The capacity of the osteoclast to resorb bone is distinctive, as is the cell's appearance. Both characteristics reflect cytoskeletal organization that yields structures such as the sealing zone and ruffled border. The unique nature of these organelles and their dependence upon contact with bone have been appreciated for some time but insights into the mechanisms by which they are generated come from more recent studies. These insights include the role of integrins, particularly αvβ3, in cytoskeletal organization and the canonical signaling pathway they activate. Investigators now appreciate that the sealing zone isolates the resorptive microenvironment from the general extracellular space, permitting secretion of matrix-degrading molecules on the bone surface. Thus, the osteoclast is a secretory cell that depends upon polarization of exocytic vesicles to the bone-apposed plasma membrane into which they insert under the aegis of vesicle/membrane fusion proteins. This process focally expands and convolutes the plasmalemma included within the sealing zone, eventuating in formation of the ruffled border. Many of these events are now better understood and are the focus of this Perspective.
Osteoclasts are polykaryons and members of the macrophage lineage with the unique capacity to degrade the inorganic and organic matrices of bone. If excessive, the bone resorptive activity of osteoclasts causes osteoporosis. Conversely, the osteoclast initiates remodeling that likely removes structurally compromised bone, thereby maintaining mechanical integrity (
Resorption is initiated by attachment of osteoclasts to bone. They then develop a compartment between their plasma membrane and the bone surface into which the cells transport matrix-degrading molecules including H+ and Cl-, which, in concert, demineralize the target bone, and cathepsin K, which degrades the exposed collagen fibers and associated proteins. To isolate this resorptive microenvironment from the general extracellular space, osteoclasts reorganize their cytoskeleton to generate an encompassing, actin-rich, gasket-like, sealing zone. A single osteoclast, being a large cell, enjoys multiple contacts with bone and therefore generates numerous sealing zones and resorptive microenvironments.
When most other cells attach to matrix they generate focal adhesions. These stable structures contain integrins and signaling and cytoskeletal molecules that, upon contact with matrix, promote formation of actin stress fibers. Consistent with the lack of actin stress fibers in mammalian osteoclasts, they lack focal adhesions but in their place develop podosomes (
Most past studies of osteoclast podosomes utilized cells resident on plastic or glass. In these circumstances, the punctuate structures initially appear in clusters but ultimately coalesce, first into an intracytoplasmic actin ring and then a peripheral actin belt (
Depending upon their state of acetylation, microtubules in osteoclasts are transient or polymerized and relatively stable (
The histone deacetylase HDAC6 depolymerizes tubulin, thereby destabilizing microtubules. Cbl proteins compete with the deacetylase for tubulin binding and thus promote polymerization (
The ruffled border is the morphological sine qua non of the resorbing osteoclast as only its presence assures that the cell is degrading bone and its absence indicates the cell is not doing so. Reflecting multiple contacts with bone and attendant sealing zones, ruffled borders are also numerous in a given osteoclast.
This complex enfolding of the plasma membrane, unique to the osteoclast, abuts and extends into the resorptive space. It is surrounded by the sealing zone and is the venue by which the cell secretes matrix-degrading molecules on the bone surface (
The ruffled border, which forms only upon contact with mineralized substrate, is initiated by transport of cathepsin K – and/or H+ATPase – and Cl- channel-bearing vesicles to the bone-apposed plasma membrane (
Ruffled border formation requires a synaptotagmin (Syt) linking the vesicle and target plasma membrane. Fifteen Syt isoforms have been identified in mammalian cells but Syt VII generates ruffled borders and is essential for bone degradation (
Autophagy is a cellular degradative process by which cells recycle organelles and long-lived proteins. Autophagosomes, which are double-membrane bound vesicles, envelop and then deliver cellular components to lysosomes for degradation. While the process promotes survival in starved or stressed cells as well as maintenance of organelle quality (
Skeletal resorption requires osteoclast-bone recognition that is mediated by α/β heterodimers known as integrins. β1-containing heterodimers probably participate in the process but αv/β3 is the key integrin regulating skeletal degradation (
In keeping with its governance of osteoclast function, absence of αv/β3, globally and conditionally in osteoclasts, increases bone mass and protects against estrogen-deficient osteoporosis (
αv/β3 signaling in osteoclasts is initiated by changing the integrin's conformation from a low to a high affinity state by outside-in or inside-out activation (
Resorption is a cyclical event wherein a portion of the osteoclast migrates to a candidate bone resorptive site and forms an actin ring and ruffled border. Following matrix degradation, the cell detaches and re-initiates the cycle. Prior to bone recognition, the integrin is predominantly in a low affinity state and confined to podosomes within the sealing zone (
Integrins serve as attachment molecules but their intracellular transmission of matrix-derived signals is at least as important. For example, αv/β3 substrate robustly activates ERKs in wild-type osteoclastic cells but not those lacking the integrin (
In 1991, Soriano et al. determined that c-src deletion eventuates in severe osteopetrosis due to osteoclast dysfunction (
Because c-src- and αv/β3-deficient osteoclasts share qualitatively similar cytoskeletal features, the kinase presents as a mediator of integrin signaling. In fact, under the aegis of phospholipase Cγ (PLCγ2) (
Syk is another non-receptor tyrosine kinase mediating αv/β3 signaling in osteoclasts. Upon integrin occupancy it binds the β3 cytoplasmic domain close to c-src, which activates it (
The ITAM-bearing adaptors, Dap 12 and FcRγ, are expressed by osteoclasts and their combined, but not individual deletion prompts severe osteopetrosis (
Syk-mediated organization of the osteoclast cytoskeleton involves Vav3. This guanine nucleotide exchange factor (GEF) is uniquely expressed in abundance in the cell and activated upon αv/β3 occupancy in a SLP-76-dependent manner (
Vavs are Rac GEFs and it is therefore not surprising that this Rho GTPase regulates the osteoclast cytoskeleton in an αv/β3-dependent manner (
RANK ligand (RANKL) and M-CSF are the requisite osteoclastogenic cytokines but each also promotes the resorptive activity of the mature polykaryon. In the case of M-CSF, the cytokine interacting with its receptor, c-fms, stimulates a signaling pathway remarkably similar to that induced by αv/β3, thereby organizing the cytoskeleton (
The magnitude of bone resorption reflects osteoclast number and function of the individual cell, the latter dependent upon cytoskeletal organization. The osteoclast cytoskeleton is a unique structure whose conversion to its active state depends upon contact with mineralized matrix (
Proposed mechanism organizing the cytoskeleton of resorbing osteoclasts. 1). M-CSF occupying its receptor, c-fms, stimulates inside-out αvβ3 activation by inducing talin association with the β3 cytoplasmic domain that binds c-src constitutively. 2). Clustering of the integrin by RGD ligand increases avidity as well as affinity by outside-in activation. The liganded integrin activates c-src as evidenced by Y416 phosphorylation. Activated c-src tyrosine phosphorylates ITAM proteins that recruit Syk to the integrin by binding Syk-SH2 domains. c-src activates β3-associated Syk that phosphorylates Vav3 in the context of SLP-76. Vav3 then shuttles Rac-GDP to its activated GTP-associated state. 3). Rac-GTP prompts association of lysosome-derived secretory vesicles with microtubules (MTs) that deliver them to the bone-apposed plasma membrane into which they insert under the influence of Syt VII and LC3. Rac-GTP and MTs also promote sealing zone (SZ) formation. Secretory vesicle fusion focally expands the plasma membrane forming the ruffled border and eventuating in discharge of cathepsin K (CTK) and HCl into the resorptive microenvironment.
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