Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid

Wiskott Aldrich Syndrome protein (WASP) has a unique regulatory role in sealing ring formation and bone resorption in osteoclasts. Here, using the TAT-transduction method, we show the possible role of WASP domain(s) in sealing ring formation and bone resorption. Transduction of TAT-fused full-length WASP peptide induced Arp2/3 complex formation, F-actin content, sealing ring formation and bone resorption. Transduction of WASP peptides containing basic, verpolin-central, pTyr294, and proline-rich regions inhibited the processes listed above at various levels. The ability to resorb bone by WASP peptides containing basic, verpolin-central, and proline-rich regions was reduced and the resorbed area matched the size of the sealing ring. However, osteoclasts transduced with WASP peptide containing pTyr294aa demonstrated the following: a) a considerable decrease in the interaction and phosphorylation of c-Src with endogenous WASP; b) total loss of sealing ring-like structures; c) formation of actin-rich patches at the peripheral edge that contains filopodia-like projections; d) reduced capacity for bone resorption in vitro. These findings suggest that modulation of phosphorylation state of pTyr294aa assists in integrating multiple signaling molecule and pathways that partake in the assembly of sealing ring

Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid-7

Its on multiple dentine slices from three osteoclast preparations are shown. ***p < 0.0001 vs. PBS-treated as well as HA-TAT, Hsv-TK, and FL-WASP transduced osteoclasts; **p < 0.001; * p < 0.05 vs. PBS-treated as well as HA-TAT, Hsv-TK transduced osteoclasts.<p><b>Copyright information:</b></p><p>Taken from "Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid"</p><p>http://www.jmolecularsignaling.com/content/3/1/4</p><p>Journal of Molecular Signaling 2008;3():4-4.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2266921.</p><p></p

Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid-8

P, GTPase binding domain and proline rich domain; Pro, proline-rich region; Verpolin-like, central, and acidic domain [VCA]; Verpolin-like, and central domain [VC]) are cloned separately into the HA-TAT expression vector. The number within the parentheses indicates the first and last amino acid of the corresponding WASP peptide. . . TAT-fused proteins were subjected to 8% (lanes 1–3) and 15% (lanes 4–9) SDS-PAGE and stained with Coomassie blue. The numbers on the left of each panel represent the standard molecular weight (MW) markers (kDa). The numbers on the top of each lane indicate the apparent molecular mass (kDa) of the purified protein.<p><b>Copyright information:</b></p><p>Taken from "Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid"</p><p>http://www.jmolecularsignaling.com/content/3/1/4</p><p>Journal of Molecular Signaling 2008;3():4-4.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2266921.</p><p></p

Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid-9

N antibody to HA to determine the levels of transduced proteins (Top panel). The immunoblot shown in the top panel was stripped and blotted with a GAPDH antibody for normalization (bottom panel). The results shown are representative of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid"</p><p>http://www.jmolecularsignaling.com/content/3/1/4</p><p>Journal of Molecular Signaling 2008;3():4-4.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2266921.</p><p></p

Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid-3

R HA (C and D) antibody. WASP immunoprecipitates were first immunoblotted with an antibody to Arp2 (A) and subsequently stripped and blotted with an antibody to WASP (B). HA-immunoprecipitates were divided into two halves; one half was immunoblotted with an Arp 2 antibody (C) and the other half was subjected to 8% (D, lane 1) and 15% (D, lanes 2–5) SDS-PAGE. Immunoblotting was performed with a HA-antibody (D) to detect the levels of transduced proteins immunoprecipitated in each lane. Immunoprecipitation with a nonimmune serum is shown in lane 6 (A-D). The results represent one of three experiments performed from three separate osteoclast preparations.<p><b>Copyright information:</b></p><p>Taken from "Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid"</p><p>http://www.jmolecularsignaling.com/content/3/1/4</p><p>Journal of Molecular Signaling 2008;3():4-4.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2266921.</p><p></p

Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid-0

P, GTPase binding domain and proline rich domain; Pro, proline-rich region; Verpolin-like, central, and acidic domain [VCA]; Verpolin-like, and central domain [VC]) are cloned separately into the HA-TAT expression vector. The number within the parentheses indicates the first and last amino acid of the corresponding WASP peptide. . . TAT-fused proteins were subjected to 8% (lanes 1–3) and 15% (lanes 4–9) SDS-PAGE and stained with Coomassie blue. The numbers on the left of each panel represent the standard molecular weight (MW) markers (kDa). The numbers on the top of each lane indicate the apparent molecular mass (kDa) of the purified protein.<p><b>Copyright information:</b></p><p>Taken from "Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid"</p><p>http://www.jmolecularsignaling.com/content/3/1/4</p><p>Journal of Molecular Signaling 2008;3():4-4.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2266921.</p><p></p

Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid-5

Sts stained for actin are shown. Sealing ring was observed in osteoclasts treated with PBS (A) or transduced with HA-TAT (B), FL-WASP (C), and HSV-TK. Sealing ring formation is reduced in osteoclasts transduced with WASP peptides consisting of BR (D), Proline-rich region (E), pTyr amino acid (F), and VC domain (G). The areas that are magnified in E' and F' are shown with a white box in Figs E and F. Arrows and arrowheads indicate small ring-like structures and actin aggregates or patches, respectively (D, E, F, E', F', and G'). Scale Bar: 50 μm. The results shown are representative of three independent osteoclast preparations and experiments.<p><b>Copyright information:</b></p><p>Taken from "Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid"</p><p>http://www.jmolecularsignaling.com/content/3/1/4</p><p>Journal of Molecular Signaling 2008;3():4-4.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2266921.</p><p></p

Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid-1

N antibody to HA to determine the levels of transduced proteins (Top panel). The immunoblot shown in the top panel was stripped and blotted with a GAPDH antibody for normalization (bottom panel). The results shown are representative of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid"</p><p>http://www.jmolecularsignaling.com/content/3/1/4</p><p>Journal of Molecular Signaling 2008;3():4-4.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2266921.</p><p></p

Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid-4

B) or HA (C and D) antibody. Immunoprecipitates were first immunoblotted with an antibody to Src pTy418 (A and C). Subsequently, blots were stripped and blotted with an antibody to c-Src (B and D) to detect the levels of c-Src coprecipitated with WASP or HA immunoprecipitates. Immunoprecipitation with a non-immune serum (NI) is shown in lane 5 (A-D). E and F: HA-immunoprecipitates were subjected to 15% SDS-PAGE. Immunoblotting was performed with a phosphotyrosine (p-Tyrosine; panel E) to detect the phosphorylation levels of transduced proteins. Subsequently, blot was stripped and blotted with a HA- antibody (F) to determine the levels of transduced proteins immunoprecipitated in each lane. An asterisk in Fig. 5E indicates coprecipitation of a non-specific protein with HA and non-immune (NI) immunoprecipitates (lanes 1–4). Immunoprecipitation with a non-immune serum (NI) is shown in lane 1 (E and F). The results represent one of three experiments performed from three separate osteoclast preparations.<p><b>Copyright information:</b></p><p>Taken from "Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid"</p><p>http://www.jmolecularsignaling.com/content/3/1/4</p><p>Journal of Molecular Signaling 2008;3():4-4.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2266921.</p><p></p

Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid-2

Esults presented are mean ± SE for three experiments. *** p < 0.0001 versus PBS-treated as well as HA-TAT, Hsv-TK, and FL-WASP transduced osteoclasts; p < 0.001, ** p < 0.01, *p < 0.05 versus PBS-treated as well as HA-TAT and Hsv-TK transduced osteoclasts.<p><b>Copyright information:</b></p><p>Taken from "Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid"</p><p>http://www.jmolecularsignaling.com/content/3/1/4</p><p>Journal of Molecular Signaling 2008;3():4-4.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2266921.</p><p></p