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A Ras-dependent pathway regulates RNA polymerase II phosphorylation in cardiac myocytes: implications for cardiac
M Abdellatif1, S E Packer, L H Michael
1Molecular Cardiology Unit, Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA. mahaa@bcm.tmc.edu
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.
Insights
Ras signaling regulates cardiac cell growth by controlling RNA polymerase II phosphorylation and expression of cyclin-dependent kinase 7. This pathway is implicated in cardiac hypertrophy.
Area of Science:
- Molecular Biology
- Cardiovascular Biology
- Cell Signaling
Background:
- Ras signaling is implicated in cardiac muscle hypertrophy, but the underlying mechanisms remain unclear.
- Previous studies showed Ras up-regulates gene expression in neonatal cardiac myocytes via Ras GTPase-activating protein (GAP).
Purpose of the Study:
- To elucidate the mechanisms by which Ras influences gene expression and cell size in cardiac myocytes.
- To investigate the role of Ras in regulating RNA polymerase II (pol II) phosphorylation and associated kinases in cardiac hypertrophy.
Main Methods:
- Utilized recombinant adenoviruses expressing dominant-negative Ras (17N Ras) or the N-terminal domain of GAP (nGAP) in neonatal cardiac myocytes.
- Assessed [3H]uridine and [3H]phenylalanine incorporation, cell size, pol II C-terminal domain (CTD) phosphorylation, and cyclin-dependent kinase 7 (Cdk7) expression.
- Employed cotransfection with constitutively active Ras (12R Ras) and wild-type or mutant pol II to determine CTD dependence.
- Examined effects of alpha1-adrenergic stimulation and pressure overload on these parameters in cardiac hypertrophy models.
Main Results:
- Inhibition of endogenous Ras reduced RNA and protein synthesis, cell size, pol II CTD phosphorylation, and Cdk7 expression.
- nGAP administration produced opposite effects, increasing these parameters.
- Ras-mediated transcriptional activation was dependent on the pol II CTD.
- Alpha1-adrenergic stimulation mimicked Ras effects, while pressure overload hypertrophy increased hyper- and hypophosphorylated pol II and Cdk7 levels, all modulated by Ras inhibition.
Conclusions:
- Ras signaling directly impacts cardiac myocyte growth and gene expression through regulation of pol II CTD phosphorylation and Cdk7.
- This Ras-dependent pathway plays a significant role in the development of cardiac myocyte hypertrophy.
- Targeting this pathway could offer therapeutic strategies for cardiac hypertrophy.