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A role for the p38 mitogen-activated protein kinase pathway in myocardial cell growth, sarcomeric organization, and cardiac-specific gene expression.

Zechner D, Thuerauf DJ, Hanford DS, McDonough PM, Glembotski CC - J. Cell Biol. (1997)

Bottom Line: While activation of JNK and/or ERK with MEKK1COOH or Raf-1 BXB, respectively, augmented cell size and effected relatively modest increases in NP and alpha-SkA promoter activities, neither upstream kinase conferred sarcomeric organization.However, transfection with MKK6 (Glu), which specifically activated p38, augmented cell size, induced NP and alpha-Ska promoter activities by up to 130-fold, and elicited sarcomeric organization in a manner similar to PE.Moreover, all three growth features induced by MKK6 (Glu) or PE were blocked with the p38-specific inhibitor, SB 203580.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Molecular Biology Institute, San Diego State University, California 92182, USA.

ABSTRACT
Three hallmark features of the cardiac hypertrophic growth program are increases in cell size, sarcomeric organization, and the induction of certain cardiac-specific genes. All three features of hypertrophy are induced in cultured myocardial cells by alpha1- adrenergic receptor agonists, such as phenylephrine (PE) and other growth factors that activate mitogen- activated protein kinases (MAPKs). In this study the MAPK family members extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 were activated by transfecting cultured cardiac myocytes with constructs encoding the appropriate kinases possessing gain-of-function mutations. Transfected cells were then analyzed for changes in cell size, sarcomeric organization, and induction of the genes for the A- and B-type natriuretic peptides (NPs), as well as the alpha-skeletal actin (alpha-SkA) gene. While activation of JNK and/or ERK with MEKK1COOH or Raf-1 BXB, respectively, augmented cell size and effected relatively modest increases in NP and alpha-SkA promoter activities, neither upstream kinase conferred sarcomeric organization. However, transfection with MKK6 (Glu), which specifically activated p38, augmented cell size, induced NP and alpha-Ska promoter activities by up to 130-fold, and elicited sarcomeric organization in a manner similar to PE. Moreover, all three growth features induced by MKK6 (Glu) or PE were blocked with the p38-specific inhibitor, SB 203580. These results demonstrate novel and potentially central roles for MKK6 and p38 in the regulation of myocardial cell hypertrophy.

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Effects of PE or MKK6 (Glu) on MEF2C-dependent  luciferase production in myocardial cells. Myocardial cells were  transfected with pG5E1bLuc and either Gal4 (codes for Gal4  DNA–binding domain only), MEF2C/Gal4 (codes for MEF2C  fused to the Gal4 DNA–binding domain), or MEF2C-S/Gal4  (codes for mutant [Ser to Ala 387] MEF2C fused to the Gal4  DNA–binding domain). All cells were also transfected with  CMV–β-galactosidase for normalization purposes. (A) Cultures  were maintained with or without PE (10 μM) + propranolol (1  μM), as shown, and then extracted and assayed for luciferase and  β-galactosidase reporter activities, as described in Materials and  Methods. (B) Cultures were also transfected with the control vector, pCEP, or with the test construct, MKK6 (Glul), maintained  in control media for 24 h, and then extracted and assayed for luciferase and β-galactosidase reporter activities, as described in  Materials and Methods. Luciferase enzyme units were normalized to β-galactosidase, and the values obtained with each treatment were normalized to MEF2C/Gal 4 + PE (A) or MEF2C/ Gal 4 + MKK6 (Glu) (B). Values are means ± SE, n = 3 cultures.
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Figure 9: Effects of PE or MKK6 (Glu) on MEF2C-dependent luciferase production in myocardial cells. Myocardial cells were transfected with pG5E1bLuc and either Gal4 (codes for Gal4 DNA–binding domain only), MEF2C/Gal4 (codes for MEF2C fused to the Gal4 DNA–binding domain), or MEF2C-S/Gal4 (codes for mutant [Ser to Ala 387] MEF2C fused to the Gal4 DNA–binding domain). All cells were also transfected with CMV–β-galactosidase for normalization purposes. (A) Cultures were maintained with or without PE (10 μM) + propranolol (1 μM), as shown, and then extracted and assayed for luciferase and β-galactosidase reporter activities, as described in Materials and Methods. (B) Cultures were also transfected with the control vector, pCEP, or with the test construct, MKK6 (Glul), maintained in control media for 24 h, and then extracted and assayed for luciferase and β-galactosidase reporter activities, as described in Materials and Methods. Luciferase enzyme units were normalized to β-galactosidase, and the values obtained with each treatment were normalized to MEF2C/Gal 4 + PE (A) or MEF2C/ Gal 4 + MKK6 (Glu) (B). Values are means ± SE, n = 3 cultures.

Mentions: Recent studies have established that the transcription factor, MEF2C, serves as a specific substrate for p38, such that phosphorylation on serine 387 confers MEF2C-mediated transcriptional activation in RAW 264.1 cells (Han et al., 1997). Accordingly, the abilities of PE or MKK6 (Glu) to activate MEF2C-dependent transcription in cardiac myocytes were tested. Myocardial cells were cotransfected with a reporter plasmid possessing GAL4 DNA–binding sites cloned upstream of luciferase and a construct encoding a fusion protein comprised of the MEF2C transactivation domain and the GAL4 DNA–binding domain. Using this system, the activation of MEF2C after phosphorylation on serine 387 can be studied in the cardiac myocytes without interference from any endogenous MEF2 family members. Treatment with PE or transfection with MKK6 (Glu)–enhanced MEF2C-mediated luciferase production (Fig. 9, MEF2C/Gal4). When cells were transfected with an altered MEF2C/Gal4 chimera, in which serine 387 was mutated to alanine, neither PE nor MKK6 (Glu) conferred luciferase induction (Fig. 9, MEF2C-S/Gal4). These results indicate that like MKK6 (Glu), PE can activate MEF2C in cardiac myocytes, an event that requires phosphorylation at serine 387 by p38. This result further supports the notion that in part, PE enhances myocardial cell growth, sarcomeric organization, and the related gene expression through a pathway involving p38 or a very similar kinase.


A role for the p38 mitogen-activated protein kinase pathway in myocardial cell growth, sarcomeric organization, and cardiac-specific gene expression.

Zechner D, Thuerauf DJ, Hanford DS, McDonough PM, Glembotski CC - J. Cell Biol. (1997)

Effects of PE or MKK6 (Glu) on MEF2C-dependent  luciferase production in myocardial cells. Myocardial cells were  transfected with pG5E1bLuc and either Gal4 (codes for Gal4  DNA–binding domain only), MEF2C/Gal4 (codes for MEF2C  fused to the Gal4 DNA–binding domain), or MEF2C-S/Gal4  (codes for mutant [Ser to Ala 387] MEF2C fused to the Gal4  DNA–binding domain). All cells were also transfected with  CMV–β-galactosidase for normalization purposes. (A) Cultures  were maintained with or without PE (10 μM) + propranolol (1  μM), as shown, and then extracted and assayed for luciferase and  β-galactosidase reporter activities, as described in Materials and  Methods. (B) Cultures were also transfected with the control vector, pCEP, or with the test construct, MKK6 (Glul), maintained  in control media for 24 h, and then extracted and assayed for luciferase and β-galactosidase reporter activities, as described in  Materials and Methods. Luciferase enzyme units were normalized to β-galactosidase, and the values obtained with each treatment were normalized to MEF2C/Gal 4 + PE (A) or MEF2C/ Gal 4 + MKK6 (Glu) (B). Values are means ± SE, n = 3 cultures.
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Related In: Results  -  Collection

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Figure 9: Effects of PE or MKK6 (Glu) on MEF2C-dependent luciferase production in myocardial cells. Myocardial cells were transfected with pG5E1bLuc and either Gal4 (codes for Gal4 DNA–binding domain only), MEF2C/Gal4 (codes for MEF2C fused to the Gal4 DNA–binding domain), or MEF2C-S/Gal4 (codes for mutant [Ser to Ala 387] MEF2C fused to the Gal4 DNA–binding domain). All cells were also transfected with CMV–β-galactosidase for normalization purposes. (A) Cultures were maintained with or without PE (10 μM) + propranolol (1 μM), as shown, and then extracted and assayed for luciferase and β-galactosidase reporter activities, as described in Materials and Methods. (B) Cultures were also transfected with the control vector, pCEP, or with the test construct, MKK6 (Glul), maintained in control media for 24 h, and then extracted and assayed for luciferase and β-galactosidase reporter activities, as described in Materials and Methods. Luciferase enzyme units were normalized to β-galactosidase, and the values obtained with each treatment were normalized to MEF2C/Gal 4 + PE (A) or MEF2C/ Gal 4 + MKK6 (Glu) (B). Values are means ± SE, n = 3 cultures.
Mentions: Recent studies have established that the transcription factor, MEF2C, serves as a specific substrate for p38, such that phosphorylation on serine 387 confers MEF2C-mediated transcriptional activation in RAW 264.1 cells (Han et al., 1997). Accordingly, the abilities of PE or MKK6 (Glu) to activate MEF2C-dependent transcription in cardiac myocytes were tested. Myocardial cells were cotransfected with a reporter plasmid possessing GAL4 DNA–binding sites cloned upstream of luciferase and a construct encoding a fusion protein comprised of the MEF2C transactivation domain and the GAL4 DNA–binding domain. Using this system, the activation of MEF2C after phosphorylation on serine 387 can be studied in the cardiac myocytes without interference from any endogenous MEF2 family members. Treatment with PE or transfection with MKK6 (Glu)–enhanced MEF2C-mediated luciferase production (Fig. 9, MEF2C/Gal4). When cells were transfected with an altered MEF2C/Gal4 chimera, in which serine 387 was mutated to alanine, neither PE nor MKK6 (Glu) conferred luciferase induction (Fig. 9, MEF2C-S/Gal4). These results indicate that like MKK6 (Glu), PE can activate MEF2C in cardiac myocytes, an event that requires phosphorylation at serine 387 by p38. This result further supports the notion that in part, PE enhances myocardial cell growth, sarcomeric organization, and the related gene expression through a pathway involving p38 or a very similar kinase.

Bottom Line: While activation of JNK and/or ERK with MEKK1COOH or Raf-1 BXB, respectively, augmented cell size and effected relatively modest increases in NP and alpha-SkA promoter activities, neither upstream kinase conferred sarcomeric organization.However, transfection with MKK6 (Glu), which specifically activated p38, augmented cell size, induced NP and alpha-Ska promoter activities by up to 130-fold, and elicited sarcomeric organization in a manner similar to PE.Moreover, all three growth features induced by MKK6 (Glu) or PE were blocked with the p38-specific inhibitor, SB 203580.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Molecular Biology Institute, San Diego State University, California 92182, USA.

ABSTRACT
Three hallmark features of the cardiac hypertrophic growth program are increases in cell size, sarcomeric organization, and the induction of certain cardiac-specific genes. All three features of hypertrophy are induced in cultured myocardial cells by alpha1- adrenergic receptor agonists, such as phenylephrine (PE) and other growth factors that activate mitogen- activated protein kinases (MAPKs). In this study the MAPK family members extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 were activated by transfecting cultured cardiac myocytes with constructs encoding the appropriate kinases possessing gain-of-function mutations. Transfected cells were then analyzed for changes in cell size, sarcomeric organization, and induction of the genes for the A- and B-type natriuretic peptides (NPs), as well as the alpha-skeletal actin (alpha-SkA) gene. While activation of JNK and/or ERK with MEKK1COOH or Raf-1 BXB, respectively, augmented cell size and effected relatively modest increases in NP and alpha-SkA promoter activities, neither upstream kinase conferred sarcomeric organization. However, transfection with MKK6 (Glu), which specifically activated p38, augmented cell size, induced NP and alpha-Ska promoter activities by up to 130-fold, and elicited sarcomeric organization in a manner similar to PE. Moreover, all three growth features induced by MKK6 (Glu) or PE were blocked with the p38-specific inhibitor, SB 203580. These results demonstrate novel and potentially central roles for MKK6 and p38 in the regulation of myocardial cell hypertrophy.

Show MeSH
Related in: MedlinePlus