In vitro cardiac myocyte hypertrophy is characterized by increased cell size, sarcomere organization, and induction of several genes including atrial natriuretic factor (ANF). The hypertrophic growth program has been associated with activation of various mitogen-activated protein kinase (MAP) kinase family members, one of which is a stress kinase, p38. In this study, we found that the p38-specific inhibitor SB-203580 failed to inhibit phenylephrine-induced ANF-driven gene expression in low-density myocyte cultures but did inhibit gene expression in higher density cultures. Dense myocyte cultures also had a higher metabolic activity and contraction rate than cells plated at low density. We found that mimicking this effect by rapid electrical pacing activated ANF-driven gene expression and that this expression was inhibited by inactivation of p38. However, addition of SB-203580 at time points ranging between 1 and 72 h suggests that the effect of p38 on the ANF promoter may be both direct and indirect. Electrical pacing induced a small, but consistent, increase in p38 phosphorylation (phospho-p38) at time points ranging from 30 min to 4 h, but at later times phospho-p38 levels were reduced. When myocytes were treated with phenylephrine or electrically paced in the presence of the p38 inhibitor, there was little discernible change in morphology or rates of protein synthesis from DMSO-treated cells at 48 or 72 h. These data indicate that cell density and myocyte contraction may modulate p38-dependent pathways for ANF gene expression, but these pathways may not be direct and have limited effects on hypertrophic morphology.
- atrial natriuretic factor
in response to physiological and pathological stimuli, the mammalian heart undergoes hypertrophic growth. This hypertrophy is associated with a number of phenotypic changes including increased expression of cardiac-specific genes such as atrial natriuretic factor (ANF). In vitro, induction of the hypertrophic program and activation of the ANF promoter in neonatal ventricular myocytes can be induced by agonists such as phenylephrine or angiotensin II, which activate G protein-coupled receptors (15, 24, 27, 30) and increase muscle cell contraction rate (18). These stimuli also enhance the organization of contractile proteins into sarcomeres and significantly increase the size of cardiac myocytes in culture.
A number of studies have indicated that the intracellular signaling mechanisms that mediate cardiac hypertrophy require one or more members of the mitogen-activated protein kinase (MAPK) cascades (2-5, 7,22, 23, 25, 28, 34, 38). The most recently described member of the MAPK family is the stress-activated protein kinase p38. Activation of p38 normally occurs as a result of cellular stresses such as ultraviolet irradiation and osmotic shock. Oxidative stress and ischemia-reperfusion can also activate p38 in perfused rat hearts (6).
Activation of the p38 MAPK pathway is sufficient to stimulate the ANF promoter in cardiac myocytes (21, 35, 36, 38). Overexpression of MEK6 (the direct upstream activator of p38) has been shown to protect myocytes from apoptosis induced by anisomycin or MEKK-1 (37) and to activate ATF6, which may cooperate with serum response factor to induce expression from the ANF promoter (35). p38 can also phosphorylate and activate the transcription factor MEF2C (11), which has been shown to be important in cardiovascular development and a likely candidate for involvement in cardiac hypertrophy. Whereas there has been some agreement that p38 can be involved in gene expression changes associated with hypertrophy, there are conflicting reports for the role of p38 in mediating the morphological changes of hypertrophy. Infection of cardiomyocytes with adenoviral vectors expressing activated mutants of MEK6 (MKK6DD) or transient transfection of expression constructs for MEK6 caused increased cell size and sarcomere organization (36, 38). However, Clerk et al. (7) showed that inhibition of p38 failed to prevent the hypertrophic morphology induced by phenylephrine and endothelin-1 between 4 and 24 h, although the inhibitor did decrease organization and cell profile at 48 h. Thus it is unclear whether p38 plays a central or minor role in the regulation of morphological and biochemical markers of myocardial hypertrophy.
We examined the role of p38 during induction of the ANF promoter and morphological hypertrophy in response to phenylephrine. Surprisingly, phenylephrine-induced ANF-driven gene expression did not require p38 in low-density myocyte cultures. However, high-density cultures required p38 activity for gene expression. High-density cultures spontaneously contracted at a significantly higher rate than isolated myocytes. Mimicking this effect by rapid electrical pacing of the myocytes induced ANF-driven reporter plasmids, and this induction was sensitive to the p38 inhibitor. Rapid pacing of cultures resulted in small increases in phosphorylated p38 (phospho-p38) levels, and at later time points the kinase was decreased in paced samples. Although p38 appeared to play a role in ANF-driven gene expression in certain conditions, we failed to find that p38 activity was necessary for the morphological features of hypertrophy. These data indicate that p38 may regulate cardiac hypertrophic morphology and gene expression only in certain cell culture conditions.
Cell culture and transfections.
Cardiac muscle cells were cultured and transfected as previously described (12, 32, 33). For low-density myocyte cultures, cells were seeded at a density of 260 cells/mm2, and for high-density cultures, cells were seeded at a density of 1,300 cells/mm2. For electrical pacing experiments, the cells were plated at 1,000–1,700 cells/mm2 in 24-well culture plates, which is required for efficient pacing in vitro (unpublished observations). Myocytes were transiently transfected in duplicate or triplicate 24 h after the cell isolation procedure for all gene expression assays. Approximately 15 h after the calcium phosphate transfection procedure was commenced, cells were washed with serum-free medium and treated with appropriate agonists and inhibitors. For pacing experiments, myocytes were subjected to electrical pacing at this time. Other transfections were conducted in duplicate or triplicate in 3.5-cm tissue culture dishes using the calcium phosphate precipitation method. Before transfection, all plasmids were purified by alkaline lysis followed by polyethylene glycol precipitation. Luciferase and β-galactosidase activities were assayed on a Dynex MLX luminometer 48 h after transfection by harvesting cells in reporter lysis buffer (Promega E397a, Madison, WI) and incubating with the appropriate light-emission accelerator [Promega E148a or Tropix (Bedford, MA) reaction buffer, respectively].
Cells were treated as required with phenylephrine (50 μM) (Sigma, St. Louis, MO) and the p38 inhibitor SB-203580 (20 μM; Calbiochem, La Jolla, CA) and kept from direct light to prevent degradation. Propranolol (1 μM) was included in culture medium used in these experiments to block β-receptors. For electrical stimulation to induce muscle cell contraction, the cells were plated in 24-well plates that were electrically connected via an agarose salt bridge and stimulated with a custom-built electric stimulator. In this case, controls were from the same plate of cells but from wells that were not electrically connected. Media were changed every 24 h for all experiments. Electrical pacing experiments were conducted in a manner similar to those described by McDonough et al. (18). Briefly, either paced or unpaced cardiac cells were kept in serum-free medium while electric pulses were delivered at a frequency of 2–3 Hz for 24–72 h. The polarity of electrodes was reversed every other pulse to prevent the accumulation of net electric charge and pH drift of medium.
p38 Western blots.
Assays for p38 activity were performed using the PhosphoPlus p38 MAP Kinase Antibody Kit (New England BioLabs, Beverly, MA). Cardiac cells were kept in serum-free medium for 24 h before agonist addition or pacing. The medium was first changed again to serum-free medium (±inhibitors), and cells were then allowed to equilibrate undisturbed for 6 h. At that time, either phenylephrine (50 μM) was added or electrical pacing at 2–3 Hz was begun. Cells were scraped in ice-cold phosphate-buffered saline (PBS) and resuspended in radioimmunoprotective assay buffer containing protease inhibitors (10 mM Tris, pH 7.4, 0.15 M NaCl, 1% Nonidet P-40, 1% SDS, and 10 μg/ml each of aprotinin, leupeptin, and pepstatin with 1 mM Na-vanadate). Protein content from cell extracts was quantified using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Sample aliquots (25–40 μg cellular protein) were subjected to SDS-PAGE and Western blotting. For pacing experiments, this represented protein from ∼4–16 wells from 24-well culture dishes. Phospho-p38 and total p38 were detected by chemiluminescence as described by the manufacturer. Real-time quantitation of band intensity was analyzed on a Lumi-Imager (Boehringer Mannheim, Indianapolis, IN), and the images were quantitated using the Lumi-Analyst program. Quantitative densitometry was conducted on a ScanMaker IISP and analyzed using Kodak Digital Science Image Analysis software.
p38 kinase assays.
Cardiac cells were first kept in serum-free medium as described inp38 Western blots. Approximately 1.6 × 106cardiac cells were either unpaced or paced at 2 Hz for 5 min to 4 h in 24-well plates. Total p38 was immunoprecipitated from whole cell extracts by first washing paced or unpaced cells with ice-cold PBS. Protein was harvested for immunoprecipitation kinase assays and incubated with 15 μl of anti-p38 antibody in kinase buffer in a protocol similar to that described previously (31, 32). Immunoprecipitates were finally used in kinase assays with myelin basic protein (MBP; Sigma). Reactions were stopped by addition of 2× Laemmli buffer, and phosphorylated products were subjected to SDS-PAGE and autoradiography.
The MEK6 expression plasmid (MKK6) was provided by Bernd Stein (Signal Pharmaceuticals, La Jolla, CA). Luciferase reporter plasmids were normalized to a cotransfected RSV-LacZ plasmid provided by Michael Kapiloff (Oregon Health Services University, Portland, OR). CL100 was provided by Steve Keyse (Imperial Cancer Research Fund, University of Dundee, Nethergate, Dundee, Scotland). ANF-luciferase is a 638-bp fragment of the rat ANF promoter cloned into pGL3 basic. pFA-CHOP and pFR-luciferase were obtained from Stratagene (La Jolla, CA).
Measurement of contraction rate.
Low- or high-density myocyte cultures were treated with 50 μM phenylephrine and 1 μM propranolol for 24 h in serum-free medium. Contraction was measured visually on a Zeiss Axiovert microscope in a preequilibrated 37°C, 10% CO2stage. Myocyte cultures were at equilibrium conditions during these measurements and were viewed under low-intensity light to prevent heating. Thirty measurements at 1 min each were made for isolated cells, and ten independent measurements were made for five cell clusters and dense cultures.
[3H]phenylalanine protein measurements.
Rates of protein synthesis were conducted by a method similar to that of Abdellatif et al. (1). Briefly, cardiac myocytes were serum starved for 24 h after isolation and then exposed to 0.4 mM unlabeled phenylalanine for 30 min to expand intracellular pools of the amino acid. The medium was then removed and replaced with medium containing 2 μCi/mll-[ring-2,3,4,5,6-3H]phenylalanine and appropriate agonists (pacing 2 Hz or 50 μM phenylephrine) or inhibitors (20 μM SB-203580) along with 1 μM propranolol. The medium was changed every 24 h. Total protein/RNA and DNA were simultaneously harvested using the RNA/DNA extraction kit (Qiagen, Santa Clarita, CA). Protein pellets were washed twice in ice-cold trichloroacetic acid (10%) and resuspended in 0.3 N NaOH, and radioactivity was quantitated by liquid scintillation counting in 5.0 ml of Opti-Fluor reagent (Packard, Meriden, CT). DNA pellets were resuspended in distilled H20 and quantitated by spectrophotometry with absorbance at 260 nm. Newly synthesized protein (in counts/min) was normalized to DNA yields. For pacing experiments, the 3H-labeled protein levels were also normalized to cell number by direct counting of the number of living cells per high-power field. Cells were counted from three random fields for each sample. Each data point represents two samples, thus resulting in six independent measurements of cell number per data point. 3H-labeled protein was also harvested in TRIzol reagent (GIBCO, Grand Island, NY) with similar results. In this case, 300 μl EtOH was added to reduce quenching of the samples during scintillation counting.
Time-course p38-inhibition studies.
To inhibit p38 activity during various time points during cell culture, cardiac myocytes were transiently transfected as described in Cell culture and transfections and washed with serum-free medium. Cells were then treated with SB-203580 (20 μM) for the first 24 h of culture (hours 1–24) or the last 24 h of culture (hours 48–72), or the p38 inhibitor was kept in the medium chronically (hours 1–72). All media were changed every 24 h. ANF-luciferase gene expression assays were conducted at the 72-h time point after the end of treatment. Cells treated with p38 inhibitor for the first 24 h were washed thoroughly with serum-free medium before the addition of medium containing either DMSO or SB-203580.
The data presented for each experiment represent the mean ± SE for a single experiment. All transfection experiments presented were conducted in either duplicate or triplicate. These experiments were representative of other experiments conducted during the study. When appropriate, statistical methods were applied including ANOVA (single factor). Two sample t-tests for independent means, assuming equal variances, were also performed where required, indicating significance at P< 0.05. For all experimental data points, significance has been indicated either between unstimulated controls and stimulated values or between nonactivated and activated cultures within similar drug treatment categories or between drug groups. Bonferroni’s method was used for making multiple pairwise comparisons when the hypothesis of the equality of several means was rejected.
p38 activity is not required for ANF-driven gene expression stimulated by phenylephrine in low-density cultures but is required in dense cultures.
To determine whether the MAP kinase p38 was involved in induction of the ANF promoter in cardiac myocytes, we performed transient transfection assays in primary ventricular myocyte cultures using an ANF-luciferase reporter plasmid. Figure1 A shows that inhibition of p38 had no effect on phenylephrine-induced ANF-luciferase expression. These cells were plated at a density of 260 cells/mm2, which is that of our routine cultures. The inhibitor failed to prevent ANF-luciferase expression even at the limiting doses of phenylephrine treatment, which did not induce maximal gene expression. Figure1 B shows a similar experiment in which active MEK6, which induces p38 but not other MAP kinases, can induce ANF expression, confirming results from others (21, 36, 38), and the p38 inhibitor SB-203580 strongly inhibits this gene expression.
Previous studies have indicated that inhibition of p38 activity by SB-203580 attenuates phenylephrine-induced ANF gene expression and morphology (38). Because we were unable to reproduce these findings (Fig. 1 A) and because controls for inhibitor function were working as expected (Fig.1 B), we reasoned that differences in cell culture preparations might lead to different results with respect to p38 activity. One notable difference between culture conditions in different laboratories is myocyte cell density. Various laboratories have reported using cells densities of 260 cells/mm2 (33), 400 cells/mm2 (9), 1,040 cells/mm2 (35, 38), or 1,400 cells/mm2 (7). These different culture conditions lead to a variation in culture density of up to fivefold between laboratories. When we increased the culture density of our cells from 260 to 1,300 cells/mm2, we found that the p38 inhibitor inhibited phenylephrine-induced gene expression (Fig.1 C). These myocytes were still subconfluent, although significantly more dense than our routine cultures. In three of three experiments, densely cultured myocytes treated with phenylephrine and p38 inhibitor had lower ANF-luciferase expression than those without the inhibitor.
Increasing contraction rate by rapid electrical pacing induces ANF gene expression that requires p38 activity.
To determine whether cell density affected metabolic activity when cells were treated with phenylephrine, we measured the pH of the medium of low- and high-density cultures with or without phenylephrine treatment. We reasoned that the pH of the medium might differ with both cell density and phenylephrine treatment because of the liberation of lactic acid. Figure2 A shows that this is the case. When low-density cultures were treated with 50–100 μM phenylephrine, no discernible change from untreated controls in pH of the medium was measured. However, high-density cultures showed a marked acidification of the medium in cells treated with phenylephrine, presumably due to the increased contraction rate and metabolic activity of these cells. Interestingly, the acidification of the medium was most apparent in cells treated with 50 rather than 100 μM phenylephrine. Similar chronotropic effects of phenylephrine have been observed previously (9). When the contraction rate of phenylephrine-treated cells in low- and high-density cultures was measured directly, we found that high-density myocyte cultures contracted at a significantly higher rate than those at low density (Fig. 2 B). Isolated myocytes contracted at a mean of 5.7 beats/min (median 0, range 58, SD 15,n = 30), five myocytes grouped together contracted at a mean of 55.7 beats/min (median 58, range 79, SD 23.5, n = 10), and cells in a field of dense culture contracted at a mean of 115 beats/min (median 114, range 20, SD 5.9, n =10). Although occasional isolated myocytes had high contraction rates, the majority of cells displayed virtually no contractile activity, as evidenced by the value of the median (0) in these measurements.
To determine whether ANF gene expression induced by increased contraction rate requires p38 activity, we set up an apparatus to electrically stimulate the cardiac muscle cells to induce contraction at a defined rate. Figure 2 C shows that ANF-luciferase expression is increased in paced (2 Hz) compared with unpaced cells, confirming previous results from others (17, 18). Contraction-induced expression of ANF-luciferase was inhibited by the p38 inhibitor SB-203580.
Rapid electrical pacing induces phosphorylation and activation of p38.
Because pacing-induced ANF-luciferase expression was inhibited by SB-203580, we wanted to determine whether increasing the contraction rate was sufficient to activate p38. Figure3 A shows that this is the case. Cardiac myocytes driven to contract at 2 Hz showed increased phosphorylation of p38 after 30 min to 2 h by Western blot using a phospho-specific antibody. Three independent pacing experiments are shown. This activation was small compared with phenylephrine (Fig. 3 A) or anisomycin controls (data not shown). However, in six of seven experiments, the level of phospho-p38 was higher in paced samples from 30 min to 1 h and was generally increased approximately twofold when assayed by a chemiluminescence imager (data not shown) or densitometry [30-min activation was 2.3-fold (median 2.15, range 1.6–3.2, SD 0.61, SE 0.3, n = 5) and 1-h activation was 2.4-fold (median 1.46, range 1.37–5.8, SD 1.65, SE 0.6, n = 7)]. We also performed in vitro kinase assays using anti-p38 antibody to immunoprecipitate p38 from cell extracts. We then tested the ability of the immunoprecipitated kinase to phosphorylate the MBP substrate. As shown in Fig. 3 B, paced samples from 5 min to 4 h had only small increases in p38 activity compared with controls. This result confirmed the small but consistent increase in phospho-p38 detected in Western blots. We also tested the levels of phosphorylated and nonphosphorylated forms of p38 at later time points. Surprisingly, as shown in Fig. 3 C, in four of four experiments, both the phosphorylated and nonphosphorylated forms of p38 were reduced at all time points after 24 h. In most cases, there was a more significant decrease in phospho-p38 in paced cells, compared with total p38 (see, e.g., 72-h time point). The same blot was also probed for total actin, confirming approximately equal loadings of total protein and that paced samples containing hypertrophied myocytes showed the expected increases in actin levels.
Inhibition of p38 during pacing suggests both direct and indirect actions of p38 on ANF promoter.
The experiments shown in Fig. 3 show that myocyte contraction induces small increases in p38 phosphorylation at early time points and that the kinase is then downregulated starting after ∼24 h of pacing. We then tested whether inhibition of p38 during the first 24 h would affect the activity of the ANF promoter when assayed at 72 h of electrically driven contraction. Figure4 A shows a time-course activation of the ANF promoter by pacing. Activation of the promoter occurs after ∼24 h. Virtually no activation of the promoter was seen at the 6-h time point. Interestingly, inhibition of p38 with SB-203580 during hours 1–24 of contraction had no effect on ANF expression when assayed at 72 h (Fig.4 B). Likewise, inhibition of p38 during hours 48–72 of contraction also had no effect on ANF promoter activity. Only the continuous presence of SB-203580 during hours 1–72 inhibited ANF-luciferase induced by contraction. We then tested the ability of SB-203580 to inhibit ANF-luciferase activation by constitutively active MEK6 (MKK6DD). As shown in Fig. 4 C, MEK6 induction of the ANF promoter is inhibited by the p38 inhibitor when included in the medium at all time points. Inhibition of p38 chronically resulted in the most significant inhibition. These data suggest that the effect of p38 on ANF gene expression may be both direct and indirect.
Inhibition of p38 fails to prevent increases in protein synthesis and morphological features of hypertrophy induced by electrical pacing or phenylephrine.
To determine whether p38 activity was necessary for cardiac myocytes to increase protein synthesis rates induced by phenylephrine and pacing, we measured the change in [3H]phenylalanine incorporation in cells that were treated with phenylephrine or electrical pacing in the presence or absence of the p38 inhibitor. These experiments are shown in Figs. 5,A andB. It was determined that in both low-density (250 cells/mm2) and high-density (1,240 cells/mm2) cultures an increase in protein synthesis occurred in response to phenylephrine, even in the presence of the inhibitor SB-203580. In several experiments we noted that the p38 inhibitor decreased the protein content in unstimulated control samples. However, even in these cases, the change from basal protein incorporation rates to the increased hypertrophied state in response to phenylephrine still occurred. This is particularly apparent in Fig.5 B, where, although the p38 inhibitor decreased basal protein metabolism in the 72-h time point, these cells still markedly hypertrophied in response to electrically driven contraction. It was also noted that in sparse myocytes at the 24-h time point the p38 inhibitor seemed to reduce the basal and induced levels of protein synthesis. However, these myocytes still markedly hypertrophied at the 48-h time point.
To determine whether p38 activity is necessary for the morphological features of cardiac myocyte hypertrophy, we inhibited p38 activity with SB-203580 in cultures treated with phenylephrine or subjected to electrical pacing at 2 Hz. Figure 6,A–F, show low-power views of fields of myocytes treated with either DMSO as control (Figs. 6,A–C) or SB-203580 (Figs. 6,D–F). As shown in these photomicrographs, the majority of myocytes undergo significant hypertrophy in response to phenylephrine or electrical pacing, which is not affected by SB-203580.
To determine whether the organization of contractile proteins still occurred normally in these myocyte cultures, high-power views of the same cells were analyzed after they were stained with FITC-conjugated phalloidin (Fig. 7,A–F). Inhibition of p38 does not prevent the increased formation of apparently normal sarcomeres in cells treated with phenylephrine (Fig.7 E) or electrically paced (Fig.7 F).
As an additional method of inhibiting p38, the MAP kinase phosphatase CL100 was used. CL100 dephosphorylates the tyrosine/threonine residues on the MAP kinases ERK, JNK, and p38 (10). Expression plasmids encoding CL100 and green fluorescent protein (GFP) were cotransfected into cardiac myocytes, and the cells were treated with 50 μM phenylephrine. GFP (Fig.8 A) and phalloidin (Fig. 8 B) fluorescence intensities are shown for a typical transfected myocyte treated with phenylephrine. Myocytes containing GFP expression plasmids and CL100 appear organized and were indistinguishable from surrounding myocytes on the basis of phalloidin staining. Before fixation, the GFP-containing cells retained the ability to contract when observed microscopically, suggesting that cells in which p38 (and other MAP kinases) is inhibited possess functional muscle fibers. Thus we conclude that p38 activity is not necessary for cardiac myocytes to undergo morphological hypertrophy or show increased myofibrillar organization in response to phenylephrine or electrical pacing in these cell culture conditions.
To be sure that the CL100 construct was inhibiting p38-dependent events, we cotransfected cells with CL100 and the p38-responsive transcription factor GAL4-CHOP (which drives a luciferase reporter plasmid containing multiple GAL4 sites) along with constitutively active MEK6. As shown in Fig. 8 C, CL100 inhibited p38 activation of CHOP-driven transcription of the GAL4-luciferase reporter. Thus we conclude that inhibition of p38 does not prevent morphological hypertrophy or myofibrillar organization in response to phenylephrine or electrical pacing.
A number of diverse stimuli induce hypertrophy of neonatal rat ventricular cardiac myocytes in tissue culture. Some of these include mechanical stretch and angiotensin II (23, 26), α1-adrenergic agonists (16, 30), growth factors such as endothelin (14, 29), rapid electrical pacing (18, 19), and calcineurin-dependent pathways (20). Recent data from a number of groups have shown that p38 can stimulate the ANF promoter (21, 35, 36, 38). However, it is not clear whether all the above stimuli require p38 for morphological changes and induction of ANF gene expression. Despite the diversity of these stimuli, the end phenotype is very similar, i.e., increased myofibrillar organization and cell size along with induction of fetal cardiac genes such as ANF.
We investigated the role of p38 in induction of gene expression and morphology in response to two physiological stimuli, phenylephrine and rapid electrical pacing. Our results indicate that p38 is required for ANF-driven gene expression stimulated by phenylephrine only in higher density cultures. We speculated that one explanation for our observations is that cells in dense myocyte cultures contract more frequently than in low-density cultures. When we investigated cell cultures that were electrically paced to induce contraction, we found that contraction caused small increases in phosphorylation of p38 but that p38 activity was required for ANF-driven gene expression when assayed 72 h later. Whereas we have found that phenylephrine can activate p38, confirming the results from other investigators (7), our data suggest that chronotropic effects of the drug might also contribute to p38 activation. Sugden and colleagues (7) have found that p38 is induced in varying degrees by different hypertrophic agonists. For example, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate only slightly elevates phospho-p38 levels but strongly activates the ANF promoter and hypertrophic organization. Endothelin also activated p38 but did so less efficiently than phenylephrine. It is possible, therefore, that the different potencies of p38 activation by hypertrophic agonists dictate the precise molecular response of the cell. Nevertheless, it is difficult to reconcile how different degrees of p38 activation can lead to very similar phenotypes if the cellular response is simply a direct result of p38 activity.
Because induction of the ANF promoter by contraction was sensitive to the p38 inhibitor, p38 may be important for gene expression mediated by contraction. Under some conditions, i.e., relatively dense cells that increase their contraction rate in response to phenylephrine treatment, activation of ANF gene expression is highly dependent on the contractile activity, as noted recently by Samarel and his colleagues (8, 9) and as indicated by our unpublished data. Thus we believe that the discrepancy between our results (Fig. 1) and those of other investigators regarding the requirement for p38 activity for phenylephrine-induced ANF expression might be reconciled by concluding that some p38 sensitivity could be mediated through an increased contraction rate. This conclusion implies that, in sparse cultures, the signaling pathways that regulate phenylephrine-induced ANF gene expression might be distinct from those that regulate contraction-induced expression, at least in regard to their dependence on p38. Recent data from our laboratory supports this view, because we have found that some promoter elements that are required for p38-dependent ANF gene expression can be differentiated from those that are required for phenylephrine-induced expression (13).
It is shown in Fig. 4 that, although blocking p38 in contracting cells with the p38 inhibitor reduced ANF-driven gene expression, this inhibition was critically dependent on the time points when SB-203580 was included in the medium. A chronic presence of the inhibitor duringhours 1–72 was required to reduce pacing-induced ANF gene expression. Including the p38 inhibitor during times when p38 was increased in its activity (hours 1–24) failed to decrease ANF gene expression when assayed 48 h after the cessation of p38 inhibition. The half-lives of firefly-luciferase mRNA and protein are 6 and 3 h, respectively. If contraction activated a p38-dependent kinase cascade and activation of this cascade was required for gene expression 72 h later, we should find that inhibition in the first 24 h would be sufficient to block ANF expression. However, as shown in Fig. 4, this did not appear to be the case. Alternatively, the ANF promoter might be directly activated by contraction [e.g., by causing the phosphorylation of a transcription factor that is p38 dependent (35)]. If this were responsible for ANF expression, we would find that including the p38 inhibitor for the last 24 h of gene expression would decrease contraction-induced ANF-luciferase expression. However, this was also not the case. Interestingly, although ANF gene expression was unaffected by the p38 inhibitor at these early and late time points in paced cells, a different result was seen in MEK6-transfected cells. Cells transfected with constitutively active MEK6 and the ANF-luciferase construct showed inhibition of ANF-driven gene expression at all time points (early, late, and chronic). Together, these data suggest that p38 activity induced by MKK6 may have both direct and indirect effects on the ANF promoter. Thus, although the activity of p38 is required for gene expression by pacing, it might not be a direct requirement. This model is also supported by our observations that levels of both phospho-p38 and total p38 are decreased in paced samples from hours 24–72. It is difficult to imagine how p38 could be directly activating transcription factors on the ANF promoter when the kinase is present at a low level in the cells.
In this study we also showed that inhibition of p38 using the inhibitor SB-203580 did not significantly alter cell shape and morphological hypertrophy in response to phenylephrine or electrical pacing at any of the cell densities tested. Similar results were obtained when we transfected the potent MAP kinase phosphatase CL100 into the cells. Because two distinct methods of inhibiting p38 both failed to significantly inhibit the hypertrophic morphology of the cells, we conclude that p38 is either dispensable or at best a minor component of the regulatory pathway that controls this phenotype in both electrically paced and phenylephrine-treated cells.
We are grateful to various colleagues who provided plasmids used in this work and thank Christophe Montessuit for comments on the manuscript. We are also grateful to Matt Topham for assistance with data imaging and analysis.
Address for reprint requests and other correspondence: A. Thorburn, Huntsman Cancer Institute, Dept. of Oncological Sciences, 15 N 2030 E, Rm. 4160b, Univ. of Utah, Salt Lake City, UT 84112 (E-mail:).
This research was supported by National Heart, Lung, and Blood Institute Grant HL-52010, the Thomas D. Dee Fellowship in Human Genetics (to W. A. Hines), and funds from the Huntsman Cancer Institute and was completed in partial fulfillment of a Ph.D. in human genetics (W. A. Hines).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
- Copyright © 1999 the American Physiological Society