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Molecular Endocrinology, doi:10.1210/me.2004-0212
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Molecular Endocrinology 19 (3): 621-631
Copyright © 2005 by The Endocrine Society

11ß-Hydroxysteroid Dehydrogenase Expression and Glucocorticoid Synthesis Are Directed by a Molecular Switch during Osteoblast Differentiation

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

Department of Internal Medicine (M.E., F.H.d.J., A.G.U., H.A.P.P., J.P.T.M.v.L.), Erasmus Medical Centre, 3000 DR Rotterdam, The Netherlands; Division of Medical Science (M.H., M.S.C., P.M.S.), Institute of Biomedical Research, The University of Birmingham, Birmingham B15 2TT, United Kingdom; and Department of Pathology (H.C.), Sapporo Medical College, Sapporo 060-8556, Japan

Address all correspondence and requests for reprints to: Dr. J. P. T. M. van Leeuwen, Erasmus Medical Center, Department Internal Medicine, Room Ee526, P.O Box 1738, 3000 DR, Rotterdam, The Netherlands. E-mail: j.vanleeuwen{at}erasmusmc.nl.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) plays an important role in the prereceptor regulation of corticosteroids by locally converting cortisone into active cortisol. To investigate the impact of this mechanism on osteoblast development, we have characterized 11ß-HSD1 activity and regulation in a differentiating human osteoblast cell line (SV-HFO). Continuous treatment with the synthetic glucocorticoid dexamethasone induces differentiation of SV-HFO cells during 21 d of culture. Using this cell system, we showed an inverse relationship between 11ß-HSD1 activity and osteoblast differentiation. 11ß-HSD1 mRNA expression and activity were low and constant in differentiating osteoblasts. However, in the absence of differentiation (no dexamethasone), 11ß-HSD1 mRNA and activity increased strongly from d 12 of culture onward, with a peak around d 19. Promoter reporter studies provided evidence that specific regions of the 11ß-HSD1 gene are involved in this differentiation controlled regulation of the enzyme. Functional implication of these changes in 11ß-HSD1 is shown by the induction of osteoblast differentiation in the presence of cortisone. The current study demonstrates the presence of an intrinsic differentiation-driven molecular switch that controls expression and activity of 11ß-HSD1 and thereby cortisol production by human osteoblasts. This efficient mechanism by which osteoblasts generate cortisol in an autocrine fashion to ensure proper differentiation will help to understand the complex effects of cortisol on bone metabolism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GLUCOCORTICOIDS EXERT DIVERSE effects on bone metabolism. In human and rat bone marrow stromal cells, glucocorticoids are crucial for the induction of osteoblast differentiation and formation of a mineralized extracellular matrix (1, 2, 3). Paradoxically, glucocorticoids in pharmacological doses cause osteoporosis, mainly by suppressing bone formation but also through effects on bone resorption (4, 5, 6). Overall, the action of glucocorticoids on bone is complex, poorly understood, and is dependent on the duration and concentration of glucocorticoid treatment and the differentiation stage of both osteoblasts and osteoclasts (7, 8, 9, 10).

At a molecular level, glucocorticoid signaling is mediated via the glucocorticoid receptor (GR{alpha}). GR{alpha} is expressed in almost all cell types including osteoblasts (11), in which it regulates gene expression by binding to glucocorticoid responsive elements in the regulatory regions of several target genes, including osteocalcin, collagenI{alpha}1, and transforming growth factor-ß1 (12, 13, 14). Regulation of glucocorticoid responsiveness may occur at several levels, including changes in GR{alpha} expression (15) and at a postreceptor level through variation in GR{alpha} accessory proteins (16). Glucocorticoid action has also been shown to be regulated at prereceptor level by isozymes of 11ß-hydroxysteroid dehydrogenase (11ß-HSD), which catalyze tissue-specific synthesis and metabolism of GR{alpha} ligands (17). 11ß-HSD type 1 (11ß-HSD1) is found in almost all glucocorticoid target tissues and primarily displays reductase activity, converting cortisone into the biologically active cortisol (18, 19). 11ß-HSD2 converts cortisol into inactive cortisone and is found primarily in mineralocorticoid target tissues, in which it protects the nonselective mineralocorticoid receptor from activation by glucocorticoids (20, 21). In bone, both isozymes of 11ß-HSD (mRNA, protein, and enzyme activity) have been demonstrated, with 11ß-HSD1 being the most prominent isozyme in human osteoblasts whereas 11ß-HSD2 is barely detectable (22, 23). Reductase activity of 11ß-HSD1 in osteoblasts provides an efficient mechanism for the local activation of glucocorticoids in bone. As a consequence, the regulation of 11ß-HSD1 activity in osteoblasts is likely to be an important autocrine determinant of osteoblast proliferation, differentiation, and function (24, 25). Previous studies have highlighted the potential importance of osteoblastic 11ß-HSD1 in predicting the detrimental effects of corticosteroids on bone (26, 27), but, as yet, the role of 11ß-HSD1 in normal osteoblast development remains unclear. In studies presented here, we have used a nonneoplastic human osteoblast cell line to study the regulation of 11ß-HSD1 and the specific impact on osteoblast differentiation and mineralization. Data indicate that regulation of 11ß-HSD1 expression and activity are integral features of osteoblast differentiation, providing a potent mechanism for autocrine regulation of bone formation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoids Induce Osteoblast Differentiation and Mineralization
During SV-HFO culture, osteoblast differentiation was monitored by measuring DNA levels, alkaline phosphatase (ALP) activity, and mineralization. In basal untreated cultures, ALP activity remained very low throughout the entire 23-d culture period. In contrast, cultures that were treated continuously with dexamethasone (DEX) showed a dose-dependent increase in ALP activity, which peaked around d 14 (see Figs. 1AGo and 4AGo). In these DEX-treated cultures, mineralization was initiated around d 14 of culture and increased up to d 19, after which it seemed to level off (Fig. 1BGo). In the absence of DEX, cultures showed no evidence of osteoblast differentiation and mineralization (Fig. 1Go, A and B). Both DEX-treated and non-DEX-treated cultures showed a continuous increase in DNA content during culture, although differentiating cultures showed a stronger increase in DNA content (Fig. 1CGo). DEX-induced differentiation and mineralization was completely blocked by the GR antagonist mifepristone (2 µM) (data not shown). Throughout the remainder of the study, DEX-treated (1 µM) and non-DEX-treated cultures were referred as differentiating and nondifferentiating osteoblasts, respectively.



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  		Fig. 1. ALP Activity (A), Mineralization (B), and DNA Content (C) during SV-HFO Culture in DEX-Treated (1 µM) (Differentiating) and Nontreated (Nondifferentiating) Cells

Values shown are the results of one typical SV-HFO culture.

 


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  		Fig. 4. Inverse Relation between DEX-Induced Differentiation and 11ß-HSD1 Activity

SV-HFO cells were cultured for 19 days in the presence of 0, 2, 4, 8, 16, 63, and 250 nM and 1 µM DEX. Subsequently, ALP activity, 11ß-HSD1 activity (A), and mineralization (B) were measured. Values are means ± SEM (n = 4).

 
GR mRNA Expression in SV-HFO Cells
In both differentiating and nondifferentiating cultures, GR{alpha} mRNA could be easily detected and mRNA expression was constant during the entire culture period (d 5–23). However, at all days of culture differentiating osteoblasts showed lower GR{alpha} mRNA levels (0.7 ± 0.1) compared with their nondifferentiating counterparts (Fig. 2Go).



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  		Fig. 2. GR{alpha} mRNA Expression in Differentiating and Nondifferentiating SV-HFO Cells

Results are presented as expression relative to control at wk 1, 2, and 3 of culture. Values are means ± SEM (n ≥ 6). **, P < 0.01 compared with nondifferentiating cultures.

 
11ß-HSD1 mRNA Expression in Differentiating and Nondifferentiating Osteoblasts
At the start of culture (d 5–9), 11ß-HSD1 mRNA was expressed at similar levels in both differentiating and nondifferentiating SV-HFO cultures. However, after d 12, 11ß-HSD1 mRNA expression increased strongly in nondifferentiating osteoblasts. This resulted in an up-regulation of about 2-fold at d 12, increasing to a 15-fold higher expression at d 19 (Fig. 3AGo). By contrast, 11ß-HSD1 mRNA expression in differentiating cultures remained constant during the entire culture period. Induction of differentiation using 10 nM DEX resulted in similar levels of 11ß-HSD1 mRNA when compared with treatment with 1 µM DEX (data not shown). To demonstrate that increased 11ß-HSD1 mRNA expression was not due to differentiation into adipocytes, we tested SV-HFO cultures for Oil-Red-O staining as well as mRNA expression of the adipocyte marker adipocyte lipid-binding protein (aP2). Cultures were negative for Oil-Red-O staining and, after 40 cycles of RT-PCR, we could not detect any aP2 expression in both differentiating and nondifferentiating cultures (data not shown).



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  		Fig. 3. Relative 11ß-HSD1 mRNA Expression (A) and 11ß-HSD1 Activity (B) in Differentiating and Nondifferentiating SV-HFO Cultures

For incubation protocols, see insets and Materials and Methods. Values are means ± SEM (n = 4). C, Inhibition of 11ß-HSD1 activity by GA in SV-HFO cells. After 19 d of culture, 11ß-HSD1 activity was measured in absence and presence of 2.5 µM GA. Values are means ± SEM (n = 4). **, P < 0.01 compared with nondifferentiating osteoblasts. #, P < 0.05 compared with levels at d 7 of nondifferentiating osteoblasts.

 
11ß-HSD1 Activity in Differentiating and Nondifferentiating Osteoblasts
Next we examined 11ß-HSD1 activity in relation to osteoblast differentiation and mineralization. To demonstrate 11ß-HSD1 reductase activity in SV-HFO cells, we analyzed cortisone to cortisol conversion after addition of 1 µM cortisone to the culture medium. In differentiating cultures, 11ß-HSD1 activity was constant (86 ± 11 pmol/µg·24 h; mean ± SEM), whereas nondifferentiating cultures showed significantly higher 11ß-HSD1 activity: 341 ± 48 and 391 ± 36 pmol/µg·24 h at d 7 and 12, which increased to 701 ± 49 and 748 ± 56 pmol/µg·24 h at d 14 and 19, respectively (Fig. 3BGo). This activity profile shows similarity to the 11ß-HSD1 mRNA expression profile in differentiating and nondifferentiating cultures. 11ß-HSD1 activity was completely blocked by addition of the 11ß-HSD inhibitor 18ß-glycyrrhetinic acid (GA) (Fig. 3CGo). Finally, the presence or absence of ß-glycerophosphate had no effect on 11ß-HSD1 activity in either differentiating and nondifferentiating cultures (data not shown).

Inverse Relationship between Osteoblast Differentiation and 11ß-HSD1 Activity
The data presented thus far suggested an inverse relationship between osteoblast differentiation and 11ß-HSD1 expression and activity. To investigate this in more detail, SV-HFO cells were cultured for 19 d with various concentrations of DEX. DEX treatment caused a dose-dependent increase in ALP activity and mineralization, which coincided with a dose-dependent decrease in 11ß-HSD1 activity (Fig. 4Go, A and B), substantiating the inverse relation between 11ß-HSD1 activity and differentiation. This inverse relationship between ALP and 11ß-HSD1 activity was tightly coupled as indicated by the DEX EC50 values of 3 ± 0.5 and 5 ± 0.5 nM, respectively. Mineralization was induced by DEX with an EC50 value of 9 ± 1 nM DEX.

Effects of Short-Term DEX Treatment on 11ß-HSD1 Expression and Activity
In the study so far, osteoblast differentiation was induced by continuous DEX treatment, resulting in a low and constant 11ß-HSD1 mRNA expression and activity. These results are in contrast to those of other studies using a variety of cell types in which DEX treatment stimulated 11ß-HSD1 activity. However, these results were obtained after short-term DEX incubation. Therefore, we studied the effect of short-term DEX incubation on the regulation of 11ß-HSD1 mRNA expression and activity in nondifferentiating SV-HFO osteoblasts.

At d 7 of culture, 11ß-HSD1 mRNA expression decreased after short-term DEX treatment (1 µM, 24 h). However, at later time points during SV-HFO culture (d 14 and 19), 11ß-HSD1 mRNA expression was not affected by short-term DEX treatment (Fig. 5AGo). To study short-term DEX effects on 11ß-HSD1 activity, cells were exposed for 48 h to DEX (1 µM); within this 48 h, the final 24 h was used to measure 11ß-HSD1 activity. This resulted in decreased 11ß-HSD1 activities at d 7 matching the observed data at the mRNA level. At later time points, 11ß-HSD1 activity was not decreased by short-term DEX treatment. At d 12 and 14, even a significant increase was observed (Fig. 5BGo). These data show that short-term DEX-treatment does not decrease 11ß-HSD1 expression and activity at later stages of culture in contrast to DEX-induced differentiation.



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  		Fig. 5. Short-Term Effects of DEX on 11ß-HSD1 mRNA Expression and Activity during SV-HFO Culture

A, Effects of 24 h DEX treatment (1 µM) on 11ß-HSD1 mRNA expression. Results are presented as expression relative to control at d 7, 14, and 19 of culture. For incubation protocols see inset. Values are means ± SEM (n ≥ 4). B, 11ß-HSD1 activity after 24–48 h of DEX treatment (1 µM DEX) at d 7, 12, 14, and 19 of culture. For incubation protocols see insets. Values are means ± SEM (n = 4). *, P < 0.05; **, P < 0.01 compared with control.

 
11ß-HSD1 Promoter Activity in Differentiating and Nondifferentiating Cultures
To gain more insight into the background of the differentiation-driven regulation of 11ß-HSD1 expression, we used a luciferase reporter gene under control of the 11ß-HSD1 promoter (HSD11B1). We used several lengths of the HSD11B1 promoter containing the –261 to +77, –301 to +77, –804 to +77, –1382 to +77, and –2506 to +77 region of the promoter. During SV-HFO culture, these reporter constructs were transiently transfected at d 7, 12, 14, or 19 of culture. At d 7 of culture, all the constructs showed similar promoter activity in differentiated and nondifferentiated cultures. Activity of the short –261 to +77 region of the HSD11B1 promoter was identical at all time points in differentiating and nondifferentiating cultures (Fig. 6AGo). However, reporter constructs containing more upstream regions of the HSD11B1 promoter were sensitive to induction in the absence of differentiation from d 12 onward (Fig. 6Go, B, C, D, and E). The reporter construct containing the –1382 to +77 region of the HSD11B1 promoter was the most sensitive to induction in the absence of differentiation (Fig. 6DGo). Further increasing the length of the promoter up to –2506 resulted in a lower induction (Fig. 6EGo). These results show that the HSD11B1 promoter is induced from d 12 onward in the absence of osteoblast differentiation. Moreover, important differentiation-dependent regulatory elements are located in region –1382 to –261 of the HSD11B1 promoter. To show that promoter activity is not silenced shortly after DEX exposure, we analyzed promoter activity after 24 and 48 h of DEX exposure (1 µM) at d 7, 12, 14, and 19. We studied both the –804 to +77 (data not shown) and –1382 to +77 (Fig. 6FGo) region of the HSD11B1 promoter in relation to short-term DEX treatment. These two promoters showed a clear suppression after DEX-induced differentiation (Fig. 6Go, C and D); however, short-term DEX treatment did not chance promoter activity (Fig. 6FGo). These results support the mRNA and activity data obtained after short-term DEX treatment.



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  		Fig. 6. HSD11B1 Promoter Activity in Differentiating and Nondifferentiating SV-HFO Cultures

Cultures were transiently transfected with luciferase reporter constructs containing the –261 to +77 (A), –301 to +77 (B), –804 to +77 (C), –1382 to +77 (D) and –2506 to +77 (E) region of the HSD11B1 promoter at d, 7, 12, 14, and 19 of culture. Values are means ± SEM (n ≥ 5). *, P < 0.05; **, P < 0.01 compared with differentiating cultures. F, Promoter activity of the –1382 to +77 region after short-term DEX treatment (1 µM, 24 and 48 h) at d 7, 12, 14, and 19 of culture.

 
CAAT/Enhancer Binding Protein {alpha} (C/EBP{alpha}) and C/EBPß mRNA Expression in Differentiating and Nondifferentiating Osteoblasts
Two important candidates for HSD11B1 promoter regulation are C/EBP{alpha} and C/EBPß. The presence of both C/EBP{alpha} and C/EBPß mRNA was demonstrated in SV-HFO cells. However, no difference in mRNA expression between differentiating and nondifferentiating osteoblasts was found (data not shown).

Functional Consequences of Osteoblastic 11ß-HSD1 Activity
To demonstrate a functional role of 11ß-HSD1 in osteoblast differentiation, we cultured SV-HFO cells in the continuous presence of 100 or 1000 nM cortisone. To prevent total removal of osteoblast-produced cortisol at every medium replacement, medium was only partially replaced. Treatment with cortisone resulted in a dose-dependent increase in ALP activity up to 3.2-fold at d 30 (Fig. 7AGo). Despite this clear induction of ALP activity, cortisone did not induce mineralization. To further study the effect on mineralization, we cotreated the cells with 1{alpha},25-dihydroxyvitamin D3 [1{alpha},25-(OH)2D3] as a facilitator of mineralization. Treatment with 10 nM 1{alpha},25-(OH)2D3 alone increased ALP activity, whereas it was not sufficient to induce mineralization. However, cotreatment of 1000 nM cortisone with 1{alpha},25-(OH)2D3 resulted in a strong induction of mineralization and ALP activity after 30 d of culture (Fig. 7Go, C and D). This effect on mineralization and on ALP activity was completely blocked by the addition of the GR antagonist mifepristone (2 µM) (Fig. 7Go, C and D). To prove a specific role for 11ß-HSD in cortisone-induced ALP activity, cells were incubated with the 11ß-HSD inhibitor GA. Cortisone-induced ALP activity was completely blocked by the addition of 2.5 µM GA. Addition of GA had no effect on either basal ALP activity or cortisol-induced ALP activity (Fig. 7BGo).



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  		Fig. 7. Functional Consequences of Osteoblastic 11ß-HSD1 Expression

A, Induction of ALP activity after cortisone incubation. Cells were cultured in the continuous presence of 100 or 1000 nM cortisone. Results are presented as ALP activity compared with non treated controls at d 7, 14, 21, and 30 of culture. Values are means ± SEM (n = 6). **, P < 0.01 compared with nondifferentiating osteoblasts. B, Inhibition of cortisone induced ALP activity by GA. SV-HFO cells were cultured for 21 d in the presence of cortisone (100 and 1000 nM), cortisol (100 and 1000 nM), and vehicle with or without 2.5 µM GA. Values are means ± SEM (n = 3). **, P < 0.01 compared with control without GA. C and D, Cortisone induced ALP activity and mineralization in the presence or absence of 1{alpha},25-(OH)2D3 (10 nM) and the GR antagonist mifepristone (RU) (2 µM) after 30 d of culture.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The current study demonstrates the presence of an intrinsic differentiation-driven molecular switch that controls the expression and activity of 11ß-HSD1 and thereby cortisol production in human osteoblasts. In the absence of exogenously added glucocorticoids and osteoblast differentiation, 11ß-HSD1 expression and activity are strongly induced. By contrast, cells induced to differentiate and mineralize in the presence of glucocorticoids show low levels 11ß-HSD1 activity. In other words, 11ß-HSD1 acts to generate cortisol in an autocrine fashion to facilitate proper differentiation and mineralization. Conversely, when sufficiently high levels of active glucocorticoids (cortisol, dexamethasone, prednisolone) are available to induce osteoblast differentiation, endogenous glucocorticoid activation via 11ß-HSD1 remains minimal. An additional benefit of this latter phenomenon is that unwanted and detrimental increases in local glucocorticoid concentrations is prevented. This mutual regulation provides an elegant and efficient mechanism by which osteoblast differentiation and local glucocorticoid levels are regulated in a balanced manner.

Under the influence of glucocorticoids, SV-HFO cultures proceed through a tightly controlled process of differentiation resulting in an extracellular protein matrix that fully mineralizes. Without glucocorticoid treatment, SV-HFO cultures do not differentiate and show no mineralization, demonstrating the importance of glucocorticoids for human osteoblast differentiation and mineralization. As a consequence, the regulation of 11ß-HSD1 activity in osteoblasts is likely to be an important autocrine determinant of osteoblast proliferation, differentiation, and function (24, 25)

The actions of glucocorticoids are mediated via GR{alpha}, which is expressed in almost all cell types, including osteoblasts (11). In SV-HFO cells, GR{alpha} mRNA was clearly expressed and, in addition to increased 11ß-HSD1 activity, nondifferentiated osteoblasts expressed GR{alpha} at a higher level than differentiating osteoblasts. Together, these observations indicate that absence of glucocorticoid-induced differentiation results in a cellular sensitization to the glucocorticoid endocrine system.

In agreement with previous studies using primary cultures of human osteoblasts (22, 23), 11ß-HSD1-mediated cortisol generation appears to be the predominant form of glucocorticoid metabolism in SV-HFO cells. Regulation of 11ß-HSD1 expression and activity by glucocorticoids have been reported for hepatocytes, adipocytes, and fibroblast and primary osteoblasts, all of which showed enhanced 11ß-HSD1 activity after glucocorticoid exposure (26, 28, 29, 30), resulting in a "feed-forward" mechanism after glucocorticoid exposure. In vivo, Jamieson et al. (31) demonstrated that glucocorticoids regulate 11ß-HSD1 mRNA in a complex tissue- and time-specific manner, suggesting a reduction of 11ß-HSD1 by DEX. Glucocorticoid-induced differentiation (continuous DEX treatment) of SV-HFO osteoblasts suppressed 11ß-HSD1 mRNA expression and activity, whereas short-term treatment with DEX did not suppress 11ß-HSD1 activities, particularly at later stages of culture. Moreover an increase in 11ß-HSD1 activity was measured after short-term DEX treatment. This demonstrates that, in osteoblasts, glucocorticoids can regulate 11ß-HSD1 in two different ways. Firstly, short-term glucocorticoid exposure directly increases 11ß-HSD1 activity, resulting in a feed-forward of glucocorticoid action. Secondly, we show that, in osteoblasts, continuous exposure to glucocorticoid indirectly regulates 11ß-HSD1 expression by inducing a differentiation-driven switch that represses 11ß-HSD1 activity. In the first week of culture, short-term DEX treatment resulted in the suppression of 11ß-HSD1 activity and mRNA expression. In this first week of culture, SV-HFO cells are very sensitive to the induction of differentiation by glucocorticoids. Therefore, 24 h of DEX exposure may be sufficient to initiate osteoblast differentiation and, as a result, suppression of 11ß-HSD1 levels.

In the absence of glucocorticoid-induced differentiation, 11ß-HSD1 mRNA expression was strongly induced in the second week of culture. Interestingly, at this same time point of culture, differentiating cultures showed peak ALP activity and initiated the process of mineralization. Why this induction of 11ß-HSD1 expression is initiated after exactly 2 wk of culture is unclear. Promoter reorganization or differential expression of transcription factors at this time point might mediate this differentiation-controlled switch in 11ß-HSD1 expression. In rat osteoblasts, it has been demonstrated that, in more differentiated osteoblasts, osteocalcin expression is strongly induced after the osteocalcin gene is opened by remodeling of the chromatin structure (32, 33). Luciferase reporter-plasmids under the control of various HSD11B1 promoter fragments provided evidence that specific regions of the HSD11B1 promoter are sensitive to activation in the absence of osteoblast differentiation. This indicates that regulation of the HSD11B1 promoter during this switch is at least partly regulated by differences in transcription factor binding to the HSD11B1 promoter in differentiating and nondifferentiating osteoblasts. The absence of promoter induction by the short –261 +77 promoter indicates that important differentiation dependent regulatory elements driving expression in nondifferentiating cells are located in region –1382 to –261. Two important candidates for 11ß-HSD1 promoter regulation are C/EBP{alpha} and C/EBPß. These proteins are known to regulate 11ß-HSD1 promoter activity in liver cells, in which C/EBP{alpha} promotes and C/EBPß represses 11ß-HSD1 transcription (34). Furthermore, these genes are known to be regulated by cortisol (35, 36). Both C/EBP{alpha} and C/EBPß are expressed in SV-HFO cells. However, mRNA expression of these genes was identical in nondifferentiating and nondifferentiating SV-HFO osteoblasts.

The current study shows that 11ß-HSD1 activity is tightly related to osteoblast differentiation and has significant consequences for osteoblast differentiation and function. In the presence of sufficient amounts of cortisone, SV-HFO osteoblasts induce, in an autocrine/paracrine way, differentiation by generating biologically active levels of cortisol. Autocrine regulation of glucocorticoids action has also been shown in adipocytes and is a potentially important target for obesity therapy (37). Freshly isolated adipose stromal cells mainly convert cortisol into cortisone. However, when adipocyte differentiation is initiated, 11ß-HSD1 dehydrogenase activity is switched into reductase activity generating cortisol and thereby promoting adipocyte differentiation (38). This shows that both osteoblast and adipocytes use 11ß-HSD1 activity to regulate their differentiation in an autocrine manner. However, in osteoblasts there is an inverse relation between 11ß-HSD1 activity and differentiation, whereas adipocytes increase their cortisol production during further differentiation.

In vivo glucocorticoids are bound to cortisol binding globulin. Cortisol binding globulin binds cortisol with approximately 10-fold higher affinity than cortisone (39), resulting in higher free levels of the 11ß-HSD1 substrate cortisone than the active glucocorticoid cortisol. Therefore, local glucocorticoid metabolism might indeed play a significant role in regulating osteoblast differentiation and function in vivo. This prereceptor hormone regulation is not unique for glucocorticoids. Local levels of androgens and estrogens are also regulated by enzymes present in target tissues (40). Our previous work has shown that human osteoblasts express all the enzymes involved in the production of estradiol, e.g. aromatase, sulfatase, and 17ß-HSD isoenzymes (41). This means that human osteoblasts have the capacity to produce active estrogens and glucocorticoids locally. Moreover, production of these hormones is regulated in a differentiation dependent manner.

This study demonstrates the importance of autocrine 11ß-HSD1 action for proper osteoblast differentiation and function. However, the 11ß-HSD1 knockout (–/–) mouse does not show a clear change in bone phenotype (42); the authors of this study highlighted important caveats why this 11ß-HSD1–/– mouse might not be necessarily a good model to study the effects of local glucocorticoid metabolism on human bone. Firstly, the global loss of 11ß-HSD1 leads to a profound alteration in the hypothalamic-pituitary-adrenal axis, which may completely alter the set-point of circulating glucocorticoid effects. Secondly, the background strain of the 11ß-HSD1–/– mouse shows expression of both 11ß-HSD1 and 11ß-HSD2 in bone, which is in direct contrast to humans, which show only expression of 11ß-HSD1. A bone-specific knockout of 11ß-HSD1 will clearly provide some answers to the issues outlined above. Evidence that endogenous glucocorticoid metabolism has significant effects in vivo on bone is illustrated by the 11ß-HSD2 bone specific transgenic mouse (43). These animals are essentially the bone-specific, transgenic equivalents of the 11ß-HSD1–/– mice and show changes in a variety of bone parameters when compared with wild-type controls.

In conclusion, the current study demonstrates a tight control of 11ß-HSD1 activity during osteoblast differentiation, thereby supplying adequate amounts of cortisol to support osteoblast differentiation and preventing excessive and detrimental amounts of cortisol in mature osteoblasts. Furthermore, the data presented indicate the presence of a molecular switch that regulates HSD11B1 promoter activity dependent on the state of cellular differentiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture
SV-HFO (44) cells were cultured in {alpha}MEM (GIBCO, Paisley, UK) supplemented with 20 mM HEPES, pH 7.5 (Sigma, St. Louis, MO); streptomycin/penicillin; 1.8 mM CaCl2 (Sigma); and heat-inactivated FCS (GIBCO) at 37 C and 5% CO2 in a humidified atmosphere. DEX, cortisone, and mifepristone (RU 38486) were purchased from Sigma; 1{alpha},25-(OH)2D3 was generously provided by Dr. L. Binderup (Leo Pharmaceuticals, Ballerup, Denmark). Thawed cells were precultured for at least 1 wk in the presence of 10% FCS. In this preculture, cells were seeded in a density of 5 x 103 vital cells per centimeter squared and were subcultured every week. During this preculture, SV-HFO cells remained in an undifferentiated stage and cells were only used between passages 8 and 13. After preculturing, cells were seeded in a density of 10 x 103 vital cells per centimeter squared in the presence of 2% charcoal-treated FCS supplemented with 10 mM ß-glycerophosphate (Sigma) (added as a phosphate donor to facilitate the mineralization process) and were grown without subculturing. Medium freshly supplemented with hormones or other additives was replaced every 2–3 d. In experiments in which medium was partially replaced, replacements were as follows: d 2 and 5, 60%; d 7 and 9, 75%; d 12, 14, 19, 21, 26, and 28, 86%; d 16 and 23, 100% fresh medium was added. For analysis, medium was stored at –20 C and cells were scraped form the culture dish in PBS containing 0.1% Triton X-100 and stored at –80 C. Before analysis, cell lysates were sonicated on ice in a sonifier cell disrupter for 2 x 15 sec.

DNA Content
For DNA measurements, 100-µl SV-HFO cell lysates were treated with 200 µl heparin (8 IU/ml in PBS) and 100 µl ribonuclease A (50 µg/ml in PBS) for 30 min at 37 C. This was followed by adding 100 µl ethidium bromide solution (25 µg/ml in PBS). Samples were analyzed on the Wallac 1420 victor2 (PerkinElmer, Wellesley, MA) using an extinction filter of 340 nm of and emission filter of 590 nm. For standards, calf thymus DNA (Sigma) was used.

ALP Activity
ALP activity was assayed by determining the release of paranitrophenol from paranitrophenylphosphate (20 mM in 1 M diethanolamine buffer supplemented with 1 mM MgCl2 at pH 9.8) in the SV-HFO cell lysates for 10 min at 37 C. The reaction was stopped by adding 0.06 M NaOH. Absorption was measured at 405 nm. Results were adjusted for DNA content of the corresponding cell-lysates.

Mineralization
SV-HFO cell lysates were incubated overnight in 0.24 M HCl at 4 C. Calcium content was colorimetrically determined with a calcium assay kit (Sigma) according to the manufacturer’s description. Results were adjusted for DNA content of the corresponding cell lysates

Oil-Red O Staining
Cell cultures were fixed for 10 min with 10% formalin in PBS. After fixation, cells were washed with PBS and stained for at least 20 min with Oil-Red-O solution (2:3 vol/vol H2O: n-propanol containing 0.5% Oil-Red-O). After staining, cells were washed twice with demineralized water and examined under the microscope for staining.

11ß-HSD1 Activity during SV-HFO Culture
Cells were cultured in the absence or presence of DEX and at various days during culture 1 µM cortisone was added to the culture medium. After 24 h, culture medium was collected and cortisol concentrations were analyzed by a chemoluminescence-based immunoassay on the Immulite 2000 (Diagnostic Products Corp., Los Angeles, CA).

Human HSD11B1 Promoter-Reporter Constructs
A yeast artificial chromosome clone of chromosome 1 (kindly provided by Prof. J. Adamski, GSF-National Research Center for Environment and Health, Institute of Experimental Genetics, Neuherberg, Germany) was used to sequence the promoter for the 11ß-HSD1 gene (HSD11B1) (GenBank accession no. AL031316). The resulting DNA was then used to produce smaller DNA fragments corresponding to different lengths of the HSD11B1 promoter including a 77-bp region of 5' untranslated region (exon 1) downstream of the ATG site at 2506 bp. The promoter fragments were generated by PCR amplification using a single reverse primer (+77 to +65 bp, 5'-TCACCCGGGTCCTCGTTTGCAG-3') in conjunction with various forward primers specific for different regions of the human HSD11B1 promoter/exon 1: promoter region –261 to +77, forward primer (–261 to –249 bp), 5'-TCAGGTACCGTCTCCTCTTGCT-3'; promoter region –301 to +77, forward primer (–301 to –289 bp), 5'-TCAGGTACCGAATCCAGTCCTG-3'; promoter region –804 to +77, forward primer (–804 to –791 bp) 5'-GCTCAGGTACCTTTACAAGACCCAG-3'; promoter region –1382 to +77, forward primer (-1382 to –1370 bp), 5'-TCAGGTACCGGCCTTTGTTGAC-3'; promoter region –2506 to +77, forward primer (–2506 to –2494 bp), 5'-TCAGGTACCGAGAACCAGCCAT-3'. Each primer contained restriction sites shown in italics to facilitate insertion into the pGEM-T Vector (Promega, Madison, WI): reverse primer, SmaI (CCCGGG); and forward primers, KpnI (GGTACC). After ligation, the promoter constructs were transformed in DH5{alpha} competent cells, selected (Luria broth + ampicillin), and DNA was extracted by midi-prep (Wizard plus SV Minipreps Purification System by Promega). The resulting HSD11B1 promoter constructs were then digested with SmaI and KpnI, separated on 1% agarose gels, reextracted, and recloned into SmaI- and KpnI-digested luciferase pGL3-enhancer vector (Promega). Ampicillin-selected clones were used to produce DNA, which was then sequenced to confirm the identity of each HSD11B1 promoter-reporter fragment.

Luciferase Activity
Four hours after medium replacement, SV-HFO cultures were transiently transfected with 200 ng pGL3 luciferase reporter plasmid, using FuGENE6 transfection reagent according to the manufacturer’s protocol (Roche, Basel, Switzerland). One day after transfection, SV-HFO cultures were lysed in 100–200 µl 1x lysis buffer (Promega) for 20 min with gentle shaking. During the final stages of differentiation when the culture was mineralized, a cell scraper was needed to release the cells and initiate cell lysis. Luciferase activity was measured using 25 µl cell lysate and the Steady-Glo Luciferase Assay System (Promega). Luciferase values were corrected for luciferase activity of empty pGL3-enhancer vector.

Quantification of mRNA Expression
Total RNA was isolated using RNA-Bee solution (Tel-Test, Friendwood, TX) according to the manufacturer’s protocol. To remove calcium (derived from extracellular matrix), RNA was precipitated by overnight incubation with 4 M LiCl and 50 mM EDTA at –20 C. After precipitation and centrifugation for 30 min at 14,000 rpm and 4 C, the RNA pellet was washed four times with 70% EtOH and dissolved in H2O. The total amount of RNA was quantified using the RiboGreen RNA Quantitation Kit (Molecular Probes, Eugene, OR). One microgram total RNA was reverse transcribed into cDNA using a cDNA synthesis kit according to the protocol of the manufacturer (MBI Fermentas, St. Leon-Rot, Germany), using 0.5 µg oligo(dT)18 and 0.2 µg random hexamer primers.

Quantitative real-time PCR was carried out using an ABI 7700 sequence detection system (Applied Biosystems, Foster City, CA). Reactions were performed in 25 µl volumes using a qPCR core kit (Eurogentec, Seraing, Belgium). Reaction mixes contained 20 ng cDNA, 5 mM MgCl2, 200 µM dNTPs, and 0.025 U/µl Hot GoldStar enzyme. Primer and probe sets were designed, using the Primer Express software (version 1.5; Applied Biosystems). Primer and probe concentrations and sequences were as described as in Table 1Go. Cycling conditions were 50 C for 2 min, 95 C for 10 min, followed by 40 cycles of 95 C for 15 sec and 60 C for 1 min. The amount of human glyceraldehyde-3-phosphate dehydrogenase mRNA was used as internal control to normalize for possible differences in RNA extraction and degradation as well as efficiency of the cDNA synthesis. The expression of this gene was not significantly affected by hormonal treatment and during osteoblast differentiation. Data were presented as relative mRNA levels calculated by the equation 2–{Delta}Ct ({Delta} Ct = Ct of target gene minus Ct of glyceraldehyde-3-phosphate dehydrogenase).


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  		Table 1. Primer and Probe Concentrations and Sequences

 
For semiquantitative RT-PCR, 80 ng of cDNA was amplified in a 25 µl reaction mix containing 200 µM dNTPs, 2.5 mM MgCl2, 400 nM of each primer, and 1.25 U Amplitaq Gold (Applied Biosystems). Cycling conditions were as follows: 95 C for 10 min followed by 40 cycles of 95 C for 15 sec, 56 C for 30 sec, and 72 C for 30 sec and a final extension step of 5 min at 72 C. Primer sequences for aP2 are described in Table 1Go.

Statistics
Data presented are the results of at least two independent experiments performed in triplicate. Values are the means ± SEM. Significance was calculated using the Student’s t test


   FOOTNOTES
 
First Published Online December 9, 2004

Abbreviations: aP2, Adipocyte lipid-binding protein; ALP, alkaline phosphatase; C/EBP, CAAT/enhancer binding protein; DEX, dexamethasone; 1{alpha},25-(OH)2D3, 1{alpha},25-dihydroxyvitamin D3; GA, 18ß-glycyrrhetinic acid; GR, glucocorticoid receptor; 11ß-HSD1, 11ß-hydroxysteroid dehydrogenase type 1.

Received for publication May 25, 2004. Accepted for publication December 1, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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Nuclear Receptors:   GR
Ligands:   Calcitriol  |  Dexamethasone  |  Hydrocortisone  |  RU486



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