Characterization of Osteoclasts from Patients Harboring a G215R Mutation in ClC-7 Causing Autosomal Dominant Osteopetrosis Type II
2004; Elsevier BV; Volume: 164; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)63712-1
ISSN1525-2191
AutoresKim Henriksen, Jeppe Gram, Sophie Schaller, Bjarne Dahl, Morten Hanefeld Dziegiel, Jens Bollerslev, M.A. Karsdal,
Tópico(s)Connective tissue disorders research
ResumoAutosomal dominant osteopetrosis II (ADOII) is a relatively benign disorder caused by a missense mutation in the ClCN7 gene. In this study, we characterize the osteoclasts from patients with ADOII, caused by a G215R mutation, and investigate the effect on osteoclast function in vitro. Osteoclasts from ADOII patients and healthy age- and sex-matched controls, were used to evaluate osteoclastogenesis, cell fusion, acidification, and resorptive activity. ADOII osteoclasts in vivo have increased number and size. However, in vitro we observed no significant changes in the osteoclast formation rate, the morphology, and the expression of markers, such as cathepsin K and tartrate-resistant acid phosphatase. When mature ADOII osteoclasts were investigated on mineralized bone, they degraded the bone material, however only to 10 to 20% of the level in controls. We show by acridine orange, that the reduced chloride transport leads to reduced acidification. We show that the residual activity is sensitive to inhibitors of cathepsins and chloride channels, confirming that resorption is reduced but present. In conclusion, this is the first functional in vitro study of human ADOII osteoclasts. We show normal osteoclastogenesis in ADOII osteoclasts. However, the residual activity of the ClC-7 channel in ADOII osteoclasts does not allow sufficient acidification and thereby resorption. Autosomal dominant osteopetrosis II (ADOII) is a relatively benign disorder caused by a missense mutation in the ClCN7 gene. In this study, we characterize the osteoclasts from patients with ADOII, caused by a G215R mutation, and investigate the effect on osteoclast function in vitro. Osteoclasts from ADOII patients and healthy age- and sex-matched controls, were used to evaluate osteoclastogenesis, cell fusion, acidification, and resorptive activity. ADOII osteoclasts in vivo have increased number and size. However, in vitro we observed no significant changes in the osteoclast formation rate, the morphology, and the expression of markers, such as cathepsin K and tartrate-resistant acid phosphatase. When mature ADOII osteoclasts were investigated on mineralized bone, they degraded the bone material, however only to 10 to 20% of the level in controls. We show by acridine orange, that the reduced chloride transport leads to reduced acidification. We show that the residual activity is sensitive to inhibitors of cathepsins and chloride channels, confirming that resorption is reduced but present. In conclusion, this is the first functional in vitro study of human ADOII osteoclasts. We show normal osteoclastogenesis in ADOII osteoclasts. However, the residual activity of the ClC-7 channel in ADOII osteoclasts does not allow sufficient acidification and thereby resorption. The osteoclasts ability to dissolve the inorganic phase of bone is essential for the degradation of the organic bone matrix, and thereby for the maintenance of the skeleton. The inorganic phase of bone consists of hydroxyapatite crystals, and the dissolution of this matrix requires a decrease in pH, which is facilitated by active transport of protons into the resorption lacunae.1Baron R Neff L Louvard D Courtoy PJ Cell-mediated extracellular acidification and bone resorption: evidence for a low pH in resorbing lacunae and localization of a 100-kD lysosomal membrane protein at the osteoclast ruffled border.J Cell Biol. 1985; 101: 2210-2222Crossref PubMed Google Scholar The proton transport is mediated by a specialized osteoclastic V-ATPase.2Blair HC Teitelbaum SL Ghiselli R Gluck S Osteoclastic bone resorption by a polarized vacuolar proton pump.Science. 1989; 245: 855-857Crossref PubMed Scopus (727) Google Scholar To preserve the electroneutrality in the resorption compartment a passive transport of chloride through chloride channels takes place.3al Awqati Q Chloride channels of intracellular organelles.Curr Opin Cell Biol. 1995; 7: 504-508Crossref PubMed Scopus (96) Google Scholar Chloride channels in osteoclasts have been studied extensively, and recently it was demonstrated that the chloride channel ClC-7 is involved in bone resorption. ClC-7 is a member of the chloride channel (CLC) family of voltage-gated chloride ion channels, which have been shown to have 16 transmembrane domains, and to form structural dimers.4Dutzler R Campbell EB Cadene M Chait BT MacKinnon R X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity.Nature. 2002; 415: 287-294Crossref PubMed Scopus (1334) Google Scholar These channels are involved in the regulation of membrane potentials, in various processes such as secretion and absorption of salts and organellar acidification.5Jentsch TJ Stein V Weinreich F Zdebik AA Molecular structure and physiological function of chloride channels.Physiol Rev. 2002; 82: 503-568Crossref PubMed Scopus (1055) Google Scholar Recent studies have shown that ClC-7 is mainly expressed in the osteoclasts in the bones and in neurons in the brain.6Kornak U Kasper D Bosl MR Kaiser E Schweizer M Schulz A Friedrich W Delling G Jentsch TJ Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man.Cell. 2001; 104: 205-215Abstract Full Text Full Text PDF PubMed Scopus (800) Google Scholar, 7Karsdal MA Schaller S Henriksen K Sveigaard S Heegaard A Christophersen P Stahlhut M Hélix M Engsig MT Foged N Delaissé J The chloride channel inhibitor NS3736 prevents bone resorption in ovariectomized rats, while preserving bone formation.J Bone Miner Res. 2003; 18: M233AGoogle Scholar The major phenotype of the ClC-7-deficient mice is the complete absence of bone resorption by the osteoclasts.6Kornak U Kasper D Bosl MR Kaiser E Schweizer M Schulz A Friedrich W Delling G Jentsch TJ Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man.Cell. 2001; 104: 205-215Abstract Full Text Full Text PDF PubMed Scopus (800) Google Scholar The absence of bone resorption was shown to lead to a severe osteopetrosis, very similar to that observed in patients with autosomal recessive osteopetrosis (ARO).6Kornak U Kasper D Bosl MR Kaiser E Schweizer M Schulz A Friedrich W Delling G Jentsch TJ Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man.Cell. 2001; 104: 205-215Abstract Full Text Full Text PDF PubMed Scopus (800) Google Scholar In a few cases of ARO, the phenotype was shown to be caused by mutations in ClC-7.6Kornak U Kasper D Bosl MR Kaiser E Schweizer M Schulz A Friedrich W Delling G Jentsch TJ Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man.Cell. 2001; 104: 205-215Abstract Full Text Full Text PDF PubMed Scopus (800) Google Scholar, 8Cleiren E Benichou O Van Hul E Gram J Bollerslev J Singer FR Beaverson K Aledo A Whyte MP Yoneyama T deVernejoul MC Van Hul W Albers-Schonberg disease (autosomal dominant osteopetrosis, type II) results from mutations in the ClCN7 chloride channel gene.Hum Mol Genet. 2001; 10: 2861-2867Crossref PubMed Google Scholar The osteopetrosis was speculated to be caused by an intrinsic defect in the osteoclasts, which correlates with the expression pattern. ClC-7 was shown to be expressed at high levels at the ruffled border of the osteoclasts, where it participates in the secretion of acid into the resorption lacunae.6Kornak U Kasper D Bosl MR Kaiser E Schweizer M Schulz A Friedrich W Delling G Jentsch TJ Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man.Cell. 2001; 104: 205-215Abstract Full Text Full Text PDF PubMed Scopus (800) Google ScholarTwo other forms of osteopetrosis related to defects in ClC-7 have been found. One of these is the intermediate form of autosomal recessive osteopetrosis (IARO), which is a less severe form of osteopetrosis than ARO. However, still caused by a mutation in ClC-7 and with the same hereditary pattern.9Campos-Xavier AB Saraiva JM Ribeiro LM Munnich A Cormier-Daire V Chloride channel 7 (CLCN7) gene mutations in intermediate autosomal recessive osteopetrosis.Hum Genet. 2003; 112: 186-189Crossref PubMed Scopus (78) Google Scholar The other is the autosomal dominant osteopetrosis type II (ADOII), which is a clinically less severe form often discovered by coincidental radiography.8Cleiren E Benichou O Van Hul E Gram J Bollerslev J Singer FR Beaverson K Aledo A Whyte MP Yoneyama T deVernejoul MC Van Hul W Albers-Schonberg disease (autosomal dominant osteopetrosis, type II) results from mutations in the ClCN7 chloride channel gene.Hum Mol Genet. 2001; 10: 2861-2867Crossref PubMed Google Scholar, 10Grodum E Gram J Brixen K Bollerslev J Autosomal dominant osteopetrosis: bone mineral measurements of the entire skeleton of adults in two different subtypes.Bone. 1995; 16: 431-434PubMed Scopus (24) Google Scholar, 11Bollerslev J Grontved A Andersen Jr, PE Autosomal dominant osteopetrosis: an otoneurological investigation of the two radiological types.Laryngoscope. 1988; 98: 411-413Crossref PubMed Google Scholar The ADOII phenotype is thought to be caused by a dominant-negative effect, related to the dimeric property of the ClC-7 channel.4Dutzler R Campbell EB Cadene M Chait BT MacKinnon R X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity.Nature. 2002; 415: 287-294Crossref PubMed Scopus (1334) Google Scholar, 8Cleiren E Benichou O Van Hul E Gram J Bollerslev J Singer FR Beaverson K Aledo A Whyte MP Yoneyama T deVernejoul MC Van Hul W Albers-Schonberg disease (autosomal dominant osteopetrosis, type II) results from mutations in the ClCN7 chloride channel gene.Hum Mol Genet. 2001; 10: 2861-2867Crossref PubMed Google Scholar The ADOII phenotype has been characterized in detail with respect to histology, response to hormonal treatments, and expression of both osteoclast and osteoblast markers. Among the characteristics are a marked increase in bone mass, increased numbers of abnormally large osteoclasts that express unusually high levels of tartrate-resistant acid phosphatase (TRAP) and creatine kinase BB (CKBB), and that have accumulations of lysosomal vesicles containing an unidentified material.12Bollerslev J Marks Jr, SC Pockwinse S Kassem M Brixen K Steiniche T Mosekilde L Ultrastructural investigations of bone resorptive cells in two types of autosomal dominant osteopetrosis.Bone. 1993; 14: 865-869Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 13Bollerslev J Steiniche T Melsen F Mosekilde L Structural and histomorphometric studies of iliac crest trabecular and cortical bone in autosomal dominant osteopetrosis: a study of two radiological types.Bone. 1989; 10: 19-24Abstract Full Text PDF PubMed Scopus (58) Google Scholar, 14Gram J Antonsen S Horder M Bollerslev J Elevated serum levels of creatine kinase BB in autosomal dominant osteopetrosis type II.Calcif Tissue Int. 1991; 48: 438-439Crossref PubMed Scopus (19) Google Scholar, 15Waguespack SG Hui SL White KE Buckwalter KA Econs MJ Measurement of tartrate-resistant acid phosphatase and the brain isoenzyme of creatine kinase accurately diagnoses type II autosomal dominant osteopetrosis but does not identify gene carriers.J Clin Endocrinol Metab. 2002; 87: 2212-2217Crossref PubMed Scopus (29) Google Scholar, 16Semba I Ishigami T Sugihara K Kitano M Higher osteoclastic demineralization and highly mineralized cement lines with osteocalcin deposition in a mandibular cortical bone of autosomal dominant osteopetrosis type II: ultrastructural and undecalcified histological investigations.Bone. 2000; 27: 389-395Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar In contrast, osteoblast markers and activity appeared to be normal.11Bollerslev J Grontved A Andersen Jr, PE Autosomal dominant osteopetrosis: an otoneurological investigation of the two radiological types.Laryngoscope. 1988; 98: 411-413Crossref PubMed Google Scholar, 17Brockstedt H Bollerslev J Melsen F Mosekilde L Cortical bone remodeling in autosomal dominant osteopetrosis: a study of two different phenotypes.Bone. 1996; 18: 67-72Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 18Bollerslev J Andersen Jr, PE Radiological, biochemical and hereditary evidence of two types of autosomal dominant osteopetrosis.Bone. 1988; 9: 7-13Abstract Full Text PDF PubMed Scopus (131) Google Scholar, 19Bollerslev J Nielsen HK Larsen HF Mosekilde L Biochemical evidence of disturbed bone metabolism and calcium homeostasis in two types of autosomal dominant osteopetrosis.Acta Med Scand. 1988; 224: 479-483Crossref PubMed Scopus (20) Google Scholar Some ADOII patients also develop premature osteoarthritis of unknown reason.20Benichou OD Laredo JD de Vernejoul MC Type II autosomal dominant osteopetrosis (Albers-Schonberg disease): clinical and radiological manifestations in 42 patients.Bone. 2000; 26: 87-93Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar Moreover, cranial nerve compression is observed in some ADOII patients, however this is a secondary effect caused by the increased thickness of the skull.21Andersen Jr, PE Bollerslev J Heterogeneity of autosomal dominant osteopetrosis.Radiology. 1987; 164: 223-225PubMed Google ScholarIn this study we dissect the effect of the G215R mutation in ClC-7 on osteoclastic processes. We show that osteoclastogenesis and expression of markers are normal in the ADOII patients, and the expression and localization of ClC-7 are unchanged in ADOII osteoclasts as well. We demonstrate that ADOII osteoclasts have decreased acidification leading to reduced resorption of mineralized bone.Materials and MethodsPatients and EthicsPatients were ascertained from a Danish family with ADOII previously shown to be caused by a G215R mutation in the ClCN-7 chloride channel gene.8Cleiren E Benichou O Van Hul E Gram J Bollerslev J Singer FR Beaverson K Aledo A Whyte MP Yoneyama T deVernejoul MC Van Hul W Albers-Schonberg disease (autosomal dominant osteopetrosis, type II) results from mutations in the ClCN7 chloride channel gene.Hum Mol Genet. 2001; 10: 2861-2867Crossref PubMed Google Scholar Nine mutation-positive members (four women and five men) aged 28 to 60 years were included as well as nine age- and sex-matched controls. The study was approved by the Danish Regional Ethical Committee (registration number 2473-03).Drugs and ChemicalsThe chloride channel inhibitor NS5818 (N-(3,5-dichloro-phenyl)-N′-[2-(1H-tetrazol-5-yl)-biphenyl-4-yl-4′-carboxylic acid dimethylamide] urea; patent reference, WO 0024707) is a close, but more potent, analog of the previously described NS3736 (1-[4-bromo-2-(1H-tetrazol-5-yl)-phenyl]-3-(4-chloro-3-trifluoromethyl-phenyl)-urea),7Karsdal MA Schaller S Henriksen K Sveigaard S Heegaard A Christophersen P Stahlhut M Hélix M Engsig MT Foged N Delaissé J The chloride channel inhibitor NS3736 prevents bone resorption in ovariectomized rats, while preserving bone formation.J Bone Miner Res. 2003; 18: M233AGoogle Scholar which was synthesized at Neurosearch A/S, Ballerup, Denmark. Bafilomycin A1 and E64 were purchased from Calbiochem (San Diego, CA, USA). The broad-spectrum matrix metalloproteinase (MMP) inhibitor GM6001 was from AM Scientific (Pleasant Hill, CA, USA). The remaining chemicals were from Sigma-Aldrich (Copenhagen, Denmark) and the culture media were from Life Technologies (Taastrup, Denmark) unless specified.Cell CultureIsolation of CD14+ Human MonocytesThe CD14+ isolation was performed as previously described.22Karsdal MA Hjorth P Henriksen K Kirkegaard T Nielsen KL Lou H Delaisse JM Foged NT TGF-beta controls human osteoclastogenesis through the p38 MAP kinase and regulation of RANK expression.J Biol Chem. 2003; 278: 44975-44987Crossref PubMed Scopus (123) Google Scholar Briefly, the monocytes were isolated from peripheral blood by centrifugation on a Ficoll-Paque gradient (Amersham Pharmacia, Hillerød, Denmark), and magnetically sorted using a CD14+ magnetic bead isolation kit (Dynabeads M-450; Dynal Biotech, Oslo, Norway). The cells were then seeded in 75-cm2 flasks, and cultured in α-minimal essential medium containing 10% serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 25 ng/ml of macrophage-colony stimulating factor (M-CSF) (R&D Systems) for 3 days, then they were lifted, reseeded, and cultured until day 10 in the presence of 25 ng/ml of M-CSF and 25 ng/ml of receptor activator of nuclear factor-κB ligand (RANKL) (R&D Systems, Minneapolis, MN, USA) unless stated.Cell FusionCell fusion was determined by seeding equal numbers of CD14+ monocytes isolated from either the ADOII patients or healthy controls, and culturing them for 5, 7, 9, or 11 days in the presence of RANKL and M-CSF. The conditioned media were removed and stored for measurement of TRAP activity. The cells were fixed using 3.7% formaldehyde in phosphate-buffered saline (PBS) for 15 minutes, and subsequently in pure methanol for 5 minutes. The nuclei of the cells were visualized using Ehrlich's hematoxylin (BDH Laboratory Supplies, Poole, England) for 2 minutes, followed by washing in tap water. Fusion scorings were performed using an Olympus IX-70 light microscope, and a video camera linked to a computer using CAST-Grid software (Olympus, Glostrup, Denmark). Osteoclast fusion was detected by the formation of cells with three or more nuclei.TRAP Activity MeasurementsThe TRAP activities were measured using a colorimetric assay. The conditioned media were tested for TRAP activity by addition of 6 mmol/L 4-nitrophenylphosphate and 25 mmol/L sodium tartrate at pH 5.5. The reaction products were quantified by measuring absorbance at 405 nm using an optical plate reader.ImmunoblottingTotal cell lysates were prepared by lysing the cells in RIPA+++ buffer22Karsdal MA Hjorth P Henriksen K Kirkegaard T Nielsen KL Lou H Delaisse JM Foged NT TGF-beta controls human osteoclastogenesis through the p38 MAP kinase and regulation of RANK expression.J Biol Chem. 2003; 278: 44975-44987Crossref PubMed Scopus (123) Google Scholar for 5 minutes. The lysates were centrifuged at 15,000 × g for 30 minutes to remove any leftover cell debris. Protein concentrations were measured using the Bio-Rad DC protein measurement assay (Bio-Rad Laboratories, Hercules, CA, USA).Thirty μg of total protein in sample buffer containing 40 mmol/L dithiothreitol was loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and then electroblotted onto nitrocellulose membranes. The quality of the protein loading was always checked by Ponceau Red (Sigma-Aldrich) staining. The membranes were then blocked in 50 mmol/L Tris-base, pH 7.5, 100 mmol/L NaCl, 0,1% Tween-20 containing 5% skim milk powder for 1 hour at ambient temperature. This was followed by overnight incubation at 4°C with the correct dilution of the primary antibodies against either CKBB (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), TRAP (Zymed, South San Francisco, CA, USA), cathepsin K (Chemicon International, Temecula, CA, USA), ClC-7 (Nordic Bioscience A/S, Merlev, Denmark), and p38 MAPK (Cell Signaling Technology, Beverly, MA, USA), which was used as a constant control.22Karsdal MA Hjorth P Henriksen K Kirkegaard T Nielsen KL Lou H Delaisse JM Foged NT TGF-beta controls human osteoclastogenesis through the p38 MAP kinase and regulation of RANK expression.J Biol Chem. 2003; 278: 44975-44987Crossref PubMed Scopus (123) Google Scholar This was followed by incubation with the corresponding horseradish peroxidase-conjugated secondary antibody for 1 hour at ambient temperature. Finally, the results were visualized using the ECL kit (Amersham Pharmacia Biotech).ImmunocytochemistryMature CD14+ isolated osteoclasts were seeded on Osteologic coverslips (BD Biosciences, Brøndby, Denmark), and cultured for 2 days, fixated in 3.7% formaldehyde in PBS for 20 minutes, and then washed thoroughly in PBS. The coverslips were blocked in Tris-buffered saline containing 2.5% casein and 0.1% Triton X-100 for 30 minutes at ambient temperature. This was followed by incubation with the primary antibody (ClC-7) or the corresponding preimmune serum overnight at 4°C in a moist atmosphere, after washing in Tris-buffered saline the coverslips were incubated in Rabbit EnVision (DakoCytomation, Glostrup, Denmark) for 30 minutes at ambient temperature. Finally, the peroxidase activity was visualized using DAB+, and the nuclei were counterstained using Ehrlich's hematoxylin. The coverslips were then dehydrated through a gradient of alcohol (70 to 99%) and toluene, and mounted in DPX. The pictures were taken using an Olympus BX-60 light microscope equipped with an Olympus C-2000 Zoom digital camera.Acridine Orange AccumulationMature CD14+ sorted human osteoclasts were seeded on Osteologic coverslips, allowed to attach, and were cultured for 2 days. The cells were then incubated with acridine orange [3,6-bis(dimethylamine)acridine, 2 μg/ml] and the additives described in the figure legends for 45 minutes, the dye was then removed by intensive washing, and the accumulation was visualized using an Olympus IX-70 microscope equipped with Olympus U-MWB filter, a ×20 objective and an Olympus C-2000 Zoom digital camera.Area and Frequency of Resorption Pits on Osteologic CoverslipsMature CD14+ sorted human osteoclasts were seeded on the coverslips, and they were cultured for 48 hours in the presence of RANKL and M-CSF, and then fixed in 3.7% formaldehyde in PBS for 20 minutes at room temperature followed by extensive washing. Finally, the coverslips were stained with Ehrlich's hematoxylin for 2 minutes, and washed in running tap water for 5 minutes. The coverslips were dehydrated through a gradient of alcohol (70 to 99%) and toluene, and mounted in DPX. The frequency of resorption was scored using an Olympus IX-70 equipped with a video camera. The frequency was determined as the number of osteoclasts that had formed a visible pit in percentage of the total number of osteoclasts (∼1000 osteoclasts per condition). The area of the resorption pits was determined using an Olympus BX-60 microscope, a ×20 objective, and an Olympus C-2000 Zoom digital camera. Osteoclasts with more than three nuclei, that had formed a visible resorption area, were selected. The resorbed area and the osteoclast area were measured using Imagepro (Media Cypernetics, Silver Spring, MD, USA). The results are shown as resorption area relative to the osteoclast area, to avoid any influence from the size of the osteoclasts (no significant differences in size between the ADOII and the control osteoclasts were observed at these time points).Resorption AssaysBone resorption was measured as formation of resorption pits on cortical bovine bone slices. Mature osteoclasts (day 10) were cultured for the indicated time periods and at the end of the culture period, adherent cells were removed using a cotton swab. The bone slices were washed in distilled water, and the pits visualized by staining with Mayer's hematoxylin (Bie & Berntsen A/S, Rødovre, Denmark), followed by washing in water. The resorbed area was measured using CAST-Grid (Olympus). The results are shown as resorbed area relative to the total area of bone. The release of the c-terminal type I collagen fragments (CTX) from mineralized bone slices was determined using the CrossLaps for Culture kit (Nordic Bioscience Diagnostics), which was used according to the manufacturer's instructions. In all of the resorption assays, the dye AlamarBlue (Trek Diagnostics Systems Inc., West Sussex, England) was used as a control for equal cell numbers, and we observed no significant differences in cell numbers during the 10-day resorption experiments.StatisticsStatistical analyses were performed using an unpaired Student's t-test, assuming normal distribution with equal variance. Statistical significance is shown by the number of asterisks, P < 0.05 = *, P < 0,01 = **, and P < 0001 = ***.ResultsOsteoclastic Fusion Is Normal in ADOII OsteoclastsBecause ADOII patients have increased TRAP activity in serum and a higher number of osteoclast profiles,12Bollerslev J Marks Jr, SC Pockwinse S Kassem M Brixen K Steiniche T Mosekilde L Ultrastructural investigations of bone resorptive cells in two types of autosomal dominant osteopetrosis.Bone. 1993; 14: 865-869Abstract Full Text PDF PubMed Scopus (70) Google Scholar we tested whether the fusion of osteoclasts cultured from CD14+-isolated peripheral blood monocytes was changed. We measured the formation of multinucleated osteoclasts as seen in Figure 1a. We did not detect differences in the formation of osteoclasts between the ADOII cells and controls. However, the TRAP activity levels measured in the conditioned media were slightly higher in the controls than the ADOII cells (Figure 1b). We observed no morphological differences between ADOII and control osteoclasts, neither with respect to formation of actin rings nor with respect to the localization of the nuclei (data not shown).Expression of TRAP and CKBB Is Normal in Differentiating ADOII OsteoclastsTRAP and CKBB activity levels have previously been found to be elevated in ADOII patients.12Bollerslev J Marks Jr, SC Pockwinse S Kassem M Brixen K Steiniche T Mosekilde L Ultrastructural investigations of bone resorptive cells in two types of autosomal dominant osteopetrosis.Bone. 1993; 14: 865-869Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 14Gram J Antonsen S Horder M Bollerslev J Elevated serum levels of creatine kinase BB in autosomal dominant osteopetrosis type II.Calcif Tissue Int. 1991; 48: 438-439Crossref PubMed Scopus (19) Google Scholar, 15Waguespack SG Hui SL White KE Buckwalter KA Econs MJ Measurement of tartrate-resistant acid phosphatase and the brain isoenzyme of creatine kinase accurately diagnoses type II autosomal dominant osteopetrosis but does not identify gene carriers.J Clin Endocrinol Metab. 2002; 87: 2212-2217Crossref PubMed Scopus (29) Google Scholar The expression of CKBB was examined in CD14+ cells stimulated either with a combination of RANKL and M-CSF, or with M-CSF alone. The expression of CKBB was induced by osteoclastic differentiation as indicated before.23Sakai D Tong HS Minkin C Osteoclast molecular phenotyping by random cDNA sequencing.Bone. 1995; 17: 111-119Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 24Bollerslev J Ueland T Landaas S Marks Jr, SC Serum creatine kinase isoenzyme BB in mammalian osteopetrosis.Clin Orthop. 2000; Aug: 241-247Crossref Scopus (12) Google Scholar Subsequently, we tested the expression of TRAP and CKBB in differentiating osteoclasts from both ADOII patients and controls. As seen in Figure 2, the expression of both TRAP and CKBB increases with osteoclast differentiation, as does the expression of the osteoclastic marker cathepsin K. We observed some person-to-person variance (data not shown). However, we did not observe significant differences in the expression levels of these markers between the ADOII and control cells, indicating that the differentiation of the ADOII osteoclasts is normal in vitro. Furthermore, these data show that CKBB can be used as an osteoclast marker.Figure 2Expression of TRAP and CKBB appears normal in ADOII osteoclasts. Peripheral blood was obtained from ADOII patients and age-matched control individuals, and the monocytes were isolated by Ficoll-Paque gradient centrifugation and CD14 magnetic bead isolation. The monocytes were cultured for 3 days in the presence of 25 ng/ml of M-CSF and then lifted and reseeded at a cell density of 100,000 cells/cm2 and cultured for 5, 7, or 9 days with M-CSF and 25 ng/ml of RANKL. This was followed by lysis of the cells in RIPA+++ buffer at the indicated time points. Thirty μg of total cell lysate was subjected to sodium dodecyl sulfate gel-electrophoresis and immunoblotting with antibodies against creatine kinase BB, TRAP, cathepsin K, and the p38 MAPK as described in the Materials and Methods section. The immunoblots are representative of five different individuals in each group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Expression and Localization of ClC-7 Are Unchanged in ADOII OsteoclastsADOII patients have defective resorption because of mutations in the ClC-7N gene. However, whether the mutation affects the expression, the localization, or the transport activity of ClC-7, has not previously been investigated. Hence, we studied the effect of the G215R mutation with respect to the expression and localization of ClC-7 in ADOII osteoclasts. We found that the level of ClC-7 expression is unchanged in ADOII osteoclasts (Figure 3a). Thus, indicating that the mutation does not affect protein expression. The localization of ClC-7 was studied in osteoclasts seeded on calcium phosphate-covered coverslips, where osteoclasts have been shown to actively resorb the calcium phosphate material.25Masuda R Sakiyama H Nonaka T Kwan A Nakagawa K Moriya H Imajoh-Ohmi S Honjo M Yoshida K Establishment and characterization of tartrate-resistant acid phosphatase and alkaline phosphatase double positive cell lines.Cell Tissue Res. 2001; 304: 351-359Crossref PubMed Scopus (7) Google Scholar We found that ClC-7 localizes in a gradient toward the resorption zone in both ADOII and normal osteoclasts (Figure 3b, 1 and 2) as expected for normal osteoclasts.6Kornak U Kasper D Bosl MR Kaiser E Schweizer M Schulz A Friedrich W Delling G Jentsch TJ Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man.Cell. 2001; 104: 205-215Abstract Full Text Full Text PDF PubMed Scopus (800) Google Scholar, 7Karsdal MA Schaller S Henriksen K Sveigaard S Heegaard A Christophersen P Stahlhut M Hélix M Engsig MT Foged N Delaissé J The chloride channel inhibitor NS3736 prevents bone resorption in ovariectomized rats, while preserving bone formation.J Bone Miner Res. 2003; 18: M233AGoogle Scholar
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