Superoxide Mediates Shock Wave Induction of ERK-dependent Osteogenic Transcription Factor (CBFA1) and Mesenchymal Cell Differentiation toward Osteoprogenitors
2002; Elsevier BV; Volume: 277; Issue: 13 Linguagem: Inglês
10.1074/jbc.m104587200
ISSN1083-351X
AutoresFeng Sheng Wang, Ching Jen Wang, Shyr Ming Sheen‐Chen, Yur Ren Kuo, Rong‐Fu Chen, Kuender D. Yang,
Tópico(s)Paraoxonase enzyme and polymorphisms
ResumoExtracorporeal shock wave (ESW) is an alternative non-invasive method for the promotion of bone growth and tendon repair. In an animal model, we have reported that ESW promoted bone marrow osteoprogenitor growth through transforming growth factor-β1 induction. We have further explored the mechanism for the ESW promotion of osteogenesis. Results showed that an optimal ESW treatment at 0.16 mJ/mm2 for 500 impulses rapidly induced a higher O2− and ONOO− production associated with a decrease of nitric oxide level in 1 h, and induced a higher transforming growth factor-β1 production in 24 h, and a higher colony-forming units-osteoprogenitor formation in 12 days. The colony-forming units-osteoprogenitor colonies revealed positive staining of bone alkaline phosphatase and turned into bone nodules in 21 days. Early scavenging of O2− but not Ca2+, H2O2, or prostaglandin E2 suppressed osteoprogenitor cell growth and maturation. Scavenging of O2− by superoxide dismutase raised the nitric oxide level back to the basal level and suppressed ESW-promoted osteoprogenitor cell growth, whereas inhibition of ONOO− by urate or NO by N-nitro-l-arginine methyl ester did not affect ESW promotion of osteogenesis, indicating that O2− acted as an early signal for ESW-induced cell growth. Further studies demonstrated that ESW induced ERK activation, and blockage of O2− production or inhibition of tyrosine kinase, but not protein kinase A and C inhibitors, suppressed ESW-induced ERK activation. In support that O2− mediated the ESW-induced ERK activation and osteogenic differentiation, we further demonstrated that scavenging of O2− by superoxide dismutase and inhibition of ERK activation by PD98059 decreased specific osteogenic transcription factor, core binding factor A1 activation, and decreased osteocalcin expression. Taken together, we showed that ESW-induced O2− production followed by tyrosine kinase-mediated ERK activation and core binding factor A1 activation resulted in osteogenic cell growth and maturation. Thus, an appropriate modulation of redox reaction by ESW may have some positive effect on the bone regeneration. Extracorporeal shock wave (ESW) is an alternative non-invasive method for the promotion of bone growth and tendon repair. In an animal model, we have reported that ESW promoted bone marrow osteoprogenitor growth through transforming growth factor-β1 induction. We have further explored the mechanism for the ESW promotion of osteogenesis. Results showed that an optimal ESW treatment at 0.16 mJ/mm2 for 500 impulses rapidly induced a higher O2− and ONOO− production associated with a decrease of nitric oxide level in 1 h, and induced a higher transforming growth factor-β1 production in 24 h, and a higher colony-forming units-osteoprogenitor formation in 12 days. The colony-forming units-osteoprogenitor colonies revealed positive staining of bone alkaline phosphatase and turned into bone nodules in 21 days. Early scavenging of O2− but not Ca2+, H2O2, or prostaglandin E2 suppressed osteoprogenitor cell growth and maturation. Scavenging of O2− by superoxide dismutase raised the nitric oxide level back to the basal level and suppressed ESW-promoted osteoprogenitor cell growth, whereas inhibition of ONOO− by urate or NO by N-nitro-l-arginine methyl ester did not affect ESW promotion of osteogenesis, indicating that O2− acted as an early signal for ESW-induced cell growth. Further studies demonstrated that ESW induced ERK activation, and blockage of O2− production or inhibition of tyrosine kinase, but not protein kinase A and C inhibitors, suppressed ESW-induced ERK activation. In support that O2− mediated the ESW-induced ERK activation and osteogenic differentiation, we further demonstrated that scavenging of O2− by superoxide dismutase and inhibition of ERK activation by PD98059 decreased specific osteogenic transcription factor, core binding factor A1 activation, and decreased osteocalcin expression. Taken together, we showed that ESW-induced O2− production followed by tyrosine kinase-mediated ERK activation and core binding factor A1 activation resulted in osteogenic cell growth and maturation. Thus, an appropriate modulation of redox reaction by ESW may have some positive effect on the bone regeneration. Oxidative stress induced by superoxide has been implicated in the induction of certain cell injury (1.Durot I. Maupoil V. Ponsar B. Cordelet C. Vergely-Vandriesse C. Rochette L. Athias P. Free Radic. Biol. Med. 2000; 29: 846-857Crossref PubMed Scopus (24) Google Scholar, 2.Warren M.C. Bump E.A. Medeiros D. Braunhut S.J. Free Radic. Biol. 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J. 1996; 318: 805-812Crossref PubMed Scopus (63) Google Scholar). Several physical factors such as heat (9.Souren J.E. van Aken H. van Wijk R. Biochem. Biophys. Res. Commun. 1996; 227: 816-821Crossref PubMed Scopus (12) Google Scholar), electrical field (10.Sauer H. Rahimi G. Hescheler J. Wartenberg M. J. Cell. Biochem. 1999; 75: 710-723Crossref PubMed Scopus (172) Google Scholar), pulsatile stretch (11.Hishikawa K. Oemar B.S. Yang Z. Luscher T.F. Circ. Res. 1997; 81: 797-803Crossref PubMed Scopus (173) Google Scholar), and laser irradiation (12.Grossman N. Schneid N. Reuveni H. Halevy S. Lubart R. Laser Surg. Med. 1998; 22: 212-218Crossref PubMed Scopus (252) Google Scholar) can stimulate cell proliferation through the involvement of superoxide. It is not known whether superoxide can regulate osteoprogenitor cell growth and differentiation. Extracorporeal shock wave (ESW) 1The abbreviations used are: ESWextracorporeal shock waveTGF-β1transforming growth factor-β1CFU-Ocolony-forming unit-osteoprogenitorERKextracellular signal-regulated kinaseCBFA1core binding factor A1DMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumSODsuperoxide dismutasel-NAMEN-nitro-l-arginine methyl esterPKAprotein kinase APKCprotein kinase CMBPmyelin basic proteinPGE2prostaglandin E2BAPTA1,2-bis(2-aminophenoxy)ethane-N, N, N′, N′-tetraacetic acidPBSphosphate-buffered saline is created by a high voltage spark discharge under water causing an explosive evaporation of water and producing high energy acoustic waves. The acoustic waves are focused on a semi-ellipsoid reflector and therefore can be transmitted into a specific tissue site (13.Ligeman J.E. Nephron. 1996; 16: 487-498Google Scholar). ESW treatment has been divergently applied for eukaryotic and prokaryotic biology systems. It is well known that ESW provides a non-invasive biophysical strategy for breaking renal stones with minimal side effects (13.Ligeman J.E. Nephron. 1996; 16: 487-498Google Scholar). Evidence also suggests that shock waves can potentially enhance gene transfer (14.Lauer U. Burgelt E. Squire Z. Messmer K. Hofschneider P.H. Greogor M. Delius M. Gene Ther. 1997; 4: 710-715Crossref PubMed Scopus (132) Google Scholar), suppress tumor growth (15.Delius M. Adams G. Cancer Res. 1999; 59: 5227-5232PubMed Google Scholar), and promote the bactericidal effect of microorganisms (16.Von Eiff C. Overbeck J. Haupt G. Hermann M. Winckler S. Richter K.D. Peters G. Spiegel H.U. J. Med. Microbiol. 2000; 49: 709-712Crossref PubMed Scopus (38) Google Scholar). extracorporeal shock wave transforming growth factor-β1 colony-forming unit-osteoprogenitor extracellular signal-regulated kinase core binding factor A1 Dulbecco's modified Eagle's medium fetal bovine serum superoxide dismutase N-nitro-l-arginine methyl ester protein kinase A protein kinase C myelin basic protein prostaglandin E2 1,2-bis(2-aminophenoxy)ethane-N, N, N′, N′-tetraacetic acid phosphate-buffered saline Recently, we and others (17.Wang C.J. Huang H.Y. Chen H.S. Pai C.H. Yang K.D. Clin. Orthop. Relat. Res. 2001; 387: 112-118Crossref PubMed Scopus (90) Google Scholar, 18.Wang C.J. Chen H.S. Chen C.E. Yang K.D. Clin. Orthop. Relat. Res. 2001; 387: 95-101Crossref PubMed Scopus (195) Google Scholar, 19.Beutler S. Regel G. Pape H. Machtens S. Weinberg A.M. Kremeike I. Jonas U. Tscherne H. Unfallchirung. 1999; 102: 839-847Crossref PubMed Scopus (44) Google Scholar, 20.Kazuo I. Katsuro T. Kazuyoshi T. J. Trauma. 1999; 47: 946-950Crossref PubMed Scopus (94) Google Scholar) have shown that ESW treatment has a promising effect on the promotion of bone fracture healing and repair of tendinitis. The mechanism by which ESW enhances fracture healing and repair of tendinitis remains to be determined. The fact that ESW treatment enhances both bone and tendon regeneration suggests that ESW may induce a certain signal for growth and maturation of the mesenchymal progenitors from bone marrow. It has been well clarified that the differentiation and maturation of bone marrow mesenchymal osteoprogenitor cells into osteoblastic lineage is involved in bone regeneration (21.Liu P. Oyajobi B.O. Russell R.G. Scutt A. Calcif. Tissue Int. 1999; 65: 173-180Crossref PubMed Scopus (111) Google Scholar, 22.Yoo J.U. Barthel T.S. Nishimura K. Solchaga L. Calpan A.I. Goldberg V.M. Johnstone B. J. Bone Jt. Surg. Am. 1998; 80: 1745-1757Crossref PubMed Scopus (783) Google Scholar, 23.Long M.W. Robinson J.A. Ashcraft A.E. Mann K.G. J. Clin. Investig. 1997; 95: 3-12Google Scholar). In support of the hypothesis, we have recently shown ESW treatment to be able to promote bone marrow stromal cell growth and differentiation toward osteogenic lineage, presumably through TGF-β1 induction (24.Wang, F. S., Yang, K. D., Wang, C. J., Sheen-Chen, S. M., and Chen, R. F. (2002) @@[email protected]@J. Bone Jt. Surg.,@@/[email protected]@ in pressGoogle Scholar). Accumulated evidence suggests that ESW induces a cavitation effect to increase membrane permeability and the influx of biological substances (25.Zhong P. Lin H. Xi X. Zhu S. Bhogte E.S. J. Acoust. Soc. Am. 1999; 105: 1997-2009Crossref PubMed Scopus (51) Google Scholar, 26.Huber P. Debus J. Jochle K. Simiantonakis I. Jenne J. Rastert R. Spoo J. Lorenz W.J. Wannenmacher M. Phys. Med. Biol. 1999; 44: 1427-1437Crossref PubMed Scopus (53) Google Scholar), which are usually implicated in cell and tissue damage (15.Delius M. Adams G. Cancer Res. 1999; 59: 5227-5232PubMed Google Scholar,27.Gambihler S. Delius M. Brendel W. Ultrasound Med. Biol. 1990; 16: 587-594Abstract Full Text PDF PubMed Scopus (62) Google Scholar). There is limited evidence showing that ESW promotes cell growth rather than cell damage. We hypothesized in this study that an optimal ESW treatment promoted osteoprogenitor cell growth and maturation via a rapid induction of oxygen radicals for a signal transduction from ERK to specific osteogenic transcription factor activation, followed by osteogenesis. Thus, we have sought to investigate which species of oxygen radicals could be induced by ESW treatment, how early the oxygen radicals transmitted the signal cascade, when the growth factor TGF-β1 was induced, and when the growth and osteogenic maturation was reached. Three-month-old Sprague-Dawley rats (National Experimental Animals Production Center, Taipei, Taiwan) were anesthetized with intraperitoneal injection of pentobarbital sodium (50 mg/kg; Nembutal® sodium, Abbott). Each rat was placed in supine position with four limbs abducent fixation. The ESW treatment with 0, 250, 500, and 1000 impulses at 0.16 mJ/mm2 (Ossatron®; HMT High Medical Technologies GmbH, Kreuzlingen, Switzerland) was applied to the left distal femur 10 mm above the knee joint as in our previous study (24.Wang, F. S., Yang, K. D., Wang, C. J., Sheen-Chen, S. M., and Chen, R. F. (2002) @@[email protected]@J. Bone Jt. Surg.,@@/[email protected]@ in pressGoogle Scholar). After ESW treatment for 1 h, bone marrow cells were harvested from the bone marrow of femurs with ESW treatment. Bone marrow cells from the femurs without ESW treatment were run as controls. The distal end of the femur bone was excised at the level of 5 mm above the knee joint. Bone marrow blood (0.4 ml) was aspirated with 20-gauge needle into a 1-ml syringe containing 20 units/ml heparin. The bone marrow mononuclear cells in the marrow blood were harvested from the interface of the Ficoll-Paque density gradient (d = 1.007 g/ml, Amersham Biosciences AB) at 500 × g for 30 min as described previously (28.Yang K.D. Chao C.Y. Shaio M.F. Acta Haematol. (Basel). 1998; 99: 191-199Crossref PubMed Scopus (8) Google Scholar). Cell number and viability were determined with a hemocytometer after staining with 0.4% trypan blue in ammonium chloride. Culture of the osteoprogenitors from bone marrow mononuclear cells (2 × 105 cells/well; 24-well plate) was raised in osteogenic medium containing Dulbecco's modified Eagle medium (DMEM; Invitrogen) with 10% fetal bovine serum (FBS), 10−8mdexamethasone, 50 μg/ml l-ascorbic acid, and 10−2m β-glycerophosphate (Sigma). After washing out nonadherent hematopoietic cells, total stromal cells were cultured for 12 days at 5% CO2 and incubated at 37 °C. The cultured supernatant was harvested and replaced with fresh osteogenic medium every 3 days. Colony-forming unit-osteoprogenitor (CFU-O) in bone marrow stromal cell culture was assessed as described previously (29.Aubin J.E. J. Cell. Biochem. 1999; 72: 396-410Crossref PubMed Scopus (207) Google Scholar). After incubation and removal of medium, the cell culture in each well was fixed with citrate/acetone/formaldehyde and subjected to bone alkaline phosphatase staining according to the instructions for the use of Sigma alkaline phosphatase histocytochemistry assay kit (Sigma). Total colonies found to have more than 32 segregate cells were recognized and calculated as positive bone alkaline phosphatase staining. The cells (1 × 104 cells/well; 96-well plate) from CFU-O colonies were subjected to determination of the osteoblastic lineage with bone alkaline phosphatase activity (30.Lennon D.P. Haynesworth S.E. Young R.G. Dennis J.E. Calpan A.I. Exp. Cell Res. 1995; 219: 211-222Crossref PubMed Scopus (267) Google Scholar). The reactions were incubated with 0.2 ml of substrate buffer containing 50 mmglycine, 1 mm magnesium chloride, pH 10.5, and 2.5 mm p-nitrophenyl phosphate (Sigma) at 37 °C for 30 min and stopped with 0.1 ml of 1 n sodium hydroxide. Results were read at A405 nm by a microplate reader (Dyn-Ex Technologies Inc.). Alkaline phosphatase activity was expressed as μm p-nitrophenol/well (31.Cassiede P. Dennis J.D. Ma F. Calpan A.I. J. Bone Miner. Res. 1996; 11: 1264-1273Crossref PubMed Scopus (148) Google Scholar). To confirm further the osteogenic formation, we also prolonged the primary osteoprogenitor cell culture to 21 days. The long term cultured CFU-O colonies were fixed with neutral buffered formaldehyde for 5 min, pH 7.4, rinsed with distilled water, and then stained using the von Kossa method with 0.3 ml of 5% freshly prepared silver nitrate. After this procedure, the size of bone nodules ≥2 mm2 showing positive von Kossa staining were counted under an inverted microscope as described previously (32.Bellows C.G. Aubin J.E. Heersche J.N.M. Endocrinology. 1987; 121: 1985-1992Crossref PubMed Scopus (272) Google Scholar). Rat bone marrow stromal cells harvested from rat femurs were cultured in DMEM with 10% FBS. After washing out nonadherent hematopoietic cells, total stromal cells were cultured for 5 days in a 5% CO2, 37 °C incubator into log phase growth before harvest. To study the role of Ca2+ in ESW-promoted cell growth, cells (1 × 106 cells/ml) were treated with the extracellular Ca2+ chelator, 1 mm EGTA, or the intracellular Ca2+ chelator, 40 μm BAPTA (Sigma), for 30 min. To study the role of superoxide or hydrogen peroxide in the ESW-promoted cell growth, cells were treated for 30 min with 500 units/ml SOD or catalase (Sigma) to scavenge superoxide and hydrogen peroxide (33.Yang K.D. Chen M.Z. Teng R.J. Yang M.Y. Liu H.C. Chen R.F. Hsu T.Y. Shaio M.F. Pediatr. Res. 2000; 48: 829-834Crossref PubMed Scopus (16) Google Scholar). To elucidate whether prostaglandin E2 was involved in the ESW-enhanced cell growth, cells were treated with 10 μm indomethacin to suppress PGE2 production by inhibiting cyclooxygenase-2 activity (34.Zaman G. Suswillo R.F. Cheng M.Z. Tavares I.A. Lanyon L.E. J. Bone Miner. Res. 1997; 12: 769-777Crossref PubMed Scopus (86) Google Scholar). In studies of the ESW-induced signal transduction of cell growth, cells were treated with 20 μm PD98059, a MEK inhibitor (Calbiochem), 50 μm calphostain C, a protein kinase A inhibitor, 100 μm (Rp)-cAMP, a protein kinase C inhibitor, and 20 μm genistein (Sigma), a tyrosine kinase inhibitor (35.Aikawa R. Komuro I. Yamazaki T. Zou Y. Kudoh S. Tanaka M. Shiojima I. J. Clin. Investig. 1997; 100: 1813-1821Crossref PubMed Scopus (630) Google Scholar), for 60 min before studies. After these treatments, cells were washed and resuspended with PBS. Cell suspensions were subjected to ESW treatment with modification as described previously (36.Wang F.S. Wang C.J. Huang H.J. Chung H. Chen R.F. Yang K.D. Biochem. Biophys. Res. Commun. 2001; 287: 648-655Crossref PubMed Scopus (159) Google Scholar). Briefly, cells (1 × 106 cells/ml) were suspended in a 5-ml polystyrene round-bottom tube (Falcon®, Becton Dickinson) containing 5 ml of PBS at pH 7.4 and exposed to ESW at 0.16 mJ/mm2 for 500 impulses. The duration of ESW treatment took 10 min. After ESW treatment, cells were cultured for 1 h and 2 days as indicated in the cell proliferation and Western blot assays. In some experiments, cells (1 × 105 cell/well, 24-well plate) were cultured in osteogenic medium for 12 and 21 days in order to elucidate the osteoprogenitor maturation as demonstrated by osteocalcin expression and bone nodule formation. Cultured medium was harvested and replaced with fresh medium every 3 days. The long term cell cultures in each well were subjected to Western blot assay of osteocalcin production and von Kossa staining of bone nodule formations. To detect kinetic change of superoxide production, ESW-treated cells (1 × 105 cells/well, 96-well plate) were incubated with phenol red-free DMEM for 1, 6, 24, and 48 h. After incubation, each well was added with 50 μm horse heart cytochrome c (Sigma) and followed by incubation in the dark at 37 °C for 1 h. The O2− production was determined by the difference of cytochrome creduction in the absence and presence of SOD. The cytochrome c reduction was monitored at A550 nm, and O2− concentration was calculated from the molar extinction coefficient of 0.0282 μm−1 cm−1 to calculate O2− production (33.Yang K.D. Chen M.Z. Teng R.J. Yang M.Y. Liu H.C. Chen R.F. Hsu T.Y. Shaio M.F. Pediatr. Res. 2000; 48: 829-834Crossref PubMed Scopus (16) Google Scholar). To elucidate whether O2− was an early or late signal for ESW promotion of bone marrow stromal cell growth, cells in each well were added with 500 units/ml SOD or vehicle (PBS) for 30 min at 0, 6, and 24 h after ESW treatment and subjected to O2− production assay. To elucidate whether O2− was an early or late signal for ESW promotion of TGF-β1 induction and bone marrow stromal cell growth, cells in each well were treated with 500 units/ml SOD or vehicle for 30 min at 0, 6, and 24 h after ESW treatment. The supernatant of the reactions was harvested for TGF-β1 production in 1 day, and the cells were subjected to the cell proliferation assay in 2 days. The changes of O2− production were correlated to the stromal cell growth, TGF-β1 production, and bone nodule formations as well as signal transduction as described below. To elucidate the role of NO or ONOO− in the ESW-promoted cell growth, cells were treated for 30 min with and without 100 μm urate or 100 μml-NAME (Sigma) to inhibit ONOO− or NO production (37.Hikiji H. Shin W.S. Koizumi T. Takato T. Susami T. Koizumi Y. Okai-Matsuo Y. Toyo-Oka T. Am. J. Physiol. 2000; 278: E1031-E1037Crossref PubMed Google Scholar, 38.Smalt R. Mitchel F.T. Howard R.L. Chambers T.J. Am. J. Physiol. 1997; 273: E751-E758PubMed Google Scholar). After washing and resuspension in PBS, cells were subjected to ESW treatment. ESW-treated cells (1 × 105 cells/well, 96-well plate) were incubated with phenol red-free DMEM for 1 h. Cultured supernatant was harvested for assessment of NO production. Cells were collected for determination of nitrotyrosine, a marker for ONOO− formation. In some experiments, the supernatants of the reactions were harvested for TGF-β1 production in 1 day, and the cells were subjected to von Kossa assay in 21 days. We measured nitrite and nitrate levels to reflect NO production as described previously (39.Schulz K. Kerber S. Kelm M. Nitric Oxide. 1999; 3: 225-234Crossref PubMed Scopus (134) Google Scholar). Briefly, the culture supernatants were determined in triplicate by injecting the 50-μl aliquot into a custom impinger with a Teflon-linked septum containing 0.4 m vanadium chloride in glacial acetic acid. The vanadium chloride reduced nitrate and nitrite to NO gas, which passed into a stream of helium and entered the nitrogen oxide analyzer (NOA280; Sievers Inc., Denver, CO) as described previously (39.Schulz K. Kerber S. Kelm M. Nitric Oxide. 1999; 3: 225-234Crossref PubMed Scopus (134) Google Scholar). The nitrite and nitrate levels in each sample were determined by interpolation calculated from a series of well known potassium nitrate concentrations. Protein concentration in each sample was measured by a Bio-Rad protein assay kit (Bio-Rad). Results were normalized with protein concentration in each sample. To investigate the interaction of O2− and NO, we measured the nitrotyrosine level, which is a marker for ONOO−formation, as described previously (40.Oyama J.I. Shimokawa H. Momii H. Chen X. Fukuyama N. Arai Y. Egashira K. Nakazawa H. Takeshita A. J. Clin. Investig. 1998; 101: 2207-2214Crossref PubMed Scopus (99) Google Scholar). The nitrotyrosine levels in each sample were determined by high performance liquid chromatography (LC-10AD, Shimadze, Tokyo, Japan) equipped with a reverse phase column (4.6 mm internal diameter × 250 mm length; TSK-gel, ODS-80TM). The column was eluted with 50 mmNaH2PO3·H3PO4 (pH 2.5) with methanol (60:40, v/v) at a flow rate of 2 ml/min through an isocratic pump. The peaks were measured with an electrochemical detector (Coulochem II, Bedford, MA) at a guard cell potential of −250 mV. The nitrotyrosine concentration in each sample was integrated from the retention time and area under the eluting peak. The exact concentration was determined by an interpolation calculated from a series of well known standard concentrations of 3-nitrotyrosine (Calbiochem). Results were normalized with protein concentration in each sample (40.Oyama J.I. Shimokawa H. Momii H. Chen X. Fukuyama N. Arai Y. Egashira K. Nakazawa H. Takeshita A. J. Clin. Investig. 1998; 101: 2207-2214Crossref PubMed Scopus (99) Google Scholar). The cultured supernatants were harvested for measurement of TGF-β1 by centrifuging at 500 × g for 5 min and then stored at −70 °C until studied. The TGF-β1 production was determined by an enzyme-linked immunosorbent assay (Quantikine®, R & D Systems Inc.). Briefly, acid-activated culture supernatants (0.2 ml) were added to each polystyrene microwell pre-coated with recombinant human TGF-β-soluble receptor type II for 3 h. The reactions were next incubated with a horseradish peroxidase-conjugated TGF-β1 polyclonal antibody for 1.5 h. After washing, the reactions were incubated with substrate solution containing 0.1 ml of stabilized hydrogen peroxide and 0.1 ml of stabilized tetramethylbenzidine for 30 min. The reaction was stopped with 0.05 ml of 2 n sulfuric acid. Data were read at A450 nm with a microplate reader. Results were calculated by an interpolation from a standard curve made by a series of TGF-β1 concentrations. Bone marrow stromal cell proliferation was determined by nuclear [3H]thymidine uptake as modified from a measurement of osteoblast proliferation described previously (36.Wang F.S. Wang C.J. Huang H.J. Chung H. Chen R.F. Yang K.D. Biochem. Biophys. Res. Commun. 2001; 287: 648-655Crossref PubMed Scopus (159) Google Scholar). Bone marrow stromal cells with and without ESW treatment (2 × 104cells/well, 96-well plate) were cultured for 24 h before the addition of 1 μCi of [3H]thymidine/well (Amersham Biosciences AB) for an additional 24-h culture. At the end of the culture period, cells in each culture well were released from the plates by trypsinization and processed for [3H]thymidine uptake determination by a liquid scintillation analyzer (Tri-Crab 2100TR, Packard Instrument Co.). Bone marrow stromal cells were lysed with 200 μl of ice-cold buffer containing 10 mm Tris, pH 7.9, 10 mm KCl, 2 mmMgCl2, 0.1 mm EDTA, 0.7% Nonidet P-40 on ice for 10 min and centrifuged at 500 × g for 5 min. The cytosolic extracts were harvested to measure ERK activation. The nuclear pellets were further lysed with buffer containing 40 mm Tris, pH 7.9, 350 mm NaCl, 2 mmMgCl2, 1 mm EDTA, 0.2 mm EGTA, 20% glycerol, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 2 μm dithiothreitol, 2 μg/ml leupeptin, and 1 μg/ml aprotinin on ice for 20 min and harvested to determine osteogenic transcription factor and core binding factor A1 (CBFA1) activation by centrifugation at 12,000 × g, 4 °C, for 10 min. Protein concentrations in cytosolic and nuclear extracts were determined by Bio-Rad assay kit (Bio-Rad). The cytosolic extracts were incubated with anti-ERK antibody (1:100; Upstate Biotechnology, Inc.) for 1 h at 4 °C. After incubation, the immune complexes were precipitated with protein A (Sigma). The immunoprecipitate (20 μg) was reacted with the substrate buffer containing 20 μg of myelin basic protein (MBP), 15 mm MgCl2, 100 μm ATP, and 5 μm protein kinase inhibitor for 30 min at 30 °C. The reaction was stopped with the Laemmli buffer containing 200 mm Tris, pH 6.8, 10% glycerol, 4% SDS, 50 μm dithiothreitol, and 0.05% bromphenol blue for 5 min at 95 °C. The mixtures were subject to Western blot assay. The phosphorylated MBP on the blot was recognized by a specific mouse anti-phospho-MBP (1:1000) antibody, followed by goat anti-mouse horseradish peroxidase-conjugated IgG (1:3000) as the second antibody. The ERK activity was reflected on the phosphorylated MBP visualized with chemiluminescence agents. The nuclear extracts were incubated with anti-CBFA1 antibody (1:100; Santa Cruz Biotechnology) for 1 h at 4 °C. After incubation, the immune complexes were precipitated with protein A (Sigma). The immunoprecipitate (20 μg) was mixed with Laemmli buffer for 5 min at 95 °C. The mixtures were subject to Western blot assay. The total CBFA1 on the blot was recognized by a rabbit anti-CBFA1 antibody at 1:500 dilution, followed by goat anti-rabbit horseradish peroxidase-conjugated IgG (1:2000) as the second antibody. The phosphorylated CBFA1 on the blot was further recognized by a specific mouse anti-phosphotyrosine (1:1000) antibody, followed by goat anti-mouse horseradish peroxidase-conjugated IgG (1:2000) as the second antibody. The CBFA1 activity was reflected on the phosphorylated CBFA1 visualized with chemiluminescence agents. To confirm ESW promotion of osteoprogenitor maturation, cytosolic extracts were subjected to determination of osteocalcin production with immunoblot assay. The osteocalcin on the blot was recognized by a specific mouse anti-osteocalcin (1:500) antibody, followed by goat anti-mouse horseradish peroxidase-conjugated IgG (1:3000) as the second antibody. The osteogenic activity was reflected on the osteocalcin expression visualized with chemiluminescence agents. Data were analyzed with a non-parametric one-way analysis of variance followed by Student's t test to determine significance between treatments. p < 0.05 was considered statistically significant. Certain doses of ESW treatments applied to rat femoral bone promoted the CFU-O formation of bone marrow stromal cells. As shown in Fig. 1, the ESW treatment with 0.16 mJ/mm2 for 250 impulses minimally enhanced CFU-O formation, and the treatment for 500 impulses had the best effect. However, the treatment with 1000 impulses brought about a suppressing effect. The CFU-O colonies were confirmed to be osteoblastic lineage, as demonstrated by an increase in alkaline phosphatase activity in the cells from CFU-O colonies (Fig. 2A). The cells from the CFU-O colonies matured into bone nodules after a 21-day long term culture as shown in Fig. 2B. The bone nodule formations were significantly higher in the ESW treatment with 500 impulses than those without ESW treatment (Fig. 2B).Figure 2ESW promoted bone growth as determined by alkaline phosphatase activity and bone nodule formations. A, ESW promotion of bone alkaline phosphatase activities. Cells (1 × 104 cells/well, 96-well plate) sub-cultured from the CFU-O colonies with and without ESW treatment at 0.16 mJ/mm2 for 500 impulses were subjected to assessment of alkaline phosphatase activity. * indicates a significant difference between both groups (p < 0.001). B, ESW promotion of bone nodule formations as determined by von Kossa staining. Bone marrow stromal cells (2 × 10
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