Abstract
Actin ring formation is a prerequisite for osteoclast bone resorption. Although gelsolin null osteoclasts failed to exhibit podosomes, actin ring was observed in these osteoclasts. Wiscott-Aldrich syndrome protein (WASP) was observed in the actin ring of gelsolin null osteoclast. Osteoclasts stimulated with osteopontin simulated the effects of Rho and Cdc42 in phosphatidylinositol 4,5-bisphosphate (PIP2) association with WASP as well as formation of podosomes, peripheral microfilopodia-like structures, and actin ring. To explore the potential functions of Rho and Cdc42, TAT-mediated delivery of Rho proteins into osteoclasts was performed. Although Rho and Cdc42 are required for actin ring formation, transduction of either one of the proteins alone is insufficient for this process. Addition of osteopontin to osteoclasts transduced with Cdc42Val12 or transduction of osteoclasts with both RhoVal14 and Cdc42Val12 augments the formation of WASP-Arp2/3 complex and actin ring. Neomycin, an antibiotic, blocked the effects of osteopontin or TAT-RhoVal14 on PIP2 interaction with WASP. WASP distribution was found to be cytosolic in these osteoclasts. Depletion of WASP by short interfering RNA-mediated gene silencing blocked actin polymerization as well as actin ring formation in osteoclasts. These results suggest that Rho-mediated PIP2 interaction with WASP may contribute to the activation and membrane targeting of WASP. Subsequent interaction of Cdc42 and Arp2/3 with WASP may enhance cortical actin polymerization in the process of actin ring formation in osteoclasts.
Highlights
IntroductionThese cells exhibited a block in actin assembly as well as motility (6 – 8)
In the present study, our objective is to determine the intracellular pathways and the mechanisms by which the Wiscott-Aldrich syndrome protein (WASP)-mediated actin ring organization is induced in response to OPN/␣v3 signaling in osteoclasts
Previous studies from our laboratory and other laboratories have demonstrated the role of several signaling molecules in osteoclast cytoskeletal organization, podosomes assembly, actin ring formation, and bone resorption [27, 28, 35, 37, 53,54,55,56,57, 59, 62,63,64]. It is not known at this time, how these signaling molecules regulate cytoskeletal remodeling toward actin ring formation
Summary
These cells exhibited a block in actin assembly as well as motility (6 – 8) We have used these peptides to delineate phosphoinositides-mediated signaling in actin reorganization, podosomes assembly/disassembly, and bone resorption. Transduction of PBD peptides of gelsolin into osteoclasts produced clusters of podosomes and disrupted the formation of the actin ring. These osteoclasts were hypomotile and less resorptive [9]. We have used neomycin to determine the role of PIP2and TAT-mediated delivery of Rho GTPases, such as Rho and Cdc in the activation of WASP, as well as cortical actin remodeling in the formation of the actin ring in osteoclasts. A cooperative interaction between Rho and Cdc has been shown to be required in the process of actin ring formation in osteoclasts
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