help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2003-0655
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/2/922    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sher, L. B.
Right arrow Articles by Kream, B. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sher, L. B.
Right arrow Articles by Kream, B. E.
Endocrinology Vol. 145, No. 2 922-929
Copyright © 2004 by The Endocrine Society

Transgenic Expression of 11ß-Hydroxysteroid Dehydrogenase Type 2 in Osteoblasts Reveals an Anabolic Role for Endogenous Glucocorticoids in Bone

Lorin B. Sher, Henning W. Woitge, Douglas J. Adams, Gloria A. Gronowicz, Zygmunt Krozowski, John R. Harrison and Barbara E. Kream

Departments of Medicine (L.B.S., H.W.W., B.E.K.) and Orthopaedic Surgery (D.J.A., G.A.G.), School of Medicine, and Orthodontics (J.R.H.), School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030; and Laboratory of Molecular Hypertension (Z.K.), Baker Medical Research Institute, Melbourne 8008, Victoria, Australia

Address all correspondence and requests for reprints to: Barbara E. Kream, Ph.D., Department of Medicine, MC-1850, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06030. E-mail: kream{at}nso1.uchc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoid excess leads to bone loss, primarily by decreasing bone formation. However, a variety of in vitro models show that glucocorticoids can promote osteogenesis. To elucidate the role of endogenous glucocorticoids in bone metabolism, we developed transgenic (TG) mice in which a 2.3-kb Col1a1 promoter fragment drives 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) expression in mature osteoblasts. 11ß-HSD2 should metabolically inactivate endogenous glucocorticoids in the targeted cells, thereby reducing glucocorticoid signaling. The inhibitory effect of 300 nM hydrocortisone on percent collagen synthesis was blunted in TG calvariae, demonstrating that the transgene was active. Collagen synthesis rates were lower in TG calvarial organ cultures compared with wild-type. Trabecular bone parameters measured by microcomputed tomography were reduced in L3 vertebrae, but not femurs, of 7- and 24-wk-old TG females. These changes were also not seen in males. In addition, histomorphometry showed that osteoid surface was increased in TG female vertebrae, suggesting that mineralization may be impaired. Our data demonstrate that endogenous glucocorticoid signaling is required for normal vertebral trabecular bone volume and architecture in female mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CHRONIC, HIGH-DOSE glucocorticoid (GC) treatment leads to bone loss in vivo (1, 2), due primarily to an inhibition of bone formation (3, 4). Reduced proliferation and function of osteoblasts, as well as enhanced osteoblast apoptosis, contribute to the bone loss (5, 6). GCs may also affect osteoclastic bone resorption. However, the effect on resorption is likely indirect (7).

In contrast to their catabolic effects, GCs also promote osteogenesis in vitro under certain experimental conditions. For example, calvarial and marrow stromal cell cultures treated with low concentrations of GCs show increased mineralized nodule size and number (8, 9) and increased expression of osteoblastic markers (10). In addition, collagen synthesis (9) and IGF-I action (11) are enhanced in rat calvarial organ cultures. Although several factors, including the type and concentration of hormone used, as well as the various types of culture conditions employed, complicate the data from in vitro models, they collectively suggest that GCs may play an anabolic role in osteogenesis.

GCs signal through the classical steroid hormone-receptor pathway (12). GCs freely enter the cell where they bind to GC receptors (GRs) in the cytoplasm. Together the hormone-receptor complex translocates to the nucleus where it binds to GC response elements and regulates the transcription of target genes. GCs also bind to the mineralocorticoid receptor (MR) with equal affinity to the GR (13). GR knockout mice have a high degree of perinatal lethality (14). The surviving mice have severe lung, heart, adrenal cortex, and liver complications. Therefore, an alternative approach to the GR knockout mice is required to study the role of GCs in postnatal bone.

The enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) is an NAD+-dependent dehydrogenase that catalyzes the inactivation of cortisol to cortisone (15). 11ß-HSD2 is expressed in several tissues, including the distal nephron of the kidney (16), colon (17), fetal bone (18), osteosarcoma cells (19, 20), sweat glands (21), salivary glands (22), and at low levels in human adult bone (23). In kidney, 11ß-HSD2 protects the MR from activation by GCs (24). 11ß-HSD2 knockout mice have a syndrome similar to humans known as apparent mineralocorticoid excess (25). This syndrome is characterized by severe hypertension due to illicit activation of the MR by GCs.

Our strategy was to overexpress 11ß-HSD2 in osteoblasts of TG mice, thus avoiding potential complications associated with global transgene expression. 11ß-HSD2 should decrease GC signaling upstream of the receptor, thereby blunting the actions of GCs. We previously cloned and characterized Col2.3-HSD2, a construct in which 2.3 kb of the rat Col1a1 promoter was used to drive expression of 11ß-HSD2 in mature osteoblasts (26). ROS 17/2.8 cells transfected with the Col2.3-HSD2 construct showed diminished GC-dependent effects on cell growth, osteoblast mRNA expression, and induction of a mouse mammary tumor virus (MMTV) promoter-reporter construct (26).

In this study, we report the initial characterization of Col2.3-HSD2 TG mice. Three lines of mice were obtained with varying levels of transgene mRNA expression. The transgene was expressed in calvariae, long bone, vertebrae, and tail. In bone, transgene protein expression was restricted to osteoblasts and osteocytes. Ex vivo studies showed TG calvariae had compromised collagen production. Trabecular bone volume (BV) was lower in the vertebrae of 11ß-HSD2 female TG mice. In addition, osteoid surface was increased in TG female vertebrae. The results suggest that endogenous GCs may play a physiological role in the maintenance of bone mass in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation and breeding of Col2.3-HSD2 TG mice
As previously described, the rat 11ß-HSD2 cDNA was cloned downstream of a 2.3-kb fragment of the rat Col1a1 promoter and upstream of the bovine GH polyadenylation sequence to produce Col2.3-HSD2 (Fig. 1AGo) (26). TG founders were developed in the CD-1 outbred background at the University of Connecticut Health Center Transgenic Animal Facility using pronuclear injection. Founder mice were bred to wild-type (WT) CD-1 mice to establish three independent TG lines. Hemizygous males were bred with WT females to produce hemizygous TG and WT littermates. For line 515, adult age-matched WT mice were derived from a timed-mated, WT breeding unit. The Institutional Animal Care and Use Committee at the University of Connecticut Health Center approved all animal protocols.



View larger version (86K):
[in this window]
[in a new window]
  		FIG. 1. Schematic representation of the Col2.3-HSD2 construct and transgene expression. A, Rat 11ß-HSD2 cDNA was cloned downstream of a 2.3-kb fragment of the rat Col1a1 promoter; BGH-PA, bovine GH polyadenylation sequence. The 11ß-HSD2 cDNA probe was generated by PCR with the p5' (forward) and p3' (reverse) primers. B, RNA was extracted from 6- to 8-d-old calvariae and assessed for 11ß-HSD2 and ß-actin expression by Northern blot analysis. Data for three independent founder lines are shown.

 
Genotype analysis
Tail DNA was extracted using reagents from the Wizard genomic DNA purification kit (Promega, Madison, WI) according to the manufacturer’s protocol. Genotyping was performed by PCR of 50–100 ng of tail DNA. The PCR included a 5-min hot start (94 C), followed by 25 cycles (94 C for 30 sec, 65 C for 30 sec, and 72 C for 2 min), and then ending with 72 C for 2 min. The sequence of the forward primer, within a portion of the HSD2 gene, was 5'-ACC TTA GCC CCG TTG TAG-3'. The reverse primer sequence, within the BGH region, was 5'-G AGG GGC AAA GAA GAA CAG ATG-3'.

RNA extraction and Northern blot analysis
Total RNA was extracted from tissues using the TRIZOL reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s protocol. The following tissues were dissected from 6-wk-old TG and WT animals and homogenized in TRIZOL: calvariae, femur, vertebrae, kidney, brain, liver, lung, skin, and tail. Ten micrograms of RNA were separated on a 1% agarose/6% formaldehyde gel and immobilized on a nylon filter. The 11ß-HSD2 transgene probe was made by PCR with the genotyping primers as previously described (26). All cDNA probes were labeled using random primers and [32P]deoxy-GTP (PerkinElmer, Boston, MA). Filters were prehybridized for 2 h and then hybridized for 24 h with 5–6 x 106 cpm/ml of each probe. After hybridization, the filters were washed, exposed to photographic film, and quantified using a phosphoimager. All filters were probed with actin or glyceraldehyde-3-phosphate dehydrogenase for normalization of the signals.

Immunohistochemistry
Vertebrae and femurs from 4-wk-old mice were fixed in 4% paraformaldehyde, decalcified overnight with 5% nitric acid, embedded in paraffin, and cut into 5-µm sections, which were mounted onto charged, precleaned slides. The slides were incubated with xylene and then decreasing (100, 90, 70, and 50%) concentrations of ethanol to remove the paraffin and rehydrate the tissue. Endogenous peroxidases were quenched with 30% H2O2 dissolved in methanol. BSA (0.5%) was used as a blocking buffer. The tissues were unmasked in a solution containing 1 M sodium citrate, 1 M citric acid monohydrate, and H2O2. A polyclonal rabbit antirat 11ß-HSD2 primary antibody (RAH23) was used at 1.13 µg/ml (27) overnight at 4 C in a moist, covered chamber. The slides were rinsed with PBS, incubated with biotinylated antirabbit IgG secondary antibody (1:200) (Vector Laboratories, Burlingame, CA) for 1 h at room temperature in a moist, covered chamber. The slides were rinsed again and incubated with horseradish peroxidase-streptavidin (1:100) (Zymed, South San Francisco, CA) for 45 min. After rinsing, the 3-amino-9-ethyl carbonate (Sigma Chemical Co., St. Louis, MO), dissolved in 0.1 M acetate buffer (pH 5.2) and 3% H2O2, was added as a chromagen. After immunostaining, all slides were counterstained for 5 sec with hematoxylin and mounted with crystal mount (Biomeda Corp., Foster City, CA).

Thin layer chromatography
11ß-HSD2 activity was assessed by measuring the conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone by thin layer chromatography. Calvariae were incubated in serum-free medium containing 5 nM [3H]corticosterone (91 Ci/mmol) and 100 nM corticosterone for 24 h at 37 C. Medium was extracted with 1 ml methylene chloride. After centrifugation for 10 min at room temperature, the organic phase was evaporated. Dried extracts were dissolved in 50 µl acetone, spotted on silica gel plates, and developed in chloroform/acetone (82:18 vol/vol) for 2 h. Appropriate areas of the silica plates were scraped into scintillation vials, each containing 1 ml isopropanol, and radioactivity was quantitated in a liquid scintillation counter.

Organ culture and assay of collagen synthesis
Hemi-calvariae were removed from 6- to 8-d-old animals and cultured separately in 35-mm tissue culture wells for 96 h in {alpha}-MEM supplemented with 100 µg/ml ascorbic acid and 1 mg/ml BSA. One hemi-calvariae from each animal was treated with 300 nM hydrocortisone (diluted in medium at 1:1000 from a stock prepared in 70% ethanol), whereas the other was treated with an equivalent volume of vehicle. The media was changed every 24 h. In the final 2 h of culture, 10 µl of [3H]proline (PerkinElmer) was added to the cultures. After organ culture, the incorporation of [3H]proline into collagen-digestible protein (CDP labeling), noncollagen protein (NCP labeling), and the percent collagen synthesis (PCS) were determined as previously described (28).

Microcomputed tomography
Trabecular morphometry within the metaphyseal region of distal femurs and centrum of the third lumbar vertebrae (L3) from 7- and 24-wk-old mice was quantified using x-ray microcomputed tomography (µCT20, Scanco Medical AG, Bassersdorf, Switzerland). Three-dimensional images were reconstructed using standard convolution back-projection algorithms with Shepp and Logan filtering and rendered at a discrete density of 171,468 voxels/mm3 (isometric 18-µm voxels). Threshold segmentation of bone from marrow and soft tissue was performed in conjunction with a constrained Gaussian filter to reduce noise. Volumetric analysis regions were selected within the endosteal borders to include the central 80% of vertebral height and secondary spongiosa of femoral metaphyses located 760 µm (~5% of length) from the growth plate. Trabecular morphometry was characterized by measuring BV density [(BV/total volume (TV)], connectivity density, bone surface (BS) density (BS/TV), trabecular thickness (TbTh), trabecular number (TbN), and trabecular spacing (TbSp).

Static and dynamic histomorphometry
For static measurements, vertebrae (L3) from 6- and 12-wk-old WT and hemizygous TG females were fixed in 4% paraformaldehyde at 4 C, dehydrated in increasing concentrations of ethanol, cleared in xylene, and embedded in methyl methacrylate. The vertebrae were sectioned sagittally with a Jung polycut microtome (Reichert-Jung, Heidelberg, Germany), and 5-µm-thick sections were deplasticized and stained with modified Masson-Goldner Trichrome (Sigma).

For dynamic measurements, 6-wk-old WT and hemizygous TG females were ip injected (10 mg/kg body weight) with calcein (Sigma). After 5 ds, 90 mg/kg xylenol orange (Sigma) was ip injected. Both the calcein and xylenol orange were dissolved in 2% sodium bicarbonate (pH 7.4). The animals were euthanized 48 h after the xylenol orange injection. Vertebrae were fixed, dehydrated, cleared, embedded, and sectioned as in static histomorphometry. Sections were deplasticized and evaluated by fluorescence microscopy. Additional slides were also evaluated for static parameters. All static and dynamic parameters were measured according to the Report of the American Society of Bone and Mineral Research Histomorphometry Nomenclature Committee (29).

Statistics
All values were expressed as the mean ± SEM. Unpaired t tests were used to determine statistical significance. Comparisons with P < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Three Col2.3-HSD2 TG lines (515, 516 and 529) were established having varying levels of transgene expression (Fig. 1BGo). When breeding the animals, the litter sizes were normal and the transgene was inherited in the expected Mendelian ratio. TG animals were approximately 10% smaller (4.2 ± 0.1 g) than their WT littermates (4.8 ± 0.1 g) at 6–8 d, and in females, this size difference was seen at 24 wk of age.

The transgene was expressed in calvariae, long bone, vertebrae, and tail of TG mice (Fig. 2AGo). Transgene expression was not detected in tissues from WT animals (Fig. 2BGo) or in brain, lung, liver, kidney, or skin of TG animals (Fig. 2AGo). Endogenous 11ß-HSD2 expression was seen in the kidney as a slightly larger band than the transgene. To determine whether there were differences in transgene expression at different sites or between the sexes, we compared transgene mRNA levels using Northern blot analysis. There were no significant differences in transgene expression normalized to GAPDH mRNA, between sexes or among skeletal sites at 6–8 wk of age (n = 5 experiments). Expression was not significantly different in females compared with males in calvariae (0.34 ± 0.15 vs. 0.16 ± 0.03), femurs (0.15 ± 0.07 vs. 0.13 ± 0.04), and vertebrae (0.11 ± 0.05 vs. 0.08 ± 0.02); however, expression tended to be higher in females than in males, and higher in calvariae than in femurs or vertebrae.



View larger version (58K):
[in this window]
[in a new window]
  		FIG. 2. Expression of 11ß-HSD2 in various tissues of TG and WT mice and transgene protein expression in osteoblasts and osteocytes of cortical and trabecular bone. Total RNA was extracted from tissues of 6-wk-old line 516 TG (A) and WT (B) mice and assayed for 11ß-HSD2 expression by Northern blot analysis. Loading of RNA was assessed by ethidium bromide (EtBr) staining of the gel to visualize the 28S and 18S rRNAs. Endogenous 11ß-HSD2 mRNA, which was slightly larger than transgene mRNA, was detected in kidney of both WT and TG. C, Paraffin-embedded vertebral (L3) sections from female WT (left) and TG (right) mice were immunostained with the RAH23 rabbit antirat 11ß-HSD2 antibody.

 
11ß-HSD2 protein was expressed in vertebrae of TG mice (Fig. 2CGo). Expression was localized to osteoblasts and osteocytes, both in cortical and trabecular bone. Calvariae, femurs, and vertebrae had similar spatial patterns of 11ß-HSD2 protein expression (data not shown). Sections of WT bone had low levels of background staining, as did sections from TG bone incubated without primary antibody (data not shown).

To determine whether TG 11ß-HSD2 had enzymatic activity in vivo, the conversion of [3H]corticosterone to 11-dehydrocorticosterone was measured by thin layer chromatography in 24-h calvarial cultures derived from two Col2.3-HSD2 lines (515 and 516). Conversion in WT calvariae ranged from 20–25%, whereas conversion in TG calvariae was 53% and 83%, respectively, in lines 515 and 516 (Fig. 3AGo).



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 3. The Col2.3-HSD2 transgene was active in bone ex vivo. A, Calvariae from two independent TG lines (515 and 516) were cultured for 24 h in the presence or absence of 105 nM [3H]corticosterone. Conversion to [3H]11-dehydrocorticosterone was measured by thin layer chromatography as described in Materials and Methods. Each value is the mean ± SEM of four to 10 calvariae. B, Calvariae were cultured for 96 h in the presence or absence of 300 nM hydrocortisone and labeled for 2 h with [3H]proline before the end of culture. The PCS was assayed as described in Materials and Methods. The data from a single experiment are shown. This experiment was repeated at least three times. Each value is the mean ± SEM for six WT and six TG calvariae. *, P < 0.005 for significant effect of cortisol.

 
To determine whether the transgene affected GC action in bone, we examined the ability of hydrocortisone to inhibit collagen synthesis in calvarial organ cultures, a well established GC response. In both WT and TG calvariae treated with 300 nM hydrocortisone, there was a significant reduction in the PCS relative to untreated controls. However, the ratio of PCS in hydrocortisone-treated bone to vehicle-treated bone was significantly higher in TG cultures (0.6 ± 0.07 for TG compared with 0.23 ± 0.03 for WT; P < 0.01), indicating that the inhibitory effect of hydrocortisone was blunted in TG calvariae by approximately 60% (Fig. 3BGo). At a lower concentration of hydrocortisone (10 nM; data not shown), the inhibitory effect on PCS was not seen in either WT or TG calvariae. At a higher concentration of hydrocortisone (1000 nM), there was an equivalent inhibition of collagen synthesis in TG and WT calvariae (data not shown). This high concentration of hydrocortisone likely exceeded the metabolic capacity of TG 11ß-HSD2.

In addition to demonstrating that the transgene was functional, the organ culture experiments showed that the 11ß-HSD2 transgene compromised the ability of the calvariae to produce collagen. The labeling of CDP was lower in the TG animals, whereas the labeling of NCP was higher. As a result of these changes in CDP and NCP labeling, TG calvariae had 32% lower PCS relative to WT (Fig. 4Go).



View larger version (8K):
[in this window]
[in a new window]
  		FIG. 4. TG calvariae had lower rates of collagen synthesis rates than WT calvariae. Calvariae were cultured for 96 h and labeled for 2 h with [3H]proline before the end of culture. CDP labeling, NCP labeling, and PCS were measured as described in Materials and Methods. Each value is the mean ± SEM of 37 WT and 28 TG calvariae. *, P < 0.003 compared with WT.

 
The 11ß-HSD2 transgene markedly affected the bone microarchitecture of vertebrae in female mice. There was a significant reduction in the percent BV (BV/TV), connective density, and TbN in female TG vertebrae (line 516) at both 7 and 24 wk of age (Table 1Go). Conversely, TbSp was increased in female TG vertebrae, whereas TbTh was unchanged. In line 515, which had lower TG 11ß-HSD2 expression, BV/TV was also significantly reduced in female vertebrae (data not shown). The lower BV in TG female L3 vertebrae compared with female WT vertebrae was apparent in the microcomputed tomography images shown in Fig. 5Go. The changes in BV and trabecular parameters were not seen in femurs of female TG mice or in vertebrae and femurs of male TG mice at 7 wk. Also, in TG line 515, there was no change in trabecular BV or structural parameters in female TG femurs (data not shown).


View this table:
[in this window]
[in a new window]
  		TABLE 1. BV and trabecular parameters in male and female TG and WT mice

 


View larger version (49K):
[in this window]
[in a new window]
  		FIG. 5. Microcomputed tomography images of vertebrae of WT mice and two TG lines. Representative vertebrae were chosen that had BV equivalent to the mean of their respective groups.

 
To examine the cellular mechanism for the reduction in BV, female vertebrae were analyzed using static and dynamic histomorphometry (Table 2Go). Both 6- and 12-wk-old TG vertebrae showed a decreasing trend in tissue area/total tissue area (TA/TTA) and TbN, similar to the microcomputed tomography. TA/TTA was significantly decreased at 12 wk, and TbSp was significantly increased at 6 wk (Table 2Go). There was a significant increase in osteoid surface in the TG vertebrae at both time points (Fig. 6Go). The amount of double-labeled surface was greatly decreased at 6 wk of age.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Dynamic and static parameters in 6- and 12-wk-old TG and WT female mice

 


View larger version (109K):
[in this window]
[in a new window]
  		FIG. 6. Osteoid surface/BS (arrows) was increased in vertebrae of female TG mice. Sections were evaluated with modified Masson-Goldner Trichrome stain. Representative 12-wk-old vertebrae were chosen that had BV equal to the mean of their respective groups.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Col2.3-HSD2 TG mice were developed to provide insight into the role of endogenous GCs in mature osteoblasts. Three lines of Col2.3-HSD2 mice were obtained that expressed the transgene. Transgene RNA was detected in skeletal tissues (calvariae, femurs, vertebrae, and tail) but not in nonskeletal tissues (brain, lung, liver, skin, and kidney). Protein expression was seen in osteoblasts and osteocytes at each skeletal site. TG calvariae were protected from cortisol-induced reductions in collagen synthesis. Moreover, TG calvariae had lower basal collagen synthesis rates than their WT littermates. Female vertebrae showed a significant reduction in BV and TbN. These results were not seen in femurs or in males. Osteoid surface was increased whereas double-labeled surface was decreased in TG female vertebrae. These data suggest that GC signaling in mature osteoblasts is required to maintain normal levels of bone mass and mineralization in female vertebrae.

Several in vitro studies have shown enhanced osteogenesis with GC treatment. Lower dose and shorter treatment protocols may better mimic the endogenous circadian-regulated pulsatile release of cortisol. Physiological levels of GCs have been shown to increase the number of bone nodules formed from rat calvarial cells (9) and promote the differentiation of human bone marrow stromal cells (30). In addition, in chick periosteal cultures, GCs enhanced alkaline phosphatase activity and proliferation (8). As with many in vitro data, the species, the bone cell type, treatment, and the culture conditions differ among studies. In the aforementioned chick study, the increase in alkaline phosphatase activity depended on the stage of differentiation of the culture. Regardless, many studies support an anabolic effect of GCs.

Treatment of organ cultures with 300 nM GCs resulted in a protective effect in TG calvariae. These findings are in accordance with another TG mouse model in which 11ß-HSD2 is driven by the osteocalcin-promoter fragment (OG-2) (31). In this model, bone mineral density, bone strength, and osteoblast apoptosis are protected in TG mice receiving exogenous GCs. Our model differs in that the rat 2.3-kb Col1a1 promoter is expressed earlier in development than the OG-2 promoter, in a broader population of osteoblasts, and at a stronger level (32).

In human adult bone (23) and in primary osteoblast cultures (19), 11ß-HSD1 is the predominant 11ß-HSD isoform. These studies show that 11ß-HSD1 acts primarily as a reductase responsible for GC activation but also has varying levels of dehydrogenase activity. In the present study, WT mice exhibited 20–25% dehydrogenase activity. To determine the source of this baseline activity, we examined endogenous 11ß-HSD1 and 11ß-HSD2 mRNA levels by conventional RT-PCR. We detected 11ß-HSD1 mRNA in both WT and TG calvariae at approximately equal levels, but there was virtually no detectable endogenous 11ß-HSD2 expression in WT or TG animals (data not shown). These data suggest that the baseline dehydrogenase activity in calvariae is due to 11ß-HSD1.

In the 11ß-HSD2 model, females exhibited a clear bone phenotype. A potential explanation for the possible sex-specific effects seen in our study is that GCs may interact with hormone pathways including sex hormones, steroid metabolizing enzymes, or downstream signaling mediators. In addition to traditional endocrine hormones, sex steroid metabolizing enzymes are expressed in the rat tibial growth plate, a source for local sex-steroid formation (33). The aromatase enzyme is an interesting candidate because it converts androgen to estrogen in the periphery of both males and females. In addition, GCs have been shown to regulate the aromatase gene promoter in adipose tissue (34). If aromatase was involved in the 11ß-HSD2 model, one may expect males to be more severely affected, because peripheral conversion by aromatase is the primary source of estrogen in males. However, female aromatase knockout mice had lower bone mineral density and BV and enhanced bone turnover compared with WT females (35, 36), suggesting a possible role for aromatase in females.

Other TG models have been shown to have sex- or site-specific phenotypes involving the IGF-I pathway. For instance, female heterozygous IGF-I receptor null mice have a prolonged lifespan and a greater resistance to oxidative stress than males (37). The authors proposed that the sexual dimorphism was due to differences in tissue sex hormone levels together with sex-specific regulation of GH and IGF-I signaling. In addition, IGF-I and IGF binding protein-4 and -6 mRNA levels are higher in vertebrae-compared with femur-derived rat marrow stromal cell cultures (38, 39).

TG calvarial collagen synthesis rates and trabecular BV were approximately 20% lower than WT animals. The magnitude of these results supports a permissive, regulatory role for GCs in bone formation or remodeling. Moreover, these data are in accordance with calvarial models in which brief treatment with a low concentration of GCs stimulates collagen synthesis (40). IGF-I may be a potential downstream target for the endogenous GCs on bone cells in these animals. GCs enhance the actions of IGF-I in calvarial organ cultures (11). The addition of IGF-binding protein-2 to calvarial cultures to sequester IGFs lowered collagen synthesis rates in both vehicle- and GC-treated cultures to the same level (41).

Calvariae from males and females were pooled for the assay of collagen synthesis before sexual maturation of the animals. Interestingly, the collagen synthesis data and the size difference at 6–8 d suggest that cortisol may be important in prenatal bone formation. Because 11ß-HSD2, GR, and MR are expressed in fetal bone cells (18), GCs may play a role in bone development. Additional studies are necessary to determine the relative role of GCs in bone in fetal and adult mice.

TbTh was unchanged in Col2.3-HSD2 animals. This differs from GC-induced osteoporosis models in which TbTh is reduced (1). In GC-treated osteoblast cell cultures, osteoclast formation (42) and activity (43) are increased. In addition, RANK ligand and osteoprotegerin expression are increased and decreased, respectively (42). If endogenous GCs play a role in the expression of these cytokines, we would have predicted a decrease in osteoclast formation and bone resorption in Col2.3-HSD2 mice, perhaps resulting in an increase in TbTh. The unchanged TbTh in Col2.3-HSD2 TG mice may reflect the absence of an effect on osteoclast formation and activity.

Female Col2.3-HSD2 TG vertebrae had considerably more osteoid than WT vertebrae at 6 and 12 wk. Double-labeled surface/trabecular BS was significantly lower in the TGs. Although we did not see a statistically significant difference in mineral apposition rate or bone formation rate, the decrease in double-labeled surface coupled with the increase in osteoid surface raises the possibility that mineralization may be affected in these mice. There are several candidate genes that may be responsible for the altered mineralization process in these animals. Phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) is a GC-responsive gene (44) that is expressed in mature osteoblasts and osteocytes (45). Loss of function mutations of the PHEX gene in mice (46) and humans (47) leads to X-linked hypophosphatemic rickets (XLH), a phosphate homeostasis disorder, in which there is an impairment of bone mineralization (48). Another possible candidate is osteocalcin, which is a GC-regulated gene in vivo (49, 50). Although osteocalcin knockout mice have been reported to be normal in mineral content (51), further analysis showed that they have an impairment of mineral maturation (52). Finally, IGF-I is another potential candidate, because mice with a complete knockout of the Igf1 gene (53) or with an osteoblast-specific knockout of the IGF-I receptor (54) have delayed or impaired mineralization. Interestingly, the anabolic response of GCs in fetal rat calvarial organ cultures requires the IGF pathway (41).

The Col2.3-HSD2 TG model demonstrates that disruption of GC signaling selectively in mature osteoblasts leads to a reduction in vertebral BV in vivo, supporting an anabolic role for the endogenous GCs in bone metabolism. Collectively, these data suggest there are site- and may be sex-specific differences in the skeletal responses to GC signaling. We speculate that disruption of GC signaling in vivo impairs osteoblast differentiation and/or function, including mineralization. Additional studies are underway to address the cellular mechanism in this model.


   Footnotes
 
This work was supported by Grant P01 AR38933 to B.E.K. from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS). L.B.S. received support from the institutional Skeletal, Craniofacial, and Oral Biology training Grant 5T32 DE07302 from the National Institute of Dental and Craniofacial Research. H.W.W. received support from the Deutsche Forschungsgemeinschaft Grant Wo 729/1-1. We also acknowledge support of the Core Center for Musculoskeletal Disorders Grant P30 AR46026 from NIAMS and the University of Connecticut Health Center Microcomputed Tomography facility.

Abbreviations: BS, Bone surface; BV, bone volume; CDP, collagen-digestible protein; GC, glucocorticoid; GR,GC receptor; HSD, hydroxysteroid dehydrogenase; MR, mineralocorticoid receptor; NCP, noncollagen protein; PCS, percent collagen synthesis; TA/TTA, tissue area/total tissue area; TbN, trabecular number; TbSp, trabecular spacing; TbTh, trabecular thickness; TG, transgenic; TV, total volume; WT, wild-type.

Received May 28, 2003.

Accepted for publication November 3, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Dempster DW 1989 Bone histomorphometry in glucocorticoid-induced osteoporosis. J Bone Miner Res 4:137–141[Medline]
  2. Advani S, LaFrancis D, Bogdanovic E, Taxel P, Raisz LG, Kream BE 1997 Dexamethasone suppresses in vivo levels of bone collagen synthesis in neonatal mice. Bone 20:41–46[Medline]
  3. Canalis E, Giustina A 2001 Glucocorticoid-induced osteoporosis: summary of a workshop. J Clin Endocrinol Metab 86:5681–5685[Free Full Text]
  4. Canalis E, Delany AM 2002 Mechanisms of glucocorticoid action in bone. Ann NY Acad Sci 966:73–81[Abstract/Free Full Text]
  5. Gohel AR, McCarthy MB, Gronowicz, G 1999 Estrogen prevents glucocorticoid-induced apoptosis in osteoblasts in vivo and in vitro. Endocrinology 140:5339–5347[Abstract/Free Full Text]
  6. Weinstein RS, Jilka RL, Parfitt AM, Manolagas SC 1998 Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone. J Clin Invest 102:274–282[Medline]
  7. Hofbauer LC, Heufelder AE 2001 Role of receptor activator of nuclear factor-{kappa}B ligand and osteoprotegerin in bone cell biology. J Mol Med 79:243–253[CrossRef][Medline]
  8. Tenenbaum HC, Heersche JN 1985 Dexamethasone stimulates osteogenesis in chick periosteum in vitro. Endocrinology 117:2211–2217[Abstract]
  9. Bellows CG, Aubin JE, Heersche JN 1987 Physiological concentrations of glucocorticoids stimulate formation of bone nodules from isolated rat calvaria cells in vitro. Endocrinology 121:1985–1992[Abstract]
  10. Rickard DJ, Sullivan TA, Shenker BJ, Leboy PS, Kazhdan I 1994 Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2. Dev Biol 161:218–228[CrossRef][Medline]
  11. Kream BE, Petersen DN, Raisz LG 1990 Cortisol enhances the anabolic effects of insulin-like growth factor I on collagen synthesis and procollagen messenger ribonucleic acid levels in cultured 21-day fetal rat calvariae. Endocrinology 126:1576–1583[Abstract]
  12. Cato AC, Nestl A, Mink S 2002 Rapid actions of steroid receptors in cellular signaling pathways. Sci STKE 2002:RE9
  13. Funder JW 2000 Aldosterone and mineralocorticoid receptors: orphan questions. Kidney Int 57:1358–1363[CrossRef][Medline]
  14. Cole TJ, Blendy JA, Monaghan AP, Krieglstein K, Schmid W, Aguzzi A, Fantuzzi G, Hummler E, Unsicker K, Schutz G 1995 Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation. Genes Dev 9:1608–1621[Abstract/Free Full Text]
  15. Stewart PM, Krozowski ZS 1999 11ß-Hydroxysteroid dehydrogenase. Vitam Horm 57:249–324[Medline]
  16. Mercer WR, Krozowski ZS 1992 Localization of an 11ß-hydroxysteroid dehydrogenase activity to the distal nephron: evidence for the existence of two species of dehydrogenase in the rat kidney. Endocrinology 130:540–543[Abstract]
  17. Stewart PM, Whorwood CB, Mason JI 1995 Type 2 11ß-hydroxysteroid dehydrogenase in foetal and adult life. J Steroid Biochem Mol Biol 55:465–471[CrossRef][Medline]
  18. Condon J, Gosden C, Gardener D, Nickson P, Hewison M, Howie AJ, Stewart PM 1998 Expression of type 2 11ß-hydroxysteroid dehydrogenase and corticosteroid hormone receptors in early human fetal life. J Clin Endocrinol Metab 83:4490–4497[Abstract/Free Full Text]
  19. Bland R, Worker CA, Noble BS, Eyre LJ, Bujalska IJ, Sheppard MC, Stewart PM, Hewison M 1999 Characterization of 11ß-hydroxysteroid dehydrogenase activity and corticosteroid receptor expression in human osteosarcoma cell lines. J Endocrinol 161:455–464[Abstract]
  20. Eyre LJ, Rabbitt EH, Bland R, Hughes SV, Cooper MS, Sheppard MC, Stewart PM, Hewison M 2001 Expression of 11ß-hydroxysteroid dehydrogenase in rat osteoblastic cells: pre-receptor regulation of glucocorticoid responses in bone. J Cell Biochem 81:453–462[CrossRef][Medline]
  21. Smith RE, Maguire JA, Stein-Oakley AN, Sasano H, Takahashi K, Fukushima K, Krozowski ZS 1996 Localization of 11ß-hydroxysteroid dehydrogenase type II in human epithelial tissues. J Clin Endocrinol Metab 81:3244–3248[Abstract]
  22. Roland BL, Funder JW 1996 Localization of 11ß-hydroxysteroid dehydrogenase type 2 in rat tissues: in situ studies. Endocrinology 137:1123–1128[Abstract]
  23. Cooper MS, Walker EA, Bland R, Fraser WD, Hewison M, Stewart PM 2000 Expression and functional consequences of 11ß-hydroxysteroid dehydrogenase activity in human bone. Bone 27:375–381[Medline]
  24. Brown RW, Diaz R, Robson AC, Kotelevtsev YV, Mullins JJ, Kaufman MH, Seckl JR 1996 The ontogeny of 11ß-hydroxysteroid dehydrogenase type 2 and mineralocorticoid receptor gene expression reveal intricate control of glucocorticoid action in development. Endocrinology 137:794–797[Abstract]
  25. Kotelevtsev Y, Brown RW, Fleming S, Kenyon C, Edwards CR, Seckl JR, Mullins JJ 1999 Hypertension in mice lacking 11ß-hydroxysteroid dehydrogenase type 2. J Clin Invest 103:683–689[Medline]
  26. Woitge H, Harrison J, Ivkosic A, Krozowski Z, Kream B 2001 Cloning and in vitro characterization of {alpha}1(I)-collagen 11ß-hydroxysteroid dehydrogenase type 2 transgenes as models for osteoblast-selective inactivation of natural glucocorticoids. Endocrinology 142:1341–1348[Abstract/Free Full Text]
  27. Smith RE, Li KX, Andrews RK, Krozowski Z 1997 Immunohistochemical and molecular characterization of the rat 11ß-hydroxysteroid dehydrogenase type II enzyme. Endocrinology 138:540–547[Abstract/Free Full Text]
  28. Kream BE, Smith MD, Canalis E, Raisz LG 1985 Characterization of the effect of insulin on collagen synthesis in fetal rat bone. Endocrinology 116:296–302[Abstract]
  29. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR 1987 Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 2:595–610[Medline]
  30. Cheng SL, Yang JW, Rifas L, Zhang SF, Avioli LV 1994 Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone. Endocrinology 134:277–286[Abstract]
  31. Weinstein RS, O’Brien CA, Crawford JA, Manolagas SC 2002 Osteoblast specific expression of 11ß-HSD2 in transgenic mice abrogates steroid-induced apoptosis and attenuates loss of BMD and strength. Program of the 24th Meeting of the American Society of Bone and Mineral Research, San Antonio, TX, p S131 (Abstract 1025)
  32. Kalajzic Z, Liu P, Kalajzic I, Du Z, Braut A, Mina M, Canalis E, Rowe DW 2002 Directing the expression of a green fluorescent protein transgene in differentiated osteoblasts: comparison between rat type I collagen and rat osteocalcin promoters. Bone 31:654–660[Medline]
  33. Van Der Eerden BC, Van De Ven J, Lowik CW, Wit JM, Karperien M 2002 Sex steroid metabolism in the tibial growth plate of the rat. Endocrinology 143:4048–4055[Abstract/Free Full Text]
  34. Zhao Y, Mendelson CR, Simpson ER 1995 Characterization of the sequences of the human CYP19 (aromatase) gene that mediate regulation by glucocorticoids in adipose stromal cells and fetal hepatocytes. Mol Endocrinol 9:340–349[Abstract]
  35. Oz OK, Hirasawa G, Lawson J, Nanu L, Constantinescu A, Antich PP, Mason RP, Tsyganov E, Parkey RW, Zerwekh JE, Simpson ER 2001 Bone phenotype of the aromatase deficient mouse. J Steroid Biochem Mol Biol 79:49–59[CrossRef][Medline]
  36. Oz OK, Zerwekh JE, Fisher C, Graves K, Nanu L, Millsaps R, Simpson ER 2000 Bone has a sexually dimorphic response to aromatase deficiency. J Bone Miner Res 15:507–514[CrossRef][Medline]
  37. Holzenberger M, Dupont J, Ducos B, Leneuve P, Geloen A, Even PC, Cervera P, Le Bouc Y 2003 IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice. Nature 421:182–187[CrossRef][Medline]
  38. Milne M, Quail JM, Baran DT 1998 Dexamethasone stimulates osteogenic differentiation in vertebral and femoral bone marrow cell cultures: comparison of IGF-I gene expression. J Cell Biochem 71:382–391[CrossRef][Medline]
  39. Milne M, Quail JM, Rosen CJ, Baran DT 2001 Insulin-like growth factor binding proteins in femoral and vertebral bone marrow stromal cells: expression and regulation by thyroid hormone and dexamethasone. J Cell Biochem 81:229–240[CrossRef][Medline]
  40. Canalis E 1983 Effect of glucocorticoids on type I collagen synthesis, alkaline phosphatase activity, and deoxyribonucleic acid content in cultured rat calvariae. Endocrinology 112:931–939[Abstract]
  41. Kream BE, Tetradis S, Lafrancis D, Fall PM, Feyen JH, Raisz LG 1997 Modulation of the effects of glucocorticoids on collagen synthesis in fetal rat calvariae by insulin-like growth factor binding protein-2. J Bone Miner Res 12:889–895[CrossRef][Medline]
  42. Hofbauer LC, Gori F, Riggs BL, Lacey DL, Dunstan CR, Spelsberg TC, Khosla S 1999 Stimulation of osteoprotegerin ligand and inhibition of osteoprotegerin production by glucocorticoids in human osteoblastic lineage cells: potential paracrine mechanisms of glucocorticoid-induced osteoporosis. Endocrinology 140:4382–4389[Abstract/Free Full Text]
  43. Hirayama T, Sabokbar A, Athanasou NA 2002 Effect of corticosteroids on human osteoclast formation and activity. J Endocrinol 175:155–163[Abstract]
  44. Hines ER, Collins JF, Jones MD, Serey SH, Ghishan FK 2002 Glucocorticoid regulation of the murine PHEX gene. Am J Physiol Renal Physiol 283:F356–F363
  45. Ruchon AF, Tenenhouse HS, Marcinkiewicz M, Siegfried G, Aubin JE, DesGroseillers L, Crine P, Boileau G 2000 Developmental expression and tissue distribution of Phex protein: effect of the Hyp mutation and relationship to bone markers. J Bone Miner Res 15:1440–1450[CrossRef][Medline]
  46. Eicher EM, Southard JL, Scriver CR, Glorieux FH 1976 Hypophosphatemia: mouse model for human familial hypophosphatemic (vitamin D-resistant) rickets. Proc Natl Acad Sci USA 73:4667–4671[Abstract/Free Full Text]
  47. Scriver CR, Tenenhouse HS 1992 X-linked hypophosphataemia: a homologous phenotype in humans and mice with unusual organ-specific gene dosage. J Inherit Metab Dis 15:610–624[CrossRef][Medline]
  48. Xiao ZS, Crenshaw M, Guo R, Nesbitt T, Drezner MK, Quarles LD 1998 Intrinsic mineralization defect in Hyp mouse osteoblasts. Am J Physiol 275:E700–E708
  49. Peretz A, Praet JP, Bosson D, Rozenberg S, Bourdoux P 1989 Serum osteocalcin in the assessment of corticosteroid induced osteoporosis. Effect of long and short term corticosteroid treatment. J Rheumatol 16:363–367[Medline]
  50. Cooper MS, Blumsohn A, Goddard PE, Bartlett WA, Shackleton CH, Eastell R, Hewison M, Stewart PM 2003 11ß-Hydroxysteroid dehydrogenase type 1 activity predicts the effects of glucocorticoids on bone. J Clin Endocrinol Metab 88:3874–3877[Abstract/Free Full Text]
  51. Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C, Smith E, Bonadio J, Goldstein S, Gundberg C, Bradley A, Karsenty G 1996 Increased bone formation in osteocalcin-deficient mice. Nature 382:448–452[CrossRef][Medline]
  52. Boskey AL, Gadaleta S, Gundberg C, Doty SB, Ducy P, Karsenty G 1998 Fourier transform infrared microspectroscopic analysis of bones of osteocalcin-deficient mice provides insight into the function of osteocalcin. Bone 23: 187–196
  53. Baker J, Liu JP, Robertson EJ, Efstratiadis A 1993 Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75:73–82[CrossRef][Medline]
  54. Zhang M, Xuan S, Bouxsein ML, von Stechow D, Akeno N, Faugere MC, Malluche H, Zhao G, Rosen CJ, Efstratiadis A, Clemens TL 2002 Osteoblast-specific knockout of the insulin-like growth factor (IGF) receptor gene reveals an essential role of IGF signaling in bone matrix mineralization. J Biol Chem 277:44005–44012[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
F.-S. Wang, J.-Y. Ko, D.-W. Yeh, H.-C. Ke, and H.-L. Wu
Modulation of Dickkopf-1 Attenuates Glucocorticoid Induction of Osteoblast Apoptosis, Adipocytic Differentiation, and Bone Mass Loss
Endocrinology, April 1, 2008; 149(4): 1793 - 1801.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Zhou, W. Mak, Y. Zheng, C. R. Dunstan, and M. J. Seibel
Osteoblasts Directly Control Lineage Commitment of Mesenchymal Progenitor Cells through Wnt Signaling
J. Biol. Chem., January 25, 2008; 283(4): 1936 - 1945.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
A. Derfoul, G. L. Perkins, D. J. Hall, and R. S. Tuan
Glucocorticoids Promote Chondrogenic Differentiation of Adult Human Mesenchymal Stem Cells by Enhancing Expression of Cartilage Extracellular Matrix Genes
Stem Cells, June 1, 2006; 24(6): 1487 - 1495.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
J. E. Phillips, C. A. Gersbach, A. M. Wojtowicz, and A. J. Garcia
Glucocorticoid-induced osteogenesis is negatively regulated by Runx2/Cbfa1 serine phosphorylation
J. Cell Sci., February 1, 2006; 119(3): 581 - 591.
[Abstract] [Full Text] [PDF]

Home page
 IBMS BoneKEyHome page
R. S. Weinstein
11{beta}-HSD: Guardian or Gate Crasher?
IBMS BoneKEy, September 1, 2005; 2(9): 6 - 13.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
J. M. Paterson, J. R. Seckl, and J. J. Mullins
Genetic manipulation of 11{beta}-hydroxysteroid dehydrogenases in mice
Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2005; 289(3): R642 - R652.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
F.-S. Wang, C.-L. Lin, Y.-J. Chen, C.-J. Wang, K. D. Yang, Y.-T. Huang, Y.-C. Sun, and H.-C. Huang
Secreted Frizzled-Related Protein 1 Modulates Glucocorticoid Attenuation of Osteogenic Activities and Bone Mass
Endocrinology, May 1, 2005; 146(5): 2415 - 2423.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
M. Eijken, M. Hewison, M. S. Cooper, F. H. de Jong, H. Chiba, P. M. Stewart, A. G. Uitterlinden, H. A. P. Pols, and J. P. T. M. van Leeuwen
11{beta}-Hydroxysteroid Dehydrogenase Expression and Glucocorticoid Synthesis Are Directed by a Molecular Switch during Osteoblast Differentiation
Mol. Endocrinol., March 1, 2005; 19(3): 621 - 631.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
C. A. O'Brien, D. Jia, L. I. Plotkin, T. Bellido, C. C. Powers, S. A. Stewart, S. C. Manolagas, and R. S. Weinstein
Glucocorticoids Act Directly on Osteoblasts and Osteocytes to Induce Their Apoptosis and Reduce Bone Formation and Strength
Endocrinology, April 1, 2004; 145(4): 1835 - 1841.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/2/922    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sher, L. B.
Right arrow Articles by Kream, B. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sher, L. B.
Right arrow Articles by Kream, B. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent P