Extracellular ATP Determines 11-Hydroxysteroid Dehydrogenase Type 2 Activity via Purinergic Receptors
Bert Kadereit,
Pierre Fustier,
Kushiar Shojaati,
Brigitte M. Frey,
Felix J. Frey and
Markus G. Mohaupt
Department of Nephrology/Hypertension, University of Berne, Berne, Switzerland
Address correspondence to: Dr. Markus G. Mohaupt, University Hospital Berne, Department of Nephrology/Hypertension, Freiburgerstr. Inselspital, 3010 Berne, Switzerland. Phone: +41-31-632-9731; Fax: +41-31-632-9734; E-mail: markus.mohaupt{at}insel.ch
Received for publication January 26, 2005.
Accepted for publication September 9, 2005.
Hypertension and sodium retention are features of a diminished11-hydroxysteroid dehydrogenase type 2 (11-HSD2). The activityof this enzyme is reduced in various disease states with abnormalrenal sodium retention and hypertension, including preeclampsia.ATP release to the extracellular compartment is observed withshear stress, inflammation, and placental ischemia. It was hypothesizedthat ATP downregulates 11-HSD2 activity. For that purpose, celllines from different tissues that previously were used to studythe regulation of 11-HSD2 were investigated: JEG-3, a vasculartrophoblastic; LLCPK1, a renal tubular; and SW620, a colonicepithelial cell line. The 11-HSD2 activity, assessed by theconversion of 3H-cortisol to cortisone, was reversibly reducedduring incubation with ATP or its stable analogue ATPS in intactJEG-3 and LLCPK1, but not in SW620 cells. In JEG-3 cells, thepurinergic antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonicacid but not suramin reversed the inhibition. Incubation withUTP and ADP and their degradation products including adenosineand ,-methylene-ATP did not inhibit 11-HSD2 activity. In contrast,11-HSD2 activity increased almost 2.5-fold after incubationwith 2'-methylthio-ATP. This indicates a bidirectional regulationby nucleotides via purinergic receptors. In JEG-3 cells, ATP/ATPSdid not alter 11-HSD2 promoter activity but reduced 11-HSD2protein and mRNA concentration and half-life, suggesting a posttranscriptionalregulation. In conclusion, ATP inhibits cell type specificallyvia purinergic receptors the expression and activity of the11-HSD2 by a posttranscriptional mechanism.
Blood pressure (BP) is controlled by the intravascular fluidvolume via renal sodium handling and by vasoconstriction. Onemechanism involved in BP control is the intracellular availabilityof cortisol. High intracellular cortisol levels induce vasoconstriction(1–3) and enhance renal sodium retention (4). Intracellularcortisol availability is regulated in renal, vascular, colonicepithelial, and placental tissues by 11-hydroxysteroid dehydrogenasetype 2 (11-HSD2), an enzyme that converts biologically activecortisol into inactive cortisone (1–5). Several clinicallyrelevant factors, including hypoxia, TNF-, angiotensin II, shearstress, and methylation status, have been described to control11-HSD2 activity (6–10).
Nucleotides are intracellularly present at a concentration rangingfrom approximately 2 to 5 mmol/L (11). Cellular injury but alsonumerous biologic processes without cellular damage cause arelease of mononucleotides (e.g., ATP) into the extracellularcompartment (12,13). Nucleotides bind to purinergic cell surfacereceptors; either ATP-gated channels named P2X or heptahelicalmetabotropic receptors named P2Y. P2 receptors divide into anexpanding number of members, currently seven for the P2X and15 for the P2Y receptor group (11,14). Numerous cell types,including trophoblasts and renal tubular and colonic epithelialcells, have been shown to exhibit P2-related signals and expressthese receptors (15–21). In trophoblast cells, P2X7 (16)and P2Y6 (17) receptors have been detected and P2Y2 regulationhas been demonstrated by agonist profiling (15,18,19). Despitethe disclosure of intracellular signals involved in the activationof purinergic receptors, their functional consequences remainto be determined in many tissues.
Placental lesions such as arteriolopathy, hypermaturity of villi,intervillous thrombi, and central infarction are more frequentand severe in preeclampsia, a disease characterized by hypertensionand renal damage with proteinuria (22–24). Placental ischemiaand inflammatory responses during preeclampsia and increasedshear stress lead to the release of ATP (9,25–27). Thecortisol-inactivating enzyme 11-HSD2 is highly expressed introphoblasts and the trophoblast cell line JEG-3 (9,10,22).One important function of the 11-HSD2 in placental tissue isto protect the fetus from high maternal cortisol concentrations.So far, the effect of mononucleotides on 11-HSD2 activity wasanalyzed only in one study. Yang et al. (28) incubated placentalmicrosomal extracts that contained 11-HSD2 activity with ATP.They observed an enhanced 11-HSD2 activity independent of thesite for substrate binding.
The reduction of 11-HSD2 activity in the presence of high ATPlevels observed in preeclampsia (22,23) prompted us to determinewhether ATP might contribute to a reduced 11-HSD2 activity introphoblasts and to elucidate whether intact cells are neededto modulate the 11-HSD2 activity by extracellular mononucleotides.To explore this hypothesis, we investigated the well-characterizedhuman choriocarcinoma cell line JEG-3 displaying endothelialproperties and two cell lines with epithelial features: LLCPK1,a renal tubular, and SW620, a human colonic cell line. All celllines have previously been used to study the regulation of 11-HSD2activity (7,8,29).
Material and Cell Lines
Cell culture material was from Becton Dickinson Labware (Basel,Switzerland) and Corning (Bodenheim, Germany). Cortisol, glycyrrhetinicacid, minimal essential medium Eagle (MEME), DMEM, Bradfordreagent, 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 chromatographyplates (silica gel 60F254) were from Macherey-Nagel (Oensingen,Switzerland), and FCS was from Biological Industries (Noisyle Grand, France). TaqMan Gene Expression Assays and Assayson Demand were from Applied Biosystems, and primers were fromMicrosynth (Balgach, Switzerland). All nucleotides including3'-O-(4'-benzoyl)-benzoyl-ATP (BzATP), 2'-methylthio-ATP (MeSATP)and ,-methylene-ATP (APCPP), suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulphonicacid (PPADS), Ro-31-8220, and cycloheximide were from SigmaAldrich (Fluka AG, Buchs, Switzerland). The human choriocarcinomacell 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/mlstreptomycin. LLCPK1 and SW620 cells were maintained in DMEMwith 0.45% glucose. Cells were passaged up to 20 times withtrypsin/EDTA, plated in cell culture dishes, grown to confluence,and washed twice with PBS (pH 7.4) at 37°C before the experimentswere started. At the completion of the final incubation period,cells again were washed twice with ice-cold PBS and the experimentproceeded according to subsequent protocols. Incubations wereperformed for 24 h, if not otherwise indicated. Cell viabilitywas assessed by measuring lactate dehydrogenase concentrationsin the supernatant, by performing FACS analysis for annexinV, 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 assayand the bicinchoninic acid method (Pierce, Rockford, IL).
3H-Cortisol/Cortisone Conversion Assay in Cell Homogenates to Determine 11-HSD2 Activity
The cortisol/cortisone conversion was used to measure oxidationat C-11 by 11-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 adeninedinucleotide in sucrose buffer. The reaction was stopped byadding 20 µl of 1 mg/ml unlabeled cortisol and cortisonein 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 (TriCarb 2000CA; United Technologies, Hartford, CT). Specific activitywas expressed as picomoles per milligrams protein per hour.The assay was repeated up to four times using different proteinconcentrations within each individual experiment.
Protein Extraction and Western Blot Analysis
Cells were lysed with cold lysis buffer (100 mM NaCl, 50 mMsodium fluoride, 0.1% SDS, 1% sodium deoxycholate, 1% TritonX-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, vortexedvigorously, and centrifuged at 10,000 x g for 20 min at 4°C.The supernatant was collected, and the protein content was determinedas described above.
For Western blot analysis, total protein (40 µg) was loadedon a denaturing 10% polyacrylamide gel. Benchmark prestainedprotein 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 witha constant voltage of 60 V for 1 h on ice. The membrane wasblocked overnight in 5% nonfat dry milk in TBS-T (0.1% Tween20 and 0.1% in TBS), washed with TBS-T, and incubated for 2h with rabbit polyclonal antibody for 11-HSD2 (1:500; SantaCruz Biotechnology, Santa Cruz, CA), P2Y1, and P2Y11 (1:500;Alomone Laboratories, Jerusalem, Israel) in 0.5% nonfat drymilk in TBS-T. Negative control experiments with the controlpeptide (preincubation with the same amount of antibody for1 h at room temperature) used for immunization were done tocheck specificity of the P2Y1 and P2Y11 primary antibodies.The polyvinylidene difluoride membrane was washed with TBS-Tand incubated for 1 h at room temperature with a goat anti-rabbitIgG 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 chemiluminescencekit (Amersham Biosciences). The filter then was exposed to x-rayfilm (Eastman Kodak Co., Rochester, NY) for 1 to 5 min. Densitometrywas performed on the radiographs, and the level of 11-HSD2 proteinwas expressed as arbitrary units.
Extraction of Total RNA and Preparation of cDNA Pools
Cell lines (JEG-3, LLCPK1, and SW620) were rinsed with ice-coldPBS and collected by centrifugation (3000 x g at 4°C for3 min). Following the Promega SV (Wallisellen, Switzerland)total RNA isolation system, cells were resuspended in 175 µlof SV RNA lysis buffer followed by the addition of 350 µlof SV RNA dilution buffer. After mixing and heating (70°Cfor 3 min), the samples were centrifuged (14,000 x g for 3 min),the clear lysates were transferred to a fresh tube, and 200µl of 95% ethanol was added. RNA was collected by spinningfor 1 min at 14,000 x g and washed using 600 µl of SVRNA washing solution. DNaseI treatment for 15 min with consecutiveaddition of SV DNase stop solution and repeated washing (SVRNA wash solution) was performed. RNA concentration was determinedafter final elution of RNA with ddH2O by using a spin column(14,000 x g for 1 min), and the RNA was stored at –70°C.One microgram of total RNA of cells that were grown with andwithout the pharmacologic agents as indicated in the Resultssection were reverse transcribed using random hexamers and ImpromII reverse transcriptase according to the Improm II Promegaprotocol.
TaqMan Gene Expression Assays for P2X and P2Y Receptors and for 11-HSD2
Homology-based TaqMan PCR was performed with assay-on-demandprimers and probes to identify and quantify P2 receptors and11-HSD2 mRNA expression. Equal amplification efficiencies wereverified. Negative controls for the reverse transcriptase andthe PCR reagents remained negative. Primer sequences for thehuman P2X and P2Y receptors as well as for 11-HSD2 and 18S-RNAare presented in Table 1. All PCR were normalized for the respective18S-RNA content, and P2X5 was arbitrarily chosen to be the calibratorin the comparative analysis, an approach described previouslyfor the analysis of P2 receptor mRNA expression (30).
Plasmid Construction, Transfections, Luciferase, and -Galactosidase Assays to Identify 11-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 withFuGENE 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 wereincubated in serum-free medium for 15 min before applicationon subconfluent JEG-3 cells in 24-well plates. The reporterplasmids were co-transfected with 0.05 µg of either apCMV-LacZ or pSV-LacZ control plasmid to correct for transfectionefficiency. After a 24-h incubation, the medium was changedagainst new MEME and the cells were treated as indicated for24 h. Finally, the medium was removed, and the cells were washedtwice with PBS, lysed in 100 µl of lysis buffer, and assayedfor luciferase and -galactosidase activity using the AppliedBiosystems dual-light reporter gene assay system. Chemiluminescencewas measured with a MediatorsPhL luminometer (Mediators DiagnosticSystems, Vienna, Austria). The normalized values were from triplicatesamples.
Statistical Analyses
All data are presented as means ± SEM. To test for statisticallysignificant differences, we used t test, ANOVA, or multiplecomparisons versus control group (Tukey test), as appropriate.Significance was assigned at P < 0.05. All statistical analyseswere performed using SYSTAT Version 10 (SPSS Inc., Chicago,IL).
Effect of ATP or Its Stable Analogue ATPS on 11-HSD2 Activity in JEG-3, LLCPK1, and SW620 Cells
In JEG-3 cell homogenates, 11-HSD2 enzyme activity, assessedby the conversion of 3H-cortisol to 3H-cortisone, was present(1.46 ± 0.15 pmol/mg protein per h) as has been shownpreviously (9,10,31). This conversion was almost completelyinhibited when glycyrrhetinic acid (10–6 mol/L) was addedto the cell culture medium. Adding Triton-X100 to the 11-HSDassay mixture abolished the enzyme activity, an observationindicating that the activity is attributable to 11-HSD2 andnot to 11-HSD1 enzyme, a contention supported by the expressionof 11-HSD2 but not 11-HSD1 mRNA (9,32). Addition of ATP (Figure 1A)or its more stable analogue ATPS (Figure 1B) to JEG-3 cellsfor 24 h consistently reduced 11-HSD2 activity. To determinewhether 11-HSD2 inhibition by ATP is cell type specific, LLCPK1and SW620 cell lines, both previously investigated for 11-HSD2regulation (7,8,29), were also studied. ATP or ATPS inhibited11-HSD2 activity in LLCPK1 but not in SW620 cells (Figure 1),suggesting a cell type specific response. The 11-HSD2 activitywas 1.35 ± 0.06 and 1.63 ± 0.26 pmol/mg proteinper h, respectively, in cultured LLCPK1 and SW620 cells.
Figure 1. Cell type–specific reduction of 11-hydroxysteroid dehydrogenase type 2 (11-HSD2) activity in JEG-3, LLCPK1 and SW620 cells after 24 h of incubation with ATP (A) and ATPS (10–4 mmol/L; B) as measured by 3H-cortisol/cortisone conversion assay. Activity of 11-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.
In JEG-3 and LLCPK1 cells, the inhibition by ATPS was time dependent(Figure 2A). Reversibility was demonstrated by removing ATPSfrom the cell culture supernatant after 24 h of incubation andmeasuring 11-HSD2 activity at 48 h without ATPS in JEG-3 cells(Figure 2B). ATP ("apparent IC50": 6.7 x 10–5 mol/L) andATPS ("apparent IC50": 2.3 x 10–6 mol/L) dose-dependentlyreduced 11-HSD2 activity (Figure 3).
Figure 2. (A) Incubation with ATPS (10–4 M) time-dependently reduced 11-HSD2 activity as measured by 3H-cortisol/cortisone conversion assay in JEG-3 and LLCPK1 cells. Activity of 11-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 ATPS (10–4 M). Activity of 11-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 ATPS dose-dependently reduced 11-HSD2 activity as measured by 3H-cortisol/cortisone conversion assay in JEG-3 cells. Activity of 11-HSD2 is given in % of control. Means ± SEM (n = 4) are given. #P < 0.01 versus control. *P < 0.001 versus control.
Regulation of 11-HSD2 Activity Requires Intact Cells
An earlier report suggested a direct effect of ATP on cellularextracts containing 11-HSD2 enzyme (28). For evaluating whetherthe 11-HSD2 response requires intact cells, ATP or ATPS wereincubated with cultured JEG-3 (Figure 4A) and LLCPK1 (Figure 4B)cells or added to the cell homogenates from these cells.The 11-HSD2 activity was inhibited by ATP or ATPS in intactcells but not when added to homogenates.
Figure 4. ATP or its stable analogue ATPS reduced 11-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 11-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.
Characterization of Purinergic Receptors with Impact on 11-HSD2 Activity
At equimolar doses (10–4 mol/L), the activity of 11-HSD2in cultured JEG-3 and LLCPK1 cells was strongly inhibited byATPS, less by ATP, whereas neither the ATP degradation productsADP, AMP, and adenosine nor UTP or its degradation productswere effective, except ADP in LLCPK1 cells (Figure 5). The relativeresponse was more pronounced in JEG-3 cells when compared withLLCPK1 cells. PPADS but not the purinergic antagonist suraminprevented ATPS-mediated inhibition of 11-HSD2 (Figure 6).
Figure 5. Response of 11-HSD2 activity as measured by 3H-cortisol/cortisone conversion assay in JEG-3 and LLCPK1 cells during incubation with ATP/ATPS (A) or UTP (B) and the respective degradation products at 10–4 mmol/L. Activity of 11-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 11-HSD2 activity by ATPS after 24 h of co-incubation. The percentage of control 11-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 ATPS (10–4 mmol/L). Suramin did not but PPADS did restore 11-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 ATPS; #P < 0.01 versus no ATPS.
To characterize further the P2Y receptors involved, we incubatedJEG-3, LLCPK1, and SW620 cells with equimolar doses (10–4mol/L) of ATPS, MeSATP, BzATP, and APCPP (Figure 7). As expected,11-HSD2 activity was not inhibited by any of these nucleotidesin SW620 cells. A minor but consistent activation by ATP (Figure 1),MeSATP, BzATP, and APCPP was present (Figure 7C). The 11-HSD2activity was clearly inhibited in LLCPK1 cells by ATPS but didnot respond to any of the other nucleotides (Figure 7B). Likewise,the activity of 11-HSD2 in JEG-3 cells was strongly inhibitedby ATPS and less by BzATP (Figure 7A). In contrast to the ATPS-inducedinhibition, incubation with MeSATP increased the enzyme activity(Figure 7A).
Figure 7. The activity of 11-HSD2 in response to 24-h treatment with the purinergic agonists ATPS, 2'-methylthio-ATP (MeSATP), 3'-O-(4'-benzoyl)-benzoyl-ATP (BzATP), and ,-methylene-ATP (APCPP) at 10–4 mmol/L was measured by 3H-cortisol/cortisone conversion assay. Activity of 11-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.
Using the known sequences of human P2 receptors, we assessedthe corresponding mRNA expression as a measure for the presenceof these receptors (11) (Figure 8). In JEG-3 cells, the P2Y12receptor was absent. Minor expression was found for P2X1, 2,3, 5, and 7 receptors, whereas larger amounts of mRNA of theP2X4 and P2Y1, 2, 4, 6, and 11 receptors were present in JEG-3cells. The most abundant P2Y receptor was the P2Y11 subtype.In SW620 cells, P2X4 and 5 and P2Y2, 4, and 11 mRNA was welldetectable. No P2X2 and P2Y12 receptor mRNA was found in thiscell line. All other receptors were present in minor quantitiesin these cells (Figure 8A). The largest differences in expressionwere detected for P2X5 (SW620>JEG-3 cells) and P2Y6 (JEG-3>SW620cells) receptors. P2Y1 and P2Y11 receptor proteins were detectedin both JEG-3 and SW620 cells (Figure 8B).
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).
Cellular Responses to Purinergic Stimulation
Incubation of JEG-3 cells with ATPS for up to 24 h reduced 11-HSD2activity (Figure 2A), a reduction prevented by hindering proteinkinase C (PKC) signaling with the PKC inhibitor Ro31–8220(P < 0.006). This coincided with a progressively reduced11-HSD2 mRNA content (Figure 9A). In contrast, the increased11-HSD2 activity in the presence of MeSATP was paralleled byan enhanced 11-HSD2 mRNA availability as depicted in Figure 9B.Protein expression of 11-HSD2 changed accordingly (Figure 9C).Reporter gene analysis of the human 11-HSD2 promoter indicatedthat no transcriptional regulation was responsible for the alteredcumulative mRNA (Figure 10A), thus suggesting posttranscriptionalmechanisms (8). TNF- and phorbol 12-myristate 13-acetate areknown to reduce 11-HSD2 promoter activity and served as controls(7,33). The assumption of a posttranscriptional regulation inthe presence of ATPS is supported by the observation of an enhanceddegradation of 11-HSD2 mRNA measured during transcriptionalinhibition by actinomycin D (Figure 10B) and by a cycloheximide-promotedstabilization of 11-HSD2 mRNA during coincubation with ATP/ATPS(data not shown).
Figure 9. (A and B) Time course of 11-HSD2 mRNA expression in JEG-3 cells in response to ATPS (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 ATPS; #P < 0.01 versus experiments in the absence of ATPS. (C) Protein expression of 11-HSD2 in JEG-3 cells in response to 24-h incubation with ATPS (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 11-HSD2 promoter transfected in JEG-3 cells in response to nucleotides, which inhibited (ATP, ATPS) or stimulated (MeSATP) 11-HSD2 activity and mRNA expression (all nucleotides at 10–4 M). TNF- (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 ATPS (10–4 mol/L) on 11-HSD2 mRNA stability in JEG-3 cells that were co-incubated with Act D (5 µg/ml) with () or without (•) ATPS for the periods indicated. Quantitative analysis of 11-HSD2 mRNA was normalized for 18s rRNA expression in % of baseline control. Means ± SEM (n = 3) are given. +P < 0.05 versus no ATPS.
The 11-HSD2 enzyme protects the mineralocorticoid receptor (MR)from inappropriate activation by cortisol. Thus, it is a prereceptorenzyme that conveys selectivity of the MR to aldosterone. Evena subtotal reduction of the 11-HSD2 activity has been shownto enhance MR activation by cortisol (4,6–8,10,34). Thisstudy identifies a significant downregulation of 11-HSD2 activityby ATP. Inhibition of 11-HSD2 activity by ATP or its stableanalogue ATPS was reversible and dose and time dependent. Giventhe potential of rapid degradation of ATP, we applied the analogueATPS, which has been demonstrated to be significantly more stableand to mimic the ATP response (35). In addition, we controlledfor an effect of degradation products of extracellular ATP,such as AMP and adenosine, which did not imitate the inhibitoryeffect of ATP.
Yang et al. (28) investigated a putative posttranslational regulationof 11-HSD2 by adding ATP to microsomal extracts. The exact mechanismremained uncertain but was independent of phosphorylation andenergy supply by ATP and mimicked by ADP or AMP but not by othernucleotides. The approach used did not allow us to study purinergicreceptor–mediated processes. In contrast, our observationsin intact cells indicate that purinergic receptors are necessaryto promote the changes in 11-HSD2 activity.
Provided the regulation of 11-HSD2 by mononucleotides requiresintact cells, a cellular response to ATP should be detectable.Such a response was observed. The reduced 11-HSD2 protein expressionand activity were paralleled by a reduction of 11-HSD2 mRNA,indicating a transcriptional or posttranscriptional regulationof transcript availability. Reporter gene analysis and the reducedhalf-life of 11-HSD2 mRNA during incubation with ATP/ATPS revealedthat the reduction of 11-HSD2 mRNA was not transcriptionallybut rather posttranscriptionally mediated, a contention supportedby the stabilization of the mRNA using the translational inhibitorcycloheximide. Incubation with ATP/ATPS thus would induce adestabilizing protein, which interferes with 11-HSD2 mRNA longevity.The presence of such a mechanism is plausible and indirectlysupported by the observations that, first, angiotensin II destabilized11-HSD2 mRNA (10); second, 11-HSD2 mRNA is posttranscriptionallyregulated in trophoblasts (36); and, third, mononucleotidesinterfere with the mRNA stability of other genes in the presenceof cytokines in mesangial cells (37).
Experiments were designed to define the receptors involved inthe regulation of 11-HSD2. A whole array of different P2 receptorshas been described in recent years. As a consequence of an almostcomplete lack of specific antagonists, the agonistic potencyof different mononucleotides had to be studied and the resultscompared with the pattern of responses that are known to belinked with activation of a specific receptor subtype, an approachused by other investigators for defining purinergic regulationof other genes (11,15,18,19,38–41). The inhibition of11-HSD2 activity by ATP in JEG-3 cells was neither reversedby suramin, a concentration-dependent inhibitor of P2 receptors,nor mimicked by the P2X receptor agonists MeSATP or APCPP. Withrespect to the low known IC50 values of suramin and PPADS forP2X1, 2, 3, and 5 receptors as well as to the rank order ofagonist potency, the inhibition of 11-HSD2 by ATP in our experimentsis most likely not due to P2X receptors. The lacking responseto UTP and UDP excluded P2Y2, P2Y4, and P2Y6 receptors, andthat to ADP, AMP, and UDP excluded P2Y12 through P2Y15 receptors(11,14). P2Y3 receptors are thought to represent the avian homologueof the mammalian P2Y6 receptor (42). P2Y5 receptors solely representfunctional inactive nucleotide binding proteins (43), and P2Y7receptors are not considered part of the P2Y receptor family(44). These considerations, including the mild inhibition byBzATP, led us to conclude that the inhibition of 11-HSD2 activityis possibly due to an activation of the P2Y11 receptor. In additionto the response observed in JEG-3 cells, ADP was inhibitoryand BzATP was inactive in LLCPK1 cells. Both findings are compatiblewith an activation of P2Y11 receptors by ATP/ATPS. The P2Y11receptor is present in our cells as clearly shown by mRNA andprotein levels. Although many P2Y11 events are seen early afterthe addition of ATP in cultured cells, some cellular modificationsrelated to the P2Y11 receptor have also been observed at 24and 48 h (45,46), as it is the case for the 11-HSD2 downregulationin this study. Of interest, the effect of ATPS was abolishedby PKC inhibition with Ro-31-8220, a similar PKC dependencethat was shown previously for P2Y-mediated regulation of inducednitric oxide production in mesangial cells (40).
Surprising, MeSATP excited a profound stimulatory response on11-HSD2 activity paralleled by an increase in 11-HSD2 mRNA inJEG-3 but not in LLCPK1 cells. This response suggests the presumedinvolvement of P2Y1 receptors. Thus, mononucleotides bidirectionallyregulate 11-HSD2 activity. In SW620 cells, no inhibition of11-HSD2 could be observed. A slight but consistent stimulatoryeffect was present on incubation with BzATP, MeSATP, and APCPPbut not with ATPS, which we could not attribute definitely toa specific receptor subtype. Given the minor response, no furtherinvestigations were performed.
Despite the presence of purinergic receptor mRNA potentiallymediating the regulation of 11-HSD2 in both JEG-3 and SW620cells, only JEG-3 cells demonstrated an inhibition by ATP. Thissuggests a cell-specific functional response of 11-HSD2 activityto purinergic signals. In the absence of large differences inall receptors potentially involved in these two cell lines,two explanations that are responsible for the differing functionalresponse have to be to considered. First, the steady-state mRNAcontent assessed in cell culture is not representative of theexpression of the corresponding receptor, which was ruled outby demonstrating the receptor protein expression for P2Y1 andP2Y11 subtype. Second, these two cell types maintain differentsignaling pathways to mediate ATP-triggered responses (47) toselectively reduce 11-HSD2 activity in JEG-3 cells.
Of interest, our experiments indicate a significant regulationof 11-HSD2 activity by ATP in cells that contribute to the regulationof BP and fluid volume, i.e., trophoblast cells with endothelialproperties (JEG-3) and renal tubular epithelial cells (LLCPK1),but not in colonic epithelial cells (SW620). These findingsare in line with observations indicating that purinergic signalingparticipates in the regulation of salt and water reabsorptionand of BP (reviewed in 48). Because trophoblast cells also linematernal vessels and the inhibition of vascular 11-HSD2 leadsto an increased BP (3), extracellular ATP thus may affect placentalperfusion.
Recent reports revealed a role for nucleotide release and signalingin sensing renal tubular laminar flow (38,49,50). A shear stress–dependentreduction of 11-HSD2 activity was demonstrated recently by ourgroup (9). On the basis of observations by Praetorius et al.(21), a rapid increase in tubular fluid delivery might increaseATP release and according to this investigation inhibit 11-HSD2activity, thus facilitating tubular sodium retention to preventinappropriate fluid losses. In contrast to these stress responsesmediating preservation of BP and circulating fluid volume, theactivation of different P2Y receptors promote NaCl secretionin colonic mucosa cells (39). Thus, the absence of an inhibitionof 11-HSD2 activity by ATP in SW620 cells seems functionallyin line with the overall effect of the P2Y-mediated intestinalloss of NaCl (20,39).
In this study, we demonstrate inhibitory and stimulatory regulationof 11-HSD2 most likely by P2Y receptors in the trophoblast cellline JEG-3. ATP inhibits 11-HSD2 activity also in the tubularepithelial cell line LLCPK1. Despite the presence of similarP2 receptors in JEG-3 and SW620 cells, 11-HSD2 is not inhibitedby the exposure of colonic epithelial SW620 cells to ATP indicatinga cell type–specific regulation. The exact mechanismsfor this cell specificity is subject of further studies. Giventhe detrimental effects of a reduced 11-HSD2 activity (4,34),it is reasonable to speculate that future successful strategiesaimed at inhibiting the ATP effect on the activity of 11-HSD2might be beneficial to control fluid retention, vasoconstriction,or fetal exposure to high maternal cortisol levels.
Acknowledgments
This work has been supported by grants for scientific researchfrom the University of Berne and the Swiss National ScienceFoundation (3200-055869.98, 3100A0-102153).
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-Hydroxysteroid Dehydrogenase Type 2 Activity via Purinergic Receptors
Top
Abstract
Introduction
-hydroxysteroid dehydrogenase type 2 (11
S in intact JEG-3 and LLCPK1, but not in SW620 cells. In JEG-3 cells, the purinergic antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid but not suramin reversed the inhibition. Incubation with UTP and ADP and their degradation products including adenosine and 
,
P < 0.02, 
, 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 ATP
dose-dependently reduced 11
). Activity of 11
