ECTS Abstracts (2015) 1 P209

Osteoclast-associated receptor (OSCAR) gene expression and protein release by human peripheral blood derived osteoclast cells in response to RANKL +/- TNF-[alpha]

Kent Algate1, Anak Dharmapatni1, Roxanne Coleman1, Michelle Lorimer1, Andrew Zannettino1, Melissa Cantley1, David Haynes1, Mihir Wechalekar2,3, Malcolm Smith2,3 & Tania Crotti1

1University of Adelaide, Adelaide, SA, Australia; 2Repatriation General Hospital, Daw Park, SA, Australia; 3Flinders University, SA, Australia.

Osteoclast-associated receptor [OSCAR] is a co-stimulatory molecule involved in osteoclast differentiation. Increased protein expression is present in synovial tissues of Rheumatoid Arthritic joints and in soft tissues adjacent to sites of peri-implant osteolysis. Soluble OSCAR is detectable in serum and synovial fluid but its role in the regulation of bone erosion is not clear. This study assessed the effect of RANKL with/out TNF-α?on the mRNA expression and protein release of OSCAR by human peripheral blood (PBMC)-derived osteoclasts. Human PBMCs pre-cultured with MCSF for 7 days were differentiated into osteoclasts with 10 ng/ml or 50 ng/ml RANKL over 10 days. TNF-α was added to 10 ng/ml RANKL treated cultures; and/or at 1 or 3 days post RANKL exposure. Media was changed every 3-4 days. After 7 days with RANKL; supernatants were assessed for OSCAR levels by ELISA, cells were stained for TRAP or RNA extracted for RT QPCR. After 10 days differentiation dentine slices were assessed for resorption pits by Scanning Electron Microscopy (SEM). Soluble OSCAR was produced by PBMC derived osteoclast cells in response to RANKL with and without TNF-α?with 50ng/ml RANKL compared with 10 ng/ml. The highest levels of OSCAR were detected when TNF-α was added pre-RANKL and on day 1. Consistent with this, OSCAR mRNA expression was significantly higher when TNF-α was added pre-RANKL and on day 1 compared with TNF-α pre-treatment only (p=0.0183) and TNF-α from day 3 (p=0.0381). The greatest resorption was observed with the TNF-α pre-treatment and RANKL at 10 ng/ml, and this was significantly greater than RANKL 10 ng/ml (p=0.0370) and RANKL 50 ng/ml (p=0.0499) without TNF-α. TRAP positive cells, osteoclast gene expression and resorption pits were evident with all treatments confirming the presence of active osteoclasts. OSCAR expression and release by osteoclast cells is mediated by RANKL and TNF-α?influenced by timing of exposure.

Disclosure: The authors declared no competing interests. This work was supported by Alan and Beryl Stephens Grant, Arthritis Australia.