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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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Morphometric analyses of the effects of Raf-1 BXB,  MEKKCOOH, or MKK6 (Glu) expression constructs on size and  sarcomeric organization in myocardial cells. (A) Photographic  images of myocardial cells transfected and treated as described in  the legend to Fig. 3, A–E, were digitized and the areas (μm2) of  ∼20–50 cells from each treatment were determined using NIH  Image software, as described in the Materials and Methods.  Shown are the mean area values for each treatment ± SE (n = 3  cultures. (B) The myofilament structure in myocardial cells transfected and treated as described in the legend to Fig. 3, A′–E′, was  evaluated using BODIPY-phalloidin to stain actin. Upon visually  inspecting 50–100 transfected (i.e., β-galactosidase–positive) cells  per treatment using a fluorescence microscope, cells were scored  for possessing organized sarcomeres (i.e., appearing similar to  cells shown in Fig. 3, B′ or E′). The number of cells transfected  with test construct that scored positive for sarcomeric organization was normalized to (divided by) the maximal values for sarcomeric organization, which were obtained by PE treating cells that  had been transfected with the empty vector control; the results  are shown as a percentage of these maximal values. Generally,  ∼25% of the cells transfected with the empty vector control and  then treated with PE displayed highly organized sarcomeres.  Shown are the mean area values for each treatment ± SE (n = 3  cultures).
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Figure 4: Morphometric analyses of the effects of Raf-1 BXB, MEKKCOOH, or MKK6 (Glu) expression constructs on size and sarcomeric organization in myocardial cells. (A) Photographic images of myocardial cells transfected and treated as described in the legend to Fig. 3, A–E, were digitized and the areas (μm2) of ∼20–50 cells from each treatment were determined using NIH Image software, as described in the Materials and Methods. Shown are the mean area values for each treatment ± SE (n = 3 cultures. (B) The myofilament structure in myocardial cells transfected and treated as described in the legend to Fig. 3, A′–E′, was evaluated using BODIPY-phalloidin to stain actin. Upon visually inspecting 50–100 transfected (i.e., β-galactosidase–positive) cells per treatment using a fluorescence microscope, cells were scored for possessing organized sarcomeres (i.e., appearing similar to cells shown in Fig. 3, B′ or E′). The number of cells transfected with test construct that scored positive for sarcomeric organization was normalized to (divided by) the maximal values for sarcomeric organization, which were obtained by PE treating cells that had been transfected with the empty vector control; the results are shown as a percentage of these maximal values. Generally, ∼25% of the cells transfected with the empty vector control and then treated with PE displayed highly organized sarcomeres. Shown are the mean area values for each treatment ± SE (n = 3 cultures).

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)

Morphometric analyses of the effects of Raf-1 BXB,  MEKKCOOH, or MKK6 (Glu) expression constructs on size and  sarcomeric organization in myocardial cells. (A) Photographic  images of myocardial cells transfected and treated as described in  the legend to Fig. 3, A–E, were digitized and the areas (μm2) of  ∼20–50 cells from each treatment were determined using NIH  Image software, as described in the Materials and Methods.  Shown are the mean area values for each treatment ± SE (n = 3  cultures. (B) The myofilament structure in myocardial cells transfected and treated as described in the legend to Fig. 3, A′–E′, was  evaluated using BODIPY-phalloidin to stain actin. Upon visually  inspecting 50–100 transfected (i.e., β-galactosidase–positive) cells  per treatment using a fluorescence microscope, cells were scored  for possessing organized sarcomeres (i.e., appearing similar to  cells shown in Fig. 3, B′ or E′). The number of cells transfected  with test construct that scored positive for sarcomeric organization was normalized to (divided by) the maximal values for sarcomeric organization, which were obtained by PE treating cells that  had been transfected with the empty vector control; the results  are shown as a percentage of these maximal values. Generally,  ∼25% of the cells transfected with the empty vector control and  then treated with PE displayed highly organized sarcomeres.  Shown are the mean area values for each treatment ± SE (n = 3  cultures).
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Figure 4: Morphometric analyses of the effects of Raf-1 BXB, MEKKCOOH, or MKK6 (Glu) expression constructs on size and sarcomeric organization in myocardial cells. (A) Photographic images of myocardial cells transfected and treated as described in the legend to Fig. 3, A–E, were digitized and the areas (μm2) of ∼20–50 cells from each treatment were determined using NIH Image software, as described in the Materials and Methods. Shown are the mean area values for each treatment ± SE (n = 3 cultures. (B) The myofilament structure in myocardial cells transfected and treated as described in the legend to Fig. 3, A′–E′, was evaluated using BODIPY-phalloidin to stain actin. Upon visually inspecting 50–100 transfected (i.e., β-galactosidase–positive) cells per treatment using a fluorescence microscope, cells were scored for possessing organized sarcomeres (i.e., appearing similar to cells shown in Fig. 3, B′ or E′). The number of cells transfected with test construct that scored positive for sarcomeric organization was normalized to (divided by) the maximal values for sarcomeric organization, which were obtained by PE treating cells that had been transfected with the empty vector control; the results are shown as a percentage of these maximal values. Generally, ∼25% of the cells transfected with the empty vector control and then treated with PE displayed highly organized sarcomeres. Shown are the mean area values for each treatment ± SE (n = 3 cultures).
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