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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 on the activities of p38, JNK, and ERK in  myocardial cells. Myocardial cells were treated for 30 min with or  without PE (10 μM) + propranolol (1 μM) and then extracted  and subjected to SDS-PAGE followed by Western analyses using  antibodies (New England Biolabs, Inc., Beverly, MA) that detect  p38, JNK, or ERK only when activated by dual phosphorylation  on Thr180 and Tyr182 (following the manufacturer's protocols).  Developed blots were then analyzed using a Molecular Dynamics  PhosphorImager. Each bar represents the mean blot intensity of  three identically treated cultures ± SE.
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Figure 6: Effects of PE on the activities of p38, JNK, and ERK in myocardial cells. Myocardial cells were treated for 30 min with or without PE (10 μM) + propranolol (1 μM) and then extracted and subjected to SDS-PAGE followed by Western analyses using antibodies (New England Biolabs, Inc., Beverly, MA) that detect p38, JNK, or ERK only when activated by dual phosphorylation on Thr180 and Tyr182 (following the manufacturer's protocols). Developed blots were then analyzed using a Molecular Dynamics PhosphorImager. Each bar represents the mean blot intensity of three identically treated cultures ± SE.

Mentions: To demonstrate that the effects of MKK6 (Glu) and PE on myocardial cell growth and gene expression involved p38, cultures were treated with the highly specific p38 inhibitor, SB 203580 (Young et al., 1993). At 20 μM, SB 203580 has been shown to block p38/MAPK, while concentrations as high as 100 μM have been shown to have no effect on 20 other protein kinases tested, including ERK and JNK (Cuenda et al., 1995). In the present study, SB 203580 (20 μM) blocked PE and MKK6 (Glu)–inducible NP promoter activity by between 40 and 70% (Fig. 5, A and B) and decreased MKK6 (Glu)–activated α-SkA by >90% (not shown). Additionally, myocardial cell p38 was shown to be activated by PE, as was ERK; however, JNK was not stimulated under these conditions (Fig. 6). Taken together, these results confirmed a central role for p38 in MKK6 (Glu) induction of cardiac gene expression and strongly suggest that the ability of PE to induce NP expression is at least partly due to the MKK6/p38 pathway.


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 on the activities of p38, JNK, and ERK in  myocardial cells. Myocardial cells were treated for 30 min with or  without PE (10 μM) + propranolol (1 μM) and then extracted  and subjected to SDS-PAGE followed by Western analyses using  antibodies (New England Biolabs, Inc., Beverly, MA) that detect  p38, JNK, or ERK only when activated by dual phosphorylation  on Thr180 and Tyr182 (following the manufacturer's protocols).  Developed blots were then analyzed using a Molecular Dynamics  PhosphorImager. Each bar represents the mean blot intensity of  three identically treated cultures ± SE.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139826&req=5

Figure 6: Effects of PE on the activities of p38, JNK, and ERK in myocardial cells. Myocardial cells were treated for 30 min with or without PE (10 μM) + propranolol (1 μM) and then extracted and subjected to SDS-PAGE followed by Western analyses using antibodies (New England Biolabs, Inc., Beverly, MA) that detect p38, JNK, or ERK only when activated by dual phosphorylation on Thr180 and Tyr182 (following the manufacturer's protocols). Developed blots were then analyzed using a Molecular Dynamics PhosphorImager. Each bar represents the mean blot intensity of three identically treated cultures ± SE.
Mentions: To demonstrate that the effects of MKK6 (Glu) and PE on myocardial cell growth and gene expression involved p38, cultures were treated with the highly specific p38 inhibitor, SB 203580 (Young et al., 1993). At 20 μM, SB 203580 has been shown to block p38/MAPK, while concentrations as high as 100 μM have been shown to have no effect on 20 other protein kinases tested, including ERK and JNK (Cuenda et al., 1995). In the present study, SB 203580 (20 μM) blocked PE and MKK6 (Glu)–inducible NP promoter activity by between 40 and 70% (Fig. 5, A and B) and decreased MKK6 (Glu)–activated α-SkA by >90% (not shown). Additionally, myocardial cell p38 was shown to be activated by PE, as was ERK; however, JNK was not stimulated under these conditions (Fig. 6). Taken together, these results confirmed a central role for p38 in MKK6 (Glu) induction of cardiac gene expression and strongly suggest that the ability of PE to induce NP expression is at least partly due to the MKK6/p38 pathway.

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