RESUMO
CARM1 is a protein arginine methyltransferase (PRMT) that has been firmly implicated in transcriptional regulation. However, the molecular mechanisms by which CARM1 orchestrates transcriptional regulation are not fully understood, especially in a tissue-specific context. We found that Carm1 is highly expressed in the mouse testis and localizes to the nucleus in spermatids, suggesting an important role for Carm1 in spermiogenesis. Using a germline-specific conditional Carm1 knockout mouse model, we found that it is essential for the late stages of haploid germ cell development. Loss of Carm1 led to a low sperm count and deformed sperm heads that can be attributed to defective elongation of round spermatids. RNA-seq analysis of Carm1-null spermatids revealed that the deregulated genes fell into similar categories as those impacted by p300-loss, thus providing a link between Carm1 and p300. Importantly, p300 has long been known to be a major Carm1 substrate. We found that CREMτ, a key testis-specific transcription factor, associates with p300 through its activator, ACT, and that this interaction is negatively regulated by the methylation of p300 by Carm1. Thus, high nuclear Carm1 levels negatively impact the p300â¢ACTâ¢CREMτ axis during late stages of spermiogenesis.
Assuntos
Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Espermatogênese , Fatores de Transcrição/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Animais , Modulador de Elemento de Resposta do AMP Cíclico/antagonistas & inibidores , Fertilidade , Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Espermátides/metabolismo , Espermatozoides/enzimologia , Testículo/anatomia & histologia , Fatores de Transcrição de p300-CBP/antagonistas & inibidoresRESUMO
Chronic ß-adrenergic stimulation is regarded as a pivotal step in the progression of heart failure which is associated with a high risk for arrhythmia. The cAMP-dependent transcription factors cAMP-responsive element binding protein (CREB) and cAMP-responsive element modulator (CREM) mediate transcriptional regulation in response to ß-adrenergic stimulation and CREM repressor isoforms are induced after stimulation of the ß-adrenoceptor. Here, we investigate whether CREM repressors contribute to the arrhythmogenic remodeling in the heart by analyzing arrhythmogenic alterations in ventricular cardiomyocytes (VCMs) from mice with transgenic expression of the CREM repressor isoform CREM-IbΔC-X (TG). Patch clamp analyses, calcium imaging, immunoblotting and real-time quantitative RT-PCR were conducted to study proarrhythmic alterations in TG VCMs vs. wild-type controls. The percentage of VCMs displaying spontaneous supra-threshold transient-like Ca(2+) releases was increased in TG accompanied by an enhanced transduction rate of sub-threshold Ca(2+) waves into these supra-threshold events. As a likely cause we discovered enhanced NCX-mediated Ca(2+) transport and NCX1 protein level in TG. An increase in I NCX and decrease in I to and its accessory channel subunit KChIP2 was associated with action potential prolongation and an increased proportion of TG VCMs showing early afterdepolarizations. Finally, ventricular extrasystoles were augmented in TG mice underlining the in vivo relevance of our findings. Transgenic expression of CREM-IbΔC-X in mouse VCMs leads to distinct arrhythmogenic alterations. Since CREM repressors are inducible by chronic ß-adrenergic stimulation our results suggest that the inhibition of CRE-dependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease.
Assuntos
Arritmias Cardíacas/metabolismo , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Potenciais de Ação , Animais , Arritmias Cardíacas/fisiopatologia , Cálcio/metabolismo , Células Cultivadas , Modulador de Elemento de Resposta do AMP Cíclico/antagonistas & inibidores , Modulador de Elemento de Resposta do AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ventrículos do Coração/fisiopatologia , Isoproterenol , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Potássio/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismoRESUMO
Substantial evidence suggests that the progressive loss of cardiomyocytes caused by apoptosis significantly contributes to the development of heart failure. beta-Adrenergic receptor activation and subsequent persistent phosphodiesterase 3A (PDE3A) downregulation and concomitant inducible cAMP early repressor (ICER) upregulation (PDE3A/ICER feedback loop) has been proposed to play a key role in the pathogenesis of cardiomyocyte apoptosis. In contrast, insulin-like growth factor-1 can activate cell survival pathways, providing protection against cell death and restoring muscle function. In this study, we found that insulin-like growth factor-1 activates extracellular signal-regulated kinase 5 (ERK5) and inhibits PDE3A/ICER feedback loop. Insulin-like growth factor-1 normalized isoproterenol-mediated PDE3A downregulation and ICER upregulation via ERK5/MEF2 activation, and also inhibited isoproterenol-induced myocyte apoptosis. To determine the physiological relevance of ERK5 activation in regulating PDE3A/ICER feedback loop, we investigated the PDE3A/ICER expression and cardiomyocyte apoptosis in transgenic mice with cardiac specific expression of a constitutively active form of mitogen-activated protein (MAP)/extracellular signal-regulated protein kinase (ERK) kinase 5alpha (MEK5alpha) (CA-MEK5alpha-Tg). In wild-type mice, pressure overload- or doxorubicin-induced significant reduction of PDE3A expression and subsequent ICER induction. Cardiac specific expression of CA-MEK5alpha rescued pressure overload- or doxorubicin-mediated PDE3A downregulation and ICER upregulation and inhibited myocyte apoptosis as well as subsequent cardiac dysfunction in vivo. These data suggest that preventing the feedback loop of PDE3A/ICER by ERK5 activation could inhibit progression of myocyte apoptosis as well as cardiac dysfunction. These data suggest a new therapeutic paradigm for end stage of heart failure by inhibiting the PDE3A/ICER feedback loop via activating ERK5.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Apoptose/fisiologia , Modulador de Elemento de Resposta do AMP Cíclico/antagonistas & inibidores , Retroalimentação Fisiológica/fisiologia , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Miócitos Cardíacos/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Animais , Apoptose/genética , Pressão Sanguínea/genética , Modulador de Elemento de Resposta do AMP Cíclico/genética , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Doxorrubicina/administração & dosagem , Doxorrubicina/antagonistas & inibidores , Ativação Enzimática/genética , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Camundongos , Camundongos Transgênicos , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/fisiologia , Miócitos Cardíacos/patologia , RatosRESUMO
In this study, we investigated the role of two inducible repressor proteins, inducible cAMP early repressor (ICER) and Fos-related antigen 2 (Fra-2) in the adrenergic induction of MAPK phosphatase-1 (MKP-1) as compared with their roles in the induction of arylalkylamine-N-acetyltransferase (AA-NAT) in rat pinealocytes. Treatment of pinealocytes with norepinephrine (NE) caused an increase in the mRNA and protein levels of MKP-1 and AA-NAT, as well as in the AA-NAT activity and melatonin production. NE stimulation also caused a simultaneous increase in the mRNA and protein levels of ICER and Fra-2. Transient knockdown of icer using adenovirus expressing small interfering RNA (siRNA) abolished the NE induction of icer expression but had little effect on the NE induction of mkp-1 or aa-nat expression. In contrast, pretreatment with adenovirus overexpressing icer was effective in reducing the NE induction of mkp-1 and aa-nat. The inhibitory effect of overexpressing icer was reversed by cotreatment with siRNA against icer. siRNA against fra-2 also abolished the NE-stimulated expression of fra-2 but had little effect on the NE induction of mkp-1 and aa-nat expression. Proteasomal inhibition, which reduced the NE-stimulated induction of aa-nat, caused a reduction of ICER and Fra-2. Together, these results indicate that whereas overexpression of ICER can suppress the NE induction of aa-nat and mkp-1, the amount of the repressors, ICER and Fra-2, present during NE induction appears insufficient to exert a significant effect in controlling the expression of these genes.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Arilalquilamina N-Acetiltransferase/biossíntese , Proteínas de Ciclo Celular/biossíntese , Modulador de Elemento de Resposta do AMP Cíclico/fisiologia , Antígeno 2 Relacionado a Fos/fisiologia , Proteínas Imediatamente Precoces/biossíntese , Lactonas/farmacologia , Norepinefrina/farmacologia , Fosfoproteínas Fosfatases/biossíntese , Glândula Pineal/metabolismo , Proteínas Tirosina Fosfatases/biossíntese , Animais , Células Cultivadas , Modulador de Elemento de Resposta do AMP Cíclico/antagonistas & inibidores , Modulador de Elemento de Resposta do AMP Cíclico/genética , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Fosfatase 1 de Especificidade Dupla , Antígeno 2 Relacionado a Fos/antagonistas & inibidores , Antígeno 2 Relacionado a Fos/genética , Antígeno 2 Relacionado a Fos/metabolismo , Técnicas de Transferência de Genes , Masculino , Glândula Pineal/citologia , Glândula Pineal/enzimologia , Inibidores de Proteassoma , Proteína Fosfatase 1 , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Oscillatory synthesis and secretion of the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), under the control of pulsatile hypothalamic gonadotropin-releasing hormone (GnRH), is essential for normal reproductive development and fertility. The molecular mechanisms by which various patterns of pulsatile GnRH regulate gonadotrope responsiveness remain poorly understood. In contrast to the alpha and LH beta subunit genes, FSH beta subunit transcription is preferentially stimulated at low rather than high frequencies of pulsatile GnRH. In this study, mutation of a cyclic AMP response element (CRE) within the FSH beta promoter resulted in the loss of preferential GnRH stimulation at low pulse frequencies. We hypothesized that high GnRH pulse frequencies might stimulate a transcriptional repressor(s) to attenuate the action of CRE binding protein (CREB) and show that inducible cAMP early repressor (ICER) fulfills such a role. ICER was not detected under basal conditions, but pulsatile GnRH stimulated ICER to a greater extent at high than at low pulse frequencies. ICER binds to the FSH beta CRE site to reduce CREB occupation and abrogates both maximal GnRH stimulation and GnRH pulse frequency-dependent effects on FSH beta transcription. These data suggest that ICER production antagonizes the stimulatory action of CREB to attenuate FSH beta transcription at high GnRH pulse frequencies, thereby playing a critical role in regulating cyclic reproductive function.