Molecular regulatory mechanisms of osteoclastogenesis through cytoprotective enzymes

It has been reported that reactive oxygen species (ROS), such as hydrogen peroxide and superoxide, take part in osteoclast differentiation as intra-cellular signaling molecules. The current assumed signaling cascade from RANK to ROS production is RANK, TRAF6, Rac1, and then Nox. The target molecules of ROS in RANKL signaling remain unclear; however, several reports support the theory that NF-κB signaling could be the crucial downstream signaling molecule of RANKL-mediated ROS signaling. Furthermore, ROS exert cytotoxic effects such as peroxidation of lipids and phospholipids and oxidative damage to proteins and DNA. Therefore, cells have several protective mechanisms against oxidative stressors that mainly induce cytoprotective enzymes and ROS scavenging. Three well-known mechanisms regulate cytoprotective enzymes including Nrf2-, FOXO-, and sirtuin-dependent mechanisms. Several reports have indicated a crosslink between FOXO- and sirtuin-dependent regulatory mechanisms. The agonists against the regulatory mechanisms are reported to induce these cytoprotective enzymes successfully. Some of them inhibit osteoclast differentiation and bone destruction via attenuation of intracellular ROS signaling. In this review article, we discuss the above topics and summarize the current information available on the relationship between cytoprotective enzymes and osteoclastogenesis.

In this review article, we have summarized the relationship between osteoclastogenesis and the protective mechanisms that work against oxidative stressors.
It is reported that TRAF6 plays a key linkage role in ROS production by RANKL [39]. We reported that dominant-interfering mutant form of TRAF6, significantly decreased ROS induction, although TRAF6 itself does not directly produce ROS. Rac, a functional downstream molecule and member of the Rho-GTPase subfamily, which is involved in the organization of the cytoskeleton, is a cytosolic component of NADPH oxidase (NOX) complex and responsible for the activation of NOXs [40]. The expression of a dominant-negative mutant of Rac1 blocks ROS production, signifying that Rac1 is responsible for regulating the generation of ROS during osteoclast differentiation [41]. In addition, NOXs have been reported as essential enzymes that produce ROS during osteoclast differentiation [42][43][44]. Taken together, the current assumed signaling cascade from RANK to ROS production is RANK, TRAF6, Rac1, and then NOX.
The target molecules of ROS in RANKL signaling remain unclear; however, several reports have suggested that MAPK, PI3K, and NF-kB activation are downstream events [45,46]. Additionally, Bharti et al. reported that curcumin, which has ROS-scavenging properties, inhibits RANKL-induced NF-κB activation, which indicates that NF-κB signaling could be the crucial downstream signaling molecule of RANKL-mediated ROS signaling [47]. Current information about the intracellular signaling cascade of RANKL is summarized in Fig. 1.

Defense mechanisms against ROS
As mentioned previously, ROS exhibit cytotoxicity [7,8]; therefore, cells have several protective mechanisms against these oxidative stressors that mainly induce cytoprotective enzymes and ROS scavenging. The mechanisms regulating cytoprotective enzymes are summarized in Table 1.
Sirtuin, which was originally identified as a protein deacetylase [59], is also a regulator of the expression of cytoprotective enzymes such as SOD [60,61] and CAT (Fig. 4) [62]. Mammalian sirtuins consist of seven members (SIRT1-7), and have been implicated in various cellular responses including aging, transcription, apoptosis, and stress resistance [63]. Among them, the functions of Sirt1 and 3 in oxidative stress responses have been reported. SIRT1 deacetylates FOXO3 and 4 [53], which results in the upregulation of Mn-SOD [64]. Furthermore, Olmos et al. reported that SIRT1-dependent upregulation of cytoprotective enzymes depended on the formation of a FOXO3a/PGC-1α complex in vascular endothelial cells [65]. However, Chen et al. reported that SIRT3 directly upregulated SOD2 [60]. Regarding the crosslinking between SIRT and Nrf2, Huang et al. reported that SIRT1 upregulated HO-1 and SOD1 via induction of Nrf2 [66]. Overall, sirtuins, especially SIRT1 and 3, directly or indirectly regulate cytoprotective enzymes.

Cytoprotective enzymes and osteoclastogenesis
Since ROS operate as intracellular signaling molecules during osteoclastogenesis, a close relationship between osteoclastogenesis and cytoprotective enzymes is to be expected. Indeed, a wellknown cytoprotective enzyme, HO-1, is a negative regulator of osteoclastogenesis [67][68][69]. Relationships between the mechanisms regulating cytoprotective enzymes and osteoclastogenesis have also been reported. Rana et al. reported that loss of Nrf2 accelerates ionizing radiation-induced bone loss in Nrf2 knockout mice [70]. Other groups have reported that Nrf2 negatively regulates osteoclastogenesis through attenuation of RANKLmediated intracellular ROS signaling by cytoprotective enzymes [71,72]. Furthermore, we previously reported that overexpression of Nrf2 induces the expression of cytoprotective enzymes, attenuates intracellular ROS signaling, and thereby inhibits osteoclastogenesis [71]. Both overexpression of Nrf2 and Nrf2 activation (induction of nuclear translocation) inhibit osteoclastogenesis [6,73,74]. These lines of evidence suggest that Nrf2 activation could be a therapeutic approach towards bone destructive diseases such as rheumatoid arthritis, osteoporosis, and periodontitis.
Another mechanism regulating cytoprotective enzyme FOXO contributes to the control of osteoclastogenesis. Bartell et al. reported that FOXO protein attenuates osteoclastogenesis via augmentation of cytoprotective enzymes [75]. Sirtuins, originally identified as protein deacetylases, have been reported as suppressors of osteoclastogenesis. SIRT1 suppresses osteoclastogenesis by the upregulation of cytoprotective enzymes via FOXOmediated transcription and subsequent attenuation of intracellular ROS signaling [76]. Lee et al. reported that the overexpression of SIRT6, an NAD (þ )-dependent deacetylase, suppresses bone destruction in a collagen-induced arthritis mouse model [77]. These lines of evidence suggest that the key molecule among the mechanisms regulating cytoprotective enzymes (Nrf2, FOXO, and Fig. 1. Summary of the current information about the intracellular signaling cascade of RANKL. Intracellular signaling molecules after RANK were identified. The current assumed signaling cascade from RANK to ROS production is also described. Some reports suggest that NF-κB is the crucial downstream molecule of RANKLmediated ROS signaling. sirtuin) negatively regulates osteoclastogenesis via attenuation of intracellular ROS signaling (Fig. 5).

Regulatory mechanisms of potential pharmacological targets for bone destructive diseases
As discussed above, osteoclasts also possess mechanisms that regulate cytoprotective enzymes, which manage the intracellular ROS levels. Since intracellular ROS play a role in RANKL-mediated osteoclastogenesis, the mechanisms that regulate cytoprotective enzymes negatively control osteoclastogenesis via ROS scavenging mediated by cytoprotective enzymes. In other words, osteoclastogenesis is controlled via interference with the mechanisms regulating cytoprotective enzymes.

Summary and perspective
In this review manuscript, we have summarized recent information about the relationship between osteoclastogenesis and the mechanisms regulating cytoprotective enzymes. Although some parts have been extensively explored, further investigations   are necessary to gain a greater understanding. In particular, crosstalk among the mechanisms regulating cytoprotective enzymes and other signaling molecules should be elucidated.
Since some of the agonists that affect the mechanisms regulating cytoprotective enzymes have been reported as inhibitors of bone destruction, these chemicals could be potential drugs for the treatment for bone destructive diseases in the near future.  [94] SRT3025 [92]