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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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Fluorescent microscopic analyses of the effects of Raf-1 BXB, MEKKCOOH, or MKK6 (Glu) expression constructs on size, sarcomeric organization, and endogenous cardiac-specific gene expression in myocardial cells. Myocardial cells were cotransfected with Raf  (Raf-1 BXB), JNKK kinase (MEKKCOOH), p38 kinase (MKK6 [Glu]), or an empty vector control (pCEP) and CMV–β-galactosidase  (A–E′) or ANP-3003GL (F–J), as described in the legend for Fig. 1. After 48 h of incubation in either serum-free control media or in the  same media containing 10 μM of the α1-adrenergic receptor agonist, phenylephrine (PE) + 1 μM propranolol (the latter to block potential binding to β-adrenergic receptors), cultures were fixed in paraformaldehyde. (A–E) β-galactosidase expression (Gal), used to identify transfected cells, was visualized with a Texas red–conjugated second antibody and photographed using a rhodamine-compatible filter.  (A′–E′) Actin organization in the same β-galactosidase–positive cells shown in A–E was assessed by staining them with BODIPY-conjugated phalloidin (Phalloidin) and photographing them using an FITC-compatible filter. (F–J) In a separate experiment, luciferase expression (Luc), used to identify transfected cells, was visualized with an FITC-conjugated second antibody and photographed using an  FITC-compatible filter. The same cells were also assessed for endogenous ANP expression (ANP), viewed with a Texas red–conjugated  second antibody, and photographed with a rhodamine-compatible filter. The digitized photographic images of luciferase- and ANP-positive  cells were overlaid using Adobe Photoshop (San Jose, CA), and the resulting montage was prepared in Claris MacDraw Pro. Bar, 50 μM.
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Figure 3: Fluorescent microscopic analyses of the effects of Raf-1 BXB, MEKKCOOH, or MKK6 (Glu) expression constructs on size, sarcomeric organization, and endogenous cardiac-specific gene expression in myocardial cells. Myocardial cells were cotransfected with Raf (Raf-1 BXB), JNKK kinase (MEKKCOOH), p38 kinase (MKK6 [Glu]), or an empty vector control (pCEP) and CMV–β-galactosidase (A–E′) or ANP-3003GL (F–J), as described in the legend for Fig. 1. After 48 h of incubation in either serum-free control media or in the same media containing 10 μM of the α1-adrenergic receptor agonist, phenylephrine (PE) + 1 μM propranolol (the latter to block potential binding to β-adrenergic receptors), cultures were fixed in paraformaldehyde. (A–E) β-galactosidase expression (Gal), used to identify transfected cells, was visualized with a Texas red–conjugated second antibody and photographed using a rhodamine-compatible filter. (A′–E′) Actin organization in the same β-galactosidase–positive cells shown in A–E was assessed by staining them with BODIPY-conjugated phalloidin (Phalloidin) and photographing them using an FITC-compatible filter. (F–J) In a separate experiment, luciferase expression (Luc), used to identify transfected cells, was visualized with an FITC-conjugated second antibody and photographed using an FITC-compatible filter. The same cells were also assessed for endogenous ANP expression (ANP), viewed with a Texas red–conjugated second antibody, and photographed with a rhodamine-compatible filter. The digitized photographic images of luciferase- and ANP-positive cells were overlaid using Adobe Photoshop (San Jose, CA), and the resulting montage was prepared in Claris MacDraw Pro. Bar, 50 μM.

Mentions: Further studies were undertaken to compare the effects of Raf BXB, MEKKCOOH, and MKK6 (Glu) with the gold standard, PE, on other features of the program, such as cell size and sarcomeric organization. Compared to cells maintained in control media (Fig. 3, A and A′), the PE-treated cells were much larger (Fig. 3 B), displaying an approximately two- to three-fold increase in area (Fig. 4 A), and they possessed a high degree of sarcomeric organization (Fig. 3 B′). In general, the PE-treated cultures possessed about 10-fold more myocytes displaying organized sarcomeres than the control cultures (Fig. 4 B). PE-treated cultures also displayed significantly increased levels of endogenous ANP expression, observed as the prototypical perinuclear staining found often in hypertrophic cardiac myocytes (Fig. 3, F [control] vs. G [PE-treated]). Interestingly, cultures transfected with Raf BXB or MEKKCOOH displayed increases in size (Figs. 3, C [BXB] and D [MEKKCOOH], and 4 A), and while the usual shape of the Raf BXB–treated cells was similar to PE-treated cells, the MEKKCOOH-treated cells were almost always very long and thin. Moreover, while either Raf BXB (Fig. 3 H [BXB]) or MEKKCOOH (Fig. 3 I [MEKK-1]) fostered the induction of endogenous ANP expression, neither construct supported sarcomeric organization (Fig. 3 C′ [BXB] and 3 D′ [MEKK-1]; also see Fig. 4 B). These results suggested that neither ERK (Raf BXB) alone nor ERK and JNK (MEKKCOOH) were sufficient to confer all the features of the hypertrophic phenotype.


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)

Fluorescent microscopic analyses of the effects of Raf-1 BXB, MEKKCOOH, or MKK6 (Glu) expression constructs on size, sarcomeric organization, and endogenous cardiac-specific gene expression in myocardial cells. Myocardial cells were cotransfected with Raf  (Raf-1 BXB), JNKK kinase (MEKKCOOH), p38 kinase (MKK6 [Glu]), or an empty vector control (pCEP) and CMV–β-galactosidase  (A–E′) or ANP-3003GL (F–J), as described in the legend for Fig. 1. After 48 h of incubation in either serum-free control media or in the  same media containing 10 μM of the α1-adrenergic receptor agonist, phenylephrine (PE) + 1 μM propranolol (the latter to block potential binding to β-adrenergic receptors), cultures were fixed in paraformaldehyde. (A–E) β-galactosidase expression (Gal), used to identify transfected cells, was visualized with a Texas red–conjugated second antibody and photographed using a rhodamine-compatible filter.  (A′–E′) Actin organization in the same β-galactosidase–positive cells shown in A–E was assessed by staining them with BODIPY-conjugated phalloidin (Phalloidin) and photographing them using an FITC-compatible filter. (F–J) In a separate experiment, luciferase expression (Luc), used to identify transfected cells, was visualized with an FITC-conjugated second antibody and photographed using an  FITC-compatible filter. The same cells were also assessed for endogenous ANP expression (ANP), viewed with a Texas red–conjugated  second antibody, and photographed with a rhodamine-compatible filter. The digitized photographic images of luciferase- and ANP-positive  cells were overlaid using Adobe Photoshop (San Jose, CA), and the resulting montage was prepared in Claris MacDraw Pro. Bar, 50 μM.
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Related In: Results  -  Collection

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

Figure 3: Fluorescent microscopic analyses of the effects of Raf-1 BXB, MEKKCOOH, or MKK6 (Glu) expression constructs on size, sarcomeric organization, and endogenous cardiac-specific gene expression in myocardial cells. Myocardial cells were cotransfected with Raf (Raf-1 BXB), JNKK kinase (MEKKCOOH), p38 kinase (MKK6 [Glu]), or an empty vector control (pCEP) and CMV–β-galactosidase (A–E′) or ANP-3003GL (F–J), as described in the legend for Fig. 1. After 48 h of incubation in either serum-free control media or in the same media containing 10 μM of the α1-adrenergic receptor agonist, phenylephrine (PE) + 1 μM propranolol (the latter to block potential binding to β-adrenergic receptors), cultures were fixed in paraformaldehyde. (A–E) β-galactosidase expression (Gal), used to identify transfected cells, was visualized with a Texas red–conjugated second antibody and photographed using a rhodamine-compatible filter. (A′–E′) Actin organization in the same β-galactosidase–positive cells shown in A–E was assessed by staining them with BODIPY-conjugated phalloidin (Phalloidin) and photographing them using an FITC-compatible filter. (F–J) In a separate experiment, luciferase expression (Luc), used to identify transfected cells, was visualized with an FITC-conjugated second antibody and photographed using an FITC-compatible filter. The same cells were also assessed for endogenous ANP expression (ANP), viewed with a Texas red–conjugated second antibody, and photographed with a rhodamine-compatible filter. The digitized photographic images of luciferase- and ANP-positive cells were overlaid using Adobe Photoshop (San Jose, CA), and the resulting montage was prepared in Claris MacDraw Pro. Bar, 50 μM.
Mentions: Further studies were undertaken to compare the effects of Raf BXB, MEKKCOOH, and MKK6 (Glu) with the gold standard, PE, on other features of the program, such as cell size and sarcomeric organization. Compared to cells maintained in control media (Fig. 3, A and A′), the PE-treated cells were much larger (Fig. 3 B), displaying an approximately two- to three-fold increase in area (Fig. 4 A), and they possessed a high degree of sarcomeric organization (Fig. 3 B′). In general, the PE-treated cultures possessed about 10-fold more myocytes displaying organized sarcomeres than the control cultures (Fig. 4 B). PE-treated cultures also displayed significantly increased levels of endogenous ANP expression, observed as the prototypical perinuclear staining found often in hypertrophic cardiac myocytes (Fig. 3, F [control] vs. G [PE-treated]). Interestingly, cultures transfected with Raf BXB or MEKKCOOH displayed increases in size (Figs. 3, C [BXB] and D [MEKKCOOH], and 4 A), and while the usual shape of the Raf BXB–treated cells was similar to PE-treated cells, the MEKKCOOH-treated cells were almost always very long and thin. Moreover, while either Raf BXB (Fig. 3 H [BXB]) or MEKKCOOH (Fig. 3 I [MEKK-1]) fostered the induction of endogenous ANP expression, neither construct supported sarcomeric organization (Fig. 3 C′ [BXB] and 3 D′ [MEKK-1]; also see Fig. 4 B). These results suggested that neither ERK (Raf BXB) alone nor ERK and JNK (MEKKCOOH) were sufficient to confer all the features of the hypertrophic phenotype.

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