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 α1- 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 α-skeletal actin (α-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 α-SkA promoter activities, neither upstream kinase conferred sarcomeric organization. However, transfection with MKK6 (Glu), which specifically activated p38, augmented cell size, induced NP and α-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.

Cardiac myocytes, which are postmitotic, increase in size during postnatal development through a well-studied hypertrophic growth program. Myocardial cell hypertrophic growth is characterized by a number of phenotypic changes, including the activation of several immediate early genes (e.g., c-fos, c-jun, and egr-1), increased expression of genes encoding certain sarcomeric proteins (e.g., α-skeletal actin, β-myosin heavy chain, and myosin light chain-2), and the induction of the genes for the A- and B-type cardiac natriuretic peptides (ANP and BNP)1 (Schneider et al., 1992; Van Bilsen and Chien, 1993; Lembo et al., 1995). Although myocardial mass in the fully developed adult does not generally undergo significant increases in size, in some pathological conditions, such as overload-induced hypertrophy, adult cardiac myocytes do reenter a hypertrophic growth program very similar to that observed in the developing neonatal heart (Schneider et al., 1992; Van Bilsen and Chien, 1993; Lembo et al., 1995; Van Heugten et al., 1995; Yamazaki et al., 1995).

Cultured neonatal rat ventricular cardiac myocytes have served as a model system for studies aimed at gaining a better understanding of this interesting mechanism of cell growth. Primary myocardial cells respond to a variety of stimuli by undergoing a hypertrophic growth program virtually identical to that observed in the developing neonate and the pathologic adult myocardium (Van Bilsen and Chien, 1993). For example, cultured myocardial cells treated with the α1-adrenergic receptor agonist, phenylephrine (PE), various other growth factors, or mechanical loading or electrical pacing of contractions display marked increases in size, enhanced sarcomeric organization, and induction of the cardiac genes associated with the hypertrophic growth program (Simpson, 1983; Komuro et al., 1990; McDonough and Glembotski, 1992; LaMorte et al., 1994; Sadoshima et al., 1995; Bogoyevitch et al., 1995b; Karns et al., 1995; Sprenkle et al., 1995; LaPointe et al., 1996; Thuerauf and Glembotski, 1997). The convergence of these diverse stimuli on the features that define myocardial cell growth suggests that a select group of intracellular signaling pathways are coordinately activated by these treatments. Accordingly, a better understanding of myocardial cell signaling pathways that contribute to hypertrophic growth is required to fully grasp normal cardiac development as well as counterproductive pathological growth.

Studies of the intracellular signaling mechanisms responsible for myocardial cell growth have focused on the mitogen-activated protein kinases (MAPK), in part because at least one of the MAPKs, extracellular signal–regulated kinase (ERK), has been implicated in mitogenic growth in a variety of cell types (Cobb et al., 1991). Typically, ERK is activated via the well-known sequential pathway sometimes referred to as a MAPK module. MAPK modules consist of three members, a MAPKKK, followed by a MAPKK and then the terminal MAPK, itself. In the case of the ERK pathway, Ras stimulates the MAPKKK, Raf-1, which activates the MAPKK known as MEK (Kyriakis et al., 1992; Davis, 1993).

In cultured cardiac myocytes, PE activates Ras, Raf, MEK, and ERK (Thorburn and Thorburn, 1994; Bogoyevitch et al., 1993a, 4b ,Clerk et al., 1994; Thorburn et al., 1994a), and transfection of constructs encoding active Ras, Raf, or MEK can induce ANP, β-MHC, α-skeletal actin (α-SkA) and/or MLC-2 promoter activities (Thorburn et al., 1993, 1994b; Abdellatif et al., 1994; Thuerauf and Glembotski, 1997) . Moreover, dominant interfering forms of Ras or Raf can inhibit PE-induced ERK and cardiac gene promoter activity (Thorburn et al., 1993, 1994b; Thorburn, 1994), consistent with a role for ERK in the cardiac growth program. However, the overexpression of active forms of either Ras or ERK does not lead to the sarcomeric organization typical of hypertrophic growth (Thorburn et al., 1994a,b), and inhibiting MEK with PD 098059 does not block PE-induced sarcomeric organization or ANP gene expression (Post et al., 1996). Additionally, while myocardial cell ERK can be activated by some agonists, such as PE, ET, or FGF, each of which can cause hypertrophic growth, it can also be activated by agonists, such as ATP or carbachol, neither of which support myocardial cell growth (Post et al., 1996). These findings suggest that the activation of ERK alone is not sufficient to confer hypertrophic growth and the related gene expression.

Another member of the MAPK family, the stress-kinase c-Jun NH2-terminal kinase (JNK) (Davis, 1994; Cano and Mahadevan, 1995; Karin, 1995) has received some attention as a potential mediator of growth in cardiac myocytes. The activation cascade for JNK is somewhat less understood than that for ERK. However, it is believed that in most cells the sequential stimulation of the Ras-like monomeric GTP-binding protein, Rac, perhaps by Ras itself, leads to the activation of MEKK1, a MAPKKK that then activates the MAPKK, MEK4, also called SEK or JNK kinase, culminating in the activation of c-jun kinase (JNK)/ MAPK (Lange-Carter et al., 1993; Derijard et al., 1994).

JNK is activated in cultured myocardial cells by the growth promoters PE and ET-1 (Bogoyevitch et al., 1995a), suggesting that it may contribute to the hypertrophic phenotype. Transfection of cultured myocardial cells with a construct encoding active MEKK1 leads to an approximately twofold increase in myocardial cell size and 5–50-fold enhancement of ANP, β-MHC, and α-SkA promoter activities (Bogoyevitch et al., 1995a, 1996). However, there have been no reports that JNK activation fosters the sarcomeric organization that is an obligate feature of the hypertrophic phenotype. Thus, it appears that like the ERK pathway, the JNK pathway alone may not be sufficient to support all of the main features associated with the hypertrophic growth program.

The third and most recently characterized member of the MAPK family is the stress-kinase, p38, also known as protein kinase HOG1 (Rouse et al., 1994; Raingeaud et al., 1995). In comparison to the JNK pathway but in contrast to ERK, the p38 pathway is not commonly activated by mitogens but is induced by cell stresses such as endotoxins, osmotic shock, or metabolic inhibitors. The upstream activators of p38 are poorly understood; however, recent studies have resulted in the cloning and characterization of members of the p38 MAPKK module, most notably MKK4, which can activate both JNK and p38 (Lin et al., 1995), and MKK3 (Derijard et al., 1995) and MKK6 (Raingeaud et al., 1996; Han et al., 1996), either of which specifically activate p38. MKK6 is also known as SAPKK3 (Cuenda et al., 1996).

In previous studies, it was shown that a p38-like kinase is induced in isolated perfused rat heart after preconditioning, which is a brief ischemic treatment known to protect cardiac tissue from damage due to subsequent ischemic episodes taking place soon thereafter (Bogoyevitch et al., 1996; Maulik et al., 1996). The possibility that p38 activation could contribute to the protective effects of preconditioning is consistent with findings that p38 can lead to the phosphorylation of heat-shock proteins (Stokoe et al., 1992b), a modification thought to enhance their cell-protective effects. However, the putative role of the p38 pathway in the hypertrophic growth of cardiac myocytes has not been assessed.

The goal of the present study was to compare the effects of activating the p38 pathway with activation of ERK and JNK on the three major features of the myocardial cell hypertrophic growth program- cell size, sarcomeric organization, and activation of cardiac gene expression. It was found that only p38 activation conferred all three features of myocardial cell growth in a manner similar to that observed using PE. These results suggest that p38 plays a central role in mediating the cardiac hypertrophic growth pathway, a concept that could also be applicable to other terminally differentiated cell types.

Materials And Methods

Cell Culture

Primary ventricular myocytes were prepared from 1–4-d-old neonatal rats as previously described (Sprenkle et al., 1995; Thuerauf and Glembotski, 1997). After the enzymatic dissociation of ventricular tissue, the cells were plated onto uncoated plastic dishes in DME/F-12 (1:1)/10% FBS for 1 h during which time most of the fibroblasts adhered to the dish. The recovered cells were then transfected (see below), plated on fibronectin-coated plastic dishes or glass slides (3 × 105 cells/mm2), and then maintained for about 18 h in DME/F-12 (1:1)/10% FBS. The cultures were then washed briefly with medium, refed with serum-free DME/F-12 (1:1), maintained for 48 h, and then either extracted for reporter enzyme assay (see below) or fixed and then analyzed by immunocytofluorescence or staining with fluorescent phallicidin.

Transfections

After preplating (see above), myocardial cells were resuspended at a density of 30 million cells/ml minimal medium (DMEM:F12 [GIBCO BRL, Gaithersburg, MD] containing 1 μg/ml BSA) and transfections were carried out as described previously (Sprenkle et al., 1995; Thuerauf and Glembotski, 1997). Briefly, for each transfection, 300 μl, or 9 million cells, were mixed with 15–30 μg of either ANP-3003GL (Sprenkle et al., 1995), BNP-2501GL (Thuerauf and Glembotski, 1997), α-SkA-394GL (MacLellan et al., 1994), or pG5E1bLuc (Raingeaud et al., 1996); 3–9 μg of cytomegalovirus–β-galactosidase (used as a normalization reporter except where the test construct is known to activate CMV promoter activity [Gillespie-Brown et al., 1995; Paradis et al., 1996; Post et al., 1996]); and in some experiments, 15–45 μg of an activated Ras, Rac, Raf-1, JNKKK, MKK6, activating transcription factor-2 (ATF2)/Gal4, or MEF2C/Gal4 expression construct (see below). The levels of plasmid used in each culture within an experiment were equalized using empty vector DNA, such as pCEP. Each 300-μl aliquot was then electroporated in a Bio-Rad (Hercules, CA) Gene Pulser at 700 V, 25 μF, 100 Ω in a 0.2-cm-gap cuvette, a protocol that allows for the selective transfection of only cardiac myocytes (Sprenkle et al., 1995). This procedure results in an ∼30% viability (Sprenkle et al., 1995); Accordingly, the 3 million viable cells were plated into fibronectin-coated 35-mm wells, at 1 × 106 cells/well, into 24-mm wells at 0.5 × 106 cells/well, or into four-chamber Lab Tek chamber slides at 0.3 × 106 cells/2 cm2 well.

Reporter Enzyme Assays

Transfected cells were maintained in DMEM:F12 supplemented with 10% FBS for ∼16 h after electroporation. The cells were then washed thoroughly and the medium was replaced with minimal medium. Unless otherwise stated, 24 h later, the medium was again replaced with minimal medium ± 50 μM phenylephrine with 1 μM propranolol added to block β-adrenergic receptors. Luciferase and β-galactosidase assays were performed as described (Sprenkle et al., 1995). Luciferase activity was measured for 30 s on a Bio Orbit 1251 Luminometer (Pharmacia Biotech. Inc., Piscataway, NJ). Data are expressed as “Relative Luciferase (Rel Luc)” = arbitrary integrated luciferase units/β-galactosidase units, representative of at least three independent experiments performed with two different plasmid preparations, and represent the mean and SEM of triplicate 35- or 24-mm wells.

Test Expression Constructs

To assess the effects of various signaling proteins, the following constructs were used: pDCR H-RasV12 (codes for activated Ha-Ras; from D. Bar-Sagi, State University of New York at Stony Brook, NY), pDCR RacV12 (codes for activated Rac; from M. Cobb and J. Frost, University of Texas Southwestern Medical Center, Dallas, TX), RSV-Raf-1 BXB (codes for activated Raf-1 kinase; from U. Rapp, University of Wurzburg, Wurzburg, Germany), pCMV5 MEKKCOOH (codes for activated MEKK-1; from G. Johnson, University of Colorado, Denver, CO), pcDNA3 MKK6 (Glu) (codes for activated MKK6, or p38/MAPKK; from R. Davis, University of Massachusetts, Worcester MA), ATF2/Gal4 (codes for the ATF2 transcriptional activation domain fused to the Gal4 DNA–binding domain; from R. Davis), MEF2C/Gal4 (codes for MEF2C fused to the Gal4 DNA– binding domain; from J. Han, The Scripps Research Institute, La Jolla, CA), MEF2C-S/Gal4 (codes for mutant [Ser to Ala 387] MEF2C fused to the Gal4 DNA–binding domain; from J. Han), pG5E1bLuc (codes for 5X Gal4 sites cloned upstream of a prolactin promoter driving luciferase expression; from R. Davis). Preliminary experiments using different concentrations of each construct verified that optimal doses were chosen.

Immunocytofluorescence: Morphometric Analyses

To study the effects of activating the MAPK pathways on cell size and sarcomeric organization, myocardial cells were cotransfected with a test expression construct or an empty vector control and CMV–β-galactosidase. Double-staining experiments demonstrated a cotransfection efficiency of ∼85%. After treatment for 48 h with or without PE, cultures were fixed, as described (McDonough and Glembotski, 1992). Transfected cardiac myocytes were identified by immunostaining for β-galactosidase using a Texas red–conjugated anti–mouse IgG. Since the β-galactosidase is cytosolic, staining was uniform throughout the myocardial cells, facilitating the determination of cell area (see below). The same samples were also stained with BODIPY-conjugated phallicidin, an actin-specific stain, to evaluate sarcomeric organization, as described (McDonough and Glembotski, 1992).

To study the effects of the test expression constructs on endogenous cardiac gene expression, myocardial cells were cotransfected with a test expression construct or an empty vector control and ANP-3003GL. After fixation, the cells were immunostained for luciferase using a rabbit anti– luciferase antiserum, and they were immunostained for ANP using a mouse monoclonal antibody to rat ANP (Glembotski et al., 1987) and visualized using differential fluorescence. Generally, positive ANP staining was visualized using a Texas red–conjugated anti–mouse IgG, and positive luciferase staining was visualized using an FITC-conjugated anti–rabbit IgG.

Morphometric Analyses

Cell Size.

Transfected myocytes that immunostained positively for β-galactosidase were microscopically visualized under fluorescent illumination and photographed. The photographic images were then digitally acquired using a scanner (model ES-1200C; Epson America, Inc., Torrance, CA) attached to a Apple Power Mac 8500 (Cupertino, CA). The area in pixels of each digitized image was determined using NIH Image software and compared to a standard image possessing an area of 1 μm2. This enabled the designation of area, in square micrometers, to each cell image; between 20 and 50 images of different cells derived from each treatment were analyzed. The values reported are the mean areas, in square micrometers ± standard error.

Sarcomeric Organization.

Transfected myocytes were identified by positive β-galactosidase immunostaining and observed using a Texas red– compatible filter. The cells were then viewed after phalloidin staining using an FITC-compatible filter and scored positively for sarcomeric organization if the myofilament alignment resembled that in cells treated with PE. Approximately 50–100 cells from each of three cultures per treatment were assessed. Generally, treating CMV–β-galactosidase/pCEP-transfected cells with PE (positive control) or without (negative control) resulted in ∼25 and 2% of the β-galactosidase–positive cells scoring for sarcomeric organization, respectively. In experimental cultures transfected with CMV–β-galactosidase and a test construct, the number of cells scoring positive for sarcomeres was normalized to (divided by) the number of cells scoring positive for sarcomeres obtained with PE, which gave maximal values, and the results are displayed as percentages of maximal values.

MAPK Assays

To test the effects of treatments on MAPK activity levels, hemagglutinin (HA)-tagged forms of ERK1 (pCEP4 HA-wt-ERK1 from M. Cobb), JNK (SRa-HA-JNK from G. Johnson) or p38 (pCEP4 HA-wt-p38Hog1 from M. Cobb) were cotransfected with the test constructs. After the appropriate times, cultures were extracted in a buffer containing 10 mM Tris, pH 7.6, 1% Triton X-100, 0.05 M NaCl, 5 mM EDTA, 2 mM o-vanadate, and 20 μg/ml aprotinin. After brief centrifugation, extracts were incubated for 2 h at 4°C with HA monoclonal antibody (12CA5; Boehringer-Mannheim Corp., Indiannapolis, IN), bound to protein A–Sepharose (Pharmacia Biotech, Inc.) and immune-complex kinase assays were carried out using the appropriate substrates, as described (Derijard et al., 1994; Post et al., 1996). Briefly, reactions were initiated by the addition of 1 μg of the appropriate substrate, MBP for ERK, GST–c-Jun for JNK, Phas-I for p38, and 6 μM [γ-32P]ATP (5,000 Ci/mmol) in a final volume of 30 μl of kinase buffer (20 mM Hepes, pH 7.4, 20 mM MgCl2, 20 mM β-glycerophosphate, 2 mM DTT, 20 μM ATP). After 30 min at 25°C, the reactions were terminated by the addition of Laemmli sample buffer, and the phosphorylation level of substrate proteins was evaluated by SDS-PAGE followed by autoradiography and phosphorimage analyses.

In each experiment, two identically treated cultures (1.5 × 106 cells/35-mm dish) were used for each treatment, and after densitometric analyses of the exposed phosphorimage plates, values for each treatment were averaged.

Results

MKK6 (Glu) Selectively Activates p38 in Myocardial Cells and Strongly Stimulates Cardiac Gene Expression

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.

The abilities of the various expression constructs to activate three cardiac genes (ANP, BNP, and α-SkA) that serve as hallmarks of the hypertrophic growth program were tested using ANP-3003GL, BNP-2501GL, or α-SkA– 394GL. These reporter constructs possess 3,003, 2,501, or 394 bp of the ANP, BNP, or α-SkA 5′-flanking sequences, respectively. As expected from previous studies (Thorburn et al., 1993; MacLellan et al., 1994; Thuerauf and Glembotski, 1997), Ras V12 served as a strong activator of both natriuretic peptide (NP) promoters, fostering up to 50-fold activation of luciferase expression (Fig. 2). The Rac V12 construct also activated these promoters, but less strongly than Ras, ∼10-fold; this may reflect the differential efficacies of ERK and JNK as inducers of the cardiac genes studied. Although Raf BXB and MEKKCOOH stimulated NP and α-SkA promoter activities by up to 20-fold, most notable were the effects of the p38-activating construct, MKK6 (Glu), which stimulated up to 130-fold (Fig. 2). These findings suggest that while each of the MAPK pathways can stimulate cardiac natriuretic peptide and α-SkA gene expression, the p38 pathway as stimulated with MKK6 (Glu) confers the strongest induction of the three genes studied.

MKK6 (Glu) Increases Cardiac Myocyte Size and Sarcomeric Organization

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.

When myocardial cells were transfected with MKK6 (Glu), they were on average four times larger than control cells (Figs. 3, E and 4,A), and notably, they displayed sarcomeric organization that was visually similar to that observed upon PE treatment (Figs. 3,E′ and 4 B). Consistent with the high degree of sarcomeric ordering was the finding that cells transfected with MKK6 (Glu) displayed spontaneous contractile activity. Moreover, like PE-treated cells, MKK6 (Glu)–transfected myocardial cells possessed significantly elevated levels of endogenous ANP (Fig. 3,J), consistent with the ability of MKK6 (Glu) to strongly activate NP promoter activities (Fig. 2). Thus, it was apparent that MKK6 (Glu), a selective p38 activator in the cardiac myocytes, was able to mimic the three hallmark features of the hypertrophic growth program.

The Effects of PE and MKK6 Are Sensitive to a p38 Inhibitor

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.

The stress-activated MAPK pathways, especially p38, are well–known stimulators of ATF2 (Gupta et al., 1995; Raingeaud et al., 1996). ATF2 can dimerize with other ATF family members (e.g., cAMP response element–binding protein [CREB] or ATF-1), Rb, NF-κB, or c-jun and enhance transcription through cAMP response elements, AP-1 sites, or NF-κB sites. Accordingly, the abilities of MKK6 (Glu) or PE to activate ATF2-dependent transcription in cardiac myocytes were tested. Myocardial cells were cotransfected with a reporter plasmid possessing GAL4 DNA–binding sites cloned upstream of luciferase, a construct encoding a fusion protein composed of the ATF2 transactivation domain and the GAL4 DNA–binding domain, and either MKK6 (Glu) or pCEP. Transfection with MKK6 (Glu) or treatment pCEP-transfected cells with PE enhanced ATF2-mediated luciferase production (Fig. 5,C). Moreover, SB 203580 served as a potent inhibitor of MKK6 (Glu)–activated ATF2, consistent with a major requirement for p38. Interestingly, PE-activated ATF2 was only partially blocked by SB 203580 (Fig. 5,C), as was PE-activated BNP transcription (Fig. 5 B), suggesting that the ability of this agonist to activate cardiac gene expression is partly dependent on p38, or a very similar kinase.

The SB 203580 compound also had potent inhibitory effects on myocardial cell size and sarcomeric organization induced by PE or MKK6 (Glu) (Fig. 7). PE- or MKK6 (Glu)–mediated sarcomeric organization and increases in cell size were reduced by >90% by SB 203580 (Fig. 8). These results are consistent with a central role for a p38-like pathway in myocardial cell growth conferred by PE or by MKK6 (Glu).

Recent studies have established that the transcription factor, MEF2C, serves as a specific substrate for p38, such that phosphorylation on serine 387 confers MEF2C-mediated transcriptional activation in RAW 264.1 cells (Han et al., 1997). Accordingly, the abilities of PE or MKK6 (Glu) to activate MEF2C-dependent transcription in cardiac myocytes were tested. Myocardial cells were cotransfected with a reporter plasmid possessing GAL4 DNA–binding sites cloned upstream of luciferase and a construct encoding a fusion protein comprised of the MEF2C transactivation domain and the GAL4 DNA–binding domain. Using this system, the activation of MEF2C after phosphorylation on serine 387 can be studied in the cardiac myocytes without interference from any endogenous MEF2 family members. Treatment with PE or transfection with MKK6 (Glu)–enhanced MEF2C-mediated luciferase production (Fig. 9, MEF2C/Gal4). When cells were transfected with an altered MEF2C/Gal4 chimera, in which serine 387 was mutated to alanine, neither PE nor MKK6 (Glu) conferred luciferase induction (Fig. 9, MEF2C-S/Gal4). These results indicate that like MKK6 (Glu), PE can activate MEF2C in cardiac myocytes, an event that requires phosphorylation at serine 387 by p38. This result further supports the notion that in part, PE enhances myocardial cell growth, sarcomeric organization, and the related gene expression through a pathway involving p38 or a very similar kinase.

Discussion

Under certain conditions, cardiac myocytes undergo nonmitotic, hypertrophic growth that is typified by dramatic increases in cell size, high degrees of sarcomeric organization, and enhanced expression of certain cardiac-specific genes. Several results from this study indicate that MKK6-activated p38 is sufficient to confer the three main features of this unique hypertrophic growth program. First, MKK6 (Glu), which amongst the MAPKs induces only p38 (Fig. 1 and Raingeaud et al., 1996), conferred sarcomeric organization, increased cell size, and increased cardiac gene expression in a manner similar to the well-characterized α1-adrenergic receptor agonist, PE. Second, when induced by MKK6, all three features of the growth program could be blocked by the p38-specific inhibitor, SB 203580. Interestingly, PE-enhanced sarcomeric organization, cell size, and, to some extent, cardiac gene induction were also blocked by SB 203580, indicating that the p38 pathway probably plays an important role in α1-adrenergic receptor signaling in myocardial cells. However, since the effects of PE on BNP transcription and ATF2-enhanced transcription were only partially blocked by SB 203580, it appears that while p38 may be central to some features of the hypertrophic response, it may play only a partial role in mediating other aspects of the growth program.

To our knowledge, this is the first report to document that the activation of the p38/MAPK pathway can mimic the morphological changes and gene inductive effects of growth factor treatment in any cell type. Thus, while p38/ MAPK is known as being a stress-activated kinase, it can apparently contribute to cell growth in a manner that may represent a compensatory response to stress. Although such a role for p38 contrasts with earlier findings that p38 induces apoptosis (Xia et al., 1995), it is consistent with recent studies indicating that this stress kinase can promote survival in certain cell types (Juo et al., 1997); in this respect, p38 appears to function in a cell-specific manner.

The mechanism by which MKK6-mediated p38 activation could lead to myocardial cell hypertrophic growth remains to be elucidated; however, recent work has revealed several downstream p38 targets that could be involved. For example, p38 phosphorylates and activates several transcription factors, such as ATF2 and Elk-1 (Gupta et al., 1995; Livingstone et al., 1995; Raingeaud et al., 1995, 1996), which could augment the expression of cardiac-specific genes induced during hypertrophy. Many of these inducible genes are known to possess relevant cis-acting sequences, including serum response elements, cAMP response elements, AP-1 sites, and NF-κB sites. Most recently, it has been demonstrated that the muscle cell–enriched transcription factor, MEF2C, a MADS box protein known to bind to A/T-rich regions of muscle-specific genes and known to be required for proper growth and development of cardiac muscle (Edmondson et al., 1994; Olson and Srivastava, 1996), serves as a substrate for p38 but not ERK or JNK (Han et al., 1997). In that report, the p38-specific phosphorylation was shown to lead to the activation of MEF2C as a transcription factor. Both the rat ANP and BNP 5′-flanking regions, as well as regulatory regions of other genes induced during the hypertrophic growth program (e.g., α-skeletal actin and β-myosin heavy chain genes), contain A/T-rich regions that are required for transcriptional induction and could bind MEF2C or related proteins (MacLellan et al., 1994; Thuerauf et al., 1994; Karns et al., 1995; Sprenkle et al., 1995). Thus, it is possible that p38 could phosphorylate and activate transcription factors that augment the expression of genes that participate in the cardiac growth program.

Alternatively, p38 may activate other downstream kinases that serve as the final steps in the signaling program. For example, it is well known that p38 can phosphorylate and activate MAP kinase–activated protein kinases (MAPKAPs)-1, -2, and -3 (MAPKAP-3 a.k.a. 3pK) (Stokoe et al., 1992a; Young et al., 1993; Rouse et al., 1994; English et al., 1995; Ludwig et al., 1996; McLaughlin et al., 1996; Sithanandam et al., 1996; Tan et al., 1996). In response to p38 activation by growth factors, MAPKAPs have been shown to phosphorylate and activate selected transcription factors, such as CREB and ATF-1, usually at protein kinase A/CaMK consensus sequences (Tan et al., 1996). Thus, it is possible that via the MAPKAPs, myocardial cell p38 stimulation could culminate with the activation of transcription factors often thought of as being downstream of non-MAPKs, e.g., protein kinase A or CaMK.

In addition to altering the function of transcription factors, p38-mediated MAPKAP/3pK activation could modulate other pathways that might favor cell survival, and in cardiac myocytes, these pathways could contribute to the development of myocardial cell-specific features, such as myofilament organization. For example, MAPKAP-2 has been shown to be activated during ischemic preconditioning of isolated rat hearts (Bogoyevitch et al., 1996; Maulik et al., 1996). Such preconditioning is known to serve as a myocardial stress adaptation, resulting in enhanced protection from ischemia-induced myocardial cell death (Murry et al., 1986; Parratt, 1994; Cumming et al., 1996; Gottlieb et al., 1996). In part, it is believed that this cardioprotection is derived from the induction and activation of heat-shock proteins (hsp's) 27 and 70 (Marber et al., 1993; Mestril et al., 1994; Parratt, 1994), both of which are known to protect cells from apoptosis (Mehlen et al., 1996; Samali and Cotter, 1996; Sharma et al., 1996). Interestingly, MAPKAP-2 and -3 have been shown to phosphorylate hsp 27, a modification known to enhance its protective properties (Ahlers et al., 1994; Huot et al., 1995). Moreover, after phosphorylation induced by either heat-shock or mitogen stimulation, hsp 27 has been shown to bind to and stabilize actin filaments in mouse fibroblasts (Lavoie et al., 1993). Such hsp 27–mediated filament stabilization in cardiac myocytes could be a major contributor to the striking sarcomeric organization observed upon MKK6-mediated p38 activation. Intracellular signaling pathways leading to hsp activation and/or phosphorylation in cardiac myocytes could conceivably extend back to α1-adrenergic receptors. Indeed, adrenergic receptor stimulation has been shown to activate hsp's in a variety of cell types; most notable is the finding that α1-adrenergic agonists activate hsp 70 in rat aortic cells (Chin et al., 1996) and in rat cardiac cells (Meng et al., 1996).

In summary, our results demonstrate a role for MKK6 and p38 in myocardial cell hypertrophic growth and gene expression. This is consistent with the view that the hypertrophic growth program represents a compensatory response of the myocardium to stress. In a physiological context, the mediators of the hypertrophic response are often hemodynamic stresses, such as increases in blood pressure or volume. Accordingly, increases in myocardial cell size and contractile function afforded by such growth could be viewed as cellular adaptations designed to counteract a physiological stress. Indeed, induction of the cardiac natriuretic peptide genes, which encode hormones that decrease blood pressure and volume, represent an endocrine compensatory response by the myocardium. The findings that the p38 pathway can mediate all three primary features of the myocardial cell growth program represent a major advancement in our understanding of the signals that regulate this important induction process. Future studies aimed at determining how other signaling pathways, perhaps even the other MAPK pathways, complement the p38 pathway and how p38 itself contributes to myocyte growth, sarcomeric organization, and cardiac gene induction, should reveal new roles for this interesting stress-kinase pathway in the heart.

Acknowledgments

This work was supported in part by National Institutes of Health Grants NS/HL-25073 (C.C. Glembotski), HL-46345 (C.C. Glembotski), HL-56861 (C.C. Glembotski) and HL-54030 (P.M. McDonough). This work was done during the tenure of a predoctoral research fellowship from the American Heart Association, California Affiliate, awarded to D.S. Hanford.

Abbreviations used in this paper

     
  • α-SkA

    α-skeletal actin

  •  
  • ANP and BNP

    A- and B-type cardiac natriuretic peptides

  •  
  • ATF

    activating transcription factor

  •  
  • CMV

    cytomegalovirus

  •  
  • ERK

    extracellular signal–regulated kinase

  •  
  • HA

    hemagglutinin

  •  
  • hsp

    heat-shock protein

  •  
  • JNK

    NH2-terminal kinase

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MAPKAP

    MAPK-activated protein kinase

  •  
  • PE

    phenylephrine

REFERENCES

REFERENCES
Abdellatif
M
,
MacLellan
WR
,
Schneider
MD
p21 Ras as a governor of global gene expression
J Biol Chem
1994
269
15423
15426
[PubMed]
Ahlers
A
,
Belka
C
,
Gaestel
M
,
Laming
N
,
Scott
C
,
Herrmann
F
,
Brach
MA
Interleukin-1-induced intracellular signaling pathways converge in the activation of mitogen-activated protein kinase and mitogen-activated protein kinase-activated protein kinase 2 and the subsequent phosphorylation of the 27-kilodalton heat shock protein in monocytic cells
Mol Pharm
1994
46
1077
1083
[PubMed]
Bogoyevitch
MA
,
Glennon
PE
,
Andersson
MB
,
Clerk
A
,
Lazou
A
,
Marshall
CJ
,
Parker
PJ
,
Sugden
PH
Endothelin-1 and fibroblast growth factors stimulate the mitogen-activated protein kinase cascade in cardiac myocytes
J Biol Chem
1993a
269
1110
1119
[PubMed]
Bogoyevitch
MA
,
Glennon
PE
,
Sugden
PH
Endothelin-1, phorbol esters and phenylephrine stimulate MAP kinase activities in ventricular cardiomyocytes
FEBS Lett
1993b
317
271
275
[PubMed]
Bogoyevitch
MA
,
Ketterman
AJ
,
Sugden
PH
Cellular stresses differentially activate c-Jun N-terminal protein kinases and extracellular signal-regulated protein kinases in cultured ventricular myocytes
J Biol Chem
1995a
270
29710
29717
[PubMed]
Bogoyevitch
MA
,
Marshall
CJ
,
Sugden
PH
Hypertrophic agonists stimulate the activities of the protein kinases c-Raf and A-Raf in cultured ventricular myocytes
J Biol Chem
1995b
270
26303
26310
[PubMed]
Bogoyevitch
MA
,
Gillespie-Brown
J
,
Ketterman
AJ
,
Fuller
SJ
,
Ben-Levy
R
,
Ashworth
A
,
Marshall
CJ
,
Sugden
PH
Stimulation of stress-activated mitogen-activated protein kinase subfamilies in perfused heart
Circ Res
1996
79
162
173
[PubMed]
Bruder
JT
,
Heidecker
G
,
Rapp
UR
Serum-, TPA-, and Ras- induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase
Genes Dev
1992
6
5454
5456
Cano
E
,
Mahadevan
LC
Parallel signal processing among mammalian MAPKs
Trends Biochem Sci
1995
20
117
122
[PubMed]
Chin
JH
,
Okazaki
M
,
Hu
ZW
,
Miller
JW
,
Hoffman
BB
Activation of heat shock protein (hsp) 70 and proto-oncogene expression by α1 adrenergic agonist in rat aorta with age
J Clin Invest
1996
97
2316
2323
[PubMed]
Clerk
A
,
Bogoyevitch
MA
,
Andersson
MB
,
Sugden
PH
Differential activation of PKC isoforms by endothelin-1 and phenylephrine and subsequent stimulation of p42 and p44 mitogen-activated protein kinases in ventricular myocytes cultured from neonatal rat hearts
J Biol Chem
1994
269
32848
32857
[PubMed]
Cobb
MH
,
Boulton
TG
,
Robbins
DJ
Extracellular signal-regulated kinases: ERKs in progress
Cell Regul
1991
2
965
978
[PubMed]
Cuenda
A
,
Rouse
J
,
Doza
YN
,
Meier
R
,
Cohen
P
,
Gallagher
TF
,
Young
PR
,
Lee
JC
SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1
FEBS Lett
1995
364
229
233
[PubMed]
Cuenda
A
,
Alonso
G
,
Morrice
N
,
Jones
M
,
Meier
R
,
Cohen
P
,
Nebreda
AR
Purification and cDNA cloning of SAPKK3, the major activator of RK/p38 in stress- and cytokine-stimulated monocytes and epithelial cells
EMBO (Eur Mol Biol Organ) J
1996
15
4293
4301
Cumming
DV
,
Heads
RJ
,
Brand
NJ
,
Yellon
DM
,
Latchman
DS
The ability of heat stress and metabolic preconditioning to protect primary rat cardiac myocytes
Basic Res Cardiol
1996
91
79
85
[PubMed]
Davis
R
The mitogen-activated protein kinase signal transduction pathway
J Biol Chem
1993
268
14553
14556
[PubMed]
Davis
R
MAPKs: new JNK expands the group
Trends Biochem Sci
1994
19
470
473
[PubMed]
Derijard
B
,
Hibi
M
,
Wu
I
,
Barrett
T
,
Su
B
,
Deng
T
,
Karin
M
,
Davis
RJ
JNK1: A protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain
Cell
1994
76
1025
1037
[PubMed]
Derijard
BJ
,
Raingeaud
J
,
Barrett
T
,
Wu
I-H
,
Han
J
,
Ulevitch
RJ
,
Davis
RJ
Independent human MAP kinase signal transduction pathways defined by MEK and MKK isoforms
Science (Wash DC)
1995
267
682
685
[PubMed]
Edmondson
DG
,
Lyons
GE
,
Martin
JF
,
Olson
EN
Mef2 gene expression marks the cardiac and skeletal muscle lineages during mouse embryogenesis
Development (Camb)
1994
120
1251
1263
[PubMed]
English
JM
,
Vanderbilt
CA
,
Xu
S
,
Marcus
S
,
Cobb
MH
Isolation of MEK 5 and differential expression of alternatively spliced forms
J Biol Chem
1995
270
28897
28902
[PubMed]
Gillespie-Brown
J
,
Fuller
SJ
,
Bogoyevitch
MA
,
Cowley
S
,
Sugden
PH
The mitogen-activated protein kinase kinase MEK1 stimulates a pattern of gene expression typical of hypertrophic phenotype in rat ventricular cardiomyocytes
J Biol Chem
1995
270
28092
28096
[PubMed]
Glembotski
CC
,
Oronzi
ME
,
Li
X
,
Shields
PP
,
Johnston
JF
,
Kallen
RG
,
Gibson
TR
The characterization of atrial natriuretic peptide (ANP) expression by primary cultures of atrial myocytes using an ANP-specific monoclonal antibody and an ANP messenger ribonucleic acid probe
Endocrinology
1987
121
843
852
[PubMed]
Gottlieb
RA
,
Gruol
DL
,
Zhu
JY
,
Engler
RL
Preconditioning rabbit cardiomyocytes: role of pH, vacuolar proton ATPase, and apoptosis
J Clin Invest
1996
97
2391
2398
[PubMed]
Gupta
S
,
Campbell
D
,
Derijard
B
,
Davis
RJ
Transcription factor ATF2 regulation by the JNK signal transduction pathway
Science (Wash DC)
1995
267
389
393
[PubMed]
Han
J
,
Lee
JD
,
Jiang
Y
,
Li
Z
,
Feng
L
,
Ulevitch
RJ
Characterization of the structure and function of a novel MAP kinase
J Biol Chem
1996
271
2886
2891
[PubMed]
Han
J
,
Jian
Y
,
Li
Z
,
Kravchenko
VV
,
Ulevitch
RJ
Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation
Nature (Lond)
1997
386
296
299
[PubMed]
Huot
J
,
Lambert
H
,
Lavoie
JN
,
Guimond
A
,
Houle
F
,
Landry
J
Characterization of 45-kDa/54k-Da HSP27 kinase, a stress-sensitive kinase which may activate phosphorylation-dependent protective function of mammalian 27-kDa heat-shock protein HSP27
Eur J Biochem
1995
227
416
427
[PubMed]
Juo
P
,
Kuo
CJ
,
Reynolds
SE
,
Konz
RF
,
Raingeaud
J
,
Davis
RJ
,
Biemann
H-P
,
Blenis
J
Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases
Mol Cell Biol
1997
17
24
35
[PubMed]
Karin
J
The regulation of AP-1 activity by MAP kinases
J Biol Chem
1995
270
16483
16486
[PubMed]
Karns
LR
,
Kariya
K
,
Simpson
PC
M-CAT, CArG, and Sp1 elements are required for α1-adrenergic induction of the skeletal α-actin promoter during cardiac myocyte hypertrophy. Transcriptional enhancer factor-1 and protein kinase C as conserved transducers of the fetal program in cardiac growth
J Biol Chem
1995
270
410
417
[PubMed]
Kolch
W
,
Heidecker
G
,
Kochs
G
,
Hummel
R
,
Vahidl
H
,
Mischak
H
,
Finkenzeller
G
,
Marme
D
,
Rapp
UR
Protein kinase C alpha activates RAF-1 by direct phosphorylation
Nature (Lond)
1993
364
249
252
[PubMed]
Komuro
I
,
Katoh
Y
,
Kaida
T
,
Shibazaki
Y
,
Kurabayashi
M
,
Takaku
F
,
Yazaki
Y
Mechanical loading stimulates cell hypertrophy and specific gene expression in cultured rat cardiac myocytes
J Biol Chem
1990
266
1265
1268
[PubMed]
Kyriakis
JM
,
App
H
,
Zhang
X
,
Banerjee
P
,
Brautigan
DL
,
Rapp
UR
,
Avruch
J
Raf-1 activates MAP kinase-kinase
Nature (Lond)
1992
358
417
420
[PubMed]
LaMorte
VJ
,
Thorburn
J
,
Absher
D
,
Spiegel
AM
,
Brown
JH
,
Chien
KR
,
Feramisco
JR
,
Knowlton
KU
Gq- and ras-dependent pathways mediate hypertrophy of neonatal rat ventricular myocytes following α1-adrenergic stimulation
J Biol Chem
1994
269
13490
13496
[PubMed]
Lange-Carter
CA
,
Pleiman
CM
,
Gardner
AM
,
Blumer
KJ
,
Johnson
GL
A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf
Science (Wash DC)
1993
260
315
319
[PubMed]
LaPointe
MC
,
Wu
G
,
Garami
M
,
Yang
X-P
,
Gardner
DG
Tissue-specific expression of the human brain natriuretic peptide gene in cardiac myocytes
Hypertension
1996
27
715
722
[PubMed]
Lavoie
JN
,
Gingras-Breton
G
,
Tanguay
RM
,
Landry
J
Induction of Chinese Hamster HSP27 gene expression in mouse cells confers resistance to heat shock: HSP27 stabilization of the microfilament organization
J Biol Chem
1993
268
3420
3429
[PubMed]
Lembo
G
,
Hunter
JJ
,
Chien
KR
Signaling pathways for cardiac growth and hypertrophy. Recent advances and prospects for growth factor therapy
Ann NY Acad Sci
1995
752
115
127
[PubMed]
Lin
A
,
Minden
A
,
Martinetto
H
,
Claret
F-X
,
Lange-Carter
C
,
Mercurio
F
,
Johnson
GL
,
Karin
M
Identification of a dual specificity kinase that activates Jun kinases and p38-Mpk2
Science (Wash DC)
1995
268
286
290
[PubMed]
Livingstone
C
,
Patel
G
,
Jones
N
ATF-2 contains a phosphorylation-dependent transcriptional activation domain
EMBO (Eur Mol Biol Organ) J
1995
14
1785
1797
[PubMed]
Ludwig
S
,
Engel
K
,
Hoffmeyer
A
,
Sithanandam
G
,
Neufeld
B
,
Palm
D
,
Gaestel
M
,
Rapp
U
3pK, a novel mitogen-activated protein (MAP) kinase-activated protein kinase, is targeted by three MAP kinase pathways
Mol Cell Biol
1996
16
6687
6697
[PubMed]
MacLellan
RW
,
Lee
TC
,
Schwartz
RJ
,
Schneider
MD
Transforming growth factor-β response elements of the skeletal α-actin gene
J Biol Chem
1994
269
16754
16760
[PubMed]
Marber
MS
,
Latchman
DS
,
Walker
JM
,
Yellon
DM
Cardiac stress protein elevation 24 hours after brief ischemia or heat stress is associated with resistance to myocardial infarction
Circulation
1993
88
1264
1272
[PubMed]
Maulik
N
,
Watanabe
M
,
Zu
YL
,
Huang
CK
,
Cordis
GA
,
Schley
JA
,
Das
DK
Ischemic preconditioning triggers the activation of MAP kinases and MAPKAP kinase-2 in rat hearts
FEBS Lett
1996
396
233
237
[PubMed]
McDonough
PM
,
Glembotski
CC
Induction of ANF and MLC-2 gene expression in cultured ventricular myocytes by electrical stimulation of contraction
J Biol Chem
1992
267
11665
11668
[PubMed]
McLaughlin
MM
,
Kumar
S
,
McDonnell
PC
,
Van Horn
S
,
Lee
JC
,
Livi
GP
,
Young
PR
Identification of mitogen-activated protein (MAP) kinase-activated protein kinase-3, a novel substrate of CSBP p38 MAP kinase
J Biol Chem
1996
271
8488
8492
[PubMed]
Mehlen
P
,
Schulze-Osthoff
K
,
Arrigo
AP
Small stress proteins as novel regulators of apoptosis. Heat shock protein 27 blocks Fas/APO-1- and staurosporine-induced cell death
J Biol Chem
1996
271
16510
16514
[PubMed]
Meng
X
,
Brown
JM
,
Ao
L
,
Banerjee
A
,
Harken
AH
Norepinephrine induces cardiac heat shock protein 70 and delayed cardioprotection in the rat through alpha 1 adrenoreceptors
Cardiovasc Res
1996
32
374
383
[PubMed]
Mestril
R
,
Chi
SH
,
Sayen
MR
,
O'Reilly
K
,
Dillman
WH
Expression of inducible stress protein 70 in rat heart myogenic cells confers protection against simulated ischemia-induced injury
J Clin Invest
1994
93
759
767
[PubMed]
Minden
A
,
Lin
A
,
McMahon
M
,
Lange-Carter
C
,
Derijard
B
,
Davis
RJ
,
Johnson
GL
,
Karin
M
Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK
Science (Wash DC)
1994
266
1719
1723
[PubMed]
Murry
CE
,
Jennings
RB
,
Reimer
KA
Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium
Circulation
1986
74
1124
1136
[PubMed]
Olson
EN
,
Srivastava
D
Molecular pathways controlling heart development
Science (Wash DC)
1996
272
671
676
[PubMed]
Paradis
P
,
MacLellan
WR
,
Belaguli
NS
,
Schwartz
RJ
,
Schneider
MJ
Serum response factor mediates AP-1-dependent induction of the skeletal α-actin promoter in ventricular myocytes
J Biol Chem
1996
271
10827
10833
[PubMed]
Parratt
JR
Proction of the heart by ischaemic preconditioning: mechanisms and possibilities for pharmacological exploitation
Trends Pharmacol Sci
1994
15
19
25
[PubMed]
Post
GR
,
Goldstein
D
,
Thuerauf
DJ
,
Glembotski
CC
,
Brown
JH
Dissociation of p44 and p42 mitogen-activated protein kinase activation from receptor-induced hypertrophy in neonatal rat ventricular myocytes
J Biol Chem
1996
271
8452
8457
[PubMed]
Raingeaud
J
,
Gupta
S
,
Rogers
J
,
Dickens
M
,
Han
J
,
Ulevitch
RJ
,
Davis
RJ
Pro-inflammatory cytokines and environmental stress cause p38 MAP kinase activation by dual phosphorylation on tyrosine and threonine
J Biol Chem
1995
270
7420
7426
[PubMed]
Raingeaud
J
,
Whitmarsh
AJ
,
Barrett
T
,
Derijard
B
,
Davis
RJ
MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway
Mol Cell Biol
1996
16
1247
1255
[PubMed]
Rouse
J
,
Cohen
P
,
Trigon
S
,
Morange
M
,
Alonso-Llamazares
A
,
Zamanillo
D
,
Hunt
T
,
Nebreda
AR
A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins
Cell
1994
78
1027
1037
[PubMed]
Sadoshima
J
,
Qiu
Z
,
Morgan
JP
,
Izumo
S
Angiotensin II and other hypertrophic stimuli mediated by G protein-coupled receptors activate tyrosine kinase, mitogen-activated protein kinase, and 90-kD S6 kinase in cardiac myocytes. The critical role of Ca2+-dependent signaling
Circ Res
1995
76
1
15
[PubMed]
Samali
A
,
Cotter
TG
Heat shock proteins increase resistance to apoptosis
Exp Cell Res
1996
223
163
170
[PubMed]
Schneider
MD
,
McLellan
WR
,
Black
FM
,
Parker
TG
Growth factors, growth factor response elements, and the cardiac phenotype
Basic Res Cardiol
1992
87
33
48
[PubMed]
Sharma, H.S., J. Stahl, D. Weisensee, and I. Low-Friedrich. 1996. Cytoprotective mechanisms of cultured cardiomyocytes. Mol. Cell. Biochem. 160–161: 217–224.
Simpson
PC
Norepinephrine-stimulated hypertrophy of cultured rat myocardial cells is an α1-adrenergic response
J Clin Invest
1983
72
732
738
[PubMed]
Sithanandam
G
,
Latif
F
,
Smola
U
,
Bernal
RA
,
Duh
F-M
,
Li
H
,
Kuzmin
I
,
Wixler
V
,
Geil
L
,
Shresta
S
et al
3pK, a new mitogen-activated protein kinase, located in the small cell lung cancer tumor suppressor gene region
Mol Cell Biol
1996
16
868
876
[PubMed]
Sprenkle
AB
,
Murray
SF
,
Glembotski
CC
Involvement of multiple cis elements in basal- and α-adrenergic agonist-inducible atrial natriuretic factor transcription. Roles for serum response elements and an SP-1-like element
Circ Res
1995
77
1060
1069
[PubMed]
Stokoe
D
,
Campbell
DG
,
Nakielny
S
,
Hidaka
H
,
Leevers
SJ
,
Marshall
C
,
Cohen
P
MAPKAP kinase-2: a novel protein kinase activated by mitogen-activated protein kinase
EMBO (Eur Mol Biol Organ) J
1992a
11
3985
3994
[PubMed]
Stokoe
D
,
Engel
K
,
Campbell
DG
,
Cohen
P
,
Gaestel
M
Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins
FEBS Lett
1992b
13
307
313
[PubMed]
Tan
Y
,
Rouse
J
,
Zhang
A
,
Cariati
S
,
Cohen
P
,
Comb
MJ
FGF and stress regulate CREB and ATF-1 via a pathway involving p38 MAP kinase and MAPKAP kinase-2
EMBO (Eur Mol Biol Organ) J
1996
15
4629
4642
[PubMed]
Thorburn
A
Ras activity is required for phenylephrine-induced activation of mitogen-activated protein kinase in cardiac muscle cells
Biochem Biophys Res Commun
1994
205
1417
1422
[PubMed]
Thorburn
J
,
Thorburn
A
The tyrosine kinase inhibitor, genistein, prevents α-adrenergic-induced cardiac muscle cell hypertrophy by inhibiting the activation of the Ras-MAP kinase signaling pathway
Biochem Biophys Res Commun
1994
202
1586
1591
[PubMed]
Thorburn
A
,
Thorburn
J
,
Chen
S-Y
,
Powers
S
,
Shubeita
HE
,
Feramisco
JR
,
Chien
KR
HRas dependent pathways can activate morphological and genetic markers of cardiac hypertrophy
J Biol Chem
1993
268
2244
2249
[PubMed]
Thorburn
J
,
Frost
JA
,
Thorburn
A
Mitogen-activated protein kinases mediate changes in gene expression, but not cytoskeletal organization associated with cardiac muscle hypertrophy
J Cell Biol
1994a
126
1565
1572
[PubMed]
Thorburn
J
,
McMahon
M
,
Thorburn
A
Raf-1 kinase activity is necessary and sufficient for gene expression changes but not sufficient for cellular morphology changes associated with cardiac myocyte hypertrophy
J Biol Chem
1994b
269
30580
30586
[PubMed]
Thuerauf
DJ
,
Glembotski
CC
Differential effects of protein kinase C, Ras and Raf-1 kinase on the induction of the cardiac B-type natriuretic peptide gene through a critical promoter-proximal M-CAT element
J Biol Chem
1997
272
7464
7472
[PubMed]
Thuerauf
DJ
,
Hanford
DS
,
Glembotski
CC
Regulation of rat BNP transcription: a potential role for the transcription factor GATA-4 in cardiac myocyte gene expression
J Biol Chem
1994
269
17772
17775
[PubMed]
Van Bilsen
M
,
Chien
KR
Growth and hypertrophy of the heart: towards an understanding of cardiac specific and inducible gene expression
Cardiovasc Res
1993
27
1140
1149
[PubMed]
Van Heugten
HAA
,
De Jonge
HW
,
Bezstarosti
K
,
Sharma
HS
,
Verdouw
PD
,
Lamers
MJ
Intracellular signaling and genetic reprogramming during agonist-induced hypertrophy of cardiac myocytes
Ann NY Acad Sci
1995
752
343
353
[PubMed]
Xia
Z
,
Dickens
M
,
Raingequd
J
,
Davis
RJ
,
Greenberg
ME
Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis
Science (Wash DC)
1995
270
1326
1331
[PubMed]
Yamazaki
T
,
Komuro
I
,
Yazaki
Y
Molecular mechanism of cardiac cellular hypertrophy by mechanical stress
J Mol Cell Cardiol
1995
27
133
140
[PubMed]
Young
P
,
McDonnel
P
,
Dunnington
D
,
Hand
A
,
Laydon
J
,
Lee
J
Pyridinyl imidazoles inhibit IL-1 and TNF production at the protein level
Agents Actions
1993
39
C67
69
[PubMed]

Address all correspondence to Christopher C. Glembotski, Department of Biology, San Diego State University, San Diego, CA 92182. Tel.: (619) 594-2959. Fax: (619) 594-6200. e-mail: cglembotski@sunstroke.sdsu.edu

SB 203580 was a generous gift from J. Lee (SKB Pharmaceuticals, King of Prussia, PA. α-SkA–394GL was a generous gift from M.D. Schneider (Baylor College of Medicine, Houston, TX).