br Materials and methods br Results br Discussion FFAR
Materials and methods
Discussion FFAR4 is a G-protein coupled free fatty Valproic acid sale receptor that has been reported to be expressed in osteoclasts and osteoblasts . In this study the role of FFAR4 on the effects of different classes of UFAs, the ω−6 PUFA, AA, the ω−3 PUFAs, DHA and EPA, the ω−7 and ω−9 MUFAs, PLA and OA respectively, were investigated on osteoclast and osteoblast cell lines. Osteoclast and osteoblast formation was evaluated as well as the activity of several signalling pathways. The purpose of this study was to elucidate the role of FFAR4 in the effects of different classes of fatty acids in bone cells. Similar to Oh et al. , we report that RAW264.7 murine macrophages express high levels of FFAR4, βarr2 and Gαq (Fig. 1C). Previous studies have shown that FFAR4 activation can inhibit osteoclast formation through inhibition of NF-κB and MAPK signalling pathways [29,30]. In vivo studies further revealed that in the presence of high levels of ω−3 fatty acids, FFAR4 activation stimulated bone formation while suppressing bone resorption . TUG891, a synthetic FFAR4 agonist, and all the UFAs used in this study inhibited RANKL-induced osteoclast formation in RAW264.7 murine macrophages (Fig. 1A). However, in the absence of FFAR4, TUG891, DHA, EPA and PLA did not reduce osteoclast formation (Fig. 1A). Silencing of the FFAR4 or βarr2 prevented the inhibitory effects of TUG891, DHA, EPA and PLA on the NF-κB and MAPK signalling pathways (Fig. 2, Fig. 3) as well their inhibitory effects on the formation of the TAK1-TAB1 complex (Fig. 3). These results indicate that TUG891, DHA, EPA and PLA may require the FFAR4-βarr2 pathway to exert their anti-osteoclastogenic effects. It may be suggested that UFAs that are naturally present in the FBS may contribute to the effects described. In this present study, all data was normalized to the vehicle control. However, when looking at the absolute values for the cell count (data not shown), there was no difference in cell numbers between control shRNA and FFAR4 shRNA cells in the vehicle treated cells. Therefore, any effect of the UFAs present in the FBS was deemed to be negligible. Oh et al. reported that the FFAR4-βarr2 signalling pathway was crucial to the anti-inflammatory effects of DHA in RAW264.7 macrophages . Interestingly, these researchers also noted that DHA, EPA, PLA and OA could activate FFAR4 while AA and saturated fatty acids could not . In the present study, FFAR4 silencing did not prevent the anti-osteoclastogenic effects of AA, and similarly did not prevent the anti-osteoclastogenic effects of OA, a known activator of the FFAR4 (Fig. 1A). However, this may indicate that AA and OA may use alternative pathways to influence osteoclast formation. Similar to FFAR4, FFAR1 preferentially binds medium to long chain UFAs and AA and OA has been shown to activate FFAR1 in rat islet beta cells [18,32,33]. Furthermore, RAW264.7 murine macrophages were shown to express FFAR1 and activation of this receptor inhibited RANKL induced osteoclast formation . Further studies are needed to investigate whether or not FFAR1 may mediate the anti-osteoclastogenic effects of AA and OA. We further report that MC3T3-E1 murine pre-osteoblasts were also shown to express FFAR4, βarr2 and Gαq (Fig. 4B). Gao et al. have reported that activation of FFAR4 can promote mineralization and osteoblast gene expression in bone marrow mesenchymal stem cells . In the present study, TUG891, DHA, EPA, PLA and OA were shown to promote the expression of pro-osteoblast genes in MC3T3-E1 pre-osteoblasts through FFAR4 (Fig. 4). OPG acts as a decoy receptor for RANKL thereby inhibiting osteoclast differentiation . A low OPG/RANKL ratio will result in increased osteoclast formation and resorption whereas a high OPG/RANKL would decrease osteoclasts and resorption. The expression of OPG was increased by TUG891, DHA, EPA, PLA and OA in control and Gαq negative MC3T3-E1 cells. However, these effects were lost in FFAR4 or βarr2 silenced MC3T3-E1 cells (Fig. 4). RANKL expression remained unchanged when exposed to any of the compounds in control plasmid, FFAR4 negative, βarr2 negative or Gαq negative cells. These results are similar to Casado-Dìaz et al. who reported that, after 7 days, DHA and EPA increased OPG expression while RANKL expression was unchanged in mesenchymal stem cells stimulated with osteogenic media . This led to an increase in the OPG/RANKL ratio. In the present study, we reported increases in the OPG/RANKL ratio in control plasmid cells when exposed to TUG891, DHA and PLA (Fig. 4). However, when FFAR4 or βarr2 was silenced, TUG891, DHA and PLA showed no effect on the OPG/RANKL ratio.