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• W2177469725 abstract "The relevance of IL-33 and its receptor ST2 for bone remodeling is not well-defined. Our aim was to assess the role and underlying mechanisms of IL-33/ST2 in mechanically induced bone remodeling. BALB/c (wild type) and ST2 deficient (St2−/−) mice were subjected to mechanical loading in alveolar bone. Microtomography, histology, and real-time quantitative PCR were performed to analyze bone parameters, apoptosis and bone cell counts, and expression of bone remodeling markers, respectively. MC3T3-E1 osteoblastic cells and bone marrow cells were used to verify if mechanical force triggered IL-33 and ST2 expression as well as the effects of IL-33 on osteoclast differentiation and activity. Mechanical loading increased the expression of IL-33 and ST2 in alveolar bone in vivo and in osteoblastic cells in vitro. St2−/− mice had increased mechanical loading-induced bone resorption, number of osteoclasts, and expression of proresorptive markers. In contrast, St2−/− mice exhibited reduced numbers of osteoblasts and apoptotic cells in periodontium and diminished expression of osteoblast signaling molecules. In vitro, IL-33 treatment inhibited osteoclast differentiation and activity even in the presence of receptor activator of NF-κB ligand. IL-33 also increased the expression of pro-apoptotic molecules, including Bcl-2-associated X protein (BAX), cell-surface Fas receptor (FAS), FASL, FAS-associated death domain, tumor necrosis factor–related apoptosis-inducing ligand, and BH3 interacting-domain death (BID). Overall, these findings suggest that IL-33/ST2 have anti-osteoclastogenic effects and reduce osteoclast formation and activity by inducing their apoptosis. The relevance of IL-33 and its receptor ST2 for bone remodeling is not well-defined. Our aim was to assess the role and underlying mechanisms of IL-33/ST2 in mechanically induced bone remodeling. BALB/c (wild type) and ST2 deficient (St2−/−) mice were subjected to mechanical loading in alveolar bone. Microtomography, histology, and real-time quantitative PCR were performed to analyze bone parameters, apoptosis and bone cell counts, and expression of bone remodeling markers, respectively. MC3T3-E1 osteoblastic cells and bone marrow cells were used to verify if mechanical force triggered IL-33 and ST2 expression as well as the effects of IL-33 on osteoclast differentiation and activity. Mechanical loading increased the expression of IL-33 and ST2 in alveolar bone in vivo and in osteoblastic cells in vitro. St2−/− mice had increased mechanical loading-induced bone resorption, number of osteoclasts, and expression of proresorptive markers. In contrast, St2−/− mice exhibited reduced numbers of osteoblasts and apoptotic cells in periodontium and diminished expression of osteoblast signaling molecules. In vitro, IL-33 treatment inhibited osteoclast differentiation and activity even in the presence of receptor activator of NF-κB ligand. IL-33 also increased the expression of pro-apoptotic molecules, including Bcl-2-associated X protein (BAX), cell-surface Fas receptor (FAS), FASL, FAS-associated death domain, tumor necrosis factor–related apoptosis-inducing ligand, and BH3 interacting-domain death (BID). Overall, these findings suggest that IL-33/ST2 have anti-osteoclastogenic effects and reduce osteoclast formation and activity by inducing their apoptosis. Bone remodeling is a strictly coordinated physiological process of bone resorption and apposition. This process, coordinated by traditional bone cells, is responsible for maintenance of homeostasis and restoration of any bone damage.1Raggatt L.J. Partridge N.C. Cellular and molecular mechanisms of bone remodeling.J Biol Chem. 2010; 285: 25103-25108Crossref PubMed Scopus (810) Google Scholar The function of bone cells is influenced by several factors, including growth factors,2Kaku M. Kohno S. Kawata T. Fujita I. Tokimasa C. Tsutsui K. Tanne K. Effects of vascular endothelial growth factor on osteoclast induction during tooth movement in mice.J Dent Res. 2001; 80: 1880-1883Crossref PubMed Scopus (64) Google Scholar hormones,3Davidovitch Z. Tooth movement.Crit Rev Oral Biol Med. 1991; 2: 411-450Crossref PubMed Scopus (171) Google Scholar diet,4Matkovic V. Calcium metabolism and calcium requirements during skeletal modeling and consolidation of bone mass.Am J Clin Nutr. 1991; 54: 245S-260SPubMed Google Scholar and cytokines.5Garlet G.P. Cardoso C.R.B. Campanelli A.P. Ferreira B.R. Avila-Campos M.J. Cunha F.Q. Silva J.S. The dual role of p55 tumour necrosis factor-α receptor in Actinobacillus actinomycetemcomitans-induced experimental periodontitis: host protection and tissue destruction.Clin Exp Immunol. 2006; 147: 128-138Google Scholar The cytokine IL-33 has been recently implicated in physiological bone remodeling.6Miller A.M. Role of IL-33 in inflammation and disease.J Inflamm (Lond). 2011; 8: 22Crossref PubMed Scopus (336) Google Scholar IL-33 belongs to the IL-1 family6Miller A.M. Role of IL-33 in inflammation and disease.J Inflamm (Lond). 2011; 8: 22Crossref PubMed Scopus (336) Google Scholar and is constitutively expressed in different organs and tissues (eg, connective, lung, endothelium, and synovial tissues).7Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. Zurawski G. Moshrefi M. Qin J. Li X. Gorman D.M. Bazan J.F. Kastelein R.A. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines.Immunity. 2005; 23: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2825) Google Scholar, 8Chackerian A.A. Oldham E.R. Murphy E.E. Schmitiz J. Pflanz S. Kastelein R.A. IL-1 receptor accessory protein and ST2 comprise the IL-33 receptor complex.J Immunol. 2007; 179: 2551-2555Crossref PubMed Scopus (435) Google Scholar, 9Ohno T. Oboki K. Kajiwara N. Morii E. Aozasa K. Flavell R.A. Okumura K. Saito H. Nakae S. Caspase-1, caspase-8, and calpain are dispensable for IL-33 release by macrophages.J Immunol. 2009; 183: 7890-7897Crossref PubMed Scopus (131) Google Scholar, 10Carriere V. Roussel L. Ortega N. Lacorre D.A. Americh L. Aguilar L. Bouche G. Girard J.P. IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nuclear factor in vivo.Proc Natl Acad Sci U S A. 2007; 104: 282-287Crossref PubMed Scopus (781) Google Scholar IL-33 is also released in response to tissue damage as an alarmin.6Miller A.M. Role of IL-33 in inflammation and disease.J Inflamm (Lond). 2011; 8: 22Crossref PubMed Scopus (336) Google Scholar IL-33 functions are associated with type 2 helper T-cell immune response,7Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. Zurawski G. Moshrefi M. Qin J. Li X. Gorman D.M. Bazan J.F. Kastelein R.A. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines.Immunity. 2005; 23: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2825) Google Scholar, 8Chackerian A.A. Oldham E.R. Murphy E.E. Schmitiz J. Pflanz S. Kastelein R.A. IL-1 receptor accessory protein and ST2 comprise the IL-33 receptor complex.J Immunol. 2007; 179: 2551-2555Crossref PubMed Scopus (435) Google Scholar, 9Ohno T. Oboki K. Kajiwara N. Morii E. Aozasa K. Flavell R.A. Okumura K. Saito H. Nakae S. Caspase-1, caspase-8, and calpain are dispensable for IL-33 release by macrophages.J Immunol. 2009; 183: 7890-7897Crossref PubMed Scopus (131) Google Scholar induction of cell division,10Carriere V. Roussel L. Ortega N. Lacorre D.A. Americh L. Aguilar L. Bouche G. Girard J.P. IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nuclear factor in vivo.Proc Natl Acad Sci U S A. 2007; 104: 282-287Crossref PubMed Scopus (781) Google Scholar apoptosis,11Na H.J. Hudson S.A. Bochner B.S. IL-33 enhances Siglec-8 mediated apoptosis on human eosinophilis.Cytokine. 2012; 57: 168-174Crossref Scopus (60) Google Scholar and regulation of bone resorption.12Saidi S. Bouri F. Lencel P. Duplomb L. Baud'huin M. Delplace S. Leterme D. Miellot F. Heymann D. Hardouin P. Palmer G. Magne D. IL-33 is expressed in human osteoblasts, but has no direct effect on bone remodeling.Cytokine. 2011; 53: 347-354Crossref PubMed Scopus (48) Google Scholar, 13Schett G. Effects of inflammatory and anti-inflammatory cytokines on the bone.Eur J Clin Invest. 2011; 41: 1361-1366Crossref PubMed Scopus (175) Google Scholar, 14Schulze J. Bickert T. Beil F.T. Zaiss M.M. Albers J. Wintges K. Streichert T. Klaetschke K. Keller J. Hissnauer T. Spiro A.S. Gessner A. Schett G. Amling M. McKenzie A.N.J. Horst A.K. Schinke T. Interleukin-33 is expressed in differentiated osteoblasts and blocks osteoclast formation from bone marrow precursor cells.J Bone Miner Res. 2011; 26: 704-717Crossref PubMed Scopus (109) Google Scholar, 15Zaiss M.M. Kurowska-Stolarska M. Bohm C. Gary R. Scholtysek C. Stolarski B. Reilly J. Kerr S. Millar N.L. Kamradt T. McInnes I.B. Fallon P.G. David J. Liew F.Y. Schett G. Interleukin (IL)-33 shifts the balance from osteoclast to alternatively- activated macrophage differentiation and protects from TNFa-mediated bone loss.J Immunol. 2011; 186: 6097-6105Crossref PubMed Scopus (86) Google Scholar IL-33 and its receptor ST216Tominaga S. A putative protein of a growth specific cDNA from BALB/c-3T3 cells is highly similar to the extracellular portion of mouse interleukin 1 receptor.FEBS Lett. 1989; 258: 301-304Abstract Full Text PDF PubMed Scopus (322) Google Scholar are expressed by osteoclasts,9Ohno T. Oboki K. Kajiwara N. Morii E. Aozasa K. Flavell R.A. Okumura K. Saito H. Nakae S. Caspase-1, caspase-8, and calpain are dispensable for IL-33 release by macrophages.J Immunol. 2009; 183: 7890-7897Crossref PubMed Scopus (131) Google Scholar, 12Saidi S. Bouri F. Lencel P. Duplomb L. Baud'huin M. Delplace S. Leterme D. Miellot F. Heymann D. Hardouin P. Palmer G. Magne D. IL-33 is expressed in human osteoblasts, but has no direct effect on bone remodeling.Cytokine. 2011; 53: 347-354Crossref PubMed Scopus (48) Google Scholar, 13Schett G. Effects of inflammatory and anti-inflammatory cytokines on the bone.Eur J Clin Invest. 2011; 41: 1361-1366Crossref PubMed Scopus (175) Google Scholar, 14Schulze J. Bickert T. Beil F.T. Zaiss M.M. Albers J. Wintges K. Streichert T. Klaetschke K. Keller J. Hissnauer T. Spiro A.S. Gessner A. Schett G. Amling M. McKenzie A.N.J. Horst A.K. Schinke T. Interleukin-33 is expressed in differentiated osteoblasts and blocks osteoclast formation from bone marrow precursor cells.J Bone Miner Res. 2011; 26: 704-717Crossref PubMed Scopus (109) Google Scholar, 15Zaiss M.M. Kurowska-Stolarska M. Bohm C. Gary R. Scholtysek C. Stolarski B. Reilly J. Kerr S. Millar N.L. Kamradt T. McInnes I.B. Fallon P.G. David J. Liew F.Y. Schett G. Interleukin (IL)-33 shifts the balance from osteoclast to alternatively- activated macrophage differentiation and protects from TNFa-mediated bone loss.J Immunol. 2011; 186: 6097-6105Crossref PubMed Scopus (86) Google Scholar, 17Mun S.H. Ko N.Y. Kim H.S. Kim J.W. Kim do K. Kim A.R. Lee S.H. Kim Y.G. Lee C.K. Lee S.H. Kim B.Y. Beaven M.A. Kim Y.M. Choi W.S. Interleukin-33 stimulates formation of functional osteoclasts from human CD14(+) monocytes.Cell Mol Life Sci. 2010; 67: 3883-3892Crossref PubMed Scopus (70) Google Scholar, 18Keller J. Catala-Lehnen P. Wintges K. Schulze J. Bickert T. Ito W. Horst A.K. Amling M. Schinke T. Transgenic over-expression of interleukin-33 in osteoblasts results in decreased osteoclastogenesis.Biochem Biophys Res Commun. 2012; 417: 217-222Crossref PubMed Scopus (42) Google Scholar osteoblasts,19Saidi S. Magne D. Interleukin-33: a novel player in osteonecrosis of the femoral head?.Joint Bone Spine. 2011; 78: 550-554Crossref PubMed Scopus (18) Google Scholar, 20Werenskiold A.K. Rossler U. Lowel M. Schmidt J. Heermeier K. Spanner M.T. Strauss P.G. Bone matrix deposition of T1, a homologue of interleukin 1 receptors.Cell Growth Differ. 1995; 6: 171-177PubMed Google Scholar and osteocytes.19Saidi S. Magne D. Interleukin-33: a novel player in osteonecrosis of the femoral head?.Joint Bone Spine. 2011; 78: 550-554Crossref PubMed Scopus (18) Google Scholar The role of IL-33/ST2 in the context of bone physiology is controversial. It may suppress bone resorption13Schett G. Effects of inflammatory and anti-inflammatory cytokines on the bone.Eur J Clin Invest. 2011; 41: 1361-1366Crossref PubMed Scopus (175) Google Scholar, 14Schulze J. Bickert T. Beil F.T. Zaiss M.M. Albers J. Wintges K. Streichert T. Klaetschke K. Keller J. Hissnauer T. Spiro A.S. Gessner A. Schett G. Amling M. McKenzie A.N.J. Horst A.K. Schinke T. Interleukin-33 is expressed in differentiated osteoblasts and blocks osteoclast formation from bone marrow precursor cells.J Bone Miner Res. 2011; 26: 704-717Crossref PubMed Scopus (109) Google Scholar, 15Zaiss M.M. Kurowska-Stolarska M. Bohm C. Gary R. Scholtysek C. Stolarski B. Reilly J. Kerr S. Millar N.L. Kamradt T. McInnes I.B. Fallon P.G. David J. Liew F.Y. Schett G. Interleukin (IL)-33 shifts the balance from osteoclast to alternatively- activated macrophage differentiation and protects from TNFa-mediated bone loss.J Immunol. 2011; 186: 6097-6105Crossref PubMed Scopus (86) Google Scholar, 21Saleh H. Eeles D. Hodge J.M. Nicholson G.C. Gu R. Pompolo S. Gillespie M.T. Quinn J.M.W. Interleukin-33, a target of parathyroid hormone and oncostatin m, increases osteoblastic matrix mineral deposition and inhibits osteoclast formation in vitro.Endocrinology. 2011; 152: 1911-1922Crossref PubMed Scopus (64) Google Scholar but also stimulate osteoclast formation, independent of the receptor activator of NF-κB (RANK) and the RANK ligand (RANKL) system.17Mun S.H. Ko N.Y. Kim H.S. Kim J.W. Kim do K. Kim A.R. Lee S.H. Kim Y.G. Lee C.K. Lee S.H. Kim B.Y. Beaven M.A. Kim Y.M. Choi W.S. Interleukin-33 stimulates formation of functional osteoclasts from human CD14(+) monocytes.Cell Mol Life Sci. 2010; 67: 3883-3892Crossref PubMed Scopus (70) Google Scholar A few studies have shown that increase of IL-33 levels in gingival crevicular fluid22Buduneli N. Özçaka Ö. Nalbantsoy A. Interleukin-33 levels in gingival crevicular fluids, saliva or plasma do not differentiate chronic periodontitis.J Periodontol. 2012; 83: 362-368Crossref PubMed Scopus (30) Google Scholar and periodontal tissues23Köseoğlu S. Hatipoğlu M. Sağlam M. Enhoş S. Esen H.H. Interleukin-33 could play an important role in the pathogenesis of periodontitis.J Periodontal Res. 2015; 50: 525-534Crossref PubMed Scopus (21) Google Scholar during inflammatory conditions is associated with alveolar bone loss. Herein, we used a model of bone remodeling induced by mechanical loading to investigate the role of IL-33/ST2 axis in the alveolar bone. We hypothesized that mechanically stressed cells in periodontium release IL-33, which functions as a stop signal for osteoclasts. Fifty 10-week-old wild-type (WT; BALB/c) and 40 ST2-deficient mice (St2−/−) from Universidade de São Paulo (São Paulo, Brazil) and Universidade Federal de Minas Gerais (Minas Gerais, Brazil) were included in this study. All animals were treated under Institutional Ethics Committee regulations for animal experiments (protocol 130/2012). For every set of experiments, five mice were used for each time point. Each animal weight was recorded throughout the experimental period, in which no significant weight loss was observed. Mice were maintained under standard conditions with a 12-hour light/dark cycle, controlled temperature (24°C ± 2°C), and free access to commercial chow and drinking water. The experimental protocol was on the basis of a previous study.24Taddei S.R.A. Moura A.P. Andrade Jr., I. Garlet G.P. Garlet T.P. Teixeira M.M. da Silva T.A. Experimental model of tooth movement in mice: a standardized protocol for studying bone remodeling under compression and tensile strains.J Biomech. 2012; 45: 2729-2735Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar Briefly, a coil was bonded between the maxillary right first molar and incisors. The force magnitude was set at 0.35 N, applied in a mesial direction. The coil was not reactivated during the experimental period. The left side of the maxilla was used as control. Orthodontic tooth movement (OTM) was obtained through the difference between the distance of the cemento-enamel junction of first and second molar of the experimental side (right hemimaxilla) in relation to the control side (left hemimaxilla) of the same animal. The measurements were obtained by microcomputed tomography (MicroCT) image and then maxillae were also submitted to histological analysis. Three measurements were conducted for each evaluation, and the variability was <5%. Maxillae were scanned with Skyscan 1176 (Bruker-MicroCT, Kontich, Belgium). A 12.45-μm camera pixel size with camera XY ratio of 0.9870, voltage of 50 kV, and current of 500 μA was used. A filter of 0.5 mm was used, and scanning trajectory was round. Results were analyzed using Dataviewer 32-bit version and CTAn 32-bit version software (Bruker-MicroCT). Maxillae were analyzed in fixed coronal and sagittal zones. Bone phenotype was evaluated in maxillae and proximal femurs on all planes. The parameters evaluated were trabecular thickness, trabecular number, trabecular separation, percentage bone volume, bone surface density, bone surface, bone mineral density, tissue mineral density, and cross-sectional thickness. We have standardized a method for including and cutting samples.24Taddei S.R.A. Moura A.P. Andrade Jr., I. Garlet G.P. Garlet T.P. Teixeira M.M. da Silva T.A. Experimental model of tooth movement in mice: a standardized protocol for studying bone remodeling under compression and tensile strains.J Biomech. 2012; 45: 2729-2735Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar The anterior maxilla fragment containing the incisors was removed with a scalpel, and the scalpel was positioned in the palatal suture separating the maxilla in right and left halves. Right and left maxilla halves, including first, second, and third molars, were dissected, fixed in 10% buffered formalin, decalcified in 14% EDTA (pH 7.4) for 21 days, and embedded in paraffin. The entire blocks containing the samples were cut into sagittal sections. After roughing the microtome approximately 120 times of 10 μm thickness, sections (4 μm thick) were obtained. The sections selected for hematoxylin and eosin, histochemistry, and immunohistochemistry presented the first and second molars, their mesial and distal-buccal root, the third molar, and adjacent structures, including the periodontal ligament and alveolar bone. At least five serial vertical sections containing the above mentioned structures were evaluated for each animal for each analysis. Immunohistochemistry was performed using maxillae sections to evaluate ST2 expression. Sections were deparaffinized, dehydrated, and rinsed in distilled water and incubated with 0.3% hydrogen peroxide twice for 15 minutes. For antigen retrieval, the slides were immersed in citric acid buffer (pH 6.0) at 96°C for 30 minutes. Sections were incubated with T1/ST2 (IL-33R) monoclonal antibody (Bioscience, San Jose, CA) at 1:100 dilution overnight. Then, the secondary antibody (kit LSAB; Dako, Glostrup, Denmark) was used according to the manufacturer's instructions. The immunolabeling was visualized after incubation with 3,3-diaminobenzidine solution (Dako). Negative controls were obtained by omission of the primary antibody, and were substituted by 0.1% phosphate-buffered saline–bovine serum albumin. The distal-buccal root of the first molar, on its coronal two-thirds of the mesial periodontal site, was used for counting osteoclasts, which were stained for tartrate-resistant acid phosphatase (TRAP; Sigma-Aldrich, St. Louis, MO). Osteoclasts were identified as TRAP-positive, multinucleated cells seated on the bone surface. The distal bone of the first molar distal-buccal root, on its mesiocoronal two-thirds, was used for osteoblast counting, stained with Masson's trichrome. Terminal deoxynucleotidyl-transferase–mediated dUTP-FITC nick-end labeling (Calbiochem, Darmstadt, Germany) was used for identification of apoptotic cells on the mesial bone of the first molar distal-buccal root, on its mesiocoronal two-thirds. By using a stereomicroscope, periodontal ligament and surrounding alveolar bone samples were extracted from the upper first molar. The samples were obtained from WT mice after 0, 12, and 72 hours of mechanical loading. Tissues were weighed and diluted in phosphate-buffered saline (0.4 mmol/L NaCl and 10 mmol/L NaPO4) containing protease inhibitors (0.1 mmol/L phenylmethylsulfonyl fluoride, 0.1 mmol/L benzethonium chloride, 10 mmol/L EDTA, and 0.01 mg/mL aprotinin A) and 0.05% Tween 20 at 1 mg/mL. The mixture was centrifuged (10,000 × g) for 10 minutes. The supernatant was collected and stored at −80°C for further analysis. The concentration (pg/mL) of IL-33 was evaluated by enzyme-linked immunosorbent assay, according to the manufacturer's protocol (R&D Systems, Minneapolis, MN). The results were expressed as picograms of cytokine per 100 mg tissue. As described above, periodontal ligament and surrounding alveolar bone samples were obtained from upper first molars. Whole alveolar samples, periodontal tissues, and alveolar bone extracted from the distal area of the distal-buccal root of the first molar were also collected in a separate set of experiments and considered samples preferentially subjected to tension forces, for measuring the expression of osteoblast markers. Similarly, the medial region of the mesial root of the first molar was collected separately, corresponding to a region of pressure strain, for measuring the expression of osteoclast markers. Samples were harvested after 0, 12, and 72 hours of mechanical loading and total RNA extracted (RNeasy FFPE kit; Qiagen Inc., Valencia, CA), according to the manufacturer’s instructions. The integrity of RNA samples was checked by analyzing 1 mg of total RNA on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The cDNA was synthesized using 3 μg of RNA through a reverse transcription reaction (Superscript III; Invitrogen Corp., Carlsbad, CA). A real-time PCR array was performed in a Viia7 instrument (Life Technologies, Rockville, MD) using TaqMan chemistry (Invitrogen Corp.) associated with inventoried optimized primer/probe sets (Invitrogen Corp.), with basic reaction conditions of (40 cycles) 95°C (10 minutes), 94°C (1 minute), 56°C (1 minute), and 72°C (2 minutes). The mean Ct values from duplicate measurements were used to calculate expression of the target gene, with normalization to an internal control (β-actin) using the 2−ΔΔCt method. Samples from osteoblast culture were also submitted to RNA extraction using Trizol (Life Technologies). The cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen Corp.), and real-time PCRs were performed in an Applied Biosystems 7500 FAST device, and detected by SYBR Green (Applied Biosystems, Foster City, CA). Briefly, reactions were performed in a 10 μL final volume containing 5 μL of SYBR Green Master mix, 1 μL of each primer (5 μmol/L), 2 μL of deionized water, and 1 μL of cDNA. The reactions were initialized with 60°C for 20 seconds, followed by a step of 95°C for 10 minutes, 40 cycles at 95°C for 10 seconds, and 60°C for 1 minute. At the end of reactions, a melting curve was performed. 18S was used as a housekeeping reference gene. Results were analyzed using the 2−ΔΔCt method, and IL-33 and ST2 were expressed as fold increase. Samples from osteoclast cultures were analyzed to quantify mRNA expression of apoptotic markers using PCR Array (SA Biosciences/Qiagen Inc., Frederick, MD) and an apoptosis focused panel (PAMM-012Z), according to the manufacturer's guidelines. The results were analyzed by RT2 profiler PCR Array Data Analysis online software version 3.5 (SA Biosciences/Qiagen Inc.; http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php, last accessed August 4, 2015), which used 2−ΔΔCt for normalizing the initial geometric average of three housekeeping genes (GAPDH, ACTB, and HPRT1) subsequently normalized by control group and expressed as fold change. The well-characterized cell line with osteoblastic phenotype MC3T3-E125Kwon R.Y. Jacobs C.R. Time-dependent deformations in bone cells exposed to fluid flow in vitro: investigating the role of cellular deformation in fluid flow-induced signaling.J Biomech. 2007; 40: 3162-3168Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar was cultured in α-minimal essential medium (Invitrogen Corp.) media containing 10% fetal bovine serum (ATCC, Manassas, VA) and 1% penicillin-streptomycin (Invitrogen Corp.) at 37°C in a 5% CO2 humidified incubator. The cells were subcultured on quartz slides coated with fibronectin 72 hours before experimentation. Approximately 1.5 × 108 beads/mL suspension of 1-μm-diameter sulfate-coated, 1-μm-diameter collagen I–coated, or 2-μm-diameter collagen I–coated fluorescent polystyrene beads (Molecular Probes, Eugene, OR) was added to each slide. The cells were incubated for approximately 1 hour to allow attachment and rinsed with phosphate-buffered saline to remove unbound beads. The beads were incubated in serum-free conditions to minimize the potential for proteins to adsorb onto the surface of beads before binding to the cell. The cells were loaded into a parallel-plate flow chamber and exposed to 10 seconds of sinusoidal oscillatory fluid flow, resulting in 1.0-Pa peak shear stress at frequencies of 0.5, 1.0, or 2.0 Hz, or 10 seconds of steady fluid flow resulting in 1.0-Pa shear stress. Real-time PCR was then performed to determinate IL-33 and ST2 mRNA expression using osteoblastic cells exposed to fluid flow (experimental group) and under static conditions (control group). Bone marrow cells were isolated from femur and tibia of BALB/c mice and cultured in Dulbecco's modified Eagle’s medium (Gibco, Carlsbad, CA), supplemented with 10% heat-inactivated fetal bovine serum, 40 U/mL penicillin, and 40 mg/mL gentamicin. Cells were grown in a humidified atmosphere containing 5% CO2 at 37°C with 100 ng/mL murine recombinant macrophage colony-stimulating factor (PeproTech Inc., Rocky Hill, NJ) for 6 days. For osteoclastic differentiation, cells were suspended in Dulbecco's modified Eagle’s medium containing 10% fetal bovine serum and then seeded at 1 × 105 cells per well in 24-well culture plates over 13-mm glass coverslips. Cells were then stimulated with 50 ng/mL murine recombinant RANKL (PeproTech Inc.) and treated with 10, 20, and 40 ng/mL of recombinant IL-33 (BioLegend, San Diego, CA) for 4 days. After 7 days of stimulus, cells were stained by TRAP using commercial kit (Sigma-Aldrich) following the manufacturer's instructions. TRAP-positive and TRAP-negative cells were counted in 10 nonconsecutive fields, and results were expressed as percentage of TRAP-positive cells per field. For the pit resorption assay, cells were plated on Corning Osteo Assay Surface (Corning Life Sciences, Corning, NY), and pit formation was evaluated after 10 days. Data were expressed as number of pits per field. Samples stimulated with RANKL or with RANKL and IL-33 were also harvested after 5 days for mRNA extraction, as described above. Results were expressed as means ± SEM. Because data sets presented a normal distribution (Kolmogorov-Smirnov), a one-way analysis of variance was used to analyze differences among groups, followed by a Newman-Keuls multiple comparison post hoc test to evaluate and compare the differences. A Kolmogorov-Smirnov test and an unpaired Student's t-test were used to evaluate and compare the differences between two groups. The data obtained from all evaluations were processed with GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA). The level of significance for all statistical tests was predetermined at 5%. To assess whether lack of ST2 interferes with bone phenotype under steady-state conditions, MicroCT was used to analyze bone parameters. Comparison of maxillary bones revealed significant reduction of bone volume percentage in St2−/− in relation to WT (Table 1). In contrast, trabecular number (μm−1), trabecular separation (μm), and bone surface (μm2) showed no significant difference (Table 1). Some parameters were modified in the proximal femurs of St2–/– mice when compared with WT mice, including trabecular number (μm−1), bone volume (%), and bone surface (μm2) (Table 1). St2−/− mice also demonstrated greater trabecular separation (μm) when compared with WT animals (Table 1). These differences are consistent with reduced bone mineral density in St2−/− mice under steady-state conditions, suggesting a role for ST2 in physiological bone homeostasis.Table 1Parameters Related to Bone Mineral Density Obtained by MicroCT of Femurs and Maxillary Bones of WT and ST2 Deficient MiceBone analyzedParametersWT, means ± SEMSt2−/−, means ± SEMP valueFemurTb.N (μm−1)1.90 × 10−2 ± 2.00 × 10−31.5 × 10−2 ± 1.00 × 10−30.0363∗P < 0.05.Tb.Sp (μm)42.92 ± 0.4947.98 ± 3.310.0100∗P < 0.05.BV/TV (%)21.96 ± 1.3616.64 ± 0.650.0036∗P < 0.05.BS (μm2)59.61 × 104 ± 5.09 × 10445.09 × 104 ± 2.46 × 1040.0113∗P < 0.05.MaxillaTb.N (μm−1)3.54 × 10−3 ± 2.75 × 10−43.27 × 10−3 ± 1.00 × 10−40.6990Tb.Sp (μm)139.80 ± 3.82120.60 ± 14.610.1200BV/TV (%)78.47 ± 0.6974.03 ± 0.370.0332∗P < 0.05.BS (μm2)15.99 × 105 ± 37.84 × 10415.57 × 105 ± 17.32 × 1040.6788BS, bone surface; BV/TV, bone volume; MicroCT, microcomputed tomography; Tb.N, trabecular number; Tb.Sp, trabecular separation; WT, wild type.∗ P < 0.05. Open table in a new tab BS, bone surface; BV/TV, bone volume; MicroCT, microcomputed tomography; Tb.N, trabecular number; Tb.Sp, trabecular separation; WT, wild type. Next, we anal" @default.
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