Growth Hormone Increases Bone Toughness and Decreases Muscle Inflammation in Glucocorticoid-Treated Mdx Mice,Model of Duchenne Muscular Dystrophy

The absence of functional dystrophin with mutations of the dystrophin-encoding gene in Duchenne muscular dystrophy (DMD) results in muscle inflammation and degeneration, as well as bone fragility. Long-term glucocorticoid therapy delays the muscular disease progression but suppresses growth hormone secretion, resulting in short stature and further deleterious effects on bone strength. This study evaluated the therapeutic potential of daily growth hormone therapy in growing mdx mice as a model of DMD. Growth hormone treatment on its own or in combination with glucocorticoids significantly improved muscle histology and function and decreased markers of inflammation in mdx mice. Glucocorticoid treatment thinned cortical bone and decreased bone strength and toughness. Despite the minimal effects of growth hormone on bone microarchitecture, it significantly improved biomechanical properties of femurs and vertebrae, even in the presence of glucocorticoid treatment. Together these studies suggest that the use of growth hormone in DMD should be considered for improvements to muscle and bone health. © 2019 American Society for Bone and Mineral Research.     

PTH Receptor Signaling in Osteoblasts Regulates Endochondral Vascularization in Maintenance of Postnatal Growth Plate

Longitudinal growth of postnatal bone requires precise control of growth plate cartilage chondrocytes and subsequent osteogenesis and bone formation. Little is known about the role of angiogenesis and bone remodeling in maintenance of cartilaginous growth plate. Parathyroid hormone (PTH) stimulates bone remodeling by activating PTH receptor (PTH1R). Mice with conditional deletion of PTH1R in osteoblasts showed disrupted trabecular bone formation. The mice also exhibited postnatal growth retardation with profound defects in growth plate cartilage, ascribable predominantly to a decrease in number of hypertrophic chondrocytes, resulting in premature fusion of the growth plate and shortened long bones. Further characterization of hypertrophic zone and primary spongiosa revealed that endochondral angiogenesis and vascular invasion of the cartilage were impaired, which was associated with aberrant chondrocyte maturation and cartilage development. These studies reveal that PTH1R signaling in osteoblasts regulates cartilaginous growth plate for postnatal growth of bone. © 2014 American Society for Bone and Mineral Research.     

Reduced Serum IGF‐1 Associated With Hepatic Osteodystrophy Is a Main Determinant of Low Cortical but Not Trabecular Bone Mass

Hepatic osteodystrophy is multifactorial in its pathogenesis. Numerous studies have shown that impairments of the hepatic growth hormone/insulin‐like growth factor‐1 axis (GH/IGF‐1) are common in patients with non‐alcoholic fatty liver disease, chronic viral hepatitis, liver cirrhosis, and chronic cholestatic liver disease. Moreover, these conditions are also associated with low bone mineral density (BMD) and greater fracture risk, particularly in cortical bone sites. Hence, we addressed whether disruptions in the GH/IGF‐1 axis were causally related to the low bone mass in states of chronic liver disease using a mouse model of liver‐specific GH‐receptor (GHR) gene deletion (Li‐GHRKO). These mice exhibit chronic hepatic steatosis, local inflammation, and reduced BMD. We then employed a crossing strategy to restore liver production of IGF‐1 via hepatic IGF‐1 transgene (HIT). The resultant Li‐GHRKO‐HIT mouse model allowed us to dissect the roles of liver‐derived IGF‐1 in the pathogenesis of osteodystrophy during liver disease. We found that hepatic IGF‐1 restored cortical bone acquisition, microarchitecture, and mechanical properties during growth in Li‐GHRKO‐HIT mice, which was maintained during aging. However, trabecular bone volume was not restored in the Li‐GHRKO‐HIT mice. We found increased bone resorption indices in vivo as well as increased basal reactive oxygen species and increased mitochondrial stress in osteoblast cultures from Li‐GHRKO and the Li‐GHRKO‐HIT compared with control mice. Changes in systemic markers such as inflammatory cytokines, osteoprotegerin, osteopontin, parathyroid hormone, osteocalcin, or carboxy‐terminal collagen cross‐links could not fully account for the diminished trabecular bone in the Li‐GHRKO‐HIT mice. Thus, the reduced serum IGF‐1 associated with hepatic osteodystrophy is a main determinant of low cortical but not trabecular bone mass. © 2017 American Society for Bone and Mineral Research.     

Osteocyte Death and Bone Overgrowth in Mice Lacking Fibroblast Growth Factor Receptors 1 and 2 in Mature Osteoblasts and Osteocytes

Fibroblast growth factor (FGF) signaling pathways have well-established roles in skeletal development, with essential functions in both chondrogenesis and osteogenesis. In mice, previous conditional knockout studies suggested distinct roles for FGF receptor 1 (FGFR1) signaling at different stages of osteogenesis and a role for FGFR2 in osteoblast maturation. However, the potential for redundancy among FGFRs and the mechanisms and consequences of stage-specific osteoblast lineage regulation were not addressed. Here, we conditionally inactivate Fgfr1 and Fgfr2 in mature osteoblasts with an Osteocalcin (OC)-Cre or Dentin matrix protein 1 (Dmp1)-CreER driver. We find that young mice lacking both receptors or only FGFR1 are phenotypically normal. However, between 6 and 12 weeks of age, OC-Cre Fgfr1/Fgfr2 double- and Fgfr1 single-conditional knockout mice develop a high bone mass phenotype with increased periosteal apposition, increased and disorganized endocortical bone with increased porosity, and biomechanical properties that reflect increased bone mass but impaired material properties. Histopathological and gene expression analyses show that this phenotype is preceded by a striking loss of osteocytes and accompanied by activation of the Wnt/β-catenin signaling pathway. These data identify a role for FGFR1 signaling in mature osteoblasts/osteocytes that is directly or indirectly required for osteocyte survival and regulation of bone mass during postnatal bone growth. © 2019 American Society for Bone and Mineral Research.     

Cortical and Trabecular Bone Benefits of Mechanical Loading Are Maintained Long Term in Mice Independent of Ovariectomy

Skeletal loading enhances cortical and trabecular bone properties. How long these benefits last after loading cessation remains an unresolved, clinically relevant question. This study investigated long‐term maintenance of loading‐induced cortical and trabecular bone benefits in female C57BL/6 mice and the influence of a surgically induced menopause on the maintenance. Sixteen‐week‐old animals had their right tibia extrinsically loaded 3 days/week for 4 weeks using the mouse tibial axial compression loading model. Left tibias were not loaded and served as internal controls. Animals were subsequently detrained (restricted to cage activities) for 0, 4, 8, 26, or 52 weeks, with ovariectomy (OVX) or sham‐OVX surgery being performed at 0 weeks detraining. Loading increased midshaft tibia cortical bone mass, size, and strength, and proximal tibia bone volume fraction. The cortical bone mass, area, and thickness benefits of loading were lost by 26 weeks of detraining because of heightened medullary expansion. However, loading‐induced benefits on bone total area and strength were maintained at each detraining time point. Similarly, the benefits of loading on bone volume fraction persisted at all detraining time points. The long‐term benefits of loading on both cortical and trabecular bone were not influenced by a surgically induced menopause because there were no interactions between loading and surgery. However, OVX had independent effects on cortical bone properties at early (4 and 8 weeks) detraining time points and trabecular bone properties at all detraining time points. These cumulative data indicate loading has long‐term benefits on cortical bone size and strength (but not mass) and trabecular bone morphology, which are not influenced by a surgically induced menopause. This suggests skeletal loading associated with physical activity may provide long‐term benefits by preparing the skeleton to offset both the cortical and trabecular bone changes associated with aging and menopause. © 2014 American Society for Bone and Mineral Research.     

Development of Hip Bone Geometry During Gender-Affirming Hormone Therapy in Transgender Adolescents Resembles That of the Experienced Gender When Pubertal Suspension Is Started in Early Puberty

Bone geometry can be described in terms of periosteal and endocortical growth and is partly determined by sex steroids. Periosteal and endocortical apposition are thought to be regulated by testosterone and estrogen, respectively. Gender-affirming hormone (GAH) treatment with sex steroids in transgender people might affect bone geometry. However, in adult transgender people, no change in bone geometry during GAH was observed. In this study, we investigated changes in bone geometry among transgender adolescents using a gonadotropin-releasing hormone agonist (GnRHa) and GAH before achieving peak bone mass. Transgender adolescents treated with GnRHa and subsequent GAH before the age of 18 years were eligible for inclusion. Participants were grouped based on their Tanner stage at the start of GnRHa treatment and divided into early, mid, and late puberty groups. Hip structure analysis software calculating subperiosteal width (SPW) and endocortical diameter (ED) was applied to dual-energy X-ray absorptiometry scans performed at the start of GnRHa and GAH treatments, and after ≥2 years of GAH treatment. Mixed-model analyses were performed to study differences over time. Data were visually compared with reference values of the general population. A total of 322 participants were included, of whom 106 were trans women and 216 trans men. In both trans women and trans men, participants resembled the reference curve for SPW and ED of the experienced gender but only when GnRHa was started during early puberty. Those who started during mid and late puberty remained within the reference curve of the gender assigned at birth. A possible explanation might be sought in the phenomenon of programming, which conceptualizes that stimuli during critical windows of development can have major consequences throughout one's life span. Therefore, this study adds insights into sex-specific bone geometry development during puberty of transgender adolescents treated with GnRHa, as well as the general population. © 2021 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.     

Osteoblast Function Is Compromised at Sites of Focal Bone Erosion in Inflammatory Arthritis

In rheumatoid arthritis (RA), synovial inflammation results in focal erosion of articular bone. Despite treatment attenuating inflammation, repair of erosions with adequate formation of new bone is uncommon in RA, suggesting that bone formation may be compromised at these sites. Dynamic bone histomorphometry was used in a murine model of RA to determine the impact of inflammation on osteoblast function within eroded arthritic bone. Bone formation rates at bone surfaces adjacent to inflammation were similar to those observed in nonarthritic bone; therefore, osteoblast activity is unlikely to compensate for the increased bone resorption at these sites. Within arthritic bone, the extent of actively mineralizing surface was reduced at bone surfaces adjacent to inflammation compared with bone surfaces adjacent to normal marrow. Consistent with the reduction in mineralized bone formation, there was a notable paucity of cells expressing the mid‐ to late stage osteoblast lineage marker alkaline phosphatase, despite a clear presence of cells expressing the early osteoblast lineage marker Runx2. In addition, several members of the Dickkopf and secreted Frizzled‐related protein families of Wnt signaling antagonists were upregulated in arthritic synovial tissues, suggesting that inhibition of Wnt signaling could be one mechanism contributing to impaired osteoblast function within arthritic bone. Together, these data indicate that the presence of inflammation within arthritic bone impairs osteoblast capacity to form adequate mineralized bone, thus contributing to the net loss of bone and failure of bone repair at sites of focal bone erosion in RA.     

Activation of Wnt Signaling by Mechanical Loading Is Impaired in the Bone of Old Mice

Aging diminishes bone formation engendered by mechanical loads, but the mechanism for this impairment remains unclear. Because Wnt signaling is required for optimal loading‐induced bone formation, we hypothesized that aging impairs the load‐induced activation of Wnt signaling. We analyzed dynamic histomorphometry of 5‐month‐old, 12‐month‐old, and 22‐month‐old C57Bl/6JN mice subjected to multiple days of tibial compression and corroborated an age‐related decline in the periosteal loading response on day 5. Similarly, 1 day of loading increased periosteal and endocortical bone formation in young‐adult (5‐month‐old) mice, but old (22‐month‐old) mice were unresponsive. These findings corroborated mRNA expression of genes related to bone formation and the Wnt pathway in tibias after loading. Multiple bouts (3 to 5 days) of loading upregulated bone formation–related genes, e.g., Osx and Col1a1, but older mice were significantly less responsive. Expression of Wnt negative regulators, Sost and Dkk1, was suppressed with a single day of loading in all mice, but suppression was sustained only in young‐adult mice. Moreover, multiple days of loading repeatedly suppressed Sost and Dkk1 in young‐adult, but not in old tibias. The age‐dependent response to loading was further assessed by osteocyte staining for Sclerostin and LacZ in tibia of TOPGAL mice. After 1 day of loading, fewer osteocytes were Sclerostin‐positive and, corroboratively, more osteocytes were LacZ‐positive (Wnt active) in both 5‐month‐old and 12‐month‐old mice. However, although these changes were sustained after multiple days of loading in 5‐month‐old mice, they were not sustained in 12‐month‐old mice. Last, Wnt1 and Wnt7b were the most load‐responsive of the 19 Wnt ligands. However, 4 hours after a single bout of loading, although their expression was upregulated threefold to 10‐fold in young‐adult mice, it was not altered in old mice. In conclusion, the reduced bone formation response of aged mice to loading may be due to failure to sustain Wnt activity with repeated loading. © 2016 American Society for Bone and Mineral Research.     

Premature Growth Plate Closure Caused by a Hedgehog Cancer Drug Is Preventable by Co-Administration of a Retinoid Antagonist in Mice

The growth plates are key engines of skeletal development and growth and contain a top reserve zone followed by maturation zones of proliferating, prehypertrophic, and hypertrophic/mineralizing chondrocytes. Trauma or drug treatment of certain disorders can derange the growth plates and cause accelerated maturation and premature closure, one example being anti-hedgehog drugs such as LDE225 (Sonidegib) used against pediatric brain malignancies. Here we tested whether such acceleration and closure in LDE225-treated mice could be prevented by co-administration of a selective retinoid antagonist, based on previous studies showing that retinoid antagonists can slow down chondrocyte maturation rates. Treatment of juvenile mice with an experimental dose of LDE225 for 2 days (100 mg/kg by gavage) initially caused a significant shortening of long bone growth plates, with concomitant decreases in chondrocyte proliferation; expression of Indian hedgehog, Sox9, and other key genes; and surprisingly, the number of reserve progenitors. Growth plate involution followed with time, leading to impaired long bone lengthening. Mechanistically, LDE225 treatment markedly decreased the expression of retinoid catabolic enzyme Cyp26b1 within growth plate, whereas it increased and broadened the expression of retinoid synthesizing enzyme Raldh3, thus subverting normal homeostatic retinoid circuitries and in turn accelerating maturation and closure. All such severe skeletal and molecular changes were prevented when LDE-treated mice were co-administered the selective retinoid antagonist CD2665 (1.5 mg/kg/d), a drug targeting retinoid acid receptor γ, which is most abundantly expressed in growth plate. When given alone, CD2665 elicited the expected maturation delay and growth plate expansion. In vitro data showed that LDE225 acted directly to dampen chondrogenic phenotypic expression, a response fully reversed by CD2665 co-treatment. In sum, our proof-of-principle data indicate that drug-induced premature growth plate closures can be prevented or delayed by targeting a separate phenotypic regulatory mechanism in chondrocytes. The translation applicability of the findings remains to be studied. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Loss of Parathyroid Hormone Receptor Signaling in Osteoprogenitors Is Associated With Accumulation of Multiple Hematopoietic Lineages in the Bone Marrow

Osteoblasts and their progenitors play an important role in the support of hematopoiesis within the bone marrow (BM) microenvironment. We have previously reported that parathyroid hormone receptor (PTH1R) signaling in osteoprogenitors is required for normal B cell precursor differentiation, and for trafficking of maturing B cells out of the BM. Cells of the osteoblast lineage have been implicated in the regulation of several other hematopoietic cell populations, but the effects of PTH1R signaling in osteoprogenitors on other maturing hematopoietic populations have not been investigated. Here we report that numbers of maturing myeloid, T cell, and erythroid populations were increased in the BM of mice lacking PTH1R in Osx-expressing osteoprogenitors (PTH1R-OsxKO mice; knockout [KO]). This increase in maturing hematopoietic populations was not associated with an increase in progenitor populations or proliferation. The spleens of PTH1R-OsxKO mice were small with decreased numbers of all hematopoietic populations, suggesting that trafficking of mature hematopoietic populations between BM and spleen is impaired in the absence of PTH1R in osteoprogenitors. RNA sequencing (RNAseq) of osteoprogenitors and their descendants in bone and BM revealed increased expression of vascular cell adhesion protein 1 (VCAM-1) and C-X-C motif chemokine ligand 12 (CXCL12), factors that are involved in trafficking of several hematopoietic populations. © 2022 American Society for Bone and Mineral Research (ASBMR).     

Administration of a Glucocorticoid With Depot Effect Counteracts the Stimulating Effect of Growth Hormone on Cancellous and Cortical Bone of the Vertebral Body in Rats

Our earlier studies have shown that growth hormone administration could not counteract decreased longitudinal growth and cortical osteopenia of rat femora induced by a glucocorticoid with depot effect. In the present study we examined the effects of glucocorticoid on vertebral bone as well as the effect of growth hormone on vertebral bone in young growing animals also given glucocorticoid injections. Five groups of female rats (3 1/2 months) were treated for 80 days as follows: (1) saline, (2) prednisolone: Delcortol 5 mg/kg/day, (3) growth hormone: 5 mg/kg/day, (4) prednisolone and growth hormone, (5) food restriction. Vertebral dimensions, histomorphometry, and mechanical competence of the vertebral bone were examined. Growth hormone administration increased body weight, vertebral height, cross-sectional area, and volume. The compressive strength of the L₄-corpus cylinder was also increased due to an increase in cancellous bone volume and an increase in the area of cortical bone surrounding the vertebral body. Glucocorticoid administration decreased body weight, height, and volume of the intact vertebrae. Histological examination revealed that glucocorticoid administration decreased the area of cortical bone surrounding the vertebral body but had no effect on the cancellous bone volume. No effect of glucocorticoid administration on mechanical strength of the L₄ corpus cylinder could be detected. In agreement with our findings in cortical bone, we found no effect of growth hormone on vertebral bone when given to animals also receiving glucocorticoid injections. Growth hormone increases longitudinal growth, cortical and cancellous bone mass, and mechanical competence of the vertebral body. Glucocorticoid administration decreases longitudinal growth of the vertebrae and cortical bone mass without affecting cancellous bone mass of the vertebral body. Despite this, administration of a glucocorticoid with depot effect totally inhibits the effect of growth hormone on vertebral bone. Received: 12 March 1997 / Accepted: 14 November 1997     

The Anti‐Osteoanabolic Function of Sclerostin Is Blunted in Mice Carrying a High Bone Mass Mutation of Lrp5

Activating mutations of the putative Wnt co‐receptor Lrp5 or inactivating mutations of the secreted molecule Sclerostin cause excessive bone formation in mice and humans. Previous studies have suggested that Sclerostin functions as an Lrp5 antagonist, yet clear in vivo evidence was still missing, and alternative mechanisms have been discussed. Moreover, because osteoblast‐specific inactivation of β‐catenin, the major intracellular mediator of canonical Wnt signaling, primarily affected bone resorption, it remained questionable, whether Sclerostin truly acts as a Wnt signaling antagonist by interacting with Lrp5. In an attempt to address this relevant question, we generated a mouse model (Col1a1‐Sost) with transgenic overexpression of Sclerostin under the control of a 2.3‐kb Col1a1 promoter fragment. These mice displayed the expected low bone mass phenotype as a consequence of reduced bone formation. The Col1a1‐Sost mice were then crossed with two mouse lines carrying different high bone mass mutations of Lrp5 (Lrp5^A170V and Lrp5^G213V), both of them potentially interfering with Sclerostin binding. Using µCT‐scanning and histomorphometry we found that the anti‐osteoanabolic influence of Sclerostin overexpression was not observed in Lrp5^A213V/A213V mice and strongly reduced in Lrp5^A170V/A170V mice. As a control we applied the same strategy with mice overexpressing the transmembrane Wnt signaling antagonist Krm2 and found that the anti‐osteoanabolic influence of the Col1a1‐Krm2 transgene was not affected by either of the Lrp5 mutations. Taken together, our data support the concept that Sclerostin inhibits bone formation through Lrp5 interaction, yet their physiological relevance remains to be established. © 2015 American Society for Bone and Mineral Research.