Effect Of α-MSH On Osteoclastogenesis And Signaling Pathway

ObjectiveTo investigate the effect and mechanism of a-Melanocyte stimulating hormone (a-MSH) on osteoclastogenesis. And to find out the key site of a-MSH on osteoclas-togenesis by studying the signaling pathway in this process.Methods1. RT-PCR was used to determine the type of melanocortin receptors in Raw264.7cells.2. Raw264.7cells were divided into Negative control group、Receptor activator of NF-kappa B ligand (RANKL) group、α-MSH group and RANKL+a-MSH group, and observed by tartrate resistant acid phosphatase (TRAP) staining; The activity of Acid phosphatase was assayed after Raw264.7cells were stimulated in different groups;3. Raw264.7cells were treated with a-MSH and its analogues respectively, and then observed by TRAP staining;4. Raw264.7cells were treated with RANKL、α-MSH and RANKL+a-MSH respectively, and phosp-c-Jun N-terminal kinases(p-JNK) were observed after Raw264.7celld were stimulated at0min、15min、60min by western blot.Result1. RT-PCR showed that MC1R～MC5R were expressed in Raw264.7cells;2. Osteoclasts were induced in the RANKL group and RANKL+a-MSH group, and the number of osteoclasts was significantly increased in the RANKL+a-MSH group (p<0.05);3. The number of TRAP-positive multinucleated cells were increased in a dose dependent manner after using different concentrations of a-MSH (10-6M～10-10M) to deal with the Raw264.7cells.4. Raw264.7cells were treated with a-MSH and its analogues (SHU9119、PT141and THIQ), the number of osteoclasts was significantly increased in the a-MSH group and SHU9119group, interestingly, the number of osteoclasts in the SHU9119+a-MSH group were more than the former two groups(p<0.05). Osteoclasts were significantly increased in the PT141group(p<0.05), but it was the same as the RANKL group(p>0.05).There was no difference between the RANKL group and THIQ group on osteoclastogenesis(p>0.05).5. Compared with the negative control group and a-MSH group, the activity of acid phosphatase was significantly increased in the RANKL group and a-MSH+RANKL group (p<0.05), but there was no difference in these two groups(p>0.05).6. In the western blot, the JNK signaling pathway was obviously activated in the a-MSH group and RANKL+a-MSH group.Conclusiona-MSH might bind with MC1R and/or MC5R to promote the fusion of pre-osteoclasts to increase osteoclastogenesis, which process might through JNK signaling pathway.