Mechanical strain activates BNP gene transcription through a p38/NF-kappaB-dependent mechanism

Abstract

Application of mechanical strain to neonatal rat ventricular myocytes in culture evokes changes in gene expression reminiscent of those that occur with hypertrophy in vivo, such as stimulation of brain natriuretic peptide (BNP) gene expression. Here, we show that a major component of strain-dependent BNP promoter activation results from stimulation of p38 mitogen-activated protein kinase (MAPK) in the cardiac myocyte. Strain increased p38 activity in a time-dependent fashion. The p38 inhibitor SB203580 led to a reduction of approximately 60% in strain-activated human BNP (hBNP) promoter activity. Cotransfection of wild-type p38 increased both basal and strain-dependent promoter activity, while cotransfection with MKK6AL, a dominant-negative inhibitor of p38 MAPK kinase, resulted in partial inhibition of either p38- or strain-activated hBNP promoter activity. p38 MAPK increased hBNP promoter activity through activation of the transcription factor NF-kappaB. Activation of the hBNP promoter by either p38 or strain was mediated by DNA elements present in the 5' flanking sequence of the gene. Mechanical strain promoted assembly of NF-kappaB components on these DNA elements in vitro. Thus, induction of the hBNP promoter by mechanical strain depends, at least in part, on stimulation of p38 and subsequent activation of NF-kappaB. This activation may play an important role in signaling the increased BNP gene expression that accompanies hemodynamic overload and cardiac hypertrophy in vivo.

Figure 1

Mechanical strain activates p38 in cultured neonatal rat ventricular myocytes. After 48 hours of culture, cells were subjected to cyclical strain for the times indicated. Cells were then collected, lysed, and assayed for p38 kinase activity using either an immune complex kinase assay (a and b) or Western blot analysis for the activated form of the enzyme (c). (a) Time course of p38 induction in conventional myocyte cultures; pooled data from 3 independent experiments. (b) p38 induction in cultures further enriched for myocytes after 48 hours of treatment with bromodeoxyuridine (BudR; 0.1 mM) or nonmyocytes (primarily fibroblasts) collected from the same neonatal hearts and expanded in culture over a 2-week period. Representative of duplicate experiments. (c) Cells were subjected to mechanical strain for varying periods of time, then assayed for active p38 using Western blot analysis. Representative of duplicate experiments. Data are presented as mean ± SD. *P < 0.01 vs. control.

Figure 2

Effects of SB203580, MKK6AL, and wild-type p38 on strain-dependent hBNP promoter activity in neonatal rat ventricular myocytes. (a) Cells were transfected with 1 μg of –1,595 hBNP luciferase. After 24 hours of culture, cells were subjected to cyclical strain for 48 hours in the presence of different concentrations of SB203580. (b) Cells were cotransfected with 1 μg of –1,595 hBNP luciferase and 2 concentrations of MKK6AL with or without 1 μg of pCMV-FLAG-p38MAPK. After 24 hours of culture, cells were subjected to cyclical strain for 48 hours. Data are presented as mean ± SD of 4 separate experiments. *P < 0.01 vs. strain control.

Figure 3

Effects of IκBα mutants on strain-dependent hBNP promoter activity in neonatal rat ventricular myocytes. Cells were cotransfected with 1 μg of –1,595 hBNP luciferase and different concentrations of either wild-type (WT) IκBα or the mutant indicated (2N, 3C, or 2N + 3C). After 24 hours of culture, cells were subjected to cyclical strain for 48 hours. A separate subgroup was treated with 10 μM SB203580 before strain. The data are expressed as mean ± SD of 4 separate experiments. *P < 0.01 vs. strain control. ^#P < 0.05 vs. strain control.

Figure 4

Effects of IκBα mutants and MKK6AL on p38-activated hBNPLuc or NF-κB TKLuc activity in neonatal rat ventricular myocytes. (a) Cells were cotransfected with 1 μg of –1,595 hBNP luciferase and 1 μg of pCMV-FLAG-p38MAPK. After 24 hours of culture, cells were treated with 10 μM SB203580 or 10 μM PD98059. (b) Cells were cotransfected with 1 μg of –1,595 hBNP luciferase, 1 μg of pCMV-FLAG-p38MAPK, and different concentrations of MKK6AL, WT IκBα, or the 2N mutant of IκBα. (c) Cells were cotransfected with 10 μg of NF-κB TKLuc, 1 μg of pCMV-FLAG-p38MAPK, and 10 μg of WT IκBα or one of the relevant mutants (2N, 3C, or 2N + 3C). A separate subgroup was treated with 10 μM SB203580. Data are presented as mean ± SD of 4 separate experiments. *P < 0.01 vs. basal control. ^#P < 0.05 vs. basal control.

Figure 5

Identification of SSRE-like structures in the 5′ flanking sequence of the hBNP gene (–904 WT). Positioning of the 3 elements relative to the transcription start site are indicated. Putative SSRE sequences are underlined. Mutated bases in each of the 3 SSRE mutant reporters (SSRE MUT) are indicated in lowercase letters.

Figure 6

Effects of mutations in putative SSRE sequences on hBNP promoter activity. (a) Wild-type –904 hBNPLuc or site-directed mutants targeted at 1 or more of the SSREs described in Figure 5 were introduced into ventricular myocytes. After 24 hours, cells were subjected to mechanical strain for 48 hours; lysates were generated and analyzed for luciferase activity. (b) One microgram of wild-type –904 hBNPLuc or the triple SSRE mutant was cotransfected into ventricular cells with 1 μg of pCMV-FLAG-p38MAPK alone or in combination with 10 μg of the 2N mutant. Where indicated, cells were treated with 10 μM SB203580 for 48 hours. (c) Cells were cotransfected with 1 μg of wild-type –904 hBNPLuc or a –904 hBNPLuc harboring a triple SSRE mutation, and 10 μg of the 2N mutant of IκBα. After 24 hours of culture, cells were subjected to mechanical strain for 48 hours in the presence or absence of 10 μM SB203580, as indicated. Seventy-two hours after transfection, cells were harvested and luciferase activity was measured. Data are presented as mean ± SD of 3 separate experiments.

Figure 7

Nuclear extracts from myocytes subjected to mechanical strain interact with SSRE-like sites in hBNP promoter. (a) Cells were subjected to strain stimulus for different periods of time. Cells were harvested and nuclear extracts were prepared. Ten micrograms of nuclear extract was incubated with ³²P-labeled oligonucleotide encoding SSRE-like sites (see Methods) and was subjected to electrophoretic mobility shift assay. (b) Ten micrograms of extract from cells subjected to 1 hour of mechanical strain was incubated with labeled oligonucleotide in the absence or presence of increasing concentrations of unlabeled oligonucleotide (10- to 100-fold excess) encoding wild-type or mutant SSRE sequence. (c) Ten micrograms of extract from myocytes subjected to strain for 1 hour was preincubated on ice for 1 hour with 1 μg of polyclonal antibody directed against p50, p65, or Sp1 before addition of labeled probe. Position of p50-p65 complex and supershift complex are indicated.