The osteoclast cytoskeleton - current understanding and therapeutic perspectives for osteoporosis.

Osteoclasts are giant multinucleated myeloid cells specialized for bone resorption, which is essential for the preservation of bone health throughout life. The activity of osteoclasts relies on the typical organization of osteoclast cytoskeleton components into a highly complex structure comprising actin, microtubules and other cytoskeletal proteins that constitutes the backbone of the bone resorption apparatus. The development of methods to differentiate osteoclasts in culture and manipulate them genetically, as well as improvements in cell imaging technologies, has shed light onto the molecular mechanisms that control the structure and dynamics of the osteoclast cytoskeleton, and thus the mechanism of bone resorption. Although essential for normal bone physiology, abnormal osteoclast activity can cause bone defects, in particular their hyper-activation is commonly associated with many pathologies, hormonal imbalance and medical treatments. Increased bone degradation by osteoclasts provokes progressive bone loss, leading to osteoporosis, with the resulting bone frailty leading to fractures, loss of autonomy and premature death. In this context, the osteoclast cytoskeleton has recently proven to be a relevant therapeutic target for controlling pathological bone resorption levels. Here, we review the present knowledge on the regulatory mechanisms of the osteoclast cytoskeleton that control their bone resorption activity in normal and pathological conditions.

Dock5 is a new regulator of microtubule dynamic instability in osteoclasts.

BACKGROUND INFORMATION: Osteoclast resorption is dependent on a podosome-rich structure called sealing zone. It tightly attaches the osteoclast to the bone creating a favourable acidic microenvironment for bone degradation. This adhesion structure needs to be stabilised by microtubules whose acetylation is maintained by down-regulation of deacetylase HDAC6 and/or of microtubule destabilising kinase GSK3ß activities. We already established that Dock5 is a guanine nucleotide exchange factor for Rac1. As a consequence, Dock5 inhibition results in a decrease of the GTPase activity associated with impaired podosome assembly into sealing zones and resorbing activity in osteoclasts. More, administration of C21, a chemical compound that directly inhibits the exchange activity of Dock5, disrupts osteoclast podosome organisation and protects mice against bone degradation in models recapitulating major osteolytic diseases. RESULTS: In this report, we show that Dock5 knockout osteoclasts also present a reduced acetylated tubulin level leading to a decreased length and duration of microtubule growth phases, whereas their growth speed remains unaffected. Dock5 does not act by direct interaction with the polymerised tubulin. Using specific Rac inhibitors, we showed that Dock5 regulates microtubule dynamic instability through Rac-dependent and -independent pathways. The latter involves GSK3ß inhibitory serine 9 phosphorylation downstream of Akt activation but not HDAC6 activity. CONCLUSION: We showed that Dock5 is a new regulator of microtubule dynamic instability in osteoclast. SIGNIFICANCE: Dock5 dual role in the regulation of the actin cytoskeleton and microtubule, which both need to be intact for bone resorption, reinforces the fact that it is an interesting therapeutic target for osteolytic pathologies.

The mineral dissolution function of osteoclasts is dispensable for hypertrophic cartilage degradation during long bone development and growth.

During long bone development and post-natal growth, the cartilaginous model of the skeleton is progressively replaced by bone, a process known as endochondral ossification. In the primary spongiosa, osteoclasts degrade the mineralized cartilage produced by hypertrophic chondrocytes to generate cartilage trabeculae that osteoblasts embed in bone matrix. This leads to the formation of the trabecular bone network of the secondary spongiosa that will undergo continuous remodeling. Osteoclasts are specialized in mineralized tissue degradation, with the combined ability to solubilize hydroxyapatite and to degrade extracellular matrix proteins. We reported previously that osteoclasts lacking Dock5 could not degrade bone due to abnormal podosome organization and absence of sealing zone formation. Consequently, adult Dock5(-/-) mice have increased trabecular bone mass. We used Dock5(-/-) mice to further investigate the different functions of osteoclast during endochondral bone formation. We show that long bones are overall morphologically normal in developing and growing Dock5(-/-) mice. We demonstrate that Dock5(-/-) mice also have normal hypertrophic cartilage and cartilage trabecular network. Conversely, trabecular bone volume increased progressively in the secondary spongiosa of Dock5(-/-) growing mice as compared to Dock5(+/+) animals, even though their osteoclast numbers were the same. In vitro, we show that Dock5(-/-) osteoclasts do present acidic compartments at the ventral plasma membrane and produce normal amounts of active MMP9, TRAP and CtsK for matrix protein degradation but they are unable to solubilize minerals. These observations reveal that contrarily to bone resorption, the ability of osteoclasts to dissolve minerals is dispensable for the degradation of mineralized hypertrophic cartilage during endochondral bone formation.

Bone resorption by osteoclasts involves fine tuning of RHOA activity by its microtubule-associated exchange factor GEF-H1.

Bone health is controlled by the balance between bone formation by osteoblasts and degradation by osteoclasts. A disequilibrium in favor of bone resorption leads to osteolytic diseases characterized by decreased bone density. Osteoclastic resorption is dependent on the assembly of an adhesion structure: the actin ring, also called podosome belt or sealing zone, which is composed of a unique patterning of podosomes stabilized by microtubules. A better understanding of the molecular mechanisms regulating the crosstalk between actin cytoskeleton and microtubules network is key to find new treatments to inhibit bone resorption. Evidence points to the importance of the fine tuning of the activity of the small GTPase RHOA for the formation and maintenance of the actin ring, but the underlying mechanism is not known. We report here that actin ring disorganization upon microtubule depolymerization is mediated by the activation of the RHOA-ROCK signaling pathway. We next show the involvement of GEF-H1, one of RHOA guanine exchange factor highly expressed in osteoclasts, which has the particularity of being negatively regulated by sequestration on microtubules. Using a CRISPR/Cas9-mediated GEF-H1 knock-down osteoclast model, we demonstrate that RHOA activation upon microtubule depolymerization is mediated by GEF-H1 release. Interestingly, although lower levels of GEF-H1 did not impact sealing zone formation in the presence of an intact microtubule network, sealing zone was smaller leading to impaired resorption. Altogether, these results suggest that a fine tuning of GEF-H1 through its association with microtubules, and consequently of RHOA activity, is essential for osteoclast sealing zone stability and resorption function.

Methods to Investigate the Role of Rho GTPases in Osteoclast Function.

The actin cytoskeleton is essential for the biology of osteoclasts, in particular during bone resorption. As key regulators of actin dynamics, the small GTPases of the Rho family are very important in the control of osteoclast activity. The study of Rho GTPase signaling pathways is essential to uncover the mechanisms of bone resorption and can have interesting applications for the treatment of osteolytic diseases. In this chapter, we describe various techniques to obtain primary osteoclasts from murine bone marrow cells, to measure Rho GTPase activation levels, to monitor bone resorption activity of osteoclasts and to introduce the expression of proteins of interest using a retroviral approach. We illustrate the different methods with experimental examples of the effect of Rac1 activation by the exchange factor Dock5 on bone resorption by osteoclasts.

Combined strategy of siRNA and osteoclast actin cytoskeleton automated imaging to identify novel regulators of bone resorption shows a non-mitotic function for anillin.

Osteoclasts are the main cells responsible for the resorption of mineralized extracellular matrices. They are the major targets for anti-resorptive therapies to manage osteoporosis, a major public health problem. Osteoclasts are giant multinucleated cells that can organize their a unique adhesion structure based on a belt of podosomes, which is the keystone of the bone resorption apparatus. We combined differential transcriptomics and siRNA screening approaches to get a broader view of cytoskeletal regulators that participate in the control of osteoclast cytoskeleton and identify novel regulators of bone resorption by osteoclasts. We identified 20 new candidate regulators of osteoclasts cytoskeleton including Fkbp15, Spire1, Tacc2 and RalA, for which we confirmed they are necessary for proper organization of the podosome belt. We also showed that Anillin, well known for its role during cytokinesis, is essential in osteoclasts for correct podosome patterning and efficient bone resorption. In particular, Anillin controls the levels of the GTPase RhoA, a known regulator of osteoclast cytoskeleton and resorption activity. Finally, we set up and validated an automated imaging strategy based on open-source software for automatic and objective measurement of actin cytoskeleton organization in osteoclasts. We provide these pipelines that are useful to automatically assess the effect of collections of siRNAs or chemical compounds on osteoclast cytoskeleton or differentiation.

Pharmacological inhibition of Dock5 prevents osteolysis by affecting osteoclast podosome organization while preserving bone formation.

Osteoporosis is caused by excessive activity of bone-degrading osteoclasts over bone-forming osteoblast. Standard antiosteolytic treatments inhibit bone resorption by inducing osteoclast loss, with the adverse effect of hindering also bone formation. Formation of the osteoclast sealing zone requires Dock5, a guanine nucleotide exchange factor for the small GTPase Rac, and C21, a chemical inhibitor of Dock5, decreases bone resorption by cultured osteoclasts. Here we show that C21 directly inhibits the exchange activity of Dock5 and disrupts osteoclast podosome organization. Remarkably, C21 administration protects mice against bone degradation in models recapitulating major osteolytic diseases: menopause, rheumatoid arthritis and bone metastasis. Furthermore, C21 administration does not affect bone formation and is not toxic. Our results validate the pharmacological inhibition of Dock5 as a novel therapeutic route for fighting osteolytic diseases while preserving bone formation.

Visualization of S100B-positive neurons and glia in the central nervous system of EGFP transgenic mice.

S100B, the EF-hand Ca(++)-binding protein with gliotrophic and neurotrophic properties implicated in the pathogenesis of Alzheimer's disease, is coined as a glial marker, despite its documented presence in rodent brain neurons. We have generated a transgenic mouse whose EGFP reporter, controlled by the -1,669/+3,106 sequence of the murine S100B gene, allows the direct microscopic observation of most S100B-expressing cells in the central nervous system (CNS). From embryonic day 13 onward, EGFP expression was targeted to selected neuroepithelial, glial, and neuronal cells, indicating that cell-specific expression of S100B is regulated at the transcriptional level during development. In adult mice, the highest level of EGFP expression was found in ependymocytes; astrocytes; and spinal, medullar, pontine, and deep cerebellar S100B neurons. Our results, thus, agree with earlier reports suggesting that S100B is not a CNS glial-specific marker. In addition, we detected EGFP and S100B in forebrain neurons previously thought not to express S100B in the mouse, including neurons of primary motor and somatosensory neocortical areas, the ventral pallidum and prerubral field. Another interesting finding was the selected EGFP targeting to neonatal S100B oligodendrocytes and adult NG2 progenitors as opposed to mature S100B oligodendrocytes. This finding suggests that, except for oligodendrocytes at the last stage of myelin maturation, the -1,669/+3,106 sequence of the S100B gene is a useful reagent for driving expression of transgenes in most S100B-expressing cells of mouse brain.

Modulation of osteoclast differentiation and bone resorption by Rho GTPases.

Bone is a dynamic tissue constantly renewed through a regulated balance between bone formation and resorption. Excessive bone degradation by osteoclasts leads to pathological decreased bone density characteristic of osteolytic diseases such as post-menopausal osteoporosis or bone metastasis. Osteoclasts are multinucleated cells derived from hematopoietic stem cells via a complex differentiation process. Their unique ability to resorb bone is dependent on the formation of the actin-rich sealing zone. Within this adhesion structure, the plasma membrane differentiates into the ruffled border where protons and proteases are secreted to demineralize and degrade bone, respectively. On the bone surface, mature osteoclasts alternate between stationary resorptive and migratory phases. These are associated with profound actin cytoskeleton reorganization, until osteoclasts die of apoptosis. In this review, we highlight the role of Rho GTPases in all the steps of osteoclasts differentiation, function, and death and conclude on their interest as targets for treatment of osteolytic pathologies.

The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts.

Osteoporosis, which results from excessive bone resorption by osteoclasts, is the major cause of morbidity for elder people. Identification of clinically relevant regulators is needed to develop novel therapeutic strategies. Rho GTPases have essential functions in osteoclasts by regulating actin dynamics. This is of particular importance because actin cytoskeleton is essential to generate the sealing zone, an osteoclast-specific structure ultimately mediating bone resorption. Here we report that the atypical Rac1 exchange factor Dock5 is necessary for osteoclast function both in vitro and in vivo. We discovered that establishment of the sealing zone and consequently osteoclast resorbing activity in vitro require Dock5. Mechanistically, our results suggest that osteoclasts lacking Dock5 have impaired adhesion that can be explained by perturbed Rac1 and p130Cas activities. Consistent with these functional assays, we identified a novel small-molecule inhibitor of Dock5 capable of hindering osteoclast resorbing activity. To investigate the in vivo relevance of these findings, we studied Dock5(-/-) mice and found that they have increased trabecular bone mass with normal osteoclast numbers, confirming that Dock5 is essential for bone resorption but not for osteoclast differentiation. Taken together, our findings characterize Dock5 as a regulator of osteoclast function and as a potential novel target to develop antiosteoporotic treatments.

ASPP2 binds Par-3 and controls the polarity and proliferation of neural progenitors during CNS development.

Cell polarity plays a key role in the development of the central nervous system (CNS). Interestingly, disruption of cell polarity is seen in many cancers. ASPP2 is a haplo-insufficient tumor suppressor and an activator of the p53 family. In this study, we show that ASPP2 controls the polarity and proliferation of neural progenitors in vivo, leading to the formation of neuroblastic rosettes that resemble primitive neuroepithelial tumors. Consistent with its role in cell polarity, ASPP2 influences interkinetic nuclear migration and lamination during CNS development. Mechanistically, ASPP2 maintains the integrity of tight/adherens junctions. ASPP2 binds Par-3 and controls its apical/junctional localization without affecting its expression or Par-3/aPKC lambda binding. The junctional localization of ASPP2 and Par-3 is interdependent, suggesting that they are prime targets for each other. These results identify ASPP2 as a regulator of Par-3, which plays a key role in controlling cell proliferation, polarity, and tissue organization during CNS development.

The Rho GTPase Wrch1 regulates osteoclast precursor adhesion and migration.

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 beta3 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 alphav beta3 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.

ASPP2: a gene that controls life and death in vivo.

The fundamental role of apoptosis in tumor prevention and the important role of p53 in this process are now universally recognized. Recently, several families of p53-binding proteins have been shown to influence p53's decision to direct the cells either into the apoptotic pathway or in cell cycle arrest. Among them, the ASPP family specifically regulate p53-dependent apoptosis. Its member ASPP2 was discovered more than 10 years ago as a binding partner of p53 and its role as a positive regulator of p53 mediated apoptosis has been clearly established in vitro. However, its physiological importance in vivo has just emerged through the generation and characterisation of the ASPP2-deficient mice. We now know that ASPP2 is a haploinsufficient tumor suppressor and an important activator of p53 during mouse development and tumor suppression in vivo. ASPP2 might be a novel target for future cancer therapy.

ASPP2 is a haploinsufficient tumor suppressor that cooperates with p53 to suppress tumor growth.

ASPP2 stimulates the apoptotic function of the p53 family in vivo. We show here that ASPP2-/- pups died before weaning. This postnatal lethality was significantly enhanced in p53+/- background and both deletions are synthetic lethal. ASPP2+/- mice developed spontaneous tumors. The tumor onset was accelerated by gamma-irradiation or in p53+/- background. Tumors derived from ASPP2+/- mice retained wild-type ASPP2 allele even though some of them lost p53. These provide the first genetic evidence that ASPP2 is a haploinsufficient tumor suppressor that shares overlapping function(s) with p53 in mouse development and tumor suppression.

Interleukin-17 inhibits tumor cell growth by means of a T-cell-dependent mechanism.

Interleukin 17 (IL-17) is a proinflammatory cytokine produced by activated CD4(+) memory T cells. We previously showed that IL-17 increased the growth rate of human cervical tumors transplanted into athymic nude mice. To address the possible role of T cells in the biologic activity of IL-17 for tumor control, we grafted 2 murine hematopoietic immunogenic tumors (P815 and J558L) transfected with a complementary DNA encoding murine IL-17 into syngeneic immunocompetent mice. We found that growth of the 2 IL-17-producing tumors was significantly inhibited compared with that of mock-transfected tumors. In contrast to the antitumor activity of IL-17 observed in immunocompetent mice, we observed no difference in the in vivo growth of IL-17-transfected or mock-transfected P815 cells (P815-IL-17 and P815-Neo, respectively) transplanted into nude mice. We then showed that IL-17 increased generation of specific cytolytic T lymphocytes (CTLs) directed against the immunodominant antigens from P815 called A, B, C, D, and E, since all mice injected with P815-IL-17 developed a P815-specific CTL response, whereas only 6 of 16 mice immunized with P815-Neo had a specific CTL response against the antigens. The induction of CTLs was associated with establishment of a tumor-protective immunity. These experiments suggest that T lymphocytes are involved in the antitumor activity of IL-17. Therefore, IL-17, like other cytokines, appears to be a pleiotropic cytokine with possible protumor or antitumor effects on tumor development, which often depends on the immunogenicity of tumor models.