargin on free [Ca²⁺]_i measured in the total cell population. Additions of 1 mm ouabain and 0.5 μM thapsigargin are shown by arrows. B, representative records of the effect of ouabain on the F₃₄₀/F₃₈₀ ratio measured in five single cells by fluorescence ratio imaging. C, free [Ca²⁺]_i in the total population of control cells and cells treated with 1 mm ouabain for 40 min. Means ± s.e.m. from six experiments are given. D, the F₃₄₀/F₃₈₀ ratio in control cells and cells treated with 1 mm ouabain for 60 min. The values of F₃₄₀/F₃₈₀ ratio in control cells was taken as 100 %. Means ± s.e.m. from the analysis of five to six single cells in three experiments are given. E, the content of exchangeable [Ca²⁺]_i in control cells and cells treated with ouabain for 6 h. Means ± s.e.m. from experiments performed in quadruplicate are given.

Figure 8. Effect of ouabain on c-Fos protein expression in calcium-free medium and in BAPTA-loaded VSMC

Quiescent VSMC were incubated for 30 min in DMEM in the absence or presence of 20 μM BAPTA-AM. This medium was then aspirated and the cells were incubated for the next 6 h in regular CaCl₂-containing DMEM, in CaCl₂-free DMEM or in CaCl₂-free DMEM containing 0.1 mm EGTA, in the absence or presence of 1 mm ouabain.

Figure 9. Effect of serum on c-Fos mRNA (A) and protein (B) expression

Above each of the graphs a representative Northern and Western blot is presented showing c-Fos mRNA (A) and protein expression (B) after stimulation of quiescent cells with 10 % serum. A, kinetics of the increment in c-Fos mRNA in serum-treated VSMC. B, kinetics of the increment in protein content in serum-treated VSMC. Since serum time-dependently augmented GAPDH mRNA, we did not use its content to normalize c-Fos mRNA expression. The value of c-Fos mRNA and protein expression in untreated cells was taken as 100 %. Means ± s.e.m. from three experiments are given.

Figure 10. Additive effect of serum and ouabain on c-Fos expression

A, representative blot showing c-Fos protein in control quiescent cells and cells treated with 1 mm ouabain and 10 % calf serum for 6 h. B, relative content of c-Fos protein after incubation with serum and/or ouabain. c-Fos content in untreated cells was taken as 1.0. Means ± s.e.m. obtained from three experiments are shown.