Induction of osteoblast apoptosis stimulates macrophage efferocytosis and paradoxical bone formation

Abstract

Apoptosis is crucial for tissue homeostasis and organ development. In bone, apoptosis is recognized to be a main fate of osteoblasts, yet the relevance of this process remains underexplored. Using our murine model with inducible Caspase 9, the enzyme that initiates intrinsic apoptosis, we triggered apoptosis in a proportion of mature osteocalcin (OCN⁺) osteoblasts and investigated the impact on postnatal bone development. Osteoblast apoptosis stimulated efferocytosis by osteal macrophages. A five-week stimulation of OCN⁺ osteoblast apoptosis in 3-week-old male and female mice significantly enhanced vertebral bone formation while increasing osteoblast precursors. A similar treatment regimen to stimulate osterix⁺ cell apoptosis had no impact on bone volume or density. The vertebral bone accrual following stimulation of OCN⁺ osteoblast apoptosis did not translate in improved mechanical strength due to disruption of the lacunocanalicular network. The observed bone phenotype was not influenced by changes in osteoclasts but was associated with stimulation of macrophage efferocytosis and vasculature formation. Phenotyping of efferocytic macrophages revealed a unique transcriptomic signature and expression of factors including VEGFA. To examine whether macrophages participated in the osteoblast precursor increase following osteoblast apoptosis, macrophage depletion models were employed. Depletion of macrophages via clodronate-liposomes and the CD169-diphtheria toxin receptor mouse model resulted in marked reduction in leptin receptor⁺ and osterix⁺ osteoblast precursors. Collectively, this work demonstrates the significance of osteoblast turnover via apoptosis and efferocytosis in postnatal bone formation. Importantly, it exposes the potential of targeting this mechanism to promote bone anabolism in the clinical setting.

References

[1] Denaxa M, Neves G, Burrone J, Pachnis V. Homeostatic regulation of interneuron apoptosis during cortical development. J. Exp. Neurosci., 2018, 12

[2] Hojo MA et al. Identification of a genomic enhancer that enforces proper apoptosis induction in thymic negative selection. Nat. Commun., 2019, 10

[3] Arandjelovic S, Ravichandran KS. Phagocytosis of apoptotic cells in homeostasis. Nat. Immunol., 2015, 16: 907-917

[4] Gerlach BD et al. Efferocytosis induces macrophage proliferation to help resolve tissue injury. Cell Metab., 2021, 33: 2445-2463.e2448

[5] Meriwether D et al. Macrophage COX2 mediates efferocytosis, resolution reprogramming, and intestinal epithelial repair. Cell. Mol. Gastroenterol. Hepatol., 2022, 13: 1095-1120

[6] Kalajzic Z et al. Use of an alpha-smooth muscle actin GFP reporter to identify an osteoprogenitor population. Bone, 2008, 43: 501-510

[7] Zhou BO, Yue R, Murphy MM, Peyer JG, Morrison SJ. Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow. Cell Stem Cell, 2014, 15: 154-168

[8] Liu Q et al. Recent advances of osterix transcription factor in osteoblast differentiation and bone formation. Front. Cell Dev. Biol., 2020, 8

[9] Amarasekara, D. S., Kim, S. & Rho, J. Regulation of osteoblast differentiation by cytokine networks. Int. J. Mol. Sci. 22, 2851 (2021).

[10] Jilka RL, Weinstein RS, Bellido T, Parfitt AM, Manolagas SC. Osteoblast programmed cell death (apoptosis): modulation by growth factors and cytokines. J. Bone Miner. Res., 1998, 13: 793-802

[11] Li H et al. Glucocorticoid receptor and sequential p53 activation by dexamethasone mediates apoptosis and cell cycle arrest of osteoblastic MC3T3-E1 cells. PLoS One, 2012, 7: e37030

[12] Yang L et al. Paeoniflorin attenuates dexamethasone-induced apoptosis of osteoblast cells and promotes bone formation via regulating AKT/mTOR/autophagy signaling pathway. Evid. Based Complement. Alternat. Med., 2021, 2021

[13] Mollazadeh S, Fazly Bazzaz BS, Kerachian MA. Role of apoptosis in pathogenesis and treatment of bone-related diseases. J. Orthop. Surg. Res., 2015, 10: 15

[14] Beltinger C et al. Herpes simplex virus thymidine kinase/ganciclovir-induced apoptosis involves ligand-independent death receptor aggregation and activation of caspases. Proc. Natl. Acad. Sci. USA, 1999, 96: 8699-8704

[15] Ruedl C, Jung S. DTR-mediated conditional cell ablation—progress and challenges. Eur. J. Immunol., 2018, 48: 1114-1119

[16] Yang J, Liu TJ, Lu Y. Effects of bicistronic lentiviral vector-mediated herpes simplex virus thymidine kinase/ganciclovir system on human lens epithelial cells. Curr. Eye Res., 2007, 32: 33-42

[17] Hu L et al. Diphtheria toxin-induced cell death triggers Wnt-dependent hair cell regeneration in neonatal mice. J. Neurosci., 2016, 36: 9479-9489

[18] Helsby NA, Ferry DM, Patterson AV, Pullen SM, Wilson WR. 2-Amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT. Br. J. Cancer, 2004, 90: 1084-1092

[19] Batoon L, Koh AJ, Kannan R, McCauley LK, Roca H. Caspase-9 driven murine model of selective cell apoptosis and efferocytosis. Cell Death Dis, 2023, 14

[20] Liu F et al. Versatile cell ablation tools and their applications to study loss of cell functions. Cell. Mol. Life Sci., 2019, 76: 4725-4743

[21] Hochweller K, Striegler J, Hämmerling GJ, Garbi N. A novel CD11c.DTR transgenic mouse for depletion of dendritic cells reveals their requirement for homeostatic proliferation of natural killer cells. Eur. J. Immunol., 2008, 38: 2776-2783

[22] Millard SM et al. Fragmentation of tissue-resident macrophages during isolation confounds analysis of single-cell preparations from mouse hematopoietic tissues. Cell Rep., 2021, 37

[23] Yurdagul A Jr et al. Macrophage metabolism of apoptotic cell-derived arginine promotes continual efferocytosis and resolution of injury. Cell Metab., 2020, 31: 518-533.e510

[24] Chistiakov DA, Killingsworth MC, Myasoedova VA, Orekhov AN, Bobryshev YV. CD68/macrosialin: not just a histochemical marker. Lab. Invest., 2017, 97: 4-13

[25] Batoon L et al. CD169⁺ macrophages are critical for osteoblast maintenance and promote intramembranous and endochondral ossification during bone repair. Biomaterials, 2019, 196: 51-66

[26] Drake MT, Khosla S. Hormonal and systemic regulation of sclerostin. Bone, 2017, 96: 8-17

[27] Kuo T-R, Chen C-H. Bone biomarker for the clinical assessment of osteoporosis: recent developments and future perspectives. Biomark. Res., 2017, 5

[28] Chubb SAP. Measurement of C-terminal telopeptide of type I collagen (CTX) in serum. Clin. Biochem., 2012, 45: 928-935

[29] Qin L et al. Amphiregulin is a novel growth factor involved in normal bone development and in the cellular response to parathyroid hormone stimulation. J. Biol. Chem., 2005, 280: 3974-3981

[30] Chandra A, Lan S, Zhu J, Siclari VA, Qin L. Epidermal growth factor receptor (EGFR) signaling promotes proliferation and survival in osteoprogenitors by increasing early growth response 2 (EGR2) expression. J. Biol. Chem., 2013, 288: 20488-20498

[31] Viola, A., Munari, F., Sánchez-Rodríguez, R., Scolaro, T. & Castegna, A. The metabolic signature of macrophage responses. Front. Immunol. 10, 1462 (2019).

[32] Pérez-Gutiérrez, L. & Ferrara, N. Biology and therapeutic targeting of vascular endothelial growth factor A. Nat. Rev. Mol. Cell Biol. 24, 816–834 (2023).

[33] Chang MK et al. Osteal tissue macrophages are intercalated throughout human and mouse bone lining tissues and regulate osteoblast function in vitro and in vivo. J. Immunol., 2008, 181: 1232-1244

[34] Mizoguchi T et al. Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development. Dev. Cell, 2014, 29: 340-349

[35] Rux DR et al. Regionally restricted Hox function in adult bone marrow multipotent mesenchymal stem/stromal cells. Dev. Cell, 2016, 39: 653-666

[36] Madel MB et al. Immune function and diversity of osteoclasts in normal and pathological conditions. Front. Immunol., 2019, 10: 1408

[37] Jilka RL et al. Decreased oxidative stress and greater bone anabolism in the aged, when compared to the young, murine skeleton with parathyroid hormone administration. Aging Cell, 2010, 9: 851-867

[38] Bellido T, Plotkin LI. Novel actions of bisphosphonates in bone: preservation of osteoblast and osteocyte viability. Bone, 2011, 49: 50-55

[39] Naot D, Musson DS, Cornish J. The activity of peptides of the calcitonin family in bone. Physiol. Rev., 2019, 99: 781-805

[40] Fairfield H et al. Targeting bone cells during sexual maturation reveals sexually dimorphic regulation of endochondral ossification. JBMR Plus, 2020, 4

[41] Yu VW et al. Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow. J. Exp. Med., 2015, 212: 759-774

[42] Moriishi T et al. Overexpression of Bcl2 in osteoblasts inhibits osteoblast differentiation and induces osteocyte apoptosis. PLoS One, 2011, 6: e27487

[43] Moriishi T et al. Overexpression of BCLXL in osteoblasts inhibits osteoblast apoptosis and increases bone volume and strength. J. Bone Miner. Res., 2016, 31: 1366-1380

[44] Mohamad SF et al. Osteomacs interact with megakaryocytes and osteoblasts to regulate murine hematopoietic stem cell function. Blood Adv., 2017, 1: 2520-2528

[45] Miron RJ, Bosshardt DD. OsteoMacs: Key players around bone biomaterials. Biomaterials, 2016, 82: 1-19

[46] Batoon L, Millard SM, Raggatt LJ, Pettit AR. Osteomacs and bone regeneration. Curr. Osteoporos. Rep., 2017, 15: 385-395

[47] Batoon L et al. Osteal macrophages support osteoclast-mediated resorption and contribute to bone pathology in a postmenopausal osteoporosis mouse model. J. Bone Miner. Res., 2021, 36: 2214-2228

[48] Pettit AR, Chang MK, Hume DA, Raggatt LJ. Osteal macrophages: a new twist on coupling during bone dynamics. Bone, 2008, 43: 976-982

[49] Xu R et al. Impaired efferocytosis enables apoptotic osteoblasts to escape osteoimmune surveillance during aging. Adv. Sci., 2023, 10

[50] Lin D et al. Efferocytosis and its associated cytokines: a light on non-tumor and tumor diseases? Mol. Ther. Oncolytics, 2020, 17: 394-407

[51] Hume DA. The many alternative faces of macrophage activation. Front. Immunol., 2015, 6: 370

[52] Murray PJ. Macrophage polarization. Annu. Rev. Physiol., 2017, 79: 541-566

[53] Xue J et al. Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity, 2014, 40: 274-288

[54] Sanin DE et al. A common framework of monocyte-derived macrophage activation. Sci. Immunol, 2022, 7

[55] Schultze JL, Freeman T, Hume DA, Latz E. A transcriptional perspective on human macrophage biology. Semin. Immunol., 2015, 27: 44-50

[56] Nahrendorf M, Swirski FK. Abandoning M1/M2 for a network model of macrophage function. Circ. Res., 2016, 119: 414-417

[57] Grosso A et al. VEGF dose controls the coupling of angiogenesis and osteogenesis in engineered bone. NPJ Regen. Med., 2023, 8: 15

[58] Okada E, Nakata H, Yamamoto M, Kasugai S, Kuroda S. Indirect osteoblast differentiation by liposomal clodronate. J. Cell. Mol. Med., 2018, 22: 1127-1137

[59] Fernandes TJ et al. Cord blood-derived macrophage-lineage cells rapidly stimulate osteoblastic maturation in mesenchymal stem cells in a glycoprotein-130 dependent manner. PLoS One, 2013, 8: e73266

[60] Lu LY et al. Pro-inflammatory M1 macrophages promote osteogenesis by mesenchymal stem cells via the COX-2-prostaglandin E2 pathway. J. Orthop. Res., 2017, 35: 2378-2385

[61] Nicolaidou V et al. Monocytes induce STAT3 activation in human mesenchymal stem cells to promote osteoblast formation. PLoS One, 2012, 7: e39871

[62] Pirraco RP, Reis RL, Marques AP. Effect of monocytes/macrophages on the early osteogenic differentiation of hBMSCs. J. Tissue Eng. Regen. Med., 2013, 7: 392-400

[63] Vi L et al. Macrophages promote osteoblastic differentiation in vivo: Implications in fracture repair and bone homeostasis. J. Bone Miner. Res., 2015, 30: 1090-1102

[64] Michalski MN, Koh AJ, Weidner S, Roca H, McCauley LK. Modulation of osteoblastic cell efferocytosis by bone marrow macrophages. J. Cell. Biochem., 2016, 117: 2697-2706

[65] Zhang Z et al. TGF‑β1 promotes the osteoinduction of human osteoblasts via the PI3K/AKT/mTOR/S6K1 signalling pathway. Mol. Med. Rep., 2019, 19: 3505-3518

[66] Wu M, Chen G, Li Y-P. TGF-β and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease. Bone Res., 2016, 4

[67] Mehrotra P, Ravichandran KS. Drugging the efferocytosis process: concepts and opportunities. Nat. Rev. Drug Discov., 2022, 21: 601-620

[68] Myers KV, Amend SR, Pienta KJ. Targeting Tyro3, Axl and MerTK (TAM receptors): implications for macrophages in the tumor microenvironment. Mol. Cancer, 2019, 18

[69] Engelmann J et al. Regulation of bone homeostasis by MERTK and TYRO3. Nat. Commun., 2022, 13

[70] Bala Y et al. Cortical porosity identifies women with osteopenia at increased risk for forearm fractures. J. Bone Miner. Res., 2014, 29: 1356-1362

[71] Zhang J, Link DC. Targeting of mesenchymal stromal cells by Cre-recombinase transgenes commonly used to target osteoblast lineage cells. J. Bone Miner. Res., 2016, 31: 2001-2007

[72] Dallas SL, Xie Y, Shiflett LA, Ueki Y. Mouse Cre models for the study of bone diseases. Curr. Osteoporos. Rep., 2018, 16: 466-477

[73] Roforth MM et al. Effects of age on bone mRNA levels of sclerostin and other genes relevant to bone metabolism in humans. Bone, 2014, 59: 1-6

[74] Chang MK et al. Disruption of Lrp4 function by genetic deletion or pharmacological blockade increases bone mass and serum sclerostin levels. Proc. Natl. Acad. Sci. USA, 2014, 111: E5187-E5195

[75] Delgado-Calle J, Sato AY, Bellido T. Role and mechanism of action of sclerostin in bone. Bone, 2017, 96: 29-37

[76] Durosier C et al. Association of circulating sclerostin with bone mineral mass, microstructure, and turnover biochemical markers in healthy elderly men and women. J. Clin. Endocrinol. Metab., 2013, 98: 3873-3883

[77] Ma YH et al. Circulating sclerostin associated with vertebral bone marrow fat in older men but not women. J. Clin. Endocrinol. Metab., 2014, 99: E2584-E2590

[78] Mödder UI et al. Relation of age, gender, and bone mass to circulating sclerostin levels in women and men. J. Bone Miner. Res., 2011, 26: 373-379

[79] Garnero P, Sornay-Rendu E, Munoz F, Borel O, Chapurlat RD. Association of serum sclerostin with bone mineral density, bone turnover, steroid and parathyroid hormones, and fracture risk in postmenopausal women: the OFELY study. Osteoporos. Int., 2013, 24: 489-494

[80] Gruver-Yates AL, Cidlowski JA. Tissue-specific actions of glucocorticoids on apoptosis: a double-edged sword. Cells, 2013, 2: 202-223

[81] Sun, J. et al. A vertebral skeletal stem cell lineage driving metastasis. Nature 621, 602–609 (2023).

[82] DeFalco J et al. Virus-assisted mapping of neural inputs to a feeding center in the hypothalamus. Science, 2001, 291: 2608-2613

[83] Madisen L et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci., 2010, 13: 133-140

[84] Koh AJ et al. An irradiation-altered bone marrow microenvironment impacts anabolic actions of PTH. Endocrinology, 2011, 152: 4525-4536

[85] Batoon L et al. Treatment with a long-acting chimeric CSF1 molecule enhances fracture healing of healthy and osteoporotic bones. Biomaterials, 2021, 275

[86] Dempster DW et al. Standardized nomenclature, symbols, and units for bone histomorphometry: A 2012 update of the report of the ASBMR Histomorphometry Nomenclature Committee. J. Bone Miner. Res., 2013, 28: 2-17

[87] Smith L, Bigelow EM, Jepsen KJ. Systematic evaluation of skeletal mechanical function. Curr. Protoc. Mouse Biol., 2013, 3: 39-67

[88] Wu CA et al. Responses in vivo to purified poly(3-hydroxybutyrate-co-3-hydroxyvalerate) implanted in a murine tibial defect model. J. Biomed. Mater. Res. A, 2009, 91: 845-854

[89] Dole NS, Yee CS, Schurman CA, Dallas SL, Alliston T. Assessment of osteocytes: techniques for studying morphological and molecular changes associated with perilacunar/canalicular remodeling of the bone matrix. Methods Mol. Biol., 2021, 2230: 303-323

[90] Mendoza-Reinoso V et al. Bone marrow macrophages induce inflammation by efferocytosis of apoptotic prostate cancer cells via HIF-1alpha stabilization. Cells, 2022, 11: 3712

[91] Dobin A et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics, 2013, 29: 15-21

[92] Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc., 2009, 4: 44-57

[93] Sherman BT et al. DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update). Nucleic Acids Res., 2022, 50: W216-w221

Funding

U.S. Department of Health & Human Services | National Institutes of Health (NIH)(AR077539)

U.S. Department of Health & Human Services | National Institutes of Health (NIH)

U.S. Department of Health & Human Services | National Institutes of Health (NIH)

Department of Health | National Health and Medical Research Council (NHMRC)(APP1143802)

Mater Foundation