Transcriptional Down-Regulation of the Human a2C- Adrenergic Receptor by cAMP

The heterologous regulation of the a2C-adrenergic receptor (a2C-AR) was investigated in the HepG2 cell line. Binding of [H]MK912 (a2-antagonist) to membranes from cells submitted to various treatments showed that exposure to insulin, phorbol 12-myristate 13-acetate, or dexamethasone did not affect receptor density. On the other hand, treatment with forskolin resulted in a large reduction of a2C-AR number. The effect of forskolin was mimicked by 8-br-cAMP and was abolished by the protein kinase A inhibitor, H89. The action of cAMP was slow (t1/2 5 23 h), dose-dependent, and additive to the receptor down-regulation elicited by the a2-agonist, UK14304. Furthermore, the diminution of receptor was not caused by an increased rate of its degradation but resulted from a decrease in the steady state amounts of a2C4-mRNA. As assessed by experiments in the presence of actinomycin D, the stability of a2C4-mRNA was not affected by 8-br-cAMP or forskolin. By contrast, the activity of a luciferase construct containing the entire promoter region of the a2C4 gene (1.9 kilobase pairs) was inhibited, indicating that the primary mechanism of action of the two compounds is at the transcriptional level. Deletions in the 59-end of this construct showed that the elements responsible for cAMP responsiveness lie within a 242-base-pair fragment of the gene promoter (nucleotides 2236/16 relative to transcription start). Band-shift experiments indicated that nuclear factors bind to this region in a cAMP-dependent manner. The determination of the actual cisand trans-acting elements involved will be the object of future investigation, but the present study provides evidence for transcriptional regulation of human a2C-AR by cAMP. The a2-adrenergic receptors (a2-ARs) are G protein-coupled receptors that play a key role in the control of numerous physiological functions, such as renal Na-reabsorption, insulin secretion, platelet aggregation, or neurotransmitter release at sympathetic nerve endings (for review, see Ruffolo et al., 1993). Molecular cloning has now definitively established that this receptor family consists of three highly homologous subtypes encoded by distinct intronless genes. In man, the genes coding the a2A-, a2B-, and a2C-AR subtypes were designated a2C10, a2C2, and a2C4, respectively (Kobilka et al., 1987; Regan et al., 1988; Lomasney et al., 1990). In addition to differences in affinity for various ligands (Bylund et al.,1994), a2-AR subtypes also diverge from each other in their tissue distribution, intracellular trafficking, and subcellular targeting as well as in their ability to undergo desensitization. All these discrepancies are consistent with growing evidence indicating that each receptor subtype is endowed with discrete functions in vivo (Link et al., 1996; MacMillan et al., 1996). The precise roles of the a2C-AR are still unclear in man, but it fulfills all the above-cited criteria of discrimination. From a pharmacological point of view, a2C-AR is distinguishable from a2A and a2B by its sensitivity to prazosin (Bylund et al., 1994) and its remarkably high affinity for MK912 (Schaak et al., 1997a). According to binding studies and to measurement of a2C4-mRNA level, its expression is primarily restricted to a limited number of tissues including brain, kidney, aorta, and spleen (De Vos et al.,1992; Perala et al., 1992; Berkowitz et al., 1994). Finally, in contrast to a2A and a2B, which are strictly membrane-located and rapidly desensitized on exposure to agonist, a2C-AR is refractory to desensitization (Eason and Liggett, 1992) and exhibits both membranous and intracellular localization (Von Zastrow et al., 1993). As assessed by transfection experiments, this peculiar behavior is likely the consequence of the incapacity of G protein-coupled receptor kinase to phosphorylate a2C-subtype (Jewell-Motz and Liggett, 1996). This work was supported by the BIOMED 2 program PL963373 (European Commission, Brussels, Belgium). ABBREVIATIONS: AR, adrenergic receptor; AP-1, activator protein-1; bp, base pair(s); PMA, phorbol 12-myristate 13-acetate; 8-br-cAMP, 8-bromo-cAMP; 8-br-cGMP, 8-bromo-cGMP; H89, N-[2-((p-bromoccinamyl)amino)ethyl]-5-isoquinolinesulfonamide; FCS, fetal calf serum; DMEM, Dulbecco’s modified Eagle’s medium; pKS1, pBlueScriptII KS1; RPA, RNase protection assay; TM buffer, Tris/MgCl2 buffer; CRE, cAMP response element. 0026-895X/00/040821-07$3.00/0 MOLECULAR PHARMACOLOGY Vol. 58, No. 4 Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics 125/849161 Mol Pharmacol 58:821–827, 2000 Printed in U.S.A. 821 at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from Compared with our knowledge of the regulation of the a2C-AR at the post-transductional level, our understanding of the mechanisms responsible for its tissue-specific distribution and for the control of its expression at the transcriptional level is virtually nonexistent. As yet, the lack of a suitable in vitro cellular system natively expressing this receptor subtype has been a major stumbling block in analyzing the molecular mechanisms controlling a2C4 gene transcription. The recent recognition that the human hepatoma HepG2 cell line exhibits a2C-ARs now allows investigation in this direction (Schaak et al., 1997a). The use of this cell line already permitted us to define some characteristics of a2C4 gene organization (Schaak et al., 1997b). Its transcription is initiated at a unique start site located 891 bases upstream of the ATG start codon through the activity of a promoter region that contains a nonconventional TATA box and several Sp1 sites, but lacks a CAAT box. Analysis of the sequence upstream of this region also indicated the presence of putative sites for other transcription factors, including upstream stimulatory factor (USF) or activator protein-1 (AP-1). The functional importance of these elements, however, remains to be demonstrated. More recently, HepG2 was also used to reexamine the homologous regulation of the a2C-AR (Cayla et al., 1999). In agreement with previous observations in transfected cells, the receptor was found refractory to desensitization after short-term exposure to a2-agonist. However, longlasting treatment induced a sharp down-regulation because of an increased rate of receptor degradation. The aim of the present work was to study the heterologous regulation of the a2C-AR in HepG2. We show that cell exposure to forskolin or cAMP analogs causes a significant reduction of receptor expression. This effect is correlated with a decrease of the amount of a2C4-mRNA ,which, according to measurement of the activity of a luciferase construct containing the promoter region of the a2C4 gene, is the consequence of an inhibition of gene transcription. Transfection experiments with constructs containing different fragments of the 59-flanking region of a2C4-gene demonstrates that the cisacting elements responsible for cAMP-dependent regulation are located within a 242-base-pair (bp) fragment of the promoter. Taken together, these observations bring new insights into the mechanisms whereby the expression of the human a2C-AR is transcriptionally regulated. Materials and Methods Drugs and Reagents. [H]MK912 (79–80.5 Ci/mmol) was from New England Nuclear (Boston, MA). [a-P]UTP and [a-P]dATP were purchased from ICN (Costa Mesa, CA). Phentolamine and UK14304 were generously donated by Ciba-Geigy (Basel, Switzerland) and Pfizer (Sandwich, UK), respectively. Dexamethasone, human recombinant insulin, phorbol 12-myristate 13-acetate (PMA), forskolin, 8-bromo-cAMP (8-br-cAMP), 8-bromo-cGMP (8-br-cGMP), actinomycin D, and all other chemicals were from Sigma (St. Louis, MO). N-[2-((p-bromoccinamyl)amino)ethyl]-5-isoquinolinesulfonamide (H89) was from Calbiochem (San Diego, CA), fetal calf serum (FCS) from Gibco-BRL (Cergy Pontoise, France). The pCRE-Luc construct was obtained from Stratagene (La Jolla, CA). The vectors, pGL3Basic and pGL3-Promoter, were from Promega (Madison, WI). Cell Culture and Treatments. The human hepatocarcinoma cell line HepG2 was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 25 mM glucose, 100 mg/ml streptomycin, 100 IU/ml penicillin, 2 mM glutamine, and 10% FCS. Unless otherwise specified, all treatments were performed on confluent attached cells and in the absence of FCS. At zero time of the treatment, the hormone or drug to be tested was added to the culture from frozen stock solution. At the indicated time, the medium was removed, the cell-layers rinsed twice with PBS, and rapidly frozen at 280°C until analysis. Synthesis of a2C4 and b-Actin Riboprobe. The probe for the detection of a2C4 mRNAs was obtained by subcloning a 370-bp fragment (SmaI-MaeIII), corresponding to nucleotides 1014/1382 of the a2C4 coding region, into pBlueScriptII KS1 (pKS1; Stratagene). The b-actin probe was obtained by polymerase chain reaction and cloned into the EcoRV site of pKS1, the amplified fragment (236 bp) corresponds to nucleotides 415/650 of the cDNA (exon 3). For synthesis of the radiolabeled probes, the two plasmids were linearized with the appropriate restriction enzyme and antisense RNAs were synthesized in the presence of [a-P]UTP using T3 RNA polymerase (Promega). RNA Preparation and RNase Protection Assays (RPA). Cellular RNAs were isolated using the guanidinium isothiocyanate/ phenol-chloroform extraction method (Chomczynski and Sacchi, 1987). RPA were performed as described previously but with slight modifications (Schaak et al., 1997b). Lyophilized RNAs (100 mg) were taken up in 30 ml of hybridization buffer (80% deionized formamide, 0.4 M NaCl, 1 mM EDTA, 40 mM 1,4-piperazinediethanesulfonic acid, pH 6.7) containing an excess of P-labeled riboprobe. The samples were heated to 95°C for 5 min and then immediately placed at 55°C for 14 h. Nonhybridized probe was eliminated by the addition of 0.3 ml of Tris/EDTA/NaCl buffer (10 mM TriszHCl, 5 mM EDTA, 300 mM NaCl, pH 7.5) containing RNase A (40 mg/ml) and RNase T1 (2 mg/ml). After 2 h at 37°C, 5 ml of proteinase K (10 mg/ml) were added and the samples further incubated for 15 min at 37°C. Carrier tRNA (10 mg) and 0.3 ml of solution D (4 M guanidinium isothiocyanate, 0.1 M 2-mercaptoethanol, 0.5% w/v sarkosyl, 25 mM sodium citrate, pH 7.0) were added to each tube and protected hybrids were precipitated with isopropyl alcohol. After washing with 70% ethanol, RNA pellets were dissolved in 10 ml of sample buffer (97% deionized formamide, 0.1% SDS, 10 mM TriszHCl, pH 7.0) and loaded onto a 5% acrylamide gel containing 7 M urea. The amounts of protected probe were quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Receptor Quantification. The number of a2-ARs was measured on crude membrane preparations using [H]MK912 (Pettibone et al., 1989). Frozen cells were harvested in 25 ml of Tris/EDTA buffer (50 mM TriszHCl, 5 mM EDTA, pH 7.5), then disrupted using a Dounce homogenizer and centrifuged at 39,000g for 10 min. The particulate fraction was washed in Tris/EDTA buffer and the final crude membrane pellet was taken up in the appropriate volume of Tris/MgCl2 (TM) buffer (50 mM TriszHCl, 0.5 mM MgCl2, pH 7.5). Total binding was measured by incubating 100 ml of cell membrane with the radioligand in a total volume of 400 ml of TM buffer. After a 45-min incubation at 25°C, bound and free radioactivity were separated by filtration through GF/C Whatman filters using a Millipore Manifold Sampling unit. The filters were rapidly washed with ice-cold TM buffer and membrane-bound radioactivity was determined by liquid spectrometry. Specific binding was defined as the difference between total and nonspecific binding measured in the presence of 10 M phentolamine. For saturation studies, the final concentrations of [H]MK912 ranged from 0.04 to 3 nM. Saturation isotherms were analyzed using the EBDA-LIGAND computer programs (McPherson, 1985). Protein concentration was estimated according to Bradford’s method using BSA as standard (Bradford, 1976). Reporter Gene Constructs. The a2C4-promoter/luciferase constructs are numbered relative to the translation start site of the a2C4 gene. They were generated from the promoterless vector pGL3-Basic, as follows. The BamHI-PstI fragment, corresponding to nucleotides 22806/2886 and ending six bases downstream of the transcription start site, was first subcloned into the BamHI and PstI sites of pKS1. It was then excised with BamHI-HindIII and inserted into the BglII822 Schaak et al. at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from HindIII sites of pGL3-Basic, generating the construct named pGL3C4 22806/2886. The series of 59-end deleted constructs (pGL3C4 21799/ 2886, pGL3C4 21633/2886 and pGL3C4 21340/2886) was obtained using the SmaI site in the polylinker of pGL3C4 22806/2886 and the SmaI, StuI, or DraI blunt sites located in the a2C4 fragment. Similarly, deletion by SacI resulted in the generation of a construct termed pGL3C4 21127/2886. The shortest construct in this study (pGL3C4 21044/-886) was generated by polymerase chain reaction using the sense primer 59-CATGGTACCCCGAGCCGCCCGTGCTGC-39, creating a KpnI site at position 21044 and an antisense primer located in the luciferase sequence. The amplification product was cut with KpnI and PstI and ligated into pGL3 digested with the same enzymes. Mutated versions of the pGL3C4 21127/2886 construct having the TGCCATCA sequence deleted or mutated into a canonical cAMP response element (CRE) were generated using the Quick Change Site-directed Mutagenesis kit from Stratagene. All constructs were verified by sequencing. Cell Transfection and Measurement of Luciferase Activity. HepG2 and JEG-3 cells were transfected using either the calcium phosphate method (Ausubel et al., 1994) or the Fugene-6 transfection reagent (Boehringer-Mannheim, Meylan, France). Transfections were performed in 30-mm diameter dishes. Cells were kept in the presence of the precipitate or Fugene/DNA complex for 8 h. They were placed in fresh medium, grown for an additional 24-h period and finally treated with 8-br-cAMP or not. Cells were harvested 12 h after treatment and luciferase activity was measured using Promega’s luciferase assay. Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assays. Nuclear extracts were prepared basically as described previously (Schreiber et al., 1989). The following oligonucleotides were used: oligonucleotide A (59-CTGCGAGAGGTTGCTCTGCCATCAGGCCATGGACCCCGAG-39) spans the region 21076/21037 of the a2C4 gene sequence. Oligonucleotide B (59GGTTGCTCTGCCATCAGGCCAT-39) corresponds to nucleotide 21068/21047. Oligonucleotide M (59-GGTTGCTCTCAGCACAGGCCAT-39) is identical with B but with the sequence TGCCATCA mutated into TCAGCACA. Oligonucleotide R (59-TGGAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGT-39) corresponds to the polylinker of the pRS series and was used as a nonspecific competitor of random sequence. Oligonucleotides A, B, or M (100 ng) were labeled in 15 ml of TriszHCl buffer (50 mM, pH 7.5) containing 10 mM MgCl2, 5 mM dithiothreitol, 50 mg/ml BSA, 20 mCi of [aP]dATP, and 2 IU of Klenow polymerase. Nuclear proteins (4 mg) and labeled double-strand probe (20,000 cpm) were incubated for 15 min at room temperature in a 15-ml final volume of HEPES buffer (40 mM, pH 7.9) containing 75 mM KCl, 0.4 mM EDTA, 1.5 mM dithiothreitol, 6% Ficoll 400, 1.5 mM MgCl2 and 1.5 mg of poly(dI-dC). DNA/protein complexes were separated on a 5% polyacrylamide gel and shifted probes were detected by autoradiography for 1 to 3 days. For competition assays, the cold competitor was added together with the labeled probe. Results Effect of Various Culture Conditions on a2-AR Expression. Studies carried out on various cellular models of human or rodent origin have demonstrated that the expression of the a2A-AR subtype is affected by different agents such as forskolin (Sakaue and Hoffman, 1991), insulin (Devedjian et al., 1991), PMA (Reutter et al., 1997), and dexamethasone (Hamamdzic et al., 1995). As a first effort to define the environmental factors that may interfere with a2C-AR expression, HepG2 cells were exposed to these four compounds. Measurement of [H]MK912 binding on membrane prepared from cells treated for 48 h indicated that neither insulin (100 nM), dexamethasone (10 mM), nor PMA (100 ng/ml) modified receptor density (not shown). In contrast, exposure to forskolin (10 mM) induced a significant reduction of [H]MK912 Bmax (Fig. 1). To verify that the effect of forskolin was cAMP-mediated, HepG2 cells were incubated in the presence of the cell-permeable cyclic-nucleotide analogs 8-br-cAMP and 8-br-cGMP. As shown in the right panel of Fig. 1, treatment with 8-br-cAMP but not 8-br-cGMP provoked a dose-dependent decrease in receptor density. Moreover, pretreatment with the PKA inhibitor H89 abolished the effect of 8-br-cAMP. In HepG2, the a2C-AR was recently demonstrated to undergo down-regulation in response to agonist exposure (Cayla et al., 1999). Cells were thus exposed to forskolin in combination with UK14304 (a2agonist) to see whether the effect of the two compounds were additive (Table 1). After 48 h of treatment, forskolin and UK14304 caused decreases of 35 6 9 and 51 6 12% in receptor number, respectively. A more pronounced reduction (68 6 4%) was observed when the two were combined. Additivity was also observed when the a2-agonist was combined with 8-br-cAMP. Taken together, these data demonstrate that the attenuation of a2C-AR expression by forskolin is mediated via the classical cAMP/PKA signaling pathway. They also suggested that forskolin and the a2-agonist act via independent mechanisms. In an effort to elucidate these mechanisms, we first analyzed the kinetics of 8-br-cAMP action. Kinetics of Receptor Decrease and Measurement of a2C4 mRNA Levels. The time course of the onset of receptor decrease induced by 8-br-cAMP is depicted in Fig. 2. In clear contrast to that observed with 10 mM UK14304, which caused a 50% down-regulation within 6 h, the effect of 8-brFig. 1. Effect of various treatments on a2C-AR density. Left, HepG2 cells were incubated for 48 h in serum-free DMEM containing 10 mM forskolin (f) or not (E). Membranes were prepared and used for [H]MK912 binding assay as described under Materials and Methods. The amount of specifically bound radioligand was determined using 10 M phentolamine to estimate nonspecific binding. The data from a typical experiment, in which each point is the mean of duplicate determinations, are displayed as Scatchard plots. Right, HepG2 cells were treated as above but with 10 mM forskolin, 8-br-cAMP (0.01 to 1 mM), 1 mM 8-br-cGMP, 10 mM H89, or 10 mM H89 plus 1 mM 8-brcAMP. Membranes were prepared and assayed for their capacity to bind [H]MK912. The values of receptor density were derived from analysis of saturation isotherms according to a one-component model and are expressed as femtomoles per milligram of protein. Results are the means 6 S.E.M. of four determinations (pp and p indicate values significantly different from controls at P , .01 and P , .05, respectively). Regulation of a2C-Adrenergic Receptor in HepG2 Cells 823 at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from cAMP was apparent only for periods of incubation longer than 12 h. The maximal decrease occurred after 36 h of treatment, 23 h being necessary for the effect of 8-br-cAMP to reach its half-maximum. Identical kinetics were observed with 10 mM forskolin (not shown). The down-regulation of a2C-AR induced by UK14304 is the consequence of an increased rate of degradation of the receptor protein without modification of the steady-state level of its mRNA (Cayla et al., 1999). Receptor half-life and the amounts of a2C4-mRNA were measured to see if the cAMP effect resulted from a similar mechanism. Receptor stability was examined as follows: after incubating the cells for 12 h with 1 mM 8-brcAMP, the protein synthesis inhibitor, cycloheximide (50 mg/ ml) was added and the disappearance of a2C-AR was appreciated by measuring [H]MK912 binding over a period of 24 h. It was seen that the a2C-AR half-life in cells treated with the cAMP analog (12.2 6 2.5 h) was not significantly different from that in control cells (13.5 6 1.5 h) (data not shown). The steady-state amounts of the a2C4-mRNA were measured by RPA on cellular RNA extracted from HepG2 cells incubated or not for 24 h in the presence of the different drugs. As shown in Fig. 3, a significant decrease was observed in cells exposed to forskolin or 8-br-cAMP. On the basis of four determinations and after normalization versus b-actin, the fall in a2C4-mRNA represented 30% for 10 mM forskolin and 44% for 1 mM 8-br-cAMP. The effect of 8-brcAMP was dose-dependent; it can be noted that the extent of the mRNA decrease closely matched the decrease in receptor expression as assessed by binding studies. As expected, UK14304 alone did not alter the amounts of mRNA. Furthermore, when applied in combination with 8-br-cAMP, it did not cause any additional reduction in the a2C4-mRNA level. Forskolin and 8-br-cAMP Inhibit a2C4 Gene Transcription. To clarify the mechanisms whereby the steady state levels of receptor mRNA are altered, we first examined the effect of 8-br-cAMP on the half-life of the a2C4 transcripts. To do so, HepG2 cells were incubated in medium containing the transcription inhibitor actinomycin D, either alone or in combination with 1 mM 8-br-cAMP; the disappearance of a2C4 mRNA was then monitored over a 4-h period. As illustrated in Fig. 4, the degradation rate of a2C4 mRNA was not affected, the half-life being 3.4 6 0.4 h in cAMP-treated cells versus 2.9 6 0.2 h in control cells (mean 6 S.E.M., n 5 3). The transcriptional activity of the a2C4 gene promoter was also investigated by transfecting HepG2 cells with a luciferase construct (pGL3C4 22806/ 2886) containing 1921 bases of the promoter region of a2C4 gene. As shown in Fig. 5, treatment of the cells for 12 h with 8-br-cAMP strongly inhibited the luciferase activity. Such an effect was observed neither with the corresponding promoterless vector pGL3-Basic nor with the pGL3-Promoter. Under identical experimental conditions, 8-br-cAMP induced a huge increase of the activity of the pCRE-Luc, a vector in which the reporter gene under the control of a promoter that comprises a TATA-box and four canonical CREs. Similar results were obtained with JEG3 cells, demonstrating that the observations made on HepG2 were not restricted to this specific cell line. Additional experiments indicated 1) that TABLE 1 Effect of combined exposure of HepG2 cells to UK14304 with forskolin or cAMP HepG2 cells were exposed to 10 mM forskolin, 1 mM 8-br-cAMP, 1 mM UK14304, 10 mM forskolin plus 1 mM UK14304, or 1 mM 8-br-cAMP plus 1 mM UK14304. After a 48-h period of treatment, membranes were prepared and assayed for their capacity to bind [H]MK912. The values of receptor density were derived from analysis of saturation isotherms according to a one-component model. Results are the means 6 S.E.M. of three determinations. Statistical analysis was performed using Student’s t test. Treatment [H]MK912 Bmax 21 mM UK14304 11 mM UK14304