*4.4. Phosphatase Regulation of p38 Signaling in Osteoclasts*

Mice lacking *DUSP1* exhibit drastic osteoclast activation in response to local LPS injection, and osteoclast precursors derived from *DUSP1*−/<sup>−</sup> mice show increased cell-cell fusion to multinucleated osteoclasts and osteoclastic bone-resorptive activity [119]. Further, DUSP1 is expressed by RANKL stimulation, is localized into the nucleus, and preferentially dephosphorylates the threonine and tyrosine residues of activated p38 and JNK over those of ERK, thus inducing transient p38 and JNK activation in response to stress and negatively regulating osteoclast formation and function by inactivating p38 MAPK-dependent signaling [13,119].

#### **5. Distinct Kinetics of MAPKs and Crosstalk between MAPKs**

The strength and duration of the response of MAPKs to different exogenous stimuli determines the biological outcome of the response. For instance, epidermal growth factor-induced transient ERK activation via the Raf-MEK-ERK axis induces proliferation in PC12 neuroendocrine cells, whereas nerve growth factor-induced prolonged ERK activation via the Raf-MEK-ERK axis leads to the differentiation of PC12 cells into sympathetic neuron-like cells [120,121]. Interestingly, prolonged ERK activation by epidermal growth factor in PC12 cells overexpressing the epidermal growth factor receptor switches cell fate from proliferation to differentiation [120]. Thus, cell fate decision is regarded a consequence of the duration of ERK activation. A recent report indicated that in osteoclast precursors, MAPK signaling induced by M-CSF or RANKL differed in terms of the extent and duration of ERK, p38, and JNK phosphorylation, as well as the selective phosphorylation of JNK isoforms [8]: (i) M-CSF induced more pronounced and sustained ERK phosphorylation than RANKL, (ii) RANKL induced more and longer p38 phosphorylation than M-CSF, (iii) M-CSF favorably phosphorylated JNK1 rather than JNK2 or JNK3, whereas RANKL had no such preference, and iv) M-CSF induced immediate monophasic activation (5 to 20 min) of MAPKs, whereas RANKL induced biphasic immediate (5 to 20 min) and delayed activation (8 to 24 h) of MAPKs. The different kinetics of MAPK activation by M-CSF or RANKL are considered to be related to cell fate decision of osteoclast precursor proliferation or differentiation [8].

#### *5.1. ERK Kinetics in Osteoclast Metabolism*

RANKL induces sustained ERK phosphorylation and maintains increased protein levels of c-Fos, resulting in enhanced osteoclastogenesis [53]. In contrast, the TLR9 ligand CpG-ODN leads to the transition of RANKL-induced sustained ERK activation into transient ERK activation by enhancing the expression of phosphatase PP2A, thereby decreasing the c-Fos level by degrading c-Fos mRNA and protein, and consequently inhibiting osteoclastogenesis. Moreover, when osteoclast precursors were pretreated with okadaic acid, a phosphatase inhibitor, RANKL-induced transient ERK activation by CpG-ODN was reverted to persistent activation, consequently inducing c-Fos expression. Additionally, 17β-estradiol triggers osteoclast apoptosis via transient ERK activation, peaking at 5 min after

estrogen administration and returning to the basal level by 30 min, but blocks osteoblast apoptosis via long-lasting ERK phosphorylation for at least 24 h [122,123]. The opposite effects of ERK activation on apoptosis were accounted as a result of the differential duration of ERK phosphorylation in the osteoclasts and osteoblasts [122,123]. Collectively, the switch between RANKL-induced transient and persistent ERK activation is able to be regulated by tuning the activity of phosphatase and TLR-mediated signaling in the osteoclast precursors or mature osteoclasts.
