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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 SB 203580 on ANP-, BNP-, or ATF2-dependent luciferase production in myocardial cells. Myocardial cells  were cotransfected with MKK6 (Glu) or an empty vector control  (pCEP and pCEP + PE) and either ANP-3003GL, BNP-2501GL,  or pG5E1bLuc reporter constructs. In C, cells were also transfected with ATF2/GAL4 (codes for the ATF2 transcriptional activation domain fused to the Gal4 DNA–binding domain). All  cells were also transfected with CMV–β-galactosidase for normalization purposes. Cultures were then maintained for 48 h with  or without SB 203580 (20 μM) or with or without DMSO (vehicle  control) and 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. Luciferase enzyme units were normalized to β-galactosidase,  and the values obtained with each treatment were normalized to  pCEP + PE. Values are means ± SE, n = 3 cultures.
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Figure 5: Effects of SB 203580 on ANP-, BNP-, or ATF2-dependent luciferase production in myocardial cells. Myocardial cells were cotransfected with MKK6 (Glu) or an empty vector control (pCEP and pCEP + PE) and either ANP-3003GL, BNP-2501GL, or pG5E1bLuc reporter constructs. In C, cells were also transfected with ATF2/GAL4 (codes for the ATF2 transcriptional activation domain fused to the Gal4 DNA–binding domain). All cells were also transfected with CMV–β-galactosidase for normalization purposes. Cultures were then maintained for 48 h with or without SB 203580 (20 μM) or with or without DMSO (vehicle control) and 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. Luciferase enzyme units were normalized to β-galactosidase, and the values obtained with each treatment were normalized to pCEP + PE. Values are means ± SE, n = 3 cultures.

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 SB 203580 on ANP-, BNP-, or ATF2-dependent luciferase production in myocardial cells. Myocardial cells  were cotransfected with MKK6 (Glu) or an empty vector control  (pCEP and pCEP + PE) and either ANP-3003GL, BNP-2501GL,  or pG5E1bLuc reporter constructs. In C, cells were also transfected with ATF2/GAL4 (codes for the ATF2 transcriptional activation domain fused to the Gal4 DNA–binding domain). All  cells were also transfected with CMV–β-galactosidase for normalization purposes. Cultures were then maintained for 48 h with  or without SB 203580 (20 μM) or with or without DMSO (vehicle  control) and 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. Luciferase enzyme units were normalized to β-galactosidase,  and the values obtained with each treatment were normalized to  pCEP + PE. Values are means ± SE, n = 3 cultures.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Effects of SB 203580 on ANP-, BNP-, or ATF2-dependent luciferase production in myocardial cells. Myocardial cells were cotransfected with MKK6 (Glu) or an empty vector control (pCEP and pCEP + PE) and either ANP-3003GL, BNP-2501GL, or pG5E1bLuc reporter constructs. In C, cells were also transfected with ATF2/GAL4 (codes for the ATF2 transcriptional activation domain fused to the Gal4 DNA–binding domain). All cells were also transfected with CMV–β-galactosidase for normalization purposes. Cultures were then maintained for 48 h with or without SB 203580 (20 μM) or with or without DMSO (vehicle control) and 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. Luciferase enzyme units were normalized to β-galactosidase, and the values obtained with each treatment were normalized to pCEP + PE. Values are means ± SE, n = 3 cultures.
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