We used RAW 264.7 cells to evaluate the direct effects of CRP on osteoclast differentiation. We carried out alkaline phosphatase (ALP) and bone nodule formation assays using MC3T3-E1 cells to evaluate osteoblast differentiation. Expression of osteoclast-specific and osteoblast-specific genes and effects on cell signaling pathways associated with cell differentiation were analyzed by reverse transcription polymerase chain reaction and Western blotting.
CRP significantly and dose-dependently inhibited TRAP-positive multinucleated cell formation in RANKL-induced RAW 264.7 cell cultures. We observed suppression of p38, ERK and AKT mitogen-activated protein kinases induced by RANKL in Western blots after CRP treatment of RAW 264.7 cells. CRP also suppressed ALP activity and mineralization by Alizarin red S staining of MC3T3-E1 cell cultures. CRP suppressed osteoclast-specific and osteoblast-specific genes. Furthermore, CRP increased interferon beta (IFN-β) mRNA expression and protein levels in RAW 264.7 and MC3T3-E1 cells, and these effects were suppressed by oxPAPC, an inhibitor of Toll-like receptor (TLR) signaling.
These data indicated that CRP may have a direct role on osteoclast and osteoblast differentiation via TLR signaling pathways.