| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Division of Medical Sciences, Institute of Clinical Research, Queen Elizabeth Hospital, The University of Birmingham, Birmingham B15 2TH, UK
1Correspondence: Department of Medicine, Institute of Clinical Research, Queen Elizabeth Hospital, The University of Birmingham, Birmingham B15 2TH, UK. E-mail: M.Hewison{at}bham.ac.uk
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: 11ß-HSD • glucocorticoid metabolism • glucocorticoid receptor • intracrine
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 2)
. In common with other steroid hormones, glucocorticoids achieve their effects by binding to cognate intracellular glucocorticoid receptors (GR) (3
, 4)
. The resulting nuclear complex acts as a ligand-dependent trans-activator either by binding as a homodimer to specific target gene response elements or by protein–protein interaction with other transcriptional regulators. For example, recent studies have shown physical interaction between GR and the p65 subunit of nuclear factor 
B (NF-B) (5)
. This appears to be central to the functional antagonism between NF-B and glucocorticoid signaling pathways that are characteristic of inflammatory and anti-inflammatory responses in the immune system (6
, 7)
. Another important physical interaction occurs between GR and the fos-jun subunits of AP-1 (8)
. Once again, liganded GR complexes antagonize the proinflammatory activity of AP-1 but, as with NF-B, this mechanism may have a more widespread effect on cell proliferation and differentiation (9)
.
‘Prereceptor’ or ‘intracrine’ regulatory mechanisms have been described for several steroid hormones and involve target tissue activation or inactivation of the circulating hormone. Glucocorticoid tissue-specific metabolism is catalyzed by two isozymes of 11ß-hydroxysteroid dehydrogenase (11ß-HSD) (10)
. In humans, the type 1 isozyme (11ß-HSD1) is predominantly involved in generating the active glucocorticoid, cortisol. 11ß-HSD1 is located in tissues that express relatively high levels of GR, such as liver, adipose, and gonads, and appears to function by increasing local concentrations of cortisol (11)
. In contrast, the type 2 isozyme (11ß-HSD2) converts active cortisol to inactive cortisone and is located predominantly in tissues with relatively high levels of mineralocorticoid receptor (MR) expression such as the kidney and colon. In these tissues, 11ß-HSD2 acts to protect the MR from illicit occupancy by cortisol, which binds to MR with a similar affinity to the natural MR ligand aldosterone (12)
. However, our studies and those by others have described relatively high levels of 11ß-HSD2 expression in osteosarcoma and breast cancer cell lines and pituitary tumors (13
14
15
16
17
18)
. In each case, the resulting inactivation of cortisol occurred in the presence of high levels of GR rather than MR, suggesting an alternative function for 11ß-HSD2 in these cells. In bone and pituitary, the presence of 11ß-HSD2 in neoplastic tissue contrasted with their normal tissue equivalents, which expressed only 11ß-HSD1 (17
, 18)
. We have therefore hypothesized that in tumors, expression of 11ß-HSD2 acts as an autocrine pro-proliferative mechanism by decreasing the local availability of ligand for the GR. This in turn is likely to have profound effects on cellular function, bearing in mind the ubiquitous expression of the GR and its fundamental role in directing antiproliferative/anti-inflammatory trans-regulatory signaling. Here we present data using transient and stable transfection models to demonstrate the divergent effects of 11ß-HSD1 and 11ß-HSD2 on cell proliferation. In particular, the ability of 11ß-HSD2 to stimulate cell proliferation independent of any changes in GR expression suggests a novel oncogenic function for this enzyme.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Production of 11ß-HSD isozyme transfectant cell variants
Stable transfections were carried out using pcDNA3 expression constructs containing full-length coding region cDNAs for either 11ß-HSD1 or 11ß-HSD2, as well as a pcDNA3 vector-only control construct. These constructs were similar to those used previously to transfect HEK-293 cells (19)
. Further stable transfectant variants were produced using a pcDNA3 expression construct containing a mutant 11ß-HSD2 cDNA (Y226N) previously shown to be associated with decreased cortisol inactivation leading to apparent mineralocorticoid excess (AME) (unpublished data). Expression construct cDNAs were transfected into ROS 17/2.8 cells using methods based on the DMRIE-C reagent protocol (Gibco BRL). Aliquots of control vector or 11ß-HSD isozyme expression construct (2 µg) were diluted in a mixture of serum-free medium and DMRIE-C reagent (1vol:1 vol). The resulting mixture was added to 50% confluent cultures of ROS 17/2.8 cells and incubated for 24 h at 37°C. Transfection medium was then removed and the cells allowed to recover for another 48 h. Pooled populations of stable transfectant variants of ROS 17/2.8 were isolated by selection with the antibiotic G418 (10 µM) for more than six passages. Antibiotic resistant pools of cells were maintained permanently in 10 µM G418. Transient transfection studies were carried out using pCR3 expression constructs containing the full-length coding region cDNAs for either 11ß-HSD2 or the Y226N mutant form of 11ß-HSD2. Empty vector cDNA (pCR3) was used as a further control. 11ß-HSD null recipient HEK-293 cells were transfected for 1 h with 1 µg cDNA using Transfast Transfection ReagentsTM according to the manufacturer’s protocols (Promega, Southampton, UK). The cells were then incubated for a further 48 h in normal growth medium to allow expression of protein for 11ß-HSD2. The resulting cultures were used to isolate RNA and assessed for glucocorticoid metabolism and cell proliferation.
Reverse transcription of mRNA
Cells were grown in 80 cm2 flasks to 80–100% confluency and total RNA extracted using RNeasy RNA extraction columns according to manufacturer’s protocols (Qiagen, Chatsworth, CA). DNA contamination was reduced by the use of QiaShredder columns (Qiagen). RNA was reverse transcribed using a Promega Reverse Transcription System; 1 µg of total RNA and 0.5 µg of oligo dT(15) were incubated at 70°C for 5 min in a final volume of 10 µl. Primer extension was then performed at 42°C for 60 min after the addition of 1x (final concentration) reaction buffer containing 50 mM Tris-HCl (pH 8.3), 50 mM KCl, 10 mM MgCl2, 10 nM dithiothreitol, and 0.5 mM spermidine, with 1 mM (final concentration) of each dNTP, 80 U of RNasin ribonuclease inhibitor, and 50 U AMV reverse transcriptase in a final volume of 50 µl.
RT-PCR analysis of 11ß-HSD isozymes
Aliquots (5 µl) of cDNA from RT reactions were used in polymerase chain reactions (PCR) to amplify specific cDNAs. PCR primers were as follows: human 11ß-HSD type 1 5' TCA GAC CAG AAA TGC TCC AG, 3' AGG AGA TGA TGG CAA TGC TG; human 11ß-HSD type 2 5' GAC TAA TGT GAS CCT CTG GGA G, 3' TCA GTG CTC GGG GTA GAS GGT G. Reactions were performed using 1x (final concentration) PCR buffer containing 50 mM KCl, 10 mM Tris-HCl (pH9.0) and 0.1% Triton X-100, with 1 mM MgCl2, 0.2 mM of each dNTP, and primers at a final concentration of 0.3 µM (11ß-HSD1) or 0.2 µM (11ß-HSD2) with 1 U Taq DNA polymerase in a final volume of 20 µl. Samples were amplified using an initial denaturation cycle of 95°C for 5 min, 30 cycles of 95°C (1 min), 50°C (1 min) (11ß-HSD1), or 60°C (1 min) (11ß-HSD2), followed by a final elongation step of 72°C for 7 min. 11ß-HSD2 mRNA expression in HEK-293 cells was compared with the housekeeping gene 18S rRNA using primers and protocols supplied by the manufacturer (Ambion, Austin, TX).
Analysis of 11ß-HSD activity
Cells were characterized for their ability to metabolize the predominant human glucocorticoids (cortisol and cortisone), as well as the predominant rat glucocorticoids (corticosterone and 11-dehydrocorticosterone). Oxidative (dehydrogenase) 11ß-HSD activity was determined using unlabeled cortisol and corticosterone (10–1000 nM for both) and tracer amounts (<1 nM) of [1,2,6,7]-3H-cortisol or [1,2,6,7]-3H-corticosterone (specific activity for both: 70–100 Ci/mmol; DuPont, Boston, MA) for 5 h. Steroids were extracted from growth medium using dichloromethane (5–10 vol) and separated by thin-layer chromatography (TLC) using ethanol:chloroform (8:92) as the mobile phase. TLC plates were analyzed using a Bioscan imaging detector (Bioscan, Washington, DC) and the fractional conversion of steroids was calculated. 11-Oxo-reductase activity was similarly assessed by incubation of cells with various concentrations of unlabeled cortisone or 11-dehydrocorticosterone (50–2000 nM) and tracer amounts of 3H-[1,2,6,7]-3H-cortisone or 3H-[1,2,6,7]-11-dehydrocorticosterone (both synthesized in-house; 19
) for a period of 5 h. In each case, cell monolayers were lysed and total protein concentration was assessed using a Bio-Rad protein assay (Bio-Rad, Melville, NY). Results were expressed as pmol product/h/mg protein. Enzyme kinetics for dose-response assays was determined by linearization of data using Hanes-Woolf plots (substrate concentration/enzyme activity vs. substrate concentration).
Glucocorticoid binding assays
GR expression in transfectant cells was assessed using whole cell steroid hormone binding assays as described previously (14)
. Confluent cells were trypsinized and washed twice in phosphate-buffered saline (PBS). Cell pellets were resuspended in serum-free medium to give a final concentration of 1 x 107 cells/ml. Aliquots (200µl) of cell suspension were then incubated for 1 h at 37°C in glass tubes with 3H-dexamethasone (3H-dex) (0.8 nM-50 nM; specific activity 82Ci/mmol, Amersham, Little Chalfont, UK) with or without a 200-fold excess of cold dex to assess binding to GR. Cells were then washed twice in cold PBS, resuspended in 500 µl water and bound radioactivity was analyzed by scintillation counting. Binding kinetics for dex were determined using Scatchard analyses. Data were linearized by plotting specifically bound hormone divided by free hormone (total minus specifically bound) against specifically bound hormone. The slope of the resulting plot corresponded to the binding affinity value (dissociation constant, Kd) and the intercept with the x axis corresponded to the total saturable binding value (maximal binding, Bmax). By using the latter together with Avogadro’s constant, it was possible to determine the number of GR/cell.
Analysis of alkaline phosphatase activity
Osteoblastic function was assessed by measuring alkaline phosphatase activity following an adaptation of a Sigma protocol. Cell monolayers were washed twice with a 0.01% salt solution, followed by a 1 h incubation at 4°C in solubilization solution (0.01% salt solution+0.1% Triton X-100). Enzyme activity was then assessed at 37°C by the release of p-nitrophenol from p-nitrophenyl during a 15 min incubation step. The reaction was stopped by the addition of 1 M NaOH and liberated p-nitrophenol measured at 405 nm. Total protein concentration of each incubate was assessed using a Bio-Rad protein assay reagent according to the manufacturer’s protocol. Results were expressed as µmol p-nitrophenol produced/h/mg protein.
Analysis of cell proliferation
The rate of proliferation of transfectant cells was assessed by measurement of nuclear 3H-thymidine incorporation. After specific treatments, cells were incubated with 0.2 µCi 3H-thymidine (specific activity 80 Ci/mmol; Amersham) for the last 6 h of culture incubation. Cells were then washed twice in PBS, followed by 1 ml of cold 5% trichloroacetic acid (TCA) to precipitate proteins, and left on ice for 20 min. The liquid layer was then removed and drained. An aliquot (250 µl) of 0.1 M sodium hydroxide (NaOH) was added to the cells and left at room temperature for 30 min on a shaker. The resulting solubilized nuclear material was then transferred to 4 ml of scintillant and radioactive counts were determined by scintillation counting.
Cell cycle analyses
Cells were grown as described for analysis of cell proliferation, but using 6-well plates. At each time point monolayers were washed with PBS and then incubated in buffer containing 0.1 mM sodium chloride, 1% sodium citrate, 0.1% Triton X-100, and 10 µg/ml propidium iodide for 30 min at 4°C. The resulting cell lysates were transferred to plastic tubes and analyzed using a FACS IV Flow Cytometer at a wavelength of 488 nm. Analyses were carried out using 8000 cells per injection for n = 4 replicates.
Statistics
For enzyme activity, alkaline phosphatase, cell cycle, and 3H-thymidine studies data are the mean ± SD of n = 3 values from different experiments. Scatchard plots are shown as a representative experiment that was repeated three times. Statistical analysis was performed using one-way ANOVA linked to Tukey-Kramer multiple comparison posttests (Instat version 2.04a, GraphPad Software, San Diego, CA).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 17)
. The lowest levels of 11ß-HSD activity were observed in ROS 17/2.8 rat osteosarcoma cells; consequently, these cells were used for subsequent expression studies. After transfection, pooled clones of cells expressing the empty vector (ROS17/2.8
) or cDNA for 11ß-HSD1 (ROS 17/2.8ß1) or 11ß-HSD2 (ROS 17/2.8ß2) were isolated. The transfectant variants were analyzed initially by RT-PCR to confirm the presence of mRNA for human 11ß-HSD1 or 2 in each of the transfectant variants (Fig. 1
B). No amplification products were detectable in parental ROS 17/2.8 cells, confirming RT-PCR specificity for human 11ß-HSD mRNA. In parallel enzyme activity assays (Fig. 1A
), ROS 17/2.8ß1 cells demonstrated exclusive reductive activity (cortisone to cortisol conversion) with an apparent substrate affinity value (Km) of 246 nM (data not shown). In contrast, ROS 17/2.8ß2 showed exclusive dehydrogenase activity (cortisol to cortisone conversion) with a Km of 
103 nM (data not shown). Similar patterns of metabolism were observed when the predominant rodent glucocorticoids corticosterone and 11-dehydrocorticosterone were used as substrates, and none of the cell lines demonstrated metabolism of dexamethasone (data not shown). Irrespective of the direction of metabolism, the overall capacity for glucocorticoid metabolism (maximal enzyme activity, Vmax) was similar for each transfectant cell line (72 pmol/h/mg protein for ROS 17/2.8ß1 and 86 pmol/h/mg protein for ROS 17/2.8ß2).
|
Additional studies were then carried out to assess the effects of 11ß-HSD isozyme transfection on GR expression in the ROS 17/2.8 cells. Typical Scatchard plot analyses shown in Fig. 2
indicated that 3H-dex binding kinetics were similar for each of the transfectant cell lines. Mean values for binding capacity (Bmax, intercept with x axis) of ROS 17/2.8 cells (26,000 GR/cell), were not significantly different from ROS 17/2.8ß1 (28,000 GR/cell) or ROS 17/2.8ß2 cells (29,000 GR/cell). For each cell line, binding affinity values (Kd, slope of plot) were of the order of 20 nM dex.
|
Effects of 11ß-HSD isozyme expression on glucocorticoid regulation of cell proliferation and differentiation
Alkaline phosphatase activity, a marker of osteoblast differentiation, was used to assess the functional impact of 11ß-HSD isozyme transfection. Data shown in Fig. 3
highlight different patterns of sensitivity to glucocorticoids in ROS 17/2.8ß1 and ß2 even though both cell lines expressed similar numbers of GR. Consistent with their low levels of endogenous oxidative 11ß-HSD2 activity, parental ROS 17/2.8 and control transfectant ROS 17/2.8 cells showed up-regulation of alkaline phosphatase activity in the presence of naturally occurring active glucocorticoids such as cortisol. Similar results were obtained in the presence of corticosterone (data not shown) whereas inactive steroids such as cortisone or 11-dehydrocorticosterone had no effect on either of the control cells. Analysis of ROS 17/2.8ß1 cells showed that treatment with either active glucocorticoids such as cortisol or inactive glucocorticoids such as cortisone stimulated alkaline phosphatase activity. Similar results were observed with corticosterone and 11-dehydrocorticosterone (data not shown). In contrast, ROS 17/2.8ß2 were insensitive to all natural glucocorticoids. To confirm that GR-mediated signaling remained intact in each transfectant, parallel studies were carried out using dexamethasone. The synthetic glucocorticoid, which is poorly metabolized by 11ß-HSD2, potently stimulated alkaline phosphatase activity in all of the cells, including parental ROS 17/2.8.
|
Effects of 11ß-HSD isozyme expression on cell proliferation
Despite their differential sensitivity to exogenous glucocorticoids, the transfectant cells and parental ROS 17/2.8 cells showed similar basal levels of alkaline phosphatase expression. However, cell proliferation analyses revealed strikingly different patterns of growth, which appeared to be dependent on 11ß-HSD isozyme expression (Fig. 4
). Transfectant cells seeded at identical concentrations showed similar levels of 3H-thymidine incorporation at 24 h. However, at 48 and 72 h, the rate of proliferation of ROS 17/2.8ß2 cells was significantly higher than either control ROS 17/2.8 or ROS 17/2.8ß1 cells. This effect was abrogated when ROS 17/2.8ß2 cells were treated with the 11ß-HSD inhibitor 18ß-glycyrrhetinic acid. Likewise, cells transfected with cDNA for 11ß-HSD2 containing an enzyme inactivating mutation (ROS 17/2.8ß2mut) showed levels of 3H-thymidine incorporation that were not significantly different from controls. In contrast, cell proliferation in ROS 17/2.8ß1 cells was significantly lower than all the other cells. The functional association between 11ß-HSD isozyme expression and transfectant cell proliferation was confirmed in further 3H-thymidine studies using exogenously added glucocorticoids (Fig. 5
). Control ROS 17/2.8 cells showed inhibition of proliferation when treated with cortisol but not cortisone. Despite their low basal levels of proliferation, ROS 17/2.8ß1 cells showed decreased incorporation of 3H-thymidine after treatment with either cortisol or cortisone. In contrast, ROS 17/2.8ß2 cells were unresponsive to cortisol or cortisone even though they had the highest basal level of cell proliferation. All control and transfectant cell lines showed inhibition of proliferation after treatment with synthetic dexamethasone (data not shown).
|
|
Flow cytometry after nuclear incorporation of propidium iodide revealed subtle changes in the cell cycle profiles of the transfectant cells (Table 1
). ROS 17/2.8ß2 cells showed a significantly higher proportion of cells in S phase at 48 h, but with a significantly lower number at 72 h. The relatively low rate of proliferation in ROS 17/2.8ß1 cells was associated with increased numbers of cells in G1/G0 phase at 72 h, although this was also observed with ROS 17/2.8ß2 cells at the same time point. Cell cycle profiles also suggested that transfection of 11ß-HSD1 or 2 had no effect on the numbers of apoptotic cells (<1 n) (data not shown). This was endorsed by parallel DNA fragmentation assays that provided no evidence for apoptosis in ROS 17/2.8ß1 or ROS 17/2.8ß2 cells (data not shown).
|
Proliferative effects of 11ß-HSD2 in nontransformed cells
The pro-proliferative potential of 11ß-HSD2 was further demonstrated in transient expression studies using nontransformed 11ß-HSD null HEK-293 cells (Fig. 6
). Expression of wild-type 11ß-HSD2 for 48 h (293ß2WT) produced levels of enzyme expression and activity (51±3 pmol/h/mg protein) similar to those described for ROS 17/2.8ß2 cells (Fig. 6A, B
). The Y226N mutant form of 11ß-HSD2 was also well expressed (293ß2MUT) (Fig. 6A
) but, as with the plasmid-only control, showed no detectable enzyme activity (Fig. 6B
). Parallel proliferation analyses showed a 50% increase in 3H-thymidine incorporation in 293ß2WT when compared with the empty vector control or cells expressing the Y226N mutant form of 11ß-HSD2 (Fig. 6C
). Similar studies were also carried out using HEK-293 cells transfected with cDNA for 11ß-HSD1 (data not shown). In each case, the resulting variants showed lower levels of cell proliferation, but the degree with which this occurred varied from experiment to experiment. This was because unlike the ROS models, HEK-293 11ß-HSD1 transfectants showed both oxidative (cortisol inactivation) and reductive (cortisone activation) enzyme activities, with the ratio of these activities varying between different transfections.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 21)
. The net effect of these observations is that, unlike the estrogen receptor in breast cancer and the retinoic acid receptor in leukemias, it is difficult to define a cancer lesion that is specifically associated with GR function. Thus, although GR-mediated mechanisms have shown considerable potential in vitro, their use in vivo has achieved limited success (22)
. To address this, we have identified an intracrine mechanism independent of GR expression that appears to be the rate-limiting determinant of glucocorticoid effects on cell proliferation.
Prereceptor or intracrine regulation of glucocorticoid metabolism via isozymes of 11ß-HSD is a pivotal determinant of tissue-specific responsiveness to these hormones. The type 1 isozyme of 11ß-HSD is most strongly expressed in GR-positive tissues such as liver and adipose, where the enzyme enhances local conversion of inactive cortisone to cortisol (11)
. This is most clearly illustrated by recent studies of 11ß-HSD1-expressing adipose stromal cells in which we showed that cortisone was as effective as cortisol in stimulating adipocyte differentiation (23)
. Although the predominant reductive activity of 11ß-HSD1 supports GR-mediated signaling, the oxidative activity of 11ß-HSD2 acts as an attenuator of local glucocorticoid responses (12)
. Mutations in the gene for 11ß-HSD2 have been shown to result in the clinical disorder known as apparent mineralocorticoid excess, or AME (10)
. In patients with AME, peripheral inactivation of cortisol is impaired most notably in key MR-expressing tissues such as the kidney and colon. This in turn leads to inappropriate MR-mediated responses including increased sodium retention, hypokalemia, and hypertension (24
, 25)
. Collectively these observations have suggested distinct, tissue-specific patterns of expression for 11ß-HSD1 and 2 in normal endocrinology. However, recent studies of bone and the pituitary have shown that both isozymes may be expressed at these sites. We have described a shift from predominant 11ß-HSD1 expression in normal tissue to 11ß-HSD2 in tumors (13
, 14
, 17
, 18)
. In each case, the presence of 11ß-HSD2 in neoplastic cells was associated with GR rather than MR expression, suggesting an alternative function for the isozyme distinct from its classical role in MR-rich tissues such as the colon or kidney. In view of the fundamental trans-regulatory role of the GR and its potential effect on cell proliferation and differentiation, we have hypothesized that abnormal expression of 11ß-HSD2 in GR-rich tissues will confer growth advantage and may be an important component of tumor initiation.
In data presented here, we have addressed the prereceptor or intracrine function of 11ß-HSD isozymes using ROS 17/2.8 cells stably transfected with cDNAs for 11ß-HSD1 or 2. Analysis of enzyme activity in ROS 17/2.8ß1 and ROS 17/2.8ß2 cells has confirmed the substrate specificity for each of the isozymes. In intact ROS 17/2.8ß1 cells, there was no dehydrogenase activity even though 11ß-HSD1 is able, at least in theory, to act as a bidirectional enzyme. This is consistent with previous studies indicating that the capacity for reductase activity in 11ß-HSD1 stable transfectants is 5- to 10-fold higher than any dehydrogenase activity (19)
. Transfection with either 11ß-HSD1 or 11ß-HSD2 did not appear to have any effect on binding or response to the synthetic glucocorticoid dexamethasone, which is a poor substrate for 11ß-HSD2 (26)
. This confirmed there was an intact and functional GR signaling pathway in each of the transfectant cell lines. Although the ROS 17/2.8ß1 and ß2 cell lines had a similar capacity for glucocorticoid metabolism, they exhibited entirely opposite patterns of enzyme activity. In this way, the transfectant cells allowed us to demonstrate that prereceptor regulation can act to both stimulate and attenuate GR-mediated responses. On the one hand, transfection of 11ß-HSD2 resulted in complete insensitivity to active cortisol whereas transfection of 11ß-HSD1 produced cells that were sensitive to ‘inactive’ cortisone. It was interesting to note that there was greater induction of the differentiation marker alkaline phosphatase when ROS 17/2.8ß1 cells were treated with cortisone than with cortisol. This suggests that endogenous activation of glucocorticoids may be a more potent activator of GR-mediated responses than exogenous treatment with cortisol itself.
The potential importance of prereceptor regulation of glucocorticoid signaling is most clearly illustrated by the divergent effects of 11ß-HSD 1 and 2 on cell proliferation. Analysis of wild-type and mutant cDNA transfectants as well as cells treated with 18ß-glycyrrhetinic acid shows clearly that the pro-proliferative effects of 11ß-HSD2 are due to the increased capacity for local inactivation of cortisol. The proportion of ROS 17/2.8ß2 cells in S phase of the cell cycle was raised at 24 h of culture but was statistically significant only after 48 h of culture (the point of greatest difference in cell proliferation as assessed by 3H-thymidine incorporation). That the number of ROS 17/2.8ß2 cells in S phase was decreased at 72 h is most probably due to the high degree of confluency (and, as a consequence, the slower growth) in these cultures compared with ROS 17/2.8 or ROS 17/2.8ß1 cells. This is supported by the fact that both ROS 17/2.8ß1 and ROS 17/2.8ß2 showed increased numbers of cells in G1/G0 phase at this point. Transient transfection studies using HEK-293 cells showed again that the pro-proliferative effects of 11ß-HSD2 specific for this enzyme were not due to selection artifacts and not restricted to neoplastic cells. In this context, 11ß-HSD2 displays attributes consistent with oncogenic status. Both ROS 17/2.8 (Fig. 2)
and HEK-293 cells (27)
show predominant GR rather than MR expression. We can therefore postulate that dysregulated expression of 11ß-HSD2 in normal GR-expressing cells may constitute a novel and potentially important facet of tumor initiation.
Although it is now clear that glucocorticoids regulate the transcription of a diverse array of target genes, the precise mechanisms by which GR-mediated changes in cell proliferation and differentiation occur are still far from clear (1
2
3
4
5
6
7
8
9)
. Among the most prominent glucocorticoid target genes are the cyclin-dependent kinases (CDKs) and their corresponding CDK inhibitors (CDIs) (28)
. Some, such as the Cip/Kip family of CDIs (particularly p57kip2), are rapidly regulated by glucocorticoids and are central to the resultant accumulation of cells in G1 phase of the cell cycle (29)
. This appears to be due to direct trans-activation of CDI gene expression, although it seems likely that parallel GR-mediated trans-repression of NF-B signaling is also important in controlling cell cycle progression (6
7
8)
. Glucocorticoids may also alter cell cycling by modulating growth factor-mediated changes in tyrosine kinase signaling either by direct effects on membrane receptor expression or by indirect regulation of protein phosphorylation (30)
. With this in mind, we propose that the transfectant cell variants described here will provide an important new model for determining the relative impact of the divergent GR-mediated pathways on cell growth.
Data presented here emphasize the pivotal role of 11ß-HSD isozymes as prereceptor determinants of GR-mediated signal transduction. However, another important facet of this study is that it highlights the possible therapeutic application of 11ß-HSD modulators in the treatment of cancer. Previous reports have described the antiproliferative properties of the liquorice derivatives glycyrrhizin and glycyrrhetinic acid (31)
. Glycyrrhizin is a key ingredient in Japanese herbal medicine ‘Sho-saiko-to’ that has been reported to have potent anticancer properties (32)
. However, liquorice derivatives also have potent effects on 11ß-HSD2 in classical tissues such as the kidney (10
, 15)
. As such, they are able to reproduce the symptoms of AME, leading in turn to abnormal sodium retention and increased blood pressure. Further analysis of the kinetics of these and other 11ß-HSD inhibitors may help to determine whether 11ß-HSD2 will provide a suitable target for specific therapeutic intervention in the treatment of cancer. A more effective approach may be to enhance 11ß-HSD1 expression, possibly through targeted gene delivery. In either case, continued analysis of the ROS 17/2.8ß1 or ROS 17/2.8ß2 cells will help to provide a further insight on the role of local glucocorticoid metabolism in normal cell growth and the pathophysiology of cancer.
|
ACKNOWLEDGMENTS |
|---|
Received for publication August 7, 2001.
Revision received October 9, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  F. Buttgereit, H. Zhou, and M. J Seibel Arthritis and endogenous glucocorticoids: the emerging role of the 11{beta}-HSD enzymes Ann Rheum Dis, September 1, 2008; 67(9): 1201 - 1203. [Full Text] [PDF]
|
|
|
|
 |
|
 B. Mariniello, V. Ronconi, S. Rilli, P. Bernante, M. Boscaro, F. Mantero, and G. Giacchetti Adipose tissue 11{beta}-hydroxysteroid dehydrogenase type 1 expression in obesity and Cushing's syndrome Eur. J. Endocrinol., September 1, 2006; 155(3): 435 - 441. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. U Onyimba, N. Vijapurapu, S J. Curnow, P. Khosla, P. M Stewart, P. I Murray, E. A Walker, and S. Rauz Characterisation of the prereceptor regulation of glucocorticoids in the anterior segment of the rabbit eye. J. Endocrinol., August 1, 2006; 190(2): 483 - 493. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. D. Giraud, S. Louey, S. Jonker, J. Schultz, and K. L. Thornburg Cortisol Stimulates Cell Cycle Activity in the Cardiomyocyte of the Sheep Fetus Endocrinology, August 1, 2006; 147(8): 3643 - 3649. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Draper and P. M Stewart 11{beta}-Hydroxysteroid dehydrogenase and the pre-receptor regulation of corticosteroid hormone action J. Endocrinol., August 1, 2005; 186(2): 251 - 271. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Y. Zhang, X. Ding, and R. A. Daynes The Expression of 11{beta}-Hydroxysteroid Dehydrogenase Type I by Lymphocytes Provides a Novel Means for Intracrine Regulation of Glucocorticoid Activities J. Immunol., January 15, 2005; 174(2): 879 - 889. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. Tomlinson, E. A. Walker, I. J. Bujalska, N. Draper, G. G. Lavery, M. S. Cooper, M. Hewison, and P. M. Stewart 11{beta}-Hydroxysteroid Dehydrogenase Type 1: A Tissue-Specific Regulator of Glucocorticoid Response Endocr. Rev., October 1, 2004; 25(5): 831 - 866. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. De Bosscher, W. Vanden Berghe, and G. Haegeman The Interplay between the Glucocorticoid Receptor and Nuclear Factor-{kappa}B or Activator Protein-1: Molecular Mechanisms for Gene Repression Endocr. Rev., August 1, 2003; 24(4): 488 - 522. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Cooper, A. Blumsohn, P. E. Goddard, W. A. Bartlett, C. H. Shackleton, R. Eastell, M. Hewison, and P. M. Stewart 11{beta}-Hydroxysteroid Dehydrogenase Type 1 Activity Predicts the Effects of Glucocorticoids on Bone J. Clin. Endocrinol. Metab., August 1, 2003; 88(8): 3874 - 3877. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Shafqat, B. Elleby, S. Svensson, J. Shafqat, H. Jornvall, L. Abrahmsen, and U. Oppermann Comparative Enzymology of 11beta -Hydroxysteroid Dehydrogenase Type 1 from Glucocorticoid Resistant (Guinea Pig) Versus Sensitive (Human) Species J. Biol. Chem., January 10, 2003; 278(3): 2030 - 2035. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. Tomlinson, B. Sinha, I. Bujalska, M. Hewison, and P. M. Stewart Expression of 11{beta}-Hydroxysteroid Dehydrogenase Type 1 in Adipose Tissue Is Not Increased in Human Obesity J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5630 - 5635. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
