## 4 Discussion
JNK regulates a variety of cellular functions including cell growth, differentiation, survival, and cell death [50; 51]. [PERSON] et al. [52] found that blocking of the JNK pathway by JNK inhibitors inhibits osteoclast differentiation and that JNK is an essential signaling molecule for osteoclast differentiation. However, another study found that a substance called glutaredoxin2 isoform b (Glrx2b) promotes the osteoclast differentiation process, but the expression of JNK remains unchanged [53]. Thus far, the effects of LLLT on MAPK and osteoclast formation have shown that JNK expression is not induced in osteoblasts or skeletal muscle cells after Er:YAG laser and He-Ne laser treatment, respectively [54]. We found that in RAW 264.7 cells not treated with RANKL, JNK was not phosphorylated after LLLT treatment. However, in the group to which LLLT was applied after treatment with RANKL, phosphorylation of JNK was found. Based on other studies and our own results, we conclude that JNK is not affected by LLLT when it is in the mononuclear form, whereas in the case of cells whose osteoclast differentiation signal is initiated by RANKL, it is affected by LLLT. Like JNK, p38 MAPK is important during the early stages of osteoclast formation and has been shown to induce cathepsin K gene expression [55], while LLLT has been reported to enhance p38 MAPK signaling in osteoblast pulp cells and fibroblasts [48; 54]. Research on osteoclast formation and p38 MAPK pathway by investigating LLLT is still lacking [56]. Collectively, our data suggest that LLLT stimulates osteoclast differentiation through the activation of ERK and the p38 pathways but not via JNK-containing pathways. Further studies will be needed to investigate the portions of the MAPK pathway that impact osteoclastogenesis after LLLT. c-Fos, which forms a heterodimer with c-Jun, resulting in the formation of the activator protein-1 (AP-1) complex, induces NFATc1 activation during osteoclastogenesis [57]. Mice lacking c-Fos develop osteoporosis as a primary pathology. It is rapidly and transiently induced within 15 min of stimulation in the fibroblast cell line by growth factors [58]. In this study, expression of c-Fos was maximal by 30 min post-LLLT treatment in the presence of RANKL. A variety of stimuli, including serum, growth factors, tumor promoters, cytokines, and UV radiation, are known to induce expression of c-Fos. Whether laser irradiation affects the expression of c-Fos or not, LLLT stimulates the formation of osteoclast-like cells via RANK expression. In our data, c-Fos expression was enhanced when treated with LLLT for 15 s. These data suggest that LLLT stimulates osteoclast differentiation through the c-Fos pathway. NFATc1 acts as a master modulator for osteoclast activation, fusion, and maturation [15]. The activation of NFATc1 is dependent on the transcription factors AP-1 (containing c-Fos) and NF-kB. NFATc1 translocates to the nucleus and induces various osteoclast-specific genes: TRAP, calcitonin receptor, and cathepsin K genes are all regulated by NFATc1 [59]. Taken together, these results indicate that the expression of NFATc1 and of osteoclast-specific genes is increased, suggesting that NFATc1 is activated by laser irradiation. The mechanism by which LLLT promotes osteoclast formation is still unclear, but researchers have suggested several hypotheses. The effect of LLLT on cells is closely related to mitochondria, and it has been reported that it is mediated by the cytochrome c oxidase present therein [60]. It has also been reported that stimulation of cell proliferation by LLLT may be due to activation of signaling pathways such as the MAPK cascade or the PI3K/Akt pathway, which regulate the regulation of gene expression [61]. [PERSON] et al. [9] used a 50 mW, 810 nm wavelength laser with a 5 mm diameter fiber. They treated osteoblasts and osteoclasts at the 810 nm wavelength band, but reported that neither treatment had an effect on cell differentiation. [PERSON] et al.