The regulation of osteoclast function and bone resorption by small GTPases

Abstract

Osteoclasts are multinucleated cells that are responsible for resorption of bone, and increased activity of these cells is associated with several common bone diseases, including postmenopausal osteoporosis. Upon adhesion to bone, osteoclasts become polarized and reorganise their cytoskeleton and membrane to form unique domains including the sealing zone (SZ), which is a dense ring of F-actin-rich podosomes delimiting the ruffled border (RB), where protons and proteases are secreted to demineralise and degrade the bone matrix, respectively. These processes are dependent on the activity of small GTPases. Rho GTPases are well known to control the organization of F-actin and adhesion structures of different cell types, affecting subsequently their migration. In osteoclasts, RhoA, Rac, Cdc42, RhoU and also Arf6 regulate podosome assembly and their organization into the SZ. By contrast, the formation of the RB involves vesicular trafficking pathways that are regulated by the Rab family of GTPases, in particular lysosomal Rab7. Finally, osteoclast survival is dependent on the activity of Ras GTPases. The correct function of almost all these GTPases is absolutely dependent on post-translational prenylation, which enables them to localize to specific target membranes. Bisphosphonate drugs, which are widely used in the treatment of bone diseases such as osteoporosis, act by preventing the prenylation of small GTPases, resulting in the loss of the SZ and RB and therefore inhibition of osteoclast activity, as well as inducing osteoclast apoptosis. In this review we summarize current understanding of the role of specific prenylated small GTPases in osteoclast polarization, function and survival.

Figure 1

Alterations in membrane domains during osteoclast activation. Unpolarized, inactive osteoclasts present dispersed podosomes. During osteoclast activation, these podosomes coalesce into a peripheral belt and subsequently into distinct “actin ring” that forms the sealing zone where the osteoclast adheres tightly to the bone surface. Following this, trafficking of late endosomes/lysosomes toward the bone surface result in formation of the ruffled border, the resorptive organelle of the osteoclast. Finally, a membrane domain known as the functional secretory domain forms at the top of the cell, to which transcytotic vesicles formed at the ruffled border are targetted.

Figure 2

Bisphosphonate drugs inhibit the prenylation of small GTPases. (A) Inhibition of protein prenylation by nitrogen-containing bisphosphonates can be demonstrated in vitro by culturing cells with [¹⁴C]mevalonate, which becomes incorporated into ¹⁴C-labeled, prenylated proteins. Radiolabelled, prenylated proteins (21–26 kD, boxed region) can then be detected by autoradiography following electrophoretic separation. Treatment with the bisphosphonate drugs alendronate (ALN), ibandronate (IBA), incadronate (INC) and risedronate (RIS) clearly inhibits prenylation compared with control (Ctrl) cells. Reproduced from Luckman et al. with permission of the American Society for Bone and Mineral Research. (B) Inhibition of protein prenylation by bisphosphonate drugs results in the accumulation of the unprenylated form of small GTPases in osteoclasts. The accumulation of unprenylated Rap1A (red) can be determined by protein gel blotting, for example after treatment of cultured cells with ≥10 µM zoledronate (image kindly provided by Gemma Shay). The unprenylated form (red) is of higher molecular mass than the prenylated form (green) due to lack of cleavage of the terminal tripepetide. (C) Inhibition of protein prenylation by bisphosphonate drugs alters the subcellular distribution of small GTPases such as Rab6. Multinucleated osteoclasts were immunostained for Rab6, which localizes to the perinuclear golgi in the untreated osteoclast (left) but has a cytosolic distribution in the osteoclast treated for 48 h with the bisphosphonate risedronate (right).

Figure 3

Role of small GTPases in signaling pathways regulating the polarization of osteoclasts. Upon adhesion of osteoclasts to the bone matrix, engagement of the vitronectin receptor (α_vβ₃) and CD44 activates several downstream signaling pathways to promote the formation of the sealing zone and osteoclast polarization. These pathways involve a variety of small GTPases (blue), GAPs (red), GEFs (pink) and GTPase effectors (green) and can also be activated via other receptors such as the M-CSF receptor and the G protein-coupled receptor GPR55.

Figure 4

Hypothetical model for the transport of late endosomes/lysosomes to the ruffled border in osteoclasts. Rab7 is localized to late endosomes/lysosomes and to the peripheral area of the ruffled border (close to the sealing zone) in osteoclasts and regulates the trafficking of these vesicles toward the plus end of microtubules. The other mediators of this process remain unknown, although the Rab7-binding protein Plekhm1, which is recruited to late endosomes/lysosomes by Rab7, is likely to be involved, since osteoclasts from osteopetrosis patients with mutations in this protein have defective ruffled borders. One possibility is that Plekhm1 may bridge Rab7 and a kinesin motor to enable trafficking to occur on microtubules. Rab7 has been shown to interact directly with Rac1 close to the sealing zone, and it has been postulated that this interaction may mediate the transfer of the late endosomes/lysosomes from the microtubule network to the cortical actin network prior to fusion with the ruffled border acceptor membrane and the release of cathepsin K and acid at the periphery of the RB into the resorption lacuna. This process also serves to insert the V-ATPase, ClC-7 and Rab7 into the ruffled border membrane. The V-ATPase itself may also have a role in this process, since subunits of the pump have been shown to bind to actin microfilaments. Transcytotic vesicles, which are involved in the further degradation and removal of collagen fragments and are trafficked on microtubules to the functional secretory domain (FSD), originate from the central region of the ruffled border; as yet the Rab GTPases governing this pathway remain unknown.