Abstract
Rho plays a regulatory role in the formation of actin stress fibers and focal adhesions, and it is also involved in integrin-mediated signaling events. To study the role of Rho in alpha(v)beta(3)/gelsolin-dependent signaling, the HIV-Tat peptide, hemagglutinin (HA)-tagged Rho(Val-14) (constitutively active) and Rho(Asn-19) (dominant negative) were transduced into avian osteoclasts. Protein transduction by HA-Tat was highly efficient, and 90-100% of the cells were transduced with HA-tagged proteins. We demonstrate here that Rho(Val-14) transduction (100 nM) stimulated gelsolin-associated phosphatidylinositol 3-kinase activity, podosome assembly, stress fiber formation, osteoclast motility, and bone resorption, mimicking osteoclast stimulation by osteopontin/alpha(v)beta(3.) The effects of Rho(Val-14) transduction stimulation was time-dependent. C3 exoenzyme blocked the effects of Rho(Val-14) and induced podosome disassembly, loss of motility, and inhibition of bone resorption. Transduction of Rho(Asn-19) produced podosome disassembly, and blocked osteopontin stimulation. These data demonstrate that integrin-dependent activation of phosphoinositide synthesis, actin stress fiber formation, podosome reorganization for osteoclast motility, and bone resorption require Rho stimulation.
Highlights
Rho, which plays a critical and regulatory role in the formation of actin stress fibers and focal adhesions, is involved in integrin-mediated signaling events [1] and has been implicated in osteoclast function
The integrin-mediated recruitment of signaling molecules required for motility and bone resorption in response to osteopontin (OP)1 is absent in gelsolin null osteoclasts [20]
Using HIV-TAT-mediated delivery [26] of Rho proteins into osteoclasts, we demonstrated that constitutively active Rho transduction was sufficient for podosome assembly but that it mimicked the effects of OP by stimulating actin stress fiber formation, motility, and bone resorption
Summary
[␥-32P]ATP and rainbow molecular weight markers for proteins were obtained from Amersham Pharmacia Biotech. Fluorescent labeling was done with either HA antibody or Rhodamine phalloidin after osteoclasts were treated with HA-Tat proteins as described above for the indicated periods. After 3– 4 days in culture, chicken osteoclasts were washed twice with PBS and incubated at 37 °C with cell dissociation buffer (Sigma) for 20 min. After cells attached to the membrane, TAT fusion proteins were added to a final concentration of 100 nM in the upper chamber in a serum-free ␣-MEM. After 3 days in culture, avian osteoclasts were washed with PBS, and treated with cell dissociation buffer for 15 min at 37 °C. Osteoclast suspensions (2 ϫ 104 cells/well) were added to dentine slices that had been washed with ␣-MEM containing 1% BSA. Pits were viewed under ϫ40 objective in a phase-contrast microscope and photographed
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