Expression of Receptor Activator of NF-kB Ligand (RANKL) and Formation of Osteoclast in Cultured Synovial Fibroblasts

Joo, Yeong Shil; Cheong, Myeong A; Sheen, Dong Hyuk; Lim, Mi Kyoung; Shim, Seung Cheol; Chung, Duke Whan

Abstract

OBJECTIVE
A number of soluble factors,which play important role in the pathophysiology of rheumatoid synovitis are also known to be involved in osteoclast differentiation and activation through RANKL (Receptor activator of NF-kB ligand). To investigate the importance of RANKL in the pathogenesis of bone erosion in rheumatoid arthritis (RA) patients, we analyzed the expression of RANKL and Osteoprotegerin (OPG) and examined the formation of osteoclasts in rheumatoid synovial fibroblasts under the influence of various osteotropic factors.
METHODS
Primary culture synoviocytes or fibroblast-like synoviocytes isolated from synovial tissues of 8 RA patients were cultured and treated with IL-1beta (2 ng/ml), TNF-alpha (2 ng/ml), INF-gamma(1000 micro/ml), IL-15 (10 ng/ml), IL-12 (10 ng/ml), dexamethasone (10(-9) M), PMA (10 ng/ml) or 1,25 (OH)2D3 (10(-9) M) for 18 hours. Expression RANKL or OPG mRNA was measured by semiquantitative RT-PCR within linear amplification condition. TRAP (+) MNC (tartrate resistant acid phosphatase-positive multinucleated cell) formation was induced from primary culture synoviocytes or in coculture system of synovial fibroblasts with PBMCs in the presence of M-CSF and 1,25 (OH)2D3.
RESULTS
1. The intensity of base-line expression was different from patient to patient. Primary culture synoviocytes and synovial fibroblasts express RANKL and OPG mRNA with decreasing intensity when they are passaged. 2. Expresssion of RANKL mRNA was significantly increased by 1,25 (OH)2D3 and IL-1beta (158.8+/-21% and 197.2+/-17% of controls, p<0.05 and p<0.005, respectively), while decreased significantly by dexamethasone (25.6+/-4.6% of controls, p<0.005). Expression of RANKL mRNA was significantly increased by IL-1beta and decreased by dexamethasone, in a dose- and time-dependant manner. 3. TRAP (+) MNCs are formed from primary culture synoviocytes or in coculture system of synovial fibroblasts and PBMC in the presence of M-CSF and 1,25 (OH)2D3. Dexamethasone clearly inhibited TRAP (+) MNCs formation from synovial cells.
CONCLUSION
The regulatory mechanism for the expression of RANKL or OPG in rheumatoid synoviocytes might be different from that in bone marrow cells. Modulating the expression of these molecules could have potential therapeutic implication targeting bone destruction in RA.