RESUMO
Immortalized human chondrocytes were established by transfection of primary cultures of juvenile costal chondrocytes with vectors encoding simian virus 40 large T antigen and selection in suspension culture over agarose. Stable cell lines were generated that exhibited chondrocyte morphology, continuous proliferative capacity (> 80 passages) in monolayer culture in serum-containing medium, and expression of mRNAs encoding chondrocyte-specific collagens II, IX, and XI and proteoglycans in an insulin-containing serum substitute. They did not express type X collagen or versican mRNA. These cells synthesized and secreted extracellular matrix molecules that were reactive with monoclonal antibodies against type II collagen, large proteoglycan (PG-H, aggrecan), and chondroitin-4- and chondroitin-6-sulfate. Interleukin-1 beta (IL-1 beta) decreased the levels of type II collagen mRNA and increased the levels of mRNAs for collagenase, stromelysin, and immediate early genes (egr-1, c-fos, c-jun, and jun-B). These cell lines also expressed reporter gene constructs containing regulatory sequences (-577/+3,428 bp) of the type II collagen gene (COL2A1) in transient transfection experiments, and IL-1 beta suppressed this expression by 50-80%. These results show that immortalized human chondrocytes displaying cartilage-specific modulation by IL-1 beta can be used as a model for studying normal and pathological repair mechanisms.
Assuntos
Cartilagem/fisiologia , Linhagem Celular/fisiologia , Colágeno/biossíntese , Interleucina-1/farmacologia , Antígenos Virais de Tumores/isolamento & purificação , Cartilagem/citologia , Cartilagem/efeitos dos fármacos , Linhagem Celular/efeitos dos fármacos , Transformação Celular Viral , Sulfatos de Condroitina/isolamento & purificação , Colágeno/genética , Cicloeximida/farmacologia , Imunofluorescência , Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Fenótipo , Proteoglicanas/isolamento & purificação , RNA Mensageiro/análise , Costelas/citologia , Costelas/fisiologia , Vírus 40 dos Símios/genéticaRESUMO
Type II collagen is one of the predominant extracellular matrix macromolecules in cartilage responsible for maintenance of integrity of this specialized tissue. We showed previously that interleukin-1 (IL-1) and interferon-gamma (IFN-gamma) are capable of decreasing the levels of alpha 1(II) procollagen mRNA and suppressing the synthesis of type II collagen in cultured human chondrocytes. Data reported here show that these effects of IL-1 and IFN-gamma on the expression of the human type II collagen gene (COL2A1) are mediated primarily at the transcriptional level. This conclusion is based on three types of experimental evidence: (1) in nuclear run-off assays, preincubation of chondrocytes with either IL-1 or IFN-gamma decreased COL2A1 transcription; (2) experiments with the protein synthesis inhibitor cycloheximide and the transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) indicated that the suppression of alpha 1(II) procollagen mRNA by IL-1 could not be ascribed to decreased mRNA stability; and (3) a plasmid (pCAT-B/4.0) containing 4.0 kb of 5'-flanking sequences of COL2A1 (-577/+3428), encompassing the promoter, exon 1 and the putative enhancer sequence in the first intron, linked to the chloramphenicol acetyltransferase (CAT) reporter gene, was transfected in human chondrocytes. A high level of expression of pCAT-B/4.0 was observed in human chondrocytes incubated with an insulin-containing serum substitute that is permissive for expression of the COL2A1 gene. Expression of pCAT-B/4.0 in these cells was inhibited by either IL-1 or IFN-gamma. Furthermore, expression of pCAT-B/4.0 was not detected in human dermal fibroblasts. When the putative enhancer fragment in the first intron was removed, the expression in chondrocytes was greatly reduced. These studies demonstrate that expression of COL2A1 is tissue specific and that suppression by either IL-1 or IFN-gamma is mediated primarily at the transcriptional level.
Assuntos
Cartilagem/metabolismo , Colágeno/genética , Expressão Gênica , Interferon gama/farmacologia , Interleucina-1/farmacologia , Transcrição Gênica , Animais , Northern Blotting , Cicloeximida/farmacologia , Diclororribofuranosilbenzimidazol/farmacologia , Humanos , Plasmídeos , Pró-Colágeno/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/farmacologia , TransfecçãoRESUMO
Interleukin 1, a product predominantly of monocytes, increases the synthesis and release of procollagenase and prostaglandin E2 by mesenchymal target cells such as synovial fibroblasts and articular chondrocytes, an effect mimicked by some phorbol esters. In order to determine the mechanisms underlying these responses primary cultures of human articular chondrocytes were preincubated with recombinant human interleukin 1 beta or the phorbol ester, phorbol 12-myristate 13-acetate, in the presence or absence of the cyclooxygenase inhibitor, indomethacin. Interleukin 1 beta or phorbol ester increased the levels of procollagenase (assayed after trypsin activation) and the labeling of several medium proteins by cells incubated with [35S]methionine, independent of prostaglandin synthesis. The labeling of a 55 kD protein immunocomplexed with antibodies to procollagenase was also increased. The increased synthesis of procollagenase was paralleled by increased cellular levels of procollagenase mRNA, determined with a cDNA probe coding for human procollagenase. Thus the increased synthesis of procollagenase in response to the inflammatory mediator, interleukin 1, is controlled at a pretranslational level, possibly at the level of transcription.
Assuntos
Cartilagem Articular/enzimologia , Colagenases , Precursores Enzimáticos/biossíntese , Interleucina-1/farmacologia , Colagenase Microbiana/biossíntese , RNA Mensageiro/genética , Proteínas Recombinantes/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Células Cultivadas , Indução Enzimática , Precursores Enzimáticos/genética , Fibroblastos/enzimologia , Humanos , Indometacina/farmacologia , Colagenase Microbiana/genética , RNA Mensageiro/biossíntese , Transcrição Gênica/efeitos dos fármacosRESUMO
Collagenase (matrix metalloproteinase 1) cleaves type I, II, and III collagen helices at a specific site between Gly-Ile or Gly-Leu bonds (residues 775 and 776, P1-P1'). To understand the mechanism of collagen processing, mutations around the cleavage site have been introduced into the cloned murine pro alpha 1(I) collagen (Col1a1) gene. These mutant constructs have been transfected into homozygous Mov13 fibroblasts that do not express the endogenous Col1a1 gene due to a retroviral insertion. Secreted triple-helical type I collagens containing substitutions of Pro for Ile (position 776) (P1') were not cleaved by human rheumatoid synovial collagenase, whereas those containing substitutions of Met for Ile (position 776) were cleaved. Type I collagens containing double substitutions of Pro for Gln-774 (P2) and Ala-777 (P2') were not cleaved regardless of whether they contained the wild-type residue Ile at position 776 or the substitution of Met for Ile at position 776. The wild-type alpha 2(I) chains derived from the endogenous Col1a2 gene were also resistant to enzyme digestion when they were complexed with the mutant alpha 1(I) chains, indicating that the presence of normal alpha 1(I) sequences is critical for cleavage of the alpha 2(I) chains in the type I heterotrimer.
Assuntos
Colágeno/metabolismo , Genes , Colagenase Microbiana/metabolismo , Mutação , Pró-Colágeno/genética , Sequência de Aminoácidos , Animais , Artrite Reumatoide/enzimologia , Sequência de Bases , Células Cultivadas , Células Clonais , Fibroblastos/enzimologia , Humanos , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Mapeamento de Peptídeos , Conformação Proteica , Desnaturação Proteica , Mapeamento por Restrição , Especificidade por Substrato , Líquido Sinovial/enzimologia , TransfecçãoRESUMO
BACKGROUND: Tissues undergoing a chronic inflammatory process, such as the synovium in rheumatoid arthritis, are characterized by the infiltration of lymphocytes of different subsets and activation of monocyte/macrophages. Interleukin-1 (IL-1), a monocyte/ macrophage product that stimulates synovial fibroblasts to produce matrix metalloproteinases (MMPs), prostaglandins, and other cytokines, also has profound effects on the synthesis of extracellular matrix components such as type I collagen. In previous studies, we have shown that synovial fibroblasts and chondrocytes isolated from human joint tissues are particularly sensitive to prostaglandins, which modulate the effects of IL-1 on collagen gene expression in an autocrine manner. MATERIALS AND METHODS: BALBc/3T3 fibroblasts were treated with IL-1 and prostaglandins in the absence and presence of indomethacin to inhibit endogenous prostaglandin biosynthesis. Collagen synthesis was analyzed by SDS-PAGE as [3H]proline-labeled, secreted proteins, and prostaglandin production and cyclic adenosine 3',5'-cyclic monophosphate (camp) content were assayed. The expression of type I collagen gene (Col1a1) promoter-reporter gene constructs was examined in transient transfection experiments, and the binding of nuclear factors to the Col1a1 promoter region spanning -222 bp/+ 116 bp was analyzed by DNase I footprinting and electrophoretic mobility shift (EMSA) assays. RESULTS: IL-1 increased the synthesis of type I and type III collagens in BALBc/3T3 fibroblasts; greater increases were observed when IL-1-stimulated synthesis of PGE2 was blocked by indomethacin. Transient transfection experiments demonstrated dose-dependent inhibition of the-222 bp Col1a1 promoter by exogenously added prostaglandins with the order of potency of PGF2alpha > PGE2 > PGE1 DNase I footprinting showed increased protection, which extended from the region immediately upstream of the TATA box, owing to the binding of nuclear factors from PGE2- or PGE1-treated BALBc/3T3 cells. EMSA analysis showed zinc-dependent differences in the binding of nuclear factors from untreated and prostaglandin-treated cells to the -84 bp/-29 bp region of the Col1a1 promoter. CONCLUSIONS: These results show that the inhibition of Col1a1 expression by IL-1 in fibroblasts is mediated by prostaglandins at the transcriptional level and suggest that PGE-responsive factors may interact directly or indirectly with basal regulatory elements in the proximal promoter region of the Col1a1 gene.
Assuntos
Colágeno/genética , Prostaglandinas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Alprostadil/farmacologia , Animais , Células Cultivadas , DNA/genética , DNA/metabolismo , Pegada de DNA , Proteínas de Ligação a DNA/análise , Desoxirribonuclease I/metabolismo , Dinoprostona/biossíntese , Dinoprostona/farmacologia , Regulação para Baixo/efeitos dos fármacos , Fibroblastos , Genes Reporter/genética , Indometacina/farmacologia , Interleucina-1/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Misoprostol/farmacologia , Regiões Promotoras Genéticas/genética , Prostaglandinas/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
We previously identified the angiogenesis inhibitor angiostatin. Using a similar strategy, we have identified endostatin, an angiogenesis inhibitor produced by hemangioendothelioma. Endostatin is a 20 kDa C-terminal fragment of collagen XVIII. Endostatin specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth. By a novel method of sustained release, E. coli-derived endostatin was administered as a nonrefolded suspension. Primary tumors were regressed to dormant microscopic lesions. Immunohistochemistry revealed blocked angiogenesis accompanied by high proliferation balanced by apoptosis in tumor cells. There was no toxicity. Together with angiostatin data, these findings validate a strategy for identifying endogenous angiogenesis inhibitors, suggest a theme of fragments of proteins as angiogenesis inhibitors, and demonstrate dormancy therapy.