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The PDE4 Inhibitors Roflumilast and Rolipram Rescue ADO2 Osteoclast Resorption Dysfunction

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Abstract

Autosomal Dominant Osteopetrosis type II (ADO2) is a rare bone disease of impaired osteoclastic bone resorption caused by heterozygous missense mutations in the chloride channel 7 (CLCN7). Adenylate cyclase, which catalyzes the formation of cAMP, is critical for lysosomal acidification in osteoclasts. We found reduced cAMP levels in ADO2 osteoclasts compared to wild-type (WT) osteoclasts, leading us to examine whether regulating cAMP would improve ADO2 osteoclast activity. Although forskolin, a known activator of adenylate cyclase and cAMP levels, negatively affected osteoclast number, it led to an overall increase in ADO2 and WT osteoclast resorption activity in vitro. Next, we examined cAMP hydrolysis by the phosphodiesterase 4 (PDE4) proteins in ADO2 versus WT osteoclasts. QPCR analysis revealed higher expression of the three major PDE4 subtypes (4a, 4b, 4d) in ADO2 osteoclasts compared in WT, consistent with reduced cAMP levels in ADO2 osteoclasts. In addition, we found that the PDE4 antagonists, rolipram and roflumilast, stimulated ADO2 and WT osteoclast formation in a dose-dependent manner. Importantly, roflumilast and rolipram displayed a concentration-dependent increase in osteoclast resorption activity which was greater in ADO2 than WT osteoclasts. Moreover, treatment with roflumilast rescued cAMP levels in ADO2 OCLs. The key findings from our studies demonstrate that osteoclasts from ADO2 mice exhibit reduced cAMP levels and PDE4 inhibition rescues cAMP levels and ADO2 osteoclast activity dysfunction in vitro. The mechanism of action of PDE4 inhibitors and their ability to reduce the high bone mass of ADO2 mice in vivo are currently under investigation. Importantly, these studies advance the understanding of the mechanisms underlying the ADO2 osteoclast dysfunction which is critical for the development of therapeutic approaches to treat clinically affected ADO2 patients.

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References

  1. Stauber T, Weinert S, Jentsch TJ (2012) Cell biology and physiology of CLC chloride channels and transporters. Compr Physiol 2:1701–1744

    Article  PubMed  Google Scholar 

  2. Poroca DR, Pelis RM, Chappe VM (2017) ClC channels and transporters: structure, physiological functions, and implications in human chloride channelopathies. Front Pharmacol 8:151

    Article  PubMed  PubMed Central  Google Scholar 

  3. Jentsch TJ, Pusch M (2018) CLC chloride channels and transporters: structure, function, physiology, and disease. Physiol Rev 98:1493–1590

    Article  CAS  PubMed  Google Scholar 

  4. Jentsch TJ, Stein V, Weinreich F, Zdebik AA (2002) Molecular structure and physiological function of chloride channels. Physiol Rev 82:503–568

    Article  CAS  PubMed  Google Scholar 

  5. Jentsch TJ, Poët M, Fuhrmann JC, Zdebik AA (2005) Physiological functions of CLC Cl- channels gleaned from human genetic disease and mouse models. Annu Rev Physiol 67:779–807

    Article  CAS  PubMed  Google Scholar 

  6. Graves AR, Curran PK, Smith CL, Mindell JA (2008) The Cl-/H+ antiporter ClC-7 is the primary chloride permeation pathway in lysosomes. Nature 453:788–792

    Article  CAS  PubMed  Google Scholar 

  7. Guzman RE, Grieschat M, Fahlke C, Alekov AK (2013) ClC-3 is an intracellular chloride/proton exchanger with large voltage-dependent nonlinear capacitance. ACS Chem Neurosci 4:994–1003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Matsuda JJ, Filali MS, Volk KA, Collins MM, Moreland JG, Lamb FS (2008) Overexpression of CLC-3 in HEK293T cells yields novel currents that are pH dependent. Am J Physiol Cell Physiol 294:C251-262

    Article  CAS  PubMed  Google Scholar 

  9. Neagoe I, Stauber T, Fidzinski P, Bergsdorf EY, Jentsch TJ (2010) The late endosomal ClC-6 mediates proton/chloride countertransport in heterologous plasma membrane expression. J Biol Chem 285:21689–21697

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Picollo A, Pusch M (2005) Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5. Nature 436:420–423

    Article  CAS  PubMed  Google Scholar 

  11. Scheel O, Zdebik AA, Lourdel S, Jentsch TJ (2005) Voltage-dependent electrogenic chloride/proton exchange by endosomal CLC proteins. Nature 436:424–427

    Article  CAS  PubMed  Google Scholar 

  12. Lange PF, Wartosch L, Jentsch TJ, Fuhrmann JC (2006) ClC-7 requires Ostm1 as a beta-subunit to support bone resorption and lysosomal function. Nature 440:220–223

    Article  CAS  PubMed  Google Scholar 

  13. Meadows NA, Sharma SM, Faulkner GJ, Ostrowski MC, Hume DA, Cassady AI (2007) The expression of Clcn7 and Ostm1 in osteoclasts is coregulated by microphthalmia transcription factor. J Biol Chem 282:1891–1904

    Article  CAS  PubMed  Google Scholar 

  14. Leisle L, Ludwig CF, Wagner FA, Jentsch TJ, Stauber T (2011) ClC-7 is a slowly voltage-gated 2Cl(-)/1H(+)-exchanger and requires Ostm1 for transport activity. Embo J 30:2140–2152

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Weinert S, Jabs S, Hohensee S, Chan WL, Kornak U, Jentsch TJ (2014) Transport activity and presence of ClC-7/Ostm1 complex account for different cellular functions. EMBO Rep 15:784–791

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Brandt S, Jentsch TJ (1995) ClC-6 and ClC-7 are two novel broadly expressed members of the CLC chloride channel family. FEBS Lett 377:15–20

    Article  CAS  PubMed  Google Scholar 

  17. Kornak U, Kasper D, Bösl MR, Kaiser E, Schweizer M, Schulz A, Friedrich W, Delling G, Jentsch TJ (2001) Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 104:205–215

    Article  CAS  PubMed  Google Scholar 

  18. Schulz P, Werner J, Stauber T, Henriksen K, Fendler K (2010) The G215R mutation in the Cl-/H+-antiporter ClC-7 found in ADO II osteopetrosis does not abolish function but causes a severe trafficking defect. PLoS ONE 5:e12585

    Article  PubMed  PubMed Central  Google Scholar 

  19. Henriksen K, Gram J, Schaller S, Dahl BH, Dziegiel MH, Bollerslev J, Karsdal MA (2004) Characterization of osteoclasts from patients harboring a G215R mutation in ClC-7 causing autosomal dominant osteopetrosis type II. Am J Pathol 164:1537–1545

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Alam I, Gray AK, Chu K, Ichikawa S, Mohammad KS, Capannolo M, Capulli M, Maurizi A, Muraca M, Teti A, Econs MJ, Del Fattore A (2014) Generation of the first autosomal dominant osteopetrosis type II (ADO2) disease models. Bone 59:66–75

    Article  CAS  PubMed  Google Scholar 

  21. Chu K, Snyder R, Econs MJ (2006) Disease status in autosomal dominant osteopetrosis type 2 is determined by osteoclastic properties. J Bone Miner Res 21:1089–1097

    Article  CAS  PubMed  Google Scholar 

  22. Alam I, Gerard-O’Riley RL, Acton D, Hardman SL, Hong JM, Bruzzaniti A, Econs MJ (2021) Chloroquine increases osteoclast activity in vitro but does not improve the osteopetrotic bone phenotype of ADO2 mice. Bone 153:116160

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Delaisse JM, Søe K, Andersen TL, Rojek AM, Marcussen N (2021) The mechanism switching the osteoclast from short to long duration bone resorption. Front Cell Dev Biol 9:644503

    Article  PubMed  PubMed Central  Google Scholar 

  24. von Kleist L, Haucke V (2012) At the crossroads of chemistry and cell biology: inhibiting membrane traffic by small molecules. Traffic 13:495–504

    Article  Google Scholar 

  25. Rahman N, Ramos-Espiritu L, Milner TA, Buck J, Levin LR (2016) Soluble adenylyl cyclase is essential for proper lysosomal acidification. J Gen Physiol 148:325–339

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Tasken K, Aandahl EM (2004) Localized effects of cAMP mediated by distinct routes of protein kinase A. Physiol Rev 84:137–167

    Article  CAS  PubMed  Google Scholar 

  27. Taylor SS, Buechler JA, Yonemoto W (1990) cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. Annu Rev Biochem 59:971–1005

    Article  CAS  PubMed  Google Scholar 

  28. Biel M, Zong X, Ludwig A, Sautter A, Hofmann F (1999) Structure and function of cyclic nucleotide-gated channels. Rev Physiol Biochem Pharmacol 135:151–171

    Article  CAS  PubMed  Google Scholar 

  29. Finn JT, Grunwald ME, Yau KW (1996) Cyclic nucleotide-gated ion channels: an extended family with diverse functions. Annu Rev Physiol 58:395–426

    Article  CAS  PubMed  Google Scholar 

  30. Bos JL (2003) Epac: a new cAMP target and new avenues in cAMP research. Nat Rev Mol Cell Biol 4:733–738

    Article  CAS  PubMed  Google Scholar 

  31. Houslay MD, Adams DR (2003) PDE4 cAMP phosphodiesterases: modular enzymes that orchestrate signalling cross-talk, desensitization and compartmentalization. Biochem J 370:1–18

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Soderling SH, Beavo JA (2000) Regulation of cAMP and cGMP signaling: new phosphodiesterases and new functions. Curr Opin Cell Biol 12:174–179

    Article  CAS  PubMed  Google Scholar 

  33. Inoue D, Watanabe R, Okazaki R (2016) COPD and osteoporosis: links, risks, and treatment challenges. Int J Chron Obstruct Pulmon Dis 11:637–648

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kawamatawong T (2017) Roles of roflumilast, a selective phosphodiesterase 4 inhibitor, in airway diseases. J Thorac Dis 9:1144–1154

    Article  PubMed  PubMed Central  Google Scholar 

  35. Scuvee-Moreau J, Giesbers I, Dresse A (1987) Effect of rolipram, a phosphodiesterase inhibitor and potential antidepressant, on the firing rate of central monoaminergic neurons in the rat. Arch Int Pharmacodyn Ther 288:43–49

    CAS  PubMed  Google Scholar 

  36. Huang S, Eleniste PP, Wayakanon K, Mandela P, Eipper BA, Mains RE, Allen MR, Bruzzaniti A (2014) The Rho-GEF Kalirin regulates bone mass and the function of osteoblasts and osteoclasts. Bone 60:235–245

    Article  CAS  PubMed  Google Scholar 

  37. Kang KS, Hong JM, Horan DJ, Lim KE, Bullock WA, Bruzzaniti A, Hann S, Warman ML, Robling AG (2019) Induction of Lrp5 HBM-causing mutations in Cathepsin-K expressing cells alters bone metabolism. Bone 120:166–175

    Article  CAS  PubMed  Google Scholar 

  38. Chen X, Zhang K, Hock J, Wang C, Yu X (2016) Enhanced but hypofunctional osteoclastogenesis in an autosomal dominant osteopetrosis type II case carrying a c.1856C>T mutation in CLCN7. Bone Res 4:16035

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Alatalo SL, Ivaska KK, Waguespack SG, Econs MJ, Vaananen HK, Halleen JM (2004) Osteoclast-derived serum tartrate-resistant acid phosphatase 5b in Albers-Schonberg disease (type II autosomal dominant osteopetrosis). Clin Chem 50:883–890

    Article  CAS  PubMed  Google Scholar 

  40. Alam I, McQueen AK, Acton D, Reilly AM, Gerard-O’Riley RL, Oakes DK, Kasipathi C, Huffer A, Wright WB, Econs MJ (2017) Phenotypic severity of autosomal dominant osteopetrosis type II (ADO2) mice on different genetic backgrounds recapitulates the features of human disease. Bone 94:34–41

    Article  PubMed  Google Scholar 

  41. Murrills RJ, Dempster DW (1990) The effects of stimulators of intracellular cyclic AMP on rat and chick osteoclasts in vitro: validation of a simplified light microscope assay of bone resorption. Bone 11:333–344

    Article  CAS  PubMed  Google Scholar 

  42. Park YG, Kim YH, Kang SK, Kim CH (2006) cAMP-PKA signaling pathway regulates bone resorption mediated by processing of cathepsin K in cultured mouse osteoclasts. Int Immunopharmacol 6:947–956

    Article  CAS  PubMed  Google Scholar 

  43. Ramaswamy G, Kim H, Zhang D, Lounev V, Wu JY, Choi Y, Kaplan FS, Pignolo RJ, Shore EM (2017) Gsalpha controls cortical bone quality by regulating osteoclast differentiation via cAMP/PKA and beta-catenin pathways. Sci Rep 7:45140

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. McSorley T, Stefan E, Henn V, Wiesner B, Baillie GS, Houslay MD, Rosenthal W, Klussmann E (2006) Spatial organisation of AKAP18 and PDE4 isoforms in renal collecting duct principal cells. Eur J Cell Biol 85:673–678

    Article  CAS  PubMed  Google Scholar 

  45. Stefan E, Wiesner B, Baillie GS, Mollajew R, Henn V, Lorenz D, Furkert J, Santamaria K, Nedvetsky P, Hundsrucker C, Beyermann M, Krause E, Pohl P, Gall I, MacIntyre AN, Bachmann S, Houslay MD, Rosenthal W, Klussmann E (2007) Compartmentalization of cAMP-dependent signaling by phosphodiesterase-4D is involved in the regulation of vasopressin-mediated water reabsorption in renal principal cells. J Am Soc Nephrol 18:199–212

    Article  CAS  PubMed  Google Scholar 

  46. Takami M, Cho ES, Lee SY, Kamijo R, Yim M (2005) Phosphodiesterase inhibitors stimulate osteoclast formation via TRANCE/RANKL expression in osteoblasts: possible involvement of ERK and p38 MAPK pathways. FEBS Lett 579:832–838

    Article  CAS  PubMed  Google Scholar 

  47. Park H, No AL, Lee JM, Chen L, Lee SY, Lee DS, Yim M (2010) PDE4 inhibitor upregulates PTH-induced osteoclast formation via CRE-mediated COX-2 expression in osteoblasts. FEBS Lett 584:173–180

    Article  CAS  PubMed  Google Scholar 

  48. Souness JE, Griffin M, Maslen C, Ebsworth K, Scott LC, Pollock K, Palfreyman MN, Karlsson JA (1996) Evidence that cyclic AMP phosphodiesterase inhibitors suppress TNF alpha generation from human monocytes by interacting with a “low-affinity” phosphodiesterase 4 conformer. Br J Pharmacol 118:649–658

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Tenor H, Hatzelmann A, Church MK, Schudt C, Shute JK (1996) Effects of theophylline and rolipram on leukotriene C4 (LTC4) synthesis and chemotaxis of human eosinophils from normal and atopic subjects. Br J Pharmacol 118:1727–1735

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Hatzelmann A, Morcillo EJ, Lungarella G, Adnot S, Sanjar S, Beume R, Schudt C, Tenor H (2010) The preclinical pharmacology of roflumilast—a selective, oral phosphodiesterase 4 inhibitor in development for chronic obstructive pulmonary disease. Pulm Pharmacol Ther 23:235–256

    Article  CAS  PubMed  Google Scholar 

  51. Rabe KF (2011) Update on roflumilast, a phosphodiesterase 4 inhibitor for the treatment of chronic obstructive pulmonary disease. Br J Pharmacol 163:53–67

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Stauber T, Wartosch L, Vishnolia S, Schulz A, Kornak U (2023) CLCN7, a gene shared by autosomal recessive and autosomal dominant osteopetrosis. Bone 168:116639

    Article  CAS  PubMed  Google Scholar 

  53. Waguespack SG, Koller DL, White KE, Fishburn T, Carn G, Buckwalter KA, Johnson M, Kocisko M, Evans WE, Foroud T, Econs MJ (2003) Chloride channel 7 (ClCN7) gene mutations and autosomal dominant osteopetrosis, type II. J Bone Miner Res 18:1513–1518

    Article  CAS  PubMed  Google Scholar 

  54. Henriksen K, Sørensen MG, Jensen VK, Dziegiel MH, Nosjean O, Karsdal MA (2008) Ion transporters involved in acidification of the resorption lacuna in osteoclasts. Calcif Tissue Int 83:230–242

    Article  CAS  PubMed  Google Scholar 

  55. Henriksen K, Sorensen MG, Nielsen RH, Gram J, Schaller S, Dziegiel MH, Everts V, Bollerslev J, Karsdal MA (2006) Degradation of the organic phase of bone by osteoclasts: a secondary role for lysosomal acidification. J Bone Miner Res 21:58–66

    Article  CAS  PubMed  Google Scholar 

  56. Al-Bari AA, Al Mamun A (2020) Current advances in regulation of bone homeostasis. FASEB Bioadv 2:668–679

    Article  PubMed  PubMed Central  Google Scholar 

  57. Krogstad DJ, Schlesinger PH (1987) The basis of antimalarial action: non-weak base effects of chloroquine on acid vesicle pH. Am J Trop Med Hyg 36:213–220

    Article  CAS  PubMed  Google Scholar 

  58. Collins MP, Forgac M (2020) Regulation and function of V-ATPases in physiology and disease. Biochim Biophys Acta Biomembr 1862:183341

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Nicholson GC, Moseley JM, Yates AJ, Martin TJ (1987) Control of cyclic adenosine 3’,5’-monophosphate production in osteoclasts: calcitonin-induced persistent activation and homologous desensitization of adenylate cyclase. Endocrinology 120:1902–1908

    Article  CAS  PubMed  Google Scholar 

  60. Doorn J, Siddappa R, van Blitterswijk CA, de Boer J (2012) Forskolin enhances in vivo bone formation by human mesenchymal stromal cells. Tissue Eng Part A 18:558–567

    Article  CAS  PubMed  Google Scholar 

  61. Siddappa R, Mulder W, Steeghs I, van de Klundert C, Fernandes H, Liu J, Arends R, van Blitterswijk C, de Boer J (2009) cAMP/PKA signaling inhibits osteogenic differentiation and bone formation in rodent models. Tissue Eng Part A 15:2135–2143

    Article  CAS  PubMed  Google Scholar 

  62. Siddappa R, Doorn J, Liu J, Langerwerf E, Arends R, van Blitterswijk C, de Boer J (2010) Timing, rather than the concentration of cyclic AMP, correlates to osteogenic differentiation of human mesenchymal stem cells. J Tissue Eng Regen Med 4:356–365

    Article  CAS  PubMed  Google Scholar 

  63. Mongillo M, McSorley T, Evellin S, Sood A, Lissandron V, Terrin A, Huston E, Hannawacker A, Lohse MJ, Pozzan T, Houslay MD, Zaccolo M (2004) Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases. Circ Res 95:67–75

    Article  CAS  PubMed  Google Scholar 

  64. Perry SJ, Baillie GS, Kohout TA, McPhee I, Magiera MM, Ang KL, Miller WE, McLean AJ, Conti M, Houslay MD, Lefkowitz RJ (2002) Targeting of cyclic AMP degradation to beta 2-adrenergic receptors by beta-arrestins. Science 298:834–836

    Article  CAS  PubMed  Google Scholar 

  65. Ainatzoglou A, Stamoula E, Dardalas I, Siafis S, Papazisis G (2021) The effects of PDE inhibitors on multiple sclerosis: a review of in vitro and in vivo models. Curr Pharm Des 27:2387–2397

    Article  CAS  PubMed  Google Scholar 

  66. Bundschuh DS, Eltze M, Barsig J, Wollin L, Hatzelmann A, Beume R (2001) In vivo efficacy in airway disease models of roflumilast, a novel orally active PDE4 inhibitor. J Pharmacol Exp Ther 297:280–290

    CAS  PubMed  Google Scholar 

  67. Cho ES, Yu JH, Kim MS, Yim M (2004) Rolipram, a phosphodiesterase 4 inhibitor, stimulates osteoclast formation by inducing TRANCE expression in mouse calvarial cells. Arch Pharmacal Res 27:1258–1262

    Article  CAS  Google Scholar 

  68. Alam I, Hardman SL, Gerard-O'Riley RL, Acton D, Parker RS, Hong JM, Bruzzaniti A, Econs MJ (2024) Effect of Roflumilast, a Selective PDE4 Inhibitor, on Bone Phenotypes in ADO2 Mice. Calcif Tissue Int. https://doi.org/10.1007/s00223-023-01180-2. Epub ahead of print. PMID: 38300304

    Article  PubMed  Google Scholar 

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Acknowledgements

We thank previous student members of our respective laboratories for technical assistance with osteoclast assays. These studies were funded in part by funds from the school of dentistry to AB.

Funding

This work was supported by the US National Institutes of Health grant AR069583.

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Authors and Affiliations

  1. Department of Biomedical Sciences and Comprehensive Care, Indiana University School of Dentistry, 1121 West Michigan Street, DS266, Indianapolis, IN, 46202, USA

    Jung Min Hong & Angela Bruzzaniti

  2. Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, 46202, USA

    Rita L. Gerard-O’Riley, Dena Acton, Imranul Alam & Michael J. Econs

  3. Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, 46202, USA

    Michael J. Econs

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Contributions

Study design: JMH, RLG, DA, IA, MJE, and AB. Study conduct: JMH, RLG, DA, IA, and AB. Data analysis: JMH and AB. Data interpretation: JMH, RLG, DA, IA, MJE, and AB. Drafting manuscript: JMH and AB. Revising manuscript content: All authors approved of final version of manuscript.

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Correspondence to Angela Bruzzaniti.

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Jung Min Hong, Rita L. Gerard-O’Riley, Dena Acton, Imranul Alam, Michael J. Econs, and Angela Bruzzaniti declare that they have no conflict of interest.

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Animal studies were conducted following protocols approved by the Indiana University Institutional Animal Care and Use Committee (IACUC).

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223_2024_1191_MOESM1_ESM.tif

Supplementary Figure 1. Rolipram and roflumilast increase the size of ADO2 OCL. BMMs were cultured with the PDE4 inhibitors, rolipram or roflumilast, for 5-6 days to form mature multinucleated OCLs. The area (size) of TRAP-positive multi-nucleated OCLs were determined (shown as µm2 x 103) determined using ImagePro Software. OCLs were generated from 4 individual mice. OCLs in 4 replicate wells per mouse were scored, with the mean ± SEM shown in the graphs. Please see Figures 4A and 5A for comparison graphs and images of TRAP-positive OCLs in vehicle and rolipram or roflumilast treated groups, respectively. Supplementary file1 (TIF 542 kb)

223_2024_1191_MOESM2_ESM.tif

Supplementary Figure 2. Rolipram and roflumilast stimulate ADO2 OCL resorption activity. Mature multinucleated OCLs from WT and ADO2 BMMs were cultured on cortical bone slices in the presence of A) rolipram or B) roflumilast, two PDE4 antagonists. Conditioned media was assayed for CTX-I and the data normalized for the number of TRAP-positive OCLs. The data from 2-5 independent experiments was consolidated and analyzed together for statistical significance, with the 0 drug WT and ADO2 data representing 5 independent experiments. Please refer to Figures 4-5 for representative data graphs with their respective OCLs images. Graphs represent mean ± SD. Asterisks (*) indicates p < 0.05 for comparison to the vehicle control for each genotype, whereas hash (#) indicates statistical significance for comparison between genotypes at matching drug concentrations (p < 0.05). Supplementary file2 (TIF 535 kb)

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Hong, J.M., Gerard-O’Riley, R.L., Acton, D. et al. The PDE4 Inhibitors Roflumilast and Rolipram Rescue ADO2 Osteoclast Resorption Dysfunction. Calcif Tissue Int 114, 430–443 (2024). https://doi.org/10.1007/s00223-024-01191-7

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