RESUMO
OBJECTIVE: Maintenance of chondrocyte phenotype is a major issue in prevention of degeneration and repair of articular cartilage. Although the critical pathways in chondrocyte maturation and homeostasis have been revealed, the in-depth understanding is deficient and novel modifying components and interaction partners are still likely to be discovered. Our focus in this study was to characterize a novel cartilage specific gene that was identified in mouse limb cartilage during embryonic development. METHODS: Open access bioinformatics tools and databases were used to characterize the gene, predicted protein and orthologs in vertebrate species. Immunohistochemistry and mRNA expression methodology were used to study tissue specific expression. Fracture callus and limb bud micromass culture were utilized to study the effects of BMP-2 during experimental chondrogenesis. Fusion protein with C-terminal HA-tag was expressed in Cos7 cells, and the cell lysate was studied for putative glycosaminoglycan attachment by digestion with chondroitinase ABC and Western blotting. RESULTS: The predicted molecule is a small, 121 amino acids long type I single-pass transmembrane chondroitin sulfate proteoglycan, that contains ER signal peptide, lumenal/extracellular domain with several threonines/serines prone to O-N-acetylgalactosamine modification, and a cytoplasmic tail with a Yin-Yang site prone to phosphorylation or O-N-acetylglucosamine modification. It is highly conserved in mammals with orthologs in all vertebrate subgroups. Cartilage specific expression was highest in proliferating and prehypertrophic zones during development, and in adult articular cartilage, expression was restricted to the uncalcified zone, including chondrocyte clusters in human osteoarthritic cartilage. Studies with experimental chondrogenesis models demonstrated similar expression profiles with Sox9, Acan and Col2a1 and up-regulation by BMP-2. Based on its cartilage specific expression, the molecule was named Snorc, (Small NOvel Rich in Cartilage). CONCLUSION: A novel cartilage specific molecule was identified which marks the differentiating chondrocytes and adult articular chondrocytes with possible functions associated with development and maintenance of chondrocyte phenotype.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Cartilagem Articular/metabolismo , Diferenciação Celular , Condrócitos/metabolismo , Condrogênese/genética , Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Idoso , Animais , Cartilagem Articular/embriologia , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Colágeno Tipo II/metabolismo , Membro Posterior/embriologia , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteoglicanas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
1,25-Dihydroxyvitamin D3 (calcitriol), transforming growth factor-beta (TGF-beta), and insulin-like growth factors (IGFs) are all important bone regulatory factors known to affect proliferation and differentiation of human bone-forming cells (osteoblasts). We have previously shown that TGF-beta1 increased IGF-I and IGF-binding protein (IGFBP)-3 production in human bone marrow stromal (hMS) osteoblast progenitors and calcitriol stimulated IGFBP-3 and IGFBP-4 production. As interaction between signaling pathways of these factors has been reported, the present study aimed at examining the concerted actions on components of the IGF-system. We report that co-treatment with TGF-beta1 and calcitriol resulted in a synergistic increase in IGFBP-3 production, thereby suggesting that the effects of these factors on hMS osteoblast differentiation may involve the observed increase in IGFBP-3.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Calcitriol/farmacologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/biossíntese , Fator de Crescimento Transformador beta/farmacologia , Adulto , Western Blotting , DNA Complementar/química , Sinergismo Farmacológico , Humanos , Fator de Crescimento Insulin-Like I/biossíntese , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like II/biossíntese , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismoRESUMO
Two new flavones were isolated from the aerial parts of Retama raetam subsp. raetam. Their structures were established as luteolin 4'-O-neohesperidoside (1) and 5,4'-dihydroxy-(3",4"-dihydro-3", 4"-dihydroxy)-2",2"-dimethylpyrano-(5",6":7,8)-flavone (2) by means of spectroscopic methods. Also present was ephedroidin (4',5, 7-trihydroxy-8-(2-hydroxy-3-butenyl)-flavone).
Assuntos
Flavonoides/química , Extratos Vegetais/química , Plantas Medicinais/química , Humanos , LuteolinaRESUMO
Cellular senescence and limited proliferative capacity of normal diploid cells has a dominant phenotype over immortality of cancerous cells, suggesting its regulation by the expression of a set of genes. In order to isolate the genes that associate with senescence, we have employed a clonal system of conditional SV40 T antigen rat embryo fibroblast cell lines which undergo senescence upon T antigen inactivation. Construction of cDNA libraries from two conditional cell lines and application of differential screening and subtractive hybridization techniques have resulted in the cloning of eight senescence-induced genes (SGP-2/Apo J, alpha 1-procollagen, osteonectin, fibronectin, SM22, cytochrome C oxidase, GTP-alpha, and a novel gene) and a senescence-repressed gene (FRS-2). Three of these genes encode for extracellular matrix proteins, others are involved in the calcium-dependent signal transduction pathways, while the SGP-2/Apo J gene may have a cellular protective function. RNA analysis has shown that the senescence-associated genes are overexpressed in both normal rat embryonic fibroblasts and human osteoblasts cell cultures undergoing aging in vitro. In comparison, the expression of these genes in a rat fibroblast immortalized cell line (208F cells) was down-regulated after both its partial and its full transformation by ras oncogenes. Thus, cloning of senescence-associated genes opens up new ways to elucidate and/or to modulate aging and cancer.