RESUMEN
Chondrocytes constantly receive external stimuli, which regulates remodeling. An optimal level of mechanical stress is essential for maintaining chondrocyte homeostasis, however, excessive mechanical stress induces inflammatory cytokines and protease, such as matrix metalloproteinases (MMPs). Therefore, excessive mechanical stress is considered to be one of the main causes to cartilage destruction leading to osteoarthritis (OA). Integrins are well-known as cell adhesion molecules and act as receptors for extracellular matrix (ECM), and are believed to control intracellular signaling pathways both physically and chemically as a mechanoreceptor. However, few studies have focused on the roles and functions of integrins in inflammation caused by excessive mechanical stress. In this study, we examined the relationship between integrins (αVß3 and αVß5) and the expression of inflammatory factors under mechanical loading in chondrocytes by using an integrin receptor antagonist (cilengitide). Cilengitide suppressed the gene expression of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), matrix metalloproteinase-3 (MMP-3), and MMP-13 induced by excessive mechanical stress. In addition, the protein expression of IL1-ß and MMP-13 was also inhibited by the addition of cilengitide. Next, we investigated the involvement of intracellular signaling pathways in stress-induced integrin signaling in chondrocytes by using western blotting. The levels of p-FAK, p-ERK, p-JNK, and p-p38 were enhanced by excessive mechanical stress and the enhancement was suppressed by treatment with cilengitide. In conclusion, this study revealed that excessive mechanical stress may activate integrins αVß3 and αVß5 on the surface of chondrocytes and thereby induce an inflammatory reaction by upregulating the expression of IL-1ß, TNF-α, MMP-3, and MMP-13 through phosphorylation of FAK and MAPKs.
Asunto(s)
Condrocitos/metabolismo , Integrina alfaVbeta3/metabolismo , Osteoartritis/metabolismo , Receptores de Vitronectina/metabolismo , Venenos de Serpiente/farmacología , Estrés Mecánico , Animales , Línea Celular , Condrocitos/patología , Citocinas/metabolismo , RatonesRESUMEN
OBJECTIVES: Amelogenins, enamel matrix proteins secreted by ameloblasts, comprise 90% of the developing extracellular enamel matrix. Recent evidence suggests that amelogenins might induce the proliferation of various cells. However, the residues comprising the active site of amelogenin remain unclear. Therefore, this study aimed to examine the effects of a human amelogenin C-terminal peptide (amgCP) on the metabolism of osteoblasts. MATERIALS AND METHODS: Mouse calvarial osteoblastic cells (MC3T3-E1) were cultured and treated with amgCP. Cell proliferation was measured using MTS and BrdU assays. After confluence was reached, the cells were cultured in osteogenic differentiation medium and treated with 0, 100, or 1000 ng/ml amgCP. Cell differentiation activity was examined by real-time PCR, western blotting, and ALP activity. Mineralization was evaluated by Alizarin red staining. RESULTS: Cell numbers of MC3T3-E1 were significantly (P < 0.05) increased by treatment with 1000 ng/ml amgCP as compared to the control group at 4 and 6 days. In addition, the proliferative activity of MC3T3-E1 was significantly enhanced by treatment with 100 or 1000 ng/ml amgCP. The mRNA levels and protein expressions of ALP and BSP were not changed by treatment with amgCP as compared to the non-treated controls on days 7 and 14. The osteogenic differentiation of MC3T3-E1 cells was not affected by treatment with amgCP as compared with untreated controls. CONCLUSION: The C-terminus of amelogenin promotes the proliferation of MC3T3-E1 cells, indicating the possible utility of the C11 peptide in bone-tissue regeneration.
Asunto(s)
Amelogenina/química , Osteoblastos/metabolismo , Células 3T3 , Animales , Regeneración Ósea , Dominio Catalítico , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Medios de Cultivo , Relación Dosis-Respuesta a Droga , Matriz Extracelular/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Péptidos/química , Reacción en Cadena en Tiempo Real de la Polimerasa , Sales de Tetrazolio/química , Tiazoles/química , Ingeniería de Tejidos/métodosRESUMEN
Hyaluronan (HA) and superficial zone protein (SZP) distribute in joint structures and play a crucial role in joint lubrication. The aim of this study was to examine the effect of fluid flow on the synthesis of both HA and SZP in synovial membrane cells. Shear stress was applied by fluid flow to the rabbit synovial membrane cell line, HIG-82. The mRNA levels of HA synthase 2 (HAS2) , HA synthase 3 (HAS3), and SZP were examined by real-time PCR. The levels of HA and SZP protein were determined by sandwich ELISA and western blotting, respectively. The expression of SZP protein was increased by the application of low-magnitude shear stress, whereas high-magnitude shear stress decreased expression of SZP protein. Meanwhile, the level of HA protein in culture was decreased when high-magnitude shear stress was applied. The levels of both HAS2 and HAS3 mRNAs were down-regulated by high-magnitude shear stress, resulting in a significant decrease in HA concentration. In conclusion, it is shown that the application of shear stress to synovial membrane cells substantially affects the synthesis of both HA and SZP, which are inhibited if excessive stress is applied.
Asunto(s)
Condrocitos/metabolismo , Ácido Hialurónico/biosíntesis , Proteoglicanos/biosíntesis , Membrana Sinovial/metabolismo , Análisis de Varianza , Animales , Línea Celular , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , ARN Mensajero/biosíntesis , Conejos , Estrés Mecánico , Membrana Sinovial/citologíaRESUMEN
We examined the localization and boundary lubricating function of superficial zone protein (SZP) on the surface of mandibular condylar cartilage. Chondrocytes were separated from the surface layer of mandibular condylar cartilage of 6- to 9-month-old female pigs. A cyclic tensile strain of 7% or 21% cell elongation was applied to the cultured chondrocytes. Gene expression levels of cartilage matrix proteins and secretory phospholipase A(2) (sPLA(2)) were quantified by real-time polymerase chain reaction analysis. The friction coefficient of the mandibular condylar surface was measured by a friction tester before and after treatment with 0.1 U/ml sPLA(2). Significantly higher mRNA levels of SZP and type I collagen were found in chondrocytes from the superficial layer than in those in the other layers. The SZP mRNA level was up-regulated by cyclic tensile strain of 7% and 21% cell elongation. Cyclic tensile strain of 21% cell elongation up-regulated the sPLA(2) mRNA level. The friction coefficient of the condylar surface was increased significantly by treatment with sPLA(2). The removal of SZP from the surface layer of mandibular condylar cartilage by sPLA(2) resulted in a significant increase in the friction coefficient on the surface of articular cartilage.
Asunto(s)
Cartílago/fisiología , Cóndilo Mandibular/fisiología , Proteoglicanos/metabolismo , Animales , Cartílago/metabolismo , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Cóndilo Mandibular/metabolismo , Fosfolipasas A2 Secretoras/biosíntesis , Fosfolipasas A2 Secretoras/genética , Fosfolipasas A2 Secretoras/metabolismo , Proteoglicanos/biosíntesis , Proteoglicanos/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Porcinos , Líquido Sinovial/metabolismo , Líquido Sinovial/fisiologíaRESUMEN
Osteoarthritis (OA) sometimes occurs as a consequence of repeated microtrauma involved in parafunction, which may lead to microfracture in the subchondral bone. The aim of this in vitro study was to evaluate the effects of subchondral osteoblasts in loading with repeated excessive mechanical stress on the metabolism of overlying chondrocytes. A high-magnitude cyclic tensile stress of 15 kPa (30 cycles min(-1)) was applied to the cultured osteoblasts obtained from porcine mandibular condyles. The chondrocytes in alginate beads were then co-cultured with mechanically stressed or unstressed osteoblasts. Chondrocytes co-cultured with unstressed osteoblasts showed a phenotypic shift to hypertrophic chondrocytes, characterized by decreased expression of type II collagen, aggrecan, Sry-related HMG box (SOX-9), and cartilage oligomeric matrix protein (COMP) genes and increased expression of type X collagen and bone sialoprotein (BSP) genes, suggesting that the co-culture may change the chondrocyte differentiation to some extent. These changes were more distinct in chondrocytes co-cultured with excessively mechanically stressed osteoblasts. After co-culture with stressed osteoblasts, the expressions of matrix metalloproteinase (MMP)1, MMP3 and MMP13 genes were also enhanced and the synthesis of DNA, proteoglycan and collagen were significantly decreased in chondrocytes. These results demonstrate that alterations in cartilage metabolism can be induced by stressed osteoblasts, indicating a possible explanation for the onset and progression of OA.
Asunto(s)
Condrocitos/metabolismo , Osteoblastos/fisiología , Agrecanos/análisis , Fosfatasa Alcalina/análisis , Animales , Fenómenos Biomecánicos , Cartílago Articular/citología , Diferenciación Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo , Colágeno/análisis , Colágeno Tipo II/análisis , Colágeno Tipo X/análisis , ADN/análisis , Proteínas de la Matriz Extracelular/análisis , Glicoproteínas/análisis , Hipertrofia , Sialoproteína de Unión a Integrina , Cóndilo Mandibular/citología , Proteínas Matrilinas , Metaloproteinasa 1 de la Matriz/análisis , Metaloproteinasa 13 de la Matriz/análisis , Metaloproteinasa 3 de la Matriz/análisis , Fenotipo , Proteoglicanos/análisis , Factor de Transcripción SOX9/análisis , Sialoglicoproteínas/análisis , Estrés Mecánico , Porcinos , Factor de Crecimiento Transformador beta/análisisRESUMEN
Excessive mechanical stimulation is considered an important factor in the destruction of chondrocytes. Focal adhesion kinase (FAK) is non-receptor tyrosine kinase related to a number of different signaling proteins. Little is known about the function of FAK in chondrocytes under mechanical stimulation. In the present study, we investigated the function of FAK in mechanical signal transduction and the mechanism through which cyclic tensile strain (CTS) induces expression of inflammation-related factors. Mouse ATDC5 chondrogenic cells were subjected to CTS of 0.5 Hz to 10% cell elongation with an FAK inhibitor. The expression of genes encoding COX-2, IL-1ß, and TNF-α was examined using real-time RT-PCR after CTS application with FAK inhibitor. Phosphorylation of p-38, ERK, and JNK was analyzed by Western blotting. Differences in COX-2 expression following pretreatment with FAK, p-38, ERK, and JNK inhibitors were compared by Western blotting. We found that CTS increased the expression of genes encoding COX-2, IL-1ß, and TNF-α and activated the phosphorylation of FAK, p-38, ERK, and JNK. Pretreatment with an FAK inhibitor for 2 h reduced the expression of genes encoding COX-2, IL-1ß, and TNF-α induced by CTS-associated inflammation and decreased phosphorylation of FAK, p-38, ERK, and JNK. Pretreatment with FAK, p-38, ERK, and JNK inhibitors markedly suppressed COX-2 and IL-1ß protein expression. In conclusion, FAK appears to regulate inflammation in chondrocytes under CTS via MAPK pathways.
Asunto(s)
Condrocitos/metabolismo , Citocinas/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Inflamación/metabolismo , Sistema de Señalización de MAP Quinasas , Resistencia a la Tracción , Animales , Línea Celular , Citocinas/genética , Proteína-Tirosina Quinasas de Adhesión Focal/fisiología , Expresión Génica , Ratones , Fosforilación , Regulación hacia ArribaRESUMEN
AIMS: To determine the effect of low-intensity pulsed ultrasound (LIPUS) on cyclooxygenase-2 (COX-2) expression and related mechanisms by using cultured articular chondrocytes derived from porcine mandibular condyles after treatment with interleukin-1 beta (IL-1ß). METHODS: Chondrocytes were derived from porcine mandibular condylar cartilage and cultured. The cells were treated with or without 10 ng/mL IL-1ß. At the same time, the cells were exposed to LIPUS for 20 minutes. After LIPUS exposure, the conditioned medium was changed to a fresh one without IL-1ß, and the cells were incubated for 0 to 24 hours. The effects of LIPUS on IL-1ß-treated chondrocytes were examined in terms of the expression of p-integrin ß1, COX-2, and phosphorylated extracellular signal-related kinase (p-ERK) 1/2 by real-time polymerase chain reaction (PCR) and Western blot analyses. Differences in the means among multiple groups were examined by one-way analysis of variance (ANOVA) for all groups at each time point, followed by a Scheffé multiple comparison test as a post-hoc test; Student t test was also used. RESULTS: COX-2 mRNA level was upregulated by the treatment with IL-1ß and was suppressed significantly (P < .01) by LIPUS exposure. Furthermore, LIPUS enhanced gene expression and phosphorylation of integrin ß, and it inhibited the expression of p-ERK 1/2. CONCLUSION: LIPUS exposure inhibited IL-1ß-induced COX-2 expression through the integrin ß1 receptor followed by the phosphorylation of ERK 1/2. Despite the restricted duration of its effect, LIPUS is suggested to be a potential candidate as a preventive and auxiliary treatment to suppress the degradation of articular chondrocytes in temporomandibular joint osteoarthritis.
Asunto(s)
Cartílago Articular/enzimología , Condrocitos/enzimología , Ciclooxigenasa 2/análisis , Cóndilo Mandibular/enzimología , Terapia por Ultrasonido/métodos , Animales , Western Blotting , Cartílago Articular/citología , Cartílago Articular/efectos de los fármacos , Técnicas de Cultivo de Célula , Separación Celular , Células Cultivadas , Condrocitos/efectos de los fármacos , Medios de Cultivo Condicionados , Ciclooxigenasa 2/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/análisis , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Femenino , Integrina beta1/análisis , Integrina beta1/efectos de los fármacos , Interleucina-1beta/farmacología , Cóndilo Mandibular/citología , Cóndilo Mandibular/efectos de los fármacos , Fosforilación , Reacción en Cadena en Tiempo Real de la Polimerasa , Porcinos , Factores de TiempoRESUMEN
The purpose of this study was to investigate the effects of basic fibroblast growth factor (bFGF) treatment on the proliferation and apoptosis of cultured gingival fibroblasts (GFs). Human GFs were isolated from the palatal gingival tissues of 16 healthy volunteers ranging in the age from 9 to 35 years old. Cultured GFs were subjected to the analyses for cell proliferation by ELISA assay, gene expression by RT-PCR analysis, and apoptosis potency by caspase-3 assay. The cell proliferation activity and gene expression of type-I collagen and caspase-3 activity were enhanced significantly by the treatment with bFGF in cultured GFs. Furthermore, the activity of caspase-3 in cultured GFs from young subjects was significantly higher than that in GFs from adults. It is shown that bFGF significantly enhances the gene expression of type-I collagen in cultured fibroblasts from human gingival tissues. It also demonstrated that bFGF modulates the apoptosis of periodontal fibroblasts, and the effect is higher in young subjects, indicating a significant role of bFGF in the prevention of scar formation during wound healing.
RESUMEN
Extracellular matrix degradation in mandibular condylar cartilage is mediated by various cytokines in the temporomandibular joint (TMJ). Interleukin-1 beta (IL-1ß) is detected in joint structures with pathologic status, and participates in catabolic action in the extracellular matrix. The purpose of this study was to investigate the effects of IL-1ß on cyclooxygenase-2 (COX-2) expression and cartilage metabolism using cultured chondrocytes from mandibular condyle. Articular chondrocytes from the porcine mandibular condylar cartilage around the surface were cultured and treated with 0-10 ng/ml IL-1ß or 0-1000 ng/ml prostaglandin (PGE(2)) for 0-24h. The mRNA levels of COX-2, MMP-1, -3, and -13 were evaluated by real-time PCR analysis. The protein levels of PGE(2) and MMPs were examined by ELISA and Western blot analysis, respectively. The expression levels of COX-2 and PGE(2) were enhanced by exogenous IL-1ß in chondrocytes. The mRNA levels of MMP-1, -3, and -13 were up-regulated by PGE(2) treatment dose-dependently. It is shown that the expression of COX-2/PGE(2) was enhanced by IL-1ß in articular chondrocytes from mandibular condyle, and that MMP-1, -3, and -13 were induced by PGE(2), suggesting that IL-1ß-induced COX-2/PGE(2) play a crucial role in catabolic processes of mandibular condylar cartilage under inflammatory conditions.
Asunto(s)
Cartílago Articular/efectos de los fármacos , Ciclooxigenasa 2/biosíntesis , Dinoprostona/biosíntesis , Dinoprostona/farmacología , Interleucina-1beta/farmacología , Cóndilo Mandibular/metabolismo , Animales , Cartílago Articular/metabolismo , Células Cultivadas , Condrocitos/metabolismo , Inducción Enzimática , Femenino , Cóndilo Mandibular/efectos de los fármacos , Metaloproteinasa 1 de la Matriz/biosíntesis , Metaloproteinasa 13 de la Matriz/biosíntesis , Metaloproteinasa 3 de la Matriz/biosíntesis , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/farmacología , Sus scrofa , Articulación Temporomandibular/efectos de los fármacos , Articulación Temporomandibular/metabolismoRESUMEN
Osteoarthritis (OA) in the temporomandibular joint (TMJ) is a degenerative disease caused by excessive external loading. Recently, it was reported that the damage in the mineralized subchondral bone caused by traumatic impact-loading is responsible for the initiation and progression of cartilage degeneration. Thus far, we have hypothesized that cytokines released from damaged subchondral bone from impact-loading affect the cartilage catabolism under pathological conditions. An impactor of 200 gw was dropped onto the top of a porcine mandibular condyle. After organ culture for 2 days, we investigated the association between the subchondral bone and cartilage using histological and biochemical experiments. The impact-loading induced the expression of IL-1beta immunohistochemically and prominently up-regulated IL-1alpha and IL-1beta mRNA levels in subchondral bone. We confirmed a significant decrease in type II collagen and aggrecan mRNA expressions in chondrocytes by co-culture with osteoblasts after impact-loading, and significant increase in mRNA and protein expressions of IL-1beta in subchondral osteoblasts from impact-loaded subchondral bone. The mRNA expressions of type II collagen, aggrecan, and type X collagen in chondrocytes were decreased significantly by the co-culture with osteoblasts pre-treated by IL-1beta, -6, and TNF-alpha. Among them, osteoblasts pre-treated by IL-1beta affected chondrocytes most strongly. It was also shown that IL-1beta-treated osteoblasts enhanced the MMP-1 mRNA level most markedly in chondrocytes among the four cytokines. These results suggest that the TMJ subjected to impact-loading can increase directly IL-1beta synthesis in the subchondral region, subsequently altering the metabolism of adjacent cartilage and may eventually resulting in the onset and progression of TMJ-OA.