RESUMEN
Osteoarthritis (OA) is the most prevalent joint disease and is characterized by pain and functional loss of the joint. However, the pathogenic mechanism of OA remains unclear, and no drug therapy for preventing its progress has been established. To identify genes related to the progress of OA, the gene expression profiles of paired intact and damaged cartilage obtained from OA patients undergoing joint substitution were compared using oligo microarrays. Using functional categorization combined with gene ontology and a statistical analysis, five genes were found to be highly expressed in damaged cartilage (HBEGF, ASUS, CRLF1, LOX, CDA), whereas three genes were highly expressed in intact tissues (CHST2, PTPRD, CPAN6). Among these genes, the upregulated expression of CRLF1 was reconfirmed using real-time PCR, and the in vivo expression of CRLF1 was detected in clusters of chondrocytes and fibrocartilage-like cells in damaged OA cartilages using in situ hybridization. In vitro, the transcriptional level of CRLF1 was positively regulated by TGF-beta1 in the mouse chondrogenic cell line ATDC5. Additionally, the CRLF1/CLC complex promoted the proliferation of ATDC5 cells and suppressed the expression level of aggrecan and type II collagen. Our data suggest that the CRLF1/CLC complex disrupts cartilage homeostasis and promotes the progress of OA by enhancing the proliferation of chondrocytes and suppressing the production of cartilage matrix. A component of the complex, CRLF1, may be useful as a biomarker of OA; and the corresponding receptor is a potential new drug target for OA.
Asunto(s)
Cartílago Articular/metabolismo , Articulación de la Rodilla/metabolismo , Osteoartritis de la Rodilla/metabolismo , Receptores de Citocinas/genética , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/genética , Agrecanos/metabolismo , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Cartílago Articular/inmunología , Cartílago Articular/fisiopatología , Línea Celular , Proliferación Celular , Condrocitos/metabolismo , Colágeno Tipo II/metabolismo , Proteínas de la Matriz Extracelular/genética , Regulación de la Expresión Génica/fisiología , Humanos , Articulación de la Rodilla/inmunología , Articulación de la Rodilla/fisiopatología , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoartritis de la Rodilla/inmunología , Osteoartritis de la Rodilla/fisiopatología , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Activación Transcripcional/genética , Regulación hacia Arriba/genéticaRESUMEN
There is increasing evidence that calpain contributes to the reorganization of the cytoskeleton in the integrin-mediated signaling pathway. Osteoclastic bone resorption requires cell-matrix contact, an event mediated by integrin alphavbeta3, and subsequent cytoskeletal reorganization to form characteristic membrane domains such as the sealing zone and ruffled border. In this study, therefore, we investigated whether calpain is involved in osteoclastic bone resorption. Membrane-permeable calpain inhibitors suppress the resorption activity of human osteoclasts, but an impermeable inhibitor does not. Upon the attachment of osteoclasts to bone, micro-calpain is translocated from the cytosolic to the cytoskeletal fraction and is autolytically activated. Both the activation of micro-calpain and the formation of actin-rings, the cytoskeletal structures essential for bone resorption, are inhibited by membrane-permeable calpain inhibitors. The activated micro-calpain in osteoclasts selectively cleaves talin, which links the matrix-recognizing integrin to the actin cytoskeleton. These findings suggest that calpain is a regulator of the bone resorption activity of osteoclasts through reorganization of the cytoskeleton related to actin-ring formation.
Asunto(s)
Resorción Ósea/fisiopatología , Calpaína/fisiología , Osteoclastos/enzimología , Osteoclastos/fisiología , Actinas/metabolismo , Anciano , Huesos/fisiología , Calpaína/antagonistas & inhibidores , Células Cultivadas , Dipéptidos/farmacología , Humanos , Leucina/análogos & derivados , Leucina/farmacología , Proteínas Recombinantes de Fusión/farmacología , Talina/metabolismoRESUMEN
OBJECTIVE: To clarify the regulation of osteoprotegerin (OPG) expression in rheumatoid synovial fibroblasts by investigating the effect of tumor necrosis factor-alpha (TNF-alpha) and the mechanism of TNF-alpha-induced OPG expression. METHODS: OPG expression was examined by Northern blot hybridization and reverse transcriptase-polymerase chain reaction in synovial fibroblasts from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and subjects with no inflammatory condition. Amounts of OPG in conditioned medium were determined by ELISA. The effect of OPG on TNF-alpha-induced osteoclastogenesis was investigated in primary cultures of RA synovial cells. RESULTS: OPG was highly expressed in RA synovial fibroblasts compared to OA and noninflammatory synovial fibroblasts. Different levels of OPG expression were found among patients with RA. TNF-alpha induced OPG expression in all synovial fibroblasts, even OA and noninflammatory fibroblasts, and expression occurred to a remarkable degree in RA fibroblasts. The OPG expression was upregulated by TNF-alpha in a time- and dose-dependent manner. TNF-alpha-induced OPG expression was inhibited by hymenialdisine, a nuclear factor-kappaB inhibitor, in a dose-dependent manner, and expression was inhibited by soluble TNF receptor/Fc fusion protein I (TNFs-RI/Fc), not by TNFs-RII/Fc. In contrast, TNF-alpha-induced osteoclastogenesis in primary cultures of RA synovial cells was inhibited by the addition of OPG. CONCLUSION: These results suggest that OPG is highly expressed and is upregulated by TNF-alpha in rheumatoid synovial fibroblasts. TNF-alpha-induced OPG expression is mediated predominantly through TNF-RI. Although TNF-alpha is known to stimulate bone destruction, TNF-alpha-induced upregulation of OPG may contribute to self-protection from the bone destruction in RA.
Asunto(s)
Artritis Reumatoide/metabolismo , Glicoproteínas/biosíntesis , Receptores Citoplasmáticos y Nucleares/biosíntesis , Membrana Sinovial/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Adulto , Anciano , Antígenos CD/fisiología , Artritis Reumatoide/patología , Calcitriol/farmacología , Proteínas Portadoras/fisiología , Células Cultivadas , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Glicoproteínas de Membrana/fisiología , Persona de Mediana Edad , Osteoartritis de la Rodilla , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoprotegerina , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Receptores del Factor de Necrosis Tumoral/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral , Receptores Tipo II del Factor de Necrosis Tumoral , Membrana Sinovial/metabolismoRESUMEN
Recently, a G/C polymorphism was found at position -573 of the interleukin-6 ( IL-6) gene promoter. We investigated how this genetic polymorphism relates to IL-6 production and osteoporosis in elderly Japanese women. Genomic DNA was extracted from an aliquot of monocytes in the bone marrow; the monocytes were simultaneously used to form osteoclast-like multinucleated cells (MNCs) and to produce IL-6. Of the 47 subjects with fractures, 96% had a C allele at position -573 of the IL-6 gene. Only 2 subjects possessed homozygotes of G at that position. We investigated IL-6 levels, MNC formation in bone marrow culture, and femoral neck bone mineral density (BMD) in the subjects with the GC and CC genotypes. There were no significant differences between these genotypes as regards IL-6 levels, MNC formation, and femoral neck BMD. However, in the CC genotype, there was a negative relationship between femoral neck BMD and IL-6 levels, and between femoral neck BMD and MNC formation, whereas in the GC genotype and combined (GC + CC) genotypes, femoral neck BMD tended to be related to IL-6 levels and MNC formation. Moreover, the stromal cells in the CC genotype showed higher IL-1alpha-stimulated IL-6 production than did the stromal cells in the GC genotype. Our findings suggest that important information might be obtained not only by continued comparison of different genotypes but also by comparative study within each particular genotype.
Asunto(s)
Fracturas de Cadera/genética , Interleucina-6/genética , Osteoporosis Posmenopáusica/genética , Polimorfismo de Nucleótido Simple , Anciano , Anciano de 80 o más Años , Pueblo Asiatico , Densidad Ósea/genética , Células Cultivadas , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Femenino , Fémur/fisiología , Predisposición Genética a la Enfermedad , Humanos , Japón , Monocitos/citología , Osteoclastos/citología , Osteoclastos/fisiología , Polimorfismo de Longitud del Fragmento de Restricción , Regiones Promotoras GenéticasRESUMEN
In the present study, we demonstrate for the first time that beta-adrenergic agonists stimulate bone-resorbing activity in human osteoclast-like multinucleated cells (MNCs). Osteoclast-like MNCs constitutively expressed mRNA for alpha1B-, alpha2B- and beta2-adrenergic receptor (AR) in addition to characteristic markers of mature osteoclast, such as calcitonin receptor (CT-R), tartrate-resistant acid phosphatase (TRAP), alphaV-chain of integrin (Int alphaV), carbonic anhydrase II (CA-II) and cathepsin K (Cathe K). Epinephrine (1 microM; alpha,beta-adrenergic agonist) up-regulated expression of Int alphaV, CA-II and Cathe K in the osteoclast-like MNCs. Osteoclastic resorbing activity was markedly increased by isoprenaline (1 microM; beta-adrenergic agonist), moderately by epinephrine, but poorly by phenylephrine (1 microM; alpha1-adrenergic agonist). The actin ring, which was suggested to be correlated with bone-resorbing activity, was clearly observed in osteoclast-like MNCs treated with isoprenaline and epinephrine, but faintly in those treated with phenylephrine. These findings suggest that beta-adrenergic agonists directly stimulate bone-resorbing activity in matured osteoclasts.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Resorción Ósea/inducido químicamente , Osteoclastos/efectos de los fármacos , Células Cultivadas , Epinefrina/farmacología , Humanos , Isoproterenol/farmacología , Osteoclastos/fisiología , Fenilefrina/farmacología , ARN Mensajero/análisis , Receptores Adrenérgicos/genéticaRESUMEN
The most reliable explanation for decreasing bone mass in elderly women is an imbalance of osteoclastic resorption and osteoblastic formation resulting from a relative increase in osteoclastic resorption. However, it is not clear whether an increase in osteoclastic bone resorption with age is due to increased osteoclast formation or to osteoclastic bone resorption activity. In this study, using a human bone marrow culture system, we attempt to clarify the increase in osteoclast formation with age. The mononuclear cell-rich fraction from bone marrow, obtained from the proximal region of the femur from female elderly patients with fracture, were cultured for 14 days in the presence of 1,25 dihydroxyvitamin D(3). Tartrate-resistant acid phosphatase-positive multinucleated cells were counted as osteoclasts. In our investigation, human osteoclast formation in the bone marrow culture increased with age in elderly women (age 64-96 years). The osteoclast formation was positively correlated with macrophage-colony stimulation factor and prostaglandin E(2) production in bone marrow culture. Also, osteoclast formation ex vivo was negatively correlated with bone mineral density of the lumbar spine (L2-L4). The above results indicate that the osteoclastogenic potential of bone marrow cells increases with aging in elderly women with fracture, and suggest that a decrease in bone mass of elderly women may be due to an increase in osteoclast population associated with aging.
Asunto(s)
Envejecimiento/fisiología , Células de la Médula Ósea/fisiología , Osteoclastos/fisiología , Factores de Edad , Anciano , Anciano de 80 o más Años , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Calcitriol/farmacología , Diferenciación Celular , Células Cultivadas , Dinoprostona/biosíntesis , Dinoprostona/farmacología , Femenino , Fracturas del Fémur , Humanos , Interleucina-1/farmacología , Interleucina-6/biosíntesis , Interleucina-6/farmacología , Factor Estimulante de Colonias de Macrófagos/biosíntesis , Factor Estimulante de Colonias de Macrófagos/farmacología , Persona de Mediana Edad , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaAsunto(s)
Técnicas de Cultivo de Célula/métodos , Osteoclastos/citología , Animales , Células de la Médula Ósea/citología , Diferenciación Celular , Glicoproteínas/fisiología , Células Madre Hematopoyéticas/citología , Humanos , Osteoblastos/citología , Osteocitos/citología , Osteoprotegerina , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores del Factor de Necrosis TumoralRESUMEN
1. We examined the effects of endogenous prostaglandin E(2) (PGE(2)) on the production of interleukin-6 (IL-6), macrophage colony stimulating factor (M-CSF), and vascular endothelial growth factor (VEGF) by interleukin-1beta (IL-1beta)-stimulated human synovial fibroblasts. 2. NS-398 (1 microM), a cyclo-oxygenase-2 (COX-2) inhibitor, inhibited IL-6 and VEGF production (35+/-4% and 26+/-2%, respectively) but enhanced M-CSF production (38+/-4%) by IL-1beta (1 ng ml(-1)) in synovial fibroblasts isolated from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Exogenous PGE(2) completely abolished the effects of NS-398 on the production of each mediator by OA fibroblasts stimulated with IL-1beta. 3. 8-Bromo cyclic AMP and dibutyryl cyclic AMP, cyclic AMP analogues, mimicked the effects of PGE(2) on IL-6, M-CSF, and VEGF production by OA fibroblasts. 4. The EP(2) selective receptor agonist ONO-AE1-259 (2 nM) and the EP(4) selective receptor agonist ONO-AE1-329 (2 or 20 nM), but not the EP(1) selective receptor agonist ONO-DI-004 (1 microM) and the EP(3) selective receptor agonist ONO-AE-248 (1 microM), replaced the effects of PGE(2) on IL-6, M-CSF, and VEGF production by OA and RA fibroblasts stimulated with IL-1beta in the presence of NS-398. 5. Both OA and RA fibroblasts expressed mRNA encoding EP(2) and EP(4) but not EP(1) receptors. In addition, up-regulation of EP(2) and EP(4) receptor mRNAs was observed at 3 h after IL-1beta treatment. 6. These results suggest that endogenous PGE(2) regulates the production of IL-6, M-CSF, and VEGF by IL-1beta-stimulated human synovial fibroblasts through the activation of EP(2) and EP(4) receptors with increase in cyclic AMP.
Asunto(s)
Dinoprostona/fisiología , Factores de Crecimiento Endotelial/biosíntesis , Fibroblastos/metabolismo , Interleucina-6/biosíntesis , Linfocinas/biosíntesis , Factor Estimulante de Colonias de Macrófagos/biosíntesis , Membrana Sinovial/metabolismo , Células Cultivadas , Dinoprostona/farmacología , Fibroblastos/fisiología , Humanos , Interleucina-1/farmacología , Receptores de Prostaglandina E/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E , Subtipo EP4 de Receptores de Prostaglandina E , Membrana Sinovial/citología , Membrana Sinovial/fisiología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
A transient increase in collagen VI expression precedes the accumulation of collagen I associated with interleukin-4 (IL-4)-induced mineralization in human osteoblast-like cells. Transfection with an antisense oligonucleotide specific for alpha1(VI) collagen mRNA was shown to attenuate mRNA levels of collagens VI and I. Incubating IL-4 treated cells with anti-collagen VI antiserum decreased expression of alpha1(I) mRNA. The results suggest that collagen VI may regulate collagen I expression in the early phase of IL-4-induced mineralization.