Abstract
BackgroundGelsolin, an actin capping protein of osteoclast podosomes, has a unique function in regulating assembly and disassembly of the podosome actin filament. Previously, we have reported that osteopontin (OPN) binding to integrin αvβ3 increased the levels of gelsolin-associated polyphosphoinositides, podosome assembly/disassembly, and actin filament formation. The present study was undertaken to identify the possible role of polyphosphoinositides and phosphoinositides binding domains (PBDs) of gelsolin in the osteoclast cytoskeletal structural organization and osteoclast function.ResultsTransduction of TAT/full-length gelsolin and PBDs containing gelsolin peptides into osteoclasts demonstrated: 1) F-actin enriched patches; 2) disruption of actin ring; 3) an increase in the association polyphosphoinositides (PPIs) with the transduced peptides containing PBDs. The above-mentioned effects were more pronounced with gelsolin peptide containing 2 tandem repeats of PBDs (PBD (2)). Binding of PPIs to the transduced peptides has resulted in reduced levels of PPIs association with the endogenous gelsolin, and thereby disrupted the actin remodeling processes in terms of podosome organization in the clear zone area and actin ring formation. These peptides also exhibited a dominant negative effect in the formation of WASP-Arp2/3 complex indicating the role of phosphoinositides in WASP activation. The TAT-PBD gelsolin peptides transduced osteoclasts are functionally defective in terms of motility and bone resorption.ConclusionsTaken together, these data demonstrate that transduction of PBD gelsolin peptides into osteoclasts produced a dominant negative effect on actin assembly, motility, and bone resorption. These findings indicate that phosphoinositide-mediated signaling mechanisms regulate osteoclast cytoskeleton, podosome assembly/disassembly, actin ring formation and bone resorption activity of osteoclasts.
Highlights
Gelsolin, an actin capping protein of osteoclast podosomes, has a unique function in regulating assembly and disassembly of the podosome actin filament
Since gelsolin has a unique function in regulating assembly and disassembly of the podosome actin filament [13,14], we addressed the question whether phosphoinositides and actin binding peptides of gelsolin would be able to block actin assembly and bone resorption in osteoclasts
Transduction of phosphoinositide binding domain (PBD) containing gelsolin peptides decreases the interaction of PI3-Kinase with endogenous gelsolin We have demonstrated that OPN stimulated tyrosine phosphorylation of the p85 regulatory subunit of PI3kinase associated with gelsolin, and increased its activity as measured by PtdIns Phosphatidylinositol 3 (P3) production in immune complex phospholipid kinase assay in vitro [13,46]
Summary
An actin capping protein of osteoclast podosomes, has a unique function in regulating assembly and disassembly of the podosome actin filament. The present study was undertaken to identify the possible role of polyphosphoinositides and phosphoinositides binding domains (PBDs) of gelsolin in the osteoclast cytoskeletal structural organization and osteoclast function. Interaction of phosphoinositides with the specific binding domains helps to coordinate the assembly of one to several signaling molecules These molecules regulate cellular signal transduction processes involving stress fiber and focal adhesion formation [11,12]. Our previous studies have provided insights into the roles of phosphoinositides in gelsolin function, in the regulation of actin reorganization, and podosome assembly/disassembly in avian and mouse osteoclasts [13,14,15,16]
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