*5.2. JNK Kinetics in Osteoclast Metabolism*

Lissencephaly-1 (LIS1)-flox;LysM-Cre mice, in which LIS1, a key regulator of microtubules and the cytoplasmic dynein motor complex, is specifically deleted in myeloid cells relevant to osteoclast precursors, exhibit increased bone mass, due to defective osteoclast formation and bone resorption [124]. Consistent with these findings in vivo, osteoclast precursors derived from LIS1 conditional knockout mice exhibited impaired osteoclast formation and accelerated apoptotic cell death through the suppression of M-CSF-induced prolonged ERK activation and the induction of RANKL-induced prolonged JNK activation. Moreover, the ablation of RelA, a component of NF-κB, induced strong activation of JNK by RANKL and resulted in JNK-Bid-mediated apoptosis of osteoclast precursors [92]. Together, these results indicate that changes in the activities of ERK and JNK during M-CSF- and RANKL-mediated osteoclastogenic signaling regulate the apoptosis of osteoclast precursors.

#### *5.3. p38 Kinetics in Osteoclast Metabolism*

p38α-flox;LysM-Cre mice exhibit bone defects in an age-dependent manner, displaying osteopetrosis at 2.5 months and osteoporosis at 6 months of age [93]. When compared with the differentiation of osteoclast precursors obtained from 2.5-month-old wild-type mice, osteoclast precursors isolated from age-matched p38α-deficient mice showed increased osteoclast formation at low cell density, but decreased osteoclast formation at high cell density. Hotokezaka et al. suggested that ERK inactivation induces RANKL-induced strong p38 activation and positively regulates osteoclastogenesis via the inhibition of ERK-mediated osteoclast precursor proliferation [125]. We also reported that p38 activation via the RANKL-RANK-TRAF6 axis leads to a shift from proliferation to differentiation in osteoclast precursors [8]. Therefore, the positive role of p38 in RANKL-, but not M-CSF-induced osteoclastogenesis seems to be differential in osteoclast formation and bone remodeling, according to spatial conditions of cell-cell confluency and physiological development stage, respectively.

#### *5.4. Crosstalk between ERK and p38 in Osteoclast Metabolism*

ERK inactivation by PD98059, a specific MEK inhibitor, suppressed serum-stimulated proliferation of SaOS-2 human osteosarcoma cells, but stimulated the osteogenic differentiation of these cells via accelerated p38 activation [126]. This phenomenon could be explained by a competition and balancing of p38-induced cell differentiation and MEK/ERK-mediated cell proliferation. In accordance herewith, ERK inactivation in osteoclast precursors by treatment with MEK inhibitors (U0126 and PD98059) elevated RANKL-induced p38 activation and resulted in enhanced osteoclast [125]. In addition, treatment of osteoclast precursors with p38 inhibitors (SB203580 and PD169316) induced an increase in RANKL-induced ERK activation and led to decreased osteoclast differentiation. These results suggest that MEK/ERK and p38 pathway may be involved in the suppression and induction of osteoclastogenesis, respectively, by regulating a seesaw-like crosstalk between ERK and p38 MAPK signaling. Of note, PD98059 and U0126 were found to show off-target effects on the MEK5/ERK5 pathway at higher concentrations, suggesting the possibility that ERK5 may contribute to some of the roles ascribed to ERK1/2 in osteoclastogenesis [127].

#### **6. Conclusions**

M-CSF and RANKL act as osteoclastogenic key regulators in normal osteoclast metabolism and share ERK, JNK, and p38 as signal mediators, but exhibit differences in the extent and duration of activation and MAPK isoform specificity. Moreover, M-CSF induces monophasic activation with an immediate phosphorylation (5 to 20 min) of MAPKs; distinctively, RANKL leads to biphasic activation with both immediate (5 to 20 min) and delayed phosphorylation (8 to 24 h) of MAPKs. The timing of RANKL-induced delayed MAPK activation coincided with the onset of osteoclast differentiation. Thus, the differential MAPK signaling induced by M-CSF and RANKL is recognized to determine the osteoclast precursor proliferation and osteoclast differentiation, respectively (Figure 4) [8]. JNK and p38 activated via RANKL-RANK signaling predominantly mediate osteoclastic apoptosis and promote osteoclast differentiation and function, respectively, whereas ERK activation via M-CSF/c-Fms axis preferentially potentiates osteoclast precursor proliferation [4,6–9,128,129]. Because p38 signaling is more tightly connected to the control of osteoclast metabolism than ERK and JNK signaling, researchers have tried to apply p38 inhibitors to prevent periopathogen-induced periodontal and active alveolar bone loss with degradation of mineralized and non-mineralized tooth tissues [130–132] and to treat rheumatoid arthritis with synovial inflammation, overactive osteoclast function, cartilage degradation, and bone erosion [133]. Although p38 is currently considered as a potential therapeutic target for inflammation-mediated bone loss [134], osteoclast-specific indirect regulators of p38 rather than direct p38 inhibitors should be developed to avoid side effects to other tissues and cells. Further studies are needed to clarify the detailed molecular mechanism underlying the crosstalk between MAPKs and the regulation of MAPKs by the balancing of kinases and phosphatases, and to explore the roles of specific isoforms of JNK1/2/3 and co-modulators capable of tuning p38 MAPK cascades in osteoclast metabolism.

**Figure 4.** Osteoclast precursor proliferation by macrophage colony stimulating factor (M-CSF)/c-Fms-mediated monophasic activation of MAPKs and osteoclast differentiation by receptor activator for nuclear factor κ-B ligand (RANKL)/RANK-mediated biphasic activation of MAPKs. Arrows indicate activation of the signaling pathways, a solid line indicates the plasma membrane, and a dotted line indicates the nuclear membrane of osteoclast precursors. Green color means the signaling pathway induced by M-CSF and red color means the signaling pathway induced by RANKL.

**Author Contributions:** All authors contributed equally to this work. K.L., I.S., M.H.C., and D.J. formulated the theme, outline of the review, and wrote the manuscript.

**Funding:** This work was supported by grants from the National Research Foundation of Korea (Nos. 2016R1A2B2012108, 2015R1A5A2009124, and 2016R1A6A3A11930818).

**Conflicts of Interest:** The authors declare no conflict of interest.
