Intracrine modulation of gene expression by intracellular generation of active glucocorticoids

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Intracrine modulation of gene expression by intracellular generation of active glucocorticoids Oren Fruchter a,b,∗ , Emmanouil Zoumakis a , Salvatore Alesci a , Massimo De Martino a , George Chrousos a , Zeev Hochberg a,b a

Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA b Meyer Children’s Hospital, Rambam Medical Center, Haifa 31096, Israel

a r t i c l e

i n f o

a b s t r a c t

Article history:

Glucocorticoids (GC) by either up-regulating or down-regulating the expression of genes

Received 11 March 2006

influence cellular processes in every tissue and organ of the body. The enzyme 11␤-

Received in revised form

hydroxysteroid dehydrogenase Type-1 (11␤-HSD-1) confers bioactivity upon the inactive GC

23 July 2006

cortisone (E) and prednisone (P) by converting them to cortisol (F) and prednisolone (L),

Accepted 8 August 2006

respectively. We sought to investigate whether gene expression modulation by GC is under

Published on line 22 September 2006

the regulation of an intracrine mechanism that determines the intracellular concentration of active GC.

Keywords:

Human cell lines were transiently and stably co-transfected with an expression construct

11␤-Hydroxysteroid dehydrogenase

for 11␤-HSD-1 and a GC-responsive reporter gene and incubated with active and inactive

type-1

GC. Whereas in cells that were not transfected with the expression construct for 11␤-HSD-1

Glucocorticoids

inactive GC had no transcriptional activity, in both transiently and stably transfected cells E

Gene expression

and P demonstrated a dose-dependent transcriptional activity. This transcriptional potency of both inactive GC was effectively abolished by carbenoxolone, an 11␤-HSD-1 inhibitor, and was directly related to the concentration of transfected 11␤-HSD-1. We conclude that gene expression modulation by GC is under a decisive influence of target cell 11␤-HSD-1 that modulates the intracellular concentration of active GC. The intracrine mechanism is an under-appreciated aspect of GC activity that could be a potential target for future therapies aimed at modulating GC effects at the cellular level. Crown Copyright © 2006 Published by Elsevier Inc. All rights reserved.

1.

Introduction

Biochemical events and cellular processes in every tissue and organ of the body are influenced by glucocorticoids (GC) via the glucocorticoid receptor (GR) [1]. Upon hormone binding, the GR undergoes translocation to the nucleus, where it binds to glucocorticoid receptor-responsive element (GRE) as a partner transcription factor to communicate with the transcription machinery. As such, it can either positively or negatively reg-

∗

ulate gene expression [2]. Cellular response to GC results from an interplay between at least three factors: the intracellular concentration of free hormone, the relative affinity of the particular GC to GR, and the ability of the specific cell to receive and transduce the hormonal signal [3]. Research in the past two decades has highlighted the importance of pre-receptor metabolism of GC as a way of modulating their action [4–9]. For GC, the key gate-keeping enzymes are the 11␤-hydroxysteroid dehydrogenases (11␤-

Corresponding author at: Meyer Children’s Hospital, Rambam Medical Center, Haifa 31096, Israel. Tel.: +972 4 8542321; fax: +972 4 8542661. E-mail address: oren [email protected] (O. Fruchter).

0039-128X/$ – see front matter Crown Copyright © 2006 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2006.08.002

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HSD). Two isoforms of 11␤-HSD are now described. 11␤HSD-2 is a high affinity NAD+ -dependent enzyme located in the placenta and mineralocorticoid target tissues, such as the kidney, colonic mucosa, and salivary glands. It converts active GC (cortisol and corticosterone) into inactive 11-ketosteroids [5–10]. 11␤-HSD-1 is a low-affinity NADP(H)dependent enzyme, whereas hexose-6-phosphate dehydrogenase, an endoluminal enzyme provides reduced NADP(H) as cofactor to 11␤-HSD1 to permit reductase activity. The enzyme is expressed mainly in human liver, adipose tissue, decidua, lung, pituitary, gonads, and cerebellum. This enzyme acts in vivo mainly as an 11 oxo-reductase and converts inactive GC, cortisone and prednisone, into their biologically active metabolites, cortisol and prednisolone, respectively [11–13]. In 1991, Labrie coined the term intracrinology to denote enzymes expressed in peripheral tissues which are responsible for generation of androgens and estrogens, and allow for transcriptional activity of these steroids within the generating cell itself [14]. In previous report, we examined GC target cells (human adipocytes), and demonstrated a dose dependant generation of cortisol from cortisone that was entirely abolished when 11␤-HSD-1 activity has been blocked by carbenoxolone [15]. The concept of intracrinology has been suggested for 11␤HSD-1, but no direct association has been demonstrated between 11␤-HSD-1 activity and the most important aspect of the role of GC in human target cells, namely, the regulation of gene expression. The hypothesis of the present study was that cortisol and prednisolone are generated from inactive cortisone and prednisone and act within the same cell to modulate the transcription of GC-responsive genes. We used human cells that express both 11␤-HSD-1, the GR, and a GC-responsive reporter gene, and demonstrated transcriptional activity induced by inactive GC that were converted to their active metabolites by 11␤-HSD-1. Our findings provide direct evidence of the intracrine decisive role of 11␤-HSD-1, acting as a pre-receptor modulator of the GC-regulated gene transcription.

2.

Materials and methods

2.1.

Plasmids

2.2.

Fetal kidney 293 cells (HEK-293) that were stably transfected with the expression construct of 11␤-HSD-1 and then selected for its expression [16]. These cells were kindly provided by Professor Paul M. Stewart and Dr. Iwona J. Bujalska (University of Birmingham, Birmingham, UK). Cells were maintained as described [15]. Human hepatocellular carcinoma cells, HepG2/C3A (ATCC CRL-10741) [17] were cultivated in Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (D-MEM/F-12) containing 10% heat-inactivated FCS, 100 units/ml penicillin, and 100 ␮g/ml streptomycin at 37 ◦ C, in humidified atmosphere of 5% CO2 /air.

2.3.

Transient transfection of HEK-293 cells

Cells were transfected with various plasmid constructs at indicated DNA concentrations with Lipofectamine 2000 (Invitrogen Corp., San Diego, California) according to the manufacturer’s instructions. Cells were transfected with 1.6 ␮g/well of the MMTV-luciferase reporter, 0.3 ␮g/well of pSV40-␤-Gal, and 0.5 ␮g/well of hGR␣ coding plasmid. Effectors were added to the cells incubated in serum-free media. Cells were incubated with GC for 16 h.

2.4.

Transient transfection of HepG2/C3A cells

Cells were transfected with various plasmid constructs at indicated DNA concentrations with Lipofectamine 2000 (Invitrogen Corp., San Diego, California) according to the manufacturer’s instructions. Cells were transfected with 10 ␮g of the MMTV-luciferase reporter, 2 ␮g of pSV40-␤-Gal, 3 ␮g of hGR␣ coding plasmid and pCR3-11␤-HSD-1 at indicated DNA concentrations with or without pCR3 that was used as a control carrier DNA to yield a constant amount (24 ␮g) of transfected DNA per plate. Thirty hours after transfection, media was changed to serum-free media and cells were incubated with GC for 16 h. When carbanexalone (CBX) – a non-specific inhibitor of 11␤-HSD-1 and -2 – was used, cells were preincubated with CBX for 3 h before GC were added.

2.5.

pMMTV-Luc contains the luciferase gene under the control of the mouse mammary tumor virus (MMTV) promoter that has four functional glucocorticoid-responsive elements (GRE’s) and was a gift from Dr. G.L. Hager (National Institutes of Health, Bethesda, Maryland). pSV40-␤-Gal was purchased from Promega (Piscataway, Wyoming). PRShGR␣ contains the full-length coding region of hGRa´ under the control of the constitutively active Rous sarcoma virus promoter and was kindly donated by Dr. R. Evans (Salk Institute, La Jolla, California). We used pCR3 expression construct (pCR3-11␤-HSD-1) containing full-length coding region cDNA for human 11␤-HSD-1 [16] This plasmid was a kind gift from Professor Paul M. Stewart and Dr. Iwona J. Bujalska (University of Birmingham, Birmingham, UK). The plasmid pCR3 was purchased from Promega (Piscataway, Wyoming) and contains no functional coding region but is otherwise similar to pCR3-11␤-HSD-1.

Cell culture

ˇ-Galactosidase and luciferase activities

Measurements were performed according to the manufacturer’s instructions using Wallac 1420 Victor2 multilabel counter (Wallac Oy, Turku, Finland) for ␤-galactosidase (Galacto-Light Plus, Tropix, Bedford, Massachusetts) and luciferase (Luciferase assay reagent, Promega, Madison, Wyoming) measurements.

2.6.

11ˇ-HSD-1 activity assay

Experiments were done in triplicates. Effectors were added to the cells already incubated in basal medium overnight (for stably-transfected HEK-293 cells), or, in the case of HepG2/C3A cells, 30 h after they had been transiently transfected with pCR3-11␤-HSD-1, as described above. Media were replaced and cells were incubated with 500 nM of cortisone (Sigma, St. Louis, Missouri) for 3 h. The supernatant was collected and assayed for cortisol, using a radioimmunoassay kit (Diagnostic

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1003

Products Corporation, Los Angeles, California), as previously reported [15]. The fractional conversion of cortisone to cortisol was calculated.

2.7.

Data analysis

Relative luciferase activities were calculated by dividing the luciferase activity by the respective ␤-galactosidase activity to correct for differences in transfection efficiency. The data represents the mean ± S.E.M. obtained from three independent experiments, each performed in triplicates (for HEK-293 cells) or octaplicates (for HepG2/C3A cells). Pearson’s correlation coefficient was calculated between two related variables to study their relation. Differences in mean values were tested by using Mann–Whitney U-test or, when appropriate, by non-parametric ANOVA (Kruskal–Wallis ANOVA followed by Dunn’s post hoc test). Statistical significance was set at P < 0.05. Data were analyzed using GraphPad InStat version 3.00 for Windows 95 (GraphPad Software, San Diego, California).

3.

Results

3.1.

Studies on 11ˇ-HSD-1/HEK-293 cells

HEK-293 cells were stably transfected and selected for the expression of 11␤-HSD-1. The calculated conversion rate of cortisone to cortisol in these cells was 37 ± 12% (mean ± S.E.M.), indicating a highly efficient level of conversion by 11␤-HSD-1 in these cells. In a preliminary set of experiments, transfection of 11␤HSD-1/HEK-293 cells with pMMTV-luc reporter plasmid alone did not show luciferase activity in response to either of the active GC, cortisol or prednisolone, indicating that these cells do not express substantial amounts of functional GR (data not shown). When we co-transfected the cells with pMMTVluc and the plasmid that contained the coding region of hGR␣, incubation with cortisol at a concentration of 10−6 M resulted in a 12 ± 1.5-fold induction in luciferase activity compared to basal activation (Fig. 1). Prednisolone was significantly more potent than cortisol with respect to its transcriptional activity. The corresponding fold induction in luciferase activity when cells were incubated with prednisolone at a concentration of 10−6 M was 37 ± 6.4 (P < 0.01; for the difference in luciferase activity between cortisol and prednisolone; Mann–Whitney U-test). We observed that the transcriptional activity of cortisone at 10−6 M was significantly greater than that of its active metabolite cortisol at equimolar concentration (P < 0.01). Although prednisone tended to be more potent in terms of transcriptional activity than prednisolone at a concentration of 10−6 M (51.2 ± 0.9 versus 37.6 ± 6.4, respectively), this difference was not statistically significant. We demonstrate a dose-dependent transcriptional activity plot for both cortisone and prednisone in concentrations ranging from 10−9 M to 10−5 M. For cortisone, a trend towards a statistically significant correlation between the compound’s concentration and potency that is necessary to activate the pMMTV-luc reporter gene was observed (P = 0.058; Spearman r = 0.829) while, for prednisone, a highly significant correlation

Fig. 1 – The comparative transcriptional potencies of active and inactive glucocorticoids in 11␤-HSD-1–HEK-293 cells. Data are plotted as fold change of corrected luciferase activities from basal activation (mean ± S.E.M., n = 3). (* P < 0.05, ** P < 0.01, for the difference in fold induction of corrected luciferase activities; Mann–Whitney U-test.)

was demonstrated (P < 0.003; Spearman r = 1.0). For cortisone, we show a dose-dependent transcriptional potency (P < 0.0005, non-parametric repeated measures ANOVA). A similar dosedependent effect was noted for prednisone (P value was 0.0004). The transcriptional potency of cortisone, at a concentration of 10−5 M, was 26.9 ± 6.5-fold induction, compared to 67.9 ± 11.5 for prednisone at equimolar concentration.

3.2. cells

Studies on transiently transfected HepG2/C3A

HepG2/C3A cells were transiently transfected with the expression plasmid for 11␤-HSD1. The calculated conversion rate of cortisone to cortisol in these cells, as measured by radioimmunoassay, was 14 ± 4% (mean ± S.E.M.). In a preliminary set of experiments, no transactivation of MMTV was induced by cortisol and prednisolone, unless they were transfected with the hGR plasmid, indicating that these cells do not express substantial amounts of functional hGR (data not shown). We compared the transcriptional activity of cortisone and prednisone in HepG2/C3A cells that were transfected with increasing amounts of pCR3-11␤-HSD-1 (Fig. 2A and B). When cells were transfected only with the mock vector, pCR3 induced no transcriptional activity with cortisone or prednisone, indicating that their activation by 11␤HSD-1 is an essential step for transcriptional activity. We observed a statistically significant dose–response relationship between the amount of DNA pCR3-11␤-HSD-1 transfected and the compound’s transcriptional activities, expressed in corrected luciferase activity fold induction over baseline activity (r = 0.9562, P = 0.0167; and r = 0.9432, P = 0.0245 for cortisone and prednisone, respectively, Spearman rank correlation). When cells were co-transfected with pMMTV-luc and pCR3-11␤-HSD-1, cortisone or prednisone at concentrations ranging from 10−10 M to 10−5 M had dose-dependent transcriptional activity (Fig. 3). Cortisone achieved a maximal transcriptional activity of 59.4 ± 10.5-fold induction at 10−5 M compared

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Fig. 3 – Carbenoxolone (CBX) inhibits the transcriptional activity of prednisone in transiently transfected HepG2/C3A cells. Cells were incubated with prednisone and prednisolone with (open circles and open squares, respectively) and without CBX (solid circles and solid squares, respectively). Data are plotted as fold change of corrected luciferase activities from basal activation (mean ± S.E.M., n = 8). (P < 0.05, for the difference in fold induction in corrected luciferase activities at a given concentration of prednisone between CBX-treated and untreated cells Mann–Whitney U-test.)

whereas CBX had no effect on the transcriptional activity of either prednisolone (Fig. 3) or cortisol.

Fig. 2 – The transcriptional activity of cortisone and prednisone depends on the molar concentrations of the transfected expression vector for 11␤-HSD-1. HepG2/C3A cells were transfected with increasing concentrations of pCR3-11␤-HSD-1. Cells were incubated with cortisone at 10−7 M (solid circle) and 10−6 M (solid square) (A) or prednisone at 10−7 M (solid circle) and 10−6 M (solid square) (B). Data are plotted as fold change from basal luciferase activity (cells transfected only with the mock vector for pCR3-11␤-HSD-1) (means ± S.E.M., n = 8). (P values represent the dose–response relationship between the amount of DNA pCR3-11␤-HSD-1 transfected and the compound’s transcriptional activities, expressed in corrected luciferase activity fold induction over baseline activity (r = 0.9562, P = 0.0167; and r = 0.9432, P = 0.0245 for cortisone and prednisone, respectively), Spearman rank correlation.)

to 52.4 ± 9.9-fold for prednisone at the same concentration (P > 0.05). Half-maximal activity was at 495 nM and 154 nM for cortisone and prednisone, respectively. CBX was used at a concentration of 5 ␮M as it has been found to be the optimal concentration for blockade of 11␤-HSD-1 [15]. HepG2/C3A cells that were incubated for 16 h in the presence of CBX alone demonstrated no luciferase activity, indicating that at that concentration CBX has no transcriptional potency (data not shown). In HepG2/C3A cells, CBX effectively abolished the transcriptional potency of both prednisone (Fig. 3), and cortisone,

4.

Discussion

The present study provides direct evidence of an intracrine mechanism for the action of GC. 11␤-HSD-1 modulates glucocorticoid-responsive genes by controlling the intracellular availability of active GC–GR binding and transactivation of GRE in GC-responsive genes within the same cell. Moreover, using transfected cells that have been previously used to study steroid metabolism [16,17], we show for the first time that the most important determinant of GC biological effectiveness within a target organ is the intracellular rather than circulating concentrations of active 11␤-hydroxysteroids; at any given concentration, cortisone-generated cortisol and prednisone-generated prednisolone were more active than cortisol or prednisolone added to the culture medium. In the first set of experiments we used stably transfected HEK-293 cells constitutively expressing 11␤-HSD-1. This model was created by a selection of a clone of human fetal kidney 293 cells (devoid of endogenous 11␤-HSD activity) that had been stably transfected with 11␤-HSD-1 expression vector as previously described [16]. Hence we ensured long-term, reproducible as well as well-controlled enzyme expression. This enabled us to study the effect of enzyme inhibition in a well-defined constant intracellular environment. In the second set of experiments, the transient transfection system of HepG2/C3A cells allowed the use of increasing level of enzyme expression to demonstrate the dose-dependent pre-receptor modulation by 11␤-HSD-1. We now show that conversion of biologically inactive compounds (cortisone and prednisone) into their active 11-

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hydrosteroids results in transcriptional activity within the same cell. Furthermore, we showed that this transcriptional potency was entirely dependent on the activity of 11␤-HSD1, as it was effectively abolished when this enzyme was blocked. Carbenoxolone effectively inhibited transcriptional activity induced by cortisone and prednisone in HepG2/C3A Cells (Fig. 3). As previously reported by us and others [15,16], we used GC concentrations ranging from 10−10 M to 10−5 M. The fact that the response curves in the current report are not identical to previous reports reflects the different cellular environments created by transfection of different cell-lines by different expression constructs for 11␤-HSD-1. Surprisingly, the transcriptional activity of cortisone was greater than that of its active metabolite, cortisol, at equimolar concentration. Pre-receptor modulation by11␤-HSD-1 turns out to create an intracellular advantage, for direct access of the locally generated GC to the GR within the same cell. This form of intracrine modulation, resulting in an increase of the local concentration of biologic active 11-hydrosteroids, is a pivotal determinant of tissue-specific responsiveness to GC [4,5]. Furthermore, transient transfection of HepG2/C3A cells allowed us to test the effect of over-expressing 11␤-HSD-1. It is the mechanism thought to be involved in certain important pathological conditions. There is increasing evidence that 11␤HSD-1 activity per se, plays an important role independent of circulating GC levels in the pathogenesis of common diseases, such as obesity, insulin resistance, and essential hypertension [18–23]. Hence, 11␤-HSD-1 activity is enhanced in hypothalamic obesity [19] and seems to be an important mechanism for visceral obesity [19–24]. By transfecting HepG2/C3A cells with increasing level of enzyme expression, we demonstrated that activation of cortisone and prednisone by 11␤-HSD-1 is mandatory for their transcriptional activity and that transcriptional activity was directly related to the amount of expression vector for 11␤HSD-1 that has been used. In humans, circulating cortisol is 95% bound to corticosteroid-binding globulin, resulting in free cortisol levels of 1 nM at nadir during late evening and 100 nM during its diurnal peak [11]. In contrast, cortisone circulates at levels around 50–100 nM, largely unbound to plasma proteins and without a pronounced diurnal rhythm, thus providing a substantial pool of inert 11-ketosteroids substrate available for activation by tissue 11␤-HSD-1 [11]. Prednisone is widely used in a variety of clinical conditions both as an anti-inflammatory agent and an immunosuppressive drug, and as part of many chemotherapy protocols [25,26]. Its activity reflects the contribution of properties of the drug, its pre-receptor metabolism, the GR, and properties of the target tissues in which it acts [26–29]. Establishing the role of pre-receptor regulation by 11␤-HSD-1 is vital to its potency. Low et al. [30] studied the transcriptional activity of inactive 11-ketosteroids in the COS-7 green monkey cell line. They found that 11-dehydrocorticosterone induced MMTV luciferase expression in COS-7 cells that transiently expressed rat 11␤-HSD-1. Warrier et al. [31] used the monkey cell line CV1, which they co-transfected with human 11␤-HSD expression vector and MMTV-chloramphenicol acetyltransferase (MMTVCAT) as a reporter gene. Our current report extends the previous ones in several other aspects.

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Understanding the tissue-specific function of 11␤-HSD-1 is likely to suggest novel approaches to target experimental and therapeutic manipulations of GC action in various target organs. In prescribing GC, the clinician has to be aware of their vulnerability to 11␤-HSD metabolizing effect. The apparently active cortisol and prednisolone are inactivated by the high affinity 11␤-HSD-2, while its apparently inactive products, cortisone and prednisone, provide a more potent substrate for the target tissue’s 11␤-HSD-1. Furthermore, 11␤-HSD-1 inhibitors are currently being tested both in animal models and humans as therapeutic agents for treatment of a variety of clinical conditions and diseases [32–34]. In the current report, CBX effectively abolished the activity of GC whose transcriptional potency depends on modulation by 11␤-HSD-1 in a human liver cell line. Hence, the expression of many proteins expressed in the liver under the influence of endogenous or exogenous GC is likely to be affected by using 11␤-HSD-1 inhibitors. This observation is likely to have many desired clinical implications as well as undesirable effects yet to be studied as selective 11␤-HSD-1 inhibitors become available for clinical practice. Taken together, our results derived from two different models for the expression of 11␤-HSD-1 in human cell lines, provide evidence for the essential role of the enzyme in the intracrine regulation of GC activity within target tissues. By creating an intracellular environment in which the concentration of active GC is more important than their circulating concentrations, 11␤-HSD-1 regulates GC potency within a given tissue. We were able to provide a direct evidence that 11␤-HSD1 can modulate the most important aspect of GC role in human target cells—the regulation of gene expression. Furthermore, this pre-receptor regulatory mechanism of gene expression and protein synthesis have been demonstrated to be potentially modulated by administration of 11␤-HSD-1 inhibitors. The clinical implications of these observations are likely to be revealed in future studies.

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