RESUMO
Exercise-induced angiogenesis is a key determinant of skeletal muscle function. Here, we investigated whether the E3 ubiquitin ligase murine double minute-2 (Mdm2) exerts a proangiogenic function in exercised skeletal muscle. Mdm2 hypomorphic (Mdm2(Puro/Δ7-9)) mice have a 60% reduction in Mdm2 expression compared with that in wild-type animals. Capillary staining on muscle sections from Mdm2(Puro/Δ7-9) sedentary mice with a wild-type or knockout background for p53 revealed that deficiency in Mdm2 resulted in 20% capillary regression independently of p53 status. In response to one bout of exercise, protein expression of the proangiogenic vascular endothelial growth factor-A (VEGF-A) was increased by 64% in muscle from wild-type animals, and endothelial cell outgrowth from exercised muscle biopsy samples cultured in a 3-dimensional collagen gel was enhanced by 37%. These proangiogenic responses to exercise were impaired in exercised Mdm2(Puro/Δ7-9) mice. Prolonged exercise training resulted in increased Mdm2 protein expression (+49%) and capillarization (+24%) in wild-type muscles. However, exercise training-induced angiogenesis was abolished in Mdm2(Puro/Δ7-9) mice. Finally, exercise training restored Mdm2, VEGF-A, and capillarization levels in skeletal muscles from obese Zucker diabetic fatty rats compared with those in healthy animals. Our results define Mdm2 as a crucial regulator of capillary maintenance and exercise-induced angiogenesis in skeletal muscle.
Assuntos
Regulação da Expressão Gênica/fisiologia , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica/fisiologia , Condicionamento Físico Animal/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Animais , Capilares , Feminino , Masculino , Camundongos , Músculo Esquelético/metabolismo , Neovascularização Fisiológica/genética , Obesidade/fisiopatologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Ratos , Ratos Sprague-Dawley , Ratos Zucker , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Tumor suppressor proteins must be exquisitely regulated since they can induce cell death while preventing cancer. For example, the p19(ARF) tumor suppressor (p14(ARF) in humans) appears to stimulate the apoptotic function of the p53 tumor suppressor to prevent lymphomagenesis and carcinogenesis induced by oncogene overexpression. Here we present a genetic approach to defining the role of p19(ARF) in regulating the apoptotic function of p53 in highly proliferating, homeostatic tissues. In contrast to our expectation, p19(ARF) did not activate the apoptotic function of p53 in lymphocytes or epithelial cells. These results demonstrate that the mechanisms that control p53 function during homeostasis differ from those that are critical for tumor suppression. Moreover, the Mdm2/p53/p19(ARF) pathway appears to exist only under very restricted conditions.
Assuntos
Homeostase/fisiologia , Proteínas Nucleares , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Peso Corporal/genética , Inibidor p16 de Quinase Dependente de Ciclina , Epitélio/metabolismo , Intestino Delgado/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p14ARF/genéticaRESUMO
The function of the p53 tumor suppressor protein must be highly regulated because p53 can cause cell death and prevent tumorigenesis. In cultured cells, the p90MDM2 protein blocks the transcriptional activation domain of p53 and also stimulates the degradation of p53. Here we provide the first conclusive demonstration that p90MDM2 constitutively regulates p53 activity in homeostatic tissues. Mice with a hypomorphic allele of mdm2 revealed a heretofore unknown role for mdm2 in lymphopoiesis and epithelial cell survival. Phenotypic analyses revealed that both the transcriptional activation and apoptotic functions of p53 were increased in these mice. However, the level of p53 protein was not coordinately increased, suggesting that p90MDM2 can inhibit the transcriptional activation and apoptotic functions of p53 in a manner independent of degradation. Cre-mediated deletion of mdm2 caused a greater accumulation of p53, demonstrating that p90MDM2 constitutively regulates both the activity and the level of p53 in homeostatic tissues. The observation that only a subset of tissues with activated p53 underwent apoptosis indicates that factors other than p90(MDM2) determine the physiological consequences of p53 activation. Furthermore, reduction of mdm2 in vivo resulted in radiosensitivity, highlighting the importance of mdm2 as a potential target for adjuvant cancer therapies.
Assuntos
Regulação Neoplásica da Expressão Gênica , Linfócitos/citologia , Proteínas Nucleares , Proteínas Proto-Oncogênicas/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Alelos , Animais , Apoptose , Northern Blotting , Western Blotting , Peso Corporal/genética , Morte Celular , Células Cultivadas , Relação Dose-Resposta à Radiação , Citometria de Fluxo , Deleção de Genes , Genótipo , Células-Tronco Hematopoéticas , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Genéticos , Testes de Precipitina , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-mdm2 , RNA Mensageiro/metabolismo , Fatores de Tempo , Distribuição Tecidual , Transcrição Gênica , Ativação Transcricional , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Murine double minute 2 (Mdm2) is a critical component of the responses to both ionizing and UV radiation. The level of Mdm2 expression determines the extent to which radiation induces an increase in the activity of the p53 tumor suppressor. Mdm2 acts as a survival factor in many cell types by limiting the apoptotic function of p53. In addition, expression of mdm2 is induced in response to DNA damage, and the resulting high levels of Mdm2 protein are thought to shorten the length of the cell cycle arrest established by p53 in the radiation response. Increased levels of Mdm2 appear to ensure that the activity of p53 returns to its low basal levels in surviving cells. Decreased levels of Mdm2 sensitize cells to ionizing radiation. Thus, Mdm2 is a potential target for therapeutic intervention because its inhibition may radiosensitize the subset of human tumors expressing wild-type p53 such that radiotherapy is more efficacious.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Neoplasias/radioterapia , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Animais , Apoptose , Ciclo Celular , Humanos , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Radiação Ionizante , Células Tumorais Cultivadas/efeitos da radiação , Proteína Supressora de Tumor p53/metabolismo , Raios UltravioletaRESUMO
Disruption of cerebellar granular neuronal precursor (GNP) maturation can result in defects in motor coordination and learning, or in medulloblastoma, the most common childhood brain tumor. The Sonic Hedgehog (Shh) pathway is important for GNP proliferation; however, the factors regulating the extent and timing of GNP proliferation, as well as GNP differentiation and migration are poorly understood. The p53 tumor suppressor has been shown to negatively regulate the activity of the Shh effector, Gli1, in neural stem cells; however, the contribution of p53 to the regulation of Shh signaling in GNPs during cerebellar development has not been determined. Here, we exploited a hypomorphic allele of Mdm2 (Mdm2(puro)), which encodes a critical negative regulator of p53, to alter the level of wild-type MDM2 and p53 in vivo. We report that mice with reduced levels of MDM2 and increased levels of p53 have small cerebella with shortened folia, reminiscent of deficient Shh signaling. Indeed, Shh signaling in Mdm2-deficient GNPs is attenuated, concomitant with decreased expression of the Shh transducers, Gli1 and Gli2. We also find that Shh stimulation of GNPs promotes MDM2 accumulation and enhances phosphorylation at serine 166, a modification known to increase MDM2-p53 binding. Significantly, loss of MDM2 in Ptch1(+/-) mice, a model for Shh-mediated human medulloblastoma, impedes cerebellar tumorigenesis. Together, these results place MDM2 at a major nexus between the p53 and Shh signaling pathways in GNPs, with key roles in cerebellar development, GNP survival, cerebellar foliation, and MB tumorigenesis.
Assuntos
Neoplasias Cerebelares/patologia , Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Proteínas Hedgehog/metabolismo , Lesões Pré-Cancerosas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Envelhecimento/patologia , Alelos , Animais , Apoptose , Proliferação de Células , Sobrevivência Celular , Neoplasias Cerebelares/metabolismo , Cerebelo/anormalidades , Cerebelo/patologia , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neurônios/metabolismo , Neurônios/patologia , Receptores Patched , Receptor Patched-1 , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de ZincoRESUMO
Exquisite control of the activity of p53 is necessary for mammalian survival. Too much p53 is lethal, whereas too little permits tumorigenesis. MDM2 and MDM4 are structurally related proteins critical for the control of p53 activity during development, homeostasis, and the response to stress. These two essential proteins regulate both the activation of p53 in response to stress and the recovery of cells following resolution of the damage, yet both are oncogenic when overexpressed. Thus, multiple regulatory circuits ensure that their activities are fine-tuned to promote tumor-free survival. Numerous diverse stressors activate p53, and much research has gone into trying to find commonalities between them that would explain the mechanism by which p53 becomes active. It is now clear that although these diverse stressors activate p53 by different biochemical pathways, one common feature is the effort they direct, through a variety of means, toward disrupting the functions of both MDM2 and MDM4. This article provides an overview of the relationship between MDM2 and MDM4, features the various biochemical mechanisms by which p53 is activated through inhibition of their functions, and proposes some emerging areas for investigation of the p53-mediated stress response.
Assuntos
Regulação da Expressão Gênica , Genes p53 , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Modelos Biológicos , Ribossomos/metabolismo , Raios UltravioletaRESUMO
The prevalence of mutations that inactivate the p53 tumor suppressor gene in human cancers reveals the importance of p53 in preventing cancer. Recent progress has generated increased enthusiasm for re-activating p53 in tumors with mutant p53 proteins as well as for increasing p53 function in tumors expressing wild-type p53 that is inhibited in trans. However, excessive p53 activity can be detrimental to the host, potentially limiting the utility of p53 activation as a therapeutic strategy. For example, uncontrolled p53 activity is lethal to the murine embryo, and p53 has been associated with increased aging in people and mice. Here we review the literature linking p53 to aging and discuss reports demonstrating that p53 can suppress tumor formation without accelerating aging. We raise the possibility that activation of p53 remains a promising strategy for cancer chemoprevention and therapy even if, under some circumstances, p53 might accelerate aging.
Assuntos
Envelhecimento/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Envelhecimento/genética , Senilidade Prematura/genética , Animais , Predisposição Genética para Doença , Humanos , Neoplasias/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismoRESUMO
The p53 inhibitor murine double-minute gene 2 (Mdm2) is a target for potential cancer therapies, however increased p53 function can be lethal. To directly address whether reduced Mdm2 function can inhibit tumorigenesis without causing detrimental side effects, we exploited a hypomorphic murine allele of mdm2 to compare the effects of decreased levels of Mdm2 and hence increased p53 activity on tumorigenesis and life span in mice. Here we report that mice with decreased levels of Mdm2 are resistant to tumor formation yet do not age prematurely, supporting the notion that Mdm2 is a promising target for cancer therapeutics.