RESUMO
In various cell types certain stresses can stimulate p38 mitogen-activated protein kinase (p38 MAPK), leading to the transcriptional activation of genes that contribute to appropriate compensatory responses. In this report the mechanism of p38-activated transcription was studied in cardiac myocytes where this MAPK is a key regulator of the cell growth and the cardiac-specific gene induction that occurs in response to potentially stressful stimuli. In the cardiac atrial natriuretic factor (ANF) gene, a promoter-proximal serum response element (SRE), which binds serum response factor (SRF), was shown to be critical for ANF induction in primary cardiac myocytes transfected with the selective p38 MAPK activator, MKK6 (Glu). This ANF SRE does not possess sequences typically required for the binding of the Ets-related ternary complex factors (TCFs), such as Elk-1, indicating that p38-mediated induction through this element may take place independently of such TCFs. Although p38 did not phosphorylate SRF in vitro, it efficiently phosphorylated ATF6, a newly discovered SRF-binding protein that is believed to serve as a co-activator of SRF-inducible transcription at SREs. Expression of an ATF6 antisense RNA blocked p38-mediated ANF induction through the ANF SRE. Moreover, when fused to the Gal4 DNA-binding domain, an N-terminal 273-amino acid fragment of ATF6 was sufficient to support trans-activation of Gal4/luciferase expression in response to p38 but not the other stress kinase, N-terminal Jun kinase (JNK); p38-activating cardiac growth promoters also stimulated ATF6 trans-activation. These results indicate that through ATF6, p38 can augment SRE-mediated transcription independently of Ets-related TCFs, representing a novel mechanism of SRF-dependent transcription by MAP kinases.
Assuntos
Fator Natriurético Atrial/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Regulação da Expressão Gênica/genética , Proteínas Quinases Ativadas por Mitógeno , Miocárdio/enzimologia , Fator 6 Ativador da Transcrição , Animais , Células Cultivadas , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endotelina-1/farmacologia , Proteínas Nucleares/genética , Fenilefrina/farmacologia , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Antissenso/farmacologia , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Fator de Resposta Sérica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia , Transfecção/genética , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
In cardiac myocytes the stimulation of p38 mitogen-activated protein kinase activates a hypertrophic growth program and the induction of the cardiac-specific genes associated with this program. This study focused on determining whether these novel growth-promoting effects are accompanied by the p38-mediated inhibition of apoptosis, and if so, what signaling pathways might be responsible. Primary neonatal rat ventricular myocytes were driven into apoptosis by treatments known to induce apoptosis in other cell types, e.g. incubation with anisomycin or overexpression constitutively active MEKK-1 (MEKK-1COOH), a protein that strongly activates extracellular signal-regulated kinase and N-terminal c-Jun kinase, but not p38. Overexpression of constitutively active MKK6, MKK6 (Glu), which selectively activates p38 in cardiac myocytes, protected cells from either anisomycin- or MEKK-1COOH-induced apoptosis. This protection was blocked by SB 203580, a selective p38 inhibitor. MKK6 (Glu) also activated transcription mediated by NF-kappaB, a factor which protects other cell types from apoptosis. The activation of NF-kappaB and the protection from apoptosis mediated by MKK6 (Glu) were both blocked by SB 203580. Interestingly, overexpression of a mutant form of I-kappaBalpha, which inhibits nuclear translocation of NF-kappaB, completely blocked MKK6 (Glu)-activated NF-kappaB but had little effect on MKK6s anti-apoptotic effects. These findings suggest that, in part, the overexpression of MKK6 (Glu) may foster growth and survival of cardiac myocytes by protecting them from apoptosis in a p38-dependent manner. Additionally, while NF-kappaB is activated in myocardial cells by p38, this does not appear to be the major mechanism by which MKK6 (Glu) exerts its anti-apoptotic effects in this cell type, suggesting a novel pathway for p38-mediated protection from apoptosis.
Assuntos
Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Miocárdio/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Animais , Apoptose/genética , Células Cultivadas , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , MAP Quinase Quinase 6 , Miocárdio/patologia , NF-kappa B/genética , Ratos , Transdução de Sinais/genética , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Three hallmark features of the cardiac hypertrophic growth program are increases in cell size, sarcomeric organization, and the induction of certain cardiac-specific genes. All three features of hypertrophy are induced in cultured myocardial cells by alpha1- adrenergic receptor agonists, such as phenylephrine (PE) and other growth factors that activate mitogen- activated protein kinases (MAPKs). In this study the MAPK family members extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 were activated by transfecting cultured cardiac myocytes with constructs encoding the appropriate kinases possessing gain-of-function mutations. Transfected cells were then analyzed for changes in cell size, sarcomeric organization, and induction of the genes for the A- and B-type natriuretic peptides (NPs), as well as the alpha-skeletal actin (alpha-SkA) gene. While activation of JNK and/or ERK with MEKK1COOH or Raf-1 BXB, respectively, augmented cell size and effected relatively modest increases in NP and alpha-SkA promoter activities, neither upstream kinase conferred sarcomeric organization. However, transfection with MKK6 (Glu), which specifically activated p38, augmented cell size, induced NP and alpha-Ska promoter activities by up to 130-fold, and elicited sarcomeric organization in a manner similar to PE. Moreover, all three growth features induced by MKK6 (Glu) or PE were blocked with the p38-specific inhibitor, SB 203580. These results demonstrate novel and potentially central roles for MKK6 and p38 in the regulation of myocardial cell hypertrophy.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Regulação da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno , Miocárdio/enzimologia , Miocárdio/metabolismo , Sarcômeros/fisiologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Cardiomegalia/enzimologia , Cardiomegalia/genética , Cardiomegalia/patologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Tamanho Celular/genética , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , MAP Quinase Quinase 6 , Miocárdio/citologia , Fenilefrina/farmacologia , Piridinas/farmacologia , Ratos , Sarcômeros/efeitos dos fármacos , Sarcômeros/enzimologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Electrical stimulation of contractions (pacing) of primary neonatal rat ventricular myocytes increases intracellular calcium and activates a hypertrophic growth program that includes expression of the cardiac-specific gene, atrial natriuretic factor (ANF). To investigate the mechanism whereby pacing increases ANF, pacing was tested for its ability to regulate mitogen-activated protein kinase family members, ANF promoter activity, and the trans-activation domain of the transcription factor, Sp1. Pacing and the calcium channel agonist BAYK 8644 activated c-Jun N-terminal kinase (JNK) but not extracellular signal-regulated kinase. Pacing stimulated ANF-promoter activity approximately 10-fold. Furthermore, transfection with an expression vector for c-Jun, a substrate for JNK, also activated the ANF promoter, and the combination of pacing and c-Jun was synergystic, consistent with roles for JNK and c-Jun in calcium-activated ANF expression. Proximal serum response factor and Sp1 binding sites were required for the effects of pacing or c-Jun on the ANF promoter. Pacing and c-Jun activated a GAL4-Sp1 fusion protein by 3- and 12-fold, respectively, whereas the two stimuli together activated GAL4-Sp1 synergistically, similar to their effect on the ANF promoter. Transfection with an expression vector for c-Fos inhibited the effects of c-Jun, suggesting that c-Jun acts independently of AP-1. These results demonstrate an interaction between c-Jun and Sp1 and are consistent with a novel mechanism of calcium-mediated transcriptional activation involving the collaborative actions of JNK, c-Jun, serum response factor, and Sp1.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Ventrículos do Coração/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição Sp1/metabolismo , Animais , Fator Natriurético Atrial/genética , Células Cultivadas , Ventrículos do Coração/citologia , Proteínas Quinases JNK Ativadas por Mitógeno , Regiões Promotoras Genéticas , Ratos , Fator de Resposta SéricaRESUMO
In cardiac myocytes, B-type natriuretic peptide (BNP) expression is induced with the rapid kinetics of a primary response gene. Like many other primary response gene transcripts, the BNP mRNA possesses destabilizing elements and is believed to be short-lived. The rapid induction of a short-lived transcript could be achieved partty by agonist-mediated increases in mRNA t1/2. Accordingly, the present study was undertaken to evaluate whether the alpha 1-adrenergic receptor agonist, phenylephrine (PE), a known inducer of BNP expression, could stabilize the BNP mRNA and, if so, what signaling pathways might be involved. In primary myocardial cells treated with a transcription inhibitor, the t1/2 of the BNP mRNA was found to be about 1 h in the absence of PE; however, in the presence of PE, the t1/2 increased to 5 h. It was shown that neither the calmodulin kinase inhibitor, KN-62, nor the protein tyrosine kinase inhibitor, tyrphostin, blocked PE-mediated stabilization of the BNP mRNA. However, either the protein kinase C (PKC) inhibitor, GF 109203X, or the mitogen-activated protein kinase kinase (MAPKK) inhibitor, PD 098059, effected some blockade of the stabilizing effects of PE. While maximal doses of PD 098059 nearly completely blocked PE-activated MAPK, stabilization was only partially inhibited. Moreover, maximal doses of GF 109203X, which only partially blocked PE-activated MAPK, nearly completely inhibited stabilization. Thus, while MAPK appears to be required for maximal agonist-mediated stabilization, PKC seems to play a dominant role, participating through both MAPK-dependent and -independent pathways. These results establish roles for both the PKC and MAPK families in alpha 1-adrenergic receptor-mediated stabilization of the BNP mRNA, suggesting that the rapid induction of BNP expression might be due, in part, to this agonist-mediated increase in mRNA t1/2.
Assuntos
Fator Natriurético Atrial/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Miocárdio/metabolismo , Proteína Quinase C/fisiologia , Receptores Adrenérgicos alfa/metabolismo , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Fator Natriurético Atrial/efeitos dos fármacos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Colforsina/farmacologia , Relação Dose-Resposta a Droga , Endotelina-1/farmacologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Meia-Vida , Coração/efeitos dos fármacos , Indóis/farmacologia , Maleimidas/farmacologia , Miocárdio/citologia , Peptídeo Natriurético Encefálico , Fenilefrina/farmacologia , Proteína Quinase C/antagonistas & inibidores , Ratos , Receptores Adrenérgicos alfa/efeitos dos fármacos , Transdução de SinaisRESUMO
To better understand the molecular basis for increased atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) expression during overload-induced cardiac hypertrophy, we studied the induction of the genes in primary myocardial cells by the alpha 1-adrenergic agonist, phenylephrine (PE), a potent hypertrophic agent. PE augmented the transcription of both genes to similar extents, although the time course of mRNA accumulation differed. Increases in ANF mRNA were evident only after 6-8 h of PE exposure, when transcript levels were 2-4-fold over control. However, similar increases in BNP mRNA were observed as soon as 1 h of PE exposure. Moreover, while ANF mRNA levels continued to increase through 24 h of PE treatment, maximal levels of BNP mRNA (8-10-fold over control) were observed at 4 h, after which transcript levels declined to about 3-fold over control. The early induction of the BNP mRNA by PE was independent of protein synthesis, whereas the late induction of both genes required protein synthesis. Interestingly, the early BNP induction was only partially blocked by the transcription inhibitor, actinomycin D, indicating that, in part, the inductive effects of PE might be the result of transcript stabilization. Indeed, the BNP transcript, which was shown to possess a half-life of less than 1 h in control cells, was stabilized by the addition of PE, while the ANF transcript possessed a half-life of at least 24 h under all conditions. These data indicate that the induction of BNP by alpha 1-adrenergic agonists has characteristics of both a primary and secondary response gene, while ANF is a typical secondary response gene. Moreover, alpha 1-adrenergic stimulation enhances BNP expression through both transcriptional activation and transcript stabilization, while ANF expression is enhanced primarily transcriptionally.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 1 , Regulação da Expressão Gênica/efeitos dos fármacos , Miocárdio/metabolismo , Proteínas do Tecido Nervoso/genética , Fenilefrina/farmacologia , Animais , Fator Natriurético Atrial/biossíntese , Fator Natriurético Atrial/genética , Células Cultivadas , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Peptídeo Natriurético Encefálico , Proteínas do Tecido Nervoso/biossíntese , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Transcrição GênicaRESUMO
GATA-binding proteins are transcription factors that regulate the stage- and tissue-specific expression of globin genes in cells of the erythroid lineage. Recently, a cardiac GATA-binding protein was found to be the earliest gene expressed during cardiogenesis; however, the target genes of this transcription factor in the heart are unknown. Since brain natriuretic peptide (BNP) is activated early in cardiac growth and development, we evaluated whether it could serve as a target gene for GATA-binding protein-mediated induction. Upon isolating and sequencing 2.5 kilobases of the rat BNP 5'-flanking sequence (FS), a variety of putative transcriptional enhancer sites were identified, including several GATA consensus sequences (WGATAR), one of which apparently serves as the major promoter site. Primary myocardial cells were transfected with BNP/luciferase fusion genes; reporter expression was strongly induced by typical growth factors such as phorbol esters, serum, or alpha 1-adrenergic agonists, as well as by GATA-4 overexpression. Truncation analyses showed that inducibility mapped primarily to the proximal -116 base pair of the rat BNP 5'-FS, where there are two consensus GATA sites in addition to the GATA sequence at the TATA box. Point mutation analyses showed that at least one of the GATA sites was required to confer full GATA-4-inducible transcription. These results demonstrate that a proximal region of the rat BNP 5'-FS is required for growth factor- and GATA-inducible transcription, supporting the view that the BNP gene could serve as a target for GATA-binding proteins during early cardiac development.