Excessive bone loss in arthritic diseases is mostly due to abnormal activation of the immune system leading to stimulation of osteoclasts. While phospholipase Cγ (PLCγ) isoforms are known modulators of T and B lymphocyte-mediated immune responses, we found that blockade of PLCγ enzymatic activity also blocks early osteoclast development and function. Importantly, targeted deletion of Plcg2 in mice led to an osteopetrotic phenotype. PLCγ2, independent of PLCγ1, was required for receptor activator of NF-κB ligand-induced (RANKL-induced) osteoclastogenesis by differentially regulating nuclear factor of activated T cells c1 (NFATc1), activator protein-1 (AP1), and NF-κB. Specifically, we show that NFATc1 upregulation is dependent on RANKL-mediated phosphorylation of PLCγ2 downstream of Dap12/Fc receptor γ (Dap12/FcRγ) receptors and is blocked by the PLCγ inhibitor U73122. In contrast, activation of JNK and NF-κB was not affected by U73122 or Dap12/FcRγ deletion. Interestingly, we found that in osteoclasts, PLCγ2 formed a complex with the regulatory adapter molecule GAB2, was required for GAB2 phosphorylation, and modulated GAB2 recruitment to RANK. Thus, PLCγ2 mediates RANKL-induced osteoclastogenesis and is a potential candidate for antiresorptive therapy.

Cell-cell adhesive events affect cell growth and fate decisions and provide spatial clues for cell polarity within tissues. The complete molecular determinants required for adhesive junction formation and their function are not completely understood. LIM domain-containing proteins have been shown to be present at cellcell contact sites and are known to shuttle into the nucleus where they can affect cell fate and growth; however, their precise localization at cell-cell contacts, how they localize to these sites, and what their functions are at these sites is unknown. Here we show that, in primary keratinocytes, the LIM domain protein Ajuba is recruited to cadherin-dependent cell-cell adhesive complexes in a regulated manner. At cadherin adhesive complexes Ajuba interacts with ␣-catenin, and ␣-catenin is required for efficient recruitment of Ajuba to cell junctions. Ajuba also interacts directly with F-actin. Keratinocytes from Ajuba null mice exhibit abnormal cell-cell junction formation and/or stability and function. These data reveal Ajuba as a new component at cadherin-mediated cell-cell junctions and suggest that Ajuba may contribute to the bridging of the cadherin adhesive complexes to the actin cytoskeleton and as such contribute to the formation or strengthening of cadherin-mediated cell-cell adhesion.Cell-to-cell adhesion is important for tissue morphogenesis. During development, cell-cell contacts provide spatial clues for cell polarity and sorting, thereby ensuring proper cellular organization within tissues. Cell surface adhesion receptor proteins direct cell-cell adhesion. The cadherins, for example, are a superfamily of receptors that display calcium-dependent adhesion between the same types of proteins (i.e. homophilic interaction). E-cadherin is one of the best studied cell-cell adhesion proteins. In epithelia, E-cadherin has an important role in the generation and maintenance of the cell morphology, polarity, and function (1, 2).At adhesive contacts, E-cadherin receptors also provide cytosolic actin filaments with points of attachment to the membrane, from which tension and reorganization of the cortical cytoskeleton are initiated. E-cadherin-mediated adhesion triggers redistribution of membrane, cytoskeletal, and cytosolic signaling proteins to sites of cell-cell contacts, giving rise to multiprotein signaling complexes (1). Much investigation has been directed at understanding how these supramolecular protein complexes are formed, what proteins make up the functional complex, and what their contribution is to the strength of junction formation and remodeling of the cytoskeletal network.Proteins of the catenin family indirectly mediate the binding of actin filaments to cadherin receptors. ␤-Catenin (or ␥-catenin/plakoglobin) associates directly with the cadherin tail, and then ␣-catenin bridges the ␤-catenin-cadherin complex to actin filaments (1). ␣-Catenin is an essential component of the cadherin complex (1). It not only binds and bundles actin (3) but also provides docking sites for other cyt...

Cell migration requires extension of lamellipodia that are stabilized by formation of adhesive complexes at the leading edge. Both processes are regulated by signaling proteins recruited to nascent adhesive sites that lead to activation of Rho GTPases. The Ajuba/Zyxin family of LIM proteins are components of cellular adhesive complexes. We show that cells from Ajuba null mice are inhibited in their migration, without associated abnormality in adhesion to extracellular matrix proteins, cell spreading, or integrin activation. Lamellipodia production, or function, is defective and there is a selective reduction in the level and tyrosine phosphorylation of FAK, p130Cas, Crk, and Dock180 at nascent focal complexes. In response to migratory cues Rac activation is blunted in Ajuba null cells, as detected biochemically and by FRET analysis. Ajuba associates with the focal adhesion-targeting domain of p130Cas, and rescue experiments suggest that Ajuba acts upstream of p130Cas to localize p130Cas to nascent adhesive sites in migrating cells thereby leading to the activation of Rac.

Integrin engagement induces a cascade of signaling pathways that include tyrosine phosphorylation of numerous proteins that lead to modulation of the actin cytoskeleton. Src is a major intracellular mediator of integrin-dependent functions, but the mechanism(s) by which Src is regulated in response to integrin signals is not fully understood. Here, we demonstrate an important role for phospholipase C gamma 2 (PLC␥2) in Src activation in the osteoclast. Through analysis of primary cells from PLC␥2 ؊/؊ mice, PLC␥2 was found to be an important regulator of ␣ v ␤ 3 integrin-mediated bone osteoclast cell adhesion, migration, and bone resorption. Adhesion-induced PYK2 and Src phosphorylation is decreased in the absence of PLC␥2, and the interaction of Src with ␤ 3 integrin and PYK2 is dramatically reduced. Importantly, PLC␥2 was found to be required for proper localization of Src to the sealing actin ring, and this function required both its catalytic activity and adapter domains. Based on these results, we propose that PLC␥2 influences Src activation by mediating the localization of Src to the integrin complex and thereby regulating integrin-mediated functions in the osteoclast.

Vertebrate bone development and homeostasis requires the exquisite balance between the bone resorptive capacity of osteoclasts and bone forming capacity of osteoblasts. Perturbation of this equilibrium can have pathologic consequences. For example, pathologic bone loss, as occurs in osteoporosis, rheumatoid arthritis, Paget disease of the bone, and tumor metastasis to bone represents enhanced net osteoclastic activity. Alternatively, defective osteoclastogenesis can lead to increased bone mass or osteopetrosis. These pathologies can result from enhanced or inhibited osteoclast development, altered osteoclast function without change in number of osteoclasts, or both.The extent of bone resorption is directly related to the control of osteoclast differentiation. Osteoclasts derive from bone marrow-derived macrophages (BMDM) 3 under the influence of macrophage colony stimulating factor (M-CSF), receptor activator of NF-B ligand (RANK-L), and incompletely understood co-stimulatory factors acting through immunoreceptor tyrosine-based activation motif-containing receptors to give rise to large, motile, multinucleated, terminally differentiated osteoclasts (1-3). Other RANK-L-related inflammatory cytokines, such as TNF␣ and interleukin-1␤, also influence osteoclastogenesis and function, either independently or in synergy with RANK-L (4). Whereas M-CSF is thought to largely provide a survival/proliferative signal to macrophage precursor cells, RANK-L signals are critical for osteoclast differentiation (1).The cellular receptor for RANK-L, RANK, is a member of the TNFR superfamily that includes the interleukin-1 receptor and Toll-like receptors (5). Like other TNFRs, RANK recruits adapter proteins after ligand-induced multimerization. A central family of such adapters is the TNF receptor-associated factors or TRAFs. RANK binds multiple TRAFs but only Traf6 has been shown to be critical for osteoclast development and function (6, 7). In the absence of Traf6 or in the presence of inhibitory peptides osteoclast differentiation is blocked (7,8). TRAFs share a common C-terminal TRAF domain that serves to localize TRAFs to their target proteins and results in oligomerization of the N-terminal effector domain leading to the activation of IKK, NF-B, the MAPKs (particularly JNK and p38), and AP-1 (9 -11). Recently, Traf6 was also found to form a complex with the atypical PKC-interacting adapter protein p62 in osteoclasts (aPKC-p62-Traf6 complex), and this complex was shown to be important for the activation of NF-B by RANK-L (12). The importance of p62 to bone physiology is evident as mutations in p62 have been identified in a group of patients