Blood pressure (BP) is controlled by the intravascular fluid volume via renal sodium handling and by vasoconstriction. One mechanism involved in BP control is the intracellular availability of cortisol. High intracellular cortisol levels induce vasoconstriction (1–3) and enhance renal sodium retention (4). Intracellular cortisol availability is regulated in renal, vascular, colonic epithelial, and placental tissues by 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), an enzyme that converts biologically active cortisol into inactive cortisone (1–5). Several clinically relevant factors, including hypoxia, TNF-α, angiotensin II, shear stress, and methylation status, have been described to control 11β-HSD2 activity (6–10).
Nucleotides are intracellularly present at a concentration ranging from approximately 2 to 5 mmol/L (11). Cellular injury but also numerous biologic processes without cellular damage cause a release of mononucleotides (e.g., ATP) into the extracellular compartment (12,13). Nucleotides bind to purinergic cell surface receptors; either ATP-gated channels named P2X or heptahelical metabotropic receptors named P2Y. P2 receptors divide into an expanding number of members, currently seven for the P2X and 15 for the P2Y receptor group (11,14). Numerous cell types, including trophoblasts and renal tubular and colonic epithelial cells, have been shown to exhibit P2-related signals and express these receptors (15–21). In trophoblast cells, P2X7 (16) and P2Y6 (17) receptors have been detected and P2Y2 regulation has been demonstrated by agonist profiling (15,18,19). Despite the disclosure of intracellular signals involved in the activation of purinergic receptors, their functional consequences remain to be determined in many tissues.
Placental lesions such as arteriolopathy, hypermaturity of villi, intervillous thrombi, and central infarction are more frequent and severe in preeclampsia, a disease characterized by hypertension and renal damage with proteinuria (22–24). Placental ischemia and inflammatory responses during preeclampsia and increased shear stress lead to the release of ATP (9,25–27). The cortisol-inactivating enzyme 11β-HSD2 is highly expressed in trophoblasts and the trophoblast cell line JEG-3 (9,10,22). One important function of the 11β-HSD2 in placental tissue is to protect the fetus from high maternal cortisol concentrations. So far, the effect of mononucleotides on 11β-HSD2 activity was analyzed only in one study. Yang et al. (28) incubated placental microsomal extracts that contained 11β-HSD2 activity with ATP. They observed an enhanced 11β-HSD2 activity independent of the site for substrate binding.
The reduction of 11β-HSD2 activity in the presence of high ATP levels observed in preeclampsia (22,23) prompted us to determine whether ATP might contribute to a reduced 11β-HSD2 activity in trophoblasts and to elucidate whether intact cells are needed to modulate the 11β-HSD2 activity by extracellular mononucleotides. To explore this hypothesis, we investigated the well-characterized human choriocarcinoma cell line JEG-3 displaying endothelial properties and two cell lines with epithelial features: LLCPK1, a renal tubular, and SW620, a human colonic cell line. All cell lines have previously been used to study the regulation of 11β-HSD2 activity (7,8,29).
Materials and Methods
Material and Cell Lines
Cell culture material was from Becton Dickinson Labware (Basel, Switzerland) and Corning (Bodenheim, Germany). Cortisol, glycyrrhetinic acid, minimal essential medium Eagle (MEME), DMEM, Bradford reagent, and transferrin were purchased from Sigma Chemical (Buchs, Switzerland), and actinomycin D was purchased from Calbiochem-Novabiochem (La Jolla, CA). Amersham (Buckinghamshire, UK) provided 3H-cortisol (specific activity 2.33 TBq/mmol). Thin-layer chromatography plates (silica gel 60F254) were from Macherey-Nagel (Oensingen, Switzerland), and FCS was from Biological Industries (Noisy le Grand, France). TaqMan Gene Expression Assays and Assays on Demand were from Applied Biosystems, and primers were from Microsynth (Balgach, Switzerland). All nucleotides including 3′-O-(4′-benzoyl)-benzoyl-ATP (BzATP), 2′-methylthio-ATP (MeSATP) and α,β-methylene-ATP (APCPP), suramin, pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), Ro-31-8220, and cycloheximide were from Sigma Aldrich (Fluka AG, Buchs, Switzerland). The human choriocarcinoma cell line JEG-3 (accession HTB-36), the LLCPK1 (accession CLL-227), and the SW620 (accession CLL-101) cell line were from ATCC (Manassas, VA). All have been characterized extensively by us (8–10,29).
Cell Cultures
JEG-3 cells were cultured in MEME supplemented with 10% FCS, 2 mmol/L glutamate, 100 U/ml penicillin, and 100 μg/ml streptomycin. LLCPK1 and SW620 cells were maintained in DMEM with 0.45% glucose. Cells were passaged up to 20 times with trypsin/EDTA, plated in cell culture dishes, grown to confluence, and washed twice with PBS (pH 7.4) at 37°C before the experiments were started. At the completion of the final incubation period, cells again were washed twice with ice-cold PBS and the experiment proceeded according to subsequent protocols. Incubations were performed for 24 h, if not otherwise indicated. Cell viability was assessed by measuring lactate dehydrogenase concentrations in the supernatant, by performing FACS analysis for annexin V, and by testing for cell diploidy with propidium iodide labeling. Cells were scraped and lysed in TS2 buffer (sucrose 250 mmol/L, 100 mmol/L NaCl, 1 mmol/L EGTA, 1 mmol/L EDTA, 1 mmol/L MgCl2, and 20 mmol/L Tris-HCl [pH 7.4]) and stored at −20°C. Protein content was determined using the Bradford protein assay and the bicinchoninic acid method (Pierce, Rockford, IL).
3H-Cortisol/Cortisone Conversion Assay in Cell Homogenates to Determine 11β-HSD2 Activity
The cortisol/cortisone conversion was used to measure oxidation at C-11 by 11β-HSD2 following protocols described earlier (9,10). In brief, cell homogenates were incubated with 5 nCi [3H]cortisol, 10 nmol/L cortisol, and 200 μmol/L nicotinamide adenine dinucleotide in sucrose buffer. The reaction was stopped by adding 20 μl of 1 mg/ml unlabeled cortisol and cortisone in methanol and thin-layer chromatography developed in chloroform-methanol (90:10 vol/vol). Steroids were located using ultraviolet light, excised, and counted in a Packard scintillation counter (Tri Carb 2000CA; United Technologies, Hartford, CT). Specific activity was expressed as picomoles per milligrams protein per hour. The assay was repeated up to four times using different protein concentrations within each individual experiment.
Protein Extraction and Western Blot Analysis
Cells were lysed with cold lysis buffer (100 mM NaCl, 50 mM sodium fluoride, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.1 mM PMSF, 1 mM orthovanadate, and 50 mM Tris-HCl [pH 7.5]) for 5 min at room temperature. The total cell lysates were collected with a cell scraper, vortexed vigorously, and centrifuged at 10,000 × g for 20 min at 4°C. The supernatant was collected, and the protein content was determined as described above.
For Western blot analysis, total protein (40 μg) was loaded on a denaturing 10% polyacrylamide gel. Benchmark prestained protein ladder (Invitrogen, Carlsbad, CA) was used as a marker. The transfer of protein to polyvinylidene difluoride membrane (Hybond-P; Amersham Biosciences, Bucks, UK) was performed with a constant voltage of 60 V for 1 h on ice. The membrane was blocked overnight in 5% nonfat dry milk in TBS-T (0.1% Tween 20 and 0.1% in TBS), washed with TBS-T, and incubated for 2 h with rabbit polyclonal antibody for 11β-HSD2 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), P2Y1, and P2Y11 (1:500; Alomone Laboratories, Jerusalem, Israel) in 0.5% nonfat dry milk in TBS-T. Negative control experiments with the control peptide (preincubation with the same amount of antibody for 1 h at room temperature) used for immunization were done to check specificity of the P2Y1 and P2Y11 primary antibodies. The polyvinylidene difluoride membrane was washed with TBS-T and incubated for 1 h at room temperature with a goat anti-rabbit IgG horseradish peroxidase conjugate (Santa Cruz Biotechnology) diluted 1:10,000 in TBS-T that contained 0.5% milk. After washing, the detection was performed with the enhanced chemiluminescence kit (Amersham Biosciences). The filter then was exposed to x-ray film (Eastman Kodak Co., Rochester, NY) for 1 to 5 min. Densitometry was performed on the radiographs, and the level of 11β-HSD2 protein was expressed as arbitrary units.
Extraction of Total RNA and Preparation of cDNA Pools
Cell lines (JEG-3, LLCPK1, and SW620) were rinsed with ice-cold PBS and collected by centrifugation (3000 × g at 4°C for 3 min). Following the Promega SV (Wallisellen, Switzerland) total RNA isolation system, cells were resuspended in 175 μl of SV RNA lysis buffer followed by the addition of 350 μl of SV RNA dilution buffer. After mixing and heating (70°C for 3 min), the samples were centrifuged (14,000 × g for 3 min), the clear lysates were transferred to a fresh tube, and 200 μl of 95% ethanol was added. RNA was collected by spinning for 1 min at 14,000 × g and washed using 600 μl of SV RNA washing solution. DNaseI treatment for 15 min with consecutive addition of SV DNase stop solution and repeated washing (SV RNA wash solution) was performed. RNA concentration was determined after final elution of RNA with ddH2O by using a spin column (14,000 × g for 1 min), and the RNA was stored at −70°C. One microgram of total RNA of cells that were grown with and without the pharmacologic agents as indicated in the Results section were reverse transcribed using random hexamers and Improm II reverse transcriptase according to the Improm II Promega protocol.
TaqMan Gene Expression Assays for P2X and P2Y Receptors and for 11β-HSD2
Homology-based TaqMan PCR was performed with assay-on-demand primers and probes to identify and quantify P2 receptors and 11β-HSD2 mRNA expression. Equal amplification efficiencies were verified. Negative controls for the reverse transcriptase and the PCR reagents remained negative. Primer sequences for the human P2X and P2Y receptors as well as for 11β-HSD2 and 18S-RNA are presented in Table 1. All PCR were normalized for the respective 18S-RNA content, and P2X5 was arbitrarily chosen to be the calibrator in the comparative analysis, an approach described previously for the analysis of P2 receptor mRNA expression (30).
Plasmid Construction, Transfections, Luciferase, and β-Galactosidase Assays to Identify 11β-HSD2 Promotor Activity
The plasmid construction was as reported earlier by our group (29). Plasmid DNA was prepared using QIAfilter columns (Qiagen, Hambrechtikon, Switzerland). Transfections were performed with FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Basel, Switzerland) following the manufacturer’s recommendations. FuGENE 6 (1.2 μl) and 0.8 μg of plasmid DNA were incubated in serum-free medium for 15 min before application on subconfluent JEG-3 cells in 24-well plates. The reporter plasmids were co-transfected with 0.05 μg of either a pCMV-LacZ or pSV-LacZ control plasmid to correct for transfection efficiency. After a 24-h incubation, the medium was changed against new MEME and the cells were treated as indicated for 24 h. Finally, the medium was removed, and the cells were washed twice with PBS, lysed in 100 μl of lysis buffer, and assayed for luciferase and β-galactosidase activity using the Applied Biosystems dual-light reporter gene assay system. Chemiluminescence was measured with a MediatorsPhL luminometer (Mediators Diagnostic Systems, Vienna, Austria). The normalized values were from triplicate samples.
Statistical Analyses
All data are presented as means ± SEM. To test for statistically significant differences, we used t test, ANOVA, or multiple comparisons versus control group (Tukey test), as appropriate. Significance was assigned at P < 0.05. All statistical analyses were performed using SYSTAT Version 10 (SPSS Inc., Chicago, IL).
Results
Effect of ATP or Its Stable Analogue ATPγS on 11β-HSD2 Activity in JEG-3, LLCPK1, and SW620 Cells
In JEG-3 cell homogenates, 11β-HSD2 enzyme activity, assessed by the conversion of 3H-cortisol to 3H-cortisone, was present (1.46 ± 0.15 pmol/mg protein per h) as has been shown previously (9,10,31). This conversion was almost completely inhibited when glycyrrhetinic acid (10−6 mol/L) was added to the cell culture medium. Adding Triton-X100 to the 11β-HSD assay mixture abolished the enzyme activity, an observation indicating that the activity is attributable to 11β-HSD2 and not to 11β-HSD1 enzyme, a contention supported by the expression of 11β-HSD2 but not 11β-HSD1 mRNA (9,32). Addition of ATP (Figure 1A) or its more stable analogue ATPγS (Figure 1B) to JEG-3 cells for 24 h consistently reduced 11β-HSD2 activity. To determine whether 11β-HSD2 inhibition by ATP is cell type specific, LLCPK1 and SW620 cell lines, both previously investigated for 11β-HSD2 regulation (7,8,29), were also studied. ATP or ATPγS inhibited 11β-HSD2 activity in LLCPK1 but not in SW620 cells (Figure 1), suggesting a cell type specific response. The 11β-HSD2 activity was 1.35 ± 0.06 and 1.63 ± 0.26 pmol/mg protein per h, respectively, in cultured LLCPK1 and SW620 cells.
In JEG-3 and LLCPK1 cells, the inhibition by ATPγS was time dependent (Figure 2A). Reversibility was demonstrated by removing ATPγS from the cell culture supernatant after 24 h of incubation and measuring 11β-HSD2 activity at 48 h without ATPγS in JEG-3 cells (Figure 2B). ATP (“apparent IC50”: 6.7 × 10−5 mol/L) and ATPγS (“apparent IC50”: 2.3 × 10−6 mol/L) dose-dependently reduced 11β-HSD2 activity (Figure 3).
Regulation of 11β-HSD2 Activity Requires Intact Cells
An earlier report suggested a direct effect of ATP on cellular extracts containing 11β-HSD2 enzyme (28). For evaluating whether the 11β-HSD2 response requires intact cells, ATP or ATPγS were incubated with cultured JEG-3 (Figure 4A) and LLCPK1 (Figure 4B) cells or added to the cell homogenates from these cells. The 11β-HSD2 activity was inhibited by ATP or ATPγS in intact cells but not when added to homogenates.
Characterization of Purinergic Receptors with Impact on 11β-HSD2 Activity
At equimolar doses (10−4 mol/L), the activity of 11β-HSD2 in cultured JEG-3 and LLCPK1 cells was strongly inhibited by ATPγS, less by ATP, whereas neither the ATP degradation products ADP, AMP, and adenosine nor UTP or its degradation products were effective, except ADP in LLCPK1 cells (Figure 5). The relative response was more pronounced in JEG-3 cells when compared with LLCPK1 cells. PPADS but not the purinergic antagonist suramin prevented ATPγS-mediated inhibition of 11β-HSD2 (Figure 6).
To characterize further the P2Y receptors involved, we incubated JEG-3, LLCPK1, and SW620 cells with equimolar doses (10−4 mol/L) of ATPγS, MeSATP, BzATP, and APCPP (Figure 7). As expected, 11β-HSD2 activity was not inhibited by any of these nucleotides in SW620 cells. A minor but consistent activation by ATP (Figure 1), MeSATP, BzATP, and APCPP was present (Figure 7C). The 11β-HSD2 activity was clearly inhibited in LLCPK1 cells by ATPγS but did not respond to any of the other nucleotides (Figure 7B). Likewise, the activity of 11β-HSD2 in JEG-3 cells was strongly inhibited by ATPγS and less by BzATP (Figure 7A). In contrast to the ATPγS-induced inhibition, incubation with MeSATP increased the enzyme activity (Figure 7A).
Using the known sequences of human P2 receptors, we assessed the corresponding mRNA expression as a measure for the presence of these receptors (11) (Figure 8). In JEG-3 cells, the P2Y12 receptor was absent. Minor expression was found for P2X1, 2, 3, 5, and 7 receptors, whereas larger amounts of mRNA of the P2X4 and P2Y1, 2, 4, 6, and 11 receptors were present in JEG-3 cells. The most abundant P2Y receptor was the P2Y11 subtype. In SW620 cells, P2X4 and 5 and P2Y2, 4, and 11 mRNA was well detectable. No P2X2 and P2Y12 receptor mRNA was found in this cell line. All other receptors were present in minor quantities in these cells (Figure 8A). The largest differences in expression were detected for P2X5 (SW620>JEG-3 cells) and P2Y6 (JEG-3>SW620 cells) receptors. P2Y1 and P2Y11 receptor proteins were detected in both JEG-3 and SW620 cells (Figure 8B).
Cellular Responses to Purinergic Stimulation
Incubation of JEG-3 cells with ATPγS for up to 24 h reduced 11β-HSD2 activity (Figure 2A), a reduction prevented by hindering protein kinase C (PKC) signaling with the PKC inhibitor Ro31–8220 (P < 0.006). This coincided with a progressively reduced 11β-HSD2 mRNA content (Figure 9A). In contrast, the increased 11β-HSD2 activity in the presence of MeSATP was paralleled by an enhanced 11β-HSD2 mRNA availability as depicted in Figure 9B. Protein expression of 11β-HSD2 changed accordingly (Figure 9C). Reporter gene analysis of the human 11β-HSD2 promoter indicated that no transcriptional regulation was responsible for the altered cumulative mRNA (Figure 10A), thus suggesting posttranscriptional mechanisms (8). TNF-α and phorbol 12-myristate 13-acetate are known to reduce 11β-HSD2 promoter activity and served as controls (7,33). The assumption of a posttranscriptional regulation in the presence of ATPγS is supported by the observation of an enhanced degradation of 11β-HSD2 mRNA measured during transcriptional inhibition by actinomycin D (Figure 10B) and by a cycloheximide-promoted stabilization of 11β-HSD2 mRNA during coincubation with ATP/ATPγS (data not shown).
Discussion
The 11β-HSD2 enzyme protects the mineralocorticoid receptor (MR) from inappropriate activation by cortisol. Thus, it is a prereceptor enzyme that conveys selectivity of the MR to aldosterone. Even a subtotal reduction of the 11β-HSD2 activity has been shown to enhance MR activation by cortisol (4,6–8,10,34). This study identifies a significant downregulation of 11β-HSD2 activity by ATP. Inhibition of 11β-HSD2 activity by ATP or its stable analogue ATPγS was reversible and dose and time dependent. Given the potential of rapid degradation of ATP, we applied the analogue ATPγS, which has been demonstrated to be significantly more stable and to mimic the ATP response (35). In addition, we controlled for an effect of degradation products of extracellular ATP, such as AMP and adenosine, which did not imitate the inhibitory effect of ATP.
Yang et al. (28) investigated a putative posttranslational regulation of 11β-HSD2 by adding ATP to microsomal extracts. The exact mechanism remained uncertain but was independent of phosphorylation and energy supply by ATP and mimicked by ADP or AMP but not by other nucleotides. The approach used did not allow us to study purinergic receptor–mediated processes. In contrast, our observations in intact cells indicate that purinergic receptors are necessary to promote the changes in 11β-HSD2 activity.
Provided the regulation of 11β-HSD2 by mononucleotides requires intact cells, a cellular response to ATP should be detectable. Such a response was observed. The reduced 11β-HSD2 protein expression and activity were paralleled by a reduction of 11β-HSD2 mRNA, indicating a transcriptional or posttranscriptional regulation of transcript availability. Reporter gene analysis and the reduced half-life of 11β-HSD2 mRNA during incubation with ATP/ATPγS revealed that the reduction of 11β-HSD2 mRNA was not transcriptionally but rather posttranscriptionally mediated, a contention supported by the stabilization of the mRNA using the translational inhibitor cycloheximide. Incubation with ATP/ATPγS thus would induce a destabilizing protein, which interferes with 11β-HSD2 mRNA longevity. The presence of such a mechanism is plausible and indirectly supported by the observations that, first, angiotensin II destabilized 11β-HSD2 mRNA (10); second, 11β-HSD2 mRNA is posttranscriptionally regulated in trophoblasts (36); and, third, mononucleotides interfere with the mRNA stability of other genes in the presence of cytokines in mesangial cells (37).
Experiments were designed to define the receptors involved in the regulation of 11β-HSD2. A whole array of different P2 receptors has been described in recent years. As a consequence of an almost complete lack of specific antagonists, the agonistic potency of different mononucleotides had to be studied and the results compared with the pattern of responses that are known to be linked with activation of a specific receptor subtype, an approach used by other investigators for defining purinergic regulation of other genes (11,15,18,19,38–41). The inhibition of 11β-HSD2 activity by ATP in JEG-3 cells was neither reversed by suramin, a concentration-dependent inhibitor of P2 receptors, nor mimicked by the P2X receptor agonists MeSATP or APCPP. With respect to the low known IC50 values of suramin and PPADS for P2X1, 2, 3, and 5 receptors as well as to the rank order of agonist potency, the inhibition of 11β-HSD2 by ATP in our experiments is most likely not due to P2X receptors. The lacking response to UTP and UDP excluded P2Y2, P2Y4, and P2Y6 receptors, and that to ADP, AMP, and UDP excluded P2Y12 through P2Y15 receptors (11,14). P2Y3 receptors are thought to represent the avian homologue of the mammalian P2Y6 receptor (42). P2Y5 receptors solely represent functional inactive nucleotide binding proteins (43), and P2Y7 receptors are not considered part of the P2Y receptor family (44). These considerations, including the mild inhibition by BzATP, led us to conclude that the inhibition of 11β-HSD2 activity is possibly due to an activation of the P2Y11 receptor. In addition to the response observed in JEG-3 cells, ADP was inhibitory and BzATP was inactive in LLCPK1 cells. Both findings are compatible with an activation of P2Y11 receptors by ATP/ATPγS. The P2Y11 receptor is present in our cells as clearly shown by mRNA and protein levels. Although many P2Y11 events are seen early after the addition of ATP in cultured cells, some cellular modifications related to the P2Y11 receptor have also been observed at 24 and 48 h (45,46), as it is the case for the 11β-HSD2 downregulation in this study. Of interest, the effect of ATPγS was abolished by PKC inhibition with Ro-31-8220, a similar PKC dependence that was shown previously for P2Y-mediated regulation of induced nitric oxide production in mesangial cells (40).
Surprising, MeSATP excited a profound stimulatory response on 11β-HSD2 activity paralleled by an increase in 11β-HSD2 mRNA in JEG-3 but not in LLCPK1 cells. This response suggests the presumed involvement of P2Y1 receptors. Thus, mononucleotides bidirectionally regulate 11β-HSD2 activity. In SW620 cells, no inhibition of 11β-HSD2 could be observed. A slight but consistent stimulatory effect was present on incubation with BzATP, MeSATP, and APCPP but not with ATPγS, which we could not attribute definitely to a specific receptor subtype. Given the minor response, no further investigations were performed.
Despite the presence of purinergic receptor mRNA potentially mediating the regulation of 11β-HSD2 in both JEG-3 and SW620 cells, only JEG-3 cells demonstrated an inhibition by ATP. This suggests a cell-specific functional response of 11β-HSD2 activity to purinergic signals. In the absence of large differences in all receptors potentially involved in these two cell lines, two explanations that are responsible for the differing functional response have to be to considered. First, the steady-state mRNA content assessed in cell culture is not representative of the expression of the corresponding receptor, which was ruled out by demonstrating the receptor protein expression for P2Y1 and P2Y11 subtype. Second, these two cell types maintain different signaling pathways to mediate ATP-triggered responses (47) to selectively reduce 11β-HSD2 activity in JEG-3 cells.
Of interest, our experiments indicate a significant regulation of 11β-HSD2 activity by ATP in cells that contribute to the regulation of BP and fluid volume, i.e., trophoblast cells with endothelial properties (JEG-3) and renal tubular epithelial cells (LLCPK1), but not in colonic epithelial cells (SW620). These findings are in line with observations indicating that purinergic signaling participates in the regulation of salt and water reabsorption and of BP (reviewed in 48). Because trophoblast cells also line maternal vessels and the inhibition of vascular 11β-HSD2 leads to an increased BP (3), extracellular ATP thus may affect placental perfusion.
Recent reports revealed a role for nucleotide release and signaling in sensing renal tubular laminar flow (38,49,50). A shear stress–dependent reduction of 11β-HSD2 activity was demonstrated recently by our group (9). On the basis of observations by Praetorius et al. (21), a rapid increase in tubular fluid delivery might increase ATP release and according to this investigation inhibit 11β-HSD2 activity, thus facilitating tubular sodium retention to prevent inappropriate fluid losses. In contrast to these stress responses mediating preservation of BP and circulating fluid volume, the activation of different P2Y receptors promote NaCl secretion in colonic mucosa cells (39). Thus, the absence of an inhibition of 11β-HSD2 activity by ATP in SW620 cells seems functionally in line with the overall effect of the P2Y-mediated intestinal loss of NaCl (20,39).
In this study, we demonstrate inhibitory and stimulatory regulation of 11β-HSD2 most likely by P2Y receptors in the trophoblast cell line JEG-3. ATP inhibits 11β-HSD2 activity also in the tubular epithelial cell line LLCPK1. Despite the presence of similar P2 receptors in JEG-3 and SW620 cells, 11β-HSD2 is not inhibited by the exposure of colonic epithelial SW620 cells to ATP indicating a cell type–specific regulation. The exact mechanisms for this cell specificity is subject of further studies. Given the detrimental effects of a reduced 11β-HSD2 activity (4,34), it is reasonable to speculate that future successful strategies aimed at inhibiting the ATP effect on the activity of 11β-HSD2 might be beneficial to control fluid retention, vasoconstriction, or fetal exposure to high maternal cortisol levels.
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Figure 1: Cell type–specific reduction of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) activity in JEG-3, LLCPK1 and SW620 cells after 24 h of incubation with ATP (A) and ATPγS (10−4 mmol/L; B) as measured by 3H-cortisol/cortisone conversion assay. Activity of 11β-HSD2 is given in % of control. Means ± SEM (n = 4) are given. (A) § P < 0.02, §§ P < 0.006, and **P < 0.009 versus control. (B) *P < 0.001 versus control.
Figure 2: (A) Incubation with ATPγS (10−4 M) time-dependently reduced 11β-HSD2 activity as measured by 3H-cortisol/cortisone conversion assay in JEG-3 and LLCPK1 cells. Activity of 11β-HSD2 is given in % of control. ▪, treatment in JEG-3 cells; ▦, treatment in LLCPK1 cells for 24 h. Means ± SEM (n = 4) are given. + P < 0.05 versus 0 h; # P < 0.01 versus 0 h; *P < 0.001 versus 0 h. (B) JEG-3 cells were incubated for 24 h with ATPγS (10−4 M). Activity of 11β-HSD2 as measured by 3H-cortisol/cortisone conversion assay was completely reversible on washout with PBS and 24 h of follow-up. Means ± SEM (n = 4) are given. *P < 0.001 versus 0 h; **P < 0.001 versus 48 h.
Figure 3: Incubation for 24 h using either ATP • or ATPγS ○ dose-dependently reduced 11β-HSD2 activity as measured by 3H-cortisol/cortisone conversion assay in JEG-3 cells. Activity of 11β-HSD2 is given in % of control. Means ± SEM (n = 4) are given. # P < 0.01 versus control. *P < 0.001 versus control.
Figure 4: ATP or its stable analogue ATPγS reduced 11β-HSD2 activity as measured by 3H-cortisol/cortisone conversion assay when given at the concentrations indicated to intact JEG-3 (A) or LLCPK1 cells (B) in intact cells (▪), yet not when added to cell extracts (□). Activity of 11β-HSD2 is given in % of control. ▪, treatment in intact cells; □, treatment of cell extracts. Means ± SEM (n = 4) are given. # P < 0.01 versus treatment of intact cells; *P < 0.001 versus control; § P < 0.001 versus treatment of intact cells.
Figure 5: Response of 11β-HSD2 activity as measured by 3H-cortisol/cortisone conversion assay in JEG-3 and LLCPK1 cells during incubation with ATP/ATPγS (A) or UTP (B) and the respective degradation products at 10−4 mmol/L. Activity of 11β-HSD2 is given in % of control. ▪, treatment in JEG-3 cells; ▦, treatment in LLCPK1 cells for 24 h. Means ± SEM (n = 4) are given. # P < 0.01 versus control; *P < 0.001 versus control.
Figure 6: In JEG-3 cells pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) but not suramin reversed the inhibition of 11β-HSD2 activity by ATPγS after 24 h of co-incubation. The percentage of control 11β-HSD2 activity was determined as a function of increasing concentrations of either suramin (dark columns) or PPADS (light columns), in the absence or presence of a fixed concentration of ATPγS (10−4 mmol/L). Suramin did not but PPADS did restore 11β-HSD2 activity. The enzyme activity was measured by 3H-cortisol/cortisone conversion assay in JEG-3 cells. Means ± SEM (n = 4) are given. *P < 0.001 versus no ATPγS; # P < 0.01 versus no ATPγS.
Figure 7: The activity of 11β-HSD2 in response to 24-h treatment with the purinergic agonists ATPγS, 2′-methylthio-ATP (MeSATP), 3′-O-(4′-benzoyl)-benzoyl-ATP (BzATP), and α,β-methylene-ATP (APCPP) at 10−4 mmol/L was measured by 3H-cortisol/cortisone conversion assay. Activity of 11β-HSD2 is given in % of control. Black bars indicate treatment in JEG-3 cells (A), dark gray bars treatment in LLCPK1 cells (B), and light gray bars treatment in SW620 cells (C). Means ± SEM (n = 4) are given. *P < 0.001 versus control; # P < 0.01 versus control.
Figure 8: (A) Expression of purinergic receptors in JEG-3 (•) and SW620 cells (○). The relative quantity of P2X1, 2, 3, 4, 5, and 7 and P2Y1, 2, 4, 6, and 11 receptors is depicted normalized to the most abundant receptor P2X5 in SW620 cells. Data are means of three independent experiments with repeated TaqMan analyses provided on a semilogarithmic scale. No detectable expression was observed for P2Y12 and minor expression was found for P2X1, 2, 3, 5, and 7, whereas P2X4 and P2Y1, 2, 4, 6, and 11 were significantly transcribed in JEG-3 cells. In contrast, P2X4 and 5 and P2Y2, 4, and 11 mRNA were significantly detectable in SW620 cells. No P2X2 and P2Y12 receptor mRNA was found in this cell line. All other receptors were present in minor quantities in SW620 cells. Means ± SEM (n = 3 duplicate determinations) are given. (B) Protein expression of P2Y1 and P2Y11 receptor protein (20 μg/lane) in JEG-3 and SW620 cells. The first two lanes are incubations with primary antibody without control antigen; the last two lanes are incubations with competing receptor antigen (representative experiments, n = 4).
Figure 9: (A and B) Time course of 11β-HSD2 mRNA expression in JEG-3 cells in response to ATPγS (10−4 M; A) and MeSATP (10−4 M; B) as measured by quantitative PCR normalized for 18S rRNA expressed as % of control. Means ± SEM (n = 4) are given. *P < 0.001 versus experiments in the absence of ATPγS; # P < 0.01 versus experiments in the absence of ATPγS. (C) Protein expression of 11β-HSD2 in JEG-3 cells in response to 24-h incubation with ATPγS (10−4 M) or MeSATP (10−4 M), respectively. One of three independent representative experiments is depicted.
Figure 10: (A) Reporter gene analysis of the human 11β-HSD2 promoter transfected in JEG-3 cells in response to nucleotides, which inhibited (ATP, ATPγS) or stimulated (MeSATP) 11β-HSD2 activity and mRNA expression (all nucleotides at 10−4 M). TNF-α (10 nmol/L) and phorbol 12-myristate 13-acetate (50 ng/ml) served as control for promoter inhibition. Two different, independently co-transfected control plasmids yielded comparable results. Values are indicated in % of control. Means ± SEM (n = 3 to 10) are given. *P < 0.001 versus control; # P < 0.01 versus control. (B) Effects of ATPγS (10−4 mol/L) on 11β-HSD2 mRNA stability in JEG-3 cells that were co-incubated with Act D (5 μg/ml) with (○) or without (•) ATPγS for the periods indicated. Quantitative analysis of 11β-HSD2 mRNA was normalized for 18s rRNA expression in % of baseline control. Means ± SEM (n = 3) are given. + P < 0.05 versus no ATPγS.
Table 1: TaqMan gene expression assays
Acknowledgments
This work has been supported by grants for scientific research from the University of Berne and the Swiss National Science Foundation (3200-055869.98, 3100A0-102153).
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