Antibodies to WASP, HA, Arp2, c-Src and c-Src pTyr418 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rhodamine phalloidin was bought from Sigma-Aldrich chemicals (St.Louis, MO). Ni-NTA sepharose beads were purchased from Pharmacia chemicals. Acrylamide-bisacrylamide solution and protein estimation reagent were obtained from Bio-Rad.

C57/BL6 mice were used for in vitro osteoclast generation. Bone marrow cells isolated from five mice were cultured into 100 mm dishes with 20 ml α-MEM medium, supplemented with 10% fetal bovine serum (α-10). After culturing for 24 h, non-adhered cells were layered on histopaque-1077 (Sigma) and processed as described previously 20. Cells were cultured with the appropriate concentrations of mCSF-1 (10 ng/ml; R and D chemicals, IN) and RANK-L (55–75 ng/ml). After three days in culture, media were replaced with fresh cytokines. The multinucleated osteoclasts were seen from day four onwards.

Bacterial expression constructs coding various HIV-TAT fusion peptides of WASP were generated by PCR method. cDNA from mouse osteoclast and full length WASP in pGEX-2T were used as PCR templates. WASP constructs were generated from mouse cDNA library by using the following primers:

Full length (FL):

Escherichia coli (BL-21 strain from Invitrogen, Carlsbed, CA) was used to transform HA-TAT vector containing the above-mentioned WASP constructs. Protein was purified using Ni-NTA column as described previously 24. Purified proteins were subjected to SDS-PAGE followed by Western analysis with a hemagglutinin (HA) antibody and Coomassie blue staining to determine the molecular weight and homogeneity 23. HA-TAT vector (8–10 kDa) and Herplex simplex virus thymidine kinase (Hsv-TK; 42 kDa) proteins were used as controls for transduction experiments 24.

Following transduction with various proteins, osteoclasts were washed three times with cold PBS and lysed in RIPA buffer (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% Triton ×-100, 0.1% SDS, 1% aprotinin and 2 mM PMSF) and centrifuged at 15,000 rpm for 15 min at 4°C. Protein contents were measured using the Bio-Rad protein assay reagent. Equal amounts of lysate proteins were used for immunoprecipitation with antibody to WASP or HA. Immunoprecipitation and immunoblotting were performed as described previously 25.

Osteoclast precursors (10⁵ cells) were seeded on dentine slices and cultured for 36–48 h. Prior to staining; osteoclasts were washed three times with PBS containing 5 mM EGTA (PBS/EGTA). Osteoclasts were fixed with 3% paraformaldehyde in PBS/EGTA for 20 min. Subsequently, cells were permeablized with 0.1% Triton ×-100 in PBS/EGTA for 5 min. Osteoclasts were incubated with rhodamine phalloidin (Sigma; 1:1000 dilution) for 30 min. at 37°C or overnight at 4°C as described earlier 1. The cells on dentine slices were washed and mounted on a slide in a mounting solution (Vector laboratories, Inc., Burlingame, CA) and sealed with nail polish. Immunostained osteoclasts were photographed with a Bio-Rad confocal laser-scanning microscope. Images were stored in TIF image format and processed by the Adobe Photoshop software program (Adobe System Inc., Mountain view, CA).

On day four, osteoclasts were transduced with 100 nM TAT-fused WASP peptides and incubated for 36–48 h. After transduction, osteoclasts were rinsed three times. Subsequently, cells were fixed, permeablized, and incubated with rhodamine phalloidin (1:200) as described earlier 20. Cells were extracted with absolute methanol and the fluorescence of each sample was measured with fluorimetry (Bio-RAD spectrofluorometer). Osteoclasts untreated with rhodamine phalloidin were used to determine the background fluorescence of the cells. Ten-fold excess unlabelled phalloidin was used to determine the non-specific binding. The non-specific binding and background fluorescence were subtracted from the total binding to determine the specific binding 2627.

Osteoclasts transduced with the indicated TAT-WASP proteins were added to 48-well-containing dentine slices (2 × 10⁴ cells). After 2 h of adherence, α-MEM medium containing 10% FBS and RANK-L was added. The same medium was replaced with the respective TAT-fused WASP proteins after 24 h. After 48 h, cells were scraped from dentine, and the slices were washed twice with water. Dentine slices were stained with acid hematoxylin (Sigma) and washed with water to remove excess stain. Pits were scanned under confocal microscopy. Images were stored in TIF image format and processed by the Adobe Photoshop software program (Adobe Systems, Inc., Mountain View, CA).

All values presented are expressed as the mean ± standard error of the mean (SEM) of three or more experiments done at different times normalized to intra-experimental control values. Asterisks were used to graphically indicate the statistical significance. A value of p < 0.05 was considered significant. For statistical comparisons, analysis of variance (ANOVA) was used with the Bonferroni corrections (Instat for IBM, version 2.0; Graphpad software).

A schematic diagram of WASP constructs cloned into HA-TAT expression vector is shown in Fig. 1A. Cloning was performed as described in the Methods section. Amino acid sequence of each WASP construct is shown within the parentheses. Purified proteins were analyzed by SDS-PAGE and stained with Coomassie blue (Fig. 1B). In an 8% SDS-PAGE gel (Fig. 1B) purified TAT-fused full length (FL)-WASP (65–70 kDa; lane 1),

G

p

Based on the dose-dependent uptake experiment (data not shown), a concentration of 100 nM TAT-fused WASP protein was decided for transduction experiments given below. The uptake of TAT-fused WASP proteins were determined by immunoblotting with an antibody to HA (Fig. 2, top panel). About 200 μg of osteoclast lysate was used for immunoblotting analysis. Western blot analysis with anti-HA demonstrates the levels of transduced peptides in each lane (Fig. 2, top panel). Loading was normalized to the cellular levels of GAPDH (Fig. 2, bottom panel).

VCA domain of WASP binds actin monomers by the V domain, and the Arp2/3 complex through the C and A domains. This interaction increases actin polymerization in vivo and in vitro 2151928. We examined the effects of transduction of various TAT-fused WASP peptides on the F-actin content in osteoclasts (Fig. 3). A significant increase in the F-actin content was observed in osteoclasts transduced with FL-WASP. Osteoclasts treated with PBS- or transduced with HA-TAT and Hsv-TK exhibited the basal level F-actin content. A decrease in the F-actin content was observed in osteoclasts transduced with basic (BR), GP, pTyr294aa, Pro, and VC domains. The decrease in the F-actin content was more pronounced in osteoclasts transduced with TAT-fused WASP fragment containing pTyr294aa.

Subsequently, we proceeded to analyze the effects of various TAT-fusion proteins on Arp2 interaction with endogenous WASP (Fig. 4A) and transduced TAT proteins (Fig. 4C). Osteoclast lysates were immunoprecipitated with WASP antibody and immunoblotted with an antibody to Arp2 (Fig. 4A). Interaction of Arp2 with the immunoprecipitated endogenous WASP protein was observed in osteoclasts transduced with HA-TAT vector protein and FL-WASP (Fig. 4A, lanes 1 and 2). This interaction is considerably reduced with endogenous WASP in osteoclasts transduced with TAT-fused WASP peptides containing the following domain: proline-rich (Fig. 4A, lane 3), pTyr294aa (lane 4), and VC (lane 5). However, the decrease was more in osteoclasts transduced with WASP peptides containing pTyr294aa (lane 4) and VC (lane 5). Stripping and reprobing of this blot with an antibody to WASP demonstrated the WASP level in each immunoprecipitates (Fig. 4B).

Since VC transduced osteoclasts exhibited reduced levels of Arp2 interaction with endogenous WASP, osteoclast lysates were immunoprecipitated with a HA-antibody to determine the interaction of Arp2 with the transduced peptides. HA immunoprecipitates were divided into two halves. The first half was subjected to 8% SDS-PAGE, immunoblotted with Arp 2 antibody (Fig. 4C). Binding of Arp 2 with transduced FL-WASP (Fig. 4C, lane 1) and VC domain (4C, lane 5) was observed. The observed decrease in the F-actin content in osteoclasts transduced with VC domain (Fig. 3) may be caused by the competitive binding of Arp2 with these fragments (Fig. 4C, lanes 1 and 5). The significant increase in F-actin content in FL-WASP transduced osteoclasts (Fig. 3) may be due to the combined effects of endogenous and transduced WASP in the actin polymerization process. Transduction of WASP peptides containing pTyr294aa or Pro-rich sequences also blocked Arp-2 binding despite the fact that these peptides do not have any binding site for Arp2/3 complex. We have recently shown that the phosphorylation of WASP protein, as well as its interaction with signaling proteins, is critical for actin polymerization 20. One possible reason could be that the phosphorylation and interaction of signaling protein with endogenous WASP may be competitively blocked by these peptides. As a result of this interaction, the stability of active WASP, its interaction with Arp2 and actin polymerization (Figs. 3 and 4) may have decreased. The second half of the HA-immunoprecipitates was subjected to 8% (Fig. 4D, lane 1) and 12% (lanes 2–7) SDS-PAGE. Immunoblotting with a HA-antibody demonstrated the presence of transduced TAT-proteins in the osteoclast lysate. Immunoprecipitation with a non-immune serum is shown in lane 6 (Fig. 4A–D).

We have previously demonstrated that tyrosine phosphorylation of WASP and associated proteins are in part dependent on the c-Src kinase activity 20. We hypothesized that transduction of WASP fragments containing proline rich-sequences and pTyr294 sequence would competitively reduce the interaction of c-Src with endogenous WASP. The ability of the transduced peptides containing proline rich and pTyr294aa sequences to interfere the binding of c-Src with endogenous WASP was investigated as shown in Figure 5. Immunoprecipitates made with an antibody to WASP (Figure 5A) or HA (Figure 5C) were immunoblotted with a Src-pTyr418 antibody. Subsequently, these blots were stripped and reprobed with a c-Src antibody (Figures 5B and 5D).

Coprecipitation of c-Src was observed with WASP immunoprecipitates in osteoclasts transduced with vector (HA-TAT) and various WASP peptides (Fig. 5A and 5B). The phosphorylation (Fig. 5A) and protein (Fig 5B) levels of c-Src are more in osteoclasts transduced with FL-WASP (lane 4) than the basal level observed in osteoclasts transduced with HA-TAT (lane 3). The increase in Src level in FL-WASP transduced osteoclasts may be due to that WASP antibody precipitates both the endogenous and transduced WASP protein. Phosphorylation (A, lanes 1 and 2) and protein (B, lanes 1 and 2) levels of c-Src associated with endogenous WASP are very minimal in osteoclasts transduced with WASP fragments containing pTy294 aa and proline-rich sequences.

The reduced interaction of c-Src with endogenous WASP (Figure 5A and 5B; lane 2) in OCs transduced with TAT-fused proline sequences was parallel with an increase in the association of c-Src with the transduced peptide in the immunoprecipitation performed with a HA-antibody (Fig. 5C; lane 1). An increase in the phosphorylation (Fig. 5C, lane 1) and protein (Fig 5D, lane 2) levels of c-Src may be due to the presence of six proline-rich clusters in this fragment. Interaction of c-Src with proline-rich sequence (Figure 5D, lane 1) is more than the level observed with FL-WASP peptide (Fig. 5D, lane 2). WASP fragment containing pTyr294 has only two proline aa sequences of the first cluster. Therefore, interaction of c-Src with this fragment was very negligible or not observed (Fig. 5C and 5D, lane 3). The interaction c-Src with the transduced proline sequences and FL-WASP is specific as immunoprecipitation with a HA- antibody in osteoclasts transduced with vector protein HA-TAT failed to exhibit coprecipitation of c-Src. These observations confirm the previously demonstrated specific interaction of c-Src with WASP 20.

Inhibition of Src interaction with endogenous WASP in cells transduced with pTyr294 fragment suggests that this peptide may have the ability to block this interaction through competitive inhibition of the phosphorylation of endogenous WASP. Subsequently we addressed the question whether the transduced WASP fragment containing pTyr294 is phosphorylated in vivo (Fig. 5E). Lysates made from osteoclasts transduced with WASP peptides containing VC domain (lane 2), proline-rich region (lane 3), pTyr294aa (lane 4) were immunoprecipitated with a HA antibody and subjected to 15% SDS-PAGE. Immunoblotting analysis with a p-Tyrosine antibody demonstrated coprecipitation and phosphorylation of proteins in osteoclasts transduced with WASP peptide containing pTyr294. Protein with a molecular mass ~31 kDa (Fig. 5E lane 4, indicated by an arrow head) and the transduced pTyr294 peptide (~15.5 kDa; indicated by an arrow) were observed (Fig. 5E, lane 4). Immunoprecipitation with a non-immune serum is shown in lane 1 (E and F). Subsequently, this blot was stripped and reprobed with a HA antibody in order to characterize the transduced proteins coprecipitated with HA immunoprecipitates (Fig. 5F). WASP peptides containing VC (13.3 kDa, lane 2), proline-rich sequences (24 kDa; lane 3) and p-Tyr294 aa (15.5 kDa, lane 4) were observed. Overall, these results (Fig. 5) suggest that phosphorylation of WASP on pTyr294 is critical for the subsequent interaction of c-Src and other signaling molecules with the proline-rich region of WASP.

We then addressed the question whether increasing the intracellular levels of WASP peptides would reduce sealing ring formation (Fig. 6) and bone resorption (Fig. 7) in osteoclasts. Osteoclasts transduced with the indicated TAT-fused WASP peptides (Fig. 6) were plated on dentine slices and incubated for 36–48 h. Staining of osteoclasts with rhodamine phalloidin for actin, exhibited sealing rings in osteoclasts treated with PBS (Fig. 6A) or transduced with HA-TAT (B), FL-WASP (C), and HSV-TK (H). Sealing ring formation was either reduced or not observed at all in osteoclasts transduced with WASP peptides containing BR (D), proline-rich (E), pTyr294aa (F), and VC (G) sequences. Actin clumps and smaller size sealing rings were observed in the center and at the periphery of the osteoclasts transduced with BR (D), pro (E), and VC (G) domains of WASP. Transduction of proline-rich containing WASP peptide induced tiny nascent actin ring throughout the osteoclasts (E). However, osteoclasts transduced with the WASP peptide containing pTyr294aa (F) exhibited, instead of sealing ring, actin-enriched clusters at the periphery of the osteoclasts. These punctate adhesion-like structures enriched in actin clumps were observed at the edges of filopodia-like projections. These osteoclasts were spread out and adhered well with the dentine slices (F). Areas marked by white square (Fig. E and F) are shown at higher magnification in E' and F'. Arrows and arrowheads indicate the small ring-like structures and actin patches, respectively (D, E, F, G, E', and F'). Reduced actin staining in osteoclasts transduced with peptides containing BR (D), pro (E), pTyr294 (F), and VC (G) agree with the decreased F-actin content shown in Fig. 3. Untransduced (A) as well as transduced osteoclasts with HA-TAT (B) and HSV-TK (H) were used as controls.

We subsequently determined the bone resorption activity of osteoclasts treated with PBS (Fig. 7A) or transduced with the indicated TAT- proteins (Fig. 7, B–I). Quantification of resorption pits (expressed as pit area in μm²) generated in vitro by osteoclasts is shown in Fig. 8. Osteoclasts transduced with HA-TAT (B) or HSV-TK (I) and treated with PBS (A) were used as controls. An increase in pit area was observed in osteoclasts transduced with FL-WASP as compared to those transduced and treated with controls (A, B, and I). Inhibition of bone resorption was observed in osteoclasts transduced with TAT- WASP peptides containing BR (D), GP (E), Pro (F), pTyr294aa (G), and VCA (H). GP domain of WASP peptide contains GTPase binding, pTyr294, and pro-rich regions. This fragment was used in this assay to confirm the effects of WASP peptide containing pTyr294 aa. It has significant inhibitory effect on osteoclast bone resorption. However, the decrease was more apparent in osteoclasts transduced with WASP peptide containing pTyr294 aa (G). Bone resorption activity was observed in the following order of the pit area (in μm²): FL-WASP > PBS = HA-TAT>BR>VC>Pro>GP > pTyr294aa.