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University Department of Endocrinology and Metabolism, University Hospital of Aarhus (J.J., K.S., M.K.), and Department of Cell Biology, Institute of Anatomy, University of Aarhus (L.M.), DK-8000 Aarhus, Denmark; University Hospital of Odense (M.K.), DK-5000 Odense, Denmark; Molecular Medicine Center, Western General Hospital (M.H., J.J.M., J.R.S.), Edinburgh EH4 2XU, Scotland, United Kingdom; and Novartis Pharma AG (J.G.), CH-4057 Basel, Switzerland
Address all correspondence and requests for reprints to: Dr. Moustapha Kassem, University Department of Endocrinology, University Hospital of Odense, DK-5000 Odense, Denmark. E-mail: mkassem{at}dadlnet.dk.
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Introduction |
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B ligand) (7). At the tissue level, GC action is determined by the availability of ligand and its binding proteins (corticosteroid-binding globulin) and the tissue density of intracellular GC receptors. It is also increasingly recognized that local GC activity is regulated by the two isozymes of 11ß-hydroxysteroid dehydrogenase (11ßHSD) (8, 9), which interconvert active GC (cortisol in humans, corticosterone in mice and rats) with inert 11-keto forms [cortisone and 11-dehydrocorticosterone (11-DHC)]. The type 2 isozyme (11ßHSD2) acts as a high affinity, NAD-dependent dehydrogenase, rapidly converting active GC to inactive forms. 11ßHSD2 is expressed highly in only a few tissues, most abundantly in aldosterone target organs, e.g. kidneys, where it protects intrinsically nonselective mineralocorticoid receptors from occupation by cortisol or corticosterone (10). 11ßHSD1, in contrast, is widely expressed in GC target organs, generally correlating with high expression of glucocorticoid receptor in tissues such as liver, brain, and adipose (11, 12). In most intact cells and organs, 11ßHSD1 acts primarily as an NADPH-dependent 11ß-reductase, converting inactive cortisone (11-DHC) to active cortisol (corticosterone) (12, 13). Thus, 11ßHSD1 has been proposed to amplify local GC action, an idea supported by the reduced tissue GC levels and insulin-sensitized phenotype of 11ßHSD1-deficient mice (14, 15, 16) and the local, not systemic, GC excess-associated insulin-resistance/metabolic syndrome of transgenic mice overexpressing 11ßHSD1 in adipose tissue (17).
Bone cells express both 11ßHSD1 and 11ßHSD2. 11ßHSD1 is the predominant isoform in human adult bone and normal OB, whereas 11ßHSD2 is expressed by human and murine osteosarcoma cell lines (18, 19, 20). Some recent studies have suggested that 11ßHSD1 plays an important role in regulating GC action in bone. Overexpression of 11ßHSD1 in ROS 17.2 osteosarcoma cells increased cell differentiation and inhibited their proliferation (21). In addition, 11ßHSD1 expression is regulated by cytokines known to affect bone turnover, e.g. IL-1 and TNF
(22, 23), and the activity of 11ßHSD1 in cultured OB is positively correlated with donor age (24).
Thus, the aim of our study was to examine the physiological role of 11ßHSD1 in bone in vivo by studying the phenotype of the skeleton and bone cells of mice deficient for 11ßHSD1 (HSD1-/-) in comparison with wild-type (Wt) animals.
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Materials and Methods |
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Bone mass measurements
Bone mineral measurements of femur, tibiae, and spine were determined using dual energy x-ray absorptiometry (DEXA) scanning by PIXI-mouse (Lunar Corp., Copenhagen, Denmark). Total, cortical, and trabecular bone mass and geometry were determined using an XCT-Research SA+ (Stratec-Norland, Pforzheim, Germany) fitted with a 0.5-mm collimator (25). The following set-up was chosen for the measurements: voxel size, 0.1 x 0.1 x 0.5 mm; scan speed: scout view, 10 mm/sec; final scan, 2 mm/sec; one block; contour mode 1; peel mode 2; cortical threshold, 280 mg/cm3; and inner threshold, 280 mg/cm3. A slice located 3 mm distal from the intercondylar tubercle in the proximal tibia metaphysis was analyzed.
Histomorphometry
Embedding of bones for histomorphometry.
Tibia and two thoracic vertebrae were embedded in methylmetacrylate (Technovit 9100, Heraeus Kulzer, Wehrheim/Ts., Germany) and cut into 4-µm vertical sections for staining with von Kossa, 10-µm sections for staining with toluidine blue, and 20-µm sections that were left unstained on a Jung model K microtome (R. Jung GmbH & Co., Dierdorf-Elgert, Germany). von Kossa and toluidine blue staining was performed according to standard procedures.
Bone marrow composition.
Bone marrow composition was determined as described previously (26). For each animal, six sections were counted as well as the whole tibia, using 1–10 field of visions depending on the size of the bone and the magnification (x100 for bone volume and x400 for the remaining parameters). The first field of vision was placed just below the primary spongiosa. The point counting was performed by taking photographs of each field (digital camera, DP11, Olympus Denmark A/S, Ballerup, Denmark) and capturing on a personal computer. A custom-made software program was used for the point-counting procedure (10 x 8 points in each field). The coefficient of variation was 0.5% for bone volume per total volume (BV/TV), 0.3% for adipose tissue volume per TV, and 7.8% for sinusoid volume per TV.
Trabecular bone resorption and formation surfaces.
The counting was performed using a computer-automated line grid that was randomly placed on the field of vision and rotated 90° for a second counting of the same field of vision. Intersections between the bone surface and the lines were determined as resorptive [osteoclast surface per bone surface (OS)], formative (OB surface per BS), or resting. For each animal, six sections were counted, and six fields of vision were randomly placed in each section. A magnification of x400 was used.
Trabecular bone structure parameters.
Vertebrae and tibiae stained with von Kossa were used for calculation of structural parameters: BV/TV (percentage), BS/TV (per millimeter), trabecular thickness (micrometers), trabecular number (per millimeter), trabecular surface (millimeters), trabecular bone pattern factor (per millimeter). The study was performed using modified QUANTIMET 600 software (Leica, Cambridge, UK), taking advantage of the optical difference between the black bone and the uncolored background. Calculations of the structural parameters were performed according to the recommendations of Parfitt et al. (27). For both tibiae and vertebrae, three or four sections were stained and counted, each with one to four fields of vision. The coefficients of variation for the measured parameters were: bone area, 0.4%; bone perimeter, 18%; and trabecular area, 1.7%.
Dynamic histomorphometry.
An Axiophot photomicroscope (Zeiss, Oberkochen, Germany) linked to a camera (CF 15/4 MC, Kappa, Gleichen, Germany) and a QUANTIMET 600 image analysis system was used to calculate the amount of single-labeled surface per BS (percentage), double-labeled surface per BS (percentage), mineralized surface per BS (percentage), corrected mineral apposition rate (micrometers per day), bone formation rate (BFR) per BS (micrometers per day), and double-labeled bone formation rate per BS (micrometers per day). The calculations of the dynamic parameters were performed according to the recommendations of Parfitt et al. (27). For the vertebrae, two to four sections were counted, each with three to 12 fields of vision.
Cell culture
Bone marrow stromal cells (MSC).
One femur from each mouse was flushed with 10% fetal calf serum (FCS; Gibco/BRL, Copenhagen, Denmark) in MEM (Invitrogen, Copenhagen, Denmark) and 1% penicillin/streptomycin (Invitrogen) into 1.5-ml tubes. The next day, 2 ml 10% FCS and 1% penicillin/streptomycin in MEM were added, and the cells were seeded in slide flasks (10 cm2) without further purification. At confluence of the first passage, MSC from all HSD1-/- mice (n = 5) and Wt mice (n = 6) were pooled and used for further experiments.
Trabecular OB.
Cell cultures of OB were established from femur and skull, similar to the procedure described by Kassem et al. (28). At confluence of the first passage, OB from femur and skull from both HSD1-/- (n = 6) and Wt mice (n = 6) were pooled and used for subsequent experiments.
Examining the phenotype of the animals
RNA and RT-PCR.
RNA was isolated, and cDNA and PCRs were performed as described previously (29). The primer sequences used were: 11ßHSD1, 5'-GTCCCTGTTTGATGGCAGTTATG-3' (sense) and 5'-GTAGGGAGCAATCATAGG-3' (antisense); and 11ßHSD2, 5'-CTGGCCACAGTG TTGGATTTG-3' (sense) and 5'-TCACTGCAGCTGTCTTGGAGC-3' (antisense). The primer sequences for glyceraldehyde-3-phosphate dehydrogenase were previously reported (29). PCR products were separated in a 3% agarose gel (Cambrex Bioscience Copenhagen, ApS, Copenhagen, Denmark) and visualized by ethidium bromide staining. RNA extracted from mouse liver was used as a positive control for 11ßHSD1, and RNA extracted from human placenta was used as a positive control for 11ßHSD2.
11ßHSD reductase assay.
Cells were seeded into six-well plates (1500 cells/cm2). At confluence, the cells were incubated for 2 h with carbenoxolone (CBX; 10-4 M; Sigma-Aldrich Denmark A/S) before adding a new medium containing 2 nM [3H]11-DHC and 23 nM 11-DHC. CBX was once again added to the same wells. Half the medium was removed after 3 h, and the other half was removed after 6 h. GCs were extracted by ethylacetate, evaporated, and analyzed by thin-layer chromatography (TLC). 11-DHC (5 mg/ml) and corticosterone (5 mg/ml) were added to each sample in 60 µl absolute ethanol, and TLC was performed in duplicate. The TLC used chloroform/ethanol (92:8), and the activities of the dehydrogenase and reductase components of 11ßHSD were estimated from the radioactivity of each fraction. In a parallel experiment, an x-ray film (Fuji-Film, Santax A/S, Aarhus, Denmark) was exposed on a TLC plate for 2 months for visualization of the bands.
[3H]11-DHC was synthesized from [3H]corticosterone (Amersham Biosciences, Copenhagen, Denmark), similar to the procedure described by Low et al. (13). The sample was exposed to TLC to check the purity.
Cell proliferation
Long-term growth curves.
At confluence, the number of cells was counted using a Bürkner-Türk counting chamber, and a number of cells was seeded. The number of population doublings (PD) was calculated using the following formula: PD = (ln 
N/ln 2), where N is the resulting number of cells divided by the initial number.
Short-term growth curves.
Two methods were employed to determine cell growth.
Methylene blue assay. Third passage OBs were seeded with 2000 or 1000 cells/well in 96-well plates. After 5 h and 1, 2, 3, 5, 7, 10, 13, 15, 17, 20, 23, 27, 31, or 34 d, the cells were stained with methylene blue. Medium was aspirated, and cells were fixed in methanol at room temperature for 30 min and stained with methylene blue for 30 min at room temperature. The stain was eluted and quantitated by spectrophotometry at 650 nm and reference 405 nm. Cell number was determined from a standard curve (28, 30).
Cell counting using hemocytometer. Second passage OBs were seeded at a concentration of 10,000 cells/ml in 24-well plates and cultured for 7 h and 1, 2, 3, 4, 5, 7, 9, 11, 14, 16, 18, 21, 23, 25, or 28 d. The cells were trypsinized using trypsin-EDTA (Invitrogen; 0.5 ml) for 8 min and counted using a Bürkner-Türk counting chamber. Triplicate wells were counted at each time point.
Cell differentiation assays
Staining for alkaline phosphatase (AP).
First passage cells grown from skull or femur were seeded in eight-well chamber slides (15,000 cells/cm2) and grown to confluence. In addition, cells at later passages were seeded at densities of 5,000 and 10,000 cells/cm2 and stained for AP after 1 and 2 wk. The cultures were stained using a procedure similar to that described previously by Justesen et al. (29). The staining intensity of the cells was compared between Wt and HSD1-/-cells.
Staining for osteocalcin (OC).
First passage cells grown from skull or femur were seeded in eight-well slides (15,000 cells/cm2) and grown to confluence. The cells were fixed and incubated overnight with primary antibody (antihuman OC antibody; Biomedical Technologies, Inc., Stoughton, MA) and then with a biotinylated goat antimouse secondary antibody (DakoCytomation Norden A/S, Copenhagen, Denmark). Peroxidase-conjugated streptavidin (DakoCytomation) and 3-amino-9-ethyl-carbazole were used for detection of immunoreactivity. The cells were counterstained with Mayers hematoxylin and mounted in Glycergel (Dako).
Assay for AP activity.
Cells were seeded in 96-well plates (8000 or 4000 cells/well) from different passages (n = 7) and were incubated either in FCS (10%) or under serum-free conditions. AP activity was analyzed according to protocol supplied by the manufacturer (Sigma-Aldrich Denmark A/S). AP activity was calculated as nanomoles of p-nitrophenol phosphate and corrected for variation in cell number among different wells. The percent change in AP activity in HSD1-/- cells compared with Wt cells was calculated.
In vitro mineralization.
Cells were seeded in six-well plates (1500 cells/cm2), and after 1 wk the medium was replaced by fresh medium containing 290 nM ascorbic acid (Wako Chemicals GmbH, Neuss, Germany) and 5 mM ß-glycerophosphate (Sigma-Aldrich Denmark A/S) or vehicle. The medium was changed once a week, and after 4 wk the cells were stained for Alizarin Red as described previously (31).
In vitro adipocyte differentiation.
Second passage cells obtained from femur and skull were seeded at 3000 cells/cm2 and grown to confluence before treatment with 1) 10% FCS (control); 2) 10% FCS, 10-7 M dexamethasone, 0.5 mM isobutylmethylxanthine (IBMX; Sigma-Aldrich Denmark A/S), 0.2 nM T3 (Sigma-Aldrich Denmark A/S), and 0.1 µM insulin (Sigma-Aldrich Denmark A/S); 3) 10% FCS, 10-7 M corticosterone, 0.5 mM IBMX, and 0.06 mM indomethacin; and 4) 15% horse serum (Sigma-Aldrich Denmark A/S), 10-7 M corticosterone, 0.5 mM IBMX, and 0.06 mM indomethacin for Denmark A/S 2–3 wk.
Statistics
Values are presented as the mean ± SEM or the mean ± SD. The difference between groups was tested by t test or rank-sum test according to the distribution of the data. For the AP activity measurements, a sign test was performed.
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Results |
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Effect of age on bone mass and bone size.
Bone mass and bone size were determined in young and old female mice by DEXA (Table 1
) and peripheral quantitative computed tomography (pQCT) (Table 2). DEXA measurements of the femur, tibiae, and spine showed small and subtle differences between HSD1-/- and Wt mice. Femur BMD increased from 0.067 ± 0.001 g/cm2 in young to 0.073 ± 0.002 g/cm2 (P < 0.05) in old Wt mice, and similar changes were observed in bone mineral content (BMC) and bone area. However, these changes were absent from HSD1-/- mice due to increased bone size in young HSD1-/- mice. On the other hand, tibia BMD exhibited an age-related decrease in BMD in both Wt and HSD1-/- mice, and this change was associated with an increase in bone size in Wt, but not HSD1-/-, mice (Table 1). pQCT was more sensitive in detecting age-related decreases in total BMC, total BMD, cortical BMC, cortical thickness, and trabecular BMD in both Wt and HSD1-/- mice. However, no differences were observed in any of these parameters between the Wt type and HSD1-/- mice (Table 2).
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) and pQCT (Table 2). As expected, several differences were observed between the old male and female skeletons, but no consistent interaction was observed between gender and 11ßHSD1 phenotype.
Bone histomorphometry.
More extensive histomorphometric studies were performed in young male HSD1-/- (n = 6) and young male Wt mice (n = 6). Table 3 shows static histomorphometric parameters from tibia and vertebrae in HSD1-/- and Wt mice, which were determined in sections stained with toluidine blue and von Kossa (Fig. 1). No difference was found in BV/TV, BS/TV, trabecular number, trabecular surface, and trabecular bone pattern factor. Trabecular thickness was decreased, however, in HSD1-/- compared with Wt mice [32.63 ± 1.53 and 40.17 ± 1.56 µm for tibia(P < 0.01) and 37.36 ± 0.68 and 42.15 ± 1.14 µm for vertebrae (P < 0.01)], but no difference was found in osteoclast surface/BS or OB surface/BS between the groups. Studying the bone marrow composition revealed a total absence of bone marrow adipocytes in HSD1-/- compared with 3.1 ± 1.5% in Wt mice (P < 0.05). However, no difference was found in BV/TV, hemopoietic volume per TV, or sinusoid volume per TV between the two groups (Table 3). Dynamic histomorphometry was performed after double labeling with calcein. As shown in Table 4, no significant difference between HSD1-/- and the Wt mice was detected in any of the parameters related to bone formation rate.
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), mRNA of 11ßHSD2 was detected in HSD1-/- and Wt mice, and as expected, 11ßHSD1 mRNA was only present in cells obtained from Wt mice in both MSCs and OB from the skull and femur. Only the results obtained for MSCs are illustrated in Fig. 2A. The results of gene expression analysis were confirmed by measuring the reductase activity of 11ßHSD1 by TLC and radioactive counting (Fig. 2, B–D). Wt cells had a higher reductase activity in all cells examined compared with cells obtained from HSD1-/- mice. However, there was a difference in the levels of reductase activity between MSC and OB (presented as percent conversion of 11-DHC to corticosterone: MSC, 86 ± 1% vs. 2 ± 1%; femur OB, 11 ± 1% vs. 2 ± 1%; skull OB, 13 ± 2% vs. 0%; Fig. 2B). When the results were pooled, a significant reduction in 11ßHSD1 reductase activity from 37 ± 38% in Wt cells to 1 ± 2% in HSD1-/- cells (P < 0.05) was observed (Fig. 2C). The level of 11ßHSD1 reductase activity in Wt mice was significantly reduced (P < 0.05) when cells were incubated with CBX before the measurement, confirming the involvement of 11ßHSD (Fig. 2D).
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). These results were confirmed using two different cell densities as well as a separate experiment using direct cell counting with a hemocytometer (data not shown).
Osteoblastic differentiation.
To investigate whether 11ßHSD1 deficiency affects the osteoblastic phenotype, cells were stained for the osteoblastic markers OC and AP. OB from femur and skull obtained from HSD1-/- and Wt mice showed positive staining for OC, and both HSD1-/- and Wt cells stained positively for AP. The intensity of staining was similar in OB and MSC in all passages examined regardless of 11ßHSD1 phenotype (an example is shown in Fig. 4). Furthermore, similar levels of AP activity were detected in MSC and OB from HSD1-/- compared with Wt cells (111 ± 28%; P = NS).
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Adipocyte differentiation.
OB and MSC were able to differentiate into adipocytes under adipogenic culture conditions, and no difference in the adipocyte differentiation capacity between HSD1-/- and Wt type cells was observed (an example is shown in Fig. 4).
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Discussion |
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The absence of 11ßHSD1 expression and activity in HSD1-/- mice has been previously extensively studied (15). HSD1 mRNA and reductase activity were completely absent in several tissues in HSD1-/-mice (15). We further extended these findings in both MSC and OB using similar methodology, as mRNA for 11ßHSD1 and its reductase activity was only detectable in Wt, not in HSD1-/-, mice. A minimal level of reductase activity was observed in MSC and OB from HSD1-/-, which probably represents an artifact resulting from the preparation of radioactive 11-DHC. Our results are in agreement with a previous study demonstrating the inability of adrenalectomized HSD1-/-mice to convert 11-DHC to corticosterone in vivo (33).
The presence of 11ßHSD1 mRNA and its reductase activity in bone, as demonstrated in our present study and by previous investigators (18, 19), suggests that 11ßHSD1 plays a role in bone metabolism. However, our results did not reveal any clear skeletal phenotype of HSD1-/- mice. Previous in vitro data have demonstrated that osteoblastic overexpression of 11ßHSD1 led to decreased cell proliferation and increased cell differentiation (20). However, in our experiments 11ßHSD1-/- bone cells did not reveal any change in their proliferation or expression of osteoblastic markers. A recent study by Cooper et al. (24) demonstrated that OB 11ßHSD1 activity increases with donor age and may contribute to the age-related bone loss. Examination of the skeletal phenotype of the aged mice demonstrated a clear age-related decrease in bone mass, but this reduction was similar in Wt and 11ßHSD1-/- mice.
Tissue-specific heterogeneity in 11ßHSD1 activity has been described in 11ßHSD1-/- mice (14). We detected higher levels of 11ßHSD1 activity in MSC than OB, suggesting an important role for 11ßHSD1 in the bone marrow microenvironment. Indeed, all 11ßHSD1-deficient mice examined lacked bone marrow adipocytes. This was not associated with generalized fat atrophy (15, 34), supporting the view that bone marrow and extramedullary adipocyte differentiation processes respond differently to hormonal signals (35, 36). Cultured MSC containing preadipocytic cells from both 11ßHSD1-/- and Wt mice exhibited similar adipocyte formation capacity, suggesting that adipocyte differentiation, not the absence of preadipocytes in the bone marrow, was the cause of absence of bone marrow adipocytes. We employed a standard procedure for inducing adipocyte formation in MSC cultures (29, 37). The adipocyte-inducing medium contained dexamethasone and other adipocyte-inducing agents that may have counteracted the inhibitory effects of absent 11ßHSD1 activity. The role of 11ßHSD1 activity in promoting adipocyte formation in bone marrow is supported by two lines of evidence. First, 11ßHSD1 is highly expressed in preadipocytes during their late stages of differentiation (38), and its activity is regulated by factors known to induce adipocyte differentiation (e.g. thiazolidinediones, insulin, IGF-I, and TNF) (23, 38, 39, 40). Second, increased adipocyte formation in the visceral tissues has been demonstrated in a transgenic mouse with adipocyte-specific overexpression of 11ßHSD1 (17).
The function of bone marrow adipocytes is not known (41, 42). They may depend on the biological context, e.g. as a local energy reservoir for urgent hemopoiesis (blood loss) or osteogenesis (fracture), support hemopoietic cell differentiation or bone cell differentiation, or play a passive role filling space between hemopoietic or trabecular bone elements. HSD1-/- mice do not show any overt disturbances in hematological parameters (Seckl, J., unpublished observations), and the bone marrow cellularity and sinusoids were not affected by the absence of marrow adipocytes. Also, osteoclast differentiation from hemopoietic stem cells seemed normal, as no changes were detected in osteoclastic bone surfaces or bone resorption rates. Thus, the biological consequences of the selective absence of bone marrow adipocytes merits further investigation.
An inverse relationship between adipocyte and OB differentiation in bone marrow has been suggested (43, 44, 45). However, these studies demonstrate an inverse correlation between bone formation and adipocyte formation without proving a causal relationship. In an intervention study we have demonstrated that troglitazone (a peroxisomal proliferator-activated receptor 
agonist) treatment can induce adipogenesis in mouse bone marrow without changing trabecular bone volume (26). Our current study demonstrates the presence of normal bone volume and bone formation rate despite the complete absence of bone marrow adipocytes. In another study by Martin et al. (46) using an ovariectomized rat model, trabecular bone volume decreased after 1 month, but the increase in bone marrow adipocytes was first observed after 3 months, suggesting two independent mechanisms. These results suggest that bone marrow adipogenesis and osteoblastogenesis can be regulated independently.
Although the absence of a clear bone phenotype in 11ßHSD-deficient mice suggests a limited role for 11ßHSD in normal bone physiology, our studies have some limitations. First, the 11ßHSD1-deficient mice exhibit several abnormalities in hypothalamic-pituitary-adrenal axis. For example, the basal level of corticosterone is double that in Wt mice (14), and the HSD1-/- mice respond to stress with increased production of corticosterone compared with Wt mice (14). It is plausible that these changes may compensate for the absence of regeneration of corticosterone within the bone. In addition, it is possible that 11ßHSD1-deficient mice have systemic derangements in other hormone levels, e.g. GH/IGFs or thyroid hormones, that may, in turn, affect bone mass and bone marrow adipocyte formation. Further studies are underway to delineate the extent of the hormonal changes in 11ßHSD1-deficient mice. Second, we detected both 11ßHSD1 and 11ßHSD2 mRNA in mouse bone cells; this is different from what was observed in human OB, where 11ßHSD1 is the predominant form (19). This important species difference may limit extrapolation of the role of 11ßHSD1 in mouse bone to human bone physiology. It is also possible that the protective effects of 11ßHSD2 against high levels of GC in bone are more important for mouse bone homeostasis than the generation of active GC by 11ßHSD1. The down-regulation of 11ßHSD1 expression in OB compared with MSC (which contains progenitor cells) supports this hypothesis. However, further studies are needed to determine the relative contributions of 11ßHSD1 and 11ßHSD2 during bone development and bone turnover. Finally, we studied HSD1-/- mice under basal conditions. Previous studies with 11ßHSD1 mice revealed subtle abnormalities in glucose and lipid metabolism as well as central nervous system responses under stress. It is possible that the role of 11ßHSD1 in bone becomes apparent during stressful conditions, e.g. fracture healing. Confirmation of these hypotheses needs further studies.
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Acknowledgments |
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Footnotes |
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Abbreviations: AP, Alkaline phosphatase; BMC, bone mineral content; BMD, bone mineral density; BS, bone surface; BV, bone volume; CBX, carbenoxolone; DEXA, dual energy x-ray absorptiometry; 11-DHC, 11-dehydrocorticosterone; FCS, fetal calf serum; GC, glucocorticoid; 11ßHSD, 11ß-hydroxysteroid dehydrogenase; IBMX, isobutylmethylxanthine; MSC, bone marrow stromal cell; OB, osteoblast; OC, osteocalcin; PD, population doubling; pQCT, peripheral quantitative computed tomography; TLC, thin-layer chromatography; TV, total volume; Wt, wild-type.
Received October 22, 2003.
Accepted for publication December 26, 2003.
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and interleukin 1ß enhance the cortisone/cortisol shuttle. J Exp Med 186:189–198 ligands inhibit adipocyte 11ß-hydroxysteroid dehydrogenase type 1 expression and activity. J Biol Chem 276:12629–12635This article has been cited by other articles:
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  J. L. W. Yau, K. M. McNair, J. Noble, D. Brownstein, C. Hibberd, N. Morton, J. J. Mullins, R. G. M. Morris, S. Cobb, and J. R. Seckl Enhanced Hippocampal Long-Term Potentiation and Spatial Learning in Aged 11{beta}-Hydroxysteroid Dehydrogenase Type 1 Knock-Out Mice J. Neurosci., September 26, 2007; 27(39): 10487 - 10496. [Abstract] [Full Text] [PDF]
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 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]
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, Wt; 
, HSD1-/-.