The Inducible Nitric Oxide Synthase Pathway Promotes Osteoclastogenesis under Hypoxic Culture Conditions
2021; Elsevier BV; Volume: 191; Issue: 12 Linguagem: Inglês
10.1016/j.ajpath.2021.08.014
ISSN1525-2191
AutoresTakao Kondo, Yuto Otsuka, Hiromasa Aoki, Yoh Goto, Yohei Kawaguchi, Yuko Waguri‐Nagaya, Ken Miyazawa, Shigemi Goto, Mineyoshi Aoyama,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoBone homeostasis depends on the balance between bone resorption by osteoclasts (OCs) and bone formation by osteoblasts. Bone resorption can become excessive under various pathologic conditions, including rheumatoid arthritis. Previous studies have shown that OC formation is promoted under hypoxia. However, the precise mechanisms behind OC formation under hypoxia have not been elucidated. The present study investigated the role of inducible nitric oxide synthase (iNOS) in OC differentiation under hypoxia. Primary bone marrow cells obtained from mice were stimulated with receptor activator of NF-κB ligand and macrophage colony-stimulating factor to induce OC differentiation. The number of OCs increased in culture under hypoxia (oxygen concentration, 5%) compared with that under normoxia (oxygen concentration, 20%). iNOS gene and protein expression increased in culture under hypoxia. Addition of an iNOS inhibitor under hypoxic conditions suppressed osteoclastogenesis. Addition of a nitric oxide donor to the normoxic culture promoted osteoclastogenesis. Furthermore, insulin-like growth factor 2 expression was significantly altered in both iNOS inhibition experiments and nitric oxide donor experiments. These data might provide clues to therapies for excessive osteoclastogenesis under several hypoxic pathologic conditions, including rheumatoid arthritis. Bone homeostasis depends on the balance between bone resorption by osteoclasts (OCs) and bone formation by osteoblasts. Bone resorption can become excessive under various pathologic conditions, including rheumatoid arthritis. Previous studies have shown that OC formation is promoted under hypoxia. However, the precise mechanisms behind OC formation under hypoxia have not been elucidated. The present study investigated the role of inducible nitric oxide synthase (iNOS) in OC differentiation under hypoxia. Primary bone marrow cells obtained from mice were stimulated with receptor activator of NF-κB ligand and macrophage colony-stimulating factor to induce OC differentiation. The number of OCs increased in culture under hypoxia (oxygen concentration, 5%) compared with that under normoxia (oxygen concentration, 20%). iNOS gene and protein expression increased in culture under hypoxia. Addition of an iNOS inhibitor under hypoxic conditions suppressed osteoclastogenesis. Addition of a nitric oxide donor to the normoxic culture promoted osteoclastogenesis. Furthermore, insulin-like growth factor 2 expression was significantly altered in both iNOS inhibition experiments and nitric oxide donor experiments. These data might provide clues to therapies for excessive osteoclastogenesis under several hypoxic pathologic conditions, including rheumatoid arthritis. Bone homeostasis depends on the balance between bone resorption by osteoclasts (OCs) and bone formation by osteoblasts. Bone resorption can become excessive under various pathologic conditions, including osteoporosis, but is essential for normal bone development, growth, and remodeling.1Sims N.A. Martin T.J. Coupling the activities of bone formation and resorption: a multitude of signals within the basic multicellular unit.Bonekey Rep. 2014; 3: 481Crossref PubMed Google Scholar, 2Teitelbaum S.L. Ross F.P. Genetic regulation of osteoclast development and function.Nat Rev Genet. 2003; 4: 638-649Crossref PubMed Scopus (1289) Google Scholar, 3Horne W.C. Sanjay A. Bruzzaniti A. Baron R. The role (s) of Src kinase and Cbl proteins in the regulation of osteoclast differentiation and function.Immunol Rev. 2005; 208: 106-125Crossref PubMed Scopus (131) Google Scholar, 4Karsenty G. Kronenberg H.M. Settembre C. 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Prediction of avascular necrosis of the femoral head by measuring intramedullary oxygen tension after femoral neck fracture.J Orthop Trauma. 2007; 21: 456-461Crossref PubMed Scopus (40) Google Scholar primary bone tumors,12Dunst J. Ahrens S. Paulussen M. Burdach S. Jürgens H. Prognostic impact of tumor perfusion in MR-imaging studies in Ewing tumors.Strahlenther Onkol. 2001; 177: 153-159Crossref PubMed Scopus (30) Google Scholar cancer metastases to bone,13Hiraga T. Kizaka-Kondoh S. Hirota K. Hiraoka M. Yoneda T. Hypoxia and hypoxia-inducible factor-1 expression enhance osteolytic bone metastases of breast cancer.Cancer Res. 2007; 67: 4157-4163Crossref PubMed Scopus (202) Google Scholar periodontitis,13Hiraga T. Kizaka-Kondoh S. Hirota K. Hiraoka M. Yoneda T. Hypoxia and hypoxia-inducible factor-1 expression enhance osteolytic bone metastases of breast cancer.Cancer Res. 2007; 67: 4157-4163Crossref PubMed Scopus (202) Google Scholar and orthodontic tooth movement.13Hiraga T. Kizaka-Kondoh S. Hirota K. Hiraoka M. Yoneda T. Hypoxia and hypoxia-inducible factor-1 expression enhance osteolytic bone metastases of breast cancer.Cancer Res. 2007; 67: 4157-4163Crossref PubMed Scopus (202) Google Scholar Mean partial pressure of oxygen in bone marrow aspirates from normal volunteer donors has been reported to be 6.5%.14Harrison J.S. Rameshwar P. Chang V. Bandari P. Oxygen saturation in the bone marrow of healthy volunteers.Blood. 2002; 99: 394Crossref PubMed Scopus (37) Google Scholar In environments such as inflamed tissues, infected tissues, tumors, and wounds, partial pressure of oxygen is often considerably lower.15Lewis J.S. Lee J.A. Underwood J.C. Harris A.L. Lewis C.E. Macrophage responses to hypoxia: relevance to disease mechanisms.J Leukoc Biol. 1999; 66: 889-900Crossref PubMed Scopus (311) Google Scholar Hypoxia stimulates both the formation and the activation of OCs from feline,16Muzylak M. Price J.S. Horton M.A. Hypoxia induces giant osteoclast formation and extensive bone resorption in the cat.Calcif Tissue Int. 2006; 79: 301-309Crossref PubMed Scopus (48) Google Scholar murine,13Hiraga T. Kizaka-Kondoh S. Hirota K. Hiraoka M. Yoneda T. Hypoxia and hypoxia-inducible factor-1 expression enhance osteolytic bone metastases of breast cancer.Cancer Res. 2007; 67: 4157-4163Crossref PubMed Scopus (202) Google Scholar,17Arnett T.R. Gibbons D.C. Utting J.C. Orriss I.R. Hoebertz A. Rosendaal M. Meghji S. Hypoxia is a major stimulator of osteoclast formation and bone resorption.J Cell Physiol. 2003; 196: 2-8Crossref PubMed Scopus (222) Google Scholar,18Fukuoka H. Aoyama M. Miyazawa K. Asai K. Goto S. Hypoxic stress enhances osteoclast differentiation via increasing IGF2 production by non-osteoclastic cells.Biochem Biophys Res Commun. 2005; 328: 885-894Crossref PubMed Scopus (36) Google Scholar and human sources,19Arnett T.R. Massey H.M. Utting J.C. Orriss I.R. Flanagan A.M. Hypoxia is a major stimulator of osteoclast formation from human peripheral blood.Calcif Tissue Int. 2003; 196: 2-8Google Scholar,20Knowles H.J. Athanasou N.A. Acute hypoxia and osteoclast activity: a balance between enhanced resorption and increased apoptosis.J Pathol. 2009; 218: 256-264Crossref PubMed Scopus (82) Google Scholar including OCs from murine and human bone marrow cells (BMCs).18Fukuoka H. Aoyama M. Miyazawa K. Asai K. Goto S. Hypoxic stress enhances osteoclast differentiation via increasing IGF2 production by non-osteoclastic cells.Biochem Biophys Res Commun. 2005; 328: 885-894Crossref PubMed Scopus (36) Google Scholar,21Nomura T. Aoyama M. Waguri-Nagaya Y. Goto Y. Suzuki M. Miyazawa K. Asai K. Goto S. Tumor necrosis factor stimulates osteoclastogenesis from human bone marrow cells under hypoxic conditions.Exp Cell Res. 2014; 321: 167-177Crossref PubMed Scopus (10) Google Scholar The microenvironmental niche is important for osteoclastogenesis under hypoxic conditions.21Nomura T. Aoyama M. Waguri-Nagaya Y. Goto Y. Suzuki M. Miyazawa K. Asai K. Goto S. Tumor necrosis factor stimulates osteoclastogenesis from human bone marrow cells under hypoxic conditions.Exp Cell Res. 2014; 321: 167-177Crossref PubMed Scopus (10) Google Scholar,22Nakao K. Aoyama M. Fukuoka H. Fujita M. Miyazawa K. Asai K. Goto S. IGF2 modulates the microenvironment for osteoclastogenesis.Biochem Biophys Res Commun. 2009; 378: 462-466Crossref PubMed Scopus (25) Google ScholarPrevious studies have shown that OC formation is promoted under hypoxic environments.18Fukuoka H. Aoyama M. Miyazawa K. Asai K. Goto S. Hypoxic stress enhances osteoclast differentiation via increasing IGF2 production by non-osteoclastic cells.Biochem Biophys Res Commun. 2005; 328: 885-894Crossref PubMed Scopus (36) Google Scholar, 21Nomura T. Aoyama M. Waguri-Nagaya Y. Goto Y. Suzuki M. Miyazawa K. Asai K. Goto S. Tumor necrosis factor stimulates osteoclastogenesis from human bone marrow cells under hypoxic conditions.Exp Cell Res. 2014; 321: 167-177Crossref PubMed Scopus (10) Google Scholar However, the precise mechanism of OC formation under hypoxia has not been elucidated. Inhibitors of inducible nitric oxide synthase (iNOS) are reportedly effective in rat models of periodontitis.23Herrera B.S. Martins-Porto R. Maia-Dantas A. Campi P. Spolidorio L.C. Costa S.K.P. Dyke T.E.V. Gyurko R. Muscara M.N. iNOS-derived nitric oxide stimulates osteoclast activity and alveolar bone loss in ligature-induced periodontitis in rats.J Periodontol. 2011; 82: 1608-1615Crossref PubMed Scopus (51) Google Scholar Hypoxia-inducible factor (HIF) has also been reported to increase the expression of iNOS in cardiomyocytes under hypoxic conditions.24Jung F. Palmer L.A. Zhou N. Johns R.A. Hypoxic regulation of inducible nitric oxide synthase via hypoxia inducible factor-1 in cardiac myocytes.Circ Res. 2000; 86: 319-325Crossref PubMed Scopus (264) Google Scholar It was therefore hypothesized that hypoxic conditions might enhance OC formation by up-regulating the expression of iNOS and activating the nitric oxide synthase pathway. In this study, the mechanism underlying OC formation by iNOS activation was investigated.Materials and MethodsMice and ReagentsSix-week–old male C57BL6 mice were obtained from Shizuoka Laboratories Animal Center (Shizuoka, Japan). All animal studies were conducted after the Animal Care and Use Committee of Nagoya City University Graduate School of Medical Sciences approved the protocols. The animals were housed under a 12 hours/12 hours light/dark cycle with lights on between 6 am and 6 pm, with standard diet and water provided ad libitum. Mouse M-CSF was obtained from Pepro Tech (Cranbury, NJ). Recombinant human soluble RANKL was obtained from Oriental Yeast (Tokyo, Japan). L-NG-monomethyl arginine acetate (L-NMMA) was purchased from Dojindo Molecular Technologies (Kumamoto, Japan). Diethylenetriamine (DETA) NONOate was purchased from Cayman Chemical (Ann Arbor, MI).OC FormationMouse BMCs were obtained from the tibia and femur of C57BL6 mice. The tibia and femur were removed aseptically, bones were cut across the epiphyses, and marrow was flushed out with α-modified Eagle minimum essential medium containing 10% fetal bovine serum (Gibco, Grand Island, NY) using a 22-gauge needle. BMCs were collected in a tube and passed through a 70-μm pore filter. BMCs were cultured in α-modified Eagle minimum essential medium containing 10% fetal bovine serum, 20 ng/mL M-CSF, and 100 ng/mL RANKL in a humidified atmosphere of 5% CO2. Each reagent was added to the culture medium every 3 days so that M-CSF was equivalent to 20 ng/mL and RANKL was equivalent to 100 ng/mL. Cells were cultured for 9 days. Hypoxic exposure was performed throughout the culture period using a 9000EX CO2 incubator (Wakenbtech Co, Kyoto, Japan). After culturing for the indicated periods, cells were fixed and stained with TRAP solution and incubated at 37°C for 45 minutes. TRAP-stained cells were traced using Illustrator (Adobe Inc., San Jose, CA), and the area was quantified using ImageJ software version 1.8.0 (NIH, Bethesda, MD; http://imagej.nih.gov/ij).Quantitative RT-PCRCells were treated with pronase; cells showing high adhesion were separated as OCs, and floating cells were separated as non-OCs. Total RNA from cultured cells was extracted with RNA iso plus (TAKARA, Shiga, Japan). RNA was reverse transcribed, then quantitative PCR was performed on selected genes using Go Taq qPCR Master Mix and a Thermal Cycler Dice Real Time System TP800 (TAKARA). Amplification was performed as 50 cycles at 95°C for 15 seconds and 60°C for 1 minute. The relative quantification of target genes was normalized by endogenous β-actin mRNA abundance after confirming that cDNAs from different genes were amplified with the same efficiency. The primer pairs used for amplification were as follows: β-actin, 5′-CTAAGGCCAACCGTGAAAAG-3′ (forward) and 5′-GTACGACCAGAGGCATACAG-3′ (reverse); NFATc1, 5′-GGTAACTCTGTCTTTCTAACCTTAAGCTC-3′ (forward) and 5′-GTGATGACCCCAGCATGCACCAGTCACAG-3′ (reverse); DC-STAMP, 5′-TGTATCGGCTCATCTCCTCCAT-3′ (forward) and 5′-GACTCCTTGGGTTCCTTGCTT-3′ (reverse); RANK, 5′-TTCGTCCACAGACAAATGCAAAC-3′ (forward) and 5′-GCTGCAGACCACATCTGATTCC-3′ (reverse); IL-1β, 5′-TCCAGGATGAGGACATGAGCAC-3′ (forward) and 5′-GAACGTCACACACCAGCAGGTTA-3′ (reverse); IL-6, 5′-CAACGATGATGCACTTGCAGA-3′ (forward) and 5′-CTCCAGGTAGCTATGGTACTCCAGA-3′ (reverse); IGF-2, 5′-AGTTTGTCTGTTCGGACCGC-3′ (forward) and 5′-GGGGTATCTGGGGAAGTCGT-3′ (reverse); iNOS, 5′-ACCCTAAGAGTCACCAAAATGGC-3′ (forward) and 5′-TTGATCCTCACATACTGTGGACG-3′ (reverse).Western Blot AnalysisCells were homogenized in SDS sample buffer (62.5 mmol/L Tris-HCl, 10% glycerol, 2% SDS, 0.02% bromophenol blue, and 5% β-mercaptoethanol). Equal amounts of protein were separated by 7.5% or 10% SDS-PAGE, then electrotransferred onto Immobilon-P membranes (Millipore, Billerica, MA). Blocking was performed overnight at 4°C. The blocking buffer was 5% skim milk. Cells were exposed to primary antibody overnight at 4°C. Primary antibodies were against iNOS (MB9502; R&D, Minneapolis, MN), HIF-1α (MAB1536; R&D), NF-κB (number 3031; Cell Signaling Technology, Danvers, MA), phosphorylated NF-κB (number 8242; Cell Signaling Technology), and actin (A5060; Sigma-Aldrich, St. Louis, MO). The secondary antibody was reacted at room temperature for 1 hour. ECL Prime Western Blotting Detection Reagent (General Electric Company, Boston, MA) was used as the detection reagent for NF-κB, phosphorylated NF-κB, and actin. SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA) was used as the detection reagent for iNOS and HIF-1α. The signal intensity was quantified using ImageJ software.Statistical AnalysisAll statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria).25Kanda Y. Investigation of the freely available easy-to-use software "EZR" for medical statistics.Bone Marrow Transplant. 2013; 48: 452-458Crossref PubMed Scopus (8120) Google Scholar Comparisons of continuous data between three or more groups were performed using two-tailed analysis of variance with post hoc Bonferroni testing. Comparisons between data from the two groups were performed using two-tailed nonpaired t-tests. Data are reported as the mean ± SEM. Differences with values of P < 0.05 were considered significant.ResultsHypoxic Stimulation Promotes OC FormationTo investigate the effects of hypoxic stimulation on OC formation, mouse BMCs were cultured at O2 concentrations of 20%, 5%, and 1% for 9 days in the presence of RANKL (100 μg/mL) and M-CSF (20 μg/mL). The number of TRAP-positive multinucleated OCs increased in hypoxia (5% O2) compared with that in normoxia (20% O2) (Figure 1, A and B ). However, TRAP-positive multinucleated OCs were not found at 1% O2 concentration (Figure 1, A and B). Fusion of mononuclear OCs into multinucleated cells is an important process in OC functional differentiation.26Lee S.H. Rho J. Jeong D. Sul J.Y. Kim T. Kim N. Kang J.S. Miyamoto T. Suda T. Lee S.K. Pignolo R.J. Koczon-Jaremko B. Lorenzo J. Choi Y. v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation.Nat Med. 2006; 12: 1403-1409Crossref PubMed Scopus (437) Google Scholar,27Yagi M. Miyamoto T. Sawatani Y. Iwamoto K. Hosogane N. Fujita N. Morita K. Ninomiya K. Suzuki T. Miyamoto K. Oike Y. Takeya M. Toyama Y. Suda T. DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells.J Exp Med. 2005; 202: 345-351Crossref PubMed Scopus (670) Google Scholar TRAP-positive multinucleated OC area was measured as the area of OCs. The distribution of representative measured areas is shown in Figure 1C. Huge OCs were observed with culturing in hypoxia but not in normoxia (Figure 1C). Analysis of the average OC area indicated an increase in OC size under hypoxia as opposed to that under normoxia (Figure 1D).Hypoxic Stimulation Increases iNOS ExpressionTo understand the molecular mechanisms responsible for the enhancement of OC differentiation with hypoxic exposure, quantitative RT-PCR was performed on total RNA from separated OC and non-OC populations to determine hypoxia-induced genes. For OC-related factors, hypoxic (5% O2) stimulation increased nuclear factor of activated T cells (NFATc1) gene expression in OCs. In non-OCs, an increase in dendritic cell–specific transmembrane protein (DC-STAMP) gene expression was observed (Figure 2A). No change in inflammatory cytokines was seen in OCs. However, expression of IL-1β and IL-6 was increased in non-OCs (Figure 2A). Previous studies showed that hypoxic (5% O2) stimulation increases gene expression of insulin-like growth factor 2 (IGF2).22Nakao K. Aoyama M. Fukuoka H. Fujita M. Miyazawa K. Asai K. Goto S. IGF2 modulates the microenvironment for osteoclastogenesis.Biochem Biophys Res Commun. 2009; 378: 462-466Crossref PubMed Scopus (25) Google Scholar An increase in IGF2 in both OCs and non-OCs in the present study corroborated this finding (Figure 2A). In addition, iNOS was significantly elevated in OCs and non-OCs under hypoxic (5% O2) conditions (Figure 2A).Figure 2Effect of hypoxic stimulation on gene and protein expression. A: Quantitative RT-PCR analysis of the expression of selected genes involved in osteoclast differentiation between normoxia (20% O2) and hypoxia (5% O2). Cultured cells were separated into osteoclasts and others (nonosteoclasts) using the pronase procedure. B: Effects of normoxia and hypoxia on protein expression of NF-κB, hypoxia-inducible factor (HIF)-1α, and inducible nitric oxide synthase (iNOS) are shown by Western blot analysis. Semiquantitative analyses of NF-κB, HIF-1α, and iNOS proteins are shown. Data represent the means ± SEM (A and B). n = 3 independent experiments (A and B). ∗P < 0.05 versus normoxia (t-test). p-NF-κB, phosphorylated NF-κB.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Next, Western blot analysis was performed to confirm changes in signal activity and protein expression. Phosphorylation of NF-κB was examined because NF-κB signals are considered to play a key role in iNOS expression.28Arias-Salvatierra D. Silbergeld E.K. Acosta-Saavedra L.C. Calderon-Aranda E.S. Role of nitric oxide produced by iNOS through NF-κB pathway in migration of cerebellar granule neurons induced by lipopolysaccharide.Cell Signal. 2011; 23: 425-435Crossref PubMed Scopus (76) Google Scholar However, no change was observed with hypoxic (5% O2) stimulation (Figure 2B). Protein expression of HIF-1α (Figure 2B)29Semenza G.L. Hypoxia-inducible factor 1 (HIF-1) pathway.Sci STKE. 2007; 2007: cm8Crossref PubMed Scopus (708) Google Scholar and expression of iNOS was increased under hypoxic (5% O2) stimulation (Figure 2B).Inhibiting iNOS Suppresses OCsTo investigate the relationship between iNOS and OC differentiation, the iNOS inhibitor L-NMMA was added to cultures. L-NMMA competitively inhibits iNOS by competing with l-arginine. L-NMMA was added at the indicated concentrations to cultures, and TRAP-positive multinucleated OCs were counted. L-NMMA inhibited OC formation at high doses in normoxia and hypoxia, but at low concentrations, it inhibited OC formation only in hypoxia (Figure 3, A and B ). OC area was reduced by exposure to low concentrations in both normoxia) and hypoxia (Figure 3, A and C). Gene expression was examined in OCs and non-OCs when L-NMMA (10 μmol/L) was added under hypoxic conditions (Figure 3D). IL-6 and IGF2 gene expression was decreased in both OCs and non-OCs. NFATc1, RANK, and IL-1β gene expression was decreased in non-OCs (Figure 3D). Gene expression of iNOS was not significantly changed in either OCs or non-OCs (Figure 3D). Expression of phosphorylated NF-κB and HIF-1α proteins was unchanged. However, iNOS inhibitor stimulation decreased iNOS protein expression under hypoxic (5% O2) conditions (Figure 3E).Figure 3Effect of inducible nitric oxide synthase (iNOS) inhibitors on osteoclast formation under hypoxic conditions. L-NG-monomethyl arginine acetate (L-NMMA) was added at the indicated concentrations during culturing under normoxia (20% O2) or hypoxia (5% O2). A: Cells were cultured in normoxia and hypoxia. Cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP) activity. B: TRAP-positive multinucleated osteoclast cells [TRAP(+) MNCs] were counted. C: The area of TRAP(+) MNCs was measured, and mean areas are shown under normoxia and hypoxia. D: Quantitative RT-PCR analysis of the expression of selected genes involved in osteoclast differentiation between control (0 μmol/L) and L-NMMA treatment (10 μmol/L). Cultured cells were separated into osteoclasts and nonosteoclasts using the pronase procedure. E: Effects of L-NMMA on protein expression of NF-κB, hypoxia-inducible factor (HIF)-1α, and iNOS are shown by Western blot analysis. Semiquantitative analyses of NF-κB, HIF-1α, and iNOS proteins are shown. Data represent the means ± SEM (B–E). n = 3 independent experiments (B–E). ∗P < 0.05 versus control (0 μmol/L) (t-test). Scale bars = 1 mm (A). DETA, diethylenetriamine; p-NF-κB, phosphorylated NF-κB.View Large Image Figure ViewerDownload Hi-res image Download (PPT)OC Differentiation Is Promoted by Donating NOTo investigate the effect of nitric oxide (NO) produced by iNOS on OC differentiation, the NO donor DETA NONOate was added to the cultures. DETA NONOate naturally decomposes in a pH-dependent manner and has a long half-life, so NO is supplied to the system over a long time. With addition of DETA NONOate under normoxia, the number and the area of OCs increased significantly (Figure 4, A–C ). Addition of DETA NONOate (100 μmol/L) under normoxia increased IGF2 gene expression in OCs. No change in gene expression was observed in non-OCs (Figure 4D). No differences in phosphorylation of NF-κB were seen. In addition, protein expression of HIF-1α and iNOS did not change with the addition of DETA NONOate (Figure 4E).Figure 4Effect of nitric oxide (NO) donor on osteoclast formation. Diethylenetriamine (DETA) NONOate was added at the indicated concentrations during culturing in normoxia (20% O2). A: Cells were cultured with DETA NONOate and then fixed and stained for tartrate-resistant acid phosphatase (TRAP) activity. B: TRAP-positive multinucleated osteoclast cells [TRAP(+) MNCs] were counted. C: The area of TRAP(+) MNCs was measured, and mean areas are shown. D: Quantitative RT-PCR analysis of the expression of selected genes involved in osteoclast differentiation between control (0 μmol/L) and DETA NONOate treatment (100 μmol/L). Cultured cells were separated into osteoclasts and nonosteoclasts using the pronase procedure. E: Effects of DETA NONOate on protein expression of NF-κB, hypoxia-inducible factor (HIF)-1α, and inducible NO synthase (iNOS) are shown by Western blot analysis. Semiquantitative analyses of NF-κB, HIF-1α, and iNOS proteins are shown. Data represent the means ± SEM (B–E). n = 3 independent experiments (B–E). ∗P < 0.05 versus control (0 μmol/L) (t-test). Scale bars = 1 mm (A). p-NF-κB, phosphorylated NF-κB.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionThis study demonstrated that NO synthesis promotes osteoclastogenesis under hypoxic conditions because of iNOS activation. First, culture system was established to study the effects of hypoxia on OCs. Removal of floating cells by medium exchange is helpful in maintaining the purity of bone marrow–derived macrophages.30Maridas D.E. Rendina-Ruedy E. Le P.T. Rosen C.J. Isolation, culture, and differentiation of bone marrow stromal cells and osteoclast progenitors from mice.J Vis Exp. 2018; 131: 56750Google Scholar However, because the microenvironment of non-OCs affects the differentiation of OCs, it is important to keep some non-OCs in the culture.31Goto Y. Aoyama M. Sekiya T. Kakita H. Waguri-Nagaya Y. Miyazawa K. Asai K. Goto S. CXCR4+ CD45- cells are niche forming for osteoclastogenesis via the SDF-1, CXCL7, and CX3CL1 signaling pathways in bone marrow.Stem Cells. 2016; 34: 2733-2743Crossref PubMed Scopus (15) Google Scholar Therefore, a culture system was devised in which OCs and non-OCs coexist by collecting whole bone marrow and culturing the cells in a dish without changing the medium to retain as many non-OCs as possible. As previously reported, the number of OCs in the current culture system was higher in hypoxia than in normoxia.18Fukuoka H. Aoyama M. Miyazawa K. Asai K. Goto S. Hypoxic stress enhances osteoclast differentiation via increasing IGF2 production by non-osteoclastic cells.Biochem Biophys Res Commun. 2005; 328: 885-894Crossref PubMed Scopus (36) Google Scholar Mean OC size was also larger in hypoxia than in normoxia. On the other hand, OCs were not formed at the oxygen concentration of 1% (Figure 1). Whether O2 concentration of <1% promotes osteoclastogenesis is controversial. Most reports showing that 1% O2 or lower promotes OC formation examined intermittent hypoxia using a hypoxia/re-oxygenation schedule.16Muzylak M. Price J.S. Horton M.A. Hypoxia induces giant osteoclast formation and extensive bone resorption in the cat.Calcif Tissue Int. 2006; 79: 301-309Crossref PubMed Scopus (48) Google Scholar,17Arnett T.R. Gibbons D.C. Utting J.C. Orriss I.R. Hoebertz A. Rosendaal M. Meghji S. Hypoxia is a major stimulator of osteoclast formation and bone resorption.J Cell Physiol. 2003; 196: 2-8Crossref PubMed Scopus (222) Google Scholar,32Utting J.C. Flanagan A.M. Brandao-Burch A. Orriss I.R. Arnett T.R. Hypoxia stimulates osteoclast formation from human peripheral blood.Cell Biochem Funct. 2010; 28: 374-380Crossref PubMed Scopus (72) Google Scholar On the other hand, some reports demonstrated inhibition of OC formation when experiments were conducted understeady 1% O2 or lower.18Fukuoka H. Aoyama M. Miyazawa K. Asai K. Goto S. Hypoxic stress enhances osteoclast differentiation via increasing IGF2 production by non-osteoclastic cells.Biochem Biophys Res Commun. 2005; 328: 885-894Crossref PubMed Scopus (36) Google Scholar,33Ma Z. Yu R. Zhao J. Sun L. Jian L. Li C. Liu X. Consta
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