Identification of an intronic enhancer regulating RANKL expression in osteocytic cells

Abstract

The bony skeleton is continuously renewed throughout adult life by the bone remodeling process, in which old or damaged bone is removed by osteoclasts via largely unknown mechanisms. Osteocytes regulate bone remodeling by producing the osteoclast differentiation factor RANKL (encoded by the TNFSF11 gene). However, the precise mechanisms underlying RANKL expression in osteocytes are still elusive. Here, we explored the epigenomic landscape of osteocytic cells and identified a hitherto-undescribed osteocytic cell-specific intronic enhancer in the TNFSF11 gene locus. Bioinformatics analyses showed that transcription factors involved in cell death and senescence act on this intronic enhancer region. Single-cell transcriptomic data analysis demonstrated that cell death signaling increased RANKL expression in osteocytic cells. Genetic deletion of the intronic enhancer led to a high-bone-mass phenotype with decreased levels of RANKL in osteocytic cells and osteoclastogenesis in the adult stage, while RANKL expression was not affected in osteoblasts or lymphocytes. These data suggest that osteocytes may utilize a specialized regulatory element to facilitate osteoclast formation at the bone surface to be resorbed by linking signals from cellular senescence/death and RANKL expression.

References

[1] Bonewald LF. The amazing osteocyte. J. Bone Min. Res., 2011, 26: 229-238

[2] Nakashima T et al. Evidence for osteocyte regulation of bone homeostasis through RANKL expression. Nat. Med., 2011, 17: 1231-1234

[3] Xiong J et al. Matrix-embedded cells control osteoclast formation. Nat. Med., 2011, 17: 1235-1241

[4] Xiong J, O'Brien CA. Osteocyte RANKL: new insights into the control of bone remodeling. J. Bone Min. Res., 2012, 27: 499-505

[5] Xiong J et al. Osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling bone. PLoS One, 2015, 10: e0138189

[6] Tsukasaki M, Takayanagi H. Osteoimmunology: evolving concepts in bone-immune interactions in health and disease. Nat. Rev. Immunol., 2019, 19: 626-642

[7] Fujiwara Y et al. RANKL (Receptor Activator of NFκB Ligand) produced by osteocytes is required for the increase in B cells and bone loss caused by estrogen deficiency in mice. J. Biol. Chem., 2016, 291: 24838-24850

[8] Xiong J et al. Osteocyte-derived RANKL is a critical mediator of the increased bone resorption caused by dietary calcium deficiency. Bone, 2014, 66: 146-154

[9] Wein MN. Parathyroid hormone signaling in osteocytes. J. Bone Min. Res, 2018, 2: 22-30

[10] O'Brien CA et al. Glucocorticoids act directly on osteoblasts and osteocytes to induce their apoptosis and reduce bone formation and strength. Endocrinology, 2004, 145: 1835-1841

[11] Piemontese M, Xiong J, Fujiwara Y, Thostenson JD, O'Brien CA. Cortical bone loss caused by glucocorticoid excess requires RANKL production by osteocytes and is associated with reduced OPG expression in mice. Am. J. Physiol. Endocrinol. Metab., 2016, 311: E587-E593

[12] Weinstein RS et al. Osteoprotegerin prevents glucocorticoid-induced osteocyte apoptosis in mice. Endocrinology, 2011, 152: 3323-3331

[13] Tatsumi S et al. Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction. Cell Metab., 2007, 5: 464-475

[14] Cheung WY et al. Pannexin-1 and P2X7-Receptor are required for apoptotic osteocytes in fatigued bone to trigger RANKL production in neighboring bystander osteocytes. J. Bone Min. Res., 2016, 31: 890-899

[15] Andreev D et al. Osteocyte necrosis triggers osteoclast-mediated bone loss through macrophage-inducible C-type lectin. J. Clin. Investig., 2020, 130: 4811-4830

[16] Kim HN et al. Osteocyte RANKL is required for cortical bone loss with age and is induced by senescence. JCI Insight, 2020, 5: e138815

[17] Jilka RL et al. Dysapoptosis of osteoblasts and osteocytes increases cancellous bone formation but exaggerates cortical porosity with age. J. Bone Min. Res., 2014, 29: 103-117

[18] Onal M et al. Unique distal enhancers linked to the mouse Tnfsf11 gene direct tissue-specific and inflammation-induced expression of RANKL. Endocrinology, 2016, 157: 482-496

[19] Fu Q, Manolagas SC, O'Brien CA. Parathyroid hormone controls receptor activator of NF-kappaB ligand gene expression via a distant transcriptional enhancer. Mol. Cell Biol., 2006, 26: 6453-6468

[20] O'Brien CA. Control of RANKL gene expression. Bone, 2010, 46: 911-919

[21] Yan M et al. ETS1 governs pathological tissue-remodeling programs in disease-associated fibroblasts. Nat. Immunol., 2022, 23: 1330-1341

[22] ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).

[23] Shen Y et al. A map of the cis-regulatory sequences in the mouse genome. Nature, 2012, 488: 116-120

[24] Holwerda SJ, de Laat W. CTCF: the protein, the binding partners, the binding sites and their chromatin loops. Philos. Trans. R. Soc. Lond. B Biol. Sci., 2013, 368: 20120369

[25] Kim SK. Identification of 613 new loci associated with heel bone mineral density and a polygenic risk score for bone mineral density, osteoporosis and fracture. PLoS One, 2018, 13: e0200785

[26] Morris JA et al. An atlas of genetic influences on osteoporosis in humans and mice. Nat. Genet., 2019, 51: 258-266

[27] Kemp JP et al. Phenotypic dissection of bone mineral density reveals skeletal site specificity and facilitates the identification of novel loci in the genetic regulation of bone mass attainment. PLoS Genet., 2014, 10: e1004423

[28] Kichaev G et al. Leveraging Polygenic Functional Enrichment to Improve GWAS Power. Am. J. Hum. Genet., 2019, 104: 65-75

[29] Wang JS et al. Control of osteocyte dendrite formation by Sp7 and its target gene osteocrin. Nat. Commun., 2021, 12

[30] Kuilman T et al. Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network. Cell, 2008, 133: 1019-1031

[31] Acosta JC et al. Chemokine signaling via the CXCR2 receptor reinforces senescence. Cell, 2008, 133: 1006-1018

[32] Kang C et al. The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4. Science, 2015, 349: aaa5612

[33] Hu Y et al. RANKL from bone marrow adipose lineage cells promotes osteoclast formation and bone loss. EMBO Rep., 2021, 22: e52481

[34] Yu W et al. Bone marrow adipogenic lineage precursors promote osteoclastogenesis in bone remodeling and pathologic bone loss. J. Clin. Investig, 2021, 131: e140214

[35] Levine M. Transcriptional enhancers in animal development and evolution. Curr. Biol., 2010, 20: R754-R763

[36] St John HC et al. The osteoblast to osteocyte transition: epigenetic changes and response to the vitamin D3 hormone. Mol. Endocrinol., 2014, 28: 1150-1165

[37] Visel A, Minovitsky S, Dubchak I, Pennacchio LA. VISTA Enhancer Browser–a database of tissue-specific human enhancers. Nucleic Acids Res., 2007, 35: D88-D92

[38] Wen AY, Sakamoto KM, Miller LS. The role of the transcription factor CREB in immune function. J. Immunol., 2010, 185: 6413-6419

[39] Frost HM. In vivo osteocyte death. J. Bone Jt. Surg. Am., 1960, 42-a: 138-143

[40] Plotkin LI. Apoptotic osteocytes and the control of targeted bone resorption. Curr. Osteoporos. Rep., 2014, 12: 121-126

[41] McKenzie J et al. Osteocyte Death and Bone Overgrowth in Mice Lacking Fibroblast Growth Factor Receptors 1 and 2 in Mature Osteoblasts and Osteocytes. J. Bone Min. Res., 2019, 34: 1660-1675

[42] Mukohira H et al. Mesenchymal stromal cells in bone marrow express adiponectin and are efficiently targeted by an adiponectin promoter-driven Cre transgene. Int. Immunol., 2019, 31: 729-742

[43] Omatsu Y et al. The essential functions of adipo-osteogenic progenitors as the hematopoietic stem and progenitor cell niche. Immunity, 2010, 33: 387-399

[44] 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

[45] Seike M, Omatsu Y, Watanabe H, Kondoh G, Nagasawa T. Stem cell niche-specific Ebf3 maintains the bone marrow cavity. Genes Dev., 2018, 32: 359-372

[46] Verborgt O, Gibson GJ, Schaffler MB. Loss of osteocyte integrity in association with microdamage and bone remodeling after fatigue in vivo. J. Bone Min. Res., 2000, 15: 60-67

[47] Jilka RL, Noble B, Weinstein RS. Osteocyte apoptosis. Bone, 2013, 54: 264-271

[48] Noble BS et al. Mechanical loading: biphasic osteocyte survival and targeting of osteoclasts for bone destruction in rat cortical bone. Am. J. Physiol. Cell Physiol., 2003, 284: C934-C943

[49] Bishop KA et al. Transcriptional regulation of the human TNFSF11 gene in T cells via a cell type-selective set of distal enhancers. J. Cell Biochem., 2015, 116: 320-330

[50] Zheng Y et al. Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate. Nature, 2010, 463: 808-812

[51] Gillies SD, Morrison SL, Oi VT, Tonegawa S. A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene. Cell, 1983, 33: 717-728

[52] Kawase S et al. Identification of a novel intronic enhancer responsible for the transcriptional regulation of musashi1 in neural stem/progenitor cells. Mol. Brain, 2011, 4

[53] Tsukasaki M et al. Periosteal stem cells control growth plate stem cells during postnatal skeletal growth. Nat. Commun., 2022, 13

[54] Asano T et al. Soluble RANKL is physiologically dispensable but accelerates tumour metastasis to bone. Nat. Metab., 2019, 1: 868-875

[55] Menendez L, Yatskievych TA, Antin PB, Dalton S. Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells. Proc. Natl. Acad. Sci. USA, 2011, 108: 19240-19245

[56] Loh C et al. TNF-induced inflammatory genes escape repression in fibroblast-like synoviocytes: transcriptomic and epigenomic analysis. Ann. Rheum. Dis., 2019, 78: 1205-1214

[57] Castro-Mondragon JA et al. JASPAR 2022: the 9th release of the open-access database of transcription factor binding profiles. Nucleic Acids Res., 2022, 50: D165-d173

[58] Szklarczyk D et al. The STRING database in 2021: customizable protein-protein networks, and functional characterization of user-uploaded gene/measurement sets. Nucleic Acids Res., 2021, 49: D605-d612

[59] Ovcharenko I, Nobrega MA, Loots GG, Stubbs L. ECR Browser: a tool for visualizing and accessing data from comparisons of multiple vertebrate genomes. Nucleic Acids Res., 2004, 32: W280-W286

[60] Hao Y et al. Integrated analysis of multimodal single-cell data. Cell, 2021, 184: 3573-3587.e3529

[61] Zhou Y et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat. Commun., 2019, 10