The CCL2/CCR2 Axis Enhances Vascular Cell Adhesion Molecule-1 Expression in Human Synovial Fibroblasts

Background

Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), belongs to the CC chemokine family that is associated with the disease status and outcomes of osteoarthritis (OA). Here, we investigated the intracellular signaling pathways involved in CCL2-induced vascular cell adhesion molecule-1 (VCAM-1) expression in human OA synovial fibroblasts (OASFs).

Methodology/Principal Findings

Stimulation of OASFs with CCL2 induced VCAM-1 expression. CCL2-mediated VCAM-1 expression was attenuated by CCR2 inhibitor (RS102895), PKCδ inhibitor (rottlerin), p38MAPK inhibitor (SB203580), and AP-1 inhibitors (curcumin and tanshinone IIA). Stimulation of cells with CCL2 increased PKCδ and p38MAPK activation. Treatment of OASFs with CCL2 also increased the c-Jun phosphorylation and c-Jun binding to the AP-1 element on the VCAM-1 promoter. Moreover, CCL2-mediated CCR2, PKCδ, p38MAPK, and AP-1 pathway promoted the adhesion of monocytes to the OASFs monolayer.

Conclusions/Significance

Our results suggest that CCL2 increases VCAM-1 expression in human OASFs via the CCR2, PKCδ, p38MAPK, c-Jun, and AP-1 signaling pathway. The CCL2-induced VCAM-1 expression promoted monocytes adhesion to human OASFs.     

Cilostazol reduces MCP-1-induced chemotaxis and adhesion of THP-1 monocytes by inhibiting CCR2 gene expression

The chemotaxis and adhesion of monocytes to the injured endothelium in the early atherosclerosis is important. Cilostazol, a specific phosphodiesterase type III inhibitor, is known to exhibit anti-atherosclerotic effects mediated by different mechanisms. This study aimed to investigate the modulating effect of cilostazol on the MCP-1-induced chemotaxis and adhesion of monocytes. The gene expression of CCR2, the major receptor of MCP-1 in THP-1 monocytes, was also analyzed. The chemotaxis of monocytes toward MCP-1 was investigated using the transwell filter assay. Cilostazol dose-dependently inhibited the MCP-1-induced chemotaxis of monocytes which was shown to be cAMP-dependent. Using western blot analysis and flow cytometry method, we demonstrated the decrease of CCR2 protein at the cell membrane of monocytes by cilostazol treatment. Results from RT/real-time PCR confirmed the decrease of CCR2 mRNA expression by cilostazol which was also mediated by cAMP. Similar inhibition was also noted in human peripheral monocytes. The post-CCR2 signaling pathways including p44/42 and p38 MAPK were examined by western blot analysis. Result confirmed the inhibitory effect of cilostazol on the phosphorylation of p44/42 and p38 MAPK after MCP-1 stimulation. The activation of monocytes after MCP-1 treatment exhibited enhanced adhesion to vascular endothelial cells which was dose-dependently suppressed by cilostazol. Together, cilostazol was demonstrated, for the first time, to inhibit the CCR2 gene expression and MCP-1-induced chemotaxis and adhesion of monocytes which might therefore reduce the infiltration of monocytes during the early atherosclerosis. The present study provides an additional molecular mechanism underlying the anti-atherosclerotic effects of cilostazol.