RESUMO
The POU4F2/Brn-3b transcription factor has been identified as a potentially novel regulator of key metabolic processes. Loss of this protein in Brn-3b knockout (KO) mice causes profound hyperglycemia and insulin resistance (IR), normally associated with type 2 diabetes (T2D), whereas Brn-3b is reduced in tissues taken from obese mice fed on high-fat diets (HFD), which also develop hyperglycemia and IR. Furthermore, studies in C2C12 myocytes show that Brn-3b mRNA and proteins are induced by glucose but inhibited by insulin, suggesting that this protein is itself highly regulated in responsive cells. Analysis of differential gene expression in skeletal muscle from Brn-3b KO mice showed changes in genes that are implicated in T2D such as increased glycogen synthase kinase-3ß and reduced GLUT4 glucose transporter. The GLUT4 gene promoter contains multiple Brn-3b binding sites and is directly transactivated by this transcription factor in cotransfection assays, whereas chromatin immunoprecipitation assays confirm that Brn-3b binds to this promoter in vivo. In addition, correlation between GLUT4 and Brn-3b in KO tissues or in C2C12 cells strongly supports a close association between Brn-3b levels and GLUT4 expression. Since Brn-3b is regulated by metabolites and insulin, this may provide a mechanism for controlling key genes that are required for normal metabolic processes in insulin-responsive tissues and its loss may contribute to abnormal glucose uptake.
Assuntos
Diabetes Mellitus Tipo 2/genética , Proteínas de Homeodomínio/genética , Hiperglicemia/genética , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , RNA Mensageiro/metabolismo , Fator de Transcrição Brn-3B/genética , Animais , Peso Corporal/genética , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ácido Glucárico/farmacologia , Intolerância à Glucose/genética , Teste de Tolerância a Glucose , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Proteínas de Homeodomínio/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Immunoblotting , Insulina/farmacologia , Camundongos , Camundongos Knockout , Fibras Musculares Esqueléticas/efeitos dos fármacos , Mutação , RNA Mensageiro/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Brn-3B/efeitos dos fármacos , Fator de Transcrição Brn-3B/metabolismoRESUMO
Adult hearts respond to increased workload such as prolonged stress or injury, by undergoing hypertrophic growth. During this process, the early adaptive responses are important for maintaining cardiac output whereas at later stages, pathological responses such as cardiomyocyte apoptosis and fibrosis cause adverse remodelling, that can progress to heart failure. Yet the factors that control transition from adaptive responses to pathological remodelling in the heart are not well understood. Here we describe the POU4F2/Brn-3b transcription factor (TF) as a novel regulator of adaptive hypertrophic responses in adult hearts since Brn-3b mRNA and protein are increased in angiotensin-II (AngII) treated mouse hearts with concomitant hypertrophic changes [increased heart weight:body weight (HW:BW) ratio]. These effects occur specifically in cardiomyocytes because Brn-3b expression is increased in AngII-treated primary cultures of neonatal rat ventricular myocytes (NRVM) or foetal heart-derived H9c2 cells, which undergo characteristic sarcomeric re-organisation seen in hypertrophic myocytes and express hypertrophic markers, ANP/ßMHC. The Brn-3b promoter is activated by known hypertrophic signalling pathways e.g. p42/p44 mitogen-activated protein kinase (MAPK/ERK1/2) or calcineurin (via NFAT). Brn-3b target genes, e.g. cyclin D1, GLUT4 and Bax, are increased at different stages following AngII treatment, supporting distinct roles in cardiac responses to stress. Furthermore, hearts from male Brn-3b KO mutant mice display contractile dysfunction at baseline but also attenuated hypertrophic responses to AngII treatment. Hearts from AngII-treated male Brn-3b KO mice develop further contractile dysfunction linked to extensive fibrosis/remodelling. Moreover, known Brn-3b target genes, e.g. GLUT4, are reduced in AngII-treated Brn-3b KO hearts, suggesting that Brn-3b and its target genes are important in driving adaptive hypertrophic responses in stressed heart.
Assuntos
Doenças Cardiovasculares/genética , Hipertrofia/genética , Miocárdio/metabolismo , Fator de Transcrição Brn-3B/genética , Angiotensina II/farmacologia , Animais , Animais Recém-Nascidos , Apoptose , Calcineurina/farmacologia , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Ciclina D1/genética , Regulação da Expressão Gênica/genética , Transportador de Glucose Tipo 4/genética , Humanos , Hipertrofia/metabolismo , Hipertrofia/patologia , Camundongos , Camundongos Knockout , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Cultura Primária de Células , RNA Interferente Pequeno/genética , Ratos , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/genéticaRESUMO
The development of drug resistance following treatment with chemotherapeutic agents such as cisplatin (cis) and paclitaxel (pax) contributes to high morbidity and mortality in ovarian cancers. However, the molecular mechanisms underlying such changes are not well understood. In this study, we demonstrate that the Brn-3b transcription factor was increased in different ovarian cancer cells including SKOV3 and A2780 following treatment with cis and pax. Furthermore, sustained increases in Brn-3b were associated with survival in drug resistant cells and correlated with elevated HSP27 expression. In contrast, targeting Brn-3b for reduction using short interfering RNA (siRNA) also resulted in attenuated HSP27 expression. Importantly, blocking Brn-3b expression with siRNA in SKOV3 cells was associated with reduced cell numbers at baseline but also increased cell death after further treatment, indicating sensitization of cells. Similar results were obtained in the metastatic IP1 cell line derived from ascites of mice bearing SKOV3 tumours. These findings suggest that increased Brn-3b may confer resistance to chemotherapeutic drugs in ovarian cancer cells by regulating key target genes such as HSP27 and that targeting Brn-3b may provide a novel mechanism for treatment of drug resistant ovarian cancers.
RESUMO
Congenital heart defects contribute to embryonic or neonatal lethality but due to the complexity of cardiac development, the molecular changes associated with such defects are not fully understood. Here, we report that transcription factors (TFs) Brn-3a (POU4F1) and Brn-3b (POU4F2) are important for normal cardiac development. Brn-3a directly represses Brn-3b promoter in cardiomyocytes and consequently Brn-3a knockout (KO) mutant hearts express increased Brn-3b mRNA during mid-gestation, which is linked to hyperplastic growth associated with elevated cyclin D1, a known Brn-3b target gene. However, during late gestation, Brn-3b can cooperate with p53 to enhance transcription of pro-apoptotic genes e.g. Bax, thereby increasing apoptosis and contribute to morphological defects such as non-compaction, ventricular wall/septal thinning and increased crypts/fissures, which may cause lethality of Brn-3a KO mutants soon after birth. Despite this, early embryonic lethality in e9.5 double KO (Brn-3a-/- : Brn-3b-/-) mutants indicate essential functions with partial redundancy during early embryogenesis. High conservation between mammals and zebrafish (ZF) Brn-3b (87%) or Brn-3a (76%) facilitated use of ZF embryos to study potential roles in developing heart. Double morphant embryos targeted with morpholino oligonucleotides to both TFs develop significant cardiac defects (looping abnormalities and valve defects) suggesting essential roles for Brn-3a and Brn-3b in developing hearts.