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. 1998 Nov;18(11):6729-36.
doi: 10.1128/MCB.18.11.6729.

A Ras-dependent pathway regulates RNA polymerase II phosphorylation in cardiac myocytes: implications for cardiac hypertrophy

M Abdellatif  1 , S E Packer, L H Michael, D Zhang, M J Charng, M D Schneider
Affiliations

A Ras-dependent pathway regulates RNA polymerase II phosphorylation in cardiac myocytes: implications for cardiac hypertrophy

M Abdellatif et al. Mol Cell Biol. 1998 Nov.

Abstract

Despite extensive evidence implicating Ras in cardiac muscle hypertrophy, the mechanisms involved are unclear. We previously reported that Ras, through an effector-like function of Ras GTPase-activating protein (GAP) in neonatal cardiac myocytes (M. Abdellatif et al., J. Biol. Chem. 269:15423-15426, 1994; M. Abdellatif and M. D. Schneider, J. Biol. Chem. 272:527-533, 1997), can up-regulate expression from a comprehensive set of promoters, including both cardiac cell-specific and constitutive ones. To investigate the mechanism(s) underlying these earlier findings, we have used recombinant adenoviruses harboring a dominant negative Ras (17N Ras) allele or the N-terminal domain of GAP (nGAP), responsible for the Ras-like effector function. Inhibition of endogenous Ras reduced basal levels of [3H]uridine and [3H]phenylalanine incorporation into total RNA, mRNA, and protein, with parallel changes in apparent cell size. In addition, 17N Ras markedly inhibited phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (pol II), known to regulate transcript elongation, accompanied by down-regulation of its principal kinase, cyclin-dependent kinase 7 (Cdk7). In contrast, nGAP elicited the opposite effects on each of these parameters. Furthermore, cotransfection of constitutively active Ras (12R Ras) with wild-type pol II, rather than a truncated mutant lacking the CTD, demonstrated that Ras activation of transcription was dependent on the pol II CTD. Consistent with a potential role for this pathway in the development of cardiac myocyte hypertrophy, alpha1-adrenergic stimulation similarly enhanced pol II phosphorylation and Cdk7 expression, where both effects were inhibited by dominant negative Ras, while pressure overload hypertrophy led to an increase in both hyperphosphorylated and hypophosphorylated pol II in addition to Cdk7.

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Figures

FIG. 1
FIG. 1
Recombinant adenoviruses deliver 17N Ras or nGAP genes to ∼100% of the cultured neonatal cardiac cells and result in morphological changes. Twenty-four hours after culturing, the cells were treated, at 10 to 20 viral particles/cell, with the indicated recombinant viruses in serum-free medium. Forty-eight hours later the cells were fixed and immunostained with an anti-MHC anti-Ras or anti-GAP antibody or were incubated with FITC-phalloidin, as indicated on the left. Cells in panels A, B, E, and F, panels C, D, G, and H, and panels I and J were from three different cultures. The bar in panel A (50 μm) applies to panels A to H; the bar in panel I (50 μm) applies to panels I and J.
FIG. 2
FIG. 2
Ad.17N Ras and Ad.nGAP regulate RNA and protein synthesis. Cells were treated with Ad.SV and Ad.17N Ras (A) or Ad.CMV and Ad.nGAP (B) in serum-free medium. Twenty-four hours later, the cells were incubated with 1 μCi of either [3H]uridine or [3H]phenylalanine per ml for an additional 24 h. Total RNA and DNA were simultaneously extracted from [3H]uridine-treated cells, while protein and DNA were extracted from cells treated with [3H]phenylalanine. The 3H content of RNA or protein was measured, and the results were normalized to the DNA content of each sample and expressed in values relative to the mean of the control (Ad.SV or Ad.CMV, adjusted to 1). The insets show the 3H content in the poly(A) mRNA fraction after normalization to DNA content, expressed in values relative to the mean of the control (Ad.SV or Ad.CMV, adjusted to 1). Each data point is the mean of six samples ± standard error.
FIG. 3
FIG. 3
Ad.17N Ras, Ad.nGAP, and phenylephrine modulate pol II phosphorylation. (A) Cardiac myocytes were infected with either Ad.SV or Ad.17N Ras. Forty-eight hours later, the cells were incubated with 0.1 mCi of 32Pi per ml in phosphate-free DMEM for 24 h. The cells were then lysed, and pol II was immunoprecipitated. The precipitate was separated by SDS-PAGE on a 6% gel and transferred to a nitrocellulose membrane. The latter was first autoradiographed and then analyzed by Western blotting with anti-pol II antibody 8WG16. The experiment is a representative of two with similar results. (B) Cardiac cells were infected with Ad.CMV or Ad.nGAP and analyzed as for panel A. The experiment is representative of two with similar results. (C) Twenty-four hours after plating, cells were serum starved for 48 h before incubation with 100 μM phenylephrine or vehicle for an additional 24 h in the presence of 32Pi. Cells were then subjected to the same analysis as for panel A. The experiment is representative of two with similar results.
FIG. 4
FIG. 4
17N Ras, nGAP, and phenylephrine regulate free pol II. Cells were infected with the viruses indicated. Twenty-four hours later, they were stimulated with 0.1 μM phenylephrine (Phe), where shown, for an additional 24 h. Cellular protein was differentially extracted as described in Materials and Methods, and the different fractions were electrophoresed separately on an SDS–4 to 15% gradient polyacrylamide gel followed by immunoblotting with anti-pol II antibody 8WG16.
FIG. 5
FIG. 5
12R Ras activation of the Sp1.Inr promoter is dependent on the pol II CTD. Cardiac myocytes were cotransfected with 0.5 μg of the Sp1.Inr luciferase reporter gene, 2 μg of the pcDNA vector, CMV-driven pol II, or Δ5 pol II genes, and increasing concentrations of 12R Ras in the presence (A) or absence (B) of α-amanitin (2.5 μg/ml). Total plasmid DNA and promoter content was kept constant by using plasmid SV-sport. Cells were then analyzed for luciferase activity; data are expressed relative to the mean of control cells transfected with pcDNA, in the absence of 12R Ras and α-amanitin, adjusted to 1. Each data point is the mean of six samples ± standard error.
FIG. 6
FIG. 6
Ad.17N Ras, Ad.nGAP, and phenylephrine regulate Cdk7 expression. Cells were infected with the viruses indicated. Twenty-four hours later, they were stimulated with phenylephrine (Phe), where shown, for an additional 24 h. Cells were then lysed and separated by SDS-PAGE on a 10% gel, immunoblotted, and incubated with the indicated antibodies. The data are representative of three separate experiments with similar results.
FIG. 7
FIG. 7
Mice were subjected to aortic banding or a sham operation as indicated. Seven days after the operation, protein was extracted from the isolated hearts, and 25 μg was electrophoresed on an SDS–4 to 15% gradient polyacrylamide gel and electroblotted onto a nitrocellulose membrane. The same blot was then incubated sequentially with anti-pol II, anti-Cdk7, and anti-Erk antibodies as indicated. The experiment is representative of two with identical results.
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References

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