Chun-Hao Tsai

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.

CYR61 Regulates BMP-2-dependent Osteoblast Differentiation through the αvβ3 Integrin/Integrin-linked Kinase/ERK Pathway

Osteoporosis is one of the most common bone pathologies. A number of novel molecules have been reported to increase bone formation including cysteine-rich protein 61 (CYR61), a ligand of integrin receptor, but mechanisms remain unclear. It is known that bone morphogenetic proteins (BMPs), especially BMP-2, are crucial regulators of osteogenesis. However, the interaction between CYR61 and BMP-2 is unclear. We found that CYR61 significantly increases proliferation and osteoblastic differentiation in MC3T3-E1 osteoblasts and primary cultured osteoblasts. CYR61 enhances mRNA and protein expression of BMP-2 in a time- and dose-dependent manner. Moreover, CYR61-mediated proliferation and osteoblastic differentiation are significantly decreased by knockdown of BMP-2 expression or inhibition of BMP-2 activity. In this study we found integrin αvβ3 is critical for CYR61-mediated BMP-2 expression and osteoblastic differentiation. We also found that integrin-linked kinase, which is downstream of the αvβ3 receptor, is involved in CYR61-induced BMP-2 expression and subsequent osteoblastic differentiation through an ERK-dependent pathway. Taken together, our results show that CYR61 up-regulates BMP-2 mRNA and protein expression, resulting in enhanced cell proliferation and osteoblastic differentiation through activation of the αvβ3 integrin/integrin-linked kinase/ERK signaling pathway.

Interleukin-6 induces vascular endothelial growth factor expression and promotes angiogenesis through apoptosis signal-regulating kinase 1 in human osteosarcoma.

Osteosarcoma is characterized by a high malignant and metastatic potential. Angiogenesis is essential for the caner metastasis. Interleukin-6 (IL-6) is a multifunctional cytokine that is associated with the disease status and outcomes of cancers. However, the relationship between IL-6 and vascular endothelial growth factor (VEGF) expression in human osteosarcoma is mostly unknown. Here we found that the IL-6 and VEGF expression was correlated with tumor stage and significantly higher than that in normal bone. Incubation of osteosarcoma cells with IL-6 increased VEGF mRNA and protein expression. Pretreatment of cells with IL-6R antibody reduced IL-6-mediated VEGF production. The apoptosis signal-regulating kinase 1 (ASK1)/p38/AP-1 pathway was activated after IL-6 treatment, and IL-6-induced VEGF expression was abolished by the specific inhibitor and siRNA of ASK1, p38, and AP-1 cascades. Importantly, knockdown IL-6 reduced VEGF expression and abolished osteosarcoma conditional medium-mediated angiogenesis. Taken together, these results indicate that IL-6 occurs through ASK1 and p38, which in turn activates AP-1, resulting in the activations of VEGF expression and contributing the angiogenesis of human osteosarcoma cells.

CTGF increases vascular endothelial growth factor-dependent angiogenesis in human synovial fibroblasts by increasing miR-210 expression.

Connective tissue growth factor (CTGF, a.k.a. CCN2) is inflammatory mediator and abundantly expressed in osteoarthritis (OA). Angiogenesis is essential for OA progression. Here, we investigated the role of CTGF in vascular endothelial growth factor (VEGF) production and angiogenesis in OA synovial fibroblasts (OASFs). We showed that expression of CTGF and VEGF in synovial fluid were higher in OA patients than in controls. Directly applying CTGF to OASFs increased VEGF production then promoted endothelial progenitor cells tube formation and migration. CTGF induced VEGF by raising miR-210 expression via PI3K, AKT, ERK, and nuclear factor-κB (NF-κB)/ELK1 pathways. CTGF-mediating miR-210 upregulation repressed glycerol-3-phosphate dehydrogenase 1-like (GPD1L) expression and PHD activity and subsequently promoted hypoxia-inducible factor (HIF)-1α-dependent VEGF expression. Knockdown of CTGF decreased VEGF expression and abolished OASF-conditional medium-mediated angiogenesis in vitro as well as angiogenesis in chick chorioallantoic membrane and Matrigel-plug nude mice model in vivo. Taken together, our results suggest CTGF activates PI3K, AKT, ERK, and NF-κB/ELK1 pathway, leading to the upregulation of miR-210, contributing to inhibit GPD1L expression and prolyl hydroxylases 2 activity, promoting HIF-1α-dependent VEGF expression and angiogenesis in human synovial fibroblasts.Cell Death and Disease advance online publication, 23 October 2014; doi:10.1038/cddis.2014.453

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.

Thrombin enhanced migration and MMPs expression of human chondrosarcoma cells involves PAR receptor signaling pathway

Thrombin is a multifunctional protease that can activate hemostasis and coagulation through the cleavage of fibrinogen to form fibrin clots. Thrombin also plays a crucial role in migration and metastasis of human cancer cells. However, the effect of thrombin on migration activity in human chondrosarcoma cells is mostly unknown. Here, we found that thrombin increased the migration and expression of matrix metalloproteinase (MMP)‐2 and MMP‐13 in human chondrosarcoma cells (JJ012 and SW1353 cells). By using pharmacological inhibitors or activators or genetic inhibition by the protease‐activated receptor (PAR), we found that the PAR1 and PAR4 receptor but not PAR3 receptor are involved in thrombin‐mediated cell migration and MMPs expression. Thrombin‐mediated migration and MMPs up‐regulation was attenuated by phospholipase C (PLC), protein kinase C, and c‐Src inhibitor. Activations of PLCβ, PKCα, c‐Src, and NF‐κB pathways after thrombin treatment was demonstrated, and thrombin‐induced MMPs expression and migration activity was inhibited by the specific inhibitors and mutants of PLC, PKC, c‐Src, and NF‐κB cascades. Taken together, our results indicated that thrombin enhances the migration of chondrosarcoma cells by increasing MMP‐2 and MMP‐13 expression through the PAR/PLC/PKCα/c‐Src/NF‐κB signal transduction pathway. J. Cell. Physiol. 223:737–745, 2010.

CCL5 promotes vascular endothelial growth factor expression and induces angiogenesis by down-regulating miR-199a in human chondrosarcoma cells

Chondrosarcoma is a primary malignant bone cancer, with a potent capacity to invade locally and cause distant metastasis. Angiogenesis is a critical step in tumor growth and metastasis. Chemokine CCL5 (previously called RANTES) has been shown to facilitate tumor progression and metastasis. However, the relationship of CCL5 with vascular endothelial growth factor (VEGF) expression and angiogenesis in human chondrosarcoma is mostly unknown. In this study, CCL5 increased VEGF expression and also promoted chondrosarcoma medium-mediated angiogenesis in vitro as well as angiogenesis effects in the chick chorioallantoic membrane and Matrigel plug nude mice model in vivo. MicroRNA analysis was performed in CCL5-treated chondrosarcoma cells versus control cells to investigate the mechanism of CCL5-mediated promotion of chondrosarcoma angiogenesis. Among the miRNAs regulated by CCL5, miR-199a was the most downregulated miRNA after CCL5 treatment. In addition, co-transfection with miR-199a mimic reversed the CCL5-mediated VEGF expression and angiogenesis in vitro and in vivo. Moreover, overexpression of CCL5 increased tumor-associated angiogenesis and tumor growth by downregulating miR-199a in the xenograft tumor angiogenesis model. Taken together, these results demonstrated that CCL5 promotes VEGF-dependent angiogenesis in human chondrosarcoma cells by downregulating miR-199a.

CCN3 increases cell motility and MMP-13 expression in human chondrosarcoma through integrin-dependent pathway.

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. CCN3, also called nephroblastoma overexpressed gene (NOV), regulates proliferation and differentiation of cancer cells. However, the effect of CCN3 on migration activity in human chondrosarcoma cells is mostly unknown. Here, we found that CCN3 increased the migration and expression of matrix metalloproteinase (MMP)‐13 in human chondrosarcoma cells (JJ012 cells). αvβ3 or αvβ5 monoclonal antibody (mAb), phosphatidylinositol 3‐kinase (PI3K) inhibitors (Ly294002 and wortmannin) and Akt inhibitor inhibited the CCN3‐induced increase of the migration and MMP‐13 upregulation of chondrosarcoma cells. CCN3 stimulation increased the phosphorylation of focal adhesion kinase (FAK), PI3K, and Akt. In addition, NF‐κB inhibitors also suppressed the cell migration and MMP‐13 expression enhanced by CCN3. Moreover, CCN3 increased NF‐κB luciferase activity and binding of p65 to the NF‐κB element on the MMP‐13 promoter. Taken together, our results indicate that CCN3 enhances the migration of chondrosarcoma cells by increasing MMP‐13 expression through the αvβ3/αvβ5 integrin receptor, FAK, PI3K, Akt, p65, and NF‐κB signal transduction pathway. J. Cell. Physiol. 226: 3181–3189, 2011.

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.

Using the Growth Factors-enriched Platelet Glue in Spinal Fusion and its Efficiency

Study Design Prospective study. Objective To determine if the use of platelet glue enhances fusion in instrumented posterolateral lumbar fusion. Summary of Background Data Platelet gel is an osteoinductive material that has been used to enhance fusion rates in lumbar fusion surgery. There are questions, however, regarding the less adhesive property of platelet gel, and whether it is sufficient to ensure appropriate attachment of bone grafts. In the present study, we used fibrin gel with the adhesive property to reinforce the platelet gel structure and to deliver growth factors. We hypothesized that the platelet gel/fibrin glue composite (platelet glue) would increase fusion rates in posterolateral lumbar fusion. Methods The control group consisted of 33 consecutive patients who received instrumented posterolateral lumbar fusion with artificial bone expander and laminectomy autograft. Thirty-four patients in the study group were treated as above with the additional platelet glue. There was no significant difference between 2 groups in the demography of patients. The amount of postoperative drainage on the first and second day was recorded. Fusion rates were also assessed. Diagnosis of union was based on flexion-extension dynamic lateral radiography and fine-cut computerized tomography. All patients have been monitored for at least 2 years. Results The nonunion rate in the platelet glue group was 15% as compared with 10% in the control group. The mean postoperative drainage on the first and second day was 362 mL in the control group and 395 mL in the platelet glue group. There were no significant differences in either fusion rate or postoperative blood loss volume between the 2 groups. Conclusions In the present study, the use of a platelet gel/fibrin glue composite (platelet glue) could not be proved to increase fusion rates in instrumented lumbar posterolateral fusion. Further investigation is warranted to find an adequate carrier of growth factors for use in instrumented lumbar posterolateral fusion.