Long chain polyunsaturated fatty acids alter membrane-bound RANK-L expression and osteoprotegerin secretion by MC3T3-E1 osteoblast-like cells.
Inflammation triggers an increase in osteoclast (bone resorbing cell) number and activity. Osteoclastogenesis is largely controlled by a triad of proteins consisting of a receptor (RANK), a ligand (RANK-L) and a decoy receptor (osteoprotegerin, OPG). Whilst RANK is expressed by osteoclasts, RANK-L and OPG are expressed by osteoblasts. The long chain polyunsaturated fatty acid (LCPUFA) arachidonic acid (AA, 20:4n-6) and its metabolite prostaglandin E2 (PGE2), are pro-inflammatory and PGE2 is a potent stimulator of RANKL expression. Various LCPUFAs such as eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3) and gamma-linolenic acid (GLA, 18:3n-6) have anti-inflammatory activity. We aimed to determine if AA itself can stimulate RANKL expression and whether EPA, DHA and GLA inhibit RANKL expression in osteoblasts. MC3T3-E1/4 osteoblast-like cells were cultured under standard conditions with each of the LCPUFAs (5microg/ml) for 48h. Membrane-bound RANKL expression was measured by flow cytometry and OPG secretion measured by ELISA. In a second experiment, RANKL expression in MC3T3-E1/4 cells was stimulated by PGE2 treatment and the effect of EPA, DHA and GLA on membrane-bound RANKL expression and OPG secretion determined. The percentage of RANKL-positive cells was higher (p<0.05) than controls following treatment with AA or GLA but not after co-treatment with the cyclooxygenase inhibitor, indomethacin. DHA and EPA had no effect on membrane-bound RANKL expression under standard cell culture conditions. Secretion of OPG was lower (p<0.05) in AA-treated cells but not significantly different from controls in GLA, EPA or DHA treated cells. Treatment with prostaglandin E2 (PGE2) resulted in an increase (p<0.05) in the percentage of RANK-L positive cells and a decrease (p<0.05) in mean OPG secretion. The percentage of RANKL positive cells was significantly lower following co-treatment with PGE2 and either DHA or EPA compared to treatment with PGE2 alone. Mean OPG secretion remained lower than controls in cells treated with PGE2 regardless of co-treatment with EPA or DHA. Results from this study suggest COX products of GLA and AA induce membrane-bound RANKL expression in MC3T3-E1/4 cells. EPA and DHA have no effect on membrane-bound RANKL expression in cells cultured under standard conditions however both EPA and DHA inhibit the PGE2-induced increase in RANKL expression in MC3T3-E1/4 cells.
Poulsen
Raewyn C
RC
Institute of Food, Nutrition and Human Health, Massey University, Private Bag 11-222, Palmerston North 4442, New Zealand. R.C.Poulsen@massey.ac.nz
Wolber
Frances M
FM
Moughan
Paul J
PJ
Kruger
Marlena C
MC
D016475:3T3 Cells; D000818:Animals; D002453:Cell Cycle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology*; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D053244:Osteoprotegerin / Q000378:metabolism*; D053245:RANK Ligand / Q000378:metabolism*
D005231:Fatty Acids, Unsaturated; D053244:Osteoprotegerin; D053245:RANK Ligand; C505689:Tnfsf11 protein, mouse