Skeletal recovery after weaning does not require PTHrP

Mice lose 20% to 25% of trabecular bone mineral content (BMC) during lactation and restore it after weaning through unknown mechanisms. We found that tibial Pthrp mRNA expression was upregulated fivefold by 7 days after weaning versus end of lactation in wild‐type (WT) mice. To determine whether parathyroid hormone–related protein (PTHrP) stimulates bone formation after weaning, we studied a conditional knockout in which PTHrP is deleted from preosteoblasts and osteoblasts by collagen I promoter–driven Cre (Cre^ColI). These mice are osteopenic as adults but have normal serum calcium, calcitriol, and parathyroid hormone (PTH). Pairs of Pthrp^flox/flox;Cre^ColI (null) and WT;Cre^ColI (WT) females were mated and studied through pregnancy, lactation, and 3 weeks of postweaning recovery. By end of lactation, both genotypes lost lumbar spine BMC: WT declined by 20.6% ± 3.3%, and null decreased by 22.5% ± 3.5% (p < .0001 versus baseline; p = NS between genotypes). During postweaning recovery, both restored BMC to baseline: WT to –3.6% ± 3.7% and null to 0.3% ± 3.7% (p = NS versus baseline or between genotypes). Similar loss and full recovery of BMC were seen at the whole body and hind limb. Histomorphometry confirmed that nulls had lower bone mass at baseline and that this was equal to the value achieved after weaning. Osteocalcin, propeptide of type 1 collagen (P1NP), and deoxypyridinoline increased equally during recovery in WT and null mice; PTH decreased and calcitriol increased equally; serum calcium was unchanged. Urine calcium increased during recovery but remained no different between genotypes. Although osteoblast‐derived PTHrP is required to maintain adult bone mass and Pthrp mRNA upregulates in bone after weaning, it is not required for recovery of bone mass after lactation. The factors that stimulate postweaning bone formation remain unknown. © 2011 American Society for Bone and Mineral Research.     

Defects in mesenchymal stem cell self‐renewal and cell fate determination lead to an osteopenic phenotype in Bmi‐1 null mice

In parathyroid hormone–related protein 1‐84 [PTHrP(1‐84)] knockin mice, expression of the polycomb protein Bmi‐1 is reduced and potentially can mediate the phenotypic alterations observed. We have therefore now examined the skeletal phenotype of Bmi‐1^?/? mice in vivo and also assessed the function of bone marrow mesenchymal stem cells (BM‐MSCs) from Bmi‐1^?/? mice ex vivo in culture. Neonatal Bmi‐1^?/? mice exhibited skeletal growth retardation, with reduced chondrocyte proliferation and increased apoptosis. Osteoblast numbers; gene expression of alkaline phosphatase, type I collagen, and osteocalcin; the mineral apposition rate; trabecular bone volume; and bone mineral density all were reduced significantly; however, the number of bone marrow adipocytes and Ppar‐γ expression were increased. These changes were consistent with the skeletal phenotype observed in the PTHrP(1‐84) knockin mouse. The efficiency of colony‐forming unit fibroblast (CFU‐F) formation in bone marrow cultures was decreased, and the percentage of alkaline phosphatase–positive CFU‐F and Runx2 expression were reduced. In contrast, adipocyte formation and Ppar‐γ expression in cultures were increased, and expression of the polycomb protein sirtuin (Sirt1) was reduced. Reduced proliferation and increased apoptosis of BM‐MSCs were associated with upregulation of senescence‐associated tumor‐suppressor genes, including p16, p19, and p27. Analysis of the skeletal phenotype in Bmi‐1^?/? mice suggests that Bmi‐1 functions downstream of PTHrP. Furthermore, our studies indicate that Bmi‐1 maintains self‐renewal of BM‐MSCs by inhibiting the expression of p27, p16, and p19 and alters the cell fate of BM‐MSCs by enhancing osteoblast differentiation and inhibiting adipocyte differentiation at least in part by stimulating Sirt1 expression. Bmi‐1 therefore plays a critical role in promoting osteogenesis. © 2010 American Society for Bone and Mineral Research     

Increased IL‐6 Expression in Osteoclasts Is Necessary But Not Sufficient for the Development of Paget's Disease of Bone

Measles virus nucleocapsid protein (MVNP) expression in osteoclasts (OCLs) and mutation of the SQSTM1 (p62) gene contribute to the increased OCL activity in Paget's disease (PD). OCLs expressing MVNP display many of the features of PD OCLs. Interleukin‐6 (IL‐6) production is essential for the pagetic phenotype, because transgenic mice with MVNP targeted to OCLs develop pagetic OCLs and lesions, but this phenotype is absent when MVNP mice are bred to IL‐6^–/– mice. In contrast, mutant p62 expression in OCL precursors promotes receptor activator of NF‐κB ligand (RANKL) hyperresponsivity and increased OCL production, but OCLs that form have normal morphology, are not hyperresponsive to 1,25‐dihydroxyvitamin D₃ (1,25‐(OH)₂D₃), nor produce elevated levels of IL‐6. We previously generated p62^P394L knock‐in mice (p62KI) and found that although OCL numbers were increased, the mice did not develop pagetic lesions. However, mice expressing both MVNP and p62KI developed more exuberant pagetic lesions than mice expressing MVNP alone. To examine the role of elevated IL‐6 in PD and determine if MVNP mediates its effects primarily through elevation of IL‐6, we generated transgenic mice that overexpress IL‐6 driven by the tartrate‐resistant acid phosphatase (TRAP) promoter (TIL‐6 mice) and produce IL‐6 at levels comparable to MVNP mice. These were crossed with p62KI mice to determine whether IL‐6 overexpression cooperates with mutant p62 to produce pagetic lesions. OCL precursors from p62KI/TIL‐6 mice formed greater numbers of OCLs than either p62KI or TIL‐6 OCL precursors in response to 1,25‐(OH)₂D₃. Histomorphometric analysis of bones from p62KI/TIL‐6 mice revealed increased OCL numbers per bone surface area compared to wild‐type (WT) mice. However, micro‐quantitative CT (µQCT) analysis did not reveal significant differences between p62KI/TIL‐6 and WT mice, and no pagetic OCLs or lesions were detected in vivo. Thus, increased IL‐6 expression in OCLs from p62KI mice contributes to increased responsivity to 1,25‐(OH)₂D₃ and increased OCL numbers, but is not sufficient to induce Paget's‐like OCLs or bone lesions in vivo. © 2014 American Society for Bone and Mineral Research.     

The Notch Ligand Jagged1 Regulates the Osteoblastic Lineage by Maintaining the Osteoprogenitor Pool

Notch signaling is critical for osteoblastic differentiation; however, the specific contribution of individual Notch ligands is unknown. Parathyroid hormone (PTH) regulates the Notch ligand Jagged1 in osteoblastic cells. To determine if osteolineage Jagged1 contributes to bone homeostasis, selective deletion of Jagged1 in osteolineage cells was achieved through the presence of Prx1 promoter‐driven Cre recombinase expression, targeting mesenchymal stem cells (MSCs) and their progeny (PJag1 mice). PJag1 mice were viable and fertile and did not exhibit any skeletal abnormalities at 2 weeks of age. At 2 months of age, however, PJag1 mice had increased trabecular bone mass compared to wild‐type (WT) littermates. Dynamic histomorphometric analysis showed increased osteoblastic activity and increased mineral apposition rate. Immunohistochemical analysis showed increased numbers of osteocalcin‐positive mature osteoblasts in PJag1 mice. Also increased phenotypically defined Lin^–/CD45^–/CD31^–/Sca1^–/CD51⁺ osteoblastic cells were measured by flow cytometric analysis. Surprisingly, phenotypically defined Lin^–/CD45^–/CD31^–/Sca1⁺/CD51⁺ MSCs were unchanged in PJag1 mice as measured by flow cytometric analysis. However, functional osteoprogenitor (OP) cell frequency, measured by Von Kossa⁺ colony formation, was decreased, suggesting that osteolineage Jagged1 contributes to maintenance of the OP pool. The trabecular bone increases were not due to osteoclastic defects, because PJag1 mice had increased bone resorption. Because PTH increases osteoblastic Jagged1, we sought to understand if osteolineage Jagged1 modulates PTH‐mediated bone anabolism. Intermittent PTH treatment resulted in a significantly greater increase in BV/TV in PJag1 hind limbs compared to WT. These findings demonstrate a critical role of osteolineage Jagged1 in bone homeostasis, where Jagged1 maintains the transition of OP to maturing osteoblasts. This novel role of Jagged1 not only identifies a regulatory loop maintaining appropriate populations of osteolineage cells, but also provides a novel approach to increase trabecular bone mass, particularly in combination with PTH, through modulation of Jagged1. © 2017 American Society for Bone and Mineral Research.     

Increased mandibular condylar growth in mice with estrogen receptor beta deficiency

Temporomandibular joint (TMJ) disorders predominantly afflict women of childbearing age, suggesting a role for female hormones in the disease process. In long bones, estrogen acting via estrogen receptor beta (ERβ) inhibits axial skeletal growth in female mice. However, the role of ERβ in the mandibular condyle is largely unknown. We hypothesize that female ERβ‐deficient mice will have increased mandibular condylar growth compared to wild‐type (WT) female mice. This study examined female 7‐day‐old, 49‐day‐old, and 120‐day‐old WT and ERβ knockout (KO) mice. There was a significant increase in mandibular condylar cartilage thickness as a result of an increased number of cells, in the 49‐day‐old and 120‐day‐old female ERβ KO compared with WT controls. Analysis in 49‐day‐old female ERβ KO mice revealed a significant increase in collagen type X, parathyroid hormone–related protein (Pthrp), and osteoprotegerin gene expression and a significant decrease in receptor activator for nuclear factor κ B ligand (Rankl) and Indian hedgehog (Ihh) gene expression, compared with WT controls. Subchondral bone analysis revealed a significant increase in total condylar volume and a decrease in the number of osteoclasts in the 49‐day‐old ERβ KO compared with WT female mice. There was no difference in cell proliferation in condylar cartilage between the genotypes. However, there were differences in the expression of proteins that regulate the cell cycle; we found a decrease in the expression of Tieg1 and p57 in the mandibular condylar cartilage from ERβ KO mice compared with WT mice. Taken together, our results suggest that ERβ deficiency increases condylar growth in female mice by inhibiting the turnover of fibrocartilage. © 2013 American Society for Bone and Mineral Research.     

Endogenous parathyroid hormone–related protein compensates for the absence of parathyroid hormone in promoting bone accrual in vivo in a model of bone marrow ablation

To assess the effect of hypoparathyroidism on osteogenesis and bone turnover in vivo, bone marrow ablation (BMXs) were performed in tibias of 8‐week‐old wild‐type and parathyroid hormone–null (PTH^?/?) mice and newly formed bone tissue was analyzed from 5 days to 3 weeks after BMX. At 1 week after BMX, trabecular bone volume, osteoblast numbers, alkaline phosphatase‐positive areas, type I collagen‐positive areas, PTH receptor–positive areas, calcium sensing receptor–positive areas, and expression of bone formation–related genes were all decreased significantly in the diaphyseal regions of bones of PTH^?/? mice compared to wild‐type mice. In contrast, by 2 weeks after BMX, all parameters related to osteoblastic bone accrual were increased significantly in PTH^?/? mice. At 5 days after BMX, active tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts had appeared in wild‐type mice but were undetectable in PTH^?/? mice, Both the ratio of mRNA levels of receptor activator of NF‐κB ligand (RANKL)/osteoprotegerin (OPG) and TRAP‐positive osteoclast surface were still reduced in PTH^?/? mice at 1 week but were increased by 2 weeks after BMX. The expression levels of parathyroid hormone–related protein (PTHrP) at both mRNA and protein levels were upregulated significantly at 1 week and more dramatically at 2 weeks after BMX in PTH^?/? mice. To determine whether the increased newly formed bones in PTH^?/? mice at 2 weeks after BMX resulted from the compensatory action of PTHrP, PTH^?/?PTHrP^+/? mice were generated and newly formed bone tissue was compared in these mice with PTH^?/? and wild‐type mice at 2 weeks after BMX. All parameters related to osteoblastic bone formation and osteoclastic bone resorption were reduced significantly in PTH^?/?PTHrP^+/? mice compared to PTH^?/? mice. These results demonstrate that PTH deficiency itself impairs osteogenesis, osteoclastogenesis, and osteoclastic bone resorption, whereas subsequent upregulation of PTHrP in osteogenic cells compensates by increasing bone accrual. © 2013 American Society for Bone and Mineral Research     

Introduction of a Phe377del Mutation in ANK Creates a Mouse Model for Craniometaphyseal Dysplasia

Craniometaphyseal dysplasia (CMD) is a monogenic human disorder characterized by thickening of craniofacial bones and flaring metaphyses of long bones. Mutations for autosomal dominant CMD have been identified in the progressive ankylosis gene ANKH. Previous studies of Ank loss‐of‐function models, Ank^null/null and Ank^ank/ank mice, suggest that Ank plays a role in the regulation of bone mineralization. However, the mechanism for Ank mutations leading to CMD remains unknown. We generated the first knockin (KI) mouse model for CMD expressing a human mutation (Phe377 deletion) in ANK. Homozygous Ank knockin mice (Ank^KI/KI) replicate many typical features of human CMD including hyperostosis of craniofacial bones, massive jawbones, decreased diameters of cranial foramina, obliteration of nasal sinuses, fusion of middle ear bones, and club‐shaped femurs. In addition, Ank^KI/KI mice have increased serum alkaline phosphatase and TRACP5b, as reported in CMD patients. Biochemical markers of bone formation and bone resorption, N‐terminal propeptide of type I procollagen and type I collagen cross‐linked C‐terminal telopeptide, are significantly increased in Ank^KI/KI mice, suggesting increased bone turnover. Interestingly, Ank^KI/KI bone marrow–derived macrophage cultures show decreased osteoclastogenesis. Despite the hyperostotic phenotype, bone matrix in Ank^KI/KI mice is hypomineralized and less mature, indicating that biomechanical properties of bones may be compromised by the Ank mutation. We believe this new mouse model will facilitate studies of skeletal abnormalities in CMD at cellular and molecular levels.     

Peripheral leptin regulates bone formation

Substantial evidence does not support the prevailing view that leptin, acting through a hypothalamic relay, decreases bone accrual by inhibiting bone formation. To clarify the mechanisms underlying regulation of bone architecture by leptin, we evaluated bone growth and turnover in wild‐type (WT) mice, leptin receptor‐deficient db/db mice, leptin‐deficient ob/ob mice, and ob/ob mice treated with leptin. We also performed hypothalamic leptin gene therapy to determine the effect of elevated hypothalamic leptin levels on osteoblasts. Finally, to determine the effects of loss of peripheral leptin signaling on bone formation and energy metabolism, we used bone marrow (BM) from WT or db/db donor mice to reconstitute the hematopoietic and mesenchymal stem cell compartments in lethally irradiated WT recipient mice. Decreases in bone growth, osteoblast‐lined bone perimeter and bone formation rate were observed in ob/ob mice and greatly increased in ob/ob mice following subcutaneous administration of leptin. Similarly, hypothalamic leptin gene therapy increased osteoblast‐lined bone perimeter in ob/ob mice. In spite of normal osteoclast‐lined bone perimeter, db/db mice exhibited a mild but generalized osteopetrotic‐like (calcified cartilage encased by bone) skeletal phenotype and greatly reduced serum markers of bone turnover. Tracking studies and histology revealed quantitative replacement of BM cells following BM transplantation. WT mice engrafted with db/db BM did not differ in energy homeostasis from untreated WT mice or WT mice engrafted with WT BM. Bone formation in WT mice engrafted with WT BM did not differ from WT mice, whereas bone formation in WT mice engrafted with db/db cells did not differ from the low rates observed in untreated db/db mice. In summary, our results indicate that leptin, acting primarily through peripheral pathways, increases osteoblast number and activity. © 2013 American Society for Bone and Mineral Research     

CRMP4 Inhibits Bone Formation by Negatively Regulating BMP and RhoA Signaling

We identified the neuroprotein collapsing response mediator protein‐4 (CRMP4) as a noncanonical osteogenic factor that regulates the differentiation of mouse bone marrow skeletal stem cells (bone marrow stromal stem cells [mBMSCs]) into osteoblastic cells. CRMP4 is the only member of the CRMP1–CRMP5 family to be expressed by mBMSCs and in osteoprogenitors of both adult mouse and human bones. In vitro gain‐of‐function and loss‐of‐function of CRMP4 in murine stromal cells revealed its inhibitory effect on osteoblast differentiation. In addition, Crmp4‐deficient mice (Crmp4^–/–) displayed a 40% increase in bone mass, increased mineral apposition rate, and bone formation rate, compared to wild‐type controls. Increased bone mass in Crmp4^–/– mice was associated with enhanced BMP2 signaling and BMP2‐induced osteoblast differentiation in Crmp4^–/– osteoblasts (OBs). Furthermore, Crmp4^–/– OBs exhibited enhanced activation of RhoA/focal adhesion kinase (FAK) signaling that led to cytoskeletal changes with increased cell spreading. In addition, Crmp4^–/– OBs exhibited increased cell proliferation that was mediated via inhibiting cyclin‐dependent kinase inhibitor 1B, p27^Kip1 and upregulating cyclin D1 expression which are targets of RhoA signaling pathway. Our findings identify CRMP4 as a novel negative regulator of osteoblast differentiation. © 2016 American Society for Bone and Mineral Research.     

ADAM8 enhances osteoclast precursor fusion and osteoclast formation in vitro and in vivo

ADAM8 expression is increased in the interface tissue around a loosened hip prosthesis and in the pannus and synovium of patients with rheumatoid arthritis, but its potential role in these processes is unclear. ADAM8 stimulates osteoclast (OCL) formation, but the effects of overexpression or loss of expression of ADAM8 in vivo and the mechanisms responsible for the effects of ADAM8 on osteoclastogenesis are unknown. Therefore, to determine the effects of modulating ADAM expression, we generated tartrate‐resistant acid phosphatase (TRAP)–ADAM8 transgenic mice that overexpress ADAM8 in the OCL lineage and ADAM8 knockout (ADAM8 KO) mice. TRAP‐ADAM8 mice developed osteopenia and had increased numbers of OCL precursors that formed hypermultinucleated OCLs with an increased bone‐resorbing capacity per OCL. They also had an enhanced differentiation capacity, increased TRAF6 expression, and increased NF‐κB, Erk, and Akt signaling compared with wild‐type (WT) littermates. This increased bone‐resorbing capacity per OCL was associated with increased levels of p‐Pyk2 and p‐Src activation. In contrast, ADAM8 KO mice did not display a bone phenotype in vivo, but unlike WT littermates, they did not increase RANKL production, OCL formation, or calvarial fibrosis in response to tumor necrosis factor α (TNF‐α) in vivo. Since loss of ADAM8 does not inhibit basal bone remodeling but only blocks the enhanced OCL formation in response to TNF‐α, these results suggest that ADAM8 may be an attractive therapeutic target for preventing bone destruction associated with inflammatory disease. © 2011 American Society for Bone and Mineral Research.     

Cherubism Mice Also Deficient in c‐Fos Exhibit Inflammatory Bone Destruction Executed by Macrophages That Express MMP14 Despite the Absence of TRAP+ Osteoclasts

Currently, it is believed that osteoclasts positive for tartrate‐resistant acid phosphatase (TRAP+) are the exclusive bone‐resorbing cells responsible for focal bone destruction in inflammatory arthritis. Recently, a mouse model of cherubism (Sh3bp2^KI/KI) with a homozygous gain‐of‐function mutation in the SH3‐domain binding protein 2 (SH3BP2) was shown to develop auto‐inflammatory joint destruction. Here, we demonstrate that Sh3bp2^KI/KI mice also deficient in the FBJ osteosarcoma oncogene (c‐Fos) still exhibit noticeable bone erosion at the distal tibia even in the absence of osteoclasts at 12 weeks old. Levels of serum collagen I C‐terminal telopeptide (ICTP), a marker of bone resorption generated by matrix metalloproteinases (MMPs), were elevated, whereas levels of serum cross‐linked C‐telopeptide (CTX), another resorption marker produced by cathepsin K, were not increased. Collagenolytic MMP levels were increased in the inflamed joints of the Sh3bp2^KI/KI mice deficient in c‐Fos. Resorption pits contained a large number of F4/80+ macrophages and genetic depletion of macrophages rescued these erosive changes. Importantly, administration of NSC405020, an MMP14 inhibitor targeted to the hemopexin (PEX) domain, suppressed bone erosion in c‐Fos‐deficient Sh3bp2^KI/KI mice. After activation of the NF‐κB pathway, macrophage colony‐stimulating factor (M‐CSF)‐dependent macrophages from c‐Fos‐deficient Sh3bp2^KI/KI mice expressed increased amounts of MMP14 compared with wild‐type macrophages. Interestingly, receptor activator of NF‐κB ligand (RANKL)‐deficient Sh3bp2^KI/KI mice failed to show notable bone erosion, whereas c‐Fos deletion did restore bone erosion to the RANKL‐deficient Sh3bp2^KI/KI mice, suggesting that osteolytic transformation of macrophages requires both loss‐of‐function of c‐Fos and gain‐of‐function of SH3BP2 in this model. These data provide the first genetic evidence that cells other than osteoclasts can cause focal bone destruction in inflammatory bone disease and suggest that MMP14 is a key mediator conferring pathological bone‐resorbing capacity on c‐Fos‐deficient Sh3bp2^KI/KI macrophages. In summary, the paradigm that osteoclasts are the exclusive cells executing inflammatory bone destruction may need to be reevaluated based on our findings with c‐Fos‐deficient cherubism mice lacking osteoclasts. © 2017 American Society for Bone and Mineral Research.     

Impact of Pregnancy‐Associated Plasma Protein‐A Deletion on the Adult Murine Skeleton

Introduction : The metalloproteinase, pregnancy‐associated plasma protein‐A (PAPP‐A) functions to enhance local insulin‐like growth factor (IGF)‐I bioavailability through cleavage of inhibitory IGF binding proteins. Because IGF‐I is an important regulator of skeletal growth and remodeling and PAPP‐A is highly expressed by osteoblastic cells, we hypothesized that, in the absence of PAPP‐A, bone physiology would be compromised because of a blunting of local IGF‐I action even in the presence of normal circulating IGF‐I levels. Materials and Methods : pQCT, μCT, histomorphometry, and mechanical strength testing were performed on bones from PAPP‐A knockout (KO) mice and wildtype (WT) littermates at 2–12 mo of age. IGF‐I levels and bone formation and resorption markers were determined in sera from these animals. Results : Volumetric BMD in PAPP‐A KO mice measured by pQCT at the femoral midshaft, which is primarily cortical bone, was 10% less than WT at 2 mo. This difference was maintained at 4, 6, and 12 mo. Cortical thickness at this site was similarly decreased. On the other hand, trabecular bone at the distal femur (pQCT) and in the tibia (μCT) showed age‐progressive decreases in bone volume fraction in PAPP‐A KO compared with WT mice. Tibial μCT indicated a 46% relative decrease in trabecular bone volume/total volume (BV/TV) and a 28% relative decrease in trabecular thickness in PAPP‐A KO compared with WT mice at 6 mo. These trabecular deficiencies in PAPP‐A KO mice corresponded to a weakening of the bone. Serum markers and bone histomorphometry indicated that the primary impact of PAPP‐A is on skeletal remodeling resulting in a state of low‐turnover osteopenia in adult PAPP‐A KO mice. Circulating IGF‐I levels were not altered in PAPP‐A KO mice. Conclusions : PAPP‐A is a bone growth regulatory factor in vivo and, in its absence, mice show skeletal insufficiency in mass, density, architecture, and strength. The data suggest a primary role for PAPP‐A in modulating local IGF bioavailability for trabecular bone remodeling.     

Targeting the LRP5 Pathway Improves Bone Properties in a Mouse Model of Osteogenesis Imperfecta

The cell surface receptor low‐density lipoprotein receptor‐related protein 5 (LRP5) is a key regulator of bone mass and bone strength. Heterozygous missense mutations in LRP5 cause autosomal dominant high bone mass (HBM) in humans by reducing binding to LRP5 by endogenous inhibitors, such as sclerostin (SOST). Mice heterozygous for a knockin allele (Lrp5^p.A214V) that is orthologous to a human HBM‐causing mutation have increased bone mass and strength. Osteogenesis imperfecta (OI) is a skeletal fragility disorder predominantly caused by mutations that affect type I collagen. We tested whether the LRP5 pathway can be used to improve bone properties in animal models of OI. First, we mated Lrp5^+/p.A214V mice to Col1a2^+/p.G610C mice, which model human type IV OI. We found that Col1a2^+/p.G610C;Lrp5^+/p.A214V offspring had significantly increased bone mass and strength compared to Col1a2^+/p.G610C;Lrp5^+/+ littermates. The improved bone properties were not a result of altered mRNA expression of type I collagen or its chaperones, nor were they due to changes in mutant type I collagen secretion. Second, we treated Col1a2^+/p.G610C mice with a monoclonal antibody that inhibits sclerostin activity (Scl‐Ab). We found that antibody‐treated mice had significantly increased bone mass and strength compared to vehicle‐treated littermates. These findings indicate increasing bone formation, even without altering bone collagen composition, may benefit patients with OI. © 2014 American Society for Bone and Mineral Research.     

Disruption of LRP6 in osteoblasts blunts the bone anabolic activity of PTH

Mutations in low‐density lipoprotein receptor‐related protein 6 (LRP6) are associated with human skeletal disorders. LRP6 is required for parathyroid hormone (PTH)‐stimulated signaling pathways in osteoblasts. We investigated whether LRP6 in osteoblasts directly regulates bone remodeling and mediates the bone anabolic effects of PTH by specifically deleting LRP6 in mature osteoblasts in mice (LRP6 KO). Three‐month‐old LRP6 KO mice had a significant reduction in bone mass in the femora secondary spongiosa relative to their wild‐type littermates, whereas marginal changes were found in femoral tissue of 1‐month‐old LRP6 KO mice. The remodeling area of the 3‐month‐old LRP6 KO mice showed a decreased bone formation rate as detected by Goldner's Trichrome staining and calcein double labeling. Bone histomorphometric and immumohistochemical analysis revealed a reduction in osteoblasts but little change in the numbers of osteoclasts and osteoprogenitors/osteoblast precursors in LRP6 KO mice compared with wild‐type littermates. In addition, the percentage of the apoptotic osteoblasts on the bone surface was higher in LRP6 KO mice compared with wild‐type littermates. Intermittent injection of PTH had no effect on bone mass or osteoblastic bone formation in either trabecular and cortical bone in LRP6 KO mice, whereas all were enhanced in wild‐type littermates. Additionally, the anti‐apoptotic effect of PTH on osteoblasts in LRP6 KO mice was less significant compared with wild‐type mice. Therefore, our findings demonstrate that LRP6 in osteoblasts is essential for osteoblastic differentiation during bone remodeling and the anabolic effects of PTH. © 2013 American Society for Bone and Mineral Research.     

Dmp1Cre-directed knockdown of parathyroid hormone–related protein (PTHrP) in murine decidua is associated with a life-long increase in bone mass,width, and strength in male progeny

Parathyroid hormone–related protein (PTHrP, gene name Pthlh) is a pleiotropic regulator of tissue homeostasis. In bone, Dmp1Cre-targeted PTHrP deletion in osteocytes causes osteopenia and impaired cortical strength. We report here that this outcome depends on parental genotype. In contrast to our previous report using mice bred from heterozygous (flox/wild type) Dmp1Cre.Pthlh^f/w parents, adult (16-week-old and 26-week-old) flox/flox (f/f) Dmp1Cre.Pthlh^f/f mice from homozygous parents (Dmp1Cre.Pthlh^f/f(hom)) have stronger bones, with 40% more trabecular bone mass and 30% greater femoral width than controls. This greater bone size was observed in Dmp1Cre.Pthlh^f/f(hom) mice as early as 12 days of age, when greater bone width was also found in male and female Dmp1Cre.Pthlh^f/f(hom) mice compared to controls, but not in gene-matched mice from heterozygous parents. This suggested a maternal influence on skeletal size prior to weaning. Although Dmp1Cre has previously been reported to cause gene recombination in mammary gland, milk PTHrP protein levels were normal. The wide-bone phenotype was also noted in utero: Dmp1Cre.Pthlh^f/f(hom) embryonic femurs were more mineralized and wider than controls. Closer examination revealed that Dmp1Cre caused PTHrP recombination in placenta, and in the maternal-derived decidual layer that resides between the placenta and the uterus. Decidua from mothers of Dmp1Cre.Pthlh^f/f(hom) mice also exhibited lower PTHrP levels by immunohistochemistry and were smaller than controls. We conclude that Dmp1Cre leads to gene recombination in decidua, and that decidual PTHrP might, through an influence on decidual cells, limit embryonic bone radial growth. This suggests a maternal-derived developmental origin of adult bone strength. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Introduction of a Cys360Tyr Mutation in ANO5 Creates a Mouse Model for Gnathodiaphyseal Dysplasia

Gnathodiaphyseal dysplasia (GDD) is a rare autosomal dominant genetic disease characterized by the osteosclerosis of tubular bones and the formation of cemento-osseous lesions in mandibles. Although genetic mutations for GDD have been identified in the ANO5/TMEM16E gene, the cellular and molecular mechanisms behind the pathogenesis of GDD remain unclear. Here, we generated the first knock-in mouse model for GDD with the expression of human mutation p.Cys360Tyr in ANO5. Homozygous Ano5 knock-in mice (Ano5^KI/KI) replicated GDD-like skeletal features, including massive jawbones, bowing tibia, bone fragility, sclerosis, and cortical thickening of the femoral and tibial diaphysis. Serum alkaline phosphatase (ALP) levels were elevated in Ano5^KI/KI mice as in GDD patients with p.Cys360Tyr mutation. Calvaria-derived Ano5^KI/KI osteoblast cultures showed increased osteoblastogenesis, including hypermineralized bone matrix and enhanced bone formation-related factors expression. Interestingly, Ano5^KI/KI bone marrow-derived macrophage cultures showed decreased osteoclastogenesis, and Ano5^KI/KI osteoclasts exhibited disrupted actin ring formation, which may be associated with some signaling pathways. In conclusion, this new mouse model may facilitate elucidation of the pathogenesis of GDD and shed more light on its treatment. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Transient Exposure to PTHrP (107-139) Exerts Anabolic Effects through Vascular Endothelial Growth Factor Receptor 2 in Human Osteoblastic Cells In Vitro

Intermittent administration of the N-terminal fragment of parathyroid hormone (PTH) and PTH-related protein (PTHrP) induces bone anabolic effects. However, the effects of the C-terminal domain of PTHrP on bone turnover remain controversial. We examined the putative mechanisms whereby this PTHrP domain can affect osteoblastic differentiation, using human osteosarcoma MG-63 cells and osteoblastic cells from human trabecular bone. Intermittent exposure to PTHrP (107-139), within 10-100 nM, for only ≤24 hours during cell growth stimulated alkaline phosphatase (ALP) and Runt homology domain protein (Runx2) activities as well as osteocalcin (OC) and osteoprotegerin (OPG) expression but inhibited receptor activator of nuclear factor κB (NF-κB) ligand. Continuous exposure to this PTHrP peptide reversed these effects. The stimulatory effects of transient treatment with PTHrP (107-139) on OC mRNA and/or OPG protein expression were unaffected by a neutralizing anti-insulin-like growth factor I antibody or [Asn¹⁰, Leu¹¹, d-Trp¹²]PTHrP (7-34) in these cells. On the other hand, the former antibody and the latter PTHrP antagonist abrogated the PTHrP (1-36)-induced increase in these osteoblastic products. Transient exposure to PTHrP (107-139), in contrast to PTHrP (1-36), stimulated vascular endothelial growth factor receptor 2 (VEGFR2) mRNA levels in these cells. Moreover, induction of ALP activity as well as OC and OPG expression by PTHrP (107-139) was blunted by SU5614, a permeable tyrosine kinase inhibitor of VEGFR2. Protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) inhibitors abolished the PTHrP (107-139)-stimulated VEGFR2 and OPG mRNA levels in these cells. These results indicate that intermittent exposure to PTHrP (107-139) exerts potential anabolic effects through the PKC/ERK pathway and, subsequently, VEGFR2 upregulation in vitro in human osteoblastic cells. A. R. de Gortázar and V. Alonso contributed equally to this work. This work was presented in part at the International Conference on Progress in Bone and Mineral Research, November 27–29, 2003, Vienna, Austria (published in Bone 33:S17, 2003); at the XLI Congress of the European Renal Association, May 15–18, 2004, Lisbon, Portugal; and at the 26th Annual Meeting of the American Society for Bone and Mineral Research, October 1–5, 2004, Seattle, WA (published in J Bone Miner Res 19[suppl 1]:S194, 2004).