Differential modulation of RANKL isoforms by human osteoarthritic subchondral bone osteoblasts: influence of osteotropic factors.
BACKGROUND
Osteoarthritis (OA) is the most common human joint disease. Recent studies suggest that an abnormal subchondral bone metabolism is intimately involved in the genesis of this disease. Bone remodelling is tightly regulated by a molecular triad composed of OPG/RANK/RANKL. RANKL exists as 3 isoforms: RANKL1, 2, and 3. RANKL1 and 2 enhance osteoclastogenesis whereas RANKL3 inhibits this phenomenon. We previously reported that human OA subchondral bone osteoblasts can be discriminated into two subgroups according to their level of PGE2 [low (L) or high (H)]. Moreover, we also showed that L-OA osteoblasts express higher levels of total RANKL compared to H-OA osteoblasts. In this study, we investigated the level of membranous RANKL, comparing L- and H-OA subchondral bone osteoblasts, as well as its modulation by osteotropic factors. The impact of the modulation of RANKL1 and 3 on the membranous RANKL level was also studied.


METHODS
Gene expression was determined using real-time PCR for RANKL1 and semi-quantitative PCR for RANKL3. Membranous RANKL was measured by flow cytometry. The modulation of membranous RANKL and RANKL isoforms was monitored on the L- and H-OA osteoblasts and also following treatment with osteotropic factors, including vitamin D3 (50 nM), IL-1beta (100 pg/ml), TNF-alpha (5 ng/ml), PGE2 (500 nM), PTH (100 nM), IL-6 (10 ng/ml) and IL-17 (10 ng/ml).


RESULTS
Membranous RANKL levels were significantly increased in L-OA osteoblasts compared to normal (p<0.01) and H-OA (p<0.05). The gene expression level of the RANKL1 profile was reminiscent of the membranous RANKL level. Although RANKL3 gene expression was lower on the H-OA osteoblasts than on normal and L-OA osteoblasts (p<0.03), the overall outcome favoured RANKL1. Treatment with the tested factors showed a significant increase in membranous RANKL on the L-OA osteoblasts, with the exception of PTH and IL-17. Interestingly in this subpopulation, the RANKL3 gene expression level was significantly increased upon PTH and IL-17 treatment. No effect of the tested osteotropic factors was found on the H-OA.


CONCLUSIONS
Our findings showed that the normal, L- and H-OA subchondral bone osteoblasts differentially express membranous RANKL and RANKL isoforms, and that treatment with osteotropic factors generally favours increased membranous localization of RANKL on L-OA compared to H-OA osteoblasts. This phenomenon appears to take place through differential modulation of each RANKL isoform.
Osteoarthritis Research Unit, University of Montreal Hospital Centre, Notre-Dame Hospital, 1560 Sherbrooke Street East, Montreal, Quebec, Canada H2L 4M1.
Steeve Kwan
SK
Tat
Osteoarthritis Research Unit, University of Montreal Hospital Centre, Notre-Dame Hospital, 1560 Sherbrooke Street East, Montreal, Quebec, Canada H2L 4M1.
Jean-Pierre
JP
Pelletier
Osteoarthritis Research Unit, University of Montreal Hospital Centre, Notre-Dame Hospital, 1560 Sherbrooke Street East, Montreal, Quebec, Canada H2L 4M1.
Daniel
D
Lajeunesse
Osteoarthritis Research Unit, University of Montreal Hospital Centre, Notre-Dame Hospital, 1560 Sherbrooke Street East, Montreal, Quebec, Canada H2L 4M1.
Hassan
H
Fahmi
Pavillon des Charmilles, 1487 boulevard des Laurentides, Vimont, Quebec, Canada H7M 2Y3.
Nicolas
N
Duval
Osteoarthritis Research Unit, University of Montreal Hospital Centre, Notre-Dame Hospital, 1560 Sherbrooke Street East, Montreal, Quebec, Canada H2L 4M1. Electronic address: jm@martelpelletier.ca.
Johanne
J
Martel-Pelletier
D000368:Aged; D016723:Bone Remodeling / Q000187:drug effects; D001842:Bone and Bones / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D016207:Cytokines / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008566:Membranes / Q000187:drug effects; D010003:Osteoarthritis / Q000473:pathology*; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D053245:RANK Ligand / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D016207:Cytokines; D020033:Protein Isoforms; D053245:RANK Ligand; D012333:RNA, Messenger