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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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Activation of p38, JNK, and ERK MAP kinases in myocardial cells. Myocardial cells were cotransfected with an expression construct encoding activated Ras (Ras V12), Rac (Rac  V12), Raf (Raf-1 BXB), JNK kinase (MEKKCOOH), p38 kinase  (MKK6 [Glu]), or an empty vector control (pCEP) and either  HA-p38, HA-JNK, or HA-ERK. After a 48-h incubation in serum-free media, the cultures (∼3 × 106 cells each) were extracted  and incubated with an HA monoclonal antibody, and the appropriate kinase assay was carried out on the resulting immune complex, as described in the Materials and Methods. After exposing  the resulting SDS gel to a phosphorimager plate, each phosphorylated substrate band was digitized and printed (see the inset of  each panel). The relative density of each band was determined  using Molecular Dynamics Image Quant software (Sunnyvale,  CA). Each treatment was carried out on two identical cultures,  and the average of the band density for each treatment was then  normalized to the maximal value obtained in each experiment.  Shown is the percentage of the maximum; the average variation  between duplicate samples was 10% or less. This is representative of three identical experiments that produced similar results.
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Figure 1: Activation of p38, JNK, and ERK MAP kinases in myocardial cells. Myocardial cells were cotransfected with an expression construct encoding activated Ras (Ras V12), Rac (Rac V12), Raf (Raf-1 BXB), JNK kinase (MEKKCOOH), p38 kinase (MKK6 [Glu]), or an empty vector control (pCEP) and either HA-p38, HA-JNK, or HA-ERK. After a 48-h incubation in serum-free media, the cultures (∼3 × 106 cells each) were extracted and incubated with an HA monoclonal antibody, and the appropriate kinase assay was carried out on the resulting immune complex, as described in the Materials and Methods. After exposing the resulting SDS gel to a phosphorimager plate, each phosphorylated substrate band was digitized and printed (see the inset of each panel). The relative density of each band was determined using Molecular Dynamics Image Quant software (Sunnyvale, CA). Each treatment was carried out on two identical cultures, and the average of the band density for each treatment was then normalized to the maximal value obtained in each experiment. Shown is the percentage of the maximum; the average variation between duplicate samples was 10% or less. This is representative of three identical experiments that produced similar results.

Mentions: To characterize the effects of overexpressing various signaling proteins on each of the three MAPK family members in the cardiac context, myocardial cells were cotransfected with constructs encoding gain-of-function forms of Ras, Rac, Raf-1 kinase, JNKK kinase, or p38 kinase, and constructs encoding HA-tagged p38, JNK, or ERK. In the cardiac myocytes, Ras V12 served as a poor activator of either p38 or JNK, but as expected, it was a strong activator of ERK (Fig. 1). Rac V12 had no effect on p38 or ERK in the cardiac cells; however, it strongly activated JNK, consistent with its hypothesized ability to serve as an upstream activator of MEKK1 (Lange-Carter et al., 1993; Derijard et al., 1994). Raf BXB, which encodes an active form of Raf-1 kinase (Bruder et al., 1992; Kolch et al., 1993), served primarily as an ERK activator, while MEKKCOOH, a truncated active form of MEKK1 (Lange-Carter et al., 1993), mildly activated p38 by about fourfold, moderately activated JNK by 8–10-fold, as expected, but more strongly stimulated ERK by about 25-fold (Fig. 1). The ability of MEKKCOOH to activate both JNK and ERK is consistent with results in other cell types (Minden et al., 1994). Importantly, however, MKK6 (Glu), an activated form of the p38 kinase, MKK6 (Raingeaud et al., 1996), potently activated p38 in the cardiac myocytes by about 16-fold, with no effect on either ERK or JNK (Fig. 1). A kinase-dead form of MKK6, MKK6 (K82A) (Raingeaud et al., 1996), did not activate p38 in the cardiac cells (not shown). These results verify the utility of constructs encoding activated forms of Raf, MEKK1, and MKK6 as stimulators of the MAPK pathways, and in particular, they clearly show that MKK6 serves as a very selective p38 activator in cardiac myocytes.


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)

Activation of p38, JNK, and ERK MAP kinases in myocardial cells. Myocardial cells were cotransfected with an expression construct encoding activated Ras (Ras V12), Rac (Rac  V12), Raf (Raf-1 BXB), JNK kinase (MEKKCOOH), p38 kinase  (MKK6 [Glu]), or an empty vector control (pCEP) and either  HA-p38, HA-JNK, or HA-ERK. After a 48-h incubation in serum-free media, the cultures (∼3 × 106 cells each) were extracted  and incubated with an HA monoclonal antibody, and the appropriate kinase assay was carried out on the resulting immune complex, as described in the Materials and Methods. After exposing  the resulting SDS gel to a phosphorimager plate, each phosphorylated substrate band was digitized and printed (see the inset of  each panel). The relative density of each band was determined  using Molecular Dynamics Image Quant software (Sunnyvale,  CA). Each treatment was carried out on two identical cultures,  and the average of the band density for each treatment was then  normalized to the maximal value obtained in each experiment.  Shown is the percentage of the maximum; the average variation  between duplicate samples was 10% or less. This is representative of three identical experiments that produced similar results.
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Related In: Results  -  Collection

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Figure 1: Activation of p38, JNK, and ERK MAP kinases in myocardial cells. Myocardial cells were cotransfected with an expression construct encoding activated Ras (Ras V12), Rac (Rac V12), Raf (Raf-1 BXB), JNK kinase (MEKKCOOH), p38 kinase (MKK6 [Glu]), or an empty vector control (pCEP) and either HA-p38, HA-JNK, or HA-ERK. After a 48-h incubation in serum-free media, the cultures (∼3 × 106 cells each) were extracted and incubated with an HA monoclonal antibody, and the appropriate kinase assay was carried out on the resulting immune complex, as described in the Materials and Methods. After exposing the resulting SDS gel to a phosphorimager plate, each phosphorylated substrate band was digitized and printed (see the inset of each panel). The relative density of each band was determined using Molecular Dynamics Image Quant software (Sunnyvale, CA). Each treatment was carried out on two identical cultures, and the average of the band density for each treatment was then normalized to the maximal value obtained in each experiment. Shown is the percentage of the maximum; the average variation between duplicate samples was 10% or less. This is representative of three identical experiments that produced similar results.
Mentions: To characterize the effects of overexpressing various signaling proteins on each of the three MAPK family members in the cardiac context, myocardial cells were cotransfected with constructs encoding gain-of-function forms of Ras, Rac, Raf-1 kinase, JNKK kinase, or p38 kinase, and constructs encoding HA-tagged p38, JNK, or ERK. In the cardiac myocytes, Ras V12 served as a poor activator of either p38 or JNK, but as expected, it was a strong activator of ERK (Fig. 1). Rac V12 had no effect on p38 or ERK in the cardiac cells; however, it strongly activated JNK, consistent with its hypothesized ability to serve as an upstream activator of MEKK1 (Lange-Carter et al., 1993; Derijard et al., 1994). Raf BXB, which encodes an active form of Raf-1 kinase (Bruder et al., 1992; Kolch et al., 1993), served primarily as an ERK activator, while MEKKCOOH, a truncated active form of MEKK1 (Lange-Carter et al., 1993), mildly activated p38 by about fourfold, moderately activated JNK by 8–10-fold, as expected, but more strongly stimulated ERK by about 25-fold (Fig. 1). The ability of MEKKCOOH to activate both JNK and ERK is consistent with results in other cell types (Minden et al., 1994). Importantly, however, MKK6 (Glu), an activated form of the p38 kinase, MKK6 (Raingeaud et al., 1996), potently activated p38 in the cardiac myocytes by about 16-fold, with no effect on either ERK or JNK (Fig. 1). A kinase-dead form of MKK6, MKK6 (K82A) (Raingeaud et al., 1996), did not activate p38 in the cardiac cells (not shown). These results verify the utility of constructs encoding activated forms of Raf, MEKK1, and MKK6 as stimulators of the MAPK pathways, and in particular, they clearly show that MKK6 serves as a very selective p38 activator in cardiac myocytes.

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