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• W2034205710 abstract "We have shown that osteopontin binding to integrin αvβ3 in osteoclasts stimulates gelsolin-associated phosphatidylinositol (PtdIns) 3-hydroxyl kinase (PI 3-kinase), leading to increased levels of gelsolin-bound PtdIns 3,4-P2, PtdIns 4,5-P2, and PtdIns 3,4,5-P3, uncapping of barbed end actin, and actin filament formation. Inhibition of PI 3-kinase activity by wortmannin blocks osteopontin stimulation of actin filament formation, suggesting that activation of gelsolin-associated PI 3-kinase is an important pathway in cytoskeletal regulation. To study the mechanism of gelsolin-associated PI 3-kinase activation, we analyzed anti-gelsolin immunoprecipitates for the association of protein kinases. We demonstrated that c-Src co-immunoprecipitates with gelsolin, and that osteopontin stimulates its activity. Elimination of osteopontin-stimulated Src activity associated with gelsolin through antisense oligodeoxynucleotides blocked the stimulation of PI 3-kinase activity associated with gelsolin and the gelsolin-dependent cytoskeletal reorganization induced by osteopontin, including increased F-actin levels. In addition, treatment of osteoclasts with antisense oligonucleotides to Src reduced bone resorption. Our results demonstrate that osteopontin stimulates gelsolin-associated Src, leading to increased gelsolin-associated PI 3-kinase activity and PtdIns 3,4,5-P3 levels, which facilitate actin filament formation, osteoclast motility, and bone resorption. We have shown that osteopontin binding to integrin αvβ3 in osteoclasts stimulates gelsolin-associated phosphatidylinositol (PtdIns) 3-hydroxyl kinase (PI 3-kinase), leading to increased levels of gelsolin-bound PtdIns 3,4-P2, PtdIns 4,5-P2, and PtdIns 3,4,5-P3, uncapping of barbed end actin, and actin filament formation. Inhibition of PI 3-kinase activity by wortmannin blocks osteopontin stimulation of actin filament formation, suggesting that activation of gelsolin-associated PI 3-kinase is an important pathway in cytoskeletal regulation. To study the mechanism of gelsolin-associated PI 3-kinase activation, we analyzed anti-gelsolin immunoprecipitates for the association of protein kinases. We demonstrated that c-Src co-immunoprecipitates with gelsolin, and that osteopontin stimulates its activity. Elimination of osteopontin-stimulated Src activity associated with gelsolin through antisense oligodeoxynucleotides blocked the stimulation of PI 3-kinase activity associated with gelsolin and the gelsolin-dependent cytoskeletal reorganization induced by osteopontin, including increased F-actin levels. In addition, treatment of osteoclasts with antisense oligonucleotides to Src reduced bone resorption. Our results demonstrate that osteopontin stimulates gelsolin-associated Src, leading to increased gelsolin-associated PI 3-kinase activity and PtdIns 3,4,5-P3 levels, which facilitate actin filament formation, osteoclast motility, and bone resorption. Osteoclasts are multinucleated, giant cells, responsible for bone resorption. Osteoclast adhesion to bone leads to the formation of the osteoclast clear zone: a ring-like adhesion zone circumscribing an area of bone resorption. The cytoplasm of the clear zone contains numerous actin filaments perpendicular to the bone matrix, which are anchored in podosomes (1Marchisio P.C. Cirillo D. Naldini L. Primavera M.V. Teti A. Zambonin-Zallone A. J. Cell Biol. 1984; 99: 1696-1705Crossref PubMed Scopus (248) Google Scholar, 2Marchisio P.C. Cirillo D. Teti A. Zambonin-Zallone A. Tarone G. Exp. Cell Res. 1987; 169: 202-214Crossref PubMed Scopus (166) Google Scholar, 3Zambonin-Zallone A. Teti A. Grano M. Rubinacci A. Abbadini M. Gaboli M. Marchisio C. Exp. Cell Res. 1989; 182: 645-652Crossref PubMed Scopus (170) Google Scholar, 4Lakkakorpi P.T. Vaananen H.K. J. Bone Miner. Res. 1991; 6: 817-826Crossref PubMed Scopus (205) Google Scholar, 5Teti A. Marchisio P.C. Zambonin-Zallone A. Am. J. Physiol. 1991; 261: C1-C7Crossref PubMed Google Scholar). Podosomes are small cell processes specific to cells of monocytic origin. Podosomes contain numerous proteins observed in the focal adhesions of other cells. Although podosomes and focal adhesions are related, there are important functional differences. Podosomes are less tightly associated with the substratum and are more highly dynamic, changing in size and location and appearing and disappearing with life spans of 2–12 min (4Lakkakorpi P.T. Vaananen H.K. J. Bone Miner. Res. 1991; 6: 817-826Crossref PubMed Scopus (205) Google Scholar). Osteoclasts are highly motile cells, and podosomes appear to be a preferred cell/matrix attachment mechanism for motility in cells such as macrophages, monocytes, and osteoclasts. The adhesion of osteoclasts through podosomes involves interaction of cell surface receptors, integrins, with matrix components. In osteoclasts, integrin αvβ3 1The abbreviations used are: αvβ3, adhesion receptor αvβ3; OP, osteopontin; GRGDS, Gly-Arg-Gly-Asp-Ser cell adhesion sequence; FAK, focal adhesion kinase; PtdIns, phosphatidylinositol; ODN, oligodeoxynucleotide; PI 3-kinase, phosphatidylinositol 3-kinase; PBS, phosphate-buffered saline. is responsible for adhesion associated with bone resorption (3Zambonin-Zallone A. Teti A. Grano M. Rubinacci A. Abbadini M. Gaboli M. Marchisio C. Exp. Cell Res. 1989; 182: 645-652Crossref PubMed Scopus (170) Google Scholar, 6Davis J. Warwich J. Totty N. Philp R. Helfrich M. Horton M. J. Cell Biol. 1989; 109: 1817-1826Crossref PubMed Scopus (319) Google Scholar). The ligands of αvβ3 are many, and they contain the RGD cell adhesion sequence present in several serum and bone matrix proteins. Osteopontin (OP) is an RGD-containing bone matrix protein, which plays a key role in anchoring osteoclasts to bone surfaces. Besides its role as an anchorage protein, OP is produced by osteoclasts in large quantities (7Ikeda T. Nomura S. Yamaguchi A. Suda T. Yoshiki S. J. Histochem. Cytochem. 1992; 40: 1079-1088Crossref PubMed Scopus (152) Google Scholar),2 and its αvβ3integrin receptor, besides location in the podosomes, is found in the osteoclast membrane opposite to the bone matrix (4Lakkakorpi P.T. Vaananen H.K. J. Bone Miner. Res. 1991; 6: 817-826Crossref PubMed Scopus (205) Google Scholar, 8Horton M.A. Taylor M.L. Arnett T.R. Helfrich M.H. Exp. Cell Res. 1991; 195: 368-375Crossref PubMed Scopus (216) Google Scholar). We have shown that osteopontin binding to basolateral αvβ3 is an autocrine motility factor regulating the shape and depth of osteoclast resorption (9Chellaiah M. Kwiatkowski D. Alvarez U. Gillis M. Hruska K. J. Bone Miner. Res. 1997; 12: S137Google Scholar). The intracellular biochemical pathways that integrins regulate, and the cellular functions that they control, have recently been the focus of careful scrutiny. Association of focal adhesion kinase (FAK) with the cell surface at focal adhesions directs interactions with the cytoplasmic domains of integrins and their participation in signal transduction (10Ingber D.E. Curr. Opin. Cell Biol. 1991; 3: 841-848Crossref PubMed Scopus (579) Google Scholar, 11Kornberg L.J. Earp H.S. Turner C.E. Procop C. Juliano R.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8392-8396Crossref PubMed Scopus (633) Google Scholar, 12Juliano R.L. Haskill S. J. Cell Biol. 1993; 120: 577-585Crossref PubMed Scopus (1545) Google Scholar). Phosphorylation of FAK in response to cell adhesion and other stimuli induce the formation of complexes between FAK and other signaling molecules in vivo, including Src (13Cobb B. Schaller M. Leu T. Parsons J.T. Mol. Cell. Biol. 1994; 14: 147-155Crossref PubMed Scopus (486) Google Scholar, 14Xing Z. Chen H.C. Noelen J.K. Taylor S.J. Shaloway D. Guan J.L. Mol. Cell. Biol. 1994; 5: 413-421Crossref Scopus (285) Google Scholar), Grb2 (15Schlaepfer D.D. Hanks S.K. Hunter T. van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1448) Google Scholar), Nck (16Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Biol. 1995; 11: 549-600Crossref Google Scholar), and PI 3-kinase (17Chen H.-C. Guan J.-L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10148-10152Crossref PubMed Scopus (478) Google Scholar, 18Guinebault C. Payrastre B. Racaud-Sultan C. Mazarguil H. Breton M. Mauco G. Plantavid M. Chap H. J. Cell Biol. 1995; 129: 831-842Crossref PubMed Scopus (224) Google Scholar). A unique aspect of signaling through αvβ3is its property of responding to soluble ligands (8Horton M.A. Taylor M.L. Arnett T.R. Helfrich M.H. Exp. Cell Res. 1991; 195: 368-375Crossref PubMed Scopus (216) Google Scholar, 19Hruska K.A. Rolnick F. Huskey M. Alvarez U. Cheresh D. Endocrinology. 1995; 136: 2984-2992Crossref PubMed Google Scholar, 20Miyauchi A. Alvarez J. Greenfield R.M. Teti A. Grano M. Colucci S. Zambonin-Zallone A. Ross F.P. Teitelbaum S.L. Cheresh D. Hruska K.A. J. Biol. Chem. 1991; 266: 20369-20374Abstract Full Text PDF PubMed Google Scholar). We have previously demonstrated the mechanisms of αvβ3 signaling in response to soluble OP (19Hruska K.A. Rolnick F. Huskey M. Alvarez U. Cheresh D. Endocrinology. 1995; 136: 2984-2992Crossref PubMed Google Scholar, 21Chellaiah M. Hruska K.A. Mol. Biol. Cell. 1996; 7: 743-753Crossref PubMed Scopus (103) Google Scholar). Binding of OP- or RGD-containing peptides to αvβ3 stimulated formation of signal generating complexes consisting of FAK, c-Src, and PI 3-kinase associated with αvβ3 (19Hruska K.A. Rolnick F. Huskey M. Alvarez U. Cheresh D. Endocrinology. 1995; 136: 2984-2992Crossref PubMed Google Scholar, 21Chellaiah M. Hruska K.A. Mol. Biol. Cell. 1996; 7: 743-753Crossref PubMed Scopus (103) Google Scholar). OP stimulated PtdIns 3,4-P2 and PtdIns 4,5-P2(PtdIns-P2) and PtdIns 3,4,5-P3(PtdIns-P3) levels in osteoclasts (19Hruska K.A. Rolnick F. Huskey M. Alvarez U. Cheresh D. Endocrinology. 1995; 136: 2984-2992Crossref PubMed Google Scholar). We further defined one specialized domain of increased PtdIns 3,4-P2 and PtdIns 3,4,5-P3 levels as an actin-capping protein found in the podosome, gelsolin (21Chellaiah M. Hruska K.A. Mol. Biol. Cell. 1996; 7: 743-753Crossref PubMed Scopus (103) Google Scholar). We demonstrated that the increase of PtdIns-P2 and PtdIns-P3 associated with gelsolin, uncapped actin oligomers leading to an increase in F-actin content and actin filament formation (21Chellaiah M. Hruska K.A. Mol. Biol. Cell. 1996; 7: 743-753Crossref PubMed Scopus (103) Google Scholar). PtdIns-P2regulates several actin-binding proteins, including gelsolin (22Janmey P.A. Stossel T.P. Nature. 1987; 325: 362-364Crossref PubMed Scopus (499) Google Scholar), profilin (23Lassing I. Lindberg U. Nature. 1985; 314: 472-474Crossref PubMed Scopus (639) Google Scholar), α-actinin (24Fukami K. Furuhashi K. Inagaki M. Endo T. Hatano S. Takenawa T. Nature. 1992; 359: 150-152Crossref PubMed Scopus (305) Google Scholar), and vinculin (25Fukami K. Endo T. Imamura M. Takenawa T. J. Biol. Chem. 1994; 269: 1518-1522Abstract Full Text PDF PubMed Google Scholar, 26Johnson R.P. Craig S.W. Biochem. Biophys. Res. Commun. 1995; 210: 159-164Crossref PubMed Scopus (73) Google Scholar). Osteopontin affected PtdIns-P2 and -P3 levels associated specifically with gelsolin and not the other proteins (21Chellaiah M. Hruska K.A. Mol. Biol. Cell. 1996; 7: 743-753Crossref PubMed Scopus (103) Google Scholar). A marked increase in the level of PtdIns-P2 with gelsolin leads to the hypothesis that PtdIns-P2 synthesis may be essential for podosome assembly and disassembly. OP treatment also resulted in the increased activity of PI 3-kinase associated with gelsolin. Phosphorylation of PtdIns 4,5-P2 by PI 3-kinase associated with gelsolin leads to the formation of PtdIns 3,4,5-P3 in OP-treated cells. The physiological function of the association of PI 3-kinase with gelsolin is stimulation of actin polymerization during cell motility, and inhibition of PI 3-kinase activity with wortmannin, a specific inhibitor of PI 3-kinase, blocks the increase in F-actin and actin polymerization stimulated by osteopontin (21Chellaiah M. Hruska K.A. Mol. Biol. Cell. 1996; 7: 743-753Crossref PubMed Scopus (103) Google Scholar). Examination of the signaling pathways leading to integrin-mediated activation of PI 3-kinase suggests that members of the Src family of nonreceptor kinases may play a role (27Pleiman C.M. Hertz W.M. Cambier J.C. Science. 1994; 263: 1609-1612Crossref PubMed Scopus (394) Google Scholar, 28Escobedo J.A. Navankassatusas K. Kavanaugh W.M. Milfay D. Fried V. Williams L.T. Cell. 1991; 65: 75-82Abstract Full Text PDF PubMed Scopus (375) Google Scholar, 29Otsu M. Hiles I. Gout I. Fry M.J. Ruizlarrea F. Panayotou G. Thompson A. Dhand R. Hsuan J. Totty N. Smith A.D. Morgan S.J. Ciurtneidge S.A. Parker P.J. Waterfield M.A. Cell. 1991; 65: 91-104Abstract Full Text PDF PubMed Scopus (541) Google Scholar, 30Liu X. Marengere L.E.M. Koch C.A. Pawson T. Mol. Cell. Biol. 1993; 13: 5226-5232Google Scholar). PI 3-kinase activity is associated with c-Src, and the associated kinase activity increases quickly upon stimulation with thrombin in platelets (31Gutkind J.S. Lacal P.M. Robbins K.C. Mol. Cell. Biol. 1990; 10: 3806-3809Crossref PubMed Scopus (89) Google Scholar). Several lines of evidence reveal that PI 3-kinase is activated by c-Src (32Fukui Y. Kornbluth S. Jong S.M. Wang L.H. Hanafusa H. Oncogene Res. 1989; 4: 283-292PubMed Google Scholar, 33Fukui Y. Hanafusa H. Mol. Cell. Biol. 1991; 11: 1972-1979Crossref PubMed Google Scholar). c-Src itself plays an important role in the osteoclast. Deletion of the gene for c-Src in mice results in impaired osteoclast polarization, failure of bone resorption, and osteopetrosis (34Horne W.C. Neff L. Chatterjee D. Lomri A. Levy J.B. Baron R. J. Cell Biol. 1992; 119: 1003-1013Crossref PubMed Scopus (209) Google Scholar, 35Lowe C. Yoneda T. Boyce B.F. Chen H. Mundy G.R. Soriano P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4485-4489Crossref PubMed Scopus (289) Google Scholar), Osteoclasts from c-Src deficient mice lack ruffled borders and have impaired bone resorptive activity in vitro (36Boyce B.F. Yoneda T. Lowe C. Soriano P. Mundy G.R. J. Clin. Invest. 1992; 90: 1622-1627Crossref PubMed Scopus (519) Google Scholar). Wortmannin inhibited PI 3-kinase activity in osteoclasts both in vivo and in vitro and also ruffled border formation and bone resorption (37Nakamura I. Takahashi N. Sasaki T. Tanaka S. Udagawa N. Murakami H. Kimura K. Kubuyama Y. Kurokawa T. Suda T. Fukui Y. FEBS Lett. 1995; 361: 79-84Crossref PubMed Scopus (131) Google Scholar). These results suggest that both Src and PI3-kinase activity are important in osteoclastic bone resorption. To examine the role of c-Src in the pathway to activation of gelsolin-associated PI 3-kinase from OP/αvβ3 integrin-mediated signaling, we have utilized an antisense strategy to disrupt the function of Src in the avian osteoclast system. Our results demonstrate that c-Src activity associated with the actin-binding protein gelsolin was increased upon treatment with OP. Furthermore, this increase in Src activity was necessary for the activation of gelsolin-associated PI 3-kinase, and for the subsequent increase in F-actin content and actin filament formation stimulated by liganding of αvβ3. Thus, c-Src is upstream of gelsolin-associated PI 3-kinase, and it activates PI 3-kinase. Since the c-Src antisense oligodeoxynucleotides-treated osteoclasts failed to form an organized podosome-containing clear zone, and were deficient in bone resorption, our data address an important second issue regarding the Src knockout phenotype. Our results, at leastin vitro, demonstrate that the osteoclast defect of the Src−/− mouse is not species-specific. They suggest that the avian osteoclasts also possess cellular sites where Src function cannot be substituted for by another member of the Src superfamily. [γ-32P]ATP, rainbow molecular weight markers for proteins were obtained from Amersham Pharmacia Biotech. Herbimycin A was obtained from Life Technologies, Inc. Protein A-Sepharose, mouse IgG, anti-gelsolin antibody, phospholipid standards, and most of the chemicals were purchased from Sigma. Polyvinylidene difluoride membranes were obtained from Millipore Corp. (Bedford, MA). Protein assay reagent kit and the reagents for polyacrylamide gel electrophoresis were purchased from Bio-Rad. Antibody to c-Src was purchased from Oncogene (Uniondale, NY). Recombinant c-Src was obtained from UBI (Lake Placid, NY). RNAzol, background quencher, and hybridization solutions were purchased from Tel-Test (Friendswood, TX). Turboblotter transfer system and nylon membranes for RNA transfer were purchased from Schleicher & Schuell. Prime-A-Gene labeling kit and restriction enzymes were purchased from Promega (Madison, WI). Rhodamine-phalloidin was obtained from Molecular Probes (Eugene, OR). Avian osteoclast precursors were prepared as described previously (21Chellaiah M. Hruska K.A. Mol. Biol. Cell. 1996; 7: 743-753Crossref PubMed Scopus (103) Google Scholar, 38Alvarez J.I. Teitelbaum S.L. Blair H.C. Greenfield E.M. Athanasou N.A. Ross F.P. Endocrinology. 1991; 128: 2324-2335Crossref PubMed Scopus (72) Google Scholar). Briefly, osteoclast precursors were isolated from bone marrow of egg-laying hens maintained on Ca2+-deficient diets. Partially purified preparations of mononuclear cells were recovered from the interface of Ficoll/Hypaque gradients. Nonadherent cells were separated from the adherent population after 18–24 h in culture. The nonadherent cells were sedimented, resuspended in fresh medium (5 × 106cells/ml), and cultured in the presence of cytosine arabinoside (5 μg/ml). Multinucleated osteoclast precursor cells formed between 3 and 6 days in culture, and the preparations were 70–90% pure multinucleated tartrate-resistant acid phosphatase-positive cells. After 4 days in culture, osteoclast precursors were incubated in serum-free media for 2 h. Subsequently cells were treated with one of the following: OP (25 μg/ml for 15 min at 37 °C) or GRGDS (100 μg/ml for 15 min at 37 °C). In some cases herbimycin A was added at a concentration of 100 ng/ml for 14–16 h prior to treatment. Following treatment, cells were washed three times with ice-cold PBS and lysed in a Triton-containing lysis buffer (10 mm Tris-HCl, pH 7.05, 50 mm NaCl, 0.5% Triton X-100, 30 mm sodium pyrophosphate, 5 mm NaF, 0.1 mm Na3VO4, 5 mmZnCl2, and 2 mm phenylmethylsulfonyl fluoride. Lysates were pelleted by centrifugation (15,000 rpm, 15 min, 4 °C), and the pellets were solubilized by trituration in radioimmune precipitation buffer (10 mm Tris-HCl, pH 7.2, 150 mm NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS, 1% aprotinin, and 2 mm phenylmethylsulfonyl fluoride). Protein concentrations were measured using the Bio-Rad protein assay reagent kit, and equal amounts of protein lysates were used for immunoprecipitations. Immunoprecipitations and Western blotting were carried out as described (21Chellaiah M. Hruska K.A. Mol. Biol. Cell. 1996; 7: 743-753Crossref PubMed Scopus (103) Google Scholar). In vitro immune complex protein kinase assays were performed as described previously (39Chellaiah M. Fitzgerald C. Filardo E.J. Cheresh D.A. Hruska K.A. Endocrinology. 1996; 137: 2432-2440Crossref PubMed Google Scholar). Equal amounts of protein lysates were immunoprecipitated with anti-gelsolin or c-Src antibodies. The immune complexes collected by the addition of protein A-Sepharose were used for kinase assays. The Sepharose beads, after washing several times with the buffers described (39Chellaiah M. Fitzgerald C. Filardo E.J. Cheresh D.A. Hruska K.A. Endocrinology. 1996; 137: 2432-2440Crossref PubMed Google Scholar), were resuspended in 20 μl of kinase buffer (20 mm Hepes, pH 7.4, 5 mmMgCl2, and 0.1 mmNa3VO4) containing [γ-32P]ATP (10 μCi) and casein (1 mg/ml) as an exogenous substrate. The mixture was incubated at 25 °C for 20 min, and the reaction was stopped by the addition of SDS-sample buffer. The samples were boiled and subjected to SDS-polyacrylamide gel electrophoresis, and radiolabeled proteins were detected by autoradiography. Experiments were performed with synthetic phosphorothioate oligodeoxynucleotides (ODNs). 24-mer sequences corresponding to positions 1–24 of c-Src cDNA (40Takeya T. Hanafusa H. Cell. 1983; 32: 881-890Abstract Full Text PDF PubMed Scopus (299) Google Scholar) were constructed. The targeted sequences include the presumed translation initiation site. ODNs were made in both sense, and antisense orientations and the sequences are as follows: sense (5′-ATG GGG AGC AGC AAG AGC AAG CCC 3′) and antisense (5′- GGG CTT GCT CTT GCT GCT CCC CAT 3′). Scrambled ODNs containing the antisense nucleotides were also synthesized and used as a control: 5′-TTT GTT ATC CTC CGT GGC CTC CCG 3′. Osteoclast precursor cells, cultured for 4–6 days were used for permeabilization with streptolysin O (41Duncan R.L. Kizer N. Barry E.L.R. Friedman P.A. Hruska K.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1864-1869Crossref PubMed Scopus (47) Google Scholar,42Barry E. Gesek F. Friedman P. Biofeedback. 1993; 15: 1019-1020Google Scholar). Osteoclasts were washed twice with permeabilization buffer (120 mm KCl, 30 mm NaCl, 10 mm Hepes, pH 7.2, 10 mm EGTA, 10 mm MgCl2) (43Graves D.D. Lucas S.C. Alexander D.R. Cantrell D.A. Biochem. J. 1990; 190: 407-413Crossref Scopus (15) Google Scholar). Freshly prepared dithiothreitol (5 mm), ATP (1 mm), and 0.5 unit/ml streptolysin O and ODNs in sense, antisense, or scrambled orientation at various concentrations were added to the buffer at the time of permeabilization. Resealing was achieved by the addition of α-minimal essential medium containing 10% fetal bovine serum, and incubation was continued for 8 h. Before the end of the 8 h, the cells were serum-starved for 2 h and stimulated with OP as above. ODNs were maintained at the indicated concentration throughout the time mentioned. Additionally, control cells were permeabilized and stimulated with OP as above but in the absence of ODNs. Total RNA was isolated using RNAzol and processed according to the manufacturer's guidelines (Tel-Test). Total RNA (10 μg) was denatured, electrophoresed on 1% agarose/formaldehyde gels and transferred to nylon membranes using a Turboblotter transfer system. The membranes were exposed to UV light to cross link the RNA. 32P-Labeled pp60c-src cDNA probes (1.6 kilobase pairs) were random prime-labeled using [α-32P]dCTP. The blots were prehybridized at 37 °C in a solution containing 1 mNaCl, 1% sodium dodecyl sulfate (SDS), 50% formamide, and 1× background quencher for 1–2 h and hybridized overnight using the solution in high efficiency hybridization system, as directed by the manufacturers' instructions. Blots were then washed once at room temperature in 5× SSPE, 0.1% SDS; once at 37 °C in 1× SSPE, 0.1% SDS; and twice at 65 °C in 0.1× SSPE, 0.1% SDS. Blots were then analyzed by exposure to x-ray films. Lysate preparation and immunoprecipitations were carried out as described above. Equal amounts of proteins were used for immunoprecipitation. The immune complexes, adsorbed to protein A-Sepharose pellets were assayed for lipid kinase activity. PI 3-kinase was assayed by following the method (44Jackson T.R. Stephens L.R. Hawkins P.T. J. Biol. Chem. 1992; 267: 16627-16636Abstract Full Text PDF PubMed Google Scholar) described previously (21Chellaiah M. Hruska K.A. Mol. Biol. Cell. 1996; 7: 743-753Crossref PubMed Scopus (103) Google Scholar). Briefly, the immune complexes, adsorbed to protein A-Sepharose pellets, were washed successively as follows: once with Triton lysis buffer; twice with 0.5 m LiCl, 0.1m Tris-HCl, pH 8.0; once with 0.1 m NaCl, 1 mm EDTA, 10 mm Tris-HCl, pH 7.6; and finally once with kinase assay buffer (1 mm dithiothreitol, 20 mm Hepes/NaOH, pH 7.4 at 25 °C, 5 mmMgCl2). The pellets were then assayed for inositol lipid kinase activity. Freshly prepared lipid mix (20 μl; Ref. 44Jackson T.R. Stephens L.R. Hawkins P.T. J. Biol. Chem. 1992; 267: 16627-16636Abstract Full Text PDF PubMed Google Scholar) was added to the beads; the mix was vortexed gently and then placed at 37 °C for 5 min. Ten microliters of kinase buffer (20 mmHepes/NaOH, pH 7.4, 5 mm MgCl2, 1 mm dithiothreitol), containing [γ-32P]ATP (5 μCi/assay), and 5 μm Na2ATP was then added. The mixture was gently vortexed and the incubation continued at 37 °C for an additional 15 min. Incubations were terminated by the addition of 0.425 ml of chloroform:methanol:water (5:10:2, v/v). Lipids were then extracted as described (45Whitman M. Kaplan D.R. Schaffhausen B. Cantley L. Roberts T. Nature. 1985; 315: 239-242Crossref PubMed Scopus (557) Google Scholar) and dried under N2. The dried lipids were reconstituted in 100 μl of chloroform:methanol (1:1) and spotted on silica gel TLC plates pretreated with 1.2% potassium oxalate in methanol and water (2:3). The plates were developed in chloroform:methanol:acetic acid:acetone:water (40:15:13:12:7) and dried. Bands were visualized by autoradiography and quantitated by scanning in GS 300 Transmittance/Reflectance scanning densitometer (Hoefer Scientific Instruments, San Francisco, CA). Phospholipid standards were visualized by exposure to iodine vapors. F-actin measurement was performed as described (21Chellaiah M. Hruska K.A. Mol. Biol. Cell. 1996; 7: 743-753Crossref PubMed Scopus (103) Google Scholar, 46Cooper J.A. Carraway K.L. Carraway C.A.C. The Cytoskeleton: A Practical Approach. Oxford University Press, New York1992: 47-71Google Scholar). Cells were cultured in 24-well culture plates for 4 days, incubated with ODNs after permeabilization with streptolysin O, and treated with OP or vehicle as described above. Eight wells were used for each treatment. The cells were fixed with 1.5% formaldehyde in PBS for 15 min, then permeabilized with 0.1% Triton X-100 in PBS for 5 min. The cells were rinsed and incubated with rhodamine-phalloidin in PBS for 30 min. After washing quickly several times with PBS, the cells were extracted with absolute methanol. The fluorescence of each sample was measured using fluorimetry (Gilford Fluoro IV). To assess nonspecific binding, a 10-fold excess of unlabeled phalloidin was used. The nonspecific binding was subtracted from the total binding to get the specific binding. Actin staining was carried out as described (47Brown P.D. Benya P.D. J. Cell Biol. 1988; 106: 171-179Crossref PubMed Scopus (136) Google Scholar). Cells were rinsed briefly with PBS containing 5 mmEGTA (PBS-EGTA) and fixed in 4% (w/v) paraformaldehyde in PBS-EGTA for 20 min at 37 °C. Coverslips were immersed in 47.5% ethanol containing 5 mm EGTA for 15 min at room temperature and rinsed with several changes of PBS-EGTA before staining with 1:20 dilution of rhodamine-phalloidin in PBS-EGTA for 30 min at 37 °C. After rinsing several times with PBS-EGTA, coverslips were mounted on a mounting solution (Vector Laboratories Inc., Burlingham, CA). Cells were viewed in a Meridian ACAS 570 (Okemos, MI) with confocal option. Rhodamine-phalloidin images were recorded in 514 argon excitation line with a 40×/1.3 numerical aperture oil objective. Confocal images were processed by the Adobe Photoshop software program (Adobe Systems, Inc., Mountain View, CA). Avian osteoclasts were cultured in 24-well plates for 4–6 days. On day 5 in culture, cells were permeabilized and treated with or without ODNs as described above. Bone resorption assays were carried out as described previously (38Alvarez J.I. Teitelbaum S.L. Blair H.C. Greenfield E.M. Athanasou N.A. Ross F.P. Endocrinology. 1991; 128: 2324-2335Crossref PubMed Scopus (72) Google Scholar). Cells were incubated with ODNs and [3H]proline-labeled bone particles (2 × 104 cpm/ml) in serum-containing media at 37 °C for 12 h. For each treatment, four to six wells were used. Aliquots of supernatants (400 μl) were removed at 12 h and replaced with fresh media containing ODNs. Further aliquots were obtained after 24 h. Released isotope from [3H]proline-labeled bone particle by osteoclasts was measured by liquid scintillation counting. The cells were then washed three times with ice-cold PBS and lysed in Triton-containing lysis buffer as described above, and the protein content was determined as described above. Radioactivity was then normalized to protein content. All comparisons were made to “control,” which refers to mock permeabilized cells or mock permeabilized and vehicle-treated cells. Data presented are mean ± S.E. of experiments performed at different times, normalized to intra-experimental control values. Statistical comparisons between the treatment groups were done using analysis of variance with the Bonferroni correction for multiple comparisons. We have previously reported osteopontin stimulation of gelsolin-associated PI 3-kinase in avian osteoclasts. The activation of PI 3-kinase associated with Triton-soluble gelsolin was required for the osteopontin stimulation of F-actin (21Chellaiah M. Hruska K.A. Mol. Biol. Cell. 1996; 7: 743-753Crossref PubMed Scopus (103) Google Scholar). PI 3-kinase has been identified in a variety of systems associated with receptor tyrosine kinases (45Whitman M. Kaplan D.R. Schaffhausen B. Cantley L. Roberts T. Nature. 1985; 315: 239-242Crossref PubMed Scopus (557) Google Scholar, 48Auger K.R. Serunian L.A. Soltoff S.P. Libby P. Cantley L.C. Cell. 1989; 57: 167-175Abstract Full Text PDF PubMed Scopus (684) Google Scholar), and it is known to be a substrate for c-Src. Therefore, anti-gelsolin immunoprecipitates made from lysates of vehicle or OP-treated cells were analyzed for Src association with gelsolin by Western blotting (Fig. 1). c-Src was observed in anti-gelsolin immunoprecipitates of Triton-soluble fractions of cell lysates (Fig. 1 A,lanes 1 and 2). Negligible amounts of Src were associated with the anti-gelsolin immunoprecipitates of Triton-insoluble fractions (Fig. 1 A, lanes 3 and4). The effect of OP was an increase in ge" @default.
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