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Endocrinology, doi:10.1210/en.2003-1089
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Endocrinology Vol. 145, No. 2 913-921
Copyright © 2004 by The Endocrine Society

Effects of Loss of Steroid Receptor Coactivator-1 on the Skeletal Response to Estrogen in Mice

Ulrike I. L. Mödder, Arunik Sanyal, Ann E. Kearns, Jean D. Sibonga, Eijun Nishihara, Jianming Xu, Bert W. O’Malley, Erik L. Ritman, B. Lawrence Riggs, Thomas C. Spelsberg and Sundeep Khosla

Endocrine Research Unit (U.I.L.M., A.S., A.E.K., B.L.R., S.K.), Departments of Orthopedics (J.D.S.) and Biochemistry and Molecular Biology (T.C.S.), and Physiological Imaging Research Laboratory (E.L.S.), Department of Physiology and Biophysics, Mayo Clinic College of Medicine, Rochester, Minnesota 55905; and Department of Molecular and Cellular Biology (E.N., J.X., B.W.O.M.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Sundeep Khosla, M.D., Mayo Clinic, 200 First Street SW, 5-194 Joseph, Rochester, Minnesota 55905. E-mail: khosla.sundeep{at}mayo.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Steroid receptor coactivator (SRC)-1 is an important nuclear receptor coactivator that enhances estrogen (E) action in many tissues, but its role in mediating E effects on bone is unknown. Thus, we assessed the skeletal response to ovariectomy (ovx) and E replacement in SRC-1 knockout (KO) mice compared with wild-type (WT) littermates. Bone mineral density was measured by dual-energy x-ray absorptiometry and peripheral quantitative computed tomography at baseline and after 2 months of sham surgery, ovx, or ovx plus E replacement. Microcomputed tomography and bone histomorphometry were also performed at the end of the study. Both WT and SRC-1 KO mice lost bone at multiple sites after ovx; however, although an estradiol (E2) dose of 10 µg/kg·d completely prevented loss of cancellous bone (at the lumbar spine and tibial metaphysis) in the WT mice, it was entirely ineffective in preventing cancellous bone loss at these sites in the SRC-1 KO mice. This E2 dose was, however, equally effective on cortical bone in the tibia in the SRC-1 KO and WT mice. Moreover, a 4-fold higher dose of E2 was able to overcome the deficit in E action in cancellous bone in the SRC-1 KO mice. These findings establish that, in mice, loss of SRC-1 leads to skeletal resistance to E predominantly in cancellous bone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NUCLEAR RECEPTOR ACTION is modulated in different tissues by a host of coregulators. These can be broadly defined as cellular factors that are recruited by the nuclear receptors and that complement the function of the receptors as mediators of the cellular response to endocrine signals. Both coactivators and corepressors of nuclear receptor function have now been identified (for review, see Ref. 1). Steroid receptor coactivator (SRC)-1 was the first of these nuclear receptor coactivators to be cloned using the progesterone receptor ligand-binding domain as bait in a yeast two-hybrid system (2). Subsequently, the closely related family members, glucocorticoid receptor-interacting protein-1/TIF2/SRC-2 (3) and p300/cAMP response element-binding protein/activator of thyroid receptor/amplified in breast cancer-1/receptor-associated coactivator-3/thyroid receptor-associated molecule-1/SRC-3 (4), were also identified. These coactivators are not only related structurally but also have similar functional activities, such as acetyltransferase activity, which leads to histone acetylation and helps disrupt the interactions responsible for maintaining the promoter in a state that is inaccessible to the transcriptional apparatus (1). In addition, the SRCs play a major role in recruiting other coactivators and transcription factors, thereby enhancing the transcriptional activity of the receptor (1).

Although much is known about the structure and in vitro function of nuclear receptor coactivators, their physiological importance was first demonstrated for SRC-1 by the generation of SRC-1 knockout (KO) mice (5). The homozygous mutants are viable and fertile in both sexes but exhibit significant resistance to estrogen (E) action in a number of tissues, including the uterus and mammary gland. It appears that the relatively normal phenotype in these mice is maintained, at least in part, by elevated circulating estradiol (E2) levels (5).

Like classical reproductive tissues, the skeleton is an E-responsive tissue. E is critical in the acquisition of peak bone mass not only in females but, somewhat surprisingly, also in males (6, 7). Moreover, E deficiency is perhaps the single most important factor in the development of postmenopausal osteoporosis in women and may also play a significant role in the pathogenesis of age-related bone loss in men (6, 7). Both osteoblasts (8, 9) and osteoclasts (10) contain E receptors (ERs), and the mechanisms of E action on bone have been studied intensively both in vitro and in vivo (for reviews, see Refs. 6 and 11).

However, despite the clear importance of nuclear receptor coregulators in mediating E action in various tissues, almost nothing is known about their role in modulating E action in bone. Using transient transfection assays, our group has recently demonstrated that SRC-1, -2, and -3 have distinct preferences in enhancing the transcriptional activity of ER{alpha} and ERß in human osteoblastic cells and that these preferences are cell type specific (12). Thus, in osteoblastic cells, SRC-1 mainly enhanced the transcriptional activity of coexpressed ER{alpha}/ß or ERß alone, with little or no effect on the activity of ER{alpha} alone. In contrast, in a kidney cell line, SRC-1 enhanced the activity primarily of ER{alpha}.

In the present study, we used the SRC-1 KO mice to characterize in detail the consequences of SRC-1 deficiency for E action on bone in vivo. Recognizing that these mice have compensated E resistance (5), we not only characterized their skeletal phenotype under basal conditions but also after ovariectomy (ovx) and replacement with a dose of E that, in a preliminary dose-finding study, was found to be the minimal physiological dose necessary to prevent bone loss and maintain uterine weight after ovx in wild-type (WT) mice. Finally, to establish that the SRC-1 KO mice did indeed have E resistance, we also tested whether the defect(s) in E action at the lower dose of E could be overcome by using a significantly higher dose of E.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation and care of KO mice
The generation of the SRC-1 KO mice has been described previously (5), and the mice used in the present studies had been extensively backcrossed (for seven or more generations) into the C57BL/6 background. The animals were housed in a temperature-controlled room (22 ± 2 C) with a daily 12-h light/12-h dark schedule. During the experiment, the animals had free access to water and were pair fed a standard laboratory chow (Laboratory Rodent Diet 5001; PMI Feeds, Richmond, VA) containing 0.95% calcium. Pups were genotyped at 4–5 wk of age by PCR as described previously (5). The Institutional Animal Care and Use Committee approved all animal procedures.

Gonadectomies and E replacement
Recognizing that the SRC-1 KO mice likely have compensated E resistance under basal conditions, we initially performed an E dose response study in WT mice to identify the minimal dose of E that effectively preserved bone mineral density (BMD) and uterine weight in the WT mice. Three-month-old WT C57BL/6 mice (n = 4–5 per group) were either sham operated or ovx and received either a vehicle pellet or pellets delivering 5, 10, 20, or 40 µg/kg·d E2 (based on an average body weight of 25 g) (Innovative Research of America, Toledo, OH). BMD was measured using dual-energy x-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT) (see below) at the beginning of the study and after 30 d of the respective treatments. This preliminary study identified an E2 dose of approximately 10 µg/kg·d as the appropriate dose to use in the subsequent study because it effectively preserved BMD at multiple sites and restored uterine weight to the level of the sham mice in WT, ovx mice (data not shown). This was further verified by measuring serum E2 levels, which decreased from 19.9 ± 2.0 pmol/liter in the sham mice to 11.7 ± 0.48 pmol/liter in the ovx mice (P < 0.001) and were restored to the level in the sham mice in the ovx mice treated with the 10 µg/kg·d dose (18.6 ± 3.5 pmol/liter). In addition, as noted below, we also tested whether the resistance to E action in the SRC-1 KO mice could be overcome using a supraphysiological dose of E2; for this, we chose the 40 µg/kg·d dose, which, as expected, resulted in circulating E2 levels approximately 4-fold higher than those with the 10 µg/kg·d dose (71.3 ± 28.5 pmol/liter).

To compare the skeletal response to E in the WT vs. the SRC-1 KO mice, 3-month old female SRC-1 KO and WT littermates had baseline measurements of BMD performed (see below) and were then divided into three groups (n = 8–11 mice/group). Mice were assigned to the three groups based on the cancellous BMD measured by pQCT to assure comparable tibial BMD among groups at baseline. Subsequently, the mice were either sham operated, ovx and implanted with a vehicle pellet, or ovx and implanted with a 10 µg/kg·d E2 pellet (0.015 mg/60-d pellets). On d 48 after the pellet implantation, the mice received tetracycline (10 mg/kg) by tail vein injection, followed by a calcein (10 mg/kg) injection on d 56. BMD was again measured by DXA and pQCT on d 60 after the surgery. After this, the animals were killed by inhalation of CO2 and various tissues harvested. The uterus was excised and weighed, and the lumbar spine (L1-L4), tibias, and femurs were excised for histomorphometry and microcomputed tomography (µCT) measurements (see below).

Subsequent to this initial study, a fourth group was added to both the WT and SRC-1 KO mice (n = 9–11 for each) to test whether the defects in E action noted in the SRC-1 KO mice using an E2 dose of 10 µg/kg·d could be overcome using a high dose of E. These mice were ovx and received pellets delivering 40 µg/kg·d of E2. In our preliminary dose-finding study, this relatively high dose of E2 was clearly supraphysiological (see above) but did not induce a sclerotic response in the tibial metaphysis, as has been seen with very high doses of E2 (13). All procedures in these mice were identical to those noted for the mice treated with the 10 µg/kg·d dose.

Bone densitometry
For both the DXA and pQCT measurements, the mice were anesthetized with Avertin (2,2,2 tribromoethanol, 720 mg/kg, ip). For the DXA measurements, they were placed on the animal tray in a prone position on the Lunar PIXImus densitometer (software version 1.44.005; Lunar Corp., Madison, WI). In this position, the head is partially outside the area scanned by the machine. However, in all analyses, the bones of the skull were excluded. Calibration of the machine was performed daily using the hydroxyl apatite phantom provided by the manufacturer. After scanning, regions of interest were identified for more specific analyses. In repeatedly scanned mice (with repositioning between scans), the coefficients of variation (CV) for total body, lumbar, and femoral BMD were 4.9, 2.7, and 4.3%, respectively.

For the pQCT measurements, the mice were placed in a supine position on the gantry of the Stratec XCT Research SA Plus using software version 5.40 (Norland Medical Systems, Inc., Fort Atkinson, WI). As for the PIXImus, calibration of the machine was performed daily with the hydroxyl apatite phantom provided by the manufacturer. The mice were positioned so that the total length of the femur and tibia were visible on the scout view. The scout view speed was set at 15.0 mm/sec with a slide distance of 0.5 mm. Once the scout view was completed, the reference line for the computed tomography (CT) scans was set at the most proximal point of the tibia. Slice images were set at 1.9 mm (proximal metaphysis of the tibia). The CT speed was set at 3 mm/sec, the pixel size was 100 x 100 µm, and slice thickness was 0.5 mm. After scanning, the CT slices were analyzed using peelmode 2, cortmode 1, and contour mode 1 to evaluate trabecular and cortical parameters. To determine the cancellous bone, the threshold was set at 214 mg/cm3 and for cortical bone at 710 mg/cm3. The CV was 4.4% for the total tibial volumetric BMD.

Bone histomorphometry
The lumbar spines (L1–L4) were fixed in 70% ethanol for at least 72 h and than dehydrated in 95% ethanol for 1 d and in 100% ethanol for 6 d. The bones were then embedded without demineralization in a mixture of methylmethacrylate-2-hydroxyethyl and methylacrylate 12.5:1 to retain flourochrome labels and subsequently sectioned at a thickness of 5 µm on a Reichert-Jung Supercut 2050 microtome (Leica Microsystems Inc., Bannockbum, IL) using tungsten-carbide tipped steel knives. Sections were taken from the dorsal spine passing through the middle of the lumbar spine. All cancellous measurements were performed on unstained sections under UV and normal light using the Osteomeasure software (OsteoMetrics Inc., Atlanta, GA). All static histomorphometric measurements were obtained at the spine to correlate with the spine DXA measurements. However, because bone turnover at this site is relatively slow, bone formation rates were determined in the various groups in the femoral metaphyses.

µCT analyses
The left tibia was collected 60 d after the surgeries and stored at -80 C. Before the scan, the tibia was slowly thawed in ice-cold 70% ethanol and kept in ethanol during the scan. The whole tibia was scanned using a µCT system (Physiological Imaging Research Laboratory, Mayo Clinic, Rochester, MN) as described by Jorgensen et al. (14) with a resolution of 20 µm in all three dimensions. The raw data were reconstructed, and the resulting three-dimensional images were displayed using an image analysis software (Analyze 4.0; Biomedical Imaging Resource, Mayo Clinic). The volume of interest was 30 slices below the growth plate and covered 0.6 mm of the proximal metaphysis of the tibia. This region was chosen because it allowed for measurements of cortical and cancellous bone and it represents the comparable region that was measured by pQCT. Cortical thickness was measured at nine equally distributed sites at every second cross-section within the volume of interest. The full-width half-maximal function was used for this measurement. The trabecular bone volume was also measured in the same volume of interest. To determine trabecular bone, a threshold was chosen that represented bone in all samples. Thus, every voxel with a gray scale above the threshold was trabecular bone, and every voxel with a gray scale below the threshold was determined as marrow within the cortical shell.

Serum E2 measurements
Serum E2 was measured by RIA (Diagnostic Products Corp., Los Angeles, CA) (interassay CV, <16%).

Assessment of ER{alpha}, ERß, and SRC-2 mRNA levels
Total RNA was isolated from the lumbar vertebrae and the midshaft of the femur. The frozen bones were ground in a freezer mill together with Trizol (Invitrogen Life Technologies, Carlsbad, CA). RNA was purified using phenol/chloroform extraction and a sodium acetate/ethanol precipitation. After quantitation by UV absorption at 260 nm, 1 µg total RNA was used for the cDNA synthesis using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). The cDNA mix was diluted 5-fold and used as a source of template for the RT-PCR analysis. For conventional RT-PCR, 10 pmol forward and reverse primers were used in a total volume of 50 µl with Taq DNA polymerase (Roche Diagnostics Corp., Indianapolis, IN) and 5 µl diluted reverse transcription reaction product. The PCR was performed as follows: 94 C for 5 min (initial denaturation step), 35-cycle amplification at 94 C for 1 min, 60 C for 1 min, and 70 C for 1 min, and additive incubation at 72 C for 5 min. The following primer pairs were used in the RT-PCR analysis: SRC-2, 5'-CTACCAGCAGCCATGAGCAATC-3' and 5'-CATCGACACACTGATGTTCATGTTG-3'; ER{alpha}, 5'-GGCAAAGAGAGTGCCAGGCTTTG3' and 5'-CAGAAACGTGTACACTCCGGAATT-3'; and ERß, 5'-GCAGCACAAAGAATATCTGTGTGTG-3' and 5'-AGCGTGTGAGCATTCAGCATCTC-3'.

The real-time PCR was performed in an I-Cycler (Bio-Rad Laboratories). A 50-µl PCR mix contained 25 µl iQ SYBR Green Supermix [100 mM KCl, 40 mM Tris-HCl (pH 8.4), 0.4 mM of each deoxynucleotide triphosphate, iTaq DNA polymerase, 6 mM MgCl2, SYBR Green 1, 20 nM fluorescein, and stabilizers], the appropriate primers at 7.5 pmol and 5 µl cDNA.

Statistical analyses
All data are presented as mean ± SEM. The primary comparison in all cases was between the WT ovx plus E2 vs. the SRC-1 KO ovx plus E2 mice because the key question was the skeletal response to E in the two groups of mice. The sham and ovx plus vehicle groups were included in all cases to demonstrate that changes in the WT and SRC-1 KO mice were comparable under these conditions. Overall comparisons within groups were made using ANOVA, and individual comparisons between groups were made using nonpaired t tests. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Skeletal phenotype of SRC-1 KO mice under basal conditions
Both male and female homozygote SRC-1 KO mice were fertile and showed growth rates similar to WT animals, with virtually identical body weights, bone dimensions, and BMD at various sites at 12 wk of age (data not shown). Moreover, as shown in Fig. 1Go, the bones in the SRC-1 KO mice were histologically normal. Importantly, there was no evidence in the SRC-1 KO mouse bones of osteomalacia or impaired mineralization, which is a significant point because SRC-1 may also modulate 1,25-dihydroxyvitamin D action (15, 16). In addition, Xu et al. (5) noted that female SRC-1 KO mice have modest elevations in serum E2 (~20%) and testosterone (~50%) levels, compared with WT mice. We confirmed these findings for serum E2 levels and, by measuring serum E2 levels through various phases of the estrus cycle in pooled serum from four to eight female SRC-1 KO and WT mice, also demonstrated that during diestrus, the SRC-1 KO mice had about 60% higher serum E2 levels compared with the WT mice (data not shown). These data provided further support to the hypothesis that the SRC-1 KO mice have compensated E resistance in a number of tissues (5), including bone; the latter was studied in detail in the studies described below.



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  		FIG. 1. A and B, Goldner’s stain of lumbar vertebrae at low power (intervertebral disk is at the center) (x6) from WT (A) and SRC-1 KO (B) mice. C and D, Higher-power view (x25). Note absence of any abnormal osteoid accumulation in the SRC-1 KO mice, compared with the WT mice, consistent with the absence of any mineralization defects in these animals. E and F, Fluorescent microscopy showing incorporation of crisp double labels of tetracycline and calcein (arrows), consistent with normal mineralization in the SRC-1 KO mice, similar to the WT mice.

 
Effects of ovx and E replacement on BMD in the SRC-1 KO mice compared with the WT mice
To clearly define possible deficits in E action in the SRC-1 KO mice, we compared the effects of sham surgery, ovx with vehicle pellets, and ovx with E2 replacement (at a dose of 10 µg/kg·d) on BMD in WT vs. SRC-1 KO mice. All treatments were for 60 d in this study, and BMD was measured at baseline and at the end of the study. Figure 2AGo shows the percentage changes in spine BMD in the three groups of WT and SRC-1 KO mice. As is evident, spine BMD decreased significantly after ovx in both the WT and SRC-1 KO mice. However, whereas E2 treatment prevented this decrease in the WT mice, it was virtually ineffective in the SRC-1 KO mice. Similar findings were present at the femur, although here the deficit in E2 action did not appear to be quite as severe as at the spine (Fig. 2BGo).



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  		FIG. 2. Percentage changes in lumbar spine (A) and femur BMD (B) (both measured by DXA) in the WT and SRC-1 KO mice after either sham surgery (open bars), ovx plus vehicle pellets (solid bars), or ovx and E2 pellets (shaded bars; 10 µg/kg·d) for 60 d. The P values for the main comparison (SRC-1 KO, ovx + E2 vs. WT, ovx + E2) are as indicated. ANOVA within-group comparisons: spine WT, P = 0.001 and spine SRC-1 KO, P < 0.001; femur WT, P = 0.03 and femur SRC-1 KO, P <0.001. **, P < 0.01 and ***, P < 0.001 for direct comparison with the respective sham group.

 
Because the spine contains predominantly cancellous bone, whereas the femur contains both cancellous and cortical bone, we further explored whether the deficit in E action in the SRC-1 KO mice was present predominantly in cancellous bone. For this, we used the tibial BMD by pQCT, which can measure changes in cancellous and cortical bone combined as well as separately in the two compartments. As shown in Fig. 3AGo, combined cortical and cancellous (total) tibial BMD decreased similarly in the WT and SRC-1 KO mice, and E2 prevented this decrease in both groups, although it appeared to be somewhat less effective in the SRC-1 KO mice compared with the WT mice. However, as shown in Fig. 3BGo, similar to the spine DXA data, E2 was entirely ineffective in the cancellous compartment of the tibia in the SRC-1 KO mice, whereas it not only maintained but also modestly increased cancellous tibial BMD in the WT mice. Moreover, as shown in Fig. 3CGo, E effects on cortical BMD in the tibia were virtually identical in the WT and SRC-1 KO mice.



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  		FIG. 3. Percentage changes in tibial total volumetric (A), tibial cancellous (B), and tibial cortical (C) BMD (all measured by pQCT) in the WT and SRC-1 KO mice after sham surgery (open bars), ovx plus vehicle pellets (solid bars), or ovx and E2 pellets (shaded bars, 10 µg/kg·d) for 60 d. The P values for the main comparison (SRC-1 KO, ovx + E2 vs. WT, ovx + E2) are as indicated. ANOVA within-group comparisons: total tibia WT, P = 0.002 and total tibia SRC-1 KO, P = 0.003; cancellous WT, P < 0.001 and cancellous SRC-1 KO, P = 0.322; cortical WT and SRC-1 KO, both P = 0.002. *, P < 0.05, **, P < 0.01, and ***, P < 0.001 for direct comparison with the respective sham group.

 
µCT analysis
The above data using DXA and pQCT strongly suggested that the SRC-1 KO mice had a clear defect in E action in cancellous bone but preserved responses to E in cortical bone. To establish this more definitively, we performed detailed µCT analyses of the tibias (at the same site as used in the tibial pQCT measurements) using excised bones from the WT and KO mice. Figure 4Go shows an example of these scans from WT and SRC-1 KO mice, ovx and treated with E2, which are the key comparison groups. As is evident from the middle and right panels (and consistent with the pQCT data at this site), E treatment of ovx WT vs. SRC-1 KO mice resulted in markedly altered effects on cancellous bone, with much more cancellous bone present in the WT mice compared with the KO mice. In contrast, the amount of cortical bone present (seen best in the cross-sectional images in the right panels) was similar in the two groups after ovx and E2 treatment. This was more rigorously quantified in Fig. 5AGo. As shown, cancellous bone volume/total volume (BV/TV) was significantly lower in the WT and SRC-1 KO mice following ovx, compared with the sham mice; however, whereas BV/TV was actually increased above levels in the sham mice in the WT E-treated mice, E2 treatment at a dose of 10 µg/kg·d failed to preserve BV/TV in the SRC-1 KO mice. In contrast, as shown in Fig. 5BGo, the pattern of changes in the ovx and ovx plus E2-treated mice in cortical thickness was very similar in the WT and SRC-1 KO mice, consistent with a preservation of E action in cortical bone in the KO mice.



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  		FIG. 4. Examples of µCT analyses of tibias from WT (top) and SRC-1 KO (bottom) mice, ovx and treated with E2. The yellow band in the middle panels identifies the region of interest using a sagittal section, and the right panels show cross-sectional images through this region. Red indicates cancellous bone and arrows indicate cortical bone.

 


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  		FIG. 5. µCT analysis of tibias from WT and SRC-1 KO mice following sham surgery (open bars), ovx plus vehicle pellets (solid bars), or ovx and E2 pellets (shaded bars; 10 µg/kg·d) for 60 d. A, Cancellous BV/TV. B, Cortical thickness. The P values for the main comparison (SRC-1 KO, ovx + E2 vs. WT, ovx + E2) are as indicated. ANOVA within-group comparisons: BV/TV WT, P = 0.036 and BV/TV SRC-1 KO, P < 0.001; cortical thickness WT, P = 0.038 and cortical thickness SRC-1 KO, P = 0.06. *, P < 0.05, **, P < 0.01, and ***, P < 0.001 for direct comparison with the respective sham group.

 
Bone histomorphometry
Table 1Go shows the bone histomorphometry data in the various groups. Consistent with the spine DXA measurements, BV/TV in cancellous bone in the spine was lower in both WT and SRC-1 KO mice after ovx. However, whereas E treatment preserved cancellous BV/TV in the WT mice, it failed to do so in the KO mice. Similar results were seen with the other cancellous bone parameters (trabecular separation and trabecular number, Table 1Go). The bone formation rate was significantly higher in the ovx WT and KO mice compared with the respective sham groups; however, although E treatment of WT mice reduced bone formation rate to even below that in the sham animals, the bone formation rate in the E-treated SRC-1 KO mice was similar to that in the ovx SRC-1 KO mice and clearly different from that in the WT E-treated mice.


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  		TABLE 1. Bone histomorphometric parameters in the WT and SRC-1 KO mice after either sham surgery, ovx with vehicle pellet replacement, or ovx with E2 pellet replacement for 60 d

 
Effects on uterine weights
As shown in Fig. 6Go, the dose of E2 used in these studies was biologically meaningful because it restored uterine weight in the ovx mice to near the level of the sham mice in the WT animals. Interestingly, although this dose of E2 was clearly having at least partial effects on bone in the SRC-1 KO mice, particularly in cortical bone, it was totally ineffective at restoring uterine weight in the SRC-1 KO mice.



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  		FIG. 6. Uterine weights in the WT and SRC-1 KO mice after sham surgery (open bars), ovx plus vehicle pellets (solid bars), or ovx and E2 pellets (shaded bars, 10 µg/kg·d) for 60 d. The P values for the main comparison (SRC-1 KO, ovx + E2 vs. WT, ovx + E2) are as indicated. ANOVA within-group comparisons: WT, P = 0.002 and SRC-1 KO, P < 0.001. ***, P < 0.001 for direct comparison with the respective sham group.

 
Elimination of the defect in E action on bone in the SRC-1 KO mice using high-dose E
To establish that the defect in E action in the SRC-1 KO mice was, in fact, due to E resistance and that this could be overcome with a higher dose of E, the WT and SRC-1 KO mice were also treated with a high dose (40 µg/kg·d) of E2. As shown in Fig. 7Go (and in contrast to the findings with the lower dose of E2 shown in Fig. 2Go), a dose of 40 µg/kg·d of E2 resulted in virtually identical changes in spine and femur BMD in the WT and SRC-1 KO mice. Thus, the defect in E action present in the KO mice could be overcome using higher doses of E.



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  		FIG. 7. Percentage changes in lumbar spine (A) and femur (B) BMD (both measured by DXA) in the WT and SRC-1 KO mice after sham surgery (open bars), ovx plus vehicle pellets (solid bars), or ovx and high-dose E2 pellets (shaded bars, 40 µg/kg·d) for 60 d. Note that the sham and ovx groups are the same as shown in Fig. 2Go. The P values for the main comparison (SRC-1 KO, ovx + E2 vs. WT, ovx + E2) are as indicated. ANOVA within-group comparisons: spine WT and KO, both P < 0.001; femur WT, P = 0.003; and SRC-1 KO, P < 0.001. *, P < 0.05, **, P < 0.01, and ***, P < 0.001 for direct comparison with the respective sham group.

 
Comparison of ER{alpha} and ERß mRNA expression between cancellous and cortical bone
As noted previously, our group recently reported that in osteoblastic cells, SRC-1 seems to preferentially enhance (at least in vitro) the transcriptional activity of coexpressed ER{alpha} and ERß or the ERß homodimer to a much greater extent than that of ER{alpha} alone (12). Because we had demonstrated what appeared to be a relatively selective defect in E action in cancellous bone in the SRC-1 KO mice, we also compared the expression of ER{alpha} and ERß mRNAs in cancellous vs. cortical bone by PCR. RNA was extracted from either the lumbar vertebrae (a site rich in cancellous bone) or the midshaft of the femur (which contains exclusively cortical bone). As shown in Fig. 8Go, the expression of ER{alpha} was similar in cancellous and cortical bone; by contrast, ERß was expressed only in the cancellous bone of the lumbar spine but not in the cortical bone of the femur shaft. In addition, there was no obvious difference in the expression of either ER{alpha} or -ß in bones from the WT vs. the SRC-1 KO mice.



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  		FIG. 8. Expression of ER{alpha} and -ß mRNA, assessed by RT-PCR, in the lumbar spine (a site rich in cancellous bone) and the midshaft of the femur (which contains exclusively cortical bone).

 
Expression of SRC-2 in bones of SRC-1 KO vs. WT mice
Because at least in some tissues (such as the brain and testis) expression of SRC-2 is up-regulated in the SRC-1 KO mice and may partially compensate for loss of SRC-1 (5), we also examined SRC-2 expression in bones from WT and KO mice using both conventional (Fig. 9Go) and real-time RT-PCR. The SRC-2 mRNA was expressed in both the lumbar spine and femur shaft; however, there were no significant differences in the level of the SRC-2 mRNA in bones from the WT and SRC-1 KO mice (Fig. 9Go). By real-time RT-PCR, the copy number for SRC-2 (per 100,000 copies of glyceraldehyde-3-phosphate dehydrogenase; n = 5 per group) was 1140 ± 188 (WT) vs. 1440 ± 215 (SRC-1 KO) in the spine (P = 0.324) and 2080 ± 206 vs. 1720 ± 232 in the femur shaft (P = 0.279).



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  		FIG. 9. Expression of SRC-2 mRNA, assessed by RT-PCR, in the lumbar spine and midshaft of the femur in the WT and SRC-1 KO mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although E is clearly critical for regulating bone remodeling in both females and males (6, 7), our understanding of E signaling pathways in bone is far from complete. As in other tissues, E can regulate gene expression in bone cells through binding of the liganded ER to E response elements (EREs) upstream of target genes, such as TGFß (17). In addition, however, the ER can alter gene expression through nonclassical signaling pathways: these include protein-protein interactions with specificity protein-1 (Sp-1) and activator protein-1 (AP-1) or with components of the nuclear factor-{kappa}B complex (18) as well as recently described nongenotropic pathways involving regulation of MAPK signaling (19), which may be particularly important for E regulation of apoptosis. Finally, it appears that the effects of mechanical loading and strain on bone may also involve ER signaling, perhaps via phosphorylation of the ER, resulting in activation of the receptor (and up-regulation of the activity of EREs) even in the absence of E (20, 21, 22).

SRC-1 was the first and one of the most important nuclear receptor coactivators to be characterized and cloned (2). E signaling via the ERE is clearly dependent on SRC-1 (2); however, there is evidence that alterations in SRC-1 levels also influence the regulation by the ER of genes such as c-myc and IGF-I, which lack a classical ERE (23). Whether E signaling through nongenotropic mechanisms also involves SRC-1 at some level is at present unclear, although it is of interest that Kousteni et al. (24) recently demonstrated that E regulation of the MAPK pathways eventually results in alterations in the activity of common transcription factors, such as Elk-1, CCAAT enhancer binding protein-ß, cAMP-response element binding protein, and c-Jun/c-Fos. Because it is possible (and perhaps even likely) that one or more of these transcription factors also interact with SRC-1, loss of SRC-1 would be expected to alter E signaling not just through classical EREs but also through one or more of these other pathways. Thus, a better understanding of the role of SRC-1 in mediating E action on bone represents an important step toward further dissecting the overall mechanisms by which E regulates bone metabolism.

The generation and characterization of SRC-1 KO mice by Xu et al. (5) provided the first in vivo evidence that loss of this coactivator could result in E resistance in reproductive tissues. Using these mice, we have now characterized in detail the consequences of the loss of SRC-1 on E action on bone. Under basal conditions, these mice represent a state of compensated E resistance in a number of tissues: by maintaining higher than normal E2 levels, they exhibit normal reproductive function and also appear to have a relatively normal skeletal phenotype and BMDs, at least in young adulthood (~3 months of age). However, by ovariectomizing the animals and using a dose of E that was clearly effective in preserving BMD in the WT mice, we were able to unequivocally demonstrate a profound defect in E action on cancellous bone in the SRC-1 KO mice. Moreover, this defect could be overcome by using a 4-fold higher dose of E, consistent with skeletal E resistance. In contrast to this marked deficit in E action in cancellous bone, the effects of E (even at the low dose) on cortical bone in the SRC-1 KO mice were relatively well preserved. This was established not only using cortical BMD measurements by pQCT (which has a voxel size of ~100 µm) but also by using what is generally considered the gold standard for such studies, µCT (which has a voxel size of ~20 µm). Interestingly, although the low dose of E was effective in preserving cortical BMD and thickness in the SRC-1 KO mice, in addition to having no effect in cancellous bone, this dose of E also failed to alter uterine weight in the ovx, SRC-1 KO mice, while maintaining it at sham levels in the WT, ovx mice.

Although we used a fairly high dose of E2 (~4-fold over physiological replacement) to overcome the deficit in E action in cancellous bone in the SRC-1 KO mice, it is possible that lower doses (i.e. 2-fold or less) might also have been effective. However, we wanted to establish, in principle, that the defect in E action in the SRC-1 KO mice could be overcome, and additional dose-response studies are needed to establish the minimal dose of E2 that is capable of overcoming the deficit in E action in these mice.

There are several possible reasons that loss of SRC-1 results in a defect in E action primarily in cancellous bone. Cancellous bone does have a higher rate of turnover than cortical bone, and E deficiency clearly has a greater impact on cancellous as opposed to cortical bone in WT female mice or, for that matter, in women (6). Thus, any defect in E action (i.e. from loss of SRC-1) would be expected to perhaps be most evident in cancellous bone. Although this may be part of the explanation for the predominant defect in E action in cancellous bone in the SRC-1 KO mice, a second possibility that involves potential differential interactions of SRC-1 with ER{alpha} vs. ERß in bone cells should also be considered.

In recent studies, our group has demonstrated that, in osteoblastic cells, SRC-1 appears to preferentially enhance the transcriptional activity of either coexpressed ER{alpha} and -ß or ERß alone, without significant effects on the activity of ER{alpha} alone (12). In contrast, in nonosteoblastic (i.e. COS-7) cells, this pattern is reversed, and SRC-1 preferentially enhances the activity of ER{alpha} to a greater extent than that of ERß or coexpressed ER{alpha} and -ß. Our present findings also demonstrate that although the cancellous bone of the vertebrae contains both ER{alpha} and -ß, the cortical bone of the femur shaft contains almost exclusively ER{alpha}. Consistent with our findings, Onoe et al. (25) also found that the cancellous bone of the vertebrae and distal femoral metaphysis in rats expressed much higher levels of ERß than the cortical bone of the femoral shaft. Moreover, Bord et al. (26) examined ER{alpha} and -ß expression by immunohistochemistry in neonatal human ribs and, similar to our results, demonstrated that cortical bone contained predominantly ER{alpha}, whereas cancellous bone contained immunoreactivity for both ER{alpha} and -ß.

Collectively, our findings and the previous data indicate that cancellous bone contains both ER{alpha} and -ß (leading presumably to the formation intracellularly of ER{alpha}/ß heterodimers) (27), whereas cortical bone primarily contains ER{alpha} (resulting in the formation of ER{alpha} homodimers). Thus, based on our previous studies (12), the activity of the ER{alpha}/ß heterodimers in cancellous bone would be predicted to be most affected by loss of SRC-1, potentially explaining the predominant deficit in E action in cancellous bone we found in the SRC-1 KO mice. In contrast, the activity of the ER{alpha} homodimers in cortical bone may not be significantly altered by SRC-1 deficiency, resulting in the relatively preserved responses to E in cortical bone that we observed in the SRC-1 KO mice. Interestingly, we also found that in contrast to SRC-1, SRC-2 does preferentially enhance the transcriptional activity of ER{alpha} in osteoblastic cells to a greater extent than that of coexpressed ER{alpha} and -ß or ERß alone (12). This leads to the plausible prediction that E effects on cortical bone would be more impaired in the SRC-2 KO animals, with a relative preservation of E action in cancellous bone in these mice, i.e. exactly the opposite of what we observed in the SRC-1 KO mice. These studies are currently underway in our laboratory and should provide an additional test of this overall hypothesis.

As noted previously, the 10 µg/kg·d dose of E2 preserved uterine weight in the WT mice but failed to do so in the SRC-1 KO mice. Like cortical bone (23, 24), the uterus contains predominantly ER{alpha} (28), and this finding may seem at odds with our hypothesis that E action in cortical bone was preserved in the SRC-1 KO mice because our previous in vitro studies had suggested that SRC-1 did not significantly enhance the transcriptional activity of ER{alpha} in osteoblastic cells (12). However, in those studies, we also found that in a kidney cell line, SRC-1 did enhance the activity primarily of ER{alpha} (12). Although we have not studied endometrial cells, these data indicate that the specific interactions of SRC-1 with ER{alpha} vs. -ß are likely tissue specific, perhaps explaining why E action is impaired in the uterus but preserved in cortical bone in the SRC-1 KO mice, even though both tissues primarily contain ER{alpha}.

The relatively selective loss of E action we observed in cancellous bone in the SRC-1 KO mice is consistent with other studies indicating that the cancellous and cortical compartments of bone are differentially regulated. Thus, in contrast to the greater effects of E deficiency on cancellous as opposed to cortical bone (6), chronic PTH exposure leads to greater losses of cortical bone, with relative preservation (or even an increase) in cancellous bone (29). In addition, mice lacking plasminogen activator inhibitor 1 do not lose cancellous bone but do lose cortical bone after ovx (30). Finally, IL-6 KO mice have relatively well-preserved cancellous bone volumes but do have significant deficits in cortical bone (31).

In summary, our studies establish that loss of SRC-1 leads to a predominant defect in E action in cancellous bone, with a relative preservation of E effects on cortical bone. However, the deficit in E action in the SRC-1 KO mice can be overcome by using high doses of E, consistent with E resistance in bone. The differential expression of ER{alpha} and -ß in cancellous vs. cortical bone and the specific interactions of these receptor isoforms with SRC-1 may, in part, explain why cancellous bone is more susceptible to loss of SRC-1 than cortical bone. In addition, these findings may also have significant implications for our understanding of the variability of skeletal responses to E in humans, some of which may be due to alterations in the level or function of SRC-1 in bone.


   Acknowledgments
 
We thank Dan Fraser for the breeding and maintenance of the mouse colonies as well as help with the mouse surgeries, Jesse Lamsam and David Nagel for technical assistance, and Cheryl Collins for secretarial assistance.


   Footnotes
 
This work was supported by Grants AG 004875 (National Institute on Aging) and EB 000305 (National Institute of Biomedical Imaging and Bioengineering).

Abbreviations: BMD, Bone mineral density; BV/TV, bone volume/total volume; CT, computed tomography; µCT, microcomputed tomography; CV, coefficient of variation; DXA, dual-energy x-ray absorptiometry; E, estrogen; E2, estradiol; ER, E receptor; ERE, E response element; KO, knockout; ovx, ovariectomy or ovariectomized; pQCT, peripheral quantitative computed tomography; SRC, steroid receptor coactivator; WT, wild-type.

Received August 20, 2003.

Accepted for publication October 7, 2003.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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