RESUMO
Osteoclast inhibitory peptide 2 (OIP-2) is a novel autocrine/paracrine factor produced by osteoclasts (OCLs) that inhibits bone resorption and OCL formation in vitro and in vivo. It is identical to the asparaginyl endopeptidase legumain. During maturation of OIP-2, a signal peptide and a 17-kDa C-terminal fragment (CTF) are cleaved to produce the mature enzyme. To determine if enzyme activity is required for inhibition of OCL formation or if only the CTF is responsible for these effects, we synthesized His-tagged complementary DNA (cDNA) constructs for the CTF of OIP-2, the proform of OIP-2, and the "mature enzyme" form of OIP-2. The proform or the CTF portion of OIP-2 inhibited OCL formation in a dose-dependent manner in murine bone marrow cultures stimulated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The mature form of OIP-2, which was enzymatically active, did not inhibit OCL formation. In addition, OIP-2 inhibited OCL formation in cultures of highly purified human OCL precursor cells or RAW264.7 cells stimulated with 10 ng/ml of receptor activator of NF-kappaB (RANK) ligand. Binding studies with His-tagged OIP-2 showed expression of a putative OIP-2 receptor on RAW264.7 cells treated with RANK ligand for 4 days and human marrow cultures treated with 1,25(OH)2D3 for 3 weeks. These data show that the CTF of OIP-2, rather than the mature enzyme, mediates the inhibitory effects of OIP-2 through a putative receptor on OCL precursors.
Assuntos
Cisteína Endopeptidases/metabolismo , Osteoclastos/citologia , Proteínas de Plantas , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Reabsorção Óssea , Calcitriol/farmacologia , Proteínas de Transporte/genética , Cisteína Endopeptidases/genética , Feminino , Glicoproteínas/genética , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Mutagênese , Oligopeptídeos/genética , Osteoprotegerina , Peptídeos/genética , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Ligante RANK , Ratos , Receptor Ativador de Fator Nuclear kappa-B , Receptores Citoplasmáticos e Nucleares/genética , Receptores do Fator de Necrose TumoralRESUMO
Chorion hardening is triggered by the contents of cortical alveoli that are released upon fertilization of medaka (Oryzias latipes) eggs. We purified the chorion hardening-inducing activity as a single protein from the exudate of cortical alveoli of medaka eggs. This activity was co-purified with proteolytic activity of the chorion protein ZI-1,2. Based on the amino acid sequence of purified protein, we cloned the cDNA of this protein from a medaka ovarian cDNA library. Sequence analyses revealed typical sequence features, a zinc-binding motif and a methionine turn motif, of the astacin metalloproteinase family. We termed this protein "alveolin." Alveolin has a molecular mass of 21.5 kDa deduced by the amino acid sequence and neutral optimal pH range. Alveolin hydrolyzes ZI-1,2. Alveolin activity was strongly inhibited by metal-chelating agents but not by various proteinase inhibitors. To our knowledge, this is the first description of the isolation and identification of the chorion hardening-inducing factor from cortical alveoli exudate of teleost eggs.
Assuntos
Córion/fisiologia , Fertilização/fisiologia , Metaloendopeptidases/fisiologia , Oryzias/genética , Óvulo/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Metaloendopeptidases/genética , Metaloendopeptidases/isolamento & purificação , Dados de Sequência Molecular , Família Multigênica , Homologia de Sequência de AminoácidosRESUMO
Our previous findings suggest the activity of cytochrome P-450 aromatase (P-450arom), the enzyme which converts testosterone to estradiol-17beta, in the ovarian follicle of medaka (Oryzias latipes) is regulated at the transcriptional level. In this study, we cloned a cDNA encoding a FTZ-F1-like protein (mdFtz-F1) from ovarian follicles of medaka. In vitro translated mdFTZ-F1, and nuclear extract from medaka ovarian follicles, formed complexes with oligonucleotide probes containing putative orphan nuclear receptor binding motifs, which are present in the promoter region of the medaka P-450arom gene. The expression pattern of mdFtz-F1 transcripts during oogenesis coincides with that of P-450arom transcripts. Transfection assays further suggest a potential transcriptional regulatory activity of mdFTZ-F1 upon the medaka P-450arom promoter. Taken together, these results suggest a potential role of mdFTZ-F1 in the transcriptional regulation of P-450arom in the ovarian follicle of medaka.