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Localization and possible role of two different alpha v beta 3 integrin conformations in resting and resorbing osteoclasts

¹ Department of Human Anatomy and Histology, University of Bari, Italy
² M.I.A. Dibit-hsr et University of Milano Bicocca-Monza, Milan, Italy
³ Department of Internal Medicine, University of Bari, Italy
⁴ Department of Cell Biology and The Scripps Research Institute, La Jolla, USA

View larger version (41K): [in a new window]   Fig. 4. Localization of total vß3 in resorbing OC. A resorbing OC is schematically represented in A. It appears as a polarized cell, attached to the bone through the sealing zone (SZ), that forms a specific membrane domain, the ruffled border (RB) involved in the dissolution of the bone matrix. Primary human OCs were plated onto bone slices in the presence of 10% FBS, and stained by double immunofluorescence with the anti-ß3 mAb AP3 followed by cyanine-3-conjugated rabbit anti-mouse IgG (B,D,F) and by fluorescein-tagged phalloidin (C,E,G). Microscopic images show the three different axial sections indicated in A: (B-C) at the level of the SZ, where vß3 (B) appears as a circular line at the cell periphery that perfectly colocalizes with the actin ring (C); (D-E) in RB, vß3 and the actin show a punctuate distribution; (F-G) an optical yz-plane confirms the distribution of the integrin and of the actin in the SZ and in the RB. Bar, 5 µm.

View larger version (24K): [in a new window]   Fig. 1. Growth factor pre-treatment increases cell surface expression of `activated' vß3. (A) Unstimulated human osteoclast-like cells were stained with the anti-ß3 mAb AP5 in the presence of 1.3 mM calcium (AP5+Ca) or in calcium-free buffer (AP5-Ca). In the presence of divalent cations, AP5 recognizes the fraction of the vß3 integrin in the `activated' conformation. Incubation of cells with AP5 in calcium-free buffer shifts all the receptor into the `activated' conformation. (B,C) Pre-incubation with HGF (B) or with M-CSF (C) increases the binding of AP5 in 1.3 mM calcium buffer, shifting most of the integrin into the `activated' conformation. Mean fluorescence intensity is indicated on the abscissa; the number of fluorescence-positive cells is depicted on the ordinate.

View larger version (103K): [in a new window]   Fig. 2. Immunolocalization of total and `activated' vß3 integrin in non-resorbing human osteoclasts. Primary human OCs were plated on coverslips in the presence of 10% FBS and immunostained for total vß3 with mAb AP3 (A) or with AP5 (C), which binds specifically to the `activated' conformation. Cells were co-stained with F-PHD for actin (B,D). Total vß3 is found in rosette-like structures clustered around actin cores (Fig. 2A,B, insert) and in the motile parts of the plasma membrane (arrow). The `activated' conformation of the integrin detected by AP5 (C) is clustered in the motile region of the plasma membrane, corresponding to ruffles and lamellipodia. At the periphery, as indicated by the arrow (Fig. 2C, insert), the integrin appears as a continuous line that colocalizes with the actin fibers (Fig. 2D, insert). Bar, 10 µm.

View larger version (72K): [in a new window]   Fig. 3. Localization of `activated' vß3 integrin in live and fixed cells. Human OCs were stained before (A-C) or after (D-F) fixation with the monoclonal activating Ab WOW-1 (red), which binds to the `activated' form of vß3, and costains with fluorescein-tagged phalloidin to detect the actin organization (green). The `activated' form of vß3 (A,D, arrows) is specifically organized at the cell edge where it colocalized with cortical actin (B,E, arrows). Clusters of podosomes are also evident in both fixed and live cells, but in both cases the `activated' form of vß3 is not organized around the podosomal core of actin filaments as shown in the overlay (C,F, asterisk)

View larger version (79K): [in a new window]   Fig. 5. Localization of `activated' vß3 in resorbing OC. Primary human OCs were plated onto bone slices in the presence of 10% FBS and stained by double immunofluorescence with the anti-ß3 mAb AP5, which recognizes only the `activated' integrin conformation, followed by cyanine-3-conjugated rabbit anti-mouse IgG (A,C,E) and by fluorescein-tagged phalloidin (B,D,F). Microscopic images show the three different axial sections indicated in Fig. 3A: at the level of the sealing zone (SZ), the `activated' ß3 (A) only partially colocalizes with the actin ring (B) and displays a discrete pattern. In the ruffled border area (RB) the `activated' ß3 (C) is very abundant with a punctuate distribution similar to that of the actin microfilaments of D. In the optical yz-plane, note the presence of the actin in the sealing zone SZ (F) as a line at the edge of the cell that only partially colocalizes with the integrin detected by AP5 (E). By contrast, the `activated' receptor and the actin are both present in the ruffled border area RB. Bar, 5 µm.

View larger version (93K): [in a new window]   Fig. 6. Pre-treatment with HGF and M-CSF in resting osteoclasts promotes vß3 redistribution in membrane ruffles and lamellipodia. Primary human osteoclasts plated onto coverslips were preincubated with vehicle alone (A-C), HGF (5 ng/ml) (D-F) or M-CSF (0.5 nM) (G-J) and immunostained for total vß3 with AP3 and co-stained with FITC-phalloidin. Pseudocolor merged images from vß3 staining in red and actin in green have been examined. The typical distribution of vß3 in rosette-like structures around a core of actin filaments in the podosomes, detected by AP3 in vehicle-treated osteoclasts (A-C, arrowhead), has been replaced by a peripheral distribution in membrane ruffles and lamellipodia in (D-F, arrow) HGF- or (G-J, arrow) M-CSF-treated osteoclasts. Pre-incubation with wortmannin before growth factor treatment inhibited integrin mobilization to the membrane edges, and vß3 is still organized in rosette-like structures (K-M, arrowhead). Phalloidin staining (B,E,H,L) shows that the podosomes in all treated cells are still well organized. Wortmannin-treated cells also show filaments of cortical actin (L). Bar, 10 µm.

View larger version (59K): [in a new window]   Fig. 7. Redistribution of vß3 occurs in resorbing human OCs pre-incubated with HGF. Primary human OCs, incubated with vehicle alone (A-C) or HGF (5 ng/ml) (D-F) were plated onto hydroxyapatite-coated coverslips (BD BioCoatTM OsteologicTM) and examined by immunofluorescence, followed by staining with AP5 (A,D) and FITC phalloidin (B,E). Without exogenous growth factors, the activated vß3 is distributed with a punctuate pattern on the cell surface (A,B). A pseudocolar merged image (C) from vß3 staining in red (A) and actin in green (B) fails to reveal colocalization of the integrin with the actin ring. HGF treatment induces a change of conformation of vß3 that is found mostly in fillopodia-like extensions (D). The pseudocolor merged image (F) reveals vß3 (red) mainly localized in small protrusions at the cell periphery, external to the actin ring (green) in the sealing zone (single arrow). A partial colocalization between the integrin and the actin ring is seen in yellow (double arrows). Bar, 5 µm.

View larger version (169K): [in a new window]   Fig. 8. HGF and M-CSF enhance bone resorption. Murine bone marrow macrophages were cultured onto dentine slices (A-C) or millennium slides (D-F) for 3 days in the presence of RANKL (100 ng/ml) and M-CSF (10 ng/ml). HGF (50 ng/ml), M-CSF (100 ng/ml) or vehicle alone were added for the next 48 hours and bone resorptive activity was analyzed. (A-C) After indicated treatments, cells plated onto dentine slices were fixed, TRAP stained and subjected to light microscopy. Pits, indicated by arrows, increased in number and area in (B) HGF- and (C) M-CSF-treated OCs compared with the control (A). (D-G) Similar experiments were performed onto millennium slides. At the indicated time, osteoclasts were removed by brief treatment with bleach, and resorptive areas were analyzed by light microscopy (identified as the light blue regions). HGF (E) and M-CSF (F) increased erosion areas by 65% and 58%, respectively, compared with vehicle-treated OCs (D). G shows a control well with no cells. All images are at the same magnification.

View larger version (38K): [in a new window]   Fig. 9. Pre-incubation with HGF and M-CSF induces an increase in cell motility toward OPN, in a vß3- and a PI-3-kinase-dependent manner. (A) Migration towards OPN. Human osteoclast-like cells (open bars) preincubated with AP5 in buffer without calcium, HGF (5 ng/ml) or M-CSF (0.5 nM) are allowed to migrate for 6 hours toward OPN (10 µg/ml), coated on the bottom membrane of a transwell plate (8 µm pore size). All three treatments significantly increased (P<0.001) migration toward OPN compared with vehicle alone (CTR) (open bars). Cell migration was inhibited by the vß3- blocking mAb LM609 (hatched bars), indicating specific involvement of the integrin vß3, and by the PI 3-kinase inhibitor wortmannin (cross-hatched bars). (B) Migration toward LN. Migration of human osteoclast-like cells toward LN is not enhanced by growth factor or AP5 pre-treatment (open bars) nor decreased by LM609 (hatched bars) or wortmannin (cross-hatched bars). In all experiments cells that migrated were viewed at 300x magnification, and the number of cells per field was counted.