glucocorticoid-induced down-regulation. In rat C6 glioma cells, which express both of these subtypes of ,-adrenergic receptors, the synthetic glucocorticoid ...
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Biochem. J. (1994) 302, 397-403 (Printed in Great Britain)
Glucocorticoids down-regulate f1-adrenergic-receptor expression by suppressing transcription of the receptor gene Joan KIELY,* John R. HADCOCK,*$ Suleiman W. BAHOUTHt and Craig C. MALBON*§ Department of Molecular Pharmacology, Diabetes & Metabolic Disease Research Program, School of Medicine, Health Sciences Center, University at Stony Brook, Stony Brook, NY 11794-8651, and t Department of Pharmacology, The University of Tennessee at Memphis, Memphis, TN 38163, U.S.A. *
The expression of /2-adrenergic receptors is up-regulated by glucocorticoids. In contrast, /%-adrenergic receptors display glucocorticoid-induced down-regulation. In rat C6 glioma cells, which express both of these subtypes of ,-adrenergic receptors, the synthetic glucocorticoid dexamethasone stimulates no change in the total fi-adrenergic receptor content, but rather shifts the I1: I2 ratio from 80:20 to 50:50. Radioligand binding and immunoblotting demonstrate a sharp decline in f,i-adrenergic receptor expression. Metabolic labelling of cells with [35S]methionine in tandem with immunoprecipitation by /%adrenergic-receptor-specific antibodies reveals a sharp decline in the synthesis of the receptor within 48 h for cells challenged with glucocorticoid. Steady-state levels of P,-adrenergic-receptor
mRNA declined from 0.47 to 0.26 amol/,ug of total cellular RNA within 2 h of dexamethasone challenge, as measured by DNA-excess solution hybridization. The stability of receptor mRNA was not influenced by glucocorticoid; the half-lives of the ,61- and ,82-subtype mRNAs were 1.7 and 1.5 h respectively. Nuclear run-on assays revealed the basis for the down-regulation of receptor expression, i.e. a sharp decline in the relative rate of transcription for the fll-adrenergic-receptor gene in nuclei from dexamethasone-treated as compared with vehicle-treated cells. These data demonstrate transcriptional suppression as a molecular explanation for glucocorticoid-induced down-regulation of /1-adrenergic receptors.
INTRODUCTION
corticoids independent of those effects accompanying differentiation, we investigated regulation of fl-adrenergic receptors by glucocorticoids in terminally differentiated rat C6 glioma cells. The data are the first to provide a molecular explanation, i.e. transcriptional suppression, for the effect of glucocorticoids on the expression of fl1-adrenergic receptors.
The regulation of the expression of ,-adrenergic receptors is not thoroughly understood (Stiles et al., 1984; Wang et al., 1990). These receptors are subdivided into three subtypes, ,?-, f2- and l3-, each subtype encoded by a separate gene (for references, see Emorine et al., 1989). I8- and fl2-adrenergic receptors have been differentiated by pharmacological (Lands et al., 1967), structural (Cubero and Malbon, 1984; Graziano et al., 1985; Moxham et al., 1986; Wang et al., 1989) and genetic (Dixon et al., 1986; Frielle et al., 1987; Bahouth et al., 1988) criteria. Comparison of the sequences of the f1- and ,82-receptor cDNAs revealed only a 550% similarity in the coding region (Dixon et al., 1986; Frielle et al., 1987). This divergence in nucleotide sequence permitted the construction of subtype-specific DNA probes for use in measuring mRNA levels (Guest et al., 1990). Glucocorticoid and thyroid hormones have been shown to regulate the expression of ,f-adrenergic receptors (Malbon, 1980; Sibley and Lefkowitz, 1985; Davies and Lefkowitz, 1984; Hadcock and Malbon, 1988a; Collins et al., 1988, 1991). Similarly, cyclic AMP and retinoic acid also regulate fl-adrenergic receptor expression (Hadcock and Malbon, 1988a; Hadcock et al., 1989a; Galvin-Parton et al., 1990). fl2-Adrenergic receptor expression increases in many cell types in response to glucocorticoids (for references, see Collins et al., 1988; Hadcock and Malbon, 1988b; Malbon and Hadcock, 1988). In contrast, far less is known about the regulation of /,adrenergic receptors by steroids. Recent studies of mouse NIH 3T3-L1 and 3T3F442A cells demonstrated that the expression of /3k- and /82-adrenergic receptors is altered as these cells progress through differentiation induced by glucocorticoids in combination with other factors (Guest et al., 1990; Feve et al., 1990). To examine regulation of /ll-adrenergic receptors by gluco-
MATERIALS AND METHODS The hamster /82-adrenergic-receptor cDNA (Dixon et al., 1986) was a gift from Dr. R. A. F. Dixon, Merck, Sharp and Dohme, West Point, PA, U.S.A. The sources of all other material is detailed elsewhere (Hadcock and Malbon, 1988a) and in the text. The human fll-adrenergic-receptor cDNA was generously given by Dr. R. J. Lefkowitz, HHMI, Duke University, Durham, NC, U.S.A.
Cell culture Rat C6 glioma cells were grown to confluence as monolayers in Dulbecco's modified Eagle's medium (DMEM), supplemented with 5 % fetal-bovine serum, streptomycin (60 mg/l) and penicillin (60 mg/l), as described by Fishman et al. (1981). Stock solutions of steroids were prepared in an ethanol vehicle and then diluted directly in DMEM. Untreated (control) cells were grown in the same DMEM to which only the vehicle was added. Membrane preparation Membrane fractions were prepared as described by Fishman et al. (1981), a lysis buffer composed of 2 mM Tris/HCl (pH 7.4), 1 mM EDTA, aprotinin (5 ,tg/ml) and leupeptin (5 ,ug/ml).
Abbreviations used: DMEM, Dulbecco's modified Eagle's medium; [1251]CYP, (-)-[1251]iodocyanopindolol; GRE, glucocorticoid response element. $ Present address: American Cyanamid Co., Agricultural Research Division, Molecular & Cellular Biology Group, Princeton, NJ 08543-0400, U.S.A. § To whom all correspondence should be addressed.
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Radloligand binding ,-adrenergic receptors were measured in a crude membrane fraction of the post-nuclear supernatant, obtained by first depleting the broken-cell preparation of nuclei with a low-speed centrifugation (5000 g, 5 min), followed by a high-speed centrifugation (48 000 gav) for 20 min at 4 'C. The resultant pellet collected by this final centrifugation step was resuspended in binding-assay buffer. Equilibrium radioligand-binding analysis was performed as described by Fishman et al. (1981) with the antagonist ligand (- )-[1251]-iodocyanopindolol ([1251]CYP; 50100 pM). Non-specific [1251]CYP binding insensitive to competition by 1O #uM propranolol represented about 100% of the radioligand binding. Binding data were corrected for the nonspecific component. Specific binding of [125I]CYP to ,1-adrenergic receptors was measured by competition binding with 0.1 ,uM of the,81-selective antagonist CGP-20712A (Bahouth and Malbon, 1987). This concentration of CGP-20712A has been shown to displace virtually all #,/-receptor binding in membranes of C6 glioma cells (Zhong and Minneman, 1993).
Immunoblotting Crude membranes were solubilized in 0.125 M Tris/HCl (pH 6.8) containing 4 % SDS, 5 % sucrose and 20 mM dithiothreitol, and incubated at 37 'C for 30 min. The samples were alkylated with 45 mM iodoacetamide and the solubilized proteins were separated by electrophoresis on 10 %-acrylamide gels. The resolved proteins were transferred electrophoretically to nitrocellulose (Moxham et al., 1986). The nitrocellulose blots were stained with a /J1-adrenergic-receptor-specific antibody (SB-03) prepared against a synthetic peptide corresponding to a sequence unique to this subtype (Bahouth, 1991), at a final dilution of 1:200. The blots were then probed with phosphatase-labelled or 1251-labelled goat anti-rabbit IgG, and the immune complexes were made visible by phosphatase-catalysed staining or autoradiography, respectively (Smith et al., 1987). Characterization ofthis antibody is provided elsewhere (Bahouth, 1991).
Metabolic labelling of cells Cells, cultured as described above, were washed twice with PBS and then cultured in methionine-free DMEM containing 50% fetal-calf serum. To each batch of 107 cells, [35S]methionine (200 ,Ci) and either vehicle or dexamethasone (500 nM) was added. Cells were harvested at the times indicated, washed twice with PBS, and then used as a source for the preparation of crude membranes. Immunoprecipitation from detergent extracts of the crude membranes was performed as described by Hadcock et al. (1990).
Extraction of RNA RNA was extracted by the guanidine isothiocyanate/ethanol precipitation method (Chirgwin et al., 1979). The integrity of the RNA was assessed by gel electrophoresis on 1.2 % agarose in 3 % formaldehyde (Sambrook et al., 1989).
Synthesis of solution probes HI11O (41-receptor) and Hfl170
(#,-receptor) The solution hybridization probe (/?AR 170) was constructed as described previously (Hadcock and Malbon, 1988a; Hadcock
et al., 1989b). A DNA fragment corresponding to the sequence +265 to +376 of the human fll-adrenergic-receptor cDNA coding region was subcloned into the phagemid Bluescript SK+. The single-stranded probe, Hf1 10, was generated by the VSC M13 helper-phage procedure of Vierra and Messing (1987). The probes, H,f I10 and H/3170, were prepared as described by Williams et al. (1986), as modified by Bahouth et al. (1988). The probes were uniformly radiolabelled with [a-32P]dCTP (sp. radioactivity 222 Ci/mmol) by primer extension, followed by digestion with SmaI. Single-stranded probes were separated from doublestranded DNA by gel chromatography on hydroxyapatite using a step gradient of sodium phosphate (20-220 mM), followed by desalting on NEN-sorb columns (New England Nuclear).
DNA-excess solution-hybridization
of receptor mRNA levels Uniformly radiolabelled probe [1.5 fmol (100 pg) per sample] assay
was incubated with known amounts a standard), total cellular RNA, or
of template DNA (used as alone for 60 h at 68 °C in 20 mM Hepes (pH 7.0)/0.3 M NaCl/1 mM EDTA containing 100 #,g/ml denatured salmon sperm DNA. S1 endonuclease (150 units/ml), denatured salmon sperm DNA (50 jtg/ml) and S1-endonuclease buffer (0.28 M NaCl, 4.5 mM ZnSO4, 50 mM sodium acetate, pH 4.5) were then added to each sample, and the mixture was incubated for 90 min at 42 0C. The samples were treated with trichloroacetic acid (7.5 %) and precipitated on ice for 10 min. The Sl -endonuclease-resistant hybrids were collected by vacuum filtration on Whatman GF/C filters. The extent of internal resistance of the probe to SI-endonuclease treatment was typically less than 3 % for each assay. Data from DNAexcess solution-hybridization assays were calculated as described previously (Hadcock and Malbon, 1988a; Williams et al., 1986) and expressed either as amol of fl-adrenergic-receptor mRNA per ,ug of total cellular RNA, or '% of control'.
Receptor-mRNA stability The half-life of ,-adrenergic-receptor mRNA was determined by the method of Rodgers et al. (1985), as previously reported (Hadcock et al., 1989a). Cells were exposed to dexamethasone (500 nM) or vehicle for 2 h. Actinomycin D (5 ,tg/ml) was added thereafter, and RNA was isolated from samples taken over the next 6 h period. The content of /%- and /82-adrenergic-receptor mRNA in the total cellular RNA was quantified by DNA-excess solution hybridization.
Nuclear run-on transcription assays Cells were cultured in DMEM supplemented with 5% fetalbovine serum depleted of steroids by prior treatment with 0.1 % activated charcoal. Dexamethasone (250 nM) or vehicle was added to the culture medium for 2 h, and the cells were then washed twice with ice-cold PBS. Initial studies of transcription by the method of Greenberg and Ziff (1984) using frozen nuclei provided no clear effect of the glucocorticoid treatment in run-on assays using nuclei from steroid-treated cells. An alternative modification was employed, as described by Bahouth (1991). To each 100 mm dish, 1 ml of buffer I (10 mM Tris/HCl, pH 8.0, 10 mM NaCl, 2.5 mM MgCl2 and 5 mM dithiothreitol) was added for 10 min. A 1 ml portion of buffer I containing 0.6 M sucrose and 0.6% Triton X-100 was then added, and the cells were scraped into a chilled Dounce homogenizer. The cells were homogenized by six strokes with a type A pestle, and the homogenate was layered over 0.6 M sucrose in buffer I. Nuclei were collected by centrifugation at 2000 g.,. for IO min and
Glucocorticoid regulation of /,3-adrenergic-receptor mRNA resuspended in glycerol buffer (50 mM Tris/HCl, pH 8.3, 40 % glycerol, 5 mM MgCl2 and 0.1 mM EDTA) for immediate assay (Greenberg and Bender, 1989). Frozen nuclei were found to be unsuitable for the nuclear run-on assays. To detect nascent transcripts, nuclei [(2-3) x 107] in 200 ul were added to 200 ,l of a reaction buffer composed of 10 mM Tris/HCl, pH 8.0, 5 mM MgCl2, 0.3 M KCI, 5 mM dithiothreitol, 0.5 mM unlabelled GTP, ATP and CTP, and 10t1l of [a-32P]UTP (New England Nuclear, 800 Ci/mmol; final concn. 0.5 mM UTP) for a 60 min reaction at 30 'C. Newly transcribed labelled RNA was extracted (Greenberg and Ziff, 1984) and then hybridized for 36 h at 65 'C with plasmid DNA immobilized on nitrocellulose. After hybridization, each sample was washed twice with 2 + SSC (SSC is 150 mM NaCl/ 15 mM sodium citrate, pH 7.0) for 60 min at 65 'C. The samples were then treated with RNAase A for 30 min at 37 'C, followed by a wash with 2 x SSC at 37 'C for 60 min. The filters were dried and subjected to autoradiography for 5 days with an intensifying screen. Relative changes in transcription were assessed by scanning densitometry of the autoradiogram. The absorbance of the /,B-adrenergic receptor band was normalized against that of the a-tubulin band, to correct for any differences in hybridization. The plasmid harbouring the rat ,/1-adrenergic-receptor cDNA was generated by subcloning the 1765-bp Styl fragment of the rat 181-adrenergic-receptor gene (nucleotides -274 to + 1418 relative to the translational start site) into pBluescript SK+ (Shimomura and Terada, 1990). The rat /82-adrenergic-receptor plasmid RHB-DAR was purchased from the American Type Culture Collection (Rockville, MD, U.S.A.). This plasmid contains the sequence - 101 to + 1878 relative to the translational start site (Cocayne et al., 1987). The avian a-tubulin cDNA in plasmid pT1 was generously given by Donald W. Cleveland (HHMI, Johns Hopkins University Medical Center). The ,adrenergic-receptor plasmids were linearized by EcoRI and 10 ,ug of plasmid/lane was used. The pT1 plasmid was linearized by PstI, and 5,ug/lane was employed.
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on expression of ,-adrenergic receptors in rat C6 glioma cells
(Table 2). The oestrogen /J-oestradiol (500 nM), the progestin progesterone (500 nM) and the androgen testosterone (500 nM) altered neither the total number of f8-adrenergic receptors nor the ratio of receptor subtypes (/81:fi2). Likewise, both the progesterone antagonist RU486 (1 uM) and retinoic acid (100 nM) had no significant effect on f-adrenergic-receptor expression by these cells. Immunoblots of crude membranes prepared from C6 cells and stained with a /,%-adrenergic-receptor-specific antiserum (SB-03; Bahouth, 1991) revealed a prominent band of immunoreactivity corresponding to Mr 65000 (Figure 1, 'Blot'). Exposure of C6 glioma cells to dexamethasone (500 nM) for 48 h was accompanied by a sharp decline in the amount of immunostaining of C6 glioma cell membranes with anti-receptor antibodies (Figure 1, 'Blot'). Confirming the glucocorticoid-induced decline in 181receptor radioligand binding, the results of the immunoblotting displayed a decline in /?1-adrenergic-receptor immunoreactivity in blots prepared from glucocorticoid-treated cells. The immunoblotting data suggest a greater apparent decline in receptor immunoreactivity than that observed indirectly by radioligand binding with the fl1-receptor-selective antagonist CGP-20712A. Glucocorticoid-induced up-regulation of the expression of the fl2-adrenergic receptors has been demonstrated at the level of mRNA and protein (Hadcock and Malbon, 1988b; Collins et al., 1988; Malbon and Hadcock, 1988; Hadcock et al., 1989a).
Table 1 Glucocortlcolds down-regulate fl1- and up-regulate f12-adrenergic receptors in rat C6 glioma cells Cells were exposed to vehicle (control) or dexamethasone (500 nM) for 48 h and then employed as a source for crude membranes. ,-Adrenergic receptors were measured in these membranes by [1251]CYP binding in competition with propranolol (f1 +#2) or CGP-20712A (fl1-selective). The data presented are means+S.E.M. from 10 independent experiments, each assayed in triplicate. Difference from control values (paired Student's t test): *P = 0.026, **P = 0.003.
[1251]CYP binding (fmol/mg of protein)
Presentation of data Unless noted otherwise, data are presented as mean+ S.E.M. Statistical significance was determined by Student's t test.
RESULTS Rat C6 glioma cells express both ,81- and /32-adrenergic receptors
(Table 1). The ratio of 1/3- to /82-subtype was determined by competition with the ,ll-adrenergic-receptor-selective antagonist CGP-20712A (Bahouth and Malbon, 1987). Under standard culture conditions, C6 glioma cells at confluence displayed 235 + 50 fmol of /ll-adrenergic receptors/mg of protein (mean + S.E.M., n = 10), of which nearly 80 % (185 + 40 fmol/mg) wasf1 and the remaining 20 % (52 + 9 fmol/mg) was /32-subtype. Treatment of these cells with the synthetic glucocorticoid dexamethasone (500 nM) for 48 h shifted the ratio of 318 132 markedly, from 80:20 to 50:50. Total receptor content was unchanged (P = 0.31 for the difference between steroid-treated and control cells). Thus, glucocorticoids induce a 45 % decline (to 105+18 fmol/mg; P < 0.05) in the 81 component, whereas the ,82-receptor population increases by about 2-fold (to 102+ 18 fmol/mg; P < 0.01). The ability of glucocorticoids to up-regulate 182- and downregulate 81-adrenergic receptors prompted us to investigate the effects of steroid analogues and hormones such as retinoic acid
Total /3-adrenergic receptors
fl1-Adrenergic receptors ,82-Adrenergic receptors
Control
Dexamethasone
235 + 50 185 + 40 52 + 09
210+ 25 105+18* 102+18**
Table 2 Glucorticoids, but not oestradiol, progesterone, testosterone or retinoic acid, down-regulate 81- and up-regulate 82-adrenergic receptors C6 glioma cells were treated with vehicle alone (C) or with dexamethasone (500 nM), oestradiol (500 nM), progesterone (500 nM), retinoic acid (100 nM), testosterone (500 nM) or RU-486 (1.0 ,uM) for 48 h before harvest. fl-Adrenergic receptors were measured in crude membranes prepared from these cells, by [1251]CYP binding in competition with propranolol (/81 + #2) or CGP-20712A (,81-selective). The data presented are means+S.E.M. from 6 independent experiments, each assayed in triplicate. Abbreviation: AR, adrenergic receptors. Treatment Control Dexamethasone
Oestradiol Progesterone Retinoic acid Testosterone RU-486
Total ,8-AR (fmol/mg of protein)
lj1AR (%)
(%)
268 + 30 225 + 50 260 + 35 237 + 42 265 +105 255 + 20 268 + 26
73 + 5 48 + 2 68 + 8 67 +11 65 +10 65 + 5 65 + 4
27 + 4 52 + 3 32 + 7 33 + 6 35 + 9 35+8 35+4
82AR
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10-3 x
Mr
1
2
3
4
Metabolic labelling 7 5 6
8
No. of bases
9
No. of bases
74>
65> .......
48>DEX...
