Effect Of NRAGE On Osteoclast Differentiation And Its Mechanism

Bone metabolism homeostasis is maintained by osteoblasts and osteoclasts. Bone turnover is activated by osteoclast. Most of osteoporosis were related with osteoclast over-differentiation and enhanced activity. The control mechanism of osteoclast is very complex, multiple signaling pathways involved in a variety of receptors, adapter proteins, transcription factors, etc. Which have been proved to be directly or indirectly regulate the differentiation of osteoclasts. NRAGE (neurotrophin receptor-interacting MAGE homolog) is the melanoma-associated antigen. It has been proved that NRAGE could interact with different signaling pathways to play a role in cell survival, proliferation, apoptosis and differentiation as an intracellular adapter protein. In our previous study, the osteoclast differentiation of NRAGE knock-out mouse may be significantly promoted, but the mechanism is not clear. In view of this, two parts of this paper from the direct effects (RANKL and M-CSF-induced osteoclast differentiation) and indirect effects (osteoblasts effect osteoclasts) the NRAGE impact osteoclasts differentiation and regulation mechanism.Part one:NRAGE effect osteoclasts directly and Mechanisms (1) In our previous experiments, NRAGE knock-out could promote osteoclast differentiation that induced by RANKL and M-CSF. This paper expand sample size(n=6). TRAP staining was used to reflect that NRAGE could regulate osteoclasts directly.(2) By Q-PCR technology, we analyzed two important transcription factors during osteoclasts differentiation, c-fos and NFATc1. The results showed that NRAGE knock-out could promote c-fos and NFATcl, after RANKL was used, the experiment team was promoted highly.(3) MAPK (ERK1/2, p38 and JNK) is a classic signaling pathway to promote the osteoclast differentiation. P38 and JNK signaling pathway can upgrade c-fos and NFATc1 expression in osteoclasts. Meanwhile, in the study of apoptosis, NRAGE was shown to play a role in ERK1 /2, p38 and JNK signaling pathways. In view of this, we isolated knock-out group and wild-type group of bone marrow mononuclear cells, RANKL and M-CSF treatment different time periods (0,5,15 and 30 minutes) to collect proteins. Western blot analyzed the level of phosphorylation of ERK1/2, p38, and JNK. The results showed that in NRAGE knock-out group, the level of phosphorylation of ERK1/2, p38 and JNK did not significantly improve, indicate that the function that NRAGE promote osteoclast differentiation directly was not come via the MAPK (ERK1/2, p38 and JNK) pathway. NRAGE knockout upgrade of c-fos and NFATcl expression and osteoclast differentiation promoting effect may be through the NF-kappaB (already proved at Xu Lijuan’s paper), or not yet detected the pathway to achieve.Part two:NRAGE indirectly regulate osteoclast and MechanismsPreliminary experiments showed that, NRAGE could promote osteoblast differentiation and enhance osteoblast activity and thus promote osteoclast differentiation, but the mechanism is not clear. So this paper separate and purify newborn mouse calvarial cells, and osteogenic differentiation-inducing (ascorbic acid, the final concentration 50μg/mL+ beta-glycerophosphate, the final concentration of 10 mM) induced differentiation 7 days osteoblasts and non-induced differentiation of osteoblast gene expression profiling, Q-PCR validation showed that the gene chip reliability. FatiGO database on molecular function and biological process of the differentially expressed genes analyzed, and the literature review, NRAGE knock-out group compared with the wild group may be related to bone development or bone metabolism related genes,2-fold upgraded genes a 2-fold down-regulated genes months. Using Biocarta database differentially expressed genes signaling pathway analysis showed that differentially expressed genes were mainly concentrated on inflammatory cytokines and chemokines and the Wnt signaling pathway. Prompted by the gene chip, Wnt signaling pathway, inflammatory factors and chemokines, as well as classic RANKL/OPG ratio analysis NRAGE into bone cells on osteoclast differentiation knockout after the possible mechanism. The results showed that:(1) After Wnt5a stimulated NRAGE knockout group and wildtype group osteoclasts, cell differentiation did not change significantly, suggesting that Wnt5a-mediated signaling pathway is not involved in the process of osteoclast differentiation.(2) The expression of most inflammatory factors in NRAGE knockout group are higher than the wildtype group, and at earlier stage of osteogenic differentiation (DO) this upgrade becomes even more obvious, at later stage(D21), the expression of inflammatory factors gradually reduced, but knockout group was still significantly higher. This suggests that inflammatory factors may be involved in bone metabolism.(3) The ratio of RANKL/OPG is a important indicator for judgment whether osteoblasts is an important role in effects on osteoclast differentiation. Q-PCR and Western blot results showed that after NRAGE knockout, OPG expression in the whole process of osteogenic differentiation was significantly reduced, and RANKL in early osteogenic differentiation (DO) and late (D28) were upgraded, expression in d7 and d14 does not change significantly. The RANKL/OPG ratio was significantly upgraded in the NRAGE-/- osteoblast differentiation process, which may be the NRAGE-/- osteoblasts promote osteoclast differentiation is an important one of the mechanisms.The main conclusions:1. NRAGE knockout could promote osteoclasts differentiation significantly, the important transcription factor c-fos and NFATc1 were obviously upgraded. But this enhancement of differentiation and up-regulation of c-Fos and NFATc1 does not related to MAPK(ERK1/2, p38 and JNK)2. The higher ratio of RANKL/OPG is very important for osteoblast inducing osteoclasts differentiation.