The Rho GTPase Wrch1 regulates osteoclast precursor adhesion and migration

Abstract

An excess of osteoclastic bone resorption relative to osteoblastic bone formation results in progressive bone loss, characteristic of osteoporosis. Understanding the mechanisms of osteoclast differentiation is essential to develop novel therapeutic approaches to prevent and treat osteoporosis. We showed previously that Wrch1/RhoU is the only RhoGTPase whose expression is induced by RANKL during osteoclastogenesis. It associates with podosomes and the suppression of Wrch1 in osteoclast precursors leads to defective multinucleated cell formation. Here we further explore the functions of this RhoGTPase in osteoclasts, using RAW264.7 cells and bone marrow macrophages as osteoclast precursors. Suppression of Wrch1 did not prevent induction of classical osteoclastic markers such as NFATc1, Src, TRAP (Tartrate-Resistant Acid Phosphatase) or cathepsin K. ATP6v0d2 and DC-STAMP, which are essential for fusion, were also expressed normally. Similar to the effect of RANKL, we observed that Wrch1 expression increased osteoclast precursor aggregation and reduced their adhesion onto vitronectin but not onto fibronectin. We further found that Wrch1 could bind integrin ß3 cytoplasmic domain and interfered with adhesion-induced Pyk2 and paxillin phosphorylation. Wrch1 also acted as an inhibitor of M-CSF-induced prefusion osteoclast migration. In mature osteoclasts, high Wrch1 activity inhibited podosome belt formation. Nevertheless, it had no effect on mineralized matrix resorption. Our observations suggest that during osteoclastogenesis, Wrch1 potentially acts through the modulation of αvß3 signaling to regulate osteoclast precursor adhesion and migration and allow fusion. As an essential actor of osteoclast differentiation, the atypical RhoGTPase Wrch1/RhoU could be an interesting target for the development of novel antiresorptive drugs.

Introduction

Bone resorbing osteoclasts are post-mitotic multinucleated cells formed after the fusion of monocyte/macrophage precursors (Boyle et al., 2003). Osteoclastic bone resorption involves the differentiation of progenitors into mononuclear prefusion osteoclasts (preOCs) that fuse to generate polykaryons, and the migration of osteoclasts towards the bone resorption site. Osteoclastic differentiation requires the two cytokines M-CSF (macrophage colony stimulating factor) and RANKL (receptor activator of NFκB-ligand) (Boyle et al., 2003). Osteoclast mediated bone resorption is tightly regulated at multiple levels, excessive osteoclast activity leading to progressive reduction of bone mass and modification of bone architecture that are associated with various diseases (Roodman, 2006, Teitelbaum and Ross, 2003). In particular osteoporosis which affects post menopausal women and older men has now become a major public health problem due to general population aging (Dennison et al., 2005).

Upon RANKL treatment, osteoclast precursors undergo profound changes. They exit the cell cycle (Ogasawara et al., 2004) while a complex transcriptional program is activated. RANKL induces the expression of NFATc1, a fundamental transcription factor in osteoclastogenesis (Takayanagi et al., 2002). RANKL also activates the expression of integrin ß3, ATP6v0d2 and DC-STAMP, which are essential for osteoclast precursor differentiation and fusion (Kim et al., 2008, Lee et al., 2006, McHugh et al., 2000, Miyamoto et al., 2000). Finally, increased expression of genes necessary for bone resorbing activity is also observed during osteoclastogenesis, including cathepsin K, TRAP and the tyrosine kinase Src.

Precursors kept in semi-solid culture medium, to prevent their anchorage to the substratum, are unable to differentiate into mature osteoclasts (Miyamoto et al., 2000); therefore adhesion-dependent signaling plays an important role during osteoclastogenesis. The major adhesion structures found in osteoclasts are podosomes. These structures are formed by an F-actin core column whose orientation is perpendicular to the plasma membrane and to the extracellular matrix. The podosome core is surrounded by several adhesion molecules such as integrins, vinculin, talin and paxillin (Block et al., 2008, Saltel et al., 2008). Osteoclasts express integrins αvß3, α2ß1 and αvß1, integrin αvß3 being involved in most aspects of osteoclast biology. Integrin αvß3 is essential during osteoclast differentiation, most likely by controlling precursor migration and adhesion (Boissy et al., 1998, Kim et al., 2007, McHugh et al., 2000, Miyamoto et al., 2000). It is also necessary for osteoclast bone resorbing activity (Zou et al., 2007). Integrin αvß3 interaction with extracellular matrix proteins activates multiple signaling pathways, in particular phosphorylation cascades triggered by the tyrosine kinases Src and Pyk2 (Lakkakorpi et al., 2003, Miyazaki et al., 2004), to regulate podosome dynamics and organization, sealing zone formation and bone resorption (Destaing et al., 2008, Gil-Henn et al., 2007, Shyu et al., 2007). Much less is known about the importance of integrin signaling during osteoclast differentiation. Whereas integrin beta3 is essential for osteoclast differentiation, Src and Pyk2 are dispensable. Osteoclast precursors defective for Src or Pyk2 can differentiate into mature multinucleated osteoclasts, but they are defective for bone resorption (Gil-Henn et al., 2007, Sanjay et al., 2001). This suggests that the two kinases are not essential for differentiation although both where shown to regulate prefusion osteoclast spreading and migration (Duong et al., 2001, Lakkakorpi et al., 2003, Nakamura et al., 2001).

Osteoclast podosomes are highly dynamic and reorganize during osteoclast maturation and activity (Destaing et al., 2003). Individual podosomes are connected to their neighbors by F-actin cables. This allows podosome compaction to generate the different osteoclast specific superstructures: podosome clusters and rings in immature osteoclasts, the podosome belt at the periphery of mature osteoclasts and the sealing zone when they resorb bone mineralized matrix (Luxenburg et al., 2007). RhoGTPases are well known to control F-actin and adhesion structure organization and then cell migration in various cell types (Burridge and Wennerberg, 2004, Cernuda-Morollon and Ridley, 2006). In osteoclasts, RhoA, Rac and Cdc42 are involved in the control of podosome assembly and organization, and in the regulation of adhesion signaling (Ory et al., 2008). So far, only Rac1 and 2 were shown to be necessary for osteoclast differentiation (Wang et al., 2008). To get a better view of the function of Rho GTPase signaling pathways in osteoclasts, we established the expression profile of RhoGTPases and their activators during RANKL-induced osteoclastogenesis. We found that among all RhoGTPases, only the expression of Wrch1 (Wnt1-Responsive Cdc42 Homolog 1)/RhoU was induced by RANKL. We also showed that suppression of Wrch1 expression in osteoclast precursors impaired fusion (Brazier et al., 2006). In the present studies, we further explored the roles of Wrch1 during osteoclast differentiation. We found that Wrch1 did not interfere with the overall transcriptional program induced by RANKL, in particular genes formerly reported as essential for precursor fusion were normally expressed. We previously showed that Wrch1 associated with adhesion structures: it localized to focal adhesion in fibroblasts and to podosomes in osteoclasts (Ory et al., 2007). We (Ory et al., 2007) and others (Chuang et al., 2007) also reported that Wrch1 regulated focal adhesion assembly and cell migration in fibroblasts and epithelial cells. Given these effects, we investigated how Wrch1 impacted on the adhesive and migration properties of osteoclast precursors. We present evidence that similar to RANKL, Wrch1 favors osteoclast precursor aggregation and diminishes their adhesion onto vitronectin. We also show that Wrch1 inhibits M-CSF-induced migration and integrin αvß3 downstream signaling of prefusion osteoclasts. Although we found that Wrch1 activity inhibited podosome belt formation, it did not affect sealing zone assembly and bone resorption in differentiated osteoclasts. Taking these findings together, we suggest that the RhoGTPase Wrch1 has an essential function during osteoclastogenesis by regulating cell adhesion and migration, potentially through the modulation of integrin αvß3 downstream signaling.