The Effects of Androgens on Murine Cortical Bone Do Not Require AR or ERα Signaling in Osteoblasts and Osteoclasts

Abstract

In men, androgens are critical for the acquisition and maintenance of bone mass in both the cortical and cancellous bone compartment. Male mice with targeted deletion of the androgen receptor (AR) in mature osteoblasts or osteocytes have lower cancellous bone mass, but no cortical bone phenotype. We have investigated the possibility that the effects of androgens on the cortical compartment result from AR signaling in osteoprogenitors or cells of the osteoclast lineage; or via estrogen receptor alpha (ERα) signaling in either or both of these two cell types upon conversion of testosterone to estradiol. To this end, we generated mice with targeted deletion of an AR or an ERα allele in the mesenchymal (AR^f/y;Prx1-Cre or ERα^f/f;Osx1-Cre) or myeloid cell lineage (AR^f/y; LysM-Cre or ERα^f/f;LysM-Cre) and their descendants. Male AR^f/y;Prx1-Cre mice exhibited decreased bone volume and trabecular number, and increased osteoclast number in the cancellous compartment. Moreover, they did not undergo the loss of cancellous bone volume and trabecular number caused by orchidectomy (ORX) in their littermate controls. In contrast, AR^f/y;LysM-Cre, ERα^f/f; Osx1-Cre, or ERα^f/f;LysM-Cre mice had no cancellous bone phenotype at baseline and lost the same amount of cancellous bone as their controls following ORX. Most unexpectedly, adult males of all four models had no discernible cortical bone phenotype at baseline, and lost the same amount of cortical bone as their littermate controls after ORX. Recapitulation of the effects of ORX by AR deletion only in the AR^f/y;Prx1-Cre mice indicates that the effects of androgens on cancellous bone result from AR signaling in osteoblasts—not on osteoclasts or via aromatization. The effects of androgens on cortical bone mass, on the other hand, do not require AR or ERα signaling in any cell type across the osteoblast or osteoclast differentiation lineage. Therefore, androgens must exert their effects indirectly by actions on some other cell type(s) or tissue(s).

Introduction

Estrogens and androgens promote the acquisition of bone mass during puberty and are responsible for the sexual dimorphism of the skeleton. In addition, estrogens and androgens contribute to the maintenance of bone mass during adulthood. A deficiency of estrogens in females or both androgens and estrogens in males tilts the balance between formation and resorption in favor of the latter and contributes to the development of osteoporosis in either sex.^(1–3)

Osteoblasts and osteoclasts originate from mesenchymal and myeloid progenitors, respectively. Estrogen and androgen receptors have been identified in several cell types along the differentiation progression of both lineages. Evidence accumulated during the last 25 years has established that both estrogens and androgens acting via their receptors suppress the birth rate of osteoclasts and osteoblasts and thereby attenuate the rate of bone remodeling.^(4–7) In addition, both estrogens and androgens shorten the lifespan of osteoclasts and prolong the lifespan of osteoblasts and osteocytes.^(8–12) Conversely, estrogen or androgen deficiency unleashes osteoclastogenesis and osteoblastogenesis, thereby increasing the rate of bone remodeling; and also prolongs osteoclast and shortens osteoblast and osteocyte lifespan.

In spite of significant advances in our understanding of the role of sex steroids on skeletal homeostasis, until fairly recently the cellular targets responsible for the effects of estrogens or androgens on the male or female skeleton could be only deduced from in vitro observations. The development of mouse models with conditional deletion of the androgen receptor (AR) or estrogen receptor (ER) has provided genetic means to interrogate functionally the cellular targets of sex steroid action in vivo.^(1,2)

The seminal role of androgens in bone homeostasis in males is well-documented.⁽²⁾ Loss of androgens in men and male rodents leads to loss of BMD. Conversely, androgen administration stimulates periosteal bone formation and cortical bone accrual in both men and male rodents. Consistent with this evidence, global deletion of the AR in mice decreases both cortical and cancellous bone mass.^(13,14) However, cell-specific deletion of AR in osteoblasts or osteocytes results in lower cancellous bone mass, but it has no effect on the cortical compartment.^(15–18) These findings leave the possibility that the effects of androgens on cortical bone—the majority of the mammalian skeleton—are mediated by AR signaling in osteoprogenitors or cells of the osteoclast lineage; or via ERα signaling in either or both of these two cell types upon local conversion of testosterone to estradiol; or perhaps indirectly through actions on some other tissue(s).

In the work reported here we generated mice with AR or ERα deletion in mesenchymal or myeloid progenitors and analyzed their bone phenotype in both the androgen-replete and the androgen-deficient state. Such deletions inexorably and necessarily lead to the deletion of AR and ERα in all the descendants of the respective progenitors. Using these four models we have inquired whether the effects of androgens on the cancellous and/or cortical compartment result from AR signaling in any cell type along the differentiation progression of these two lineages, or through the conversion of testosterone to estradiol and thereby ERα signaling.

Materials and Methods

Animal experimentation

To disrupt the AR gene in the mesenchymal lineage, AR^f/f mice (C57BL/6N background)⁽¹⁹⁾ were crossed with mice expressing Prx1-Cre (C57BL/6J background).⁽²⁰⁾ The experimental mice were generated using a two-step breeding strategy. Specifically, hemizygous Cre transgenic mice were crossed with homozygous AR^f/f mice to generate heterozygous AR^f/y or AR^f/+ offspring with and without the Cre allele. These offspring were then intercrossed to generate the following: wild-type mice, mice hemizygous for the Cre allele, and mice with the AR^f/y or AR^f/f allele with and without the Cre allele. Offspring were genotyped by PCR using the following primer sequences: Cre-forward 5′-GCGATTATCTTCTATATCTTCAGG-3′, Cre-reverse 5′-GCCAATATGGATTAACATTCTCCC-3′, product size 400 bp; AR-flox-forward 5′-AGCCTGTATACTCAGTTGGGG-3′, AR-flox-reverse 5′-AATGCATCACATTAAGTTGATACC-3′, product size 855 bp (WT) and 952 bp (floxed allele). To disrupt the AR gene in the osteoclast lineage, AR^f/f mice were crossed with mice expressing LysM-Cre (C57BL/6J background),⁽²¹⁾ following a strategy similar to the one described earlier in this paragraph. (Cre-forward 5′-CCCAGAAATGCCAGATTACG-3′, Cre-reverse 5′-CTTGGGCTGCCAGAATTTCTC-3′, product size 700 bp). The generation of ERα^f/f;Osx1-Cre and ERα^f/f;LysM-Cre has been described.^(22,23) ERα^f/f and Osx1-Cre were in C57BL/6J background. Notably, mice expressing Osx1-Cre exhibit decreased body weight and femoral length and cortical bone mass.^(23,24) We have, therefore, used Osx1-Cre as controls for ERα^f/f;Osx1-Cre. Offspring from all genotypes were tail-clipped for DNA extraction at the time of weaning (21 days), and then group-housed with same-sex littermates. Mice were maintained with a constant temperature of 23°C, a 12-hour light/dark cycle, and had access to food and water ad libitum.

Mice used in ORX experiments were 20 weeks old and were stratified into sham-surgical or ORX-surgical groups according to their femoral DXA BMD. Specifically, within each genotype, mice were sorted from low to high BMD values. Mice were then assigned the numbers 1 and 2, successively. All animals with the same number were assigned to the same group. BMD means and SD for each group were calculated and compared by t-test to assure that means were similar. Surgeries were performed in the morning. After 6 weeks, animals were euthanized and the tissues dissected for further analyses. BMD measurements were performed 1 day prior to surgery and before euthanasia. Procedures were approved by Institutional Animal Care and Use Committees of the University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System.

Bone imaging

BMD measurements were performed by DXA using a PIXImus densitometer (GE Lunar, Madison, WI, USA), as described,⁽²⁵⁾ in mice sedated with 2% isoflurane. The spine window was a rectangle depending on animal body length, reaching from just below the skull to the base of the tail. The femoral window captured the entire left femur. Micro-computed tomography (mCT) analysis (μCT40; Scanco Medical, Wayne, PA, USA) was done after the bones were dissected, cleaned, fixed in 10% Millonig’s formalin, and transferred to 100% ethanol. Scans were performed at medium resolution (nominal isotropic voxel size = 12 μm) and integrated into 3D voxel images (1024 × 1024 pixel matrices for each individual planar stack). A Gaussian filter (sigma = 0.8, support = 1) was applied to all analyzed scans. Key parameters were as follows: X-ray tube potential = 55 kVp, X-ray intensity = 145 μA, integration time = 200 ms, and threshold = 200 mg/cm³. The entire vertebral body was scanned with a transverse orientation excluding any bone outside the vertebral body. In the distal femur, 151 transverse slices were taken from the epicondyles and extending toward the proximal end of the femur. Manual analysis excluded the cortical bone and the primary spongiosa from the analysis. All trabecular measurements were made by manually drawing contours every 10 to 20 slices and using voxel counting for bone volume per tissue volume and sphere filling distance transformation indices without assumptions about the bone shape as a rod or plate for trabecular microarchitecture. Image processing language scripts including the “cl_image” command were used to obtain the femoral endocortical and periosteal circumference. μCT measurements were expressed in 3D nomenclature.⁽²⁶⁾

Histology

Femurs and vertebrae were fixed in 10% Millonig’s formalin, transferred to 100% ethanol, and embedded undecalcified in methyl methacrylate. Paraffin sections of decalcified vertebrae were also examined. The histomorphometric examination was performed in longitudinal sections stained for tartrate-resistant acid phosphatase (TRAP) using the OsteoMeasure Analysis System (OsteoMetrics, Inc., Decatur, GA, USA). Histomorphometry measurements of the cancellous bone were restricted to the secondary spongiosa. One section was measured for each sample by one “blind” observer. Histomorphometry was performed in only five or six animals per group, based on our extensive experience with the technique and the animal model and power calculations showing that this number is sufficient. The selection of the animals to perform histomorphometry was done blindly and there were no outliers.

Cell culture

Bone marrow cells were obtained from femurs and tibias that were dissected free of muscle and connective tissue under sterile conditions. The proximal and distal metaphyses were cut out with a scalpel and the bone marrow flushed from the diaphysis using a syringe. Bone marrow cells from three mice of each genotype were pooled and seeded in triplicate wells. One pool of cells per genotype was used for mRNA analysis. To obtain osteoblastic cells, bone marrow cells were cultured in α-MEM culture medium containing 10% FBS, ascorbic acid 50 μg/mL, 100 U/mL penicillin, and 100 μg/mL streptomycin until they reached 90% confluence. Half of the medium was replaced every 5 days. To generate osteoclasts, bone marrow cells were suspended in α-MEM and incubated for 24 hours in the presence of 10 ng/mL M-CSF. Nonadherent cells were then collected and cultured with 30 ng/mL M-CSF and 30 ng/mL RANKL (R&D Systems, Minneapolis, MN, USA) for 4 days. Osteoclast cultures were fixed with 10% neutral buffered formalin for 15 min and stained for TRAP using the Leukocyte Acid Phosphatase Assay Kit, following the manufacturer’s instructions (Sigma–Aldrich, St. Louis, MO, USA).

Quantitative PCR

Soft tissues were removed from mice and immediately stored in liquid nitrogen. Osteocyte-enriched femoral bone shafts were obtained by removing the ends of the femurs and flushing out the bone marrow with PBS. Bones were then sectioned longitudinally and the periosteal and endocortical surfaces scraped with a scalpel, cut into small pieces, and frozen in liquid nitrogen. Total RNA was extracted from tissues or cultured cells using TRIzol Reagent (Life Technologies, Grand Island, NY, USA) and reverse-transcribed using the High-Capacity cDNA Archive Kit (Applied Biosystems, Grand Island, NY, USA), according to the manufacturer’s instructions. TaqMan quantitative PCR was performed as described⁽²⁷⁾ to determine mRNA levels using the following primers: Mm00433148_mH (ERα), Mm01238473_m1 (AR), and Mm00475528_m1 (ribosomal protein S2) manufactured by the TaqMan Gene Expression Assays service (Applied Biosystems). mRNA expression levels were normalized to the housekeeping gene ribosomal protein S2 using the delta-delta threshold cycle (ΔΔCt) method.⁽²⁸⁾

Genomic DNA was isolated from tissues and cultured cells using a QIAamp DNA Mini Kit. The efficiency of AR genomic deletion was quantified using TaqMan Assay-by-Design primer set 5′-CCCAGAAGACCTGCCTGAT-3′ and 5′-AGTGAGAGCTCCGTAGTGACA-3′. Genomic DNA expression levels were normalized to the TaqMan Copy Number Reference Assay Tfrc (Life Technologies) using the ΔCt method.

Flow cytometry analysis

Femoral bone marrow cells were treated with Red Blood Cell Lysis Buffer for 1 min and stained with 4 μg/mL anti-CD19-APC-Cy7 (BD Pharmingen, San Jose, CA, USA) to identify B cells, and 4 μg/mL anti-CD11b-PE (eBioscience, San Diego, CA, USA) to identify myeloid cells. Cells were then washed and stained for 30 min with 1 μg/mL 4,6-diamidino-2-phenylindole (DAPI) (Santa Cruz Biotechnology, Dallas, TX, USA). All samples were analyzed by flow cytometry Fortessa (BD Biosciences, San Jose, CA, USA), and the data were analyzed with FACSDiva software.

Statistical analysis

Group mean values were compared, as appropriate, by Student’s unpaired two-tailed t-test or two-way ANOVA with Holm–Sidak multiple comparison test after determining that the data were normally distributed and exhibited equivalent variances. A p value ≤0.05 was considered significant.

Results

Generation of AR^f/f;Prx1-Cre mice

Mice in which exon 2 of the AR was floxed were crossed with mice expressing the Cre recombinase under the control of regulatory elements of the Prrx1 gene. The transgene is expressed in pluripotent mesenchymal progenitors and their progeny in the appendicular, but not the axial, skeleton.⁽²⁰⁾ The effectiveness of the AR gene deletion was demonstrated by a >80% decrease in AR mRNA levels from cultured bone marrow–derived osteoblasts and osteocyte-enriched femoral shafts (Fig. 1A). AR mRNA expression in cultured osteoclasts, as well as seminal vesicle and kidney was unaffected, although the mRNA expression in osteoclasts was considerably lower than in osteoblasts. Total body weight, lean body mass, and femoral length (Fig. 1B–D) were indistinguishable in the four genotypes. The morphology of the growth plate was also unaffected by AR deletion (Supporting Fig. 1). More important, AR^f/y;Prx1-Cre mice had normal seminal vesicle weight (Fig. 1E), indicating that androgen levels were not affected.

Fig. 1.

Deletion of AR in Prx1-Cre expressing cells in males. (A) AR mRNA levels in cultured osteoblasts and osteoclasts (triplicate cultures), femur shafts, seminal vesicles, and kidney (n = 5–10/group). (B) Longitudinal body weight of one cohort of mice (n = 8–13/group). (C) Lean body mass; (D) femur length measured with calipers; and (E) seminal vesicle weight in 16-week-old mice (n = 8–13/group). Bars represent mean and SD; *p < 0.05 by Student’s t-test. Ob = osteoblast; Oc = osteoclast; n.d. = not detected.

AR^f/y;Prx1-Cre mice have low cancellous, but normal cortical bone mass

μCT analysis of the femur of male mice, euthanized at 4 months of age, revealed low cancellous bone mass in the AR^f/y;Prx1-Cre mice as compared to WT, Prx1-Cre, and AR^f/y littermates (Fig. 2A, B). In line with the fact that the Prx1-Cre transgene is not expressed in the axial skeleton, spinal cancellous bone mass was unaltered (Supporting Fig. 2), providing additional evidence for the specificity of the AR deletion. The low cancellous bone mass in the AR^f/y;Prx1-Cre mice was associated with decreased trabecular number and increased trabecular separation (Fig. 2C), whereas trabecular thickness was not affected. In a different cohort of mice, the bone phenotype was determined at 7 weeks of age. At this age also, trabecular number was reduced and trabecular separation increased in AR^f/y;Prx1-Cre mice (Table 1). Cancellous bone mass, however, was not different at this earlier age. In difference to cancellous bone, the cortical bone of AR^f/y;Prx1-Cre mice was indistinguishable from control littermates as determined by cortical thickness and the periosteal and endocortical perimeters at the midshaft (Fig. 2D). In agreement with the imaging studies, histological analysis of femoral bone sections from 7-week-old AR^f/y;Prx1-Cre mice revealed a 25% increase in osteoclast number (Fig. 2E), whereas osteoblast number was unaffected.

Fig. 2.

Deletion of AR in Prx1-Cre expressing cells decreases cancellous bone mass. (A) Representative μCT images of femoral cancellous bone in 16-week-old male mice. (B) Cancellous bone mass and (C) microarchitecture in the distal end of femurs (n = 8–13/group). (D) Cortical bone measurements in the midshaft region of femurs. (E) Osteoblast and osteoclast in cancellous bone sections of the femur (n = 5 to 6/group). Bars represent mean and SD; *p < 0.05 versus wild-type, AR^f/y, and Prx1-Cre by two-way ANOVA; #p < 0.05 by Student’s t-test. Ob = osteoblast; Oc = osteoclast; BV/TV = bone volume per tissue volume, B.Pm = bone perimeter.

Table 1.

Skeletal Phenotype of 7 Week-Old Male Mice. Micro-CT Measurements Performed in Femur. BV/TV Bone Volume/Total Volume; Tb. Trabecular; Th Thickness; Sp Separation; Ct. Cortical

	BV/TV	3D BMD (mg/cm3)	Tb. Number (/mm)	Tb. Th (μm)	Tb. Sp (mm)	Ct. Th (mm)
ARf/y (n = 11)	0.19 ± 0.05	158.97 ± 40.86	4.84 ± 0.55	57.95 ± 7.21	0.20 ± 0.03	0.18 ± 0.02
ARf/y;Prx1-Cre (n = 8)	0.18 ± 0.04	150.11 ± 35.78	4.13 ± 0.81*	62.85 ± 8.59	0.25 ± 0.05*	0.18 ± 0.02

^*p < 0.05 by Student t-test.

Unlike males, female AR^f/y;Prx1-Cre mice had no changes in cancellous bone mass (Table 2). However, BMD and trabecular number in the females were decreased and trabecular separation was increased when compared to littermate controls. Intrigued by this observation, we examined whether the expression of the AR in bone marrow-derived osteoblasts from female WT C57BL/6 mice was comparable to that of ERα. We found that AR mRNA in these ex vivo osteoblast preparations was threefold higher than ERα (Supporting Fig. 3A), whereas AR mRNA was similar in osteoblasts from males versus females (Supporting Fig. 3A, B). These differences, nonetheless, need to be interpreted cautiously considering the heterogeneous nature of the cell preparations.

Table 2.

Deletion of AR in the Mesenchymal Lineage of Female Mice Decreases Cancellous Bone BMD. Micro-CT Measurements Performed in Femurs of 16 Week-Old Mice. BV/TV Bone Volume/Total Volume; Tb. Trabecular; Th Thickness; Sp Separation; Ct. Cortical

	BV/TV	3D BMD (mg/cm3)	Tb. Number (/mm)	Tb.Th (μm)	Tb. Sp (mm)	Ct.Th (mm)
ARf/f (n = 10)	0.08 ± 0.01	80.22 ± 18.73	3.51 ± 0.21	49.78 ± 3.75	0.28 ± 0.02	0.20 ±0.01
ARf/f;Prx1-Cre (n = 8)	0.07 ± 0.01	56.68 ± 9.97*	3.22 ± 0.18*	51.39 ± 3.79	0.31 ± 0.02*	0.21 ±0.01

^*p < 0.05 by Student t-test.

Deletion of AR from mesenchymal progenitors mimics the effects of ORX on cancellous bone

We next compared the effects of the targeted deletion of the AR in the entire mesenchymal lineage with the effects of the systemic loss of androgens. To do this, we orchidectomized the AR^f/y and the AR^f/y;Prx1-Cre mice at 20 weeks of age and determined the impact of the loss of testicular function on the skeleton 6 weeks later. The effect of ORX on seminal vesicle weight was indistinguishable between the two genotypes (Fig. 3A), indicating that androgen levels declined in both strains. Orchidectomized AR^f/y mice exhibited the expected loss of femoral BMD, as determined by the absolute BMD (mg/cm²) at the end of the experiment, or as a percent change of BMD between 20 and 26 weeks (Fig. 3B, C). By comparison to the AR^f/y control mice, the effect of ORX was blunted in the AR^f/y;Prx1-Cre mice.

Fig. 3.

AR^f/f;Prx1-Cre mice do not lose cancellous bone following ORX. (A–F) Twelve-week-old mice were sham-operated or orchidectomized and euthanized 6 weeks later (n = 8–11/group). (A) Seminal vesicle weight and (B) BMD determined by DXA in 20-week-old mice (n = 8–11/group). (C) The percent change from the initial BMD was determined by DXA measurements 1 day before surgery and before death. (D) Representative images (left panel) and cancellous bone mass at the distal femur (right panel) determined by μCT (n = 8–11/group). (E) Cancellous bone microarchitecture and (F) cortical thickness at the femoral midshaft (n = 8–11/group). Bars represent mean and SD; *p < 0.05 by two-way ANOVA. ORX = orchidectomy/orchidectomized; BV/TV = bone volume per tissue volume.

We next analyzed the femurs by μCT. As expected, loss of androgens caused a decrease in cancellous BV/TV (Fig. 3D) and in trabecular number, and an increase in trabecular separation in the control mice (Fig. 3E). The effect of ORX on cancellous bone in the control mice was similar to the effects of the AR deletion from mesenchymal progenitors in androgen-sufficient mice. In other words, the targeted AR deletion in mesenchymal progenitors mimicked the effect of the loss of the hormones. The ORX-induced changes in cancellous bone, however, did not occur in the AR^f/y;Prx1-Cre mice. However, it remains possible that the lack of a reduction in cancellous bone mass in the AR^f/y; Prx1-Cre mice following ORX may simply be because of the already low bone mass, making it difficult to ascertain further reduction. Nonetheless, in contrast to the case with cancellous bone mass, the effect of ORX on cortical thickness was indistinguishable between the two genotypes (Fig. 3F), indicating that the changes in DXA BMD reflected changes in cancellous as opposed to cortical bone mass.

Deletion of AR in myeloid progenitors and osteoclasts does not alter bone mass

Having shown that direct effects of androgens on cells of the mesenchymal lineage play no role in the maintenance of cortical bone mass in male mice, we next investigated whether direct effects of androgens on osteoclasts could be responsible for their protective effects on cortical bone. To this end, we generated mice lacking AR in osteoclasts by crossing the AR floxed mice with mice expressing the Cre recombinase under the control of regulatory elements of the LysM gene. The LysM gene is expressed in cells of the monocyte/macrophage lineage and their descendants, as well as in neutrophils.⁽²¹⁾ The efficiency of the AR deletion was established by genomic DNA analysis of cultured bone marrow macrophages and osteoclasts (Fig. 4A, B). Deletion of the AR from LysM cells had no effect on cortical or cancellous bone, as determined at 12 or 26 weeks of age in male (Fig. 4C) or female (Supporting Table 1) mice by μCT. Consistent with the lack of an effect on bone mass, the number of osteoclasts in vertebral cancellous bone of AR^f/y;LysM-Cre males was unaffected (Fig. 4D). Moreover, the effect of ORX on either the cortical or cancellous bone in both the femur and the vertebra was indistinguishable between the two genotypes (Fig. 4E, F). Notably, in line with our inability to show genetically a direct role of the myeloid cell AR in bone resorption and osteoclast numbers in male mice, the levels of AR expression in osteoclasts were much lower than in osteoblasts (Fig. 1A, Supporting Fig. 3B). Because of this situation, we were not able to show a decrease in AR mRNA in the AR^f/y;LysM-Cre mice. ORX had no effect in the number of myeloid cells in the bone marrow of either genotype (Fig. 4G). Nonetheless, as others have shown before,⁽²⁹⁾ ORX caused an increase in B cell numbers, and this effect was not affected by the AR deletion in myeloid cells.

Fig. 4.

Deletion of AR in LysM-Cre-expressing cells does not alter bone mass. (A) Genomic DNA analysis of cultured BMMs. (B) Quantitative PCR of loxP-flanked genomic DNA, normalized to a control locus, isolated from cultured BMM or osteoblasts (triplicates). (C) Cancellous bone mass at the distal femur and cortical thickness and perimeters at the midshaft of 12-week-old male mice, measured by μCT (n = 6–9/group). (D) Vertebral cancellous bone mass by μCT and osteoclast number per mm of cancellous bone surface in undecalcified sections of vertebra (n = 10/group). (E, F) Twenty-week-old mice were sham-operated or orchidectomized and euthanized 6 weeks later (n = 11–13/group). (E) μCT analysis as in C (n = 8–11/group). (F) Cancellous bone mass in the 5th lumbar vertebra. (G) Myeloid (CD11b+) and B cells (CD19+) in the bone marrow quantified by flow cytometry (n = 5–8/group). Bars represent mean and SD. *p < 0.05 by two-way ANOVA; #p < 0.05 by Student’s t-test. BMM = bone marrow macrophage; BV/TV = bone volume per tissue volume; B. Pm = bone perimeter; Oc = osteoclast; ORX = orchidectomized; BM = bone marrow.

ERα signaling in osteoblasts or osteoclasts play no role in the protective effect of androgens on male bone

We investigated whether the protective effects of androgens on the cancellous or cortical bone of males result from the aromatization of androgens (ie, their conversion to estrogens), and thereby, estrogen-activated ERα signaling in mesenchymal or myeloid cells. To do this, we used two mouse models of ERα deletion in the osteoblast or osteoclast lineage, which we had used earlier to elucidate the role of ERα in females.^(22,23) Cancellous bone mass in male ERα^f/f;Osx1-Cre was indistinguishable from Osx1-Cre littermate controls at 6, 10, and 26 weeks of age (Fig. 5A–C). Cortical thickness, however, was lower in 6-week-old ERα^f/f;Osx1-Cre mice as compared to their Osx1-Cre mice littermates (Fig. 5A). No differences could be detected in the periosteal and endocortical perimeters. Nevertheless, the lower cortical thickness was no longer present at 10 or 26 weeks of age (Fig. 5B, C). The transient low cortical thickness of ERα^f/f; Osx1-Cre mice is reminiscent of the transient low cortical thickness we had seen previously in ERα^f/f;Prx1-Cre male mice.⁽²³⁾ In any event, the loss of cancellous or cortical bone mass caused by ORX was indistinguishable between ERα^f/f;Osx1-Cre and the Osx1-Cre littermates (Fig. 5C).

Fig. 5.

The effects of androgens on the bone mass of male mice are not mediated by ERα in osteoblasts or osteoclasts. (A) Cancellous bone mass at the distal femur (left panel) and cortical thickness and perimeters at the midshaft measured by μCT in 6-week-old mice (n = 6/group) and (B) in 10-week-old mice (n = 7–9/group). (C) Twenty-week-old mice were sham-operated or orchidectomized and euthanized 6 weeks later. μCT analysis as in A (n = 7–9/group). (D) Longitudinal femoral BMD by DXA (n = 12–15/group). (E) Cancellous bone mass at the distal femur (left panel) and cortical thickness and perimeters at the midshaft of 12-week-old mice (n = 6 to 7/group). (F) Longitudinal spinal BMD by DXA (n = 12–15/group). (G) Cancellous bone mass in the 5th lumbar vertebra by μCT (left panel) and osteoclast number per millimeter of cancellous bone surface (right panel) in decalcified sections of vertebra (n = 6 to 7/group). Bars represent mean and SD; *p < 0.05 by two-way ANOVA; #p < 0.05 by Student’s t-test. ORX = orchidectomized; Oc = osteoclast; BV/TV = bone volume per tissue volume; B.Pm = bone perimeter.

Male mice with ERα deletion in LysM cells (ERα^f/f;LysM-Cre) also had indistinguishable vertebral and femoral DXA BMD at 4, 8, and 12 weeks of age (Fig. 5D, F) and indistinguishable cancellous and cortical bone mass by μCT (at 12 weeks) at the spine and femur as compared to their littermate controls (ERα^f/f) (Fig. 5E, G). Furthermore, male ERα^f/f;LysM-Cre mice had similar osteoclast numbers as compared to their littermate control mice. This is different from female ERα^f/f;LysM-Cre mice, which have an increase in the number of osteoclasts.⁽²²⁾ ERα mRNA expression in osteoclasts of male mice was lower than in females (Supporting Fig. 3C). It seems unlikely, however, that this relatively small difference explains the lack of a phenotype in the ERα^f/f;LysM-Cre males. Hence, in contrast to the case with estrogens in females, osteoclast ERα signaling activated by (androgen-derived) estrogens plays no role in the male skeleton.

Discussion

In the work described here we found that male mice lacking AR in cells of the mesenchymal lineage had decreased bone volume and trabecular number and increased osteoclast number in the cancellous compartment; moreover, these mice did not lose cancellous bone volume and trabecular number following ORX. In contrast, male mice lacking AR in cells of the myeloid lineage, or ERα in both the mesenchymal and myeloid lineages, had no cancellous bone phenotype at baseline and lost the same amount of cancellous bone as their controls following ORX. Most surprisingly, males of all four models had no discernible cortical bone phenotype at baseline, and lost the same amount of cortical bone as their littermate controls following ORX.

The finding that direct actions on cells of the osteoblast lineage are responsible for the effect of androgens on cancellous bone is in line with earlier reports of others.^(15–18) Indeed, the similar cancellous bone phenotype in the mice of the present work and mice with targeted deletion of the AR in mature osteoblasts and osteocytes in the earlier reports suggests that within the mesenchymal cell lineage the functionally-relevant cell targets of androgen action on cancellous bone are the mature osteoblasts and/or osteocytes. Nevertheless, unlike the present work showing that deletion of the AR from cells of the mesenchymal lineage fully recapitulates the effects of ORX on the cancellous compartment, in those earlier reports the effects of the AR deletion was not compared with the effects of ORX. It remains, therefore, possible that the effects of androgens on cancellous bone result not only from direct actions on osteoblasts and osteocytes, but additional direct effects on mesenchymal progenitors. In any case, in agreement with earlier reports,^(15,17,18) we found that the loss of cancellous bone in male AR^f/y;Prx1-Cre mice was accompanied by an increase in osteoclast numbers in the cancellous compartment. Hence, the results from four separate studies clearly point out that the antiresorptive effects of androgens on cancellous bone result from AR-mediated actions on cells of the mesenchymal lineage.

The results of the present work reveal that the effects of androgens on cortical bone mass do not result from direct actions on any cell type in the mesenchymal lineage. This is unexpected considering the well-established effect of androgens on periosteal bone expansion and the greater cortical bone mass in males as compared to females. More surprisingly, the evidence that male mice lacking AR in the myeloid lineage exhibit no skeletal phenotype indicates that the antiresorptive effects of androgens in either compartment do not result from direct actions on osteoclasts. The in vivo evidence that AR signaling in osteoclasts plays no discernible role in the antiresorptive effect of androgens in males is at odds with earlier in vitro studies showing that non-aromatizable androgens stimulate the apoptosis of murine osteoclasts.^(10,22,30) One possible explanation that could reconcile this seeming incongruence between the in vitro and in vivo results is that direct action of androgens on osteoclast apoptosis are obscured by attenuating effects on osteoclastogenesis which result from predominant actions of androgens on cells of the osteoblast lineage.

The results of the present work also reveal that the ERα in any cell type along the osteoblast or the myeloid lineage does not mediate the effects of androgens in the bone of male mice. Therefore, direct actions in these cell types resulting from androgen aromatization do not play a role in the protective effects of androgens on cancellous or cortical bone in mice. Nevertheless, extensive evidence from humans with loss-of-function ERα mutations and humans and rodents with aromatase deficiency indicates that estrogens do indeed play an important role in male bone.⁽³⁾ Consistent with this, administration of aromatase inhibitors to adult male rodents decreases DXA BMD.⁽³¹⁾ One must, therefore, conclude that the effects of aromatizable androgens on bone result from hormonal action on cells other than committed osteoblast progenitors and their descendants or cells of the myeloid lineage, or perhaps actions on cells of tissues other than bone.

The evidence against a role of the osteoclast ERα in the effects of androgens on cortical bone in males is not surprising considering a similar lack of an effect of the osteoclast ERα in female cortical bone and the low levels of estrogens in the male. What is surprising, on the other hand, is that whereas in males the effects of androgens on cancellous bone are the result of actions on osteoblasts—a consistent result from all the AR deletion models in the osteoblast lineage,^(15–18) including the present one—in females the effect of estrogens on cancellous bone result from direct ERα signaling in osteoclasts.^(22,32) Although, at this stage, there remains disagreement on whether ERα signaling in mature osteoblasts and osteocytes also contributes to the effects of estrogens on cancellous bone. Specifically, in our earlier studies with ERα^f/f;Prx1-Cre, ERα^f/f;Osx1-Cre, or ERα^f/f;Col1-Cre mice,⁽²³⁾ and those of Windahl and colleagues⁽³³⁾ with ERα^f/f;Dmp1-Cre mice, ERα deletion had no effect on cancellous bone. In contrast, other studies with ERα^f/f; Dmp1-Cre and ERα^f/f;OCN-Cre mice have suggested that ERα deletion decreased cancellous bone.^(34–36) Windahl and colleagues⁽³³⁾ also reported that, in difference to their finding of no effect in the females, deletion of ERα in osteocytes with Dmp1-Cre decreased cancellous bone mass in males. We did not observe such an effect in our osteoblast-specific ERα deletion model (ERα^f/f;Osx1-Cre). It is possible that these seeming incongruences are due to limitations of the Cre/LoxP methodology that we may not yet fully understand.⁽³⁷⁾ An alternative explanation is that incongruences of this sort are suggestive evidence of a weak phenotype. Saying it differently, conditional knockout models and the methods of their analyses may be sensitive enough to reveal large differences in the contribution of one cell type versus another (i.e., predominant versus minor), but not sensitive enough to completely rule out a minor contribution of a particular cell type.

In any case, the present work clearly shows that the predominant, if not exclusive, cellular targets of androgen action on cancellous and cortical bone are different. Furthermore, the comparative examination of the effects of the deletion of the AR or ERα in osteoclasts suggests that in difference to estrogens in the female, androgens or estrogens in the male do not protect cancellous bone mass by direct actions on osteoclasts. Similarly, whereas the antiresorptive effects of estrogens on the endocortical surface require ERα signaling in the osteoblast lineage,⁽²³⁾ the antiresorptive effects of androgens on cortical bone require neither AR nor ERα signaling in this lineage. We had shown previously that the osteoblast progenitor ERα is responsible for periosteal bone accrual in the female,⁽²³⁾ but in the present report we found that AR or ERα signaling in the same cell types play no role in the anabolic effects of androgens in the periosteum. This result negates our earlier suggestion that AR in osteoblast progenitors was responsible for optimal periosteal bone accrual in males.

At this stage, the target cells of the indirect actions of androgens on bone are a matter of conjecture. Notably, however, androgens and estrogens attenuate B cell production both by direct actions on lymphoid cell precursors and indirectly through actions on bone marrow stromal cells.⁽³⁸⁾ Consistent with this evidence, we observed here an increase in B-lymphopoiesis following ORX. In addition, mice lacking RANKL in B-lymphocytes are partially protected from the loss of cancellous bone caused by ovariectomy.⁽³⁹⁾ Taking this all together, it is possible that androgens may exert their antiresorptive effects on the endocortical surface, at least in part, via AR-mediated actions on B-lymphocytes and/or via ERα-mediated actions (secondary to aromatization) on cells of the mesenchymal lineage upstream from the Osx1-Cre committed osteoprogenitors. The anabolic effects of androgens on the periosteal surface of cortical bone, on the other hand, may result form direct actions of androgens on muscles and thereby the mechanical strains that muscles exert on bone. Alternatively, such anabolic effects may be secondary to the effects of androgens on growth factor production from distant tissues, such as the liver.⁽²⁾

Deletion of the AR in mesenchymal progenitors in female mice decreased cancellous BMD and trabecular number in the femur (Table 2). However, these changes were less pronounced in the females than they were in the males (Fig. 2B, C). Similar observations have been reported by Maatta and colleagues⁽¹⁸⁾ in AR^f/f;OCN-Cre mice. Confirmation of the results of Maatta and colleagues⁽¹⁸⁾ adds strength to the contention that androgens acting through the AR may contribute to the acquisition or maintenance of bone mass in female mice. In support of this view, we have consistently found that administration of dihydrotestosterone (DHT), a non-aromatizable androgen, to ovariectomized mice prevents the loss of BMD.^(10,40) The results of the present report and those of Maatta and colleagues⁽¹⁸⁾ raise the possibility that the effects of androgen signaling through the AR may not only be of pharmacologic relevance to females, but of physiologic relevance as well. Whether this could also be the case in women is far less clear, because clinical evidence both in favor and against such possibility has been reported.^(41–44)

In conclusion, the results of the work reported herein suggest that as is the case for estrogens in females, the effects of androgens on the cancellous and cortical bone compartment in the male are mediated, by and large, via different cell types. Androgen signaling through the AR expressed in cells of the mesenchymal lineage mediates the protective effects of androgens on cancellous bone, by decreasing osteoclast numbers and restraining bone resorption in this compartment. Androgen signaling through the AR or the ERα expressed in cells of the mesenchymal or the myeloid lineage, on the other hand, plays no role in the effects of androgens on cortical bone. Even more intriguingly, whereas the antiresorptive effects of estrogens on cancellous bone result from direct actions on osteoclasts, the antiresorptive effects of androgens on cancellous bone are exerted indirectly.