CCL3 promotes angiogenesis by dysregulation of miR-374b/ VEGF-A axis in human osteosarcoma cells

Chondrosarcoma is the second most frequently occurring type of bone malignancy that is characterized by the distant metastasis propensity. Vascular endothelial growth factor-C (VEGF-C) is the chief lymphangiogenic mediator and makes crucial contributions to tumor lymphangiogenesis. Leptin is an adipocytokine and has been indicated to facilitate tumorigenesis, angiogenesis and metastasis. However, the effect of leptin on VEGF-C regulation and lymphangiogenesis in human chondrosarcoma has hugely remained a mystery. Our results showed a clinical correlation between leptin and VEGF-C as well as tumor stage in human chondrosarcoma tissues. We further demonstrated that leptin promoted VEGF-C production and secretion in human chondrosarcoma cells. The conditioned medium from leptin-treated chondrosarcoma cells induced lymphangiogenesis of human lymphatic endothelial cells. We also found that leptin-induced VEGF-C is mediated by the FAK, PI3K and Akt signaling pathway. Furthermore, the expression of microRNA-27b was negatively regulated by leptin via the FAK, PI3K and Akt cascade. Our study is the first to describe the mechanism of leptin-promoted lymphangiogenesis by upregulating VEGF-C expression in chondrosarcomas. Thus, leptin could serve as a therapeutic target in chondrosarcoma metastasis and lymphangiogenesis.

Synovium-induced angiogenesis is central to osteoarthritis (OA) pathogenesis and thus a promising therapeutic target. The adipokine apelin (APLN) is involved in both OA pathogenesis and angiogenesis. We examined the role of APLN in synovium-induced angiogenesis by investigating the crosstalk between APLN and vascular endothelial growth factor (VEGF) expression in human OA synovial fibroblasts (OASFs). We found higher levels of APLN and VEGF expression in OA samples compared with normal samples. APLN-induced stimulation of VEGF expression and VEGF-dependent angiogenesis in OASFs was mitigated by FAK/Src/Akt signaling. APLN also inhibited levels of microRNA-150-5p (miR-150-5p), which represses VEGF production and angiogenesis. Analyses of an OA animal model showed that shAPLN transfection of OASFs rescued pathologic changes in OA cartilage and histology. Here, we found APLN enhances VEGF expression and angiogenesis via FAK/Src/Akt cascade and via downstream suppression of miR-150-5p expression. These findings help to clarify the pathogenesis of adipokine-induced angiogenesis in OA synovium.

CCN3, otherwise known as the nephroblastoma overexpressed (NOV) protein, is a cysteine-rich protein that belongs to the CCN family and regulates several cellular functions. Osteoblasts are major bone-forming cells that undergo proliferation, mineralization, renewal, and repair during the bone formation process. We have previously reported that CCN3 increases bone morphogenetic protein 4 (BMP-4) production and bone mineralization in osteoblasts, although the role of CCN3 remains unclear with regard to osteogenic transcription factors (runt-related transcription factor 2 (Runx2) and osterix). Here, we used alizarin red-S and alkaline phosphatase staining to show that CCN3 enhances osteoblast differentiation. Stimulation of osteoblasts with CCN3 increases expression of osteogenic factors such as BMPs, Runx2, and osterix. Moreover, we found that the inhibition of miR-608 expression is involved in the effects of CCN3 and that incubation of osteoblasts with CCN3 promotes focal adhesion kinase (FAK) and Akt phosphorylation. Our results indicate that CCN3 promotes the expression of Runx2 and osterix in osteoblasts by inhibiting miR-608 expression via the FAK and Akt signaling pathways.

Inflammatory response and articular destruction are common symptoms of osteoarthritis (OA) and rheumatoid arthritis (RA). Leptin, an adipocyte-secreted hormone that centrally regulates weight control, may exert proinflammatory effects in the joint, depending on the immune response. Yet, the mechanism of leptin interacting with the arthritic inflammatory response is unclear. This study finds that leptin increased expression of oncostatin M (OSM) in human osteoblasts in a concentration- and time-dependent manner. In addition, OBRl, but not OBRs receptor antisense oligonucleotide, abolished the leptin-mediated increase of OSM expression. On the other hand, leptin inhibited miR-93 expression; an miR-93 mimic reversed leptin-increased OSM expression. Stimulation of osteoblasts with leptin promoted Akt phosphorylation, while pretreatment of cells with Akt inhibitor or siRNA reversed leptin-inhibited miR-93 expression. Our results showed that leptin heightened OSM expression by downregulating miR-93 through the Akt signaling pathway in osteoblasts, suggesting leptin as a novel target in arthritis treatment.

Over the last two decades, there have been no significant changes in patient outcomes in relation to the treatment of osteosarcoma, an aggressive malignant neoplasm. It is known that vascular endothelial growth factor-A (VEGF-A) plays a crucial role in angiogenesis and in osteosarcoma. Moreover, VEGF-A expression correlates with clinical stages of osteosarcoma. The adipokine resistin exhibits proinflammatory, proangiogenic and metastatic properties, and evidence suggests that resistin may serve as a prognostic biomarker linking obesity and inflammation to cancer. However, whether resistin has a role in osteosarcoma angiogenesis is unclear. This investigation shows that resistin promotes VEGF-A expression in human osteosarcoma cells and activates the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 signaling pathways, while ERK, JNK, and p38 inhibitors or their small interfering RNAs (siRNAs) inhibit resistin-induced VEGF-A expression as well as endothelial progenitor cell (EPC) migration and tube formation. We also found that resistin upregulates VEGF-A expression by enhancing activation of the transcription factor nuclear factor-kappa B (NF-κB). Finally, resistin promotes angiogenesis in the chick chorioallantoic membrane (CAM) model. Resistin appears to be a promising target for human osteosarcoma.