Protein Kinase G2 Is Essential for Skeletal Homeostasis and Adaptation to Mechanical Loading in Male but Not Female Mice

We previously showed that the NO/cGMP/protein kinase G (PKG) signaling pathway positively regulates osteoblast proliferation, differentiation, and survival in vitro, and that cGMP-elevating agents have bone-anabolic effects in mice. Here, we generated mice with an osteoblast-specific (OB) knockout (KO) of type 2 PKG (gene name Prkg2) using a Col1a1(2.3 kb)-Cre driver. Compared to wild type (WT) littermates, 8-week-old male OB Prkg2-KO mice had fewer osteoblasts, reduced bone formation rates, and lower trabecular and cortical bone volumes. Female OB Prkg2-KO littermates showed no bone abnormalities, despite the same degree of PKG2 deficiency in bone. Expression of osteoblast differentiation- and Wnt/β-catenin-related genes was lower in primary osteoblasts and bones of male KO but not female KO mice compared to WT littermates. Osteoclast parameters were unaffected in both sexes. Since PKG2 is part of a mechano-sensitive complex in osteoblast membranes, we examined its role during mechanical loading. Cyclical compression of the tibia increased cortical thickness and induced mechanosensitive and Wnt/β-catenin-related genes to a similar extent in male and female WT mice and female OB Prkg2-KO mice, but loading had a minimal effect in male KO mice. We conclude that PKG2 drives bone acquisition and adaptation to mechanical loading via the Wnt/β-catenin pathway in male mice. The striking sexual dimorphism of OB Prkg2-KO mice suggests that current U.S. Food and Drug Administration-approved cGMP-elevating agents may represent novel effective treatment options for male osteoporosis. © 2022 American Society for Bone and Mineral Research (ASBMR).     

Sex‐Dependent,Osteoblast Stage‐Specific Effects of Progesterone Receptor on Bone Acquisition

The role of the progesterone receptor (PR) in the regulation of sexual dimorphism in bone has yet to be determined. Here we utilized genetic fate mapping and Western blotting to demonstrate age‐dependent PR expression in the mouse femoral metaphysis and diaphysis. To define sex‐dependent and osteoblast stage–specific effects of PR on bone acquisition, we selectively deleted PR at different stages of osteoblast differentiation. We found that when Prx1‐Cre mice were crossed with PR floxed mice to generate a mesenchymal stem cell (MSC) conditional KO model (Prx1; PRcKO), the mutant mice developed greater trabecular bone volume with higher mineral apposition rate and bone formation. This may be explained by increased number of MSCs and greater osteogenic potential, particularly in males. Age‐related trabecular bone loss was similar between the Prx1; PRcKO mice and their WT littermates in both sexes. Hormone deficiency during the period of rapid bone growth induced rapid trabecular bone loss in both the WT and the Prx1; PRcKO mice in both sexes. No differences in trabecular bone mass was observed when PR was deleted in mature osteoblasts using osteocalcin‐Cre (Bglap‐Cre). Also, there were no differences in cortical bone mass in all three PRcKO mice. In conclusion, PR inactivation in early osteoprogenitor cells but not in mature osteoblasts influenced trabecular bone accrual in a sex‐dependent manner. PR deletion in osteoblast lineage cells did not affect cortical bone mass. © 2017 American Society for Bone and Mineral Research.     

The structural and hormonal basis of sex differences in peak appendicular bone strength in rats.

To identify the structural and hormonal basis for the lower incidence of fractures in males than females, sex differences in femoral mid-shaft geometry and breaking strength were studied in growth hormone (GH)-replete and -deficient male and female rats. Sexual dimorphism appeared during growth. Cortical thickening occurred almost entirely by acquisition of bone on the outer (periosteal) surface in males and mainly on the inner (endocortical) surface in females. By 8 months of age, males had 22% greater bone width and 33% greater breaking strength than females. Gonadectomy (Gx) at 6 weeks reduced sex differences in bone width to 7% and strength to 21% by halving periosteal bone formation in males and doubling it in females. Gx had no net effect on the endocortical surface in males but abolished endocortical bone acquisition in females. GH deficiency halved periosteal bone formation and had no net effect on the endocortical surface in males, but abolished bone acquisition on both surfaces in females, leaving males with 17% greater bone width and 44% greater breaking strength than females. Sex hormone deficiency produces greater bone fragility in males than females by removing a stimulator of periosteal growth in males and removing an inhibitor of periosteal growth in females. GH deficiency produces less bone fragility in males than females because males retain androgen-dependent periosteal bone formation while bone acquisition on both surfaces is abolished in females. Thus, periosteal growth is independently and additively stimulated by androgens and GH in males, inhibited by estrogen, and stimulated by GH in females. The hormonal regulation of bone surfaces establishes the amount and spatial distribution of bone and so the sexual dimorphism in its strength.     

Skeletal Site-Dependent Expression of the Androgen Receptor in Human Osteoblastic Cell Populations

There are obvious sexual differences in adult skeletal morphology which for the most part are related to differences in size. Higher androgen serum levels in males exert potent osteoanabolic effects and therefore may contribute to this sexual dimorphism of the skeleton. The presence of androgen receptors (AR) in bone cells is a prerequisite for a direct osteoanabolic action of androgens. To investigate the possibility that, in addition to gender-related differences in androgen serum levels, there are also gender-related differences in the osteoblastic expression pattern of the androgen receptor, we examined AR mRNA expression, androgen binding sites, and mitogenic responses to the androgen dihydrotestosterone (DHT) in human osteoblastic cell (HOC) populations. HOCs were isolated from bone biopsy specimens derived from different skeletal sites of healthy adult males and females (2–69 years old). We found that male and female HOCs of all examined ages express similar AR mRNA levels and similar numbers of androgen binding sites. Using whole-cell-binding assays, we observed 3129–8417 androgen binding sites per femoral HOC with apparent KDs of 1.45–2.83 nM depending on the age of the investigated HOC population. Mandibular and cortical HOC of both sexes expressed higher AR mRNA levels, significantly more androgen binding sites per cell, and exhibited significantly greater mitogenic responses to DHT than iliac crest-derived and trabecular HOC of the same skeletal system and the same skeletal-site, respectively. In early adulthood, HOCs of both sexes appear to express somewhat higher AR mRNA levels and to possess more androgen binding sites than prepubertal and senescent HOC. Because sex hormone serum levels rise in puberty, we investigated the regulation of the AR mRNA expression by various steroids. We found that dexamethasone (dexa) and in some experiments also 17β-estradiol (E2) and 1,25-dihydroxyvitamin D3 (D3) increased AR mRNA levels and androgen binding in HOC cultures. A pretreatment with dexa, E2, and D3 significantly increased the mitogenic response of HOCs to DHT. We conclude that (1) higher androgen serum levels in males together with a higher AR expression at certain skeletal sites may contribute to the development of sex-related differences in skeletal morphology, (2) glucocorticoids induce AR gene expression in HOC cultures, and (3) glucocorticoids, E2, and D3 enhance the mitogenic action of DHT. Received: 3 June 1996 / Accepted: 30 April 1997     

Effects of bone-specific physical activity,gender and maturity on tibial cross-sectional bone material distribution: a cross-sectional pQCT comparison of children and young adults aged 5–29 years

Growth is the opportune time to modify bone accrual. While bone adaptation is known to be dependent on local loading and consequent deformations (strain) of bone, little is known about the effects of sex, and bone-specific physical activity on location-specific cross-sectional bone geometry during growth. To provide more insight we examined bone traits at different locations around tibial cross sections, and along the tibia between individuals who vary in terms of physical activity exposure, sex, and pubertal status. Data from 304 individuals aged 5–29 years (172 males, 132 females) were examined. Peripheral quantitative computed tomography (pQCT) was applied at 4%, 14%, 38%, and 66% of tibial length. Maturity was established by estimating age at peak height velocity (APHV). Loading history was quantified with the bone-specific physical activity questionnaire (BPAQ). Comparisons, adjusted for height, weight and age were made between sex, maturity, and BPAQ tertile groups. Few to no differences were observed between sexes or BPAQ tertiles prior to APHV, whereas marked sexual dimorphism and differences between BPAQ tertiles were observed after APHV. Cross-sectional location-specific differences between BPAQ tertiles were not evident prior to APHV, whereas clear location-specificity was observed after APHV. In conclusion, the skeletal benefits of physical activity are location-specific in the tibia. The present results indicate that the peri- or post-pubertal period is likely a more favourable window of opportunity for enhancing cross-sectional bone geometry than pre-puberty. Increased loading during the peri-pubertal period may enhance the bone of both sexes.     

Overexpression of secreted frizzled‐related protein 1 inhibits bone formation and attenuates parathyroid hormone bone anabolic effects

Secreted frizzled‐related protein 1 (sFRP1) is an antagonist of Wnt signaling, an important pathway in maintaining bone homeostasis. In this study we evaluated the skeletal phenotype of mice overexpressing sFRP1 (sFRP1 Tg) and the interaction of parathyroid hormone (PTH) treatment and sFRP1 (over)expression. Bone mass and microarchitecture were measured by micro‐computed tomography (µCT). Osteoblastic and osteoclastic cell maturation and function were assessed in primary bone marrow cell cultures. Bone turnover was assessed by biochemical markers and dynamic bone histomorphometry. Real‐time PCR was used to monitor the expression of several genes that regulate osteoblast maturation and function in whole bone. We found that trabecular bone mass measurements in distal femurs and lumbar vertebral bodies were 22% and 51% lower in female and 9% and 33% lower in male sFRP1 Tg mice, respectively, compared with wild‐type (WT) controls at 3 months of age. Genes associated with osteoblast maturation and function, serum bone formation markers, and surface based bone formation were significantly decreased in sFRP1 Tg mice of both sexes. Bone resorption was similar between sFRP1 Tg and WT females and was higher in sFRP1 Tg male mice. Treatment with hPTH(1‐34) (40 µg/kg/d) for 2 weeks increased trabecular bone volume in WT mice (females: +30% to 50%; males: +35% to 150%) compared with sFRP1 Tg mice (females: +5%; males: +18% to 54%). Percentage increases in bone formation also were lower in PTH‐treated sFRP1 Tg mice compared with PTH‐treated WT mice. In conclusion, overexpression of sFRP1 inhibited bone formation as well as attenuated PTH anabolic action on bone. The gender differences in the bone phenotype of the sFRP1 Tg animal warrants further investigation. © 2010 American Society for Bone and Mineral Research     

Glomerulosclerosis in aging humans is not influenced by gender.

Aging male rats develop progressive glomerulosclerosis, proteinuria, and loss of renal function, whereas females are remarkably resistant to the development of these abnormalities. Although sex hormones appear to contribute to gender-related differences in the development of glomerulosclerosis in aging rats, it is not clear that sexual dimorphism characterizes glomerular obsolescence in aging humans. To study this question further, the glomerular histology of males and females ranging in age from infancy to 90 years was compared in 250 autopsy specimens. We found no differences between the sexes in the development of glomerulosclerosis in aging humans. These data disprove the hypothesis that testosterone is an important factor contributing to progressive glomerulosclerosis in aging men. Conversely, any renoprotective effects of estrogen would be limited by the onset of menopause because significant glomerulosclerosis did not develop until after the age of 50 years.     

A comparison of bone geometry and cortical density at the mid-femur between prepuberty and young adulthood using magnetic resonance imaging

In upper extremity bones, a sexual dimorphism exists in the development of periosteal and endocortical bone surfaces during growth. Little is known about developmental patterns of bone geometry at weight-bearing bones like the femur. Using MRI and dual energy X-ray absorptiometry (DXA), this study assessed the differences in mid-femoral total (TA), cortical (CA) and medullary areas (MA), cortical thickness, and cortical density (BMD(compartment)) between prepuberty and young adulthood in 145 healthy subjects (94 females) 6 to 25 years old. Additionally, agreement between mid-femoral total bone volume (TV) measurements by DXA and MRI were investigated. In both sexes, TA, CA, MA, and cortical thickness were significantly larger in adults compared to prepubertal subjects (P < 0.001), and males had greater values than females. This sex difference persisted for TA, CA, and cortical thickness (P < 0.05), but not MA, after adjusting for femur length and weight. Mean (SD) cortical BMD increased from 1.05 (0.07) and 1.09 (0.10) g/cm(3) in prepubertal children to 1.46 (0.14) and 1.42 (0.1) g/cm(3) in young adults, females and males, respectively (P < 0.001). TV measurements by DXA were significantly greater than by MRI (P < 0.001) in young adults. In conclusion, periosteal and endocortical expansion and increasing cortical BMD are the growth processes found at the mid-femur in both sexes. Our findings contrast to that in upper extremity bones, where MA is constant in females during growth. The difference in femoral bone development may be due to higher strains caused by weight bearing and genetic factors. DXA, in contrast to MRI, is inaccurate in the determination of mid-femoral TV measures.     

Sexual dimorphism in cortical bone size and strength but not density is determined by independent and time‐specific actions of sex steroids and IGF‐1: Evidence from pubertal mouse models

Although it is well established that males acquire more bone mass than females, the underlying mechanism and timing of this sex difference remain controversial. The aim of this study was to assess the relative contribution of sex steroid versus growth hormone–insulin‐like growth factor 1 (GH–IGF‐1) action to pubertal bone mass acquisition longitudinally in pubertal mice. Radial bone expansion peaked during early puberty (3 to 5 weeks of age) in male and female mice, with significantly more expansion in males than in females (+40%). Concomitantly, in 5 week old male versus female mice, periosteal and endocortical bone formation was higher (+70%) and lower (?47%), respectively, along with higher serum IGF‐1 levels during early puberty in male mice. In female mice, ovariectomy increased radial bone expansion during early puberty as well as the endocortical perimeter. In male mice, orchidectomy reduced radial bone expansion only during late puberty (5 to 8 weeks of age), whereas combined androgen and estrogen deficiency modestly decreased radial bone expansion during early puberty, accompanied by lower IGF‐1 levels. GHRKO mice with very low IGF‐1 levels, on the other hand, showed limited radial bone expansion and no skeletal dimorphism. From these data we conclude that skeletal sexual dimorphism is established during early puberty and depends primarily on GH–IGF‐1 action. In males, androgens and estrogens have stimulatory effects on bone size during late and early puberty, respectively. In females, estrogens limit bone size during early puberty. These longitudinal findings in mice provide strong evidence that skeletal dimorphism is determined by independent and time‐specific effects of sex steroids and IGF‐1. © 2010 American Society for Bone and Mineral Research     

Sex differences in the inferior parietal lobule

The inferior parietal lobule (IPL) - a neocortical region and part of the heteromodal association cortex (HASC) - has been hypothesized to exhibit sexual dimorphism, as do other HASC regions, particularly with regard to asymmetry. Using a reliable method for measuring IPL gray matter volume based upon individual sulcal-gyral landmarks, we measured this region on magnetic resonance imaging scans from a sample of 15 individually matched pairs of normal male and female subjects. Male subjects showed significantly larger left, but not right, IPL volumes when compared to females. Males also showed a leftward (left > right) asymmetry for the IPL, with a less marked opposite asymmetry in females. Such sexual dimorphisms may possibly underlie the subtle cognitive differences observed between the sexes.     

Regulation of postnatal trabecular bone formation by the osteoblast endothelin A receptor

Endothelin‐1 (ET‐1) is a potent vasoconstrictor that also stimulates cells in the osteoblast lineage by binding to the endothelin A receptor (ETAR). ET‐1 ligand is widely secreted, particularly by the vasculature. However, the contributions of ETAR signaling to adult bone homeostasis have not been defined. ETAR was inactivated in osteoblasts by crossing ETAR‐floxed and osteocalcin‐Cre mice. Histomorphometric analyses were performed on 4‐, 8‐, and 12‐week‐old osteoblast‐targeted ETAR knockout (KO) and wild‐type (WT) male and female mice. Tibial trabecular bone volume was significantly lower from 12 weeks in KO versus WT mice in both males and females. Bone‐formation rate, osteoblast density, and in vitro osteoblast differentiation were reduced by targeted inactivation of ETAR. A separate longitudinal analysis was performed between 8 and 64 weeks to examine the effect of aging and castration on bone metabolism in ETAR KO mice. Hypogonadism did not change the rate of bone accrual in WT or KO females. However, eugonadal KO males had a significantly larger increase in tibial and femoral bone acquisition than WT mice. Male mice castrated at 8 weeks of age showed the reverse: KO mice had reduced rates of tibial and femoral BMD acquisition compared with WT mice. In vitro, ET‐1 increased osteoblast proliferation, survival, and differentiation. Dihydrotestosterone also increased osteoblast differentiation using a mechanism distinct from the actions of ET‐1. These results demonstrate that endothelin signaling in osteoblasts is an important regulator of postnatal trabecular bone remodeling and a modulator of androgen effects on bone. © 2011 American Society for Bone and Mineral Research     

Effects of global or targeted deletion of the EP4 receptor on the response of osteoblasts to prostaglandin in vitro and on bone histomorphometry in aged mice

Because global deletion of the prostaglandin EP4 receptor results in neonatal lethality, we generated a mouse with targeted EP4 receptor deletion using Cre–LoxP methodology and a 2.3 kb collagen I a1 promoter driving Cre recombinase that is selective for osteoblastic cells. We compared wild type (WT), global heterozygote (G-HET), targeted heterozygote (T-HET) and knockout (KO) mice. KO mice had one targeted and one global deletion of the EP4 receptor. All mice were in a mixed background of C57BL/6 and CD-1. Although there were one third fewer G-HET or KO mice at weaning compared to WT and T-HET mice, G-HET and KO mice appeared healthy. In cultures of calvarial osteoblasts, prostaglandin E₂ (PGE₂) increased alkaline phosphatase (ALP) activity in cells from WT mice, and this effect was significantly decreased in cells from either G-HET or T-HET mice and further decreased in cells from KO mice. A selective agonist for EP4 receptor increased ALP activity and osteocalcin mRNA levels in cells from WT but not KO mice. A selective COX-2 inhibitor, NS-398, decreased osteoblast differentiation in WT but not KO cells. At 15 to 18 months of age there were no differences in serum creatinine, calcium, PTH, body weight or bone mineral density among the different genotypes. Static and dynamic histomorphometry showed no consistent changes in bone volume or bone formation. We conclude that expression of the EP4 receptor in osteoblasts is critical for anabolic responses to PGE₂ in cell culture but may not be essential for maintenance of bone remodeling in vivo.     

Gender differences in volumetric bone density: a study of opposite-sex twins

Gender difference in bone size is a potential confounder when comparing bone density between males and females. A comparison of volumetric BMD (vBMD) between men and women, which is a measure of bone mass relative to three-dimensional bone volume (g/cm³) as opposed to areal bone density (g/cm²), may be a more accurate reflection of gender differences in bone density. The aims of this study were to examine gender differences in bone mass (BMC), areal BMD (aBMD), volumetric BMD (vBMD) by comparing twins of opposite sex in whom the effects of age, genes and environment are partially controlled for. DEXA derived BMC, aBMD, vBMD at the third lumbar vertebra (L3), femoral neck (FN) and forearm (1/3 radius) were compared between 82 opposite sex pairs aged 18–80. BMC was significantly higher in males at all three sites (26–45.5%). For aBMD the gender differences remained significant at all sites except the spine. The average differences in aBMD were not as great as the differences in BMC (2.2–20.5%). The differences in vBMD, however, followed a different pattern. FN and L3 vBMD were significantly higher in females (4.8 and 0.6%, respectively), while radial BMD was not significantly different between the sexes. Comparing aBMD values between males and females, when females in general have a smaller skeleton than males may not be a true indication of gender differences in bone density. A comparison of vBMD between men and women shows only small differences in bone density between the sexes.     

Photographic Facial Soft Tissue Analysis of Healthy Turkish Young Adults: Anthropometric Measurements

Background  The purpose of this study was to establish facial soft tissue norms for Turkish young adults. Methods  Anthropometric measurements of the facial soft tissue were taken from 281 female and 149 male Turkish adults aged between 18 and 24 years. The soft tissue facial profiles were digitally analyzed using linear (17 vertical and 10 horizontal) measurements made with standardized photographic records, taken in a natural head position, to determine the average soft tissue facial profile for males and females. Results  A statistically significant difference was found between males and females in 20 of 27 measurements taken (p < 0.001). The most prominent differences between the sexes were observed in the measurements taken from the face region. Results were compared with other ethnic groups.     

Endostatin Affects Osteoblast Behavior In Vitro,but Collagen XVIII/Endostatin Is Not Essential for Skeletal Development In Vivo

Endostatin, a fragment of collagen XVIII, can inhibit vascular endothelial growth factor (VEGF) signaling. VEGF is known to be crucial for bone development. The aims of this study were to investigate the influences of endostatin on osteoblast behavior in vitro and the roles of collagen XVIII/endostatin on bone development in vivo. For the in vitro experiments, MC3T3-E1 osteoblasts were treated with VEGF-A, 2 μg/ml endostatin, 20 μg/ml endostatin, VEGF-A + 2 μg/ml endostatin, or VEGF-A + 20 μg/ml endostatin. Osteoblast proliferation and matrix mineralization were analyzed. Faxitron, pQCT, and histological analyses were performed on hindleg bones of transgenic mice overexpressing endostatin (ES-tg) and mice lacking collagen XVIII (Col18a1 ^−/−) to study bone development in vivo. Treatment of cells with endostatin decreased osteoblast proliferation. Moreover, VEGF-A together with endostatin (2 μg/ml) decreased osteoblast proliferation and matrix mineralization. In vivo, Col18a1 ^−/− and ES-tg mice displayed no differences in bone density or mineral content during bone development, but ES-tg bones grew in length more slowly compared to the controls. The formation of secondary ossification centers was delayed in Col18a1 ^−/− mice. Immunohistochemistry revealed collagen XVIII in basement membranes of periosteal and bone marrow vessels and at muscle attachment sites. In conclusion, endostatin affects osteoblast behavior in vitro, the effects being boosted by simultaneous treatment with VEGF. In vivo, Col18a1 ^−/− and ES-tg mice show mild delays in bone development. These changes are transitory and suggest that collagen XVIII/endostatin does not play an indispensable role in skeletal development.     

Sex differences in absolute rates of bone resorption in young rats: Appendicular versus axial bones

This study compares absolute rates of bone resorption and formation at the organ level in adolescent Sprague-Dawley rats as a function of sex and type of bone. Bone r’esorption and formation were quantified in rapidly growing male and female rats (4-7 weeks of age) who were multiply prelabeled with [³H]tetracycline. Ten different whole bones were compared: four cranial or appendicular bones and six axial bones. Absolure rate of bone r’esorption was measured isotopically by the loss of³H-tetracycline from each whole bone. Bone growth was quantified in terms of relative and absolute increase in bone calcium mass. When the rates of bone resorption (loss of [³H]-tetracycline as percent of whole bone per 3 weeks) were compared between sexes, the six axial bones showed significantly higher rates(P < 0.05-0.001) in males (64-73) than in females (37-66). No significant sex differences were observed in rate for the two cranial and two appendicular bones. During 4–7 weeks of age, a comparison of bone masses showed that only one bone (calvaria) gained more mass in the male and two bones (mandible and hum’erus) gained more mass in the female. In contrast, five of six axial bones gained more mass in the female. Thus, 7 out of 10 bones were larger in the female. In growing male and female rats, an inverse relationship appears between rate of bone r’esorption and mass for most of the axial bones; this relationship was not apparent for cranial or appendicular bones. Sexual dimorphism was consistently seen by greater axial bone mass in females. However, greater rates of bone resorption were seen in male axial bones but not in cranial or appendicular bones. It is apparent that the different types of bones are heterogeneous in their rates of resorption and formation during this period of growth.