Bone phenotype of the aromatase deficient mouse

Journal of Steroid Biochemistry & Molecular Biology 79 (2001) 49–59

Bone phenotype of the aromatase deficient mouse夽 Orhan K. Öz a,∗ , Gen Hirasawa a , Jonathan Lawson a , Lydia Nanu a , Anca Constantinescu a , Peter P. Antich a , Ralph P. Mason a , Edward Tsyganov a , Robert W. Parkey a , Joseph E. Zerwekh b , Evan R. Simpson c a Department of Radiology, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, USA Department of Internal Medicine, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, USA Victorian Breast Cancer Consortium, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Vic., Australia b

c

Abstract Estrogens are important for normal bone growth and metabolism. The mechanisms are incompletely understood. Thus, we have undertaken characterization of the skeletal phenotype of aromatase (ArKO) deficient mice. No abnormalities have been noted in skeletal patterning in newborns. Adult ArKO mice show decreased femur length and decreased peak Bone Mineral Density (BMD) with accelerated bone loss by 7 months of age in females. Magnetic resonance microscopy (MR) and microCT (␮CT) imaging disclosed decreased cancellous connectivity and reduced cancellous bone volume in ArKO females. Bone formation rate (BFR) is increased in ArKO females and decreased in ArKO males. Estradiol therapy reverses these changes. This anabolic effect of estradiol in the male skeleton is supported by 18-F − Positron Emission Tomography (PET) imaging, which clearly demonstrates decreased spinal uptake, but marked increase after estradiol therapy. Serum IGF-1 levels are high in young female ArKO mice but low in young ArKO males. The reduced BMD in ArKO females, despite the presence of elevated serum IGF 1, suggests that other mechanism(s) are operative. There is increased B-cell lymphopoiesis in adult female ArKO bone marrow cells. These results show that ArKO mice show the effects of estrogen deficiency on bone growth, mass, metabolism, microarchitecture and the hematopoietic microenvironment. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Aromatase; Bone; Dual energy X-ray absorptiometry; Estrogen; Histomorphometry; Insulin-like growth factor 1; Major urinary protein

1. Introduction Estrogens are known to be important in the establishment and maintenance of normal bone mass in women and men. In particular, loss of estrogen at the menopause leads to accelerated bone loss in women [1] and is also contributory to the slow bone loss seen with aging in men [1,2]. Though the mechanisms of estrogens’ actions in maintenance of bone are incompletely understood, it is clear that they act ultimately by controlling the balance between resorption or osteoclastic activity and bone formation or osteoblastic activity. While most research has focused on estrogens’ effects on osteoclastogenesis, recent evidence suggests that estrogen also stimulates osteoblast activity [3]. Cytokines and growth factors are known mediators of these effects on bone [4]. Moreover estrogens are necessary for acquisition of peak Bone Mineral Density (BMD) in the maturing skeletons of both sexes (reviewed in [5]). In terms of the accrual of bone mass to 夽 Proceedings of the Symposium: ‘Aromatase 2000 and the Third Generation’ (Port Douglas, Australia, 3–7 November 2000). ∗ Corresponding author. Fax: +1-214-648-2727. E-mail address: [email protected] (O.K. Öz).

peak BMD, the GH–IGF-1 axis is known to be important and in at least one case of estrogen deficiency, Turner’s syndrome, there are abnormalities in GH secretion. This raises the possibility of a causal relationship. Estrogens also regulate the hematopoietic component of bone. Ovariectomy, though not a pure estrogen deficient state, results in increased B-cell lymphopoiesis in the mouse bone marrow. This and the associated bone loss can be prevented by estrogen administration [6]. On the other hand, the osteosclerosis induced by high doses of estrogen [7] is preceded by suppression of hematopoiesis including B-cell lymphopoiesis [8]. The osteogenic precursor population increases, but after the effects on the hematopoietic component. Thus, estrogens’ anti-resorptive and potential pro-formation effects may involve regulation of the hematopoietic component of the marrow. Estrogens are synthesized from androgen precursors by the enzyme aromatase. In order to unravel the mechanisms underlying the multiple effects of estrogens animal models are necessary. The aromatase deficient mouse or ArKO mouse [9] is such a model. We have previously reported that ArKO have reduced cancellous bone volume and a sexually dimorphic pattern of remodeling, high turnover in females

0960-0760/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 0 7 6 0 ( 0 1 ) 0 0 1 3 0 - 3

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and low turnover in males [10]. We also reported male mice have significantly reduced femur length. Since that original study we have continued our analysis of the bone phenotype by studying longitudinal growth dynamically, bone mineral accretion, remodeling, and B-cell lymphopoiesis in additional animals. The results are reported below.

FITC-conjugated anti-B220(CD45R) (Pharmingen, San Diego, CA USA) for 15 min on ice, washed once, and fixed for 10 min with formalin. Stained cells were analyzed on a Becton Dickinson FACS system. Unstained cells were used as negative controls. 2.5. DEXA

2. Materials and methods 2.1. Animals ArKO mice and their wt littermates were produced by nonbrother-sister breeding of F6 heterozygous animals. All animals were from the hybrid 129S6/SvEv Taconic/C57Bl/6 background. Animals had unlimited access to drinking water and were maintained on standard chow. For estradiol replacement experiments, animals were injected subcutaneously with 10 ␮g/kg of estradiol in corn oil three times per week. All protocols were approved by the IACRAC of UT Southwestern Medical Center. 2.2. Skeletal staining Skin and viscera were removed from newborn mice. The carcasses were fixed overnight in 95% ethanol. The fixed skeletons were stained for 24 h in a 0.3% w/v alcian blue solution prepared in 70% ethanol. After clearing the residual stain by soaking in 95% EtOH for several hours, the skeletons were transferred to 2% KOH for 24 h or longer depending upon the size of the animal to clear any remaining flesh. The cleared skeletons were transferred to 1% KOH/0.05 Alizarin red S for overnight staining. The skeletons were then cleared for a least 2 days in 1% KOH/20% glycerol. Stained cleared skeletons were stored in glycerol: 95% EtOH (1:1). 2.3. Static and dynamic histomorphometry Static and dynamic histomorphometry were performed as previously described [10]. Bone Formation Rate (BFR) was calculated as the product of the mineral apposition rate times the fraction of osteoid surface covered by a double label. It represents the volume of mineralized new bone formed per unit area of osteoid surface per unit time. 2.4. Flow cytometry Bone marrow cells were prepared from lumbar spine and femurs of 8-month-old wt and ArKO females by crushing the bones in PBS containing 2% fetal bovine serum (FBS) and 1 mM EDTA. A single cell suspension was prepared by first filtering the resultant supernatant through a nylon mesh followed by repeated dispersion through a 22-gauge needle. The cells (2.5 × 105 ) were then incubated with

Whole body DEXA was performed using the PIXImus densitometer (Lunar Corporation, Madison, WI, USA) equipped with software version 1.43. 2.6. Urine and serum analysis Urine was collected from mice as a spontaneous void generated at the time animals were handled between 1500 h and 1730 h. The urine was diluted 1:8 and analyzed by SDS-PAGE. For plasma IGF-1 analysis, blood was collected from the retro-orbital venous plexus into heparinized pipettes. Blood was always collected at the same time each day, about 1530. Plasma was separated from clotted blood by centrifugation. IGF-1 concentration was determined by RIA (DSL, Inc. Webster, TX, USA). 2.7. Magnetic resonance and microcomputed tomography imaging MR imaging was performed using an Omega 9.4T MRI system (GE/Bruker, Fremont, CA) and a home built slotted tube resonator coil (10 mm i.d.). Shimming on the water signal gave a typical linewidth of 70 Hz. Data were acquired using a simple partial saturation 3D spin-echo sequence with hard pulses (π /2 = 55 ␮s), a repetition time (TR) of 150 ms, an echo time (TE) of 16 ms and 64 × 64 phase encode steps over the small dimensions. One hundred and twenty eight read-out points for the long axis with a typical FOV 4 × 4 × 16 mm gave a 62.5 × 62.5 × 125 ␮m resolution. Sine-bell apodization was applied prior to 3D Fourier transformation. Images were interpolated (up to 8 fold) and edited using NIH Image shareware (NIMH, Bethesda, MD) and analyzed using c language software developed in house [11]. Connectivity density was determined for regions of the metaphysis expected to be rich in trabecular structure using algorithms described previously [11]. The vertebral bodies were scanned on a ␮CT 20 system (Scanco USA, Inc. Wayne, PA) at a resolution of 18 ␮m3 . A set of images was obtained from each sample. Three-dimensional analysis was conducted on manually selected volume of interest to calculate trabecular bone morphometric parameters. 2.8. Positron emission tomography imaging Following intravenous administration of 500 ␮Ci of carrier free 18-F − , 1 h delayed whole body images were acquired on an in-house designed PET imaging system [12].

O.K. Öz et al. / Journal of Steroid Biochemistry & Molecular Biology 79 (2001) 49–59

Following reconstruction, the images were displayed as two-dimensional projections. 2.9. Statistical analysis Data are expressed as the mean ± S.D. or standard error (S.E.). In all instances P<0.05 was taken to be significant. For normally distributed data, analysis was done using the t-test. Otherwise, the Mann–Whitney test was utilized.

3. Results

51

litters we have not found any consistent differences between ArKO animals and their wt littermates. In this particular example, the cartilaginous portions of the wild-type female skull show more mineralization (blue) than the ArKO female. On the other hand, it appears that these areas are more mineralized in the ArKO male relative to wildtype control. The ArKO male also shows greater mineralization of the tibial epiphysis compared with its wt littermate. However, in other litters we have seen reversal of these relationships with no consistent trend from litter to litter. In the current example, the ArKO male demonstrates shortened femurs. Again, this pattern was not consistent in the three litters we have examined to date.

3.1. Bone patterning 3.2. Longitudinal bone growth To determine whether aromatase deficiency results in developmental abnormalities in skeletal patterning, we have examined the newborn skeleton after staining for bone and mineralized cartilage (Fig. 1). After careful study of three

Previously, we have shown that adult male ArKO mice have decreased mean femur length as compared with wt littermates. Study of the dynamics of femur growth show this

Fig. 1. Alizarin red and Alcian blue staining of newborn skeletons. A, Wt female; B, ArKO female; C, Wt male; D, ArKO male. Skeletons were cleared and stained as described in Section 2. In Panel A one femur of the newborn has been disarticulated during processing and the offset of the bones of posterior skull in the males is a processing artifact. No gross abnormalities, such as missing bones and large areas of undermineralization are present.

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Fig. 2. Longitudinal growth of the femur with age. In a cross-sectional study design femur length was followed from the time of weaning to 7 months. There is significant difference in femur length between Wt and ArKO females only at the 46–65 day range. Note male ArKO mice show an absence of the accelerated growth during puberty as compared with Wt. For time intervals after 46–65, the difference is statistically significant.

to be a consequence of the absence of rapid growth seen in male wt mice during the postpubertal period (Fig. 2). On the other hand, the female ArKO animals demonstrate significantly longer femurs (ArKO 14.8 mm vs. wt 14.5, P = 0.04) at the 46–65 day range (Fig. 2), though at the 6–7 month range the relationship between the means was reversed (wt 16.4 vs. 16.1 mm ArKO) and nearly significantly different (P = 0.06). This contrasts with the accumulation

of body weight and nasal–anal length which is significantly increased by 5 weeks of age in ArKO females as compared with wt littermates (Hirasawa and Öz, unpublished data) but not in males. We have previously reported this sexually dimorphic pattern in body size of 3–4-month-old ArKO animals [13]. IGF-1 has been shown to be an important regulator of body mass and bone growth. Therefore, we investigated

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Table 1 Serum IGF-1 levels Age

IGF-1 (ng/ml) Males

1–2 months

Females

Wt

ArKO

Wt

ArKO

675 ± 132.8 (n = 4)

474.4 ± 160.1 (n = 5)

526.7 ± 90.7 (n = 3)

733 ± 79.6 (n = 4)

ArKO significantly different from Wt, P<0.05.

IGF-1 serum levels in ArKO and wt type mice. Serum IGF-1 levels in young male ArKO mice are significantly lower than controls. On the other hand, ArKO females have significantly higher levels than wt female controls (Table 1). This correlates well with body weight results. Serum IGF-1 is mainly produced by the liver and that production is regulated by growth hormone [14]. Since growth hormone levels are difficult to measure because of rhythmic variations or

pulsatility we measured levels of the major urinary protein (MUP) as a surrogate [15]. Consistent with the low IGF-1 levels ArKO males show decreased MUP in their urine and ArKO females, which have higher serum IGF-1, show increased MUP levels relative to their controls (Fig. 3). Note there is variability in MUP levels between ArKO males of different litters but ArKO males show lower levels than wt males of the same litter.

Fig. 3. Major Urinary Protein levels in ArKO and Wt littermates. Equal volumes of diluted urine were analyzed by SDS-PAGE under reducing conditions. Lanes A–G, male animals. Lane H–M female animals. The animals are grouped by litters. Notice that ArKO males have lower levels of MUP relative to littermates, whereas female ArKO animals have higher levels than Wt littermates.

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Table 2 Lumbar spine BMD (mg/cm2 ) of female mice as a function of age Age Genotype 3 weeks 2 months 3 months 4 months 7 months 10 months 12 months

Wt

ArKO

P

Table 5 Summary of static and dynamic histomorphometry analysis of the lumbar spine Compared with wt littermate

28.8 53.6 61.3 66.8 66.4 65.6 67.2

± ± ± ± ± ± ±

2.4 2.9 5.4 5.4 4.3 5.5 5.5

30.6 51.5 51.0 52.1 47.6 46.2 45.9

± ± ± ± ± ± ±

2.2 3.5 2.9 2.9 4.4 5.8 5.3

NS NS <0.01 <0.01 <0.01 <0.01 <0.01

Table 3 Lumbar spine BMD of male mice as a function of age Age

Wt

Genotype 3 weeks 2 months 3 months 4 months 7 months 12 months

29.1 49.2 55.2 58.5 58.2 58.2

ArKO ± ± ± ± ± ±

1.6 3.4 4.1 5.1 5.1 3.8

27.3 47.8 53.1 54.3 53.4 53.6

± ± ± ± ± ±

P <0.05 NS <0.01 <0.01 <0.01 <0.01

1.4 4.1 3.8 4.4 5.3 5.3

ArKO female

ArKO male

Structural parameters BV/TV (%) C.Th (␮m)

–

Tb.Th (␮m)

Surface parameters Formation OV/BV (%)

↑

OS/BS (%)

↑

Ob.S/BS (%)

–

–

Resorption Oc.S/BS (%)

–

ES/BS (%)

–

n≥4 in each group. Dynamic parameters

3.3. Bone mass accretion and change with aging or estradiol therapy Estrogens are now known to influence bone mass accrual not only in human females but males as well. Therefore, we have used DEXA to examine the acquisition of peak BMD and maintenance with age. Irrespective of sex, as a population, ArKO animals have decreased peak BMD compared with wt controls (Tables 2 and 3). Furthermore, as the animals become older they show an accelerated loss of spinal BMD, females much more dramatically than males. The ArKO skeleton responds appropriately to estradiol as 1 month of estradiol therapy increased BMC in male and female ArKO animals relative to placebo controls (Table 4). 3.4. Bone remodeling We have previously reported sexual dimorphism in the remodeling patterns of 5–7-month-old ArKO animals [10]. These results are summarized in Table 5. Adult males show a low turnover pattern while adult females show a high turnover pattern. This is confirmed by tetracycline double label patterns in which ArKO females show increased tetracycline labeled surfaces (both single and double) as Table 4 Effect of short term estradiol therapy on ex-vivo spinal BMC

Baseline (g/cm × 10 − 3 ) Estradiol therapy Change (%)

ArKO males

ArKO females

20.3 (n = 3) 21.6 (n = 3) 6

17 (n = 3) 20.8 (n = 3) 18

MS/BS (%)

↑

MS/BS (2X%) Modified from [10]. Bold arrows indicate significant differences. Thin arrows indicate direction of trends though the differences were not significant. Horizontal lines indicate there is no trend to suggest a change.

compared with wt female or male animals and ArKO males. ArKO males show the lowest double labeled surface of any of the animal groups (Fig. 4). This is shown by dramatic elevations of BFR in ArKO females and decreased BFR in ArKO males (Table 6). Estradiol therapy, as in ovariectomized mice, decreases BFR in ArKO females. However, the same therapeutic regimen stimulated BFR in ArKO males (Table 6) as well as other histomorphometric measures of bone remodeling (data not shown). Fluorine-18 positron emission tomography (PET) is an imaging method in which F-18 is retained in the skeleton in proportion to the degree of remodeling that is occurring at Table 6 Bone formation rates Genotype

BFR (␮m3 /mm2 per year) Female

WT ArKO ArKO+E2

Mean ± S.E.M.

P

Mean ± S.E.M.

P

374.5 ± 58.4 401.9 ± 45.3 130.3 ± 22.4

– NSa <0.01b

384.5 ± 6.8 81.3 ± 30.4a 192 ± 45.9b

– <0.0001 0.006

NS, not significant. a ArKO

Male

vs. WT. vs. ArKO.

b ArKO + E2

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Fig. 4. Double labeled surfaces in spinal cancellous bone of Wt and ArKO animals. Panel A, Wt female. Panel B, ArKO female. Panel C, Wt male. Panel D, ArKO male. Notice that female mice have more label incorporation compared with males and that there are more double labeled surfaces in ArKO females compared with other groups.

the time of imaging [16]. Therefore, we tested whether male ArKO animals would show decreased F-18 retention compared with wt males and whether estradiol therapy would correct this decrease. Fig. 5 shows decreased activity in male ArKO lumbar spine (yellow vs. red) but a more normal pattern (more red) in the lumbar spine after therapy. 3.5. Bone hematopoietic microenvironment Ovariectomy has been reported to increase B-cell lymphopoiesis in bone marrow cells. Therefore, we compared the bone marrow derived from wt and ArKO mice for cells staining positive for the B-cell marker B220. A representative experiment is shown in Fig. 6. The ArKO female clearly shows higher levels of the two populations of B220 positive cells. This same pattern has been seen in three independent experiments. 3.6. Bone 3D microarchitecture To demonstrate whether there is an alteration in the 3D structure of cancellous bone we have employed MR microscopy and ␮CT on femurs and vertebral bodies,

respectively, of female wt and ArKO littermates at 4 months of age. As expected the ArKO animals have decreased bone volume in the spine (Fig. 7A and B) and femur (Fig. 7C and D). Furthermore, the connectivity is also decreased. For example, in the vertebral bodies examined by ␮CT the mean connectivity density is 115.5 mm − 3 for Wt female and 44.4 mm − 3 for the ArKO female. Femurs examined by MR microscopy show similar decreased connectivity density, 119 vs. 44. 4. Discussion and conclusion Bone is a multi-compartment dynamic organ composed of mineralized tissue, stromal, and hematopoietic marrow compartments. The present report demonstrates that estrogen insufficiency caused by aromatase deficiency in the ArKO mice results in abnormalities in bone growth, mass, remodeling, microarchitecture and the hematopoietic microenvironment. It extends our previous report [10] by showing when the abnormalities in longitudinal growth arise. Furthermore, the abnormalities are sexually dimorphic in that in the early post-pubertal period the ArKO females have longer bones

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Fig. 5. Fluorine-18 PET imaging of wt, ArKO and estrogen-treated ArKO males. Shown are planar projections of whole body images acquired 1.5 h after intravenous administration of carrier free 18-F − . The color scale of 18-F − uptake is shown on the right of the figure. Areas that are more red are indicative of greater bone remodeling, particularly mineralization. Clearly the ArKO male demonstrates less spinal remodeling than Wt. Notice the dramatic increase in uptake in the ArKO animal after estradiol therapy. This represents excellent correlation with the histological findings after estradiol therapy.

Fig. 6. Flow cytometric analysis of bone marrow derived cells of Wt and ArKO. Bone marrow cells were harvested from the lumbar spine and femurs of 8-month-old females and stained with FITC-Anti-B220, which is a marker of B-cell precursors. Bold tracings are from stained cells and thin lines are control unstained cells. Wt (A) and ArKO (B). Note the two peaks of positive staining cells under marker M1 in the (B), indicating increased B-cell lymphopoiesis in ArKO females.

than Wt littermates, while ArKO males have shorter bones. This is associated with a decrease in peak bone mass in both genders as assessed by DEXA and quantitative histomorphometry. The high BFR in ArKO females, in contrast to the low BFR in ArKO males, emphasizes the dimorphic adult

remodeling patterns. The GH/IGF-1 axis is also altered in a sexually dimorphic fashion in young ArKO mice. Finally, increased B-cell lymphopoiesis in female ArKO animals reinforces the point that estrogens have regulatory functions in all compartments of bone.

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Fig. 7. MR microscopy and ␮CT imaging of femurs or vertebrae from Wt and ArKO females. ␮CT 3D renderings (A, B). MC microscopy (C, D). A, C Wt female. B, D ArKO female. In C and D the gray scale has been inverted so that bone appears white and a part of the cortical bone has been ‘cut away’ to reveal the difference in trabecular structure. Notice the decreased trabecular bone in the femoral metaphysis of the ArKO animal.

The longitudinal bone phenotype in ArKO mice is age and sex dependent. By 7 months of age the ArKO female femurs are nearly significantly shorter (P = 0.06) while femurs from ArKO males show much more significant shortening (P = 0.0001). Both sexes of ArKO animals experience a reduced longitudinal femur growth rate after the early post-pubertal period (i.e. 46–65 days old). From this time to 7 months of age the mean femur length increased by 12.4% in wt females versus 9% in ArKO females; however, the male ArKO mice show a more dramatic difference with only a 2% increase compared with a 10.1% increase in males. The limiting step in longitudinal growth rate regulation within long bones occurs at the level of the growth plate. Therefore, these changes may be secondary to a problem(s) in the requisite osteoconduction by growth plate septae. These sexually dimorphic differences in longitudinal bone growth observed in the early post-pubertal ArKO animals could result from a number of paracrine/autocrine and/or endocrine abnormalities, including GH acting through IGF-1, Vitamin D, and thyroid hormone. For example, the altered serum IGF-1 levels observed in ArKO mice during puberty (1 month), low in males but high in females, appear to be consistent

with the observed decreased and increased bone lengths, respectively. However, recent data suggest autocrine/paracrine effects of IGF-1 in the local skeletal environment regulate growth [17] as opposed to an endocrine mode of regulation by hepatic IGF-1 [14]. It remains to be tested whether there might be local changes of IGF-1 in ArKO bone. Several other paracrine/autocrine factors are known to regulate growth plate chondrocyte proliferation and differentiation including TGF-␤, FGF-2, PTHrP, BMPs 6 and 7, and Vitamin D [18]. Of particular interest is the PTHrP/IHH regulatory feedback system. Originally identified as a critical feedback system regulating embryonic skeletal development, this system has recently been shown to be expressed in epiphyseal cartilage during the early post-pubertal period in mice [19], and the pubertal period in rats [20]. To our knowledge there are no published studies on the regulation of this feedback system by estrogens; however, estrogens have been shown to regulate PTHrP expression in breast cancer cells though the results are conflicting [21,22]. Nevertheless, these results raise the question of dysregulation of this system as the cause of the altered longitudinal growth in ArKO mice.

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A defect in growth plate function might also be the basis for the decreased peak bone mass in the lumbar spine, if trabecular number is decreased early in life. Indeed at 3 weeks of age ArKO males have lower spinal BMDs but not ArKO females. However, both sexes show reduced spinal BMD at 3 months. If local IGF-1 changes are represented by the changes in circulating levels then the differences may be secondary to changes in the GH/IGF-1 axis. The reduced MUP levels, a family of proteins produced in the liver, of ArKO males is more characteristic of the low continuous GH release seen in female mice as opposed the high MUP levels in normal male mice which release high levels of GH in a pulsatile fashion [15]. On the other hand, the high MUP levels in ArKO females is more characteristic of normal male levels. Altered MUP levels in ArKO animals suggest that estrogens or possibly the estrogen to testosterone ratio regulates the pattern of GH secretion in both sexes. Whatever the case with IGF-1, reduced peak BMD in both sexes indicates normal levels of estrogens are required to attain peak BMD. Maintenance of skeletal mass after establishment of peak BMD requires normal coupling of bone formation and resorption. While a growth plate malfunction might explain the low peak BMD it cannot explain the abnormal patterns of turnover demonstrated by tetracycline labeling and PET imaging. The loss of bone mass in aging ArKO females is consistent with increased osteoclastic activity. The low turnover pattern with little change in BMD demonstrated in ArKO males might be the result of a defect(s) in osteoblast differentiation or function. Since osteoclastogenesis is coupled to osteoblasts through the OPG/RANK/RANKL system the low osteoclastic surface may also be explained by a problem with osteoblasts/stromal cells in ArKO mice. We are currently investigating whether male ArKO animals have fewer osteoblast progenitor cells or faulty differentiation in in vitro studies. The dramatic anabolic response (high BFR and increased osteoblastic surface (data not shown)) of ArKO males to estradiol indicates the problem is a consequence of estrogen deficiency and suggests the defect is in progenitor differentiation. Why there is sexual dimorphism in the remodeling pattern remains unclear. The increased B cell lymphopoiesis demonstrated in ArKO females is similar to the results observed in ovariectomized mice [6]. However, ovariectomy does not result in estrogen deficiency alone. Our results show unequivocally that estrogen deficiency is sufficient for increased B cell lymphopoiesis in bone marrow cells. Previous studies have shown that cell-to-cell interaction between B lymphocytes and human bone marrow stromal cells triggers the production of the bone resorbing cytokines IL-1 and IL-6 [23]. Thus, the increased bone loss seen in adult ArKO females may be related to this increase in B cells in the bone microenvironment. In summary, the bone phenotype of the ArKO mouse shows many of the phenotypes associated with estrogen deficiency, decreased peak BMD in both sexes; abnormal

longitudinal growth; alterations in bone remodeling; increased B-cell lymphopoiesis; and an altered GH/IGF-1 axis. Understanding the underlying mechanisms should impact our understanding of growth plate function and regulation, bone remodeling in adults, and the health care of patients with osteopenia and osteoporosis.

Acknowledgements The authors wish to thank Dr Rasesh Kapadia and Dr Douglass Adams for ␮CT scanning and analysis, Dr Mark Wessels for MR microscopy, Dr James Richardson and John Shelton for photography of stained skeletons and Virginia Barnett for assistance with graphics. This work was supported by grants 1-K08-HDO1463-01 from the NICHD of the NIH, and in part by funds from the Effie and Woffard Cain Chair endowment, institutional funds from the Center for Mineral Metabolism and Clinical Research and a grant from the Victorian Breast Cancer Research Consortium, Inc.

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