Sfrp4 is required to maintain Ctsk-lineage periosteal stem cell niche function.

We have previously reported that the cortical bone thinning seen in mice lacking the Wnt signaling antagonist Sfrp4 is due in part to impaired periosteal apposition. The periosteum contains cells which function as a reservoir of stem cells and contribute to cortical bone expansion, homeostasis, and repair. However, the local or paracrine factors that govern stem cells within the periosteal niche remain elusive. Cathepsin K (Ctsk), together with additional stem cell surface markers, marks a subset of periosteal stem cells (PSCs) which possess self-renewal ability and inducible multipotency. Sfrp4 is expressed in periosteal Ctsk-lineage cells, and Sfrp4 global deletion decreases the pool of PSCs, impairs their clonal multipotency for differentiation into osteoblasts and chondrocytes and formation of bone organoids. Bulk RNA sequencing analysis of Ctsk-lineage PSCs demonstrated that Sfrp4 deletion down-regulates signaling pathways associated with skeletal development, positive regulation of bone mineralization, and wound healing. Supporting these findings, Sfrp4 deletion hampers the periosteal response to bone injury and impairs Ctsk-lineage periosteal cell recruitment. Ctsk-lineage PSCs express the PTH receptor and PTH treatment increases the % of PSCs, a response not seen in the absence of Sfrp4. Importantly, in the absence of Sfrp4, PTH-dependent increase in cortical thickness and periosteal bone formation is markedly impaired. Thus, this study provides insights into the regulation of a specific population of periosteal cells by a secreted local factor, and shows a central role for Sfrp4 in the regulation of Ctsk-lineage periosteal stem cell differentiation and function.

Comparative study in estrogen-depleted mice identifies skeletal and osteocyte transcriptomic responses to abaloparatide and teriparatide.

Osteocytes express parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptors and respond to the PTHrP analog abaloparatide (ABL) and to the PTH 1-34 fragment teriparatide (TPTD), which are used to treat osteoporosis. Several studies indicate overlapping but distinct skeletal responses to ABL or TPTD, but their effects on cortical bone may differ. Little is known about their differential effects on osteocytes. We compared cortical osteocyte and skeletal responses to ABL and TPTD in sham-operated and ovariectomized mice. Administered 7 weeks after ovariectomy for 4 weeks at a dose of 40 µg/kg/d, TPTD and ABL had similar effects on trabecular bone, but ABL showed stronger effects in cortical bone. In cortical osteocytes, both treatments decreased lacunar area, reflecting altered peri-lacunar remodeling favoring matrix accumulation. Osteocyte RNA-Seq revealed that several genes and pathways were altered by ovariectomy and affected similarly by TPTD and ABL. Notwithstanding, several signaling pathways were uniquely regulated by ABL. Thus, in mice, TPTD and ABL induced a positive osteocyte peri-lacunar remodeling balance, but ABL induced stronger cortical responses and affected the osteocyte transcriptome differently. We concluded that ABL affected the cortical osteocyte transcriptome in a manner subtly different from TPTD, resulting in more beneficial remodeling/modeling changes and homeostasis of the cortex.

Social isolation through single housing negatively affects trabecular and cortical bone in adult male, but not female, C57BL/6J mice.

Social isolation is a potent form of psychosocial stress and is a growing public health concern, particularly among older adults. Even prior to the onset of the COVID-19 pandemic, which has significantly increased the prevalence of isolation and loneliness, researchers have been concerned about a rising "epidemic" of loneliness. Isolation is associated with an increased risk for many physical and mental health disorders and increased overall mortality risk. In addition to social isolation, older adults are also at greater risk for osteoporosis and related fractures. While researchers have investigated the negative effects of other forms of psychosocial stress on bone, including depression and PTSD, the effects of social isolation on bone have not been thoroughly investigated. The aim of this study was to test the hypothesis that social isolation would lead to bone loss in male and female C57BL/6J mice. 16-week-old mice were randomized into social isolation (1 mouse/cage) or grouped housing (4 mice/cage) for four weeks. Social isolation significantly decreased trabecular (BV/TV, BMD, Tb. N., Tb. Th.) and cortical bone (Ct.Th., Ct.Ar., Ct.Ar./Tt.Ar., pMOI) parameters in male, but not female mice. Isolated male mice had signs of reduced bone remodeling represented by reduced osteoblast numbers, osteoblast-related gene expression and osteoclast-related gene expression. However, isolated females had increased bone resorption-related gene expression, without any change in bone mass. Overall, our data suggest that social isolation has negative effects on bone in male, but not female mice, although females showed suggestive effects on bone resorption. These results provide critical insight into the effects of isolation on bone and have key clinical implications as we grapple with the long-term health impacts of the rise in social isolation related to the COVID-19 pandemic.

Social Isolation Causes Cortical and Trabecular Bone Loss in Adult Male, but not Female, C57BL/6J Mice.

Social isolation is a potent form of psychosocial stress and is a growing public health concern, particularly among older adults. Even prior to the onset of the COVID-19 pandemic, which has significantly increased the prevalence of isolation and loneliness, researchers have been concerned about a rising "epidemic" of loneliness. Isolation is associated with an increased risk for many physical and mental health disorders and increased overall mortality risk. In addition to social isolation, older adults are also at greater risk for osteoporosis and related fractures. While researchers have investigated the negative effects of other forms of psychosocial stress on bone, including depression and PTSD, the effects of social isolation on bone have not been thoroughly investigated. The aim of this study was to test the hypothesis that social isolation would lead to bone loss in male and female C57BL/6J mice. 16-week-old mice were randomized into social isolation (1 mouse/cage) or grouped housing (4 mice/cage) for four weeks (N=16/group). Social isolation significantly decreased trabecular (BV/TV, BMD, Tb. N., Tb. Th.) and cortical bone (Ct.Th., Ct.Ar., Ct.Ar./Tt.Ar., pMOI, Ct.Por.) parameters in male, but not female mice. Isolated male mice had signs of reduced bone remodeling represented by reduced osteoblast numbers, osteoblast-related gene expression and osteoclast-related gene expression. However, isolated females had increased bone resorption-related gene expression, without any change in bone mass. Overall, our data suggest that social isolation has negative effects on bone in males, but not females, although females showed suggestive effects on bone resorption. These results provide critical insight into the effects of isolation on bone and have key clinical implications as we grapple with the long-term health impacts of the rise in social isolation related to the COVID-19 pandemic.

Marrow aspiration in aged mice: intramedullary osteogenesis, reduced mechano-adaptation, increased marrow fat.

Introduction: With age, the number of adipocytes and osteoclasts increases, the number of osteoblasts decreases, and mechano-adaptation is impaired.Objectives: Using marrow aspiration, which has a known osteogenic effect in young mice, we sought to recruit osteoblast progenitors to mediate the mechano-adaptive response to in vivo tibial loading.Methods: First, we assessed bone formation and marrow adiposity in the tibiae of old mice (>20 months) sacrificed 1, 2, and 4 weeks after unilateral marrow aspiration. Then, we examined the effects of marrow aspiration on mechano-adaptation in aged mice using tibial loading.Results: Two weeks after aspiration, aspirated tibiae had more bone than contralateral tibiae due to the formation of bone in the medullary canal. Two weeks and four weeks after marrow aspiration, the volume of marrow adipose tissue was higher in the aspirated tibiae, compared to contralateral tibiae. Histomorphometry indicated that aspiration increased non-periosteal (endosteal, intracortical, intramedullary) bone formation, compared to the contralateral tibia.  Mice with marrow aspiration had reduced periosteal bone formation in the contralateral tibia, compared to mice that had loading alone. Loading-induced periosteal bone formation was higher in mice that had loading alone, compared to mice that had aspiration + loading, indicating that aspiration further reduced the mechano-adaptive response.Conclusion: These data demonstrate that, in old mice, bone forms in the medullary canal following aspiration. Adiposity is increased following marrow aspiration, and periosteal mechano-adaptation is reduced.

Inhibition of longevity regulator PAPP-A modulates tissue homeostasis via restraint of mesenchymal stromal cells.

Pregnancy-associated plasma protein-A (PAPP-A) is a secreted metalloprotease that increases insulin-like growth factor (IGF) availability by cleaving IGF-binding proteins. Reduced IGF signaling extends longevity in multiple species, and consistent with this, PAPP-A deletion extends lifespan and healthspan; however, the mechanism remains unclear. To clarify PAPP-A's role, we developed a PAPP-A neutralizing antibody and treated adult mice with it. Transcriptomic profiling across tissues showed that anti-PAPP-A reduced IGF signaling and extracellular matrix (ECM) gene expression system wide. The greatest reduction in IGF signaling occurred in the bone marrow, where we found reduced bone, marrow adiposity, and myelopoiesis. These diverse effects led us to search for unifying mechanisms. We identified mesenchymal stromal cells (MSCs) as the source of PAPP-A in bone marrow and primary responders to PAPP-A inhibition. Mice treated with anti-PAPP-A had reduced IGF signaling in MSCs and dramatically decreased MSC number. As MSCs are (1) a major source of ECM and the progenitors of ECM-producing fibroblasts, (2) the originating source of adult bone, (3) regulators of marrow adiposity, and (4) an essential component of the hematopoietic niche, our data suggest that PAPP-A modulates bone marrow homeostasis by potentiating the number and activity of MSCs. We found that MSC-like cells are the major source of PAPP-A in other tissues also, suggesting that reduced MSC-like cell activity drives the system-wide reduction in ECM gene expression due to PAPP-A inhibition. Dysregulated ECM production is associated with aging and drives age-related diseases, and thus, this may be a mechanism by which PAPP-A deficiency enhances longevity.

Abaloparatide treatment increases bone formation, bone density and bone strength without increasing bone resorption in a rat model of hindlimb unloading.

Disuse osteoporosis can result from prolonged bed rest, paralysis, casts, braces, fractures and other conditions. Abaloparatide (ABL) is a PTHrP analog that increases bone density and strength by stimulating osteogenesis with limited effects on bone resorption. We examined skeletal responses to abaloparatide in young adult male rats with normal weight-bearing and with hindlimb unloading via a pelvic harness. Rats were allocated to four groups (10-12 per group): normal weight-bearing plus vehicle treatment (CON-VEH), normal weight-bearing plus ABL treatment (CON-ABL), hindlimb-unloading plus vehicle (HLU-VEH), or hindlimb-unloading plus ABL (HLU-ABL). Rats received ABL (25 µg/kg/day, s.c.) or vehicle throughout the 28-day unloading period and were then sacrificed, at which time HLU-VEH rats exhibited reduced bone formation and significant deficits in tibial, femoral, and vertebral bone mass compared with CON-VEH. ABL treatment increased serum osteocalcin in CON and HLU animals while having no effect on the osteoclast marker TRACP-5b. Longitudinal peripheral quantitative computed tomography (pQCT) indicated that ABL increased trabecular and cortical bone mass in the tibia. ABL was also associated with improved trabecular and cortical bone mass and architectural parameters at the femur, tibia, and vertebrae by µCT. Tibial histomorphometry indicated increased trabecular and endocortical bone formation with HLU-ABL versus HLU-VEH and with CON-ABL versus CON-VEH, and ABL was also associated with lower trabecular and endocortical osteoclast surfaces. Vertebral finite element analysis indicated higher ultimate load and stiffness for CON-ABL versus CON-VEH and for HLU-ABL versus HLU-VEH. In summary, ABL was associated with improved trabecular and cortical bone density and architecture in normal weight-bearing and hindlimb-unloaded rats, with higher bone formation and no difference in bone resorption. ABL was also associated with improved bone biomechanical parameters. These results provide rationale for investigating the ability of abaloparatide to prevent or treat disuse osteoporosis in humans.

Effects of abaloparatide and teriparatide on bone resorption and bone formation in female mice.

Intermittent administration of PTH type 1 receptor (PTH1R) agonists increases bone remodeling, with greater stimulation of bone formation relative to bone resorption causing net gains in bone mass. This pharmacodynamic feature underlies the bone-building effects of teriparatide and abaloparatide, the only PTH1R agonists approved to reduce osteoporotic fracture risk in postmenopausal women. This study in 8-week-old female mice compared bone resorption and formation responses to these agents delivered at the same 10 µg/kg dose, and a 40 µg/kg abaloparatide dose was also included to reflect its 4-fold higher approved clinical dose. Peptides or vehicle were administered by daily supra-calvarial subcutaneous injection for 12 days, and local (calvarial) and systemic (L5 vertebral and tibial) responses were evaluated by histomorphometry. Terminal bone histomorphometry data indicated that calvarial resorption cavities were similar in both abaloparatide groups versus vehicle controls, whereas the teriparatide group had more calvarial resorption cavities compared with the vehicle or abaloparatide 40 µg/kg groups. The bone resorption marker serum CTX was significantly lower in the abaloparatide 40 µg/kg group and similar in the other two active treatment groups compared with vehicle controls. Both peptides increased trabecular bone formation rate (BFR) in L5 and proximal tibia versus vehicle, and L5 BFR was higher with abaloparatide 40 µg/kg versus teriparatide. At the tibial diaphysis, periosteal BFR was higher with abaloparatide 40 µg/kg versus vehicle or teriparatide, and endocortical BFR was higher with teriparatide but not with abaloparatide 10 or 40 µg/kg versus vehicle. Few differences in structural or microarchitectural bone parameters were observed with this brief duration of treatment. In summary, calvarial bone resorption cavity counts were higher in the teriparatide group versus the vehicle and abaloparatide 40 µg/kg groups, and the abaloparatide 40 µg/kg group had lower serum CTX versus vehicle. L5 and tibial trabecular bone formation indices were higher in all three active treatment groups versus vehicle. The abaloparatide 40 µg/kg group had higher L5 trabecular BFR and tibial periosteal BFR versus teriparatide, whereas tibial endocortical BFR was higher with teriparatide but not abaloparatide. Together, these findings in female mice indicate that an improved balance of bone formation versus bone resorption is established shortly after initiating treatment with abaloparatide.

Sfrp4 repression of the Ror2/Jnk cascade in osteoclasts protects cortical bone from excessive endosteal resorption.

Loss-of-function mutations in the Wnt inhibitor secreted frizzled receptor protein 4 (SFRP4) cause Pyle's disease (OMIM 265900), a rare skeletal disorder characterized by wide metaphyses, significant thinning of cortical bone, and fragility fractures. In mice, we have shown that the cortical thinning seen in the absence of Sfrp4 is associated with decreased periosteal and endosteal bone formation and increased endocortical resorption. While the increase in Rankl/Opg in cortical bone of mice lacking Sfrp4 suggests an osteoblast-dependent effect on endocortical osteoclast (OC) activity, whether Sfrp4 can cell-autonomously affect OCs is not known. We found that Sfrp4 is expressed during bone marrow macrophage OC differentiation and that Sfrp4 significantly suppresses the ability of early and late OC precursors to respond to Rankl-induced OC differentiation. Sfrp4 deletion in OCs resulted in activation of canonical Wnt/ß-catenin and noncanonical Wnt/Ror2/Jnk signaling cascades. However, while inhibition of canonical Wnt/ß-catenin signaling did not alter the effect of Sfrp4 on OCgenesis, blocking the noncanonical Wnt/Ror2/Jnk cascade markedly suppressed its regulation of OC differentiation in vitro. Importantly, we report that deletion of Ror2 exclusively in OCs (CtskCreRor2fl/fl ) in Sfrp4 null mice significantly reversed the increased number of endosteal OCs seen in these mice and reduced their cortical thinning. Altogether, these data show autocrine and paracrine effects of Sfrp4 in regulating OCgenesis and demonstrate that the increase in endosteal OCs seen in Sfrp4-/- mice is a consequence of noncanonical Wnt/Ror2/Jnk signaling activation in OCs overriding the negative effect that activation of canonical Wnt/ß-catenin signaling has on OCgenesis.

Increased Cellular Presence After Sciatic Neurectomy Improves the Bone Mechano-adaptive Response in Aged Mice.

The mechano-adaptive response of bone to loading in the murine uniaxial tibial loading model is impaired in aged animals. Previous studies have shown that in aged mice, the amount of bone formed in response to loading is augmented when loads are applied following sciatic neurectomy. The synergistic effect of neurectomy and loading remains to be elucidated. We hypothesize that sciatic neurectomy increases cellular presence, thereby augmenting the response to load in aged mice. We examined bone adaptation in four groups of female C57BL/6J mice, 20-22 months old: (1) sham surgery + 9N loading; (2) sciatic neurectomy, sacrificed after 5 days; (3) sciatic neurectomy, sacrificed after 19 days; (4) sciatic neurectomy + 9N loading. We examined changes in bone cross sectional properties with micro-CT images, and static and dynamic histomorphometry with histological sections taken at the midpoint between tibiofibular junctions. The response to loading at 9N was not detectable with quantitative micro-CT data, but surface-specific histomorphometry captured an increase in bone formation in specific regions. 5 days following sciatic neurectomy, the amount of bone in the neurectomized leg was the same as the contralateral leg, but 19 days following sciatic neurectomy, there was significant bone loss in the neurectomized leg, and both osteoclasts and osteoblasts were recruited to the endosteal surfaces. When sciatic neurectomy and loading at 9N were combined, 3 out of 4 bone quadrants had increased bone formation, on the endosteal and periosteal surfaces (increased osteoid surface and mineralizing surface respectively). These data demonstrate that sciatic neurectomy increases cellular presence on the endosteal surface. With long-term sciatic-neurectomy, both osteoclasts and osteoblasts were recruited to the endosteal surface, which resulted in increased bone formation when combined with a sufficient mechanical stimulus. Controlled and localized recruitment of both osteoblasts and osteoclasts combined with appropriate mechanical loading could inform therapies for mechanically-directed bone formation.

Cathepsin K-deficient osteocytes prevent lactation-induced bone loss and parathyroid hormone suppression.

Lactation induces bone loss to provide sufficient calcium in the milk, a process that involves osteoclastic bone resorption but also osteocytes and perilacunar resorption. The exact mechanisms by which osteocytes contribute to bone loss remain elusive. Osteocytes express genes required in osteoclasts for bone resorption, including cathepsin K (Ctsk), and lactation elevates their expression. We show that Ctsk deletion in osteocytes prevented the increase in osteocyte lacunar area seen during lactation, as well as the effects of lactation to increase osteoclast numbers and decrease trabecular bone volume, cortical thickness and mechanical properties. In addition, Ctsk deletion in osteocytes increased bone Parathyroid Hormone related Peptide (PTHrP), prevented the decrease in serum Parathyroid Hormone (PTH) induced by lactation, but amplified the increase in serum 1,25(OH)2D. The net result of these changes is to maintain serum and milk calcium levels in the normal range, ensuring normal offspring skeletal development. Our studies confirm the fundamental role of osteocytic perilacunar remodeling in physiological states of lactation and provides genetic evidence that osteocyte-derived Ctsk contributes not only to osteocyte perilacunar remodeling, but also to the regulation of PTH, PTHrP, 1,25-Dyhydroxyvitamin D (1,25(OH)2D), osteoclastogenesis and bone loss in response to the high calcium demand associated with lactation.

Abaloparatide improves cortical geometry and trabecular microarchitecture and increases vertebral and femoral neck strength in a rat model of male osteoporosis.

Androgen deficiency is a leading cause of male osteoporosis, with bone loss driven by an inadequate level of bone formation relative to the extent of bone resorption. Abaloparatide, an osteoanabolic PTH receptor agonist used to treat women with postmenopausal osteoporosis at high risk for fracture, increases bone formation and bone strength in estrogen-deficient animals without increasing bone resorption. This study examined the effects of abaloparatide on bone formation, bone mass, and bone strength in androgen-deficient orchiectomized (ORX) rats, a male osteoporosis model. Four-month-old Sprague-Dawley rats underwent ORX or sham surgery. Eight weeks later, sham-operated rats received vehicle (saline; n = 10) while ORX rats (n = 10/group) received vehicle (Veh) or abaloparatide at 5 or 25 µg/kg (ABL5 or ABL25) by daily s.c. injection for 8 weeks, followed by sacrifice. Dynamic bone histomorphometry indicated that the tibial diaphysis of one or both abaloparatide groups had higher periosteal mineralizing surface, intracortical bone formation rate (BFR), endocortical BFR, and cortical thickness vs Veh controls. Vertebral trabecular BFR was also higher in both abaloparatide groups vs Veh, and the ABL25 group had higher trabecular osteoblast surface without increased osteoclast surface. By micro-CT, the vertebra and distal femur of both abaloparatide-groups had improved trabecular bone volume and micro-architecture, and the femur diaphysis of the ABL25 group had greater cortical thickness with no increase in porosity vs Veh. Biomechanical testing indicated that both abaloparatide-groups had stronger vertebrae and femoral necks vs Veh controls. These findings provide preclinical support for evaluating abaloparatide as an investigational treatment for male osteoporosis.

Bone adaptation compensates resorption when sciatic neurectomy is followed by low magnitude induced loading.

The uniaxial tibial loading model is commonly used to promote bone formation through mechanoadaptation in mice. Sciatic neurectomy on the other hand recruits osteoclasts, which results in bone loss. Previous studies have shown that combining sciatic neurectomy with high magnitude loading increases the amount of bone formed. Here we determine whether low-intensity loading (low magnitude and few cycles) is sufficient to maintain bone mass after sciatic neurectomy, either by promoting bone formation (balance between concurrent resorption and formation), or by preventing bone resorption altogether. We examined bone adaptation in 4 groups of female C57BL/6J mice, 19-22 weeks old: (1) sham surgery +10 N loading; (2) sham surgery +5 N loading; (3) sciatic neurectomy; (4) sciatic neurectomy +5 N loading. Left legs were kept intact as internal controls. We examined changes in bone cross sectional properties and marrow area with micro-CT images, and histomorphometric measures with histological sections at the midpoint between tibiofibular junctions. Loading at 10 N caused a significant increase in the amount of bone, but bone formation after 5 N of loading was not detectable in micro-CT images. There was significant bone loss in mice with sciatic neurectomy alone, but when combined with loading there was no significant bone loss. Histomorphometric analyses showed that loading at 5 N augmented bone formation periosteally on the lateral and posterior-medial surfaces, and reduced the number of endosteal osteoclasts on the posterior-medial surface compared to the contralateral leg. Combining sciatic neurectomy and loading at 5 N promoted faster mineral apposition on the periosteal lateral surface and augmented bone resorption on the endosteal posterior surface compared to the contralateral leg. These data demonstrate that low-intensity loading is sufficient to maintain bone mass after sciatic neurectomy, both by preventing recruitment of osteoclasts on the endosteal surface and by compensating endosteal resorption caused by disuse with periosteal formation promoted by loading. This has implications for the loading required to maintain bone mass after injury or prolonged bedrest.

Pharmacological targeting of MYC-regulated IRE1/XBP1 pathway suppresses MYC-driven breast cancer.

The unfolded protein response (UPR) is a cellular homeostatic mechanism that is activated in many human cancers and plays pivotal roles in tumor progression and therapy resistance. However, the molecular mechanisms for UPR activation and regulation in cancer cells remain elusive. Here, we show that oncogenic MYC regulates the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) branch of the UPR in breast cancer via multiple mechanisms. We found that MYC directly controls IRE1 transcription by binding to its promoter and enhancer. Furthermore, MYC forms a transcriptional complex with XBP1, a target of IRE1, and enhances its transcriptional activity. Importantly, we demonstrate that XBP1 is a synthetic lethal partner of MYC. Silencing of XBP1 selectively blocked the growth of MYC-hyperactivated cells. Pharmacological inhibition of IRE1 RNase activity with small molecule inhibitor 8866 selectively restrained the MYC-overexpressing tumor growth in vivo in a cohort of preclinical patient-derived xenograft models and genetically engineered mouse models. Strikingly, 8866 substantially enhanced the efficacy of docetaxel chemotherapy, resulting in rapid regression of MYC-overexpressing tumors. Collectively, these data establish the synthetic lethal interaction of the IRE1/XBP1 pathway with MYC hyperactivation and provide a potential therapy for MYC-driven human breast cancers.

Induced GnasR201H expression from the endogenous Gnas locus causes fibrous dysplasia by up-regulating Wnt/ß-catenin signaling.

Fibrous dysplasia (FD; Online Mendelian Inheritance in Man no. 174800) is a crippling skeletal disease caused by activating mutations of the GNAS gene, which encodes the stimulatory G protein Gαs FD can lead to severe adverse conditions such as bone deformity, fracture, and severe pain, leading to functional impairment and wheelchair confinement. So far there is no cure, as the underlying molecular and cellular mechanisms remain largely unknown and the lack of appropriate animal models has severely hampered FD research. Here we have investigated the cellular and molecular mechanisms underlying FD and tested its potential treatment by establishing a mouse model in which the human FD mutation (R201H) has been conditionally knocked into the corresponding mouse Gnas locus. We found that the germ-line FD mutant was embryonic lethal, and Cre-induced Gnas FD mutant expression in early osteochondral progenitors, osteoblast cells, or bone marrow stromal cells (BMSCs) recapitulated FD features. In addition, mosaic expression of FD mutant Gαs in BMSCs induced bone marrow fibrosis both cell autonomously and non-cell autonomously. Furthermore, Wnt/ß-catenin signaling was up-regulated in FD mutant mouse bone and BMSCs undergoing osteogenic differentiation, as we have found in FD human tissue previously. Reduction of Wnt/ß-catenin signaling by removing one Lrp6 copy in an FD mutant line significantly rescued the phenotypes. We demonstrate that induced expression of the FD Gαs mutant from the mouse endogenous Gnas locus exhibits human FD phenotypes in vivo, and that inhibitors of Wnt/ß-catenin signaling may be repurposed for treating FD and other bone diseases caused by Gαs activation.

Klotho expression in osteocytes regulates bone metabolism and controls bone formation.

Osteocytes within the mineralized bone matrix control bone remodeling by regulating osteoblast and osteoclast activity. Osteocytes express the aging suppressor Klotho, but the functional role of this protein in skeletal homeostasis is unknown. Here we identify Klotho expression in osteocytes as a potent regulator of bone formation and bone mass. Targeted deletion of Klotho from osteocytes led to a striking increase in bone formation and bone volume coupled with enhanced osteoblast activity, in sharp contrast to what is observed in Klotho hypomorphic (kl/kl) mice. Conversely, overexpression of Klotho in cultured osteoblastic cells inhibited mineralization and osteogenic activity during osteocyte differentiation. Further, the induction of chronic kidney disease with high-turnover renal osteodystrophy led to downregulation of Klotho in bone cells. This appeared to offset the skeletal impact of osteocyte-targeted Klotho deletion. Thus, our findings establish a key role of osteocyte-expressed Klotho in regulating bone metabolism and indicate a new mechanism by which osteocytes control bone formation.

Renal Fanconi Syndrome and Hypophosphatemic Rickets in the Absence of Xenotropic and Polytropic Retroviral Receptor in the Nephron.

Tight control of extracellular and intracellular inorganic phosphate (Pi) levels is critical to most biochemical and physiologic processes. Urinary Pi is freely filtered at the kidney glomerulus and is reabsorbed in the renal tubule by the action of the apical sodium-dependent phosphate transporters, NaPi-IIa/NaPi-IIc/Pit2. However, the molecular identity of the protein(s) participating in the basolateral Pi efflux remains unknown. Evidence has suggested that xenotropic and polytropic retroviral receptor 1 (XPR1) might be involved in this process. Here, we show that conditional inactivation of Xpr1 in the renal tubule in mice resulted in impaired renal Pi reabsorption. Analysis of Pi transport in primary cultures of proximal tubular cells or in freshly isolated renal tubules revealed that this Xpr1 deficiency significantly affected Pi efflux. Further, mice with conditional inactivation of Xpr1 in the renal tubule exhibited generalized proximal tubular dysfunction indicative of Fanconi syndrome, characterized by glycosuria, aminoaciduria, calciuria, and albuminuria. Dramatic alterations in the renal transcriptome, including a significant reduction in NaPi-IIa/NaPi-IIc expression, accompanied these functional changes. Additionally, Xpr1-deficient mice developed hypophosphatemic rickets secondary to renal dysfunction. These results identify XPR1 as a major regulator of Pi homeostasis and as a potential therapeutic target in bone and kidney disorders.

Regulation of Sclerostin Expression in Multiple Myeloma by Dkk-1: A Potential Therapeutic Strategy for Myeloma Bone Disease.

Sclerostin is a potent inhibitor of osteoblastogenesis. Interestingly, newly diagnosed multiple myeloma (MM) patients have high levels of circulating sclerostin that correlate with disease stage and fractures. However, the source and impact of sclerostin in MM remains to be defined. Our goal was to determine the role of sclerostin in the biology of MM and its bone microenvironment as well as investigate the effect of targeting sclerostin with a neutralizing antibody (scl-Ab) in MM bone disease. Here we confirm increased sclerostin levels in MM compared with precursor disease states like monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM. Furthermore, we found that a humanized MM xenograft mouse model bearing human MM cells (NOD-SCID.CB17 male mice injected intravenously with 2.5 million of MM1.S-Luc-GFP cells) demonstrated significantly higher concentrations of mouse-derived sclerostin, suggesting a microenvironmental source of sclerostin. Associated with the increased sclerostin levels, activated ß-catenin expression levels were lower than normal in MM mouse bone marrow. Importantly, a high-affinity grade scl-Ab reversed osteolytic bone disease in this animal model. Because scl-Ab did not demonstrate significant in vitro anti-MM activity, we combined it with the proteasome inhibitor carfilzomib. Our data demonstrated that this combination therapy significantly inhibited tumor burden and improved bone disease in our in vivo MM mouse model. In agreement with our in vivo data, sclerostin expression was noted in marrow stromal cells and osteoblasts of MM patient bone marrow samples. Moreover, MM cells stimulated sclerostin expression in immature osteoblasts while inhibiting osteoblast differentiation in vitro. This was in part regulated by Dkk-1 secreted by MM cells and is a potential mechanism contributing to the osteoblast dysfunction noted in MM. Our data confirm the role of sclerostin as a potential therapeutic target in MM bone disease and provides the rationale for studying scl-Ab combined with proteasome inhibitors in MM. © 2016 American Society for Bone and Mineral Research.

CHMP5 controls bone turnover rates by dampening NF-κB activity in osteoclasts.

Physiological bone remodeling requires that bone formation by osteoblasts be tightly coupled to bone resorption by osteoclasts. However, relatively little is understood about how this coupling is regulated. Here, we demonstrate that modulation of NF-κB signaling in osteoclasts via a novel activity of charged multivesicular body protein 5 (CHMP5) is a key determinant of systemic rates of bone turnover. A conditional deletion of CHMP5 in osteoclasts leads to increased bone resorption by osteoclasts coupled with exuberant bone formation by osteoblasts, resembling an early onset, polyostotic form of human Paget's disease of bone (PDB). These phenotypes are reversed by haploinsufficiency for Rank, as well as by antiresorptive treatments, including alendronate, zolendronate, and OPG-Fc. Accordingly, CHMP5-deficient osteoclasts display increased RANKL-induced NF-κB activation and osteoclast differentiation. Biochemical analysis demonstrated that CHMP5 cooperates with the PDB genetic risk factor valosin-containing protein (VCP/p97) to stabilize the inhibitor of NF-κBα (IκBα), down-regulating ubiquitination of IκBα via the deubiquitinating enzyme USP15. Thus, CHMP5 tunes NF-κB signaling downstream of RANK in osteoclasts to dampen osteoclast differentiation, osteoblast coupling and bone turnover rates, and disruption of CHMP5 activity results in a PDB-like skeletal disorder.

Tetraspanin CD9 and ectonucleotidase CD73 identify an osteochondroprogenitor population with elevated osteogenic properties.

Cell-based bone regeneration strategies offer promise for traumatic bone injuries, congenital defects, non-union fractures and other skeletal pathologies. Postnatal bone remodeling and fracture healing provide evidence that an osteochondroprogenitor cell is present in adult life that can differentiate to remodel or repair the fractured bone. However, cell-based skeletal repair in the clinic is still in its infancy, mostly due to poor characterization of progenitor cells and lack of knowledge about their in vivo behavior. Here, we took a combined approach of high-throughput screening, flow-based cell sorting and in vivo transplantation to isolate markers that identify osteochondroprogenitor cells. We show that the presence of tetraspanin CD9 enriches for osteochondroprogenitors within CD105(+) mesenchymal cells and that these cells readily form bone upon transplantation. In addition, we have used Thy1.2 and the ectonucleotidase CD73 to identify subsets within the CD9(+) population that lead to endochondral or intramembranous-like bone formation. Utilization of this unique cell surface phenotype to enrich for osteochondroprogenitor cells will allow for further characterization of the molecular mechanisms that regulate their osteogenic properties.