Single-cell Transcriptome Analysis Identifies Senescent Osteocytes as Contributors to Bone Destruction in Breast Cancer Metastasis.

Breast cancer bone metastases increase fracture risk and are a major cause of morbidity and mortality among women. Upon colonization by tumor cells, the bone microenvironment undergoes profound reprogramming to support cancer progression that disrupts the balance between osteoclasts and osteoblasts, leading to bone lesions. Whether such reprogramming affects matrix-embedded osteocytes remains poorly understood. Here, we demonstrate that osteocytes in breast cancer bone metastasis develop premature senescence and a distinctive senescence-associated secretory phenotype (SASP) that favors bone destruction. Single-cell RNA sequencing identified osteocytes from mice with breast cancer bone metastasis enriched in senescence and SASP markers and pro-osteoclastogenic genes. Using multiplex in situ hybridization and AI-assisted analysis, we detected osteocytes with senescence-associated distension of satellites, telomere dysfunction, and p16Ink4a expression in mice and patients with breast cancer bone metastasis. In vitro and ex vivo organ cultures showed that breast cancer cells promote osteocyte senescence and enhance their osteoclastogenic potential. Clearance of senescent cells with senolytics suppressed bone resorption and preserved bone mass in mice with breast cancer bone metastasis. These results demonstrate that osteocytes undergo pathological reprogramming by breast cancer cells and identify osteocyte senescence as an initiating event triggering bone destruction in breast cancer metastases.

Refining the identity of mesenchymal cell types associated with murine periosteal and endosteal bone.

Single-cell RNA-seq has led to novel designations for mesenchymal cells associated with bone as well as multiple designations for what appear to be the same cell type. The main goals of this study were to increase the amount of single-cell RNA sequence data for osteoblasts and osteocytes, to compare cells from the periosteum to those inside bone, and to clarify the major categories of cell types associated with murine bone. We created an atlas of murine bone-associated cells by harmonizing published datasets with in-house data from cells targeted by Osx1-Cre and Dmp1-Cre driver strains. Cells from periosteal bone were analyzed separately from those isolated from the endosteum and trabecular bone. Over 100,000 mesenchymal cells were mapped to reveal 11 major clusters designated fibro-1, fibro-2, chondrocytes, articular chondrocytes, tenocytes, adipo-Cxcl12 abundant reticular (CAR), osteo-CAR, preosteoblasts, osteoblasts, osteocytes, and osteo-X, the latter defined in part by periostin expression. Osteo-X, osteo-CAR, and preosteoblasts were closely associated with osteoblasts at the trabecular bone surface. Wnt16 was expressed in multiple cell types from the periosteum but not in cells from endocortical or cancellous bone. Fibro-2 cells, which express markers of stem cells, localized to the periosteum but not trabecular bone in adult mice. Suppressing bone remodeling eliminated osteoblasts and altered gene expression in preosteoblasts but did not change the abundance or location of osteo-X or osteo-CAR cells. These results provide a framework for identifying bone cell types in murine single-cell RNA-seq datasets and suggest that osteoblast progenitors reside near the surface of remodeling bone.

A framework for defining mesenchymal cell types associated with murine periosteal and endosteal bone.

Single-cell RNA sequencing has led to numerous novel designations for mesenchymal cell types associated with bone. Consequently, there are now multiple designations for what appear to be the same cell type. In addition, existing datasets contain relatively small numbers of mature osteoblasts and osteocytes and there has been no comparison of periosteal bone cells to those at the endosteum and trabecular bone. The main goals of this study were to increase the amount of single cell RNA sequence data for osteoblasts and osteocytes, to compare cells from the periosteum to those inside bone, and to clarify the major categories of cell types associated with murine bone. To do this, we created an atlas of murine bone-associated cells by harmonizing published datasets with in-house data from cells targeted by Osx1-Cre and Dmp1-Cre driver strains. Cells from periosteal bone were analyzed separately from those isolated from the endosteum and trabecular bone. Over 100,000 mesenchymal cells were mapped to reveal 11 major clusters designated fibro-1, fibro-2, chondrocytes, articular chondrocytes, tenocytes, adipo-CAR, osteo-CAR, pre-osteoblasts, osteoblasts, osteocytes, and osteo-X, the latter defined in part by Postn expression. Osteo-X, osteo-CAR, and pre-osteoblasts were closely associated with osteoblasts at the trabecular bone surface. Wnt16 was expressed in multiple cell types from the periosteum but not in any cells from endocortical or cancellous bone. Fibro-2 cells, which express markers of skeletal stem cells, localized to the periosteum but not trabecular bone in adult mice. Suppressing bone remodeling eliminated osteoblasts and altered gene expression in pre-osteoblasts but did not change the abundance or location of osteo-X or osteo-CAR cells. These results provide a framework for identifying bone cell types in murine single cell RNA sequencing datasets and suggest that osteoblast progenitors reside near the surface of remodeling bone.

CRISPR interference provides increased cell type-specificity compared to the Cre-loxP system.

Cre-mediated recombination is frequently used for cell type-specific loss of function (LOF) studies. A major limitation of this system is recombination in unwanted cell types. CRISPR interference (CRISPRi) has been used effectively for global LOF in mice. However, cell type-specific CRISPRi, independent of recombination-based systems, has not been reported. To test the feasibility of cell type-specific CRISPRi, we produced two novel knock-in mouse models that achieve gene suppression when used together: one expressing dCas9::KRAB under the control of a cell type-specific promoter and the other expressing a single guide RNA from a safe harbor locus. We then compared the phenotypes of mice in which the same gene was targeted by either CRISPRi or the Cre-loxP system, with cell specificity conferred by Dmp1 regulatory elements in both cases. We demonstrate that CRISPRi is effective for cell type-specific LOF and that it provides improved cell type-specificity compared to the Cre-loxP system.

Reduced osteoprotegerin expression by osteocytes may contribute to rebound resorption after denosumab discontinuation.

Denosumab is an anti-RANKL Ab that potently suppresses bone resorption, increases bone mass, and reduces fracture risk. Discontinuation of denosumab causes rapid rebound bone resorption and bone loss, but the molecular mechanisms are unclear. We generated humanized RANKL mice and treated them with denosumab to examine the cellular and molecular conditions associated with rebound resorption. Denosumab potently suppressed both osteoclast and osteoblast numbers in cancellous bone in humanized RANKL mice. The decrease in osteoclast number was not associated with changes in osteoclast progenitors in bone marrow. Long-term, but not short-term, denosumab administration reduced osteoprotegerin (OPG) mRNA in bone. Localization of OPG expression revealed that OPG mRNA is produced by a subpopulation of osteocytes. Long-term denosumab administration reduced osteocyte OPG mRNA, suggesting that OPG expression declines as osteocytes age. Consistent with this, osteocyte expression of OPG was more prevalent near the surface of cortical bone in humans and mice. These results suggest that new osteocytes are an important source of OPG in remodeling bone and that suppression of remodeling reduces OPG abundance by reducing new osteocyte formation. The lack of new osteocytes and the OPG they produce may contribute to rebound resorption after denosumab discontinuation.

The NAD salvage pathway in mesenchymal cells is indispensable for skeletal development in mice.

NAD is an essential co-factor for cellular energy metabolism and multiple other processes. Systemic NAD+ deficiency has been implicated in skeletal deformities during development in both humans and mice. NAD levels are maintained by multiple synthetic pathways but which ones are important in bone forming cells is unknown. Here, we generate mice with deletion of Nicotinamide Phosphoribosyltransferase (Nampt), a critical enzyme in the NAD salvage pathway, in all mesenchymal lineage cells of the limbs. At birth, NamptΔPrx1 exhibit dramatic limb shortening due to death of growth plate chondrocytes. Administration of the NAD precursor nicotinamide riboside during pregnancy prevents the majority of in utero defects. Depletion of NAD post-birth also promotes chondrocyte death, preventing further endochondral ossification and joint development. In contrast, osteoblast formation still occurs in knockout mice, in line with distinctly different microenvironments and reliance on redox reactions between chondrocytes and osteoblasts. These findings define a critical role for cell-autonomous NAD homeostasis during endochondral bone formation.

RETRACTED: Deletion of the scavenger receptor Scarb1 in myeloid cells does not affect bone mass

The scavenger receptor class B member 1 (SR-B1 or Scarb1) is a glycosylated cell surface receptor for high density lipoproteins (HDL), oxidized low density lipoproteins (OxLDL), and phosphocholine-containing oxidized phospholipids (PC-OxPLs). Scarb1 is expressed in macrophages and has been shown to have both pro- and anti-atherogenic properties. It has been reported that global deletion of Scarb1 in mice leads to either high or low bone mass and that PC-OxPLs decrease osteoblastogenesis and increase osteoclastogenesis. PC-OxPLs decrease bone mass in 6-month-old mice and are critical pathogenetic factors in the bone loss caused by high fat diet or aging. We have investigated here whether Scarb1 expression in myeloid cells affects bone mass and whether PC-OxPLs exert their anti-osteogenic effects via activation of Scarb1 in macrophages. To this end, we generated mice with deletion of Scarb1 in LysM-Cre expressing cells and found that lack of Scarb1 did not affect bone mass in vivo. These results indicate that Scarb1 expression in cells of the myeloid/osteoclast lineage does not contribute to bone homeostasis. Based on this evidence, and earlier studies of ours showing that Scarb1 expression in osteoblasts does not affect bone mass, we conclude that Scarb1 is not an important mediator of the adverse effects on PC-OxPLs in osteoclasts or osteoblasts in 6-month-old mice.

Osteoclasts, Master Sculptors of Bone.

Osteoclasts are multinucleated cells with the unique ability to resorb bone matrix. Excessive production or activation of osteoclasts leads to skeletal pathologies that affect a significant portion of the population. Although therapies that effectively target osteoclasts have been developed, they are associated with sometimes severe side effects, and a fuller understanding of osteoclast biology may lead to more specific treatments. Along those lines, a rich body of work has defined essential signaling pathways required for osteoclast formation, function, and survival. Nonetheless, recent studies have cast new light on long-held views regarding the origin of these cells during development and homeostasis, their life span, and the cellular sources of factors that drive their production and activity during homeostasis and disease. In this review, we discuss these new findings in the context of existing work and highlight areas of ongoing and future investigation.

Deletion of the scavenger receptor Scarb1 in osteoblast progenitors does not affect bone mass.

The scavenger receptor class B member 1 (SR-B1 or Scarb1) is a cell surface receptor for high density lipoproteins. It also binds oxidized low density lipoproteins and phosphocholine-containing oxidized phospholipids (PC-OxPL), which adversely affect bone homeostasis. Overexpression of a single chain form of the antigen-binding domain of E06 IgM-a natural antibody that recognizes PC-OxPL-increases trabecular and cortical bone mass in female and male mice by stimulating bone formation. We have previously reported that Scarb1 is the most abundant scavenger receptor for PC-OxPL in calvaria-derived osteoblastic cells. Additionally, bone marrow- and calvaria-derived osteoblasts from Scarb1 knockout mice (Scarb1 KO) are protected from the pro-apoptotic and anti-differentiating effects of OxPL. Previous skeletal analysis of Scarb1 KO mice has produced contradictory results, with some studies reporting elevated bone mass but another study reporting low bone mass. To clarify the role of Scarb1 in osteoblasts, we deleted Scarb1 specifically in cells of the osteoblast lineage using Osx1-Cre transgenic mice. We observed no difference in bone mineral density measured by DXA in either female or male Osx1-Cre;Scarb1fl/fl mice compared to wild type (WT), Osx1-Cre, or Scarb1fl/fl littermate controls. Additionally, microCT analysis of 6-month-old females and 7-month-old males did not detect any difference in trabecular or cortical bone mass between genotypes. These results indicate that expression of Scarb1 in cells of the osteoblast lineage does not play an important role in bone homeostasis and, therefore, it is not essential for the effects of PC-OxPL on these cells.

Deletion of a putative promoter-proximal Tnfsf11 regulatory region in mice does not alter bone mass or Tnfsf11 expression in vivo.

The cytokine RANKL is essential for osteoclast formation during physiological and pathological bone resorption. RANKL also contributes to lymphocyte production, development of lymph nodes and mammary glands, as well as other biological activities. Transcriptional control of the Tnfsf11 gene, which encodes RANKL, is complex and involves distant regulatory regions. Nevertheless, cell culture studies suggest that an enhancer region near the transcription start site is involved in the control of Tnfsf11 expression by hormones such as 1,25-(OH)2 vitamin D3 and parathyroid hormone, as well as the sympathetic nervous system. To address the significance of this region in vivo, we deleted the sequence between -510 to -1413 bp, relative to Tnfsf11 exon 1, from mice using CRISPR-based gene editing. MicroCT analysis of the femur and fourth lumbar vertebra of enhancer knockout mice showed no differences in bone mass compared to wild type littermates at 5 weeks and 6 months of age, suggesting no changes in osteoclast formation. RNA extracted from the tibia, fifth lumbar vertebra, thymus, and spleen at 6 months of age also showed no reduction in Tnfsf11 mRNA abundance between these groups. However, maximal stimulation of Tnfsf11 mRNA abundance in cultured stromal cells by PTH was reduced approximately 40% by enhancer deletion, while stimulation by 1,25-(OH)2 vitamin D3 was unaffected. The abundance of B and T lymphocytes in the bone marrow did not differ between genotypes. These results demonstrate that the region between -510 and -1413 does not contribute to Tnfsf11 expression, osteoclast support, or lymphocyte production in mice under normal physiological conditions but may be involved in situations of elevated parathyroid hormone.

A Uromodulin Mutation Drives Autoimmunity and Kidney Mononuclear Phagocyte Endoplasmic Reticulum Stress.

We identified a family with a UMOD gene mutation (C106F) resulting in glomerular inflammation and complement deposition. To determine if the observed phenotype is due to immune system activation by mutant uromodulin, a mouse strain with a homologous cysteine to phenylalanine mutation (C105F) in the UMOD gene was generated using CRISPR-Cas9 gene editing and the effect of this mutation on mononuclear phagocytic cells was examined. Mutant mice developed high levels of intracellular and secreted aggregated uromodulin, resulting in anti-uromodulin antibodies and circulating uromodulin containing immune complexes with glomerular deposition and kidney fibrosis with aging. F4/80+ and CD11c+ kidney cells phagocytize uromodulin. Differential gene expression analysis by RNA sequencing of F4/80+ phagocytic cells revealed activation of the activating transcription factor 5 (ATF5)-mediated stress response pathway in mutant mice. Phagocytosis of mutant uromodulin by cultured dendritic cells resulted in activation of the endoplasmic reticulum stress response pathway and markers of cell inactivation, an effect not seen with wild-type protein. Mutant mice demonstrate a twofold increase in T-regulatory cells, consistent with induction of immune tolerance, resulting in decreased inflammatory response and improved tissue repair following ischemia-reperfusion injury. The C105F mutation results in autoantibodies against aggregated misfolded protein with immune complex formation and kidney fibrosis. Aggregated uromodulin may induce dendritic cell tolerance following phagocytosis through an unfolded protein/endoplasmic reticulum stress response pathway, resulting in decreased inflammation following tissue injury.

Osteocyte RANKL is required for cortical bone loss with age and is induced by senescence.

In aging mice, osteoclast number increases in cortical bone but declines in trabecular bone, suggesting that different mechanisms underlie age-associated bone loss in these 2 compartments. Osteocytes produce the osteoclastogenic cytokine RANKL, encoded by Tnfsf11. Tnfsf11 mRNA increases in cortical bone of aged mice, suggesting a mechanism underlying the bone loss. To address this possibility, we aged mice lacking RANKL in osteocytes. Whereas control mice lost cortical bone between 8 and 24 months of age, mice lacking RANKL in osteocytes gained cortical bone during this period. Mice of both genotypes lost trabecular bone with age. Osteoclasts increased with age in cortical bone of control mice but not in RANKL conditional knockout mice. Induction of cellular senescence increased RANKL production in murine and human cell culture models, suggesting an explanation for elevated RANKL levels with age. Overexpression of the senescence-associated transcription factor Gata4 stimulated Tnfsf11 expression in cultured murine osteoblastic cells. Finally, elimination of senescent cells from aged mice using senolytic compounds reduced Tnfsf11 mRNA in cortical bone. Our results demonstrate the requirement of osteocyte-derived RANKL for age-associated cortical bone loss and suggest that increased Tnfsf11 expression with age results from accumulation of senescent cells in cortical bone.

Local Production of Osteoprotegerin by Osteoblasts Suppresses Bone Resorption.

Osteoprotegerin (OPG) inhibits the ability of receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) to stimulate the differentiation, activity, and survival of bone-resorbing osteoclasts. Genetic studies in mice show that osteocytes are an important source of RANKL, but the cellular sources of OPG are unclear. We use conditional deletion of Tnfrsf11b, which encodes OPG, from different cell populations to identify functionally relevant sources of OPG in mice. Deletion from B lymphocytes and osteocytes, two cell types commonly thought to supply OPG, has little or no impact on bone mass. By contrast, deletion of Tnfrsf11b from osteoblasts increases bone resorption and reduces bone mass to an extent similar to germline deletion, demonstrating that osteoblasts are an essential source of OPG. These results suggest that, in addition to producing new bone matrix, osteoblasts also play an active role in terminating the resorption phase of the bone remodeling cycle by suppressing RANKL activity.

Effective CRISPR interference of an endogenous gene via a single transgene in mice.

Drawbacks of conditional gene deletion in mice include the need for extensive breeding and, often, a lack of cell type-specificity. CRISPR interference (CRISPRi) is an alternative approach for loss-of-function studies that inhibits expression by guiding a transcriptional repressor to the transcription start-site of target genes. However, there has been limited exploration of CRISPRi in mice. We tested the effectiveness of a single CRISPRi transgene broadly expressing a single guide RNA and a catalytically dead Cas9 fused to the KRAB repressor domain to suppress a well-characterized target gene, Tnfsf11. The phenotype of CRISPRi transgenic mice was compared to mice with germline deletion of Tnfsf11, which are osteopetrotic and do not form lymph nodes. High transgene expression mimicked gene deletion, with failure of lymph node development and classic signs of osteopetrosis such as high bone mass and failure of tooth eruption. Mice with low transgene expression were normal and mice with medium expression displayed an intermediate phenotype. Transgene expression in tissues from these mice correlated inversely with Tnfsf11 mRNA levels. These results demonstrate that a single CRISPRi transgene can effectively suppress a target gene in mice and suggest that this approach may be useful for cell type-specific loss-of-function studies.

Loss of RANKL in osteocytes dramatically increases cancellous bone mass in the osteogenesis imperfecta mouse (oim).

Osteogenesis imperfecta (OI) is characterized by osteopenia and bone fragility, and OI patients during growth often exhibit high bone turnover with the net result of low bone mass. Recent evidence shows that osteocytes significantly affect bone remodeling under physiological and pathological conditions through production of osteoclastogenic cytokines. The receptor activator of nuclear factor kappa-B ligand (RANKL) produced by osteocytes for example, is a critical mediator of bone loss caused by ovariectomy, low-calcium diet, unloading and glucocorticoid treatment. Because OI bone has increased density of osteocytes and these cells are embedded in matrix with abnormal type I collagen, we hypothesized that osteocyte-derived RANKL contributes to the OI bone phenotype. In this study, the conditional loss of RANKL in osteocytes in oim/oim mice (oim-RANKL-cKO) resulted in dramatically increased cancellous bone mass in both the femur and lumbar spine compared to oim/oim mice. Bone cortical thickness increased significantly only in spine but ultimate bone strength in the long bone and spine was minimally improved in oim-RANKL-cKO mice compared to oim/oim mice. Furthermore, unlike previous findings, we report that oim/oim mice do not exhibit high bone turnover suggesting that their low bone mass is likely due to defective bone formation and not increased bone resorption. The loss of osteocyte-derived RANKL further diminished parameters of formation in oim-RANKL-cKO. Our results indicate that osteocytes contribute significantly to the low bone mass observed in OI and the effect of loss of RANKL from these cells is similar to its systemic inhibition.

Soluble RANKL contributes to osteoclast formation in adult mice but not ovariectomy-induced bone loss.

Receptor activator of NFkB ligand (RANKL) is a TNF-family cytokine required for osteoclast formation, as well as immune cell and mammary gland development. It is produced as a membrane-bound protein that can be shed to form a soluble protein. We created mice harboring a sheddase-resistant form of RANKL, in which soluble RANKL is undetectable in the circulation. Lack of soluble RANKL does not affect bone mass or structure in growing mice but reduces osteoclast number and increases cancellous bone mass in adult mice. Nonetheless, the bone loss caused by estrogen deficiency is unaffected by the lack of soluble RANKL. Lymphocyte number, lymph node development, and mammary gland development are also unaffected by the absence of soluble RANKL. These results demonstrate that the membrane-bound form of RANKL is sufficient for most functions of this protein but that the soluble form does contribute to physiological bone remodeling in adult mice.

Modeling Rare Bone Diseases in Animals.

PURPOSE OF REVIEW: The goal of this review is to highlight some of the considerations involved in creating animal models to study rare bone diseases and then to compare and contrast approaches to creating such models, focusing on the advantages and novel opportunities offered by the CRISPR-Cas system. RECENT FINDINGS: Gene editing after creation of double-stranded breaks in chromosomal DNA is increasingly being used to modify animal genomes. Multiple tools can be used to create such breaks, with the newest ones being based on the bacterial adaptive immune system known as CRISPR/Cas. Advances in gene editing have increased the ease and speed, while reducing the cost, of creating novel animal models of disease. Gene editing has also expanded the number of animal species in which genetic modification can be performed. These changes have significantly increased the options for investigators seeking to model rare bone diseases in animals.

The YAP/TAZ transcriptional co-activators have opposing effects at different stages of osteoblast differentiation.

The related transcriptional co-factors YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif) have been proposed to either promote or inhibit osteoblast differentiation. Here we investigated the skeletal consequences of deleting YAP and TAZ at different stages of the osteoblast lineage using Prx1-Cre, Osx1-Cre, and Dmp1-Cre transgenic mice. Prx1-Cre-mediated deletion resulted in embryonic lethality. Mice lacking both copies of TAZ and one copy of YAP in cells targeted by Prx1-Cre were viable and displayed elevated bone mass associated increased bone formation. Deletion of YAP and TAZ using Osx1-Cre mice led to perinatal lethality. Suppression of Osx1-Cre activity until 21 days of age permitted postnatal deletion of YAP and TAZ, which resulted in increased osteoblast number at 12 weeks of age but no change in bone mass. Mechanistic studies revealed that YAP and TAZ suppress canonical Wnt signaling and Runx2 activity in osteoblast progenitors. Consistent with this, deletion of YAP and TAZ from osteoprogenitor cells increased osteoblast differentiation in vitro. Deletion of YAP and TAZ from mature osteoblasts and osteocytes using Dmp1-Cre mice led to reduced osteoblast number and bone formation, as well as increased osteoclast number, but no changes in known regulators of bone turnover such as RANKL, OPG, and Sost. Together these results suggest that YAP and TAZ in osteoblast progenitors oppose differentiation towards the osteoblast lineage but in mature osteoblasts and osteocytes, they promote bone formation and inhibit bone resorption.