The calcium‐sensing receptor and 25‐hydroxyvitamin D–1α‐hydroxylase interact to modulate skeletal growth and bone turnover

We examined parathyroid and skeletal function in 3‐month‐old mice expressing the null mutation for 25‐hydroxyvitamin D–1α‐hydroxylase [1α(OH)ase^?/?] and in mice expressing the null mutation for both the 1α(OH)ase and the calcium‐sensing receptor [Casr^?/?1α(OH)ase^?/?] genes. On a normal diet, all mice were hypocalcemic, with markedly increased parathyroid hormone (PTH), increased trabecular bone volume, increased osteoblast activity, poorly mineralized bone, enlarged and distorted cartilaginous growth plates, and marked growth retardation, especially in the compound mutants. Osteoclast numbers were reduced in the Casr^?/?1α(OH)ase^?/? mice. On a high‐lactose, high‐calcium, high‐phosphorus “rescue” diet, serum calcium and PTH were normal in the 1α(OH)ase^?/? mice but increased in the Casr^?/?1α(OH)ase^?/? mice with reduced serum phosphorus. Growth plate architecture and mineralization were improved in both mutants, but linear growth of the double mutants remained abnormal. Mineralization of bone improved in all mice, but osteoblast activity and trabecular bone volume remained elevated in the Casr^?/?1α(OH)ase^?/? mice. These studies support a role for calcium‐stimulated maturation of the cartilaginous growth plate and mineralization of the growth plate and bone and calcium‐stimulated CaSR‐mediated effects on bone resorption. PTH‐mediated bone resorption may require calcium‐stimulated CaSR‐mediated enhancement of osteoclastic activity. © 2010 American Society for Bone and Mineral Research. © 2010 American Society for Bone and Mineral Research     

Estrogen receptor‐α is required for the osteogenic response to mechanical loading in a ligand‐independent manner involving its activation function 1 but not 2

Estrogen receptor‐α (ERα) is crucial for the adaptive response of bone to loading but the role of endogenous estradiol (E2) for this response is unclear. To determine in vivo the ligand dependency and relative roles of different ERα domains for the osteogenic response to mechanical loading, gene‐targeted mouse models with (1) a complete ERα inactivation (ERα^?/?), (2) specific inactivation of activation function 1 (AF‐1) in ERα (ERαAF‐1⁰), or (3) specific inactivation of ERαAF‐2 (ERαAF‐2⁰) were subjected to axial loading of tibia, in the presence or absence (ovariectomy [ovx]) of endogenous E2. Loading increased the cortical bone area in the tibia mainly as a result of an increased periosteal bone formation rate (BFR) and this osteogenic response was similar in gonadal intact and ovx mice, demonstrating that E2 (ligand) is not required for this response. Female ERα^?/? mice displayed a severely reduced osteogenic response to loading with changes in cortical area (?78% ± 15%, p < 0.01) and periosteal BFR (?81% ± 9%, p < 0.01) being significantly lower than in wild‐type (WT) mice. ERαAF‐1⁰ mice also displayed a reduced response to mechanical loading compared with WT mice (cortical area ?40% ± 11%, p < 0.05 and periosteal BFR ?41% ± 8%, p < 0.01), whereas the periosteal osteogenic response to loading was unaffected in ERαAF‐2⁰ mice. Mechanical loading of transgenic estrogen response element (ERE)‐luciferase reporter mice did not increase luciferase expression in cortical bone, suggesting that the loading response does not involve classical genomic ERE‐mediated pathways. In conclusion, ERα is required for the osteogenic response to mechanical loading in a ligand‐independent manner involving AF‐1 but not AF‐2. © 2013 American Society for Bone and Mineral Research     

The role of activation functions 1 and 2 of estrogen receptor‐α for the effects of estradiol and selective estrogen receptor modulators in male mice

Estradiol (E2) is important for male skeletal health and the effect of E2 is mediated via estrogen receptor (ER)‐α. This was demonstrated by the findings that men with an inactivating mutation in aromatase or a nonfunctional ERα had osteopenia and continued longitudinal growth after sexual maturation. The aim of the present study was to evaluate the role of different domains of ERα for the effects of E2 and selective estrogen receptor modulators (SERMs) on bone mass in males. Three mouse models lacking either ERαAF‐1 (ERαAF‐1⁰), ERαAF‐2 (ERαAF‐2⁰), or the total ERα (ERα^?/?) were orchidectomized (orx) and treated with E2 or placebo. E2 treatment increased the trabecular and cortical bone mass and bone strength, whereas it reduced the thymus weight and bone marrow cellularity in orx wild type (WT) mice. These parameters did not respond to E2 treatment in orx ERα^?/? or ERαAF‐2⁰ mirx ERαAF‐1⁰ mice were tissue‐dependent, with a clear response in cortical bone parameters and bone marrow cellularity, but no response in trabecular bone. To determine the role of ERαAF‐1 for the effects of SERMs, we treated orx WT and ERαAF‐1⁰ mice with raloxifene (Ral), lasofoxifene (Las), bazedoxifene (Bza), or vehicle. These SERMs increased total body areal bone mineral density (BMD) and trabecular volumetric BMD to a similar extent in orx WT mice. Furthermore, only Las increased cortical thickness significantly and only Bza increased bone strength significantly. However, all SERMs showed a tendency toward increased cortical bone parameters. Importantly, all SERM effects were absent in the orx ERαAF‐1⁰ mice. In conclusion, ERαAF‐2 is required for the estrogenic effects on all evaluated parameters, whereas the role of ERαAF‐1 is tissue‐specific. All evaluated effects of Ral, Las and Bza are dependent on a functional ERαAF‐1. Our findings might contribute to the development of bone‐specific SERMs in males. © 2013 American Society for Bone and Mineral Research.     

Genes with specificity for expression in the round cell layer of the growth plate are enriched in genomewide association study (GWAS) of human height

Human adult height reflects the outcome of childhood skeletal growth. Growth plate (epiphyseal) chondrocytes are key determinants of height. As epiphyseal chondrocytes mature and proliferate, they pass through three developmental stages, which are organized into three distinct layers in the growth plate: (i) resting (round), (ii) proliferative (flat), and (iii) hypertrophic. Recent genomewide association studies (GWASs) of human height identified numerous associated loci, which are enriched for genes expressed in growth plate chondrocytes. However, it remains unclear which specific genes expressed in which layers of the growth plate regulate skeletal growth and human height. To connect the genetics of height and growth plate biology, we analyzed GWAS data through the lens of gene expression in the three dissected layers of murine newborn tibial growth plate. For each gene, we derived a specificity score for each growth plate layer and regressed these scores against gene-level p values from recent height GWAS data. We found that specificity for expression in the round cell layer, which contains chondrocytes early in maturation, is significantly associated with height GWAS p values (p = 8.5 × 10⁻⁹); this association remains after conditioning on specificity for the other cell layers. The association also remains after conditioning on membership in an “Online Mendelian Inheritance in Man (OMIM) gene set” (genes known to cause monogenic skeletal growth disorders, p < 9.7 × 10⁻⁶). We replicated the association in RNA-sequencing (RNA-seq) data from maturing chondrocytes sampled at early and late time points during differentiation in vitro: we found that expression early in differentiation is significantly associated with p values from height GWASs (p = 6.1 × 10⁻¹⁰) and that this association remains after conditioning on expression at 10 days in culture and on the OMIM gene set (p < 0.006). These findings newly implicate genes highlighted by GWASs of height and specifically expressed in the round cell layer as being potentially important regulators of skeletal biology. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Female Mice Lacking Estrogen Receptor‐Alpha in Osteoblasts Have Compromised Bone Mass and Strength

Reduced bioavailability of estrogen increases skeletal fracture risk in postmenopausal women, but the mechanisms by which estrogen regulates bone mass are incompletely understood. Because estrogen signaling in bone acts, in part, through estrogen receptor alpha (ERα), mice with global deletion of ERα (ERαKO) have been used to determine the role of estrogen signaling in bone biology. These animals, however, have confounding systemic effects arising from other organs, such as increased estrogen and decreased insulin‐like growth factor 1 (IGF‐1) serum levels, which may independently affect bone. Mice with tissue‐specific ERα deletion in chondrocytes, osteoblasts, osteocytes, or osteoclasts lack the systemic effects seen in the global knockout, but show that presence of the receptor is important for the function of each cell type. Although bone mass is reduced when ERα is deleted from osteoblasts, no study has determined if this approach reduces whole bone strength. To address this issue, we generated female osteoblast‐specific ERαKO mice (pOC‐ERαKO) by crossing mice expressing a floxed ERα gene (ERα^fl/fl) with mice transgenic for the osteocalcin‐Cre promoter (OC‐Cre). Having confirmed that serum levels of estrogen and IGF‐1 were unaltered, we focused on relating bone mechanics to skeletal phenotype using whole bone mechanical testing, microcomputed tomography, histology, and dynamic histomorphometry. At 12 and 18 weeks of age, pOC‐ERαKO mice had decreased cancellous bone mass in the proximal tibia, vertebra, and distal femur, and decreased cortical bone mass in the tibial midshaft, distal femoral cortex, and L5 vertebral cortex. Osteoblast activity was reduced in cancellous bone of the proximal tibia, but osteoclast number was unaffected. Both femora and vertebrae had decreased whole bone strength in mechanical tests to failure, indicating that ERα in osteoblasts is required for appropriate bone mass and strength accrual in female mice. This pOC‐ERαKO mouse is an important animal model that could enhance our understanding of estrogen signaling in bone cells in vivo. © 2014 American Society for Bone and Mineral Research.     

Ablation of Osteopontin Improves the Skeletal Phenotype of Phospho1−/− Mice

PHOSPHO1 and tissue‐nonspecific alkaline phosphatase (TNAP) have nonredundant functions during skeletal mineralization. Although TNAP deficiency (Alpl^?/? mice) leads to hypophosphatasia, caused by accumulation of the mineralization inhibitor inorganic pyrophosphate (PP_i), comparably elevated levels of PP_i in Phospho1^?/? mice do not explain their stunted growth, spontaneous fractures, bowed long bones, osteomalacia, and scoliosis. We have previously shown that elevated PP_i in Alpl^?/? mice is accompanied by elevated osteopontin (OPN), another potent mineralization inhibitor, and that the amount of OPN correlates with the severity of hypophosphatasia in mice. Here we demonstrate that plasma OPN is elevated and OPN expression is upregulated in the skeleton, particularly in the vertebrae, of Phospho1^?/? mice. Liquid chromatography/tandem mass spectrometry showed an increased proportion of phosphorylated OPN (p‐OPN) peptides in Phospho1^?/? mice, suggesting that accumulation of p‐OPN causes the skeletal abnormalities in Phospho1^?/? mice. We also show that ablation of the OPN gene, Spp1, leads to improvements in the skeletal phenotype in Phospho1^?/? as they age. In particular, their scoliosis is ameliorated at 1 month of age and is completely rescued at 3 months of age. There is also improvement in the long bone defects characteristic of Phospho1^?/? mice at 3 months of age. Mineralization assays comparing [Phospho1^?/?; Spp1^?/?], Phospho1^?/?, and Spp1^?/? chondrocytes display corrected mineralization by the double knockout cells. Expression of chondrocyte differentiation markers was also normalized in the [Phospho1^?/?; Spp1^?/?] mice. Thus, although Alpl and Phospho1 deficiencies lead to similar skeletal phenotypes and comparable changes in the expression levels of PP_i and OPN, there is a clear dissociation in the hierarchical roles of these potent inhibitors of mineralization, with elevated PP_i and elevated p‐OPN levels causing the respective skeletal phenotypes in Alpl^?/? and Phospho1^?/? mice. © 2014 American Society for Bone and Mineral Research.     

Depletion of annexin A5, annexin A6, and collagen X causes no gross changes in matrix vesicle–mediated mineralization,but lack of collagen X affects hematopoiesis and the Th1/Th2 response

Numerous biochemical studies have pointed to an essential role of annexin A5 (AnxA5), annexin A6 (AnxA6), and collagen X in matrix vesicle–mediated biomineralization during endochondral ossification and in osteoarthritis. By binding to the extracellular matrix protein collagen X and matrix vesicles, annexins were proposed to anchor matrix vesicles in the extracellular space of hypertrophic chondrocytes to initiate the calcification of cartilage. However, mineralization appears to be normal in mice lacking AnxA5 and AnxA6, whereas collagen X–deficient mice show only subtle alterations in the growth plate organization. We hypothesized that the simultaneous lack of AnxA5, AnxA6, and collagen X in vivo induces more pronounced changes in the growth plate development and the initiation of mineralization. In this study, we generated and analyzed mice deficient for AnxA5, AnxA6, and collagen X. Surprisingly, mice were viable, fertile, and showed no obvious abnormalities. Assessment of growth plate development indicated that the hypertrophic zone was expanded in Col10a1^?/? and AnxA5^?/?AnxA6^?/?Col10a1^?/? newborns, whereas endochondral ossification and mineralization were not affected in 13‐day‐ and 1‐month‐old mutants. In peripheral quantitative computed tomography, no changes in the degree of biomineralization were found in femora of 1‐month‐ and 1‐year‐old mutants even though the diaphyseal circumference was reduced in Col10a1^?/? and AnxA5^?/?AnxA6^?/?Col10a1^?/? mice. The percentage of naive immature IgM⁺/IgM⁺ B cells and peripheral T‐helper cells were increased in Col10a1^?/? and AnxA5^?/?AnxA6^?/?Col10a1^?/? mutants, and activated splenic T cells isolated from Col10a1^?/? mice secreted elevated levels of IL‐4 and GM‐CSF. Hence, collagen X is needed for hematopoiesis during endochondral ossification and for the immune response, but the interaction of annexin A5, annexin A6, and collagen X is not essential for physiological calcification of growth plate cartilage. Therefore, annexins and collagen X may rather fulfill functions in growth plate cartilage not directly linked to the mineralization process. © 2012 American Society for Bone and Mineral Research.     

GPR30 deficiency causes increased bone mass,mineralization, and growth plate proliferative activity in male mice

Estrogen regulation of the male skeleton was first clearly demonstrated in patients with aromatase deficiency or a mutation in the ERα gene. Estrogen action on the skeleton is thought to occur mainly through the action of the nuclear receptors ERα and ERβ. Recently, in vitro studies have shown that the G protein–coupled receptor GPR30 is a functional estrogen receptor (ER). GPR30‐deficient mouse models have been generated to study the in vivo function of this protein; however, its in vivo role in the male skeleton remains underexplored. We have characterized size, body composition, and bone mass in adult male Gpr30 knockout (KO) mice and their wild‐type (WT) littermates. Gpr30 KO mice weighed more and had greater nasal‐anal length (p < .001). Both lean mass and percent body fat were increased in the KO mice. Femur length was greater in Gpr30 KO mice, as was whole‐body, spine, and femoral areal bone mineral density (p < .01). Gpr30 KO mice showed increased trabecular bone volume (p < .01) and cortical thickness (p < .001). Mineralized surface was increased in Gpr30 KO mice (p < .05). Bromodeoxyuridine (BrdU) labeling showed greater proliferation in the growth plate of Gpr30 KO mice (p < .05). Under osteogenic culture conditions, Gpr30 KO femoral bone marrow cells produced fewer alkaline phosphatase–positive colonies in early differentiating osteoblast cultures but showed increased mineralized nodule deposition in mature osteoblast cultures. Serum insulin‐like growth factor 1 (IGF‐1) levels were not different. These data suggest that in male mice, GPR30 action contributes to regulation of bone mass, size, and microarchitecture by a mechanism that does not require changes in circulating IGF‐1. © 2011 American Society for Bone and Mineral Research.     

The p27 Pathway Modulates the Regulation of Skeletal Growth and Osteoblastic Bone Formation by Parathyroid Hormone–Related Peptide

Parathyroid hormone–related peptide (PTHrP) 1–84 knock‐in mice (Pthrp KI) develop skeletal growth retardation and defective osteoblastic bone formation. To further examine the mechanisms underlying this phenotype, microarray analyses of differential gene expression profiles were performed in long bone extracts from Pthrp KI mice and their wild‐type (WT) littermates. We found that the expression levels of p27, p16, and p53 were significantly upregulated in Pthrp KI mice relative to WT littermates. To determine whether p27 was involved in the regulation by PTHrP of skeletal growth and development in vivo, we generated compound mutant mice, which were homozygous for both p27 deletion and the Pthrp KI mutation (p27^‐/‐Pthrp KI). We then compared p27^‐/‐Pthrp KI mice with p27^‐/‐, Pthrp KI, and WT littermates. Deletion of p27 in Pthrp KI mice resulted in a longer lifespan, increased body weight, and improvement in skeletal growth. At 2 weeks of age, skeletal parameters, including length of long bones, size of epiphyses, numbers of proliferating cell nuclear antigen (PCNA)‐positive chondrocytes, bone mineral density, trabecular bone volume, osteoblast numbers, and alkaline phosphatase (ALP)‐, type I collagen‐, and osteocalcin‐positive bone areas were increased in p27^‐/‐ mice and reduced in both Pthrp KI and p27^‐/‐Pthrp KI mice compared with WT mice; however, these parameters were increased in p27^‐/‐Pthrp KI mice compared with Pthrp KI mice. As well, protein expression levels of PTHR, IGF‐1, and Bmi‐1, and the numbers of total colony‐forming unit fibroblastic (CFU‐f) and ALP‐positive CFU‐f were similarly increased in p27^‐/‐Pthrp KI mice compared with Pthrp KI mice. Our results demonstrate that deletion of p27 in Pthrp KI mice can partially rescue defects in skeletal growth and osteoblastic bone formation by enhancing endochondral bone formation and osteogenesis. These studies, therefore, indicate that the p27 pathway may function downstream in the action of PTHrP to regulate skeletal growth and development. © 2015 American Society for Bone and Mineral Research.     

Cartilage‐Specific Autophagy Deficiency Promotes ER Stress and Impairs Chondrogenesis in PERK‐ATF4‐CHOP–Dependent Manner

Autophagy is activated during nutritionally depleted or hypoxic conditions to facilitate cell survival. Because growth plate is an avascular and hypoxic tissue, autophagy may have a crucial role during chondrogenesis; however, the functional role and underlying mechanism of autophagy in regulation of growth plate remains elusive. In this study, we generated ^TamCartAtg7^–/– (Atg7cKO) mice to explore the role of autophagy during endochondral ossification. Atg7cKO mice exhibited growth retardation associated with reduced chondrocyte proliferation and differentiation, and increased chondrocyte apoptosis. Meanwhile, we observed that Atg7 ablation mainly induced the PERK‐ATF4‐CHOP axis of the endoplasmic reticulum (ER) stress response in growth plate chondrocytes. Although Atg7 ablation induced ER stress in growth plate chondrocytes, the addition of phenylbutyric acid (PBA), a chemical chaperone known to attenuate ER stress, partly neutralized such effects of Atg7 ablation on longitudinal bone growth, indicating the causative interaction between autophagy and ER stress in growth plate. Consistent with these findings in vivo, we also observed that Atg7 ablation in cultured chondrocytes resulted in defective autophagy, elevated ER stress, decreased chondrocytes proliferation, impaired expression of col10a1, MMP­13, and VEGFA for chondrocyte differentiation, and increased chondrocyte apoptosis, while such effects were partly nullified by reduction of ER stress with PBA. In addition, Atg7 ablation‐mediated impaired chondrocyte function (chondrocyte proliferation, differentiation, and apoptosis) was partly reversed in CHOP^–/– cells, indicating the causative role of the PERK‐ATF4‐CHOP axis of the ER stress response in the action of autophagy deficiency in chondrocytes. In conclusion, our findings indicate that autophagy deficiency may trigger ER stress in growth plate chondrocytes and contribute to growth retardation, thus implicating autophagy as an important regulator during chondrogenesis and providing new insights into the clinical potential of autophagy in cartilage homeostasis. © 2017 American Society for Bone and Mineral Research.     

IGF‐I Signaling in Osterix‐Expressing Cells Regulates Secondary Ossification Center Formation,Growth Plate Maturation,and Metaphyseal Formation During Postnatal Bone Development

To investigate the role of IGF‐I signaling in osterix (OSX)‐expressing cells in the skeleton, we generated IGF‐I receptor (IGF‐IR) knockout mice (^OSXIGF‐IRKO) (floxed‐IGF‐IR mice × OSX promoter‐driven GFP‐labeled cre‐recombinase [^OSXGFPcre]), and monitored postnatal bone development. At day 2 after birth (P2), ^OSXGFP‐cre was highly expressed in the osteoblasts in the bone surface of the metaphysis and in the prehypertrophic chondrocytes (PHCs) and inner layer of perichondral cells (IPCs). From P7, ^OSXGFP‐cre was highly expressed in PHCs, IPCs, cartilage canals (CCs), and osteoblasts (OBs) in the epiphyseal secondary ossification center (SOC), but was only slightly expressed in the OBs in the metaphysis. Compared with the control mice, the IPC proliferation was decreased in the ^OSXIGF‐IRKOs. In these mice, fewer IPCs invaded into the cartilage, resulting in delayed formation of the CC and SOC. Immunohistochemistry indicated a reduction of vessel number and lower expression of VEGF and ephrin B2 in the IPCs and SOC of ^OSXIGF‐IRKOs. Quantitative real‐time PCR revealed that the mRNA levels of the matrix degradation markers, MMP‐9, 13 and 14, were decreased in the ^OSXIGF‐IRKOs compared with the controls. The ^OSXIGF‐IRKO also showed irregular morphology of the growth plate and less trabecular bone in the tibia and femur from P7 to 7 weeks, accompanied by decreased chondrocyte proliferation, altered chondrocyte differentiation, and decreased osteoblast differentiation. Our data indicate that during postnatal bone development, IGF‐I signaling in OSX‐expressing IPCs promotes IPC proliferation and cartilage matrix degradation and increases ephrin B2 production to stimulate vascular endothelial growth factor (VEGF) expression and vascularization. These processes are required for normal CC formation in the establishment of the SOC. Moreover, IGF‐I signaling in the OSX‐expressing PHC is required for growth plate maturation and osteoblast differentiation in the development of the metaphysis. © 2015 American Society for Bone and Mineral Research.     

Relative Skeletal Maturation and Population Ancestry in Nonobese Children and Adolescents

More rapid skeletal maturation in African‐American (AA) children is recognized and generally attributed to an increased prevalence of obesity. The objective of the present study was to evaluate the effects of population ancestry on relative skeletal maturation in healthy, non‐obese children and adolescents, accounting for body composition and sexual maturation. To do this, we leveraged a multiethnic, mixed‐longitudinal study with annual assessments for up to 7 years (The Bone Mineral Density in Childhood Study and its ancillary cohort) conducted at five US clinical centers. Participants included 1592 children, skeletally immature (45% females, 19% AA) who were aged 5 to 17 years at study entry. The primary outcome measure was relative skeletal maturation as assessed by hand‐wrist radiograph. Additional covariates measured included anthropometrics, body composition by dual‐energy X‐ray absorptiometry (DXA), and Tanner stage of sexual maturation. Using mixed effects longitudinal models, without covariates, advancement in relative skeletal maturation was noted in self‐reported AA girls (～0.33 years, p < 0.001) and boys (～0.43 years, p < 0.001). Boys and girls of all ancestry groups showed independent positive associations of height, lean mass, fat mass, and puberty with relative skeletal maturation. The effect of ancestry was attenuated but persistent after accounting for covariates: for girls, 0.19 years (ancestry by self‐report, p = 0.02) or 0.29 years (ancestry by admixture, p = 0.004); and for boys, 0.20 years (ancestry by self‐report, p = 0.004), or 0.29 years (ancestry by admixture, p = 0.004). In summary, we conclude that advancement in relative skeletal maturation was associated with AA ancestry in healthy, non‐obese children, independent of growth, body composition, and puberty. Further research into the mechanisms underlying this observation may provide insights into the regulation of skeletal maturation. © 2016 American Society for Bone and Mineral Research.     

Estrogen receptor-beta inhibits skeletal growth and has the capacity to mediate growth plate fusion in female mice.

To determine the long-term role of ER beta in the regulation of longitudinal bone growth, appendicular and axial skeletal growth was followed and compared in female ER beta-/-, ER alpha-/-, and ER alpha-/- beta-/- mice. Our results show that ER beta inhibits appendicular and axial skeletal growth and has the capacity to induce fusion of the growth plates. INTRODUCTION: Estrogen affects skeletal growth and promotes growth plate fusion in humans. In rodents, the growth plates do not fuse after sexual maturation, but prolonged treatment with supraphysiological levels of estradiol has the capacity to fuse the growth plates. It should be emphasized that the estrogen receptor (ER) alpha-/- and the ER alpha-/- beta-/-, but not the ER beta-/-, mouse models have clearly increased serum levels of estradiol. MATERIALS AND METHODS: The skeletal growth was monitored by X-ray and dynamic histomorphometry, and the growth plates were analyzed by quantitative histology, calcein double labeling, bromodeoxyuridine (BrdU) incorporation, and TUNEL assay in 4- and 18-month-old female ER beta-/-, ER alpha-/-, and ER alpha-/- beta-/- mice. RESULTS: Young adult (4-month-old) ER beta-/- mice demonstrated an increased axial- and appendicular-skeletal growth, supporting the notion that ER beta inhibits skeletal growth in young adult female mice. Interestingly, the growth plates were consistently fused in the appendicular skeleton of 18-month-old female ER alpha-/- mice. This fusion of growth plates, caused by a prolonged exposure to supraphysiological levels of estradiol in female ER alpha-/- mice, must be mediated through ER beta because old ER alpah-/- beta-/- mice displayed unchanged, unfused growth plates. CONCLUSIONS: Our results confirm that ER beta is a physiological inhibitor of appendicular- and axial-skeletal growth in young adult female mice. Furthermore, we made the novel observation that ER beta, after prolonged supraphysiological estradiol exposure, has the capacity to mediate growth plate fusion in old female mice.     

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.     

Absence of Proteoglycan 4 (Prg4) Leads to Increased Subchondral Bone Porosity Which Can Be Mitigated Through Intra‐Articular Injection of PRG4

Proteoglycan 4 (PRG4) is a mucin‐like glycoprotein important for joint health. Mice lacking Prg4 demonstrate degeneration of the cartilage and altered skeletal morphology. The purpose of this study was to examine if Prg4 deficiency leads to subchondral bone defects and if these defects could be mitigated through intra‐articular injection of recombinant human PRG4 (rhPRG4). Mice deficient in Prg4 expression demonstrated increased cartilage thickness and increased subchondral bone porosity compared with C57BL/6 controls. While the porosity of the subchondral bone of Prg4^?/? mice decreased over time with maturation, intra‐articular injection of rhPRG4 was able to forestall the increase in porosity. In contrast, neither hyaluronan (HA) nor methylprednisolone injections had beneficial effects on the subchondral bone porosity in the Prg4 knockout mice. Bone marrow progenitor cells from Prg4^?/? mice demonstrated reduced osteogenic differentiation capacity at 4 weeks of age, but not at 16 weeks of age. While most studies on PRG4/lubricin focus on the health of the cartilage, this study demonstrates that PRG4 plays a role in the maturation of the subchondral bone. Furthermore, increasing joint lubrication/viscosupplementation through injection of HA or controlling joint inflammation through injection of methylprednisolone may help maintain the cartilage surface, but had no positive effect on the subchondral bone in animals lacking Prg4. Therefore, alterations in the subchondral bone in models with absent or diminished Prg4 expression should not be overlooked when investigating changes within the articular cartilage regarding the pathogenesis of osteoarthritis/arthrosis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2077–2088, 2019     

Deficiency of Sef Is Associated With Increased Postnatal Cortical Bone Mass by Regulating Runx2 Activity

Sef (similar expression to fgf genes) is a feedback inhibitor of fibroblast growth factor (FGF) signaling and functions in part by binding to FGF receptors and inhibiting their activation. Genetic studies in mice and humans indicate an important role for fibroblast growth factor signaling in bone growth and homeostasis. We, therefore, investigated whether Sef had a function role in skeletal acquisition and remodeling. Sef expression is increased during osteoblast differentiation in vitro, and LacZ staining of Sef+/? mice showed high expression of Sef in the periosteum and chondro‐osseous junction of neonatal and adult mice. Mice with a global deletion of Sef showed increased cortical bone thickness, bone volume, and increased periosteal perimeter by micro‐computed tomography (micro‐CT). Histomorphometric analysis of cortical bone revealed a significant increase in osteoblast number. Interestingly, Sef?/? mice showed very little difference in trabecular bone by micro‐CT and histomorphometry compared with wild‐type mice. Bone marrow cells from Sef?/? mice grown in osteogenic medium showed increased proliferation and increased osteoblast differentiation compared with wild‐type bone marrow cells. Bone marrow cells from Sef?/? mice showed enhanced FGF2‐induced activation of the ERK pathway, whereas bone marrow cells from Sef transgenic mice showed decreased FGF2‐induced signaling. FGF2‐induced acetylation and stability of Runx2 was enhanced in Sef?/? bone marrow cells, whereas overexpression of Sef inhibited Runx2‐responsive luciferase reporter activity. Bone marrow from Sef?/? mice showed enhanced hematopoietic lineage‐dependent and osteoblast‐dependent osteoclastogenesis and increased bone resorptive activity relative to wild‐type controls in in vitro assays, whereas overexpression of Sef inhibited osteoclast differentiation. Taken together, these studies indicate that Sef has specific roles in osteoblast and osteoclast lineages and that its absence results in increased osteoblast and osteoclast activity with a net increase in cortical bone mass. © 2014 American Society for Bone and Mineral Research.     

An essential role for the association of CD47 to SHPS‐1 in skeletal remodeling

Integrin‐associated protein (IAP/CD47) has been implicated in macrophage‐macrophage fusion. To understand the actions of CD47 on skeletal remodeling, we compared Cd47^?/? mice with Cd47^+/+ controls. Cd47^?/? mice weighed less and had decreased areal bone mineral density compared with controls. Cd47^?/? femurs were shorter in length with thinner cortices and exhibited lower trabecular bone volume owing to decreased trabecular number and thickness. Histomorphometry revealed reduced bone‐formation and mineral apposition rates, accompanied by decreased osteoblast numbers. No differences in osteoclast number were observed despite a nonsignificant but 40% decrease in eroded surface/bone surface in Cd47^?/? mice. In vitro, the number of functional osteoclasts formed by differentiating Cd47^?/? bone marrow cells was significantly decreased compared with wild‐type cultures and was associated with a decrease in bone‐resorption capacity. Furthermore, by disrupting the CD47–SHPS‐1 association, we found that osteoclastogenesis was markedly impaired. Assays for markers of osteoclast maturation suggested that the defect was at the point of fusion and not differentiation and was associated with a lack of SHPS‐1 phosphorylation, SHP‐1 phosphatase recruitment, and subsequent dephosphorylation of non–muscle cell myosin IIA. We also demonstrated a significant decrease in osteoblastogenesis in bone marrow stromal cells derived from Cd47^?/? mice. Our finding of cell‐autonomous defects in Cd47^?/? osteoblast and osteoclast differentiation coupled with the pronounced skeletal phenotype of Cd47^?/? mice support the conclusion that CD47 plays an important role in regulating skeletal acquisition and maintenance through its actions on both bone formation and bone resorption. © 2011 American Society for Bone and Mineral Research