RESUMEN
The roles of insulin-like growth factors (IGFs) in regulating growth and their modulation by six IGF binding proteins (IGFBP) are well established. IGFBP-5, the most abundant IGFBP stored in bone, is an important regulator of bone formation via IGF-dependent and -independent mechanisms. Two new proteins, four and a half lim (FHL)-2, a transcription modulator that interacts with IGFBP-5, and a disintegrin and metalloprotease (ADAM)-9, an IGFBP-5 protease, have been identified as potential regulators of IGFBP-5 action in bone. We tested the hypothesis that agents which modulate bone formation by regulating IGFBP-5 expression would also regulate FHL-2 and ADAM-9 expression in a coordinated manner. We evaluated the expression of IGFBP-5, FHL-2, and ADAM-9 by real-time reverse transcriptase (RT)-PCR during differentiation of mouse bone marrow stromal cells into osteoblasts and in response to treatment with bone formation modulators in the LSaOS human osteosarcoma cell line. IGFBP-5 and FHL-2 increased 4.3- and 3.0-fold (P < or = 0.01), respectively, during osteoblast differentiation. Dexamethasone (Dex), an inhibitor of bone formation, decreased IGFBP-5 and FHL-2 and increased ADAM-9 in LSaOS cells (P < or = 0.05). Bone morphogenic protein (BMP)-7, a stimulator of bone formation, increased IGFBP-5 and decreased ADAM-9 (P<0.01). To determine if BMP-7 would eliminate Dex inhibition of IGFBP-5, cells were treated with Dex+BMP-7. The BMP-7-induced increase in IGFBP-5 was reduced, but not eliminated, in the presence of Dex (P < or = 0.01), indicating that BMP-7 and Dex may regulate IGFBP-5 via different mechanisms. Transforming growth factor (TGF)-beta, a stimulator of bone formation, increased IGFBP-5 and FHL-2 expression (P < or = 0.01). IGF-I and TNF-alpha decreased expression of ADAM-9 (P<0.05). In conclusion, our findings are consistent with the hypothesis that FHL-2 and ADAM-9 are important modulators of IGFBP-5 actions and are, in part, regulated in a coordinated manner in bone.
Asunto(s)
Proteínas ADAM/metabolismo , Desintegrinas/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Osteoblastos/metabolismo , Factores de Transcripción/metabolismo , Animales , Médula Ósea/metabolismo , Proteína Morfogenética Ósea 7 , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/farmacología , Diferenciación Celular , Humanos , Proteínas con Homeodominio LIM , Ratones , Células del Estroma/metabolismo , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
This study reports the molecular characterization of the nitrate-assimilation gene cluster from the opportunistic fungal pathogen Aspergillus fumigatus. A genomic fragment was isolated which contained the entire structural gene encoding nitrite reductase (niiA), plus segments of the nitrate reductase (niaD) and the nitrate transporter (crnA) genes. Nitrate-assimilation genes in A. fumigatus are physically linked and transcribed in the same direction as in A. nidulans. The nitrate-assimilation gene cluster is on the largest chromosome (5.3 Mb). The nitrite reductase (niiA) gene encodes a protein of 1110 amino acids that contains regions corresponding to FAD, NADPH, FeS and siroheme binding sites. Eight small introns interrupt the niiA open reading frame. The niaD-niiA intergenic regulatory region contains promoter consensus sequences including TATA, CAAT, and binding sites for the areA and nirA gene products. Northern analysis indicated that the expression of niaD, niiA and crnA are induced by nitrate and repressed by ammonium at the transcriptional level.
Asunto(s)
Proteínas de Transporte de Anión , Aspergillus fumigatus/genética , Mapeo Cromosómico , Proteínas Fúngicas , Genes Fúngicos/genética , Familia de Multigenes/genética , Nitrito Reductasas/genética , Secuencia de Aminoácidos , Aspergillus fumigatus/enzimología , Secuencia de Bases , Proteínas Portadoras/genética , Cromosomas Fúngicos/genética , Clonación Molecular , Regulación Fúngica de la Expresión Génica/fisiología , Intrones/genética , Datos de Secuencia Molecular , Nitrato-Reductasa , Nitrato Reductasas/genética , Transportadores de Nitrato , Nitratos/metabolismo , ARN de Hongos/análisis , ARN Mensajero/análisis , Secuencias Reguladoras de Ácidos Nucleicos/genética , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
In this paper, we report the cloning and sequencing of the C. elegans histidyl-tRNA synthetase gene. The complete genomic sequence, and most of the cDNA sequence, of this gene is now determined. The gene size including flanking and coding regions is 2230 nucleotides long. Three small introns (45-50 bp long) are found to interrupt the open reading frame. The open reading frame translates to 523 amino acids. This putative protein sequence shows extensive homology with the human and yeast histidyl-tRNA the histidyl-tRNA synthetase gene is a single copy gene. Hence, it is very likely that it encodes both the cytoplasmic and the mitochondrial histidyl-tRNA synthetases. It is likely to be trans-spliced since it contains a trans-splice site in its 5' untranslated region.