Endothelin-Dependent and -Independent Components of Strain-Activated Brain Natriuretic Peptide Gene Transcription Require Extracellular Signal Regulated Kinase and p38 Mitogen-Activated Protein Kinase
Abstract—The application of mechanical strain to cultured cardiac myocytes in vitro leads to activation of the brain natriuretic peptide (BNP) gene promoter, a marker of cardiac hypertrophy. We have previously shown that this activation results from both a direct mechanostimulatory event and an indirect autocrine/paracrine stimulation involving the sequential production of angiotensin II and endothelin (ET). In the present study, we examined the role of p38 mitogen-activated protein kinase (MAPK) and extracellular signal regulated kinase (ERK) in signaling the increase in promoter activity trafficking through each of these pathways. ET was shown to stimulate both p38 MAPK and ERK activity in these cultures and to activate human BNP (hBNP) promoter activity. Activation of the promoter was inhibited ≈45% by SB-203580, a p38 MAPK inhibitor, and ≈70% by PD98059, an inhibitor of the ERK-activating kinase MAPK kinase. The ET-independent (ie, direct) stimulation of the hBNP promoter by mechanical strain was inhibited ≈70% by SB-203580 and ≈60% by PD98059, implying that similar signaling circuitry is used, albeit to different degrees, by the direct and indirect pathways. The p38 MAPK component of both the ET-dependent and the ET-independent responses to strain appears to operate through a series of nuclear factor-κB binding, shear stress response element–like structures in the hBNP gene promoter. Collectively, these data suggest that activation of the BNP promoter by hypertrophic stimuli involves the participation of several independent signaling pathways. Such redundancy would help to guarantee generation of the full hypertrophic phenotype independently of the nature of the hypertrophic stimulus.
Terminally differentiated cardiac myocytes respond to hemodynamic overload with an increase in cell size (hypertrophy) rather than cell number. This is accompanied by structural alterations in the myocytes (eg, sarcomeric assembly) and changes in gene expression (eg, activation of the fetal gene program),1 which culminate in the generation of the full hypertrophic phenotype.
Activation of the fetal gene program is regarded as one of the earliest and most consistent markers of myocyte hypertrophy. Genes within this group (eg, atrial natriuretic peptide, brain natriuretic peptide, α-skeletal actin, and β-myosin heavy chain) are typically expressed in late embryonic and early neonatal life. Expression is quiescent in the adult unless the myocyte is subjected to a hypertrophic stimulus (eg, hemodynamic overload). Reactivation of the expression of these genes either in vivo or in vitro has come to represent a phenotypic marker for initiation of the hypertrophic process.
A number of signal transduction pathways have been invoked as playing an important role in the generation of myocyte hypertrophy. Among these, the mitogen-activated protein kinase (MAPK) family, which includes the extracellular signal regulated kinases (ERKs), the Jun amino terminal kinases (JNKs), and the p38 MAPKs, has received the most attention. In neonatal rat cardiac myocytes, all 3 members of this family have been shown to be activated by hypertrophic stimuli, and in each case, this activation has been linked to 1 or more downstream markers of hypertrophy.2 3 4 5 However, the precise role that each pathway plays in the initiation and maintenance of myocyte hypertrophy remains controversial.3 6
The application of mechanical strain (ie, stretch) to myocytes cultured on a distensible culture surface has been shown to trigger a number of signal transduction pathways.7 8 9 These, in turn, have been linked to downstream phenotypic changes associated with hypertrophy. We have recently shown that mechanical strain stimulates the activity of a transfected human brain natriuretic peptide (hBNP) gene promoter in neonatal ventricular myocytes.10 This stimulation appears to be derived in part from a direct effect on the cardiac myocyte and in part from an autocrine/paracrine pathway that involves the sequential generation of angiotensin II and endothelin (ET).11 From a mechanotransduction standpoint, strain has been shown to activate ERKs, JNKs, and p38 MAPKs in these myocyte cultures,7 8 9 10 with the latter (ie, p38 MAPK) accounting for ≈50% of the composite hBNP promoter response.12 This activity depends on interaction of the transcription factor nuclear factor (NF)-κB with 3 shear stress response element (SSRE)–like structures in the proximal hBNP promoter.
In the present study, we attempted to dissect the relative contributions of the different MAPK signaling pathways to the ET-dependent (autocrine/paracrine) and ET-independent responses to mechanical strain. Our findings suggest that p38 MAPK and ERK are required for both components of the response to strain, albeit to different degrees.
ET-1 and BQ-610 were purchased from Phoenix Pharmaceuticals, Inc. PD98059 was obtained from Research Biochemicals Inc. SB-203580 was a gift from J. Lee (SmithKline Beecham Pharmaceuticals, Inc, King of Prussia, PA). Bovine myelin basic protein was purchased from Upstate Biotechnology, Inc. Anti-p38 antibody and anti-ERK antibody were obtained from Santa Cruz Biotechnology, Inc. The luciferase assay system was purchased from Promega. [γ-32P]ATP was purchased from NEN Life Science Products. Other reagents were obtained through standard commercial suppliers.
Cell Culture and Application of Mechanical Strain
Ventricular myocytes were prepared from 1- to 2-day-old neonatal rat hearts through alternate cycles of 0.05% trypsin digestion and mechanical disruption as previously described.13 Cells (1×106) were cultured onto collagen-coated FLEX plates (Flexcell, Inc) in Dulbecco’s modified Eagle’s-H21 medium containing 10% bovine calf serum (Hyclone Laboratories, Inc), 2 mmol/L glutamine, 10 U/mL penicillin, and 100 mg/mL streptomycin. A glass cloning cylinder (1-cm diameter) was placed in the middle of each well to preclude cell attachment, thereby placing the majority of adherent cells on the outer 75% of the culture surface, where distention is maximal.14 The medium was changed 24 hours before initiation of the experiment. Cells were subjected to cyclical strain (60 cycles/min) on the Flexcell strain apparatus at a level of distention sufficient to promote ≈20% increment in surface area at the point of maximal distention on the culture surface.14
The construction of −904 and −1595 hBNP-luciferase have been described previously.15 2N, an IκBα mutant (IκB is an inhibitor of NF-κB),16 was provided by J. Hiscott. Site-directed mutagenesis of 3 shear stress response element–like sequences (SSREs) located between −160 and −155 (SSRE-1), −641 and −636 (SSRE-2), and −650 and −645 (SSRE-3) were introduced into −904 hBNP luciferase through use of the QuickChange kit (Stratagene). Details of the structures of these individual constructs have been described previously.12
Immunoprecipitation and Kinase Assay
Cells were harvested in 1 mL of lysis buffer (20 mmol/L Tris · HCl, pH 7.9, 137 mmol/L NaCl, 1% Triton X-100, 5 mmol/L EDTA, 1 mmol/L EGTA, 10% glycerol, 10 mmol/L NaF, 1 mmol/L β-glycerophosphate, 1 mmol/L PMSF, 1.5 μg/mL aprotinin, and 1 μg/mL pepstatin) and centrifuged at 12 000 cpm for 30 minutes; 200 μg supernatant protein was incubated with 1 μg anti-p38 antibody or anti-ERK antibody and 10 μL protein G-Sepharose for 2 hours at 4°C. The immunoprecipitates were recovered through centrifugation and washed twice with cell lysis buffer and once with a kinase reaction buffer (25 mmol/L HEPES, pH 7.4, 10 mmol/L MgCl2, 10 mmol/L MnCl2, 1 mmol/L sodium vanadate, and 1 mmol/L dithiothreitol) without ATP. Then, 20 μg myelin basic protein was added to the immunoprecipitates in 30 μL kinase reaction buffer containing 2 μCi [γ-32P]ATP. Reactions were incubated for 15 minutes at 30°C. Total reaction contents were electrophoresed on 15% SDS-polyacrylamide gels, which were then dried and subjected to autoradiography. Autoradiographic signals were quantified with the use of NIH Image.
Transfection and Luciferase Assay
Freshly prepared ventricular myocytes were transiently transfected with the indicated reporters and expression vectors through electroporation (Gene Pulser; Bio-Rad) at 280 mV and 250 μF. DNA content in individual cultures was normalized with pUC 18. After transfection, cells were plated and cultured as described earlier. Cells were harvested and lysed in 100 μL cell culture lysis reagent (Promega). Protein concentration of each cell extract was measured with the use of Coomassie protein reagent (Pierce Biochemicals). Cell lysates were processed (20 μg protein/sample) and assayed for luciferase activity. To ensure reproducibility, experiments were repeated 3 to 5 times.
Data were analyzed with ANOVA with the Newman-Keuls test to assess statistical significance.
Our earlier studies showed that ≈50% of the strain-dependent increment in hBNP promoter activity was dependent on the sequential stimulation of angiotensin II and ET production in the cardiac myocyte.11 In the present study, we used exogenous ET as a surrogate of the autocrine/paracrine system to probe the signaling mechanism or mechanisms responsible for this component of the strain response. As shown in Figure 1A⇓, ET effected a time-dependent increment in p38 MAPK activity that peaked at 15 minutes. This activation was inhibited by the ETA receptor antagonist BQ-610 and by the p38 MAPK inhibitor SB-203580, whereas the ERK inhibitor PD98059 had no effect (Figure 1B⇓). Next, we determined the contribution of ET to the strain-dependent activation of p38 MAPK. As shown in Figure 1C⇓, BQ-610 reduced strain-dependent activation of p38 MAPK activity by ≈40% to 45%, implying that the local generation of ET contributes a significant fraction of the total p38 MAPK induction. Predictably, SB-203580 completely blocked the strain-dependent activation of p38 MAPK, whereas PD98059 had no effect.
ET also stimulated ERK activity in these cultured myocytes. As shown in Figure 2A⇓, ET effected a time-dependent increment in ERK activity that peaked at 15 minutes. The induction was completely reversible after treatment with BQ-610 or PD98059 (Figure 2B⇓). Mechanical strain promoted a 6-fold increment in ERK activity (Figure 2C⇓) that was unaffected by SB-203580 and completely suppressed by PD98059. Approximately 50% of this induction appeared to be ET dependent based on inhibition with BQ-610.
These ET-dependent increases in ERK and p38 MAPK activity appear to have functional sequelae at the level of BNP gene transcription. ET effected a ≈3-fold increase in hBNP promoter activity, an effect that was completely blocked by the ETA receptor antagonist BQ-610 (Figure 3⇓). SB-203580 produced a 35% reduction in ET-dependent hBNP promoter activity, whereas PD98059 effected a more pronounced reduction of ≈75%. Of note, the combination of SB-203580 and PD98059 was more effective (≈85% inhibition) than the use of either agent alone, implying that the 2 operate independently, at least in part, to activate BNP gene transcription.
We next turned our attention to the ET-independent signaling cascades. As shown in Figure 4⇓, BQ-610 effected ≈50% reduction in strain-regulated BNP promoter activity, reflecting the autocrine/paracrine contribution to the strain response. Inhibition of p38 MAPK or ERK effected similar levels of inhibition (≈60% to 65% reduction in the total response to strain). Of note, the relative induction in the presence of PD98059 was reduced even further (180% versus 270% with SB-203580) due to a modest elevation in basal promoter activity. The combination of SB-203580 and PD98059 was more effective than the use of either agent alone (≈85% to 90% inhibition), again implying at least some independence in the signaling circuitry that regulates BNP promoter activity through these 2 pathways. Of greater importance, the combination of BQ-610 with either SB-230580 or PD98059 resulted in a significant decrease in strain-dependent promoter activity (85% inhibition with SB-203580 and 79% with PD98059 versus BQ-610 alone), implying that both p38 MAPK and ERK are important in signaling ET-independent components of the response to mechanical strain. Again, the inhibition of the combination of SB-230580 and PD98059, together with BQ-610, was greater than that seen with either agent alone (≈95% inhibition).
We have shown previously that the p38 MAPK-dependent component of the response to mechanical strain relies on an NF-κB–signaled event involving 3 SSREs in the proximal promoter of the hBNP gene.12 To examine the role of the NF-κB/SSRE system in signaling the ET/p38 MAPK–dependent activation of this promoter, we carried out the experiment presented in Figure 5A⇓. Inhibition of p38 MAPK with SB-203580 or suppression of NF-κB activity after transfection with a nonphosphorylatable and, therefore, constitutively active mutant of IκB (2N), which effects near-complete suppression of NF-κB activity, resulted in a similar reduction (≈30%) in ET-stimulated promoter activity. Transfection of a promoter construct mutated at each of the involved SSRE-like elements resulted in a similar fall in promoter activity, and this reduction was not further amplified with the inhibition of p38 MAPK or NF-κB activity. Collectively, these findings indicate that a minor fraction of the ET-dependent stimulatory activity traffics through the same 3 SSREs previously identified as playing a role in the strain-dependent activation of the hBNP gene promoter.
Finally, we raised the same question with regard to the ET-independent response to the strain stimulus. As shown in Figure 5B⇑, ET-independent (ie, that seen in the presence of BQ-610) promoter activity was reduced to a comparable extent by SB-203580, by the IκB mutant, or by mutation of the SSRE-like sites in the promoter (≈75% to 80% inhibition). Again, the inference is that the bulk of ET-independent activation of the p38/NF-κB pathway operates through the same SSRE-like sites in the hBNP promoter.
The results of the present study indicate that the activation of p38 MAPK and ERK is required for both ET-dependent and -independent stimulation of hBNP promoter activity by mechanical strain. Thus, it appears that both the direct response and the autocrine/paracrine response to mechanical strain use similar signaling circuitry to drive hBNP gene expression.
The nature of the signaling cascade linking hypertrophic agonists, like ET, to the phenotypic changes associated with hypertrophy is controversial. Although some features of the phenotype can be linked to activation of the ERK signal-ing cascade,2 ERK alone appears to be insufficient to trigger the complete hypertrophic response.6 The role of p38 MAPK, the JNKs, or both in this process is better supported,3 4 5 but the magnitude of their respective contributions remains unclear.
The activation of both p38 MAPK12 and ERK7 8 10 has been demonstrated after the application of mechanical strain to cultured myocytes. The involvement of these pathways extends to both the ET-dependent and -independent components of the strain response (see earlier). The relative contribution of ERK versus p38 MAPK to the observed response does, however, differ in the 2 components. Although the ERK pathway seems to be of greater importance for the response to ET in this particular model (see Figure 3⇑), p38 MAPK appears to dominate the ET-independent response. Because these pathways operate, to some extent, independently in the control of hBNP gene promoter activity, their differential use under the conditions of submaximal induction might be used to advantage to amplify the strain response.
Residual activity after the inhibition of both the ERK and the p38 MAPK pathways accounts for ≈20% of the response to ET and ≈10% of the ET-independent response to strain (see Figures 3⇑ and 4⇑). The nature of the signaling mechanism or mechanisms that account for this activity remains undefined, although the JNKs represent obvious candidates. Both ET3 and mechanical strain9 10 have been shown to stimulate JNK activity in cultured cardiac myocytes, and this stimulation has been linked to the hypertrophic phenotype. The levels of inhibition cited probably are low estimates of the true participation of this pathway (or pathways) in signaling the response to mechanical strain. There clearly is partial overlap in the response to p38 MAPK and ERK activation (ie, inhibition of p38 MAPK and ERK each independently accounts for 60% to 65% of the reduction in response, respectively, whereas simultaneous inhibition of both results in only 85% to 90% of the reduction). It is likely that similar redundancy exists between the JNKs (or other non-ERK/non-p38 effectors) and these pathways.
Interestingly, the p38 MAPK/NF-κB–mediated response to ET appears to traffic through the same SSRE-like structures in the hBNP promoter that support the ET-independent component of the response to strain. The ERK-mediated response to ET, on the other hand, does not appear to require these SSREs. The location of the sites responsible for signaling this activity remains unknown. Elucidation of the genomic element or elements and transcription factors responsible for this activity and the mechanisms underlying their interaction with the SSRE/NF-κB complex represent logical targets for future study. A schematic depicting our present understanding of the events linking mechanical strain to enhanced BNP gene promoter activity is presented in Figure 6⇓.
In summary, the ET-dependent (ie, autocrine/paracrine) and ET-independent components of the transcriptional response to mechanical strain in cardiac myocytes use similar signal transduction pathways, albeit to different degrees. ET-dependent pathways favor ERK over p38 MAPK, whereas the reverse applies for the ET-independent pathways. Both require a non-ERK, non-p38 MAPK–dependent signaling pathway or pathways (eg, JNKs) to provide optimal transduction of the strain stimulus to the transcriptional machinery. The data argue for a generalized, and perhaps redundant, activation of several different MAPK species in cardiac myocytes subjected to mechanical strain. Collectively, these kinases appear to traffic the signals that lead to increased BNP gene transcription and, ultimately, myocyte hypertrophy.
This work was supported by NIH grant HL-35753 and American Heart Association Grant-in-Aid 9950062N. Dr Liang was supported by a fellowship from the American Heart Association, Western Affiliate, during the tenure of this study. The authors are grateful to F. Roediger for preparation of the myocyte cultures used in the study.
- Received September 14, 1999.
- Revision received October 12, 1999.
- Accepted November 10, 1999.
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