RESUMO
Doxorubicin (Adriamycin) is an effective anti-cancer drug, but its clinical usage is limited by a dose-dependent cardiotoxicity characterized by widespread sarcomere disarray and loss of myofilaments. Cardiac ankyrin repeat protein (CARP, ANKRD1) is a transcriptional regulatory protein that is extremely susceptible to doxorubicin; however, the mechanism(s) of doxorubicin-induced CARP depletion and its specific role in cardiomyocytes have not been completely defined. We report that doxorubicin treatment in cardiomyocytes resulted in inhibition of CARP transcription, depletion of CARP protein levels, inhibition of myofilament gene transcription, and marked sarcomere disarray. Knockdown of CARP with small interfering RNA (siRNA) similarly inhibited myofilament gene transcription and disrupted cardiomyocyte sarcomere structure. Adenoviral overexpression of CARP, however, was unable to rescue the doxorubicin-induced sarcomere disarray phenotype. Doxorubicin also induced depletion of the cardiac transcription factor GATA4 in cardiomyocytes. CARP expression is regulated in part by GATA4, prompting us to examine the relationship between GATA4 and CARP in cardiomyocytes. We show in co-transfection experiments that GATA4 operates upstream of CARP by activating the proximal CARP promoter. GATA4-siRNA knockdown in cardiomyocytes inhibited CARP expression and myofilament gene transcription, and induced extensive sarcomere disarray. Adenoviral overexpression of GATA4 (AdV-GATA4) in cardiomyocytes prior to doxorubicin exposure maintained GATA4 levels, modestly restored CARP levels, and attenuated sarcomere disarray. Interestingly, siRNA-mediated depletion of CARP completely abolished the Adv-GATA4 rescue of the doxorubicin-induced sarcomere phenotype. These data demonstrate co-dependent roles for GATA4 and CARP in regulating sarcomere gene expression and maintaining sarcomeric organization in cardiomyocytes in culture. The data further suggests that concurrent depletion of GATA4 and CARP in cardiomyocytes by doxorubicin contributes in large part to myofibrillar disarray and the overall pathophysiology of anthracycline cardiomyopathy.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Cardiomiopatias/metabolismo , Doxorrubicina/farmacologia , Fator de Transcrição GATA4/metabolismo , Proteínas Musculares/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Sarcômeros/efeitos dos fármacos , Transdução de Sinais/fisiologia , Animais , Cardiomiopatias/induzido quimicamente , Fator de Transcrição GATA4/genética , Masculino , Proteínas Musculares/genética , Miócitos Cardíacos/metabolismo , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Proteínas Repressoras/genética , Sarcômeros/metabolismoRESUMO
The let-7 family of microRNAs (miRNAs) are important regulators of developmental timing and cell differentiation and are often misexpressed in human cancer. In C. elegans, let-7 controls cell fate transitions from larval stage 4 (L4) to adulthood by post-transcriptionally down-regulating lineage-abnormal 41 (lin-41) and hunchback-like 1 (hbl-1). Primary let-7 (pri-let-7) transcripts are up-regulated in the L3, yet little is known about what controls this transcriptional up-regulation. We sought factors that either turn on let-7 transcription or keep it repressed until the correct time. Here we report that one of let-7's targets, the transcription factor Hunchback-like 1 (HBL-1), is responsible for inhibiting the transcription of let-7 in specific tissues until the L3. hbl-1 is a known developmental timing regulator and inhibits adult development in larval stages. Therefore, one important function of HBL-1 in maintaining larval stage fates is inhibition of let-7. Indeed, our results reveal let-7 as the first known target of the HBL-1 transcription factor in C. elegans and suggest a negative feedback loop mechanism for let-7 and HBL-1 regulation.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Retroalimentação Fisiológica , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Larva , MicroRNAs/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Fatores de Tempo , Fatores de Transcrição/genéticaRESUMO
Small, inhibitory RNA molecules called microRNAs cause large decreases in target protein levels through a post-transcriptional mechanism. Until recently, it was believed this mechanism operated almost exclusively at a step in translation. However, new work has revealed that microRNAs have a second, post-transcriptional mechanism that accelerates the rate of deadenylation, the initial step of mRNA decay.
