Shan-Chi Liu

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.

Leptin Induces IL-6 Expression through OBRl Receptor Signaling Pathway in Human Synovial Fibroblasts

Background Leptin, an adipocyte-secreted hormone that centrally regulates weight control, may exert proinflammatory effects in the joint, depending on the immune response. Leptin is abundantly expressed in osteoarthritis (OA) cartilage and synovium. However, the relationship between leptin and interleukin-6 (IL-6) in OA synovial fibroblasts (OASFs) remains obscure. Methodology/Principal Findings Stimulation of OASFs with leptin induced IL-6 expression in a concentration- and time-dependent manner. OASFs expressed the long (OBRl) and short (OBRs) isoforms of the leptin receptor. However, OBRl, but not OBRs, antisense oligonucleotide (AS-ODN) abolished the leptin-mediated increase of IL-6 expression. Transfection with insulin receptor substrate (IRS)-1 siRNA decreased leptin-induced IL-6 production. In addition, pretreatment of cells with PI3K, Akt, or AP-1 inhibitor also inhibited the potentiating action of leptin. Leptin-induced AP-1 activation was inhibited by OBRl, IRS-1, PI3K, or Akt inhibitors and siRNAs. Conclusions/Significance Our results showed that leptin activates the OBRl receptor, which in turn activates IRS-1, PI3K, Akt, and AP-1 pathway, leading to up-regulation of IL-6 expression.

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.

Berberine attenuates CCN2-induced IL-1β expression and prevents cartilage degradation in a rat model of osteoarthritis

Connective tissue growth factor (CTGF; also known as CCN2) is an inflammatory mediator that is abundantly expressed in osteoarthritis (OA). Interleukin-1β (IL-1β) plays a pivotal role in OA pathogenesis. Berberine exhibits an anti-inflammatory effect, but the mechanisms by which it modulates CCN2-induced IL-1β expression in OA synovial fibroblasts (OASFs) remain unknown. We showed that CCN2-induced IL-1β expression is mediated by the activation of αvβ3/αvβ5 integrin-dependent reactive oxygen species (ROS) generation, and subsequent activation of apoptosis signal-regulating kinase 1 (ASK1), p38/JNK, and nuclear factor-κB (NF-κB) signaling pathways. This IL-1β expression in OASFs is attenuated by N-acetylcysteine (NAC), inhibitors of ASK1, p38, or JNK, or treatment with berberine. Furthermore, berberine also reverses cartilage damage in an experimental model of collagenase-induced OA (CIOA). We observed that CCN2 increased IL-1β expression via αvβ3/αvβ5 integrins, ROS, and ASK1, p38/JNK, and NF-κB signaling pathways. Berberine was found to inhibit these signaling components in OASFs in vitro and prevent cartilage degradation in vivo. We suggest a novel therapeutic strategy of using berberine for managing OA.

The novel isoflavone derivatives inhibit RANKL-induced osteoclast formation.

Isoflavones are compounds structurally similar to the mammalian estrogens and have received considerable attention for their preventive actions on bone loss. Here, we synthesized the novel isoflavone derivatives and examined their activities in bone cells. We found that the novel isoflavone derivatives markedly inhibited the receptor activator of nuclear factor kappa B ligand (RANKL) plus macrophage colony stimulating factor (M-CSF)-induced osteoclastic differentiation from bone marrow stromal cells and RAW264.7 macrophage cells. Treatment of RAW264.7 macrophages with RANKL-induced extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) phosphorylation. However, RANKL-induced p38 and JNK but not ERK phosphorylation was attenuated by isoflavone derivatives. Furthermore, RANKL-mediated increase of p65 phosphorylation at Ser⁵³⁶, NF-κB-specific DNA-protein complex formation and κB-luciferase activity was inhibited by isoflavone derivatives. On the other hand, isoflavone derivatives did not affect the cell proliferation and differentiation of human cultured osteoblasts. Our data suggest that the novel isoflavone derivatives inhibit osteoclastogenesis from bone marrow stromal cells and macrophage cells via attenuation of RANKL-induced p38, JNK and NF-κB activation.

Osteopontin inhibition of miR-129-3p enhances IL-17 expression and monocyte migration in rheumatoid arthritis

BACKGROUND Osteopontin (OPN) is an important proinflammatory cytokine in rheumatoid arthritis (RA). Levels of OPN have been shown to be significantly correlated with interleukin-17 (IL-17) production and expression of Th17 cells in the synovial fluid of RA patients. Here, we investigated the role of OPN in monocyte migration, IL-17 production and osteoblasts. METHODS OPN and IL-17 expression profiles in osteoarthritis (OA) and RA synovial fluid were determined by enzyme-linked immunosorbent assay (ELISA). The expression of the microRNA, miR-129-3p, in osteoblasts was analyzed by real-time quantitative polymerase chain reaction (qPCR). Immunoreactive proteins were spotted by Western blotting. We used the collagen-induced arthritis (CIA) mouse model to investigate the role of OPN in monocyte migration during RA. RESULTS OPN and IL-17 expression were higher in RA synovial fluid as compared to OA samples. We also found that OPN promotes IL-17 expression in osteoblasts and thereby enhances monocyte migration via the Syk/PI3K/Akt signaling pathway. miR-129-3p expression was found to be negatively regulated by OPN via the Syk/PI3K/Akt signal cascade. In contrast, lentiviral vectors expressing short hairpin RNA inhibited OPN expression and ameliorated articular swelling, cartilage erosion and monocyte infiltration in the ankle joints of CIA mice. CONCLUSION To our knowledge, our study is the first to describe how OPN promotes monocyte migration by upregulating IL-17 expression in osteoblasts in RA disease. SIGNIFICANCE These findings indicate that OPN could serve as a potential therapeutic target for the treatment of RA.

β5 integrin up-regulation in brain-derived neurotrophic factor promotes cell motility in human chondrosarcoma.

Chondrosarcoma is a primary malignant bone cancer, with a potent capacity to invade locally and cause distant metastasis; it has a poor prognosis and shows a predilection for metastasis to the lungs. Brain derived neurotrophic factor (BDNF) is a small-molecule protein from the neurotrophin family of growth factors that is associated with the disease status and outcomes of cancers. However, the effect of BDNF on migration activity in human chondrosarcoma cells is mostly unknown. Here, we found that human chondrosarcoma tissues showed significant expression of BDNF, which was higher than that in normal cartilage and primary chondrocytes. We also found that BDNF increased the migration and expression of β5 integrin in human chondrosarcoma cells. In addition, knockdown of BDNF expression markedly inhibited migratory activity. BDNF-mediated migration and β5 integrin up-regulation were attenuated by antibody, inhibitor, or siRNA against the TrkB receptor. Pretreatment of chondrosarcoma cells with PI3K, Akt, and NF-κB inhibitors or mutants also abolished BDNF-promoted migration and integrin expression. The PI3K, Akt, and NF-κB signaling pathway was activated after BDNF treatment. Taken together, our results indicate that BDNF enhances the migration of chondrosarcoma by increasing β5 integrin expression through a signal transduction pathway that involves the TrkB receptor, PI3K, Akt, and NF-κB. BDNF thus represents a promising new target for treating chondrosarcoma metastasis.

D-pinitol inhibits RANKL-induced osteoclastogenesis.

Numerous studies have indicated that inflammatory cytokines play a major role in osteoclastogenesis, leading to the bone resorption that is frequently associated with osteoporosis. D-pinitol, a 3-methoxy analogue of D-chiroinositol, was identified as an active principle in soy foods and legumes. Here we found that D-pinitol markedly inhibited the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastic differentiation from bone marrow stromal cells and RAW264.7 macrophage cells. In addition, D-pinitol also reduced RANKL-induced p38 and JNK phosphorylation. Furthermore, RANKL-mediated increase of IKK, IκBα, and p65 phosphorylation and NF-κB-luciferase activity was inhibited by D-pinitol. However, D-pinitol did not affect the proliferation and differentiation of osteoblasts. In addition, D-pinitol also prevented the bone loss induced by ovariectomy in vivo. Our data suggest that D-pinitol inhibits osteoclastogenesis from bone marrow stromal cells and macrophage cells via attenuated RANKL-induced p38, JNK, and NF-κB activation, which in turn protect bone loss from ovariectomy.

Enhancement of CCL2 expression and monocyte migration by CCN1 in osteoblasts through inhibiting miR-518a-5p: implication of rheumatoid arthritis therapy

Cysteine-rich 61 (Cyr61 or CCN1), a secreted protein from the CCN family, is an important proinflammatory cytokine. Migration and infiltration of mononuclear cells to inflammatory sites play a critical role in the pathogenesis of rheumatoid arthritis (RA). Monocyte chemoattractant protein-1 (MCP-1/CCL2) is the key chemokine that regulates migration and infiltration of monocytes. Here, we examined the role of CCN1 in monocyte migration, and CCL2 expression in osteoblasts. We found higher levels of CCN1 and CCL2 in synovial fluid from RA patients compared with levels from non-RA controls. We also found that the CCN1-induced increase in CCL2 expression is mediated by the MAPK signaling pathway and that miR-518a-5p expression was negatively regulated by CCN1 via the MAPK cascade. In contrast, inhibition of CCN1 expression with lentiviral vectors expressing short hairpin RNA ameliorated articular swelling, cartilage erosion, and infiltration of monocytes in the ankle joints of mice with collagen-induced arthritis. Our study describes how CCN1 promotes monocyte migration by upregulating CCL2 expression in osteoblasts in RA disease. CCN1 could serve as a potential target for RA treatment.

Transforming growth factor β1 enhances heme oxygenase 1 expression in human synovial fibroblasts by inhibiting microRNA 519b synthesis

Background Osteoarthritis (OA) is manifested by synovial inflammation and cartilage destruction that is directly linked to synovitis, joint swelling and pain. In the light of the role of synovium in the pathogenesis and the symptoms of OA, synovium-targeted therapy is a promising strategy to mitigate the symptoms and progression of OA. Transforming growth factor beta 1 (TGF-β1), a secreted homodimeric protein, possesses unique and potent anti-inflammatory and immune-regulatory properties in many cell types. Heme oxygenase 1 (HO-1) is an inducible anti-inflammatory and stress responsive enzyme that has been proven to prevent injuries caused by many diseases. Despite the similar anti-inflammatory profile and their involvement in the pathogenesis of arthritic diseases, no studies have as yet explored the possibility of any association between the expression of TGF-β1 and HO-1. Methodology/Principal findings TGF-β1-induced HO-1 expression was examined by HO-1 promoter assay, qPCR, and Western blotting. The siRNAs and enzyme inhibitors were utilized to determine the intermediate involved in the signal transduction pathway. We showed that TGF-β1 stimulated the synthesis of HO-1 in a concentration- and time-dependent manner, which can be mitigated by blockade of the phospholipase (PLC)γ/protein kinase C alpha (PKC)α pathway. We also showed that the expression of miRNA-519b, which blocks HO-1 transcription, is inhibited by TGF-β1, and the suppression of miRNA 519b could be reversed via blockade of the PLCγ/PKCα pathway. Conclusions/Significance TGF-β1 stimulated the expression of HO-1 via activating the PLCγ/PKCα pathway and suppressing the downstream expression of miRNA-519b. These results may shed light on the pathogenesis and treatment of OA.