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. 2000 Feb 21;148(4):665-78.
doi: 10.1083/jcb.148.4.665.

Gelsolin deficiency blocks podosome assembly and produces increased bone mass and strength

M Chellaiah  1 , N Kizer, M Silva, U Alvarez, D Kwiatkowski, K A Hruska
Affiliations

Gelsolin deficiency blocks podosome assembly and produces increased bone mass and strength

M Chellaiah et al. J Cell Biol. .

Abstract

Osteoclasts are unique cells that utilize podosomes instead of focal adhesions for matrix attachment and cytoskeletal remodeling during motility. We have shown that osteopontin (OP) binding to the alpha(v)beta(3) integrin of osteoclast podosomes stimulated cytoskeletal reorganization and bone resorption by activating a heteromultimeric signaling complex that includes gelsolin, pp(60c-src), and phosphatidylinositol 3'-kinase. Here we demonstrate that gelsolin deficiency blocks podosome assembly and alpha(v)beta(3)-stimulated signaling related to motility in gelsolin-null mice. Gelsolin-deficient osteoclasts were hypomotile due to retarded remodeling of the actin cytoskeleton. They failed to respond to the autocrine factor, OP, with stimulation of motility and bone resorption. Gelsolin deficiency was associated with normal skeletal development and endochondral bone growth. However, gelsolin-null mice had mildly abnormal epiphyseal structure, retained cartilage proteoglycans in metaphyseal trabeculae, and increased trabecular thickness. With age, the gelsolin-deficient mice expressed increased trabecular and cortical bone thickness producing mechanically stronger bones. These observations demonstrate the critical role of gelsolin in podosome assembly, rapid cell movements, and signal transduction through the alpha(v)beta(3) integrin.

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Figures

Figure 1
Figure 1
High-power examination of the actin cytoskeleton of Gsn+/+ and Gsn−/− osteoclasts. Osteoclasts cultured on glass coverslips were fixed and stained with rhodamine-phalloidin. Cells were examined by confocal microscopy. The actin cytoskeleton of Gsn−/− osteoclasts (lower panels) was distinctly altered compared with the Gsn+/+ cells (upper panels). The arrows indicate areas of the cytoskeleton shown at higher magnification (right, upper and lower panels). Bars, 100 μm (left panels); 10 μm (right panels).
Figure 2
Figure 2
The effect of OP on the actin cytoskeleton. Osteoclasts were fixed and stained with rhodamine-phalloidin. Actin filament organization in cells treated with PBS or OP is shown. Cells were examined by confocal microscopy. The actin staining of osteoclasts derived from wild-type (+/+) and null (−/−) mice are shown.
Figure 4
Figure 4
The effect of OP treatment on F-actin content of Gsn+/+ and Gsn−/− osteoclasts. F-actin content of wild-type (Gsn+/+, PBS) osteoclasts was increased threefold by treatment with OP (Gsn+/+, OP). No effect of OP on F-actin levels was seen in the gelsolin-eficient (Gsn−/−, PBS and Gsn−/−, OP) osteoclasts. Asterisks indicate that data are mean ± SEM (n = 3), P < 0.001.
Figure 5
Figure 5
The effect of OP on phosphorylation of proteins associated with gelsolin. (A) In vitro protein kinase assay without casein as exogenous substrate and (B) with casein as exogenous substrate. Phosphorylation of protein with molecular masses of 125, 85, and 60 kD is shown by arrows in A and B. Phosphorylation of casein is shown in B. Phosphorylation of casein is increased by OP treatment. (C) Effect of OP on gelsolin-associated PI3-K activity. Autoradiogram of the TLC plate is shown. OP-stimulated phosphatidylinositide 3,4,5-trisphosphate (PtdIns P3) formation is shown by arrow. Treatments are shown below the figure.
Figure 6
Figure 6
Vinculin distribution in Gsn+/+ and Gsn−/− osteoclasts. In Gsn+/+ osteoclasts, vinculin was arrayed in often dual rows bordering actin rings shown here as the unstained ring between the vinculin (+/+). Two types of vinculin distribution were observed in the Gsn−/− osteoclasts: a peripheral distribution intermixed in the actin mesh (middle panels, +/+), or a diffuse distribution (lower panels, −/−).
Figure 7
Figure 7
Effect of OP on the motility of Gsn+/+ and Gsn−/− osteoclasts. Osteoclast motility was assessed in vitro during both phagokinesis (A) and haptotaxis (B) assays. OP increased motility of Gsn+/+ osteoclasts more than twofold in both assays, and had no effect on the motility of Gsn−/− osteoclasts. Likewise vitronectin (VN) stimulated osteoclast motility, whereas GRGDS and fibronectin (FN) had no effect. None of these proteins stimulated Gsn−/− osteoclast motility. Furthermore, in the haptotaxis assays, the Gsn−/− osteoclasts were significantly hypomotile. The data in A represent three separate osteoclast preparations, and each experiment is the mean of 20–30 cell tracks. The data are mean ± SE. Asterisks indicate P < 0.0001. The data in B represent four separate osteoclast preparations, and four Transwells were counted for each experimental condition. Asterisk indicates P < 0.01.
Figure 8
Figure 8
Polarization of the osteoclast plasma membrane as detected by distribution of the vacuolar H+ ATPase. (A) Confocal microscopy of an osteoclast derived from Gsn−/− cells. The section was taken at the level of the dentine slice as shown by the reflected light (red). The arrows point to areas of concentration of the H+ ATPase detected by the E11 antibody. (B) The areas of H+ ATPase concentration overlie a multiocular resorption pit produced by the osteoclast.
Figure 9
Figure 9
Bone resorbing activity of osteoclasts isolated from Gsn+/+ and Gsn−/− mice. Osteoclasts were plated on dentine slices as described in Materials and Methods, and the formation of resorption pits was assessed after 48 h. In low-power views of dentine slices stained by acid hematoxylin, resorption pits were seen as dark spots as shown in A. In the dentine slices on which Gsn+/+ osteoclasts (+/+) were added, there were numerous resorption pits that increased in size and complexity by treatment with OP. In osteoclasts derived from cells of Gsn−/− mice (−/−), resorption pits were small and not affected by OP treatment. (B) Confocal images of the pit. Individual resorption pits were assessed by confocal microscopy for pit depth (XZ) and pit area (XY). In dentine slices on which Gsn−/− osteoclasts were plated, pit depth was diminished. Pit depth was remarkably increased by treatment with OP in Gsn+/+ osteoclasts, but it was unaffected by OP in the slices with Gsn−/− osteoclasts. Group data of pit depth (XZ scans) and pit area (XY scans) from confocal microscopy of pits on multiple dentine slices from three osteoclast preparation are shown in Table .
Figure 10
Figure 10
Histomorphometric analysis of proximal tibial sections of Gsn−/− and Gsn+/+ mice. Proximal tibial sections of bone isolated from 14-wk-old Gsn+/+ (A and B) and Gsn−/− (C and D) mice were stained for TRAP (A and C) or with toluidine blue (B and D). (A and C) TRAP staining. Numerous osteoclasts were visible in the primary spongiosa and on the surfaces of metaphyseal trabeculae in both +/+ (A) and −/− mice (C). The number of TRAP-positive osteoclasts below the growth plate in −/− mice tends to be increased, giving the appearance of a double row of cells as the trabeculae develop from the primary spongiosa (C). The trabecular bone volume in the metaphysis of the −/− mice was increased (shown by arrows; compare A and C; see Table ). (B and D) Toluidine blue staining. The metaphyseal trabeculae of −/− mice (D) had retained cartilage-derived proteoglycan.
Figure 11
Figure 11
Mechanical strength of femurs from Gsn+/+ and Gsn−/− mice. (a) Graphic representation of the data generated by four-point bending test. The slope of the line relating bending and displacement is a reflection of stiffness or rigidity, and the area under the curve is the energy required to produce failure. (b) The ultimate moment-displacement curves for +/+ and −/− femurs. The normalized displacement at failure (ultimate) was greater in the Gsn−/− femurs (see Table ). (c) The energy required to produce failure was greater in the femurs from Gsn−/− mice (see Table ).
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