RESUMO
Space travelers experience anorexia and body weight loss in a microgravity environment, and microgravity-like situations cause changes in hypothalamic activity. Hypothalamic melanocortins play a critical role in the regulation of metabolism. Therefore, we hypothesized that microgravity affects metabolism through alterations in specific hypothalamic signaling pathways, including melanocortin signaling. To address this hypothesis, the microgravity-like situation was produced by an antiorthostatic tail suspension in wild-type and agouti mice, and the effect of tail suspension on energy expenditure and hypothalamic gene expression was examined. Energy expenditure was measured using indirect calorimetry before and during the tail suspension protocol. Hypothalamic tissues were collected for gene expression analysis at the end of the 3 h tail suspension period. Tail suspension significantly increased oxygen consumption, carbon dioxide production, and heat production in wild-type mice. Tail suspension-induced increases in energy expenditure were not attenuated in agouti mice. Although tail suspension did not alter hypothalamic proopiomelanocortin (POMC) and agouti-related protein (AGRP) mRNA levels, it significantly increased hypothalamic interleukin 6 (Il-6) mRNA levels. These data are consistent with the hypothesis that microgravity increases energy expenditure and suggest that these effects are mediated through hypothalamic signaling pathways that are independent of melanocortins, but possibly used by Il-6.
Assuntos
Metabolismo Energético/fisiologia , Elevação dos Membros Posteriores/fisiologia , Melanocortinas/biossíntese , Tecido Adiposo Marrom/metabolismo , Proteína Relacionada com Agouti/metabolismo , Animais , Análise Química do Sangue , Western Blotting , Expressão Gênica/fisiologia , Hormônios/sangue , Hipotálamo/fisiologia , Interleucina-6/biossíntese , Cinética , Masculino , Melanocortinas/genética , Metabolismo/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Pró-Opiomelanocortina/metabolismo , RNA/biossíntese , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Ausência de PesoRESUMO
IGF binding protein (IGFBP)-3 inhibits cell growth and promotes apoptosis by sequestering free IGFs. In addition IGFBP-3 has IGF-independent, proapoptotic, antiproliferative effects on prostate cancer cells in vitro. Expression of the large T-antigen (Tag) under the long probasin promoter (LPB) in LPB-Tag mice results in prostate tumorigenesis. To investigate the IGF-dependent and IGF-independent effects of IGFBP-3 on prostate tumor growth, we crossed LPB-Tag mice with cytomegalovirus (CMVBP-3) and phosphoglycerate kinase (PGKBP-3) mice that overexpress IGFBP-3 under the cytomegalovirus promoter and the phosphoglycerate kinase promoter, respectively, and also I56G/L80G/L81G-mutant IGFBP-3 (PGKmBP-3) mice that express I56G/L80G/L81G-IGFBP-3, a mutant, that does not bind IGF-I but retains IGF-independent proapoptotic effects in vitro. Prostate tumor size and the steady-state level of p53 were attenuated in LPB-Tag/CMVBP-3 and LPB-Tag/PGKBP-3 mice, compared with LPB-Tag/wild-type (Wt) mice. A more marked effect was observed in LPB-Tag/CMVBP-3, compared with LPB-Tag/PGKBP-3, reflecting increased levels of transgene expression in CMVBP-3 prostate tissue. No attenuation of tumor growth was observed in LPB-Tag/PGKmBP-3 mice during the early tumor development, indicating that the inhibitory effects of IGFBP-3 were most likely IGF dependent during the initiation of tumorigenesis. At 15 wk of age, epidermal growth factor receptor expression was increased in LPB-Tag/Wt and LPB-Tag/PGKmBP-3 tissue, compared with LPB-Tag/PGKBP-3. IGF receptor was increased in all transgenic mice, but pAkt expression, a marker of downstream IGF-I action, was increased only in LPB-Tag/Wt and LPB-Tag/PGKmBP-3. After 15 wk of age, a marked reduction in tumor growth was apparent in LPB-Tag/PGKmBP-3 mice, indicating that the IGF-independent effects of IGFBP-3 may be important in inhibiting tumor progression.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/fisiologia , Fator de Crescimento Insulin-Like I/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Animais , Apoptose , Proliferação de Células , Cruzamentos Genéticos , Citomegalovirus/metabolismo , Progressão da Doença , Receptores ErbB/metabolismo , Feminino , Genótipo , Immunoblotting , Imuno-Histoquímica , Ligantes , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Fenótipo , Fosfoglicerato Quinase/metabolismo , Regiões Promotoras Genéticas , Neoplasias da Próstata/terapia , RNA/metabolismo , RNA Mensageiro/metabolismo , Análise de Regressão , Ribonucleases/metabolismo , Fatores de Tempo , Transgenes , Regulação para CimaRESUMO
The human GH family consists of five genes, including the placental chorionic somatomammotropins (CS), within a single locus on chromosome 17. Based on nuclease sensitivity, the entire GH/CS locus is accessible in pituitary chromatin, yet only GH-N is expressed. Previously, we reported a P sequence element (263P) capable of repressing placental CS promoter activity in transfected pituitary (GC) cells. Regions of protein binding within 263P include P sequence elements A and B (PSE-A and PSE-B), and we reported nuclear factor-1 (NF-1) recognition of PSE-B. We now provide evidence for multiple interactions on PSE-A, including binding of the regulatory factor X (RFX) family. Disruption of the RFX site within 263P blunts repressor activity in transfected GC cells; however, repression is only abolished when both PSE-A/RFX and PSE-B/NF-1 sites are mutated. The capacity of RFX and NF-1 to participate in a novel common complex is further suggested by coimmunoprecipitation of RFX1 and epitope-tagged NF-1 family members. Finally, we confirm the association of NF-1 and RFX1 with P sequences in human pituitary tissue by chromatin immunoprecipitation. Taken together, our data suggest that an inverse relationship exists between 263P and CS promoter histone hyperacetylation and the association of these factors in vivo.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Hormônio do Crescimento Humano/genética , Hipófise/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Fatores de Transcrição NFI , Proteínas Nucleares , Sondas de Oligonucleotídeos , Hormônios Placentários/genética , Hormônios Placentários/metabolismo , Regiões Promotoras Genéticas/fisiologia , Ratos , Fatores de Transcrição de Fator Regulador X , Fator Regulador X1 , Proteína 1 de Ligação a Y-BoxRESUMO
OBJECT: One of the current challenges in neurobiology is to ensure that neural precursor cells differentiate into specific neuron types, so that they can be used for transplantation purposes in patients with neuron loss. The goal of this study was to determine if spinal cord precursor cells could differentiate into motor neurons both in culture and following transplantation into a transected sciatic nerve. METHODS: In cultures with trophic factors, neurons differentiate from embryonic precursor cells and express motor neuronal markers such as choline acetyltransferase (ChAT), Islet-1, and REG2. Reverse transcription-polymerase chain reaction analysis has also demonstrated the expression of Islet-1 in differentiated cultures. A coculture preparation of neurospheres and skeletal myocytes was used to show the formation of neuromuscular connections between precursor cell-derived neurons and myocytes both immunohistochemically and electrophysiologically. Following various survival intervals, precursor cells transplanted distal to a transection of the sciatic nerve differentiated into neurons expressing the motor neuron markers ChAT and the alpha1 1.2 (class C, L-type) voltage-sensitive Ca++ channel subunit. These cells extended axons into the muscle, where they formed cholinergic terminals. CONCLUSIONS: These results demonstrate that motor neurons can differentiate from spinal cord neural precursor cells grown in culture as well as following transplantation into a transected peripheral nerve.