Elevated Aromatase Expression in Osteoblasts Leads to Increased Bone Mass Without Systemic Adverse Effects

Authors


  • The authors state that they have no conflicts of interest

  • Published online on February 16, 2009

Abstract

The stimulatory effects of testosterone (T) on bone can either be through a direct activation of the androgen receptor (AR) or mediated through aromatization of T to estradiol (E2), followed by activation of estrogen receptors (ERs) in bone. Aromatase expression in osteoblasts and reproductive tissues is dependent on different promoters, which are differentially regulated. To study the effect of elevated local aromatization of T to E2 in bone, we developed a transgenic mouse model (Coll-1α1-Arom) that overexpresses the human aromatase gene under the control of the osteoblast specific rat type I α I procollagen promoter. The Coll-1α1-Arom mice expressed human aromatase mRNA specifically in bone and had unaffected serum E2 and T levels. Male Coll-1α1-Arom mice had clearly increased total body BMD, trabecular BMD, cortical BMD, and cortical thickness associated with elevated osteoprotegerin mRNA levels and reduced number of osteoclasts (p < 0.01). Treatment of ovariectomized mice with T increased cortical and trabecular thickness in the Coll-1α1-Arom mice (p < 0.001) but not in the wildtype mice. In conclusion, elevated aromatase expression specifically in osteoblasts results in stimulatory estrogenic effects in bone without increasing serum E2 levels. Because osteoblast-specific aromatase expression results in an increased ER to AR activation ratio in bone, we propose that activation of ERs results in a more pronounced increase in bone mass than what is seen after activation of the AR. Development of osteoblast-specific inducers of aromatase expression might identify substances with stimulatory effects on bone without systemic adverse effects.

INTRODUCTION

Sex steroids are important for the maintenance of both the female and the male skeleton.(1–3) However, the relative contribution of androgens versus estrogens in the regulation of the skeleton is unclear. The effects of testosterone (T) can be exerted either directly through the androgen receptor (AR) or indirectly through aromatization to estrogens and further through estrogen receptor (ER)α and/or ERβ.(2,3) All these receptors are expressed both in growth plate cartilage and in bone.(4–7) ERα is the major ER responsible for the trabecular bone-sparing effect of estrogens both in males and females.(8–10) Furthermore, it has been shown that the in vivo effect of ER activation on bone is distinct from the effect of AR activation.(9–11)

The important role of aromatase for the skeleton is clearly shown from clinical studies of men with mutation in cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1), the gene that encodes aromatase, and from studies in aromatase-deficient mice, showing that deficiency of aromatase results in reduced bone mass.(12–15) Some studies showed a reduced bone size and a reduced periosteal apposition in aromatase-deficient men, suggesting that estrogen is required for a normal periosteal apposition during sexual maturation.(16–18) Furthermore, male transgenic (TG) mice with a general overexpression of human aromatase, using the human ubiquitin C promoter/human P450 aromatase fusion gene, have increased bone mass.(19) However, as a consequence of elevated serum E2 levels in this mouse model, male mice showed several systemic pathological phenotypes, including infertility, rudimentary prostate/seminal vesicles, reduced serum T, gynecomastia, and adrenal gland hyperplasia.(20–22) Thus, the role of aromatase expression specifically in osteoblasts without affected circulating E2 levels could not be determined.

In humans, the aromatase is expressed in several different tissues including in bone/osteoblasts.(3) Therefore, local levels of E2 and T in osteoblasts are not only dependent on serum levels of E2 and T but also on the aromatase activity in osteoblasts. There are some indications that endogenous aromatase activity in bone might be more important in humans than in mice. First, aromatase expression in bone is relatively higher in humans than in mice. Second, mice have no expression of CYP 17, the enzyme responsible for sex steroid synthesis, in the adrenal gland, and the gonads are the exclusive site for sex steroid production in mice.(23) The situation is different in humans that, in addition to gonadal-derived androgens, humans have androgens secreted from the adrenal gland that can be converted to E2 by aromatase in peripheral tissues such as bone.(24) The human CYP19A1 gene is located in the chromosome 15q21.2 region and is comprised of a 30-kb coding region and a 93-kb regulatory region. The unusually large regulatory region contains 10 tissue-specific promoters (I.1, I.2, I.3, I.4, I.5, I.6, I.7, 2a, I.f, and PII) that are alternatively used in various cell types.(25) Each promoter is regulated by a distinct set of regulatory sequences in DNA and transcription factors that bind to these specific sequences. Aromatase expression in ovary, which is the major source of E2 in fertile women, is mainly regulated by the PII promoter and is strongly induced by follicle-stimulating hormone (FSH).(26) In contrast, promoters I.4 and 1.6 have been described as the major promoters regulating aromatase expression in bone/osteoblasts, and it has been shown that vitamin D, dexamethasone, prostaglandin E2 (PGE2), and dehydroepiandrosterone regulate aromatase expression in cultured osteoblasts.(25, 27–32) Moreover, in estrogen-dependent pathologic tissues such as breast cancer and endometriosis, aromatase is upregulated through inappropriate activation of aberrant promoters.(25) Because the use of aromatase promoters in humans differs between tissues, one can postulate that it is possible to achieve a tissue-specific estrogenic effect (e.g., on the bone) by substances that specifically activates bone specific aromatase promoters. These substances would avoid the adverse effects in nonskeletal tissues. However, an osteoblast-specific induction of aromatase would result not only in elevated local estradiol (E2) but also reduced local T, and the effect of the resulting change in the balance between ER and AR activation in osteoblasts is unknown.

To study the importance of high local aromatization of T to E2 and thereby increased ER to AR activation ratio specifically in osteoblasts, we developed a TG mouse model (Coll-1α1-Arom mice) that overexpresses the human aromatase gene under the control of the osteoblast specific rat type I α I procollagen promoter.

MATERIALS AND METHODS

Construction of the transgene

A 2.3-kb fragment of the rat type I α I procollagen promoter was subcloned into the multiple cloning site of the pGL3-Basic Vector. This promoter fragment has earlier been shown to give efficient expression restricted to osteoblasts and osteocytes with no detectable expression in chondrocytes.(33–35) A 2.2-kb fragment coding for full-length human aromatase cDNA was subcloned into the multiple cloning site of the pGL3-Basic Vector after the promoter followed by a SV40 polyadenylation signal. The 5.4-kb-long Coll 1α1-Aromatase fragment was released from the vector backbone by digestion with NotI and HindIII enzymes.

Development of Coll-1α1-Arom transgenic founder mice

TG mice were produced by a standard technique in a C57Bl6 strain. Positive founders for the transgene were identified by Southern blot analysis of DNA obtained from tail biopsies. Briefly, genomic DNA (10 μg) was digested with XhoI and resolved by electrophoresis in 0.5% agarose gel. The DNA was blotted onto nylon membrane and prehybridized for 15 min at 65°C in hybridization buffer (1.5 × sodium chloride–sodium phosphate–EDTA buffer [SSPE], 10% polyethylene glycol [PEG] 6000, 7% wt/vol SDS, 100 μg/ml salmon sperm DNA) and with [α-32P]dCTP (Amersham Pharmacia Biotech)-labeled Coll 1α1 aromatase fragment overnight at 65°C. The membranes were washed in 1× SSC and 0.1% SDS three times at 65°C for 15 min and exposed to a PhosphorImager screen.

Establishment of a Coll-1α1-Arom transgenic mouse line

Coll-1α1-Arom mice showed normal fertility. To avoid exposure to the transgene during fetal life, all experiments were carried out on mice born from crossing a male Coll-1α1-Arom mouse with a female C57Bl6 female mouse. WT littermates were used as control groups. The mice were housed in a standard animal facility under controlled temperature (22°C) and photoperiod (12 h of light, 12 h of dark) and fed standard phytoestrogen-free pellet diet ad libitum. Animal care was in accordance with institutional guidelines. All animal experiments had been approved by the local Ethical Committees for Animal Research at the University of Gothenburg.

PCR genotyping of transgenic mice

For routine genotyping of the Coll-1α1-Arom mice, PCR analyses were carried out using DNA extracted from tail biopsies. Primer sequences are available on request.

Testosterone treatment of mice

Twelve-week-old female Coll-1α1-Arom and WT mice were either sham operated or ovariectomized (OVX). The OVX mice were treated with vehicle or T (25 μg/d) for 4 wk. The administration was done using slow release pellets (Innovative Research of America, Sarasota, FL, USA). The dose corresponds to a replacement dose giving a physiological T level in orchidectomized male mice at similar weight (unpublished data).

Quantitative real-time PCR analysis

Total RNA was prepared from retroperitoneal fat, gastrocnemius muscle, and femur (n = 4–10/group), using TriZol Reagent (Life Technologies) according to the manufacturer's instructions. The RT-PCR analysis was performed using the ABI Prism 7000 Sequence Detection System (PE Applied Biosystems). The mRNA abundance of each gene was calculated using the “standard curve method” (User Bulletin 2; PE Applied Biosystems) and adjusted for the expression of 18S. Primer and probe sequences are available on request.

DXA

Analyses of total body areal BMD (aBMD) and spine BMD were performed by DXA using the Lunar PIXImus Mouse Densitometer (Wipro GE Healthcare).

pQCT

CT scans were performed with the pQCT XCT RESEARCH M (version 4.5B; Norland), operating at a resolution of 70 μm as described previously.(36) Trabecular BMD was determined ex vivo, with metaphyseal pQCT scans of the distal femur and the proximal tibia. Bone lengths were measured with a slide caliper. The scans were positioned in the metaphysis at a distance from the growth plate corresponding to 3% of the total length of the bone, and the trabecular bone region was defined as the inner 45% of the total cross-sectional area. Cortical bone parameters (cortical BMD, cortical BMD, cortical thickness, periosteal circumference, and endosteal circumference) were analyzed in the mid-diaphyseal region of femur and tibia.(37)

μCT

μCT analyses were performed on the proximal tibia by using Skyscan 1072 scanner (Skyscan, Aartselaar, Belgium) and imaged with an X-ray tube voltage of 50 kV and current 200 FA, with a 1-mm aluminium filter.(9) The scanning angular rotation was 1801 and the angular increment was 0.451. The voxel size was 4.36 Fm isotropically. Datasets were reconstructed using a modified Feldkamp algorithm and segmented into binary images using adaptive local thresholding.(38,39) Trabecular bone distal of the proximal growth plate was selected for analyses within a conforming volume of interest (cortical bone excluded) starting at a distance of 200 Fm from the growth plate and extending a further longitudinal distance of 1.3 mm in the proximal direction. Trabecular thickness and separation were calculated by the sphere-fitting local thickness method.(40)

Histomorphometric analyses of osteoclast number

Tibias were fixed in Burckhardt's fixative (24 h), dehydrated, and embedded in methylmethacrylate (Technovit 9100 New; Heraeus Kulzer, Hanau, Germany). Four-micrometer longitudinal sections of the proximal tibia were stained for TRACP activity and counterstained with Mayer's hematoxylin (Histolab, Göteborg, Sweden). Images were captured using a Nikon Eclipse 80i light microscope connected to a Sony DXC-S500 video camera using the Osteomeasure software (OsteoMetrics, Decatur, GA, USA). Number of osteoclasts per bone surface is reported according to the recommended American Society for Bone and Mineral Research nomenclature.(41)

Measurement of serum hormone levels

Commercially available radioimmunoassay (RIA) kits were used to assess serum concentrations of T (MP Biomedicals) and E2 (Siemens Medical Solutions Diagnostics) in 3-mo-old mice.

RESULTS

Development of a transgenic mouse model with osteoblast specific aromatase overexpression

We developed a TG mouse model (Coll-1α1-Arom), which overexpresses the human aromatase gene under the control of a 2.3-kb fragment of the rat type I α I procollagen promoter (Fig. 1A). RT-PCR measurements showed that the Coll-1α1-Arom mice overexpressed aromatase specifically in bone (Fig. 1B). At 3 mo of age, Coll-1α1-Arom mice showed normal body weight (males: WT, 26.9 g; Coll-1α1-Arom, 25.8 g; females: WT, 20.2 g; Coll-1α1-Arom, 20.0 g), crown-rump length (males: WT, 53.7 mm; Coll-1α1-Arom, 52.9 mm; females: WT, 51.4 mm; Coll-1α1-Arom, 51.0 mm), and body mass index (males: WT, 9.3 kg/m2; Coll-1α1-Arom, 9.2 kg/m2; females: WT, 7.7 kg/m2; Coll-1α1-Arom, 7.7 kg/m2) compared with WT mice. Serum IGF-I levels were unchanged in Coll-1α1-Arom mice (females: WT, 358 ± 18 ng/ml; Coll-1α1-Arom, 310 ± 21 ng/ml; males: WT, 356 ± 17 ng/ml; Coll-1α1-Arom, 318 ± 19 ng/ml) compared with WT mice. Both the male and female Coll-1α1-Arom mice had normal fertility (data not shown) and normal serum E2 levels (females: 14.0 ± 2.3 versus 10.3 ± 1.0 pg/ml; males: not detectable in WT or Coll-1α1-Arom mice) and T levels (females 0.01 ± 0.001 versus 0.01 ± 0.001 ng/ml; males: 0.12 ± 0.03 versus 0.08 ± 0.02 ng/ml) compared with WT mice (n = 8–12 in all groups). In addition, the weights of some organs known to be sensitive to systemic E2 treatment (uterus weight, thymus weight and fat mass) were normal in the Coll-1α1-Arom mice, indicating that neither circulating E2 levels nor E2 levels in nonskeletal tissues were affected in the Coll-1α1-Arom mice. In addition, the weight of the seminal vesicles, which are a sensitive androgen-responsive tissue, was unaffected in the Coll-1α1-Arom mice, supporting the notion that the elevated aromatase expression in osteoblasts did not result in reduction of circulating serum T levels.

Figure Figure 1.

Development of transgenic mice with osteoblast-specific overexpression of human aromatase. (A) Development of a TG mouse model (Coll-1α1-Arom) that overexpresses the human aromatase gene under the control of a 2.3-kb fragment of the rat type I α I procollagen promoter. This promoter will direct the aromatase expression to osteoblasts in bone. (B) Bone-specific expression of human aromatase mRNA in 3-mo-old Coll-1α1-Arom mice. Values are means ± SE. ND, not detectable.

Increased aBMD in adult male Coll-1α1-Arom mice

DXA analyses before sexual maturation (5 wk) showed that there was no difference in the aBMD (total body and lumbar spine; Fig. 2) between males and females and that Coll-1α1-Arom mice did not have altered aBMD. However, after sexual maturation (3 mo old), male but not female Coll-1α1-Arom mice had clearly increased aBMD in both total body and lumbar spine compared with WT mice (Fig. 2).

Figure Figure 2.

Increased aBMD in adult male Coll-1α1-Arom mice Lumbar spine (A) and total body (B) aBMD analyzed by DXA in 5-wk-old and 3-mo-old Coll-1α1-Arom mice and WT mice (n = 8–12). Values are means ± SE. **p < 0.01 vs. WT.

Increased trabecular BMD and cortical BMC in adult male Coll-1α1-Arom mice

The trabecular and cortical bone compartments were analyzed separately using pQCT in adult Coll-1α1-Arom mice and WT mice. These analyses showed that the above-described elevated aBMD in adult male Coll-1α1-Arom mice compared with WT mice was a consequence of increased trabecular BMD (+31%), cortical BMC (+16%), cortical BMD (+5.6%), and cortical thickness (+15%; Table 1). The increased cortical thickness was a result of reduced endosteal circumference, whereas the periosteal circumference was unaffected in the male Coll-1α1-Arom mice compared with the WT mice (Table 1). The effect of elevated osteoblast specific aromatase expression on cortical thickness in male mice was confirmed by μCT analyses of the diaphyseal region of tibia, showing increased cortical thickness (+18.5 ± 4.7%, p < 0.01) and reduced endosteal circumference (−9.8 ± 3.4%, p < 0.01) in male Coll-1α1-Arom mice compared with the WT mice (n = 9 in both groups). Similar as seen using DXA, no major skeletal phenotype except a minor increase in cortical thickness (+4.3%, p < 0.05) was evident for the adult female Coll-1α1-Arom mice compared with the WT mice (Table 1).

Table Table 1.. Trabecular and Cortical Bone Parameters in Coll-1α1-Arom Mice
original image

At 3 mo of age, a more pronounced sex difference was seen in Coll-1α1-Arom than in WT mice for lumbar spine aBMD (WT, 5.4 ± 3.6%; Coll-1α1-Arom, 21.9 ± 3.6% males over females; p < 0.01, Coll-1α1-Arom versus WT; Fig. 2A), total body aBMD (WT, 8.6 ± 1.9%; Coll-1α1-Arom, 15.9 ± 2.2% males over females; p < 0.05, Coll-1α1-Arom versus WT; Fig. 2B), and cortical thickness (WT, 7.6 ± 2.1%; Coll-1α1-Arom, 18.2 ± 2.3% males over females; p < 0.01, Coll-1α1-Arom versus WT; Table 1).

Elevated OPG levels and reduced number of osteoclasts in male Coll-1α1-Arom mice

To explore the mechanism behind the increased bone mass in the male Coll-1α1-Arom mice, the mRNA levels of several genes known to be involved in bone metabolism were analyzed in bone. Interestingly, male Coll-1α1-Arom mice showed clearly increased OPG mRNA levels (+82 ± 11%, p < 0.001) and unchanged RANKL mRNA levels (+15 ± 10%, not significant) compared with WT mice. There were no significant differences in TRACP5b, cathepsin K, osteocalcin, ERα, and ERβ mRNA levels between the Coll-1α1-Arom and WT mice.

Histomorphometric analyses of the proximal metaphyseal region of tibia showed that the elevated OPG mRNA levels in the male Coll-1α1-Arom mice were associated with reduced number of osteoclasts per bone surface in the male Coll-1α1-Arom mice compared with WT mice (−58.9 ± 4.7%, p < 0.01, n = 7–8).

Effect of OVX in Coll-1α1-Arom mice

To determine the role of increased local aromatization in the absence of high endogenous ovary-derived circulating E2, 12-wk-old Coll-1α1-Arom and WT mice were OVX. There was no difference in trabecular BMD in female gonadal intact (sham operated) Coll-1α1-Arom and WT mice (Table 1). Four weeks after OVX, both Coll-1α1-Arom and WT mice had lost trabecular bone mass to the same extent (tibia trabecular BMD—Coll-1α1-Arom: sham, 180 ± 10 mg/cm3; OVX, 142 ± 9 mg/cm3; p ≤ 0.01; WT: sham, 159 ± 10 mg/cm3; OVX, 124 ± 6 mg/cm3; p ≤ 0.01). There was a small increase in cortical thickness in gonadal intact Coll-1α1-Arom compared with WT (Table 1). The cortical thickness was clearly reduced after OVX in Coll-1α1-Arom (tibia cortical thickness: sham, 218 ± 4 μm; OVX, 198 ± 4 μm; p ≤ 0.01) but not in WT (tibia cortical thickness: sham, 209 ± 3 μm; OVX, 207 ± 3 μm) mice, resulting in cortical thickness that was not significantly different between female Coll-1α1-Arom and WT mice after OVX.

Enhanced skeletal T response in female Coll-1α1-Arom mice

The minor skeletal phenotype in the female Coll-1α1-Arom mice is probably because they have low serum levels of T, which is the substrate for aromatase. To determine whether the female Coll-1α1-Arom mice showed a more pronounced skeletal T response than WT mice, exogenous T was given to OVX Coll-1α1-Arom and WT mice. Serum levels of E2 were not detectable in OVX mice before and after T treatment. DXA analyses showed that T treatment resulted in a clearly more pronounced increase in total body aBMD in the Coll-1α1-Arom mice than in the WT mice (p < 0.01; Fig. 3). Detailed bone compartment–specific analyses of the diaphyseal region of femur, using pQCT (Table 2), and of tibia, using μCT (Fig. 4), showed that T treatment increased the cortical bone mass as a consequence of increased cortical thickness and cortical BMD in the OVX Coll-1α1-Arom mice but not in the OVX WT mice compared with placebo-treated mice (Table 2; Figs. 4A and 5). The increased cortical thickness was a result of reduced cortical endosteal circumference, whereas the periosteal circumference was unaffected (Table 2; Fig. 5). T increased the trabecular BMD, as measured by pQCT (Table 2; Fig. 5), and the trabecular BV/TV and trabecular number, as analyzed by μCT (Figs. 4B, 4C, and 5), in both OVX Coll-1α1-Arom and in OVX WT mice, but the effect of T treatment was significantly more pronounced (p < 0.01) in the OVX Coll-1α1-Arom mice than in the OVX WT mice. Interestingly, T increased the trabecular thickness in OVX Coll-1α1-Arom mice, whereas it actually reduced the thickness in OVX WT mice compared with placebo treatment (Fig. 4D).

Table Table 2.. Effect of Testosterone on Trabecular and Cortical Bone Parameters in OVX Mice
original image
Figure Figure 3.

More pronounced effect of testosterone on total body aBMD in OVX Coll-1α1-Arom mice than in WT mice. Total body aBMD analyzed by DXA in 3-mo-old OVX Coll-1α1-Arom mice and WT mice that were either vehicle treated (OVX) or testosterone treated (OVX + T; n = 10–11). Values are means ± SE. *p < 0.05 and ***p < 0.001 vs. OVX. ++p < 0.01, effect of testosterone in Coll-1α1-Arom mice vs. in WT mice.

Figure Figure 4.

More pronounced effect of testosterone on cortical and trabecular bone parameters in OVX Coll-1α1-Arom mice than in WT mice. Cortical (A) and trabecular (B–D) bone parameters were analyzed by μCT analyses of proximal tibia in 3-mo-old Coll-1α1-Arom mice and WT mice that were either vehicle treated (OVX) or testosterone treated (OVX + T; n = 10–11). Values are means ± SE. BV, bone volume; TV, total volume. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. OVX. ++p < 0.01 and +++p < 0.001, effect of testosterone in Coll-1α1-Arom mice vs. in WT mice.

Figure Figure 5.

Summary of the effects of T in OVX WT mice and in OVX mice overexpressing aromatase in osteoblasts, resulting in an increased ER to AR activation in bone (Coll-1α1-Arom). For all cortical and trabecular bone parameters, the stimulatory effect of T was more pronounced in the OVX Coll-1α1-Arom mice than in OVX WT mice. Importantly, for all cortical bone parameters and for trabecular thickness, elevated aromatase levels in osteoblasts, resulting in elevated E2 levels specifically in osteoblasts and thereby increased ER activation in osteoblasts, were required for T to increase these bone parameters. Based on these data, we propose that activation of ERs results in a more pronounced increase in bone mass than what is seen after a direct T-mediated activation of AR in OVX mice. ↑, increased vs. vehicle-treated OVX; →, unchanged vs. vehicle-treated OVX; ↓, decreased vs. vehicle-treated OVX; ↑↑, significantly more pronounced effect of T in Coll-1α1-Arom mice than in WT mice.

DISCUSSION

Human aromatase is tissue specifically regulated, and in this study, we developed a TG mouse model with osteoblast specific aromatase overexpression, resulting in stimulatory estrogenic effects in bone without systemic adverse effects. Because elevated aromatase expression in osteoblasts results in increased E2 and reduced T levels in osteoblasts and, as a consequence, an increased ER to AR activation ratio in bone, we propose that activation of ERs results in a more pronounced increase in bone mass than what is seen after activation of the AR.

Ubiquitous overexpression of aromatase in male mice results in elevated serum E2 levels associated with infertility, rudimentary prostate/seminal vesicles, gynecomastia, and adrenal gland hyperplasia.(20–22) In this study, we developed a mouse model (Coll-1α1-Arom mice) with elevated osteoblast-specific expression of human aromatase with unaffected serum E2 levels using the rat type I α I procollagen promoter that has previously been shown to be exclusively active in osteoblasts and osteocytes.(33–35) Coll-1α1-Arom mice showed a normal fertility and normal serum E2 and T levels, showing that there was no significant leakage of osteoblast-derived E2 into the circulation of the Coll-1α1-Arom mice.

Adult male Coll-1α1-Arom mice had increased trabecular and cortical BMD and increased cortical thickness compared with the WT mice. This is a proof of concept model providing evidence that induction of aromatase expression specifically in osteoblasts results in bone-specific stimulatory estrogenic effects without systemic adverse effects. E2 treatment in aromatase-deficient men at the age of sexual maturation has been associated with an increased periosteal bone expansion,(16) and ERα-deficient male mice exhibit decreased periosteal bone growth, associated with decreased serum IGF-I levels during sexual maturation.(42) These finding indicated that E2 might promote periosteal bone expansion in males during sexual maturation and that this might be caused by altered endocrine serum IGF-I levels. Neither serum IGF-I levels nor periosteal circumference was affected in male Coll-1α1-Arom mice, suggesting that the high local E2 levels in the Coll-1α1-Arom mice mainly results in an endosteal contraction. However, the increased cortical endosteal contraction observed in adult male Coll-1α1-Arom mice is probably an effect of local very high pharmacological E2 levels coming from osteoblast-mediated aromatization of T. In line with this, an increased cortical endosteal contraction is also observed in OVX Coll-1α1-Arom mice treated with T. Thus, because the currently used transgenic mouse model probably results in very high local E2 levels, one can not directly interpret these data as a physiological comparison of the role of estrogens versus androgens in bone.

As expected, the female Coll-1α1-Arom mice, with low levels of endogenous serum T, and thereby, little substrate for aromatase, had no major skeletal phenotype. OVX resulted in a similar loss of trabecular bone in Coll-1α1-Arom and WT mice. However, the cortical thickness was clearly reduced after OVX in Coll-1α1-Arom but not in WT mice. The reason behind this could be that the female gonadal intact Coll-1α1-Arom mice have increased cortical thickness as a result of aromatization of low levels of endogenous ovary-derived androgens,(23) and after OVX, the substrate for osteoblast-expressed aromatase is lost, resulting in a cortical bone loss in the female Coll-1α1-Arom. For all cortical and trabecular bone parameters, the stimulatory effect of exogenous T was more pronounced in the OVX Coll-1α1-Arom mice than in the OVX WT mice. Importantly, for all cortical bone parameter and for trabecular thickness, elevated aromatase levels in osteoblasts, resulting in elevated E2 levels in osteoblast and thereby increased ER activation in osteoblasts, were required for T to increase these bone parameters. Similarly, we have previously shown that E2, activating the ERs but not dihydrotestosterone, which is a nonaromatizable androgen exclusively activating the AR, increases cortical thickness, trabecular thickness, and cortical density in orchidectomized mice.(9) Based on these data, we propose that activation of ERs results in a more pronounced increase in bone mass than what is seen after a direct T activation of the AR in both female and male gonadectomized mice.

Much of the current research on estrogens aims to separate beneficial estrogenic effects from harmful side effects. The main approach has been to develop selective estrogen receptor modulators (SERMs), and some of the clinically available SERMs show at least partial tissue selectivity in ER activity, including block of estrogen action in breast while still maintaining the beneficial effects of estrogen in other tissues such as bone. As an alternative approach, we hypothesize that it is possible to target tissue-specific local E2 synthesis, resulting in tissue-specific estrogenic effects without systemic adverse effects. The present finding that Coll-1α1-Arom mice have increased bone mass without systemic adverse effects, together with the fact that human aromatase expression in osteoblasts and reproductive tissues is dependent on differentially regulated promoters,(25,27–32) suggests that development of osteoblast-specific inducers of aromatase expression might provide means to stimulate bone mass without systemic adverse effects.

Bone resorption is dependent on RANKL, which is essential for osteoclast formation, activity, and survival. The catabolic effects of RANKL are prevented by OPG, which binds RANKL and thereby prevents activation of its receptor RANK. Thus, osteoclast activity is likely to depend on the relative balance of RANKL and OPG.(43) Importantly, in this study, the OPG levels were significantly increased, whereas the number of osteoclasts per bone surface was reduced in the male Coll-1α1-Arom mice. This suggests that the increased bone mass in these mice was a result of reduced RANK activation and thereby reduced bone resorption. However, not only osteoclasts but also osteoblasts express estrogen receptors and is affected by E2 treatment when studied in vitro.(2,3) Therefore, it is possible that the probably very high local E2 levels in the transgenic mice, besides affecting bone resorption, also might regulate osteoblast number, survival, and/or formation, resulting in the observed endosteal contraction and trabecular thickening. Furthermore, although our data of increased OPG mRNA levels in male Coll-1α1-Arom mice suggest that OPG might be involved in the estrogen action, one should interpret this finding with caution because this was not confirmed in osteoblast cultures and only at the mRNA level.

In conclusion, elevated aromatase expression specifically in osteoblasts results in stimulatory estrogenic effects in bone without systemic adverse effects. Because osteoblast-specific aromatase expression results in an increased ER to AR activation ratio in osteoblasts, we propose that activation of ERs results in a more pronounced increase in bone mass than what is seen after activation of the AR. Furthermore, because human aromatase expression in osteoblasts and reproductive tissues is dependent on differentially regulated promoters, development of osteoblast specific inducers of aromatase expression might identify substances with stimulatory effects on bone without systemic adverse effects.

Acknowledgements

This study was supported by the Swedish Research Council, the Swedish Foundation for Strategic Research, the ALF/LUA research grant in Gothenburg, the Lundberg Foundation, the Torsten and Ragnar Söderberg's Foundation, the Novo Nordisk Foundation, Magnus Bergvall Foundation, Åke Wiberg Foundation, Tore Nilson Foundation, and The Swedish Society for Medical Research.

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