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

Chellaiah M, Kizer N, Silva M, Alvarez U, Kwiatkowski D, Hruska KA - J. Cell Biol. (2000)

Bottom Line: 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.These observations demonstrate the critical role of gelsolin in podosome assembly, rapid cell movements, and signal transduction through the alpha(v)beta(3) integrin.

View Article: PubMed Central - PubMed

Affiliation: Renal Division, Department of Medicine, Barnes-Jewish Hospital, Washington University, St. Louis, Missouri 63110, USA.

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- 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- 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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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.
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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.

Mentions: We have shown previously that binding of OP to αvβ3 stimulated association signal generating molecules with gelsolin in avian osteoclasts (Chellaiah and Hruska 1996; Chellaiah et al. 1998). To see whether OP stimulation in mouse osteoclasts produced similar activation as seen in avian osteoclasts, in vitro kinase assays were performed on the immune complexes generated by using a gelsolin antibody. In vitro immune complex kinase assays for pp60c-src have demonstrated that OP induced an increase in phosphorylation of pp60c-src (Fig. 5A and Fig. B) and increased its activity towards phosphorylating an exogenous substrate, casein (Fig. 5 B). In addition, phosphorylation of two other proteins with the molecular masses of 125 kD (Fig. 5A and Fig. B) and 85 kD (Fig. 5 B) was observed. p85 protein was identified as PI3-K in avian osteoclasts (Chellaiah and Hruska 1996). Coimmunoprecipitation of PI3-K with gelsolin was further confirmed by a PI3-K assay (Fig. 5 C). OP stimulation of PI3-K activity associated with gelsolin is evident from the phosphorylation of the exogenous substrate, phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate (Fig. 5 C).


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

Chellaiah M, Kizer N, Silva M, Alvarez U, Kwiatkowski D, Hruska KA - J. Cell Biol. (2000)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169374&req=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.
Mentions: We have shown previously that binding of OP to αvβ3 stimulated association signal generating molecules with gelsolin in avian osteoclasts (Chellaiah and Hruska 1996; Chellaiah et al. 1998). To see whether OP stimulation in mouse osteoclasts produced similar activation as seen in avian osteoclasts, in vitro kinase assays were performed on the immune complexes generated by using a gelsolin antibody. In vitro immune complex kinase assays for pp60c-src have demonstrated that OP induced an increase in phosphorylation of pp60c-src (Fig. 5A and Fig. B) and increased its activity towards phosphorylating an exogenous substrate, casein (Fig. 5 B). In addition, phosphorylation of two other proteins with the molecular masses of 125 kD (Fig. 5A and Fig. B) and 85 kD (Fig. 5 B) was observed. p85 protein was identified as PI3-K in avian osteoclasts (Chellaiah and Hruska 1996). Coimmunoprecipitation of PI3-K with gelsolin was further confirmed by a PI3-K assay (Fig. 5 C). OP stimulation of PI3-K activity associated with gelsolin is evident from the phosphorylation of the exogenous substrate, phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate (Fig. 5 C).

Bottom Line: 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.These observations demonstrate the critical role of gelsolin in podosome assembly, rapid cell movements, and signal transduction through the alpha(v)beta(3) integrin.

View Article: PubMed Central - PubMed

Affiliation: Renal Division, Department of Medicine, Barnes-Jewish Hospital, Washington University, St. Louis, Missouri 63110, USA.

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- 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- 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.

Show MeSH
Related in: MedlinePlus