Chin-Jung Hsu

Osteopontin increases migration and MMP-9 up-regulation via αvβ3 integrin, FAK, ERK, and NF-κB-dependent pathway in human chondrosarcoma cells†

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and proliferation via interaction with its receptor, that is, αvβ3 integrin. However, the effect of OPN on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that OPN increased the migration and expression of matrix metalloproteinase (MMP)‐9 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the OPN‐induced increase of the migration and MMP‐9 up‐regulation of chondrosarcoma cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited OPN‐induced cell migration and MMP‐9 up‐regulation. Stimulation of JJ012 cells with OPN also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser536 phosphorylation, and κB‐luciferase activity. The OPN‐mediated increases in MMP‐9 and κB‐luciferase activities were inhibited by RGD peptide, PD98059 or FAK and ERK2 mutant. Taken together, our results indicated that OPN enhances the migration of chondrosarcoma cells by increasing MMP‐9 expression through the αvβ3 integrin, FAK, MEK, ERK and NF‐κB signal transduction pathway. J. Cell. Physiol. 221: 98–108, 2009.

CCL5/CCR5 axis promotes the motility of human oral cancer cells

CCL5 (previously called RANTES) is in the CC‐chemokine family and plays a crucial role in the migration and metastasis of human cancer cells. On the other hand, the effect of CCL5 is mediated via CCR receptor. RT‐PCR and flow cytometry studies demonstrated CCR5 but not CCR1 and CCR3 mRNA in oral cancer cell lines, especially higher in those with high invasiveness (SCC4) as compared with lower levels in HSC3 cells and SCC9 cells. Stimulation of oral cancer cells with CCL5 directly increased the migration and metalloproteinase‐9 (MMP‐9) production. MMP‐9 small interfering RNA inhibited the CCL5‐induced MMP‐9 expression and thereby significantly inhibited the CCL5‐induced cell migration. Activations of phospholipase C (PLC), protein kinase Cδ (PKCδ), and NF‐κB pathways after CCL5 treatment was demonstrated, and CCL5‐induced expression of MMP‐9 and migration activity was inhibited by the specific inhibitor of PLC, PKCδ, and NF‐κB cascades. In addition, migration‐prone sublines demonstrate that cells with increasing migration ability had more expression of MMP‐9, CCL5, and CCR5. Taken together, these results indicate that CCL5/CCR5 axis enhanced migration of oral cancer cells through the increase of MMP‐9 production. J. Cell. Physiol. 220: 418–426, 2009.

The CCL5/CCR5 axis promotes interleukin-6 production in human synovial fibroblasts

OBJECTIVE CCL5 (RANTES) was originally identified as a product of activated T cells and plays a crucial role in the inflammatory response. This study was undertaken to investigate the intracellular signaling pathways involved in CCL5-induced interleukin-6 (IL-6) production in human synovial fibroblasts. METHODS CCL5-mediated IL-6 expression was assessed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The mechanisms of action of CCL5 in different signaling pathways were studied using Western blotting. Knockdown of CCR5 and protein kinase Cδ (PKCδ) protein was achieved by transfection of small interfering RNA (siRNA). Chromatin immunoprecipitation assays were used to study in vivo binding of c-Jun to the IL-6 promoter. Transient transfection was used to examine IL-6 and activator protein 1 (AP-1) activity. RESULTS Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of CCL5 and CCR5, and expression was higher than that in normal synovial fibroblasts. Stimulation of OASFs with CCL5 induced concentration- and time-dependent increases in IL-6 production. CCL5-mediated IL-6 production was attenuated by CCR5 monoclonal antibody, CCR5 inhibitor (Met-RANTES), and CCR5 siRNA. Pretreatment with a PKCδ inhibitor (rottlerin), a c-Src inhibitor (PP2), or an AP-1 inhibitor (tanshinone IIA) also blocked the potentiating action of CCL5. Treatment of OASFs with CCL5 increased the accumulation of phosphorylated c-Jun in the nucleus, AP-1 luciferase activity, and c-Jun binding to the AP-1 element on the IL-6 promoter. CCL5-mediated AP-1 luciferase activity and c-Jun binding to the AP-1 element were inhibited by Met-RANTES, rottlerin, and PP2. CONCLUSION The present results suggest that the interaction between CCL5 and CCR5 increases IL-6 production in human synovial fibroblasts via the PKCδ/c-Src/c-Jun and AP-1 signaling pathways.

Bradykinin-induced IL-6 expression through bradykinin B2 receptor, phospholipase C, protein kinase Cδ and NF-κB pathway in human synovial fibroblasts

Bradykinin (BK) is an inflammatory mediator, and shows elevated levels in regions of severe injury and inflammatory diseases. It has been shown to induce interleukin-6 (IL-6) expression in inflammatory responses in rheumatoid arthritis. We investigated the signaling pathway involved in IL-6 production caused by BK in synovial fibroblasts. BK caused concentration- and time-dependent increases in IL-6 production. By using pharmacological inhibitors or genetic inhibition of the BK receptor, siRNA revealed that B2 but not B1 BK receptors are involved in BK-mediated up-regulation of IL-6. BK-mediated IL-6 production was attenuated by phospholipase C inhibitor (U73122), protein kinase Cdelta inhibitor (rottlerin), NF-kappaB inhibitor (PDTC), IkappaB protease inhibitor (TPCK) and NF-kappaB inhibitor peptide. Stimulation of synovial fibroblasts with BK activated IkappaB kinase alpha/beta (IKK alpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser(276), p65 and p50 translocation from the cytosol to the nucleus and kappaB-luciferase activity. BK mediated an increase of IKK alpha/beta and IkappaBalpha phosphorylation, kappaB-luciferase activity and p65 and p50 binding to the NF-kappaB element was inhibited by B2 BK receptor antagonist (HOE140), U73122 and rottlerin. Our results suggest that BK increased IL-6 production in synovial fibroblasts via the B2 BK receptor/PI-PLC/PKCdelta/and NF-kappaB signaling pathway.

The epidemiology of traumatic humeral shaft fractures in Taiwan

We retrospectively analysed 106 consecutive traumatic humeral shaft fractures over a five-year period. The mechanism of injury, age, gender, fracture types, associated injury and the presence of injury to the radial nerve were reviewed. The incidence was about 10 per 100,000 per year; most were closed fractures in young males which had been sustained as a result of traffic accidents. The age–gender distribution was characterised by gradually increased incidence from the fifth decade in women, while it reached a peak at the third decade and decreased after the fifth decade in men. The results revealed different epidemiological features from previous studies. The epidemiology differs between ethnicity and country, and updating the epidemiological features of humeral shaft fractures may provide information for appropriate treatment programmes. This study documents the epidemiology of humeral shaft fracture in Taiwan, probably for the first time in this Asian community.RésuméNous avons analysé de façon rétrospective 106 fractures traumatiques de la diaphyse humérale sur une période de 5 ans. Le mécanisme du traumatisme, l’âge, le sexe, le type de fractures, le type de traumatismes et la présence de lésion du nerf radial ont été analysés. L’incidence est, approximativement de 10 pour un million de personnes par an. La plupart sont des fractures à foyer fermé sur des sujets jeunes de sexe masculin secondaires à des accidents de la circulation. L’analyse de l’âge et du sexe permet de mettre en évidence une augmentation de l’incidence de cette fracture chez les femmes dans les 5 premières décades, avec un pic à 30 ans et une diminution après 50 ans chez l’homme. Les résultats de l’étude épidémiologique permettent un algorithme chirurgical adapté. Il s’agit probablement de la première étude asiatique, elle a été réalisée à Taiwan.

Glial Cell-Derived Neurotrophic Factor Increases Migration of Human Chondrosarcoma Cells Via ERK and NF-κB Pathways

Invasion of tumor cells is the primary cause of therapeutic failure in the treatment of malignant chondrosarcomas. Glial cell‐derived neurotrophic factor (GDNF) plays a crucial role in migration and metastasis of human cancer cells. Integrins are the major adhesive molecules in mammalian cells. Here we found that GDNF directed the migration and increased cell surface expression of αv and β3 integrin in human chondrosarcoma cells. Pretreated of JJ012 cells with MAPK kinase (MEK) inhibitors PD98059 or U0126 inhibited the GDNF‐mediated migration and integrin expression. Stimulation of cells with GDNF increased the phosphorylation of MEK and extracellular signal‐regulating kinase (ERK). In addition, NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) also inhibited GDNF‐mediated cells migration and integrin up‐regulation. Stimulation of cells with GDNF induced IκB kinase (IKKα/β) phosphorylation, IκB phosphorylation, p65 Ser536 phosphorylation, and κB‐luciferase activity. Furthermore, the GDNF‐mediated increasing of κB‐luciferase activity was inhibited by PD98059, U0126, PDTC and TPCK or MEK, ERK, IKKα, and IKKβ mutants. Taken together, these results suggest that the GDNF acts through MEK/ERK, which in turn activates IKKα/β and NF‐κB, resulting in the activations of αvβ3 integrin and contributing the migration of human chondrosarcoma cells. J. Cell. Physiol. 220: 499–507, 2009.

CCN4 induces IL-6 production through αvβ5 receptor, PI3K, Akt, and NF-κB singling pathway in human synovial fibroblasts

IntroductionOsteoarthritis (OA) is the most common degenerative joint disease that is involved in the degradation of articular cartilage. The exact etiology of OA is not completely understood. CCN4 is related to up-regulation in the cartilage of patients with osteoarthritis. Previous studies have shown that CCN4 might be associated with the pathogenesis of OA, but the exact signaling pathways in CCN4-mediated IL-6 expression in synovial fibroblasts (SF) are largely unknown. Therefore, we explored the intracellular signaling pathway involved in CCN4-induced IL-6 production in human synovial fibroblast cells.MethodsCCN4-induced IL-6 production was assessed with quantitative real-time qPCR and ELISA. The mechanisms of action of CCN4 in different signaling pathways were studied by using Western blotting. Neutralizing antibodies of integrin were used to block the integrin signaling pathway. Luciferase assays were used to study IL-6 and NF-κB promoter activity. Immunocytochemistry was used to examine the translocation activity of p65.ResultsOsteoarthritis synovial fibroblasts (OASFs) showed significant expression of CCN4 and the expression was higher than in normal SFs. OASF stimulation with CCN4 induced concentration- and time-dependent increases in IL-6 production. Pretreatment of OASFs with αvβ5 but not α5β1 and αvβ3 integrin antibodies reduced CCN4-induced IL-6 production. CCN4-mediated IL-6 production was attenuated by PI3K inhibitor (LY294002 and Wortmannin), Akt inhibitor (Akti), and NF-κB inhibitor (PDTC and TPCK). Stimulation of cells with CCN4 also increased PI3K, Akt, and NF-κB activation.ConclusionsOur results suggest that CCN4 activates αvβ5 integrin, PI3K, Akt, and NF-κB pathways, leading to up-regulation of IL-6 production. According to our results, CCN4 may be an appropriate target for drug intervention in OA in the future.

Lipoteichoic acid enhances IL-6 production in human synovial fibroblasts via TLR2 receptor, PKCδ and c-Src dependent pathways

Patients with rheumatoid arthritis (RA) are at increased risk of developing infections and appear to be particularly susceptible to septic arthritis. Lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria is an amphiphilic, negatively charged glycolipid. However, the effects of LTA on human synovial fibroblasts are largely unknown. We investigated the signaling pathway involved in IL-6 production stimulated by LTA in rheumatoid arthritis synovial fibroblasts (RASF). LTA caused concentration- and time-dependent increases in IL-6 production. LTA-mediated IL-6 production was attenuated by Toll-like receptor 2 (TLR2) monoclonal antibody or siRNA. Pretreatment with PKCdelta inhibitor (rottlerin), c-Src inhibitor (PP2), AP-1 inhibitor (tanshinone IIA) and NF-kappaB inhibitor (PDTC and TPCK) also inhibited the potentiating action of LTA. However, focal adhesion kinase (FAK) mutant and siRNA did not affect LTA-mediated IL-6 production. Stimulation of cells with LTA increased the PKCdelta and c-Src phosphorylation and kinase activity. LTA increased the accumulation of p-c-Jun and p-p65 in the nucleus, as well as AP-1 and NF-kappaB luciferase activity. LTA-mediated increase of AP-1 and NF-kappaB luciferase activity was inhibited by rottlerin and PP2 or TLR2 and PKCdelta siRNA or c-Src mutant. Our results suggest that LTA-increased IL-6 production in human synovial fibroblasts via the TLR2 receptor, PKCdelta, c-Src, AP-1 and NF-kappaB signaling pathways.

CTGF increases IL-6 expression in human synovial fibroblasts through integrin-dependent signaling pathway.

Background Connective tissue growth factor (CTGF; also known as CCN2) is an inflammatory mediator, and shows elevated levels in regions of severe injury and inflammatory diseases. CTGF is abundantly expressed in osteoarthritis (OA). However, the relationship between CTGF and IL-6 in OA synovial fibroblasts (OASFs) is mostly unknown. Methodology/Principal Findings OASFs showed significant expression of CTGF, and expression was higher than in normal SFs. OASFs stimulation with CTGF induced concentration-dependent increases in IL-6 expression. CTGF mediated IL-6 production was attenuated by αvβ5 integrin neutralized antibody and apoptosis signal-regulating kinase 1 (ASK1) shRNA. Pretreatment with p38 inhibitor (SB203580), JNK inhibitor (SP600125), AP-1 inhibitors (Curcumin and Tanshinone IIA), and NF-κB inhibitors (PDTC and TPCK) also inhibited the potentiating action of CTGF. CTGF-mediated increase of NF-κB and AP-1 luciferase activity was inhibited by SB203580 and SP600125 or ASK1 shRNA or p38 and JNK mutant. Conclusions/Significance Our results suggest that CTGF increased IL-6 production in OASFs via the αvβ5 integrin, ASK1, p38/JNK, and AP-1/NF-κB signaling pathways.

CTGF induces monocyte chemoattractant protein-1 expression to enhance monocyte migration in human synovial fibroblasts.

Connective tissue growth factor (CTGF; also known as CCN2) is an inflammatory mediator, and shows elevated levels in regions of severe injury and inflammatory diseases. CTGF is abundantly expressed in osteoarthritis (OA). Migration and infiltration of mononuclear cells to inflammatory sites are playing important roles during OA pathogenesis. Monocyte chemoattractant protein-1 (MCP-1/CCL2) is the key chemokine that regulates migration and infiltration of monocytes. However, the effect of CTGF on MCP-1 expression and monocyte migration is largely unknown. Our results showed that MCP-1 was highly expressed in OA synovial fibroblasts (OASFs) as compared with normal SFs. Directly applying OASFs with CTGF increased MCP-1 expression in a concentration- and a time-dependent manner. CTGF mediated MCP-1 production was attenuated by αvβ5 integrin neutralized antibody. Pretreatment with focal adhesion kinase (FAK), MEK, AP-1, and NF-κB inhibitors also inhibited the potentiating action of CTGF. CTGF-mediated increase of NF-κB and AP-1 luciferase activity was inhibited by FAK, MEK, and ERK inhibitors or mutants. In vitro chemotaxis assay showed that OA synovial fluid and supernatants from CTGF treated OASFs increased migration of monocyte. In addition, CTGF-mediated migration was inhibited by the FAK and MEK inhibitors. Taken together, our results indicated that CTGF enhances the migration of monocyte cells by increasing MCP-1 expression through the αvβ5 integrin, FAK, MEK, ERK, and NF-κB/AP-1 signal transduction pathway.