Bone Is Not Essential for Osteoclast Activation

Background: The mechanism whereby bone activates resorptive behavior in osteoclasts, the cells that resorb bone, is unknown. It is known that avb3 ligands are important, because blockade of avb3 receptor signaling inhibits bone resorption, but this might be through inhibition of adhesion or migration rather than resorption itself. Nor is it known whether avb3 ligands are sufficient for resorption the consensus is that bone mineral is essential for the recognition of bone as the substrate appropriate for resorption. Methodology/Principal Findings: Vitronectin- but not fibronectin-coated coverslips induced murine osteoclasts to secrete tartrate-resistant acid phosphatase, as they do on bone. Osteoclasts incubated on vitronectin, unlike fibronectin, formed podosome belts on glass coverslips, and these were modulated by resorption-regulating cytokines. Podosome belts formed on vitronectin-coated surfaces whether the substrates were rough or smooth, rigid or flexible. We developed a novel approach whereby the substrate-apposed surface of cells can be visualized in the scanning electron microscope. With this approach, supported by transmission electron microscopy, we found that osteoclasts on vitronectin-coated surfaces show ruffled borders and clear zones characteristic of resorbing osteoclasts. Ruffles were obscured by a film if cells were incubated in the cathepsin inhibitor E64, suggesting that removal of the film represents substrate-degrading behavior. Analogously, osteoclasts formed resorption-like trails on vitronectin-coated substrates. Like bone resorption, these trails were dependent upon resorbogenic cytokines and were inhibited by E64. Bone mineral induced actin rings and surface excavation only if first coated with vitronectin. Fibronectin could not substitute in any of these activities, despite enabling adhesion and cell spreading. Conclusions/Significance: Our results show that ligands avb3 are not only necessary but sufficient for the induction of resorptive behavior in osteoclasts; and suggest that bone is recognized through its affinity for these ligands, rather than by its mechanical or topographical attributes, or through a putative ‘mineral receptor’

Distinctive subdomains in the resorbing surface of osteoclasts.

We employed a novel technique to inspect the substrate-apposed surface of activated osteoclasts, the cells that resorb bone, in the scanning electron microscope. The surface revealed unexpected complexity. At the periphery of the cells were circles and crescents of individual or confluent nodules. These corresponded to the podosomes and actin rings that form a 'sealing zone', encircling the resorptive hemivacuole into which protons and enzymes are secreted. Inside these rings and crescents the osteoclast surface was covered with strips and patches of membrane folds, which were flattened against the substrate surface and surrounded by fold-free membrane in which many orifices could be seen. Corresponding regions of folded and fold-free membrane were found by transmission electron microscopy in osteoclasts incubated on bone. We correlated these patterns with the distribution of several proteins crucial to resorption. The strips and patches of membrane folds corresponded in distribution to vacuolar H+-ATPase, and frequently co-localized with F-actin. Cathepsin K localized to F-actin-free foci towards the center of cells with circular actin rings, and at the retreating pole of cells with actin crescents. The chloride/proton antiporter ClC-7 formed a sharply-defined band immediately inside the actin ring, peripheral to vacuolar H+-ATPase. The sealing zone of osteoclasts is permeable to molecules with molecular mass up to 10,000. Therefore, ClC-7 might be distributed at the periphery of the resorptive hemivacuole in order to prevent protons from escaping laterally from the hemivacuole into the sealing zone, where they would dissolve the bone mineral. Since the activation of resorption is attributable to recognition of the αVβ3 ligands bound to bone mineral, such leakage would, by dissolving bone mineral, release the ligands and so terminate resorption. Therefore, ClC-7 might serve not only to provide the counter-ions that enable proton pumping, but also to facilitate resorption by acting as a 'functional sealing zone'

CLSM localization of F-actin (green) and cathepsin K (red) in osteoclasts.

<p>A–D: Osteoclasts incubated on bone show patchy distribution of F-actin within the F-actin ring (clearly shown in panel A). In the cell with a non-migratory appearance (A–C), cathepsin K localizes towards the centre of the apex (B), in a region that is free of F-actin (A, C). In the osteoclast showing a migratory morphology (D), cathepsin K localizes to the retracting pole, also in an F-actin-free region (D, z-stacks). E: In osteoclast incubated on vitronectin, cathepsin K localizes to punctuate foci present at the cell-substrate interface (arrows) centrally to F-actin ring. C-E are counterstained with DAPI. Bar 10 µm.</p

CLSM localization of F-actin (red) and V-ATPase (green) at the apical region of osteoclasts.

<p>A–C: In osteoclasts on bone, F-actin (A) and V-ATPase (B) co-localize (C) in a patchy distribution within the F-actin rings. D–L: Like osteoclasts on bone, osteoclasts incubated on vitronectin show prominent F-actin staining in the actin ring/crescent, and noticeable F-actin staining within the ring/crescent. The F-actin within the actin ring tended to represent a greater proportion of smaller (D) than larger diameter (G, J) actin rings. V-ATPase (E, H, K) colocalizes with F-actin (F, I, L,). In all cells the F-actin/V-ATPase is present peripherally and as more central patches. C, F, I, L are counterstained with DAPI. Bar 10 µm (A–C), 5 µm (D–F, J–L) and 25 µm (G–I).</p

The apical surface of osteoclasts incubated on vitronectin- or fibronectin-coated nail-varnish.

<p>Osteoclasts were incubated on coverslips coated with nail-varnish and then vitronectin (A, C–F) or fibronectin (B), fixed, and inverted onto glass slides before removal of nail varnish and preparation for SEM. A: cell incubated on vitronectin, showing a circular arrangement of nodules, which merge in places (at top left of the cell) into ridges. These surround a central region filled with membrane folds. B: substrate-apposed surface of cell incubated on fibronectin lacks these features. C, D: cell shows raised nodules that correspond to podosomes (arrow). Within this podosome crescent are irregular, predominantly peripheral, patches of ruffled membrane. Orifices can be seen in the fold-free surface (arrowheads). E, F: well-spread cell with podosomes predominantly merged into ridges. Folded membrane is flattened against the substrate and is limited to a peripheral strip. Orifices can be seen in the apical membrane central to the peripheral ruffles (F, arrowheads). D and F are magnified portions of C and E, respectively. Bar 2 μm ( A, D, F), 5 μm (C, E) and 10 μm (B).</p

Transmission electron micrographs of osteoclasts after incubation on bone slices.

<p>A, B: osteoclast shows clear zone (CZ) at extreme left of field, and extensive ruffled border (RB) to centre and right. Between is an area, shown at higher magnification in B, in which the surface of the cell shows no membrane folds, and in which there is close approach of vesicles to the apical surface. C: view of part of an osteoclast in which zones of membrane ruffles are separated by a region of fold-free membrane. Secondary lysosomes are present just above this fold-free region. Bar 2 µm (A) and 1 µm (B, C).</p

CLSM localization of V-ATPase (green) and cathepsin K (red) in osteoclasts.

<p>A–C: Osteoclast incubated on bone shows strong immunolocalizion of V-ATPase in which a patch of cathepsin K immunolocalization is also seen, in a region free of V-ATPase. D: an osteoclast incubated on glass showing cathepsin K immunolocalized in the center of this well-spread cell, while V-ATPase is observed in small patches at the periphery, immediately adjacent to the actin ring (blue). C and D are also counterstained with DAPI to show nuclei. Bar 10 µm.</p

CLSM images showing the distribution of ClC-7, V-ATPase and F-actin in osteoclasts.

<p>A, B: In osteoclasts incubated on bone (A) or glass coverslips (B), ClC-7 (green) shows a clear circular distribution. C–F: In osteoclasts incubated on bone, ClC-7 (red) (C) is restricted to a circular strip immediately inside the F-actin ring (D) and immediately outside the central, V-ATPase-rich area (green) (E), and does not co-localize with either V-ATPase or F-actin (F). A, B, D, counterstained with DAPI to show nuclei. Bars 10 µm (A–C) and 50 µm (D).</p

Transmission electron micrographs of osteoclast after incubation on vitronectin-coated tissue culture plastic.

<p>A: low magnification of cell shown in B–E. In B, D a peripheral ‘clear zone’ (CZ), clear of organelles, is seen. This is further magnified in D and E, where it is immediately peripheral to a zone of closely-packed membrane folds (ruffled border, RB), which show a pale appearance resembling that of ‘clear zones’. C: central area is devoid of membrane folds. Many vesicles containing electron-dense material (arrows) can also be seen above this area. Bar 5 μm (A) and 1 μm (B–E).</p