The role of Rho GTPases’ substrates Rac and Cdc42 in osteoclastogenesis and relevant natural medicinal products study

Abstract Recently, Rho GTPases substrates include Rac (Rac1 and Rac2) and Cdc42 that have been reported to exert multiple cellular functions in osteoclasts, the most prominent of which includes regulating the dynamic actin cytoskeleton rearrangements. In addition, natural products and their molecular frameworks have a long tradition as valuable starting points for medicinal chemistry and drug discovery. Although currently, there are reports about the natural product, which could play a therapeutic role in bone loss diseases (osteoporosis and osteolysis) through the regulation of Rac1/2 and Cdc42 during osteoclasts cytoskeletal structuring. There have been several excellent studies for exploring the therapeutic potentials of various natural products for their role in inhibiting cancer cells migration and function via regulating the Rac1/2 and Cdc42. Herein in this review, we try to focus on recent advancement studies for extensively understanding the role of Rho GTPases substrates Rac1, Rac2 and Cdc42 in osteoclastogenesis, as well as therapeutic potentials of natural medicinal products for their properties on the regulation of Rac1, and/or Rac2 and Cdc42, which is in order to inspire drug discovery in regulating osteoclastogenesis.


Introduction
Osteoclastogenesis has been defined as a multi-step processes of osteoclast differentiation [1], which including several osteoclastic cellular biological functions; such as: migration, cellular contact, cellular fusion and cellular responding to extracellular factors [2]. Document studies demonstrated that osteoclastogenesis initially mediated by two critical cytokines, the macrophage colony stimulating factor-1 (M-CSF) and the receptor activator of nuclear factor-kappaB ligand (RANKL) [3]. In that, M-CSF binds to its receptor (cFms) present in osteoclast precursors, which stimulates their proliferation and inhibits their apoptosis. While, RANKL interact with its receptor RANK in osteoclast precursor cells, osteoclastognesis is induced [4] (Figure 1).
However, at the late stage of osteoclastogenesis, osteoclastic polarization characterized the final maturation of bone resorptive osteoclasts. Notably, during the bone resorption process, osteoclastic polarization involves rearrangement of the actin cytoskeleton, in which an filamentous (F)-actin ring that comprises a dense continuous zones of highly dynamic podosomes are formed and consequently an area of membrane that develop into the ruffled border is isolated [5,6].

Cytoskeletal rearrangement during osteoclastogenesis
It is worthy to noted that during the cytoskeletal rearrangement among the osteoclastogenesis, podosome is the most prominent cytoskeletal structure for the degradation of mineralized bone matrix and associate to the mobility of osteoclast [7]. In fact, podosome is not exclusive organelle in osteoclast, which also include endothelial cells, and cells from the monocytic lineage such as: dendritic cells (DCs) and macrophages [8].
Regardless the presenting of podosomes in various cells, podosomes patterning play a crucial and unique role for the support osteoclast final maturation [8]. As early as individual podosome form within an osteoclast, they are collectively and sequentially organized into different patterns along the life of the same cell. However, these patterns evolve along with osteoclastogenesis from monocytes/macrophages to osteoclast precursors, further to the bone resorptive matured osteoclast. In the early stage of osteoclastogenesis, podosome pattern from apparently random groups of "clusters" to circle pattern "rings" in the middle term stage [9]. Eventually, in the late stage of osteoclastogenesis, podosome pattern into much massive circular structures, i.e., either "sealing zone like structures" (SZL, also known as "belts") or "sealing zones" (SZ) [10].

arrangement during osteoclastogenesis
It has been reported that Rac1 and Rac2 are critical GTPase to osteoclast formation and maturation. In fact. Rac1 and Rac2, are intimately associated with the organization of the different types of cellular cytoskeleton, such as: osteoclast, DCs and macrophages. Notably, these two isoforms are also involved in the osteoclastic adhesive function formation and subsequent bone resorption [11,12]. However, the specific role of Rac1 and Rac2 in osteoclastogenesis still unknown. For example, osteoclasts contain NADPH diaphorase activity [13,14], and free radicals which both could influence bone resorption, however, Rac1 and Rac2 are also essential components of NADPH oxidase [15][16][17][18], the enzyme responsible for generating free radicals. Besides that, study has also demonstrated Rac1 and Rac2 could through regulating the generation of reactive oxygen species (ROS) [19] and actin remodeling participating the osteoclastogenesis regulation. Recent study has found that both Rac1 and Rac2 are required for normal RANKL induced osteoclast differentiation, but Rac1 deletion results in a more profound reduction in osteoclast formation in vitro because of its regulatory role in pre-osteoclast M-CSF mediated chemotaxis and actin assembly and RANKL-mediated reactive oxygen species generation [20]. These results speculated that Rac1 and Rac2 might function osteoclastic organelle actin dynamics regulating, such as: actin filament ends and podosomes. In fact, Rac1 and Rac2

The role of Cdc42 in regulation the podosome of osteoclast
Cdc42 is another Rho family small GTPase [33]. As a downstream signaling of RANKL, Cdc42 might interact with the Crib domain of the adaptor Par3 [34,35], Par6 and atypical PKC (aPKC) [36][37][38], which forming a quaternary complex to cascade the upper signaling transduction from RANKL and RANK binding, further stimulate the osteoclastogenesis. However, unlike Rac1 and Rac2 the definition role of Cdc42 in osteoclastogenesis is much clearly associate with its actin regulative effects, i.e the podosome regulation. Recent studies using mice with increased Cdc42 activation due to knockout of its negative regulator Cdc42GAP have shown increased sealing zone formation and bone resorption, compared to wildtype cells [27,39].  [40,41,43]. The hydrophobic core is disrupted, releasing the VCA (Verprolin Homology domain-cofilin homology domain-Acidic region) domain and enabling its interaction with the Arp2/3 complex, thereby promoting actin nucleation [44][45][46].

Natural products that targeting on the regulation of Rac1&Rac2, and Cdc42
Natural products and their molecular frameworks have a long tradition as valuable starting points for medicinal chemistry and drug discovery.
Recently, there has been a revitalization of interest in the inclusion of these chemotypes in compound collections for screening and achieving selective target modulation. Although currently there has no report on the Downloaded from https://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20200407/885857/bsr-2020-0407.pdf by guest on 01 July 2020 natural product, which could play a therapeutic role on bone loss diseases (osteoporosis and osteolysis) that through the regulation of Rac1/2 and Cdc42 during osteoclasts cytoskeletal structuring, there have several excellent studies for exploring the therapeutic potentials of various natural products in regulating cancer cells migration and function ( Table   1). Here we collected several natural products with a focus on recent advances in their properties on the regulation of Rac1, and/or Rac2 and Cdc42, and related signaling molecular, which in order to inspire drug discovery in regulating osteoclastogenesis (Figure 2).
Most recently, Jacob et al. [50] have reported Fisetin showed a significant protective effect on developmental Methyl mercury neurotoxicity in the F1 generation of MeHg exposed rats. In that, Methyl mercury is a teratogenic and neurodevelopmental toxicant in the environment.

Cdc42 regulative natural products
Cudrania cochinchinensis (Moraceae) has been reported for its potent biological activites such as: anti-inflammation [63] and neuroprotective effects [64]. Whereas, the compound Cudraxanthone-S derived from Cudrania cochinchinensis was studied for its pharmacokinetics and binding potential in treating the fungal infection of Candida albicans, which could cause several lethal infections in immune-suppressed patients and recently emerged as drug-resistant pathogens worldwide [65].
Authors found Cudraxanthone-S were exhibited ability on regulating the Cdc42 in MAPK signaling pathway.
Panacis Japonici Rhizoma (PJR), derived from dry rhizome of Panax japonicus C. A. Meyer (Araliaceae), distributes in the southwest of China [66][67][68]. As a widely used focal medicine, the PJR manifested  [74,75]. Study from Chaotham et al. [76] showing that TDB reduced such cell migration and invasion by decreasing migration-regulating proteins, including integrins αv, α4, β1, β3 and β5, as well as downstream signaling proteins, such as activated focal adhesion kinase (pFAK), activated Rac1 and Cdc42. As the presence of cellular protrusion, called filopodia, has been indicated as a Downloaded from https://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20200407/885857/bsr-2020-0407.pdf by guest on 01 July 2020 hallmark of migrating cells, we showed that the reduction of the mentioned proteins correlated well with the disappearance of filopodia. In summary, this study demonstrates the promising activity of TDB and its mechanism in the inhibition of lung cancer cell migration, which might be useful for encouraging the development of this compound for antimetastatic approaches.
Inhibition of VEGFR2 activity has been proposed as an important strategy for the clinical treatment of hepatocellular carcinoma (HCC).
Corosolic acid (CA), which exists in the root of Actinidia chinensis, as having a significant anti-cancer effect on HCC cells by decreasing the tumor cellular migration. Ku et al. [77]have extensively studied the effects of CA on its cellular regulating effects found that CA inhibits VEGFR2 kinase activity by directly interacting with the ATP binding pocket. Moreover, they found CA could decrease the VEGFR2/Src/FAK/Cdc42 axis, subsequently decreasing F-actin formation and migratory activity in vitro.
Gigantol is a bibenzyl compound derived from the Thai orchid, Dendrobium draconis. It exhibits significant cytotoxic activity against several cancer cell lines. Study conducted by Charoenrungruang et al. [78] demonstrates that gigantol suppresses the migratory cellular behavior via

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The authors declare that they have no competing interests.