RESUMO
Members of the GATA family of transcription factors play important roles in cell fate specification, differentiation, and morphogenesis during mammalian development. GATA5, the only one of the six vertebrate GATA factor genes not yet inactivated in mice, is expressed in a pattern that overlaps with but is distinct from that of other GATA factor genes. During mouse embryogenesis, GATA5 is expressed first in the developing heart and subsequently in the lung, vasculature, and genitourinary system. To investigate the function of GATA5 in vivo, we created mice homozygous for a GATA5 null allele. Homozygous mutants were viable and fertile, but females exhibited pronounced genitourinary abnormalities that included vaginal and uterine defects and hypospadias. In contrast, the genitourinary system was unaffected in male GATA5 mutants. These results reveal a specific role of GATA5 in development of the female genitourinary system and suggest that other GATA factors may have functions overlapping those of GATA5 in other tissues.
Assuntos
Proteínas de Ligação a DNA/genética , Genitália Feminina/anormalidades , Fatores de Transcrição/genética , Sistema Urinário/anormalidades , Animais , Proteínas de Ligação a DNA/metabolismo , Feminino , Fator de Transcrição GATA5 , Engenharia Genética/métodos , Genitália Feminina/anatomia & histologia , Homozigoto , Hipospadia/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fatores de Transcrição/metabolismo , Sistema Urinário/anatomia & histologia , Dedos de ZincoRESUMO
Mitogen-activated protein kinase (MAPK) signaling pathways are important regulators of cell growth, proliferation, and stress responsiveness. A family of dual-specificity MAP kinase phosphatases (MKPs) act as critical counteracting factors that directly regulate the magnitude and duration of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) activation. Here we show that constitutive expression of MKP-1 in cultured primary cardiomyocytes using adenovirus-mediated gene transfer blocked the activation of p38, JNK1/2, and ERK1/2 and prevented agonist-induced hypertrophy. Transgenic mice expressing physiological levels of MKP-1 in the heart showed (1) no activation of p38, JNK1/2, or ERK1/2; (2) diminished developmental myocardial growth; and (3) attenuated hypertrophy in response to aortic banding and catecholamine infusion. These results provide further evidence implicating MAPK signaling factors as obligate regulators of cardiac growth and hypertrophy and demonstrate the importance of dual-specificity phosphatases as counterbalancing regulatory factors in the heart.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cardiomegalia/enzimologia , Miocárdio/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , Adenoviridae/genética , Animais , Animais Recém-Nascidos , Fator Natriurético Atrial/efeitos dos fármacos , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/efeitos dos fármacos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Cardiomegalia/induzido quimicamente , Cardiomegalia/genética , Catecolaminas/farmacologia , Células Cultivadas , DNA Recombinante , Endotelina-1/farmacologia , Feminino , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Camundongos , Camundongos Transgênicos , Miocárdio/citologia , Miocárdio/metabolismo , Fenilefrina/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
The advent of conditional and tissue-specific recombination systems in gene-targeted or transgenic mice has permitted an assessment of single gene function in a temporally regulated and cell-specific manner. Here we generated transgenic mice expressing a tamoxifen-inducible Cre recombinase protein fused to two mutant estrogen-receptor ligand-binding domains (MerCreMer) under the control of the alpha-myosin heavy chain promoter. These transgenic mice were crossed with the ROSA26 lacZ-flox-targeted mice to examine Cre recombinase activity and the fidelity of the system. The data demonstrate essentially no Cre-mediated recombination in the embryonic, neonatal, or adult heart in the absence of inducing agent but >80% recombination after only four tamoxifen injections. Expression of the MerCreMer fusion protein within the adult heart did not affect cardiac performance, cellular architecture, or expression of hypertrophic marker genes, demonstrating that the transgene-encoded protein is relatively innocuous. In summary, MerCreMer transgenic mice represent a tool for temporally regulated inactivation of any loxP-targeted gene within the developing and adult heart or for specifically directing recombination and expression of a loxP-inactivated cardiac transgene in the heart.
Assuntos
Coração/embriologia , Integrases/genética , Miocárdio/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/farmacologia , Proteínas Virais/genética , Animais , Regulação da Expressão Gênica , Integrases/metabolismo , Cinética , Camundongos , Camundongos Transgênicos , Miocárdio/citologia , RNA Mensageiro/biossíntese , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão , Recombinação Genética , Transgenes , Proteínas Virais/metabolismoRESUMO
BACKGROUND: Left ventricular failure is commonly preceded by a period of hypertrophy. Intriguingly, many of the signaling pathways that have been implicated in the regulation of hypertrophy, including the 3 mitogen-activated protein kinases (MAPKs: extracellular signal-regulated kinase, stress-activated protein kinase, and p38), protein phosphatase, calcineurin, and the protein kinase Akt and its target glycogen synthase kinase-3 (GSK-3), also regulate the apoptotic response. METHODS AND RESULTS: To understand the mechanisms that might regulate the progression of heart failure, we analyzed the activity of these signaling pathways in the hearts of patients with advanced heart failure, patients with compensated cardiac hypertrophy, and normal subjects. In patients with hypertrophy, neither the MAPK nor the Akt/GSK-3 pathways were activated, and the dominant signaling pathway was calcineurin. In failing hearts, calcineurin activity was increased but less so than in the hypertrophied hearts, and all 3 MAPKs and Akt were activated (and, accordingly, GSK-3ss was inhibited), irrespective of whether the underlying diagnosis was ischemic or idiopathic cardiomyopathy. CONCLUSIONS: In the failing heart, there is a clear prohypertrophic activity profile, likely occurring in response to increased systolic wall stress and neurohormonal mediators. However, with the activation of these hypertrophic pathways, potent proapoptotic and antiapoptotic signals may also be generated. Therapies directed at altering the balance of activity of these signaling pathways could potentially alter the progression of heart failure.
Assuntos
Calcineurina/metabolismo , Cardiomegalia/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cardiomegalia/enzimologia , Feminino , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Quinase 9 Ativada por Mitógeno , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Cardiac-specific expression of an activated calcineurin protein in the hearts of transgenic (CLN) mice produces a profound hypertrophy that rapidly progresses to heart failure. While calcineurin is regulated by Ca2+, the potential effects of calcineurin on cardiac myocyte Ca2+ handling has not been evaluated. To this end, we examined L-type Ca2+ currents (I(Ca)) in left ventricular myocytes. CLN myocytes had larger (approximately 80%) cell capacitance and enhanced I(Ca) density (approximately 20%) compared with non-transgenic (NTG) littermates, but no change in the current-voltage relationship, single-channel conductance or protein levels of alpha 1 or beta 2 subunit of L-type Ca2+ channels. Interestingly, the kinetics of I(Ca) inactivation was faster (approximately two-fold) in CLN myocytes compared with NTG myocytes. Ryanodine application slowed the rate of I(Ca) inactivation in both groups and abolished the kinetic difference, suggesting that Ca2+ dependent inactivation is increased in CLN myocytes due to altered SR Ca2+ release. Treatment of CLN mice with Cyclosporine A (CsA), a calcineurin inhibitor, prevented myocyte hypertrophy and changes in I(Ca) activity and inactivation kinetics. However, there was no direct effect of CsA on I(Ca) in either NTG or CLN myocytes, suggesting that endogenous calcineurin activity does not directly regulate Ca2+ channel activity. This interpretation is consistent with the observation that I(Ca) density, inactivation kinetics and regulation by isoproterenol were normal in cardiac-specific transgenic mice expressing calcineurin inhibitory protein domains from either Cain or AKAP79. Taken together these data suggest that chronic activation of calcineurin is associated with myocyte hypertrophy and a secondary enhancement of intracellular Ca2+ handling that is tied to the hypertrophy response itself.
Assuntos
Calcineurina/metabolismo , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Cardiomegalia/metabolismo , Animais , Western Blotting , Inibidores de Calcineurina , Canais de Cálcio/genética , Ciclosporina/farmacologia , Eletrofisiologia , Isoproterenol/farmacologia , Cinética , Camundongos , Camundongos Transgênicos , Miocárdio/citologia , Miocárdio/metabolismo , Estrutura Terciária de Proteína , Rianodina/farmacologiaRESUMO
Members of the mitogen-activated protein kinase (MAPK) cascade such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 are implicated as important regulators of cardiomyocyte hypertrophic growth in culture. However, the role that individual MAPK pathways play in vivo has not been extensively evaluated. Here we generated nine transgenic mouse lines with cardiac-restricted expression of an activated MEK1 cDNA in the heart. MEK1 transgenic mice demonstrated concentric hypertrophy without signs of cardiomyopathy or lethality up to 12 months of age. MEK1 transgenic mice showed a dramatic increase in cardiac function, as measured by echocardiography and isolated working heart preparation, without signs of decompensation over time. MEK1 transgenic mice and MEK1 adenovirus-infected neonatal cardiomyocytes each demonstrated ERK1/2, but not p38 or JNK, activation. MEK1 transgenic mice and MEK1 adenovirus-infected cultured cardiomyocytes were also partially resistant to apoptotic stimuli. The results of the present study indicate that the MEK1-ERK1/2 signaling pathway stimulates a physiologic hypertrophy response associated with augmented cardiac function and partial resistance to apoptotsis.