Calcium/Calmodulin Regulation of the Rat Prolactin Gene is Conferred by the Proximal Enhancer Region
Julian R. E. Davis, Nigel Hoggard, Elizabeth M. Wilson, Maria E. Vidal, and Michael C. Sheppard Department of Medicine (J.R.E.D., N.H.) Manchester University Manchester M13 9PT, United Kingdom Department of Medicine Queen Elizabeth Hospital Edgbaston, Birmingham B15 2TH, United Kingdon
The effects of the calcium/calmodulin signaling system on expression of the rat PRL gene were studied in rat pituitary GH3 cells using two specific naphthalene sulfonamide calmodulin (CaM) antagonist drugs, W7 and a more potent and more highly specific iodo-derivative, 5-iodo-1-C8. PRL (but not GH) mRNA accumulation was markedly inhibited by W7, which in coincubations abolished the stimulation normally seen with TRH. Transient transfection assays showed that expression of the reporter gene chloramphenicol acetyl transferase (CAT) linked to 5'-flanking sequences from the PRL gene was inhibited by the calcium-channel blocker verapamil and by the two CaM antagonists. The calcium effects showed partial promoter specificity, in that transcription of PRL-CAT constructs was markedly inhibited by verapamil, but the Rous sarcoma virus-CAT construct also showed significant inhibition, whereas the pBL-CAT2 construct was unaffected. Three hundred ninety five base pairs were sufficient to confer the full inhibitory effect of calcium channel blockade or CaM antagonist seen with longer constructs. The data indicate that CaM is important for PRL gene transcription, and that the effects of CaM are exerted on DNA sequences within the proximal 395bp of prolactin 5'-flanking DNA. (Molecular Endocrinology 5: 8-12, 1991)
intracellular calcium stores and influx of extracellular calcium through voltage-gated L-type channels are involved (12, 13). The proximal 172 base pairs of 5'flanking DNA from the PRL gene are able to confer calcium-responsiveness onto a reporter gene (14). The effects of calcium on the PRL gene may be mediated either by protein kinase C or by CaM. Earlier work has suggested that CaM is involved also in regulating PRL mRNA accumulation and gene transcriptional rate (15, 16), but many of the drugs used, notably the phenothiazines, are recognized to have effects on other intracellular signaling systems including protein kinase C itself. In order to address the question of the role of CaM in PRL gene regulation we have employed two specific CaM antagonist agents: the highly specific naphthalene sulfonamide CaM antagonist W7 (A/-(6-aminohexyl)-1 naphthalene sulfonamide; IC50 ~30 ^mol/liter) which has only very slight effects on protein kinase C, and a more potent and more specific iodo-derivative of W7, 5-iodo-1-C8 (abbreviated W8 in this report, IC50 ~3 /iimol/liter) which has no detectable effect on protein kinase C (17). We have demonstrated CaM dependence of PRL mRNA levels (to a much greater extent than GH mRNA levels) in GH3 cells. Secondly, we have demonstrated that the proximal 395 bp of upstream flanking DNA from the rat PRL gene confers calcium and CaM dependence on a bacterial reporter gene, expressed in transfected GH3 cells.
INTRODUCTION PRL secretion is dependent upon calcium and calmodulin (CaM; 1-6), and the calcium-CaM signaling system is thought to mediate in part the effects of TRH (7). PRL mRNA accumulation (much more than GH mRNA) is critically dependent upon calcium (8-11), and both 0888-8809/91/0008-0012$02.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
RESULTS Hybridization Experiments The CaM antagonist W7 reduced PRL secretion, and PRL mRNA levels fell to 26% of control after 24-h incubation with 100 HM W7 (Fig. 1a). W7 (100 ^mol/ liter) reduced both PRL secretion and PRL mRNA levels
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Calmodulin Regulation of PRL Gene
NS
mRNA 0.5 (O.D. u n i t s )
lull
lOOuM Control D vi lOugnQ
release
0
lun
irxii tw7]
mRNA O.S (0.0. units)
ng/ml
lun
iooun ( W 7 1
Fig. 1. Effects of CaM antagonist W7 on GH3 cells A, Effects of treatment with 1 or 100 HM W7 for 24 h on PRL mRNA accumulation and PRL secretion, n = 6 from a single experiment. B, Effects of coincubation with 100 MM W7 and 100 nM TRH for 24 h (n = 12 replicates per group pooled from three similar experiments, P < 0.01 compared with control in each case). C, Effects of W7 on GH mRNA in the same experiments; n = 6 from the experiment shown in A, and n = 12 from the experiments shown in B. *, P < 0.03 compared to respective controls.
and prevented any stimulation by 100 nM TRH (Fig. 1 b). GH mRNA levels in these experiments showed slight reductions (to 77-92% of control) in the presence of 100 fiM W7, but these were much less marked than for PRL mRNA (Fig. 1c). High doses of the CaM antagonists caused rounding of GH3 cells and reduced adherence to the culture dishes, but did not affect cell viability as determined by trypan blue exclusion.
% OF CONTROL ACETYLATION
r395CAT
Transfection Experiments
r958CAT
RSVCAT
pBLCAT2
Fig. 2. Effects of verapamil (50 HM for 24 h) on acetylation of C-chloramphenicol by cytoplasmic extracts of GH3 cells transfected with rPRL(-395)-CAT, rPRL(-958)-CAT, RSVCAT, or pBL-CAT2 The inhibition due to verapamil is shown as a percentage of control values for each construct. Values shown are means ± SEM of triplicate determinations, and similar results were obtained in two or three independent experiments in each case. *, P < 0.01 compared to respective controls. 14
GH3 cells showed no endogenous CAT activity, while cells transfected with the Rous sarcoma virus (RSV> chloramphenicol acetyl transferase (CAT) construct had high levels of activity, with up to 90% acetylation of chloramphenicol per 100 ng protein. Transfection efficiency differed somewhat between different experiments, but in general transfection with the PRL-CAT constructs resulted in acetylation of approximately 15% of that seen with the RSV-CAT construct. In experiments to validate calcium dependence of the transfected constructs, GH3 cells were transfected with different constructs and treated with control medium or 50 nM verapamil for 24 h. Cells were transfected before being divided into treatment groups to avoid possible differences due to different transfection efficiency. Verapamil treatment reduced CAT activity from the PRL(-395)-CAT and PRL(-958)-CAT constructs to 12.5 ± 3.4% and 16.3 ± 2.1 % of control levels, respectively, but had less effect on RSV-CAT (30.1 ± 3.3% of control) and none on pBL-CAT2 (Fig. 2). On the other hand, treatment with the calcium channel agonist Bay K 8644 increased CAT activity from the PRL-CAT con-
structs but not from the other promoters, indicating a degree of promoter specificity of calcium channel blockade or activation, as has been reported previously (14, 18) (data not shown). The two CaM antagonists W7 and W8 both markedly reduced CAT activity in GH3 cells transfected with PRL(-395)-CAT (Fig. 3). W8 at 3 fiM reduced CAT activity to 73% of control levels (mean of two similar experiments), and to 14 ± 6% of control at 10 ^ M concentration (mean ± SEM from four separate experiments). W7 reduced CAT activity to 6% and 5% of control at 10 and 50 HM concentrations, respectively (mean of two similar experiments). The degree of inhi-
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MOL ENDO-1991 10
RSVCAT
50 nM W7
PRL(-395)-CAT
Fig. 3. Effects of two CaM antagonists (W7 and W8) on acetylation of 14C-chloramphenicol by cytoplasmic extracts of GH3 cells transfected with rPRL-(-395)-CAT The first lane shows cells transfected with RSV-CAT plasmid. A representative experiment is shown (see text), and cells were treated with CaM antagonists for 24 h before assay for CAT activity.
bition of CAT activity by W8 was similar using both PRL-CAT constructs: using the PRL(-395)-CAT construct activity was reduced by 10 HM W8 to 14% of control levels, and with the PRL(-958)-CAT construct to 22% of control (means of four and two independent experiments, respectively).
DISCUSSION
The data presented in this report indicate that highly specific CaM antagonists have major effects on rat PRL gene transcription in GH3 cells, and show that the mechanism for calcium-CaM action may involve the proximal enhancer region from the 5'-flanking DNA within the gene. The precise role of calcium in basal and stimulated PRL gene transcription has been a subject of some controversy. Calcium is an important cofactor for protein kinase C, which is an established stimulator of PRL gene transcription (15, 19), but it also activates the calcium-binding protein CaM. Recent evidence has shown that down-regulation of protein kinase C by chronic exposure of GH3 cells to phorboi esters does not affect the increase in PRL mRNA levels induced by calcium (20), implying that this kinase is not essential for this effect. Previous studies have suggested that CaM is important in PRL gene transcription (15, 16, 21), but much previous work has relied upon CaM antagonists whose specificity is relatively poor as they also inhibit a number of other intracellular enzymes including protein kinase C itself (17). The present results, obtained using two highly specific CaM antagonists which have only minimal effect on protein kinase
C, confirm that PRL gene expression is indeed CaM dependent, in contrast to GH mRNA. The effects of calcium and CaM appear to be mediated by effects on the 5'-flanking DNA in the PRL gene. The PRL gene contains a proximal enhancer region (up to 400 bp upstream from the cap site) and a distal region (up to 1700 bp upstream); response elements have recently been defined for several hormones and intracellular regulators within these enhancer regions (22). For example, the transcriptional response to both epidermal growth factor (EGF) and protein kinase C activation by phorboi ester is conferred by DNA sequences between positions - 3 5 and - 7 8 bp upstream from the cap site (19). A precise response element for calcium itself has not yet been identified, but the information required for a full transcriptional response to calcium is contained within the first 174 bp of DNA upstream from the cap site (14), and our results with Bay K 8644 and verapamil using the PRL(-395)CAT construct confirm these findings. The present data show that the CaM-sensitivity of the endogenous PRL gene shown by the hybridization studies is, like the calcium responsiveness, conferred onto the CAT reporter gene by the first 395 bp of the proximal enhancer region of the gene. Pharmacological manipulations of the calcium/CaM signaling system are likely to have wide-spread effects on cellular metabolism and on a variety of different genes, and indeed our data show that the transcription of CAT from the RSV promoter sequence is also substantially reduced by verapamil, whereas that with the pBL-CAT2 construct was unaffected. Likewise, three control promoter-CAT constructs tested by Jackson and Bancroft (14) in fact showed some degree of calcium induction. However these effects were much different in magnitude compared to the effects on the PRL gene, implying some degree of promoter specificity. The proximal enhancer region of the PRL gene therefore appears to contain specific CaM response elements) which are important in basal and TRH-stimulated gene transcriptional control. However the rPRL gene appears to contain multiple potential response elements, and TRH, EGF, and cAMP effects have recently been shown to be conferred onto a reporter gene by the distal as well as the proximal enhancer regions of the gene (23), and Day and Maurer (18) have recently shown that the distal enhancer also confers a transcriptional response to a pure L-type calcium channel agonist. The 174 bp calcium-responsive stretch of the PRL 5'-flanking DNA contains three of the four proximal binding sites for a pituitary-specific protein factor (termed Pit-1, PUF-1, or LSF-1) (24-27). The first such site coincides with a response region for EGF and phorboi ester (19) and also with a possible recognition sequence for the CaM-dependent enzyme DNA topoisomerase (28), inviting the speculation that these intracellular signals may exert their effect by modifying the binding of tissue-specific proteins to DNA or to other DNA-binding factors, or alternatively by modifying al-
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Calmodulin Regulation of PRL Gene
ready-bound trans factors to affect their interaction with the transcription initiation complex. So far there are few data available that address this question, but preliminary data suggest that the binding of pituitary-specific nuclear protein(s) to putative Pit-1 binding sites within the proximal enhancer region is affected by CaM antagonists (Wilson, J., I. D. Phillips, and J. R. E. Davis, manuscript in preparation). Further studies will be required to investigate in detail the mechanisms of calcium-CaM effects on expression of the PRL gene.
11
natants from the cell lysate were assayed for protein content, and stored for assay of CAT activity. Assay for CAT Activity CAT activity was assayed as described (33) in a 100 n\ reaction volume containing 100 ^g cell lysate protein, 12 mM acetyl Coenzyme A, and 0.2 nC\ 14C-labeled chloramphenicol. Reactions were incubated for 16 h at 37 C, and samples were then extracted with ethyl acetate before acetylation products were separated by TLC, and quantified by scintillation counting. Results are expressed as percent acetylation of total chloramphenicol/100 M9 protein. Reagents
MATERIALS AND METHODS GH3 Cell Culture and RIAs GH3 cells obtained from the European Collection of Animal and Cell Cultures, were grown in continuous monolayer culture in Ham's F-10 medium supplemented with 5% fetal calf serum (GIBCO, Paisley, Strathclyde, UK) and 10% horse serum (GIBCO). Cells were subcultured from single donor cultures 2 days before experiments, and then washed with serum-free medium before treatment with test substances in serum-free F-10 medium as described. PRL secretion into culture fluid was measured by RIA using reagents supplied by the National Hormone and Pituitary Program, NIADDK (Bethesda, MD); results are expressed in terms of the standard rPRL-RP3. Hybridization Studies: mRNA Measurements Relative cytoplasmic concentrations of PRL and GH mRNA were measured by cytoplasmic dot hybridization analysis (29) as described previously (30). Briefly, plasmid probes containing complementary DNA inserts specific for rat PRL or rat GH were labeled with 32P-dCTP by nick translation to a specific activity of 1-2 x 108 dpm/^g DNA. After filter hybridization and autoradiography, mRNA data were quantified by scanning densitometry and data expressed in arbitrary units of optical density (O.D.) of the developed autoradiographs. Transfection Studies GH3 cells were transfected with synthetic DNA constructs consisting of 395 bp or 958 bp of the rat PRL promoter (31) linked to the bacterial CAT gene, or as a control RSV-CAT or pBL-CAT2 (32). Transfection was carried out either by the diethylaminoethyl (DEAE)-Dextran method (CaM antagonist data) or by electroporation (verapamil data). Two days before transfection, GH3 cells were subcultured onto 100-mm Petri dishes at 4 x 106 cells per dish. For the DEAE-Dextran method, cells were washed with serum-free medium, before the addition to each dish of 10 ng plasmid DNA in 5 ml medium containing 50 ITIM Tris (pH 7.5) and 100 M9/ml DEAE dextran. After 30-60 min this medium was removed, and the cells were washed before addition of fresh medium or test substances for 24 h. For the electroporation technique, approximately 50 x 106 cells in 0.8 ml culture medium were transferred to an electroporation cuvette containing 20 ^9 plasmid DNA and then transfected by a 275 V pulse at a total capacitance of 960 nf (Electropore apparatus, SEDD, Liege, Belgium). The cells were immediately diluted in culture medium and then divided into a series of 35-mm multiwell culture dishes for treatment with test substances. At the end of the incubation period, medium was removed from the culture dishes, and the cells were washed in ice-cold PBS. Cells were then harvested by trypsinization, washed, and lysed in 0.5% Triton X-100 in 0.25 mui Tris (pH 7.8). Cell lysates were centrifuged at 15,000 x g for 4 min, and super-
Restriction enzymes and kinases were purchased from Boehringer Mannheim (Lewes, East Sussex, UK). W7 (Sigma, Poole, Dorset, UK) and W8 (17) were initially dissolved in dimethylsulfoxide and freshly diluted in F-10 culture fluid before experiments. Verapamil was purchased from Abbott Laboratories Ltd. (Queensborough, Kent, UK), and Bay K 8644 was a generous gift from Bayer Pharmaceuticals Ltd. Radiolabeled chloramphenicol was purchased from Amersham International PLC (Amersham, Bucks, UK), and DEAE-dextran from Sigma.
Acknowledgments The authors are very grateful to Prof. F. C. Bancroft, New York, New York for generous provision of the PRL(-395)CAT, PRL(-958)-CAT clones, and for the rGH cDNA probe, and to Dr. R. A. Maurer, Iowa City, Iowa, for the rPRL cDNA probe. Dr. G. M. Blackburn, Department of Chemistry, Sheffield University, UK, kindly donated the W7 derivative 5-iodo1-C8 (termed W8 in this paper). Received December 19, 1989. Revision received October 5,1990. Accepted October 5,1990 Address requests for reprints to: Dr. J. R. E. Davis, Department of Medicine, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom. These studies were supported by grants from the Central Birmingham Health Authority Endowment Fund, the University of Birmingham Faculty of Medicine Scientific Projects Committee, and the Wellcome Trust. Portions of this work were presented at the 71st meeting of The Endocrine Society, Seattle, WA, 1989 (Abstract 64).
REFERENCES 1. Tashjian Jr AH, Lomedico ME, Maina D 1978 Role of calcium in the thyrotropin-releasing hormone-stimulated release of prolactin from pituitary cells in culture. Biochem Biophys Res Commun 81:798-806 2. Merritt JE, Tomlinson S, Brown BL 1981 Phenothiazines inhibit prolactin secretion in vitro. A possible role for calmodulin in stimulus-secretion coupling in the pituitary. FEBS Letts 135:107-110 3. Schettini G, Cronin MJ, Macleod RM 1983 Adenosine 3',5'-monophosphate (cAMP) and calcium-calmodulin interrelation in the control of prolactin secretion: evidence for dopamine inhibition of cAMP accumulation and prolactin release after calcium mobilization. Endocrinology 112:1801-1807 4. Merritt JE, Brown BL 1984 An investigation of the involvement of calcium in the control of prolactin secretion: studies with low calcium, methoxyverapamil, cobalt and manganese. J Endocrinol 101:319-325
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5. Enyeart JJ, Hinkle PM 1984 The calcium agonist Bay K 8644 stimulates secretion from a pituitary cell line. Biochem Biophys Res Commun 122:991 -996 6. Martin TFJ, Kowalchyk JA 1984 Evidence for the role of calcium and diacylglycerol as dual second messengers in thyrotropin-releasing hormone action. Role of Ca2+. Endocrinology 115:1527-1536 7. Gershengorn MC 1986 Mechanisms of thyrotropin releasing hormone stimulation of pituitary hormone secretion. Annu Rev Physiol 48:515-526 8. White BA, Bauerle LR, Bancroft FC 1981 Calcium specifically stimulates prolactin synthesis and messenger RNA synthesis in GH3 cells. J Biol Chem 256:5942-5945 9. Gick GG, Bancroft FC 1985 Regulation by calcium of prolactin and growth hormone mRNA sequences in primary cultures of rat pituitary cells. J Biol Chem 260:76147618 10. Hinkle PM, Jackson AE, Thompson TM, Zavacki AM, Coppola DA, Bancroft C 1988 Calcium channel agonists and antagonists: effects of chronic treatment on pituitary prolactin synthesis and intracellular calcium. Mol Endocrinol 2:1132-1138 11. Davis JRE, Vidal ME, Wilson EM, Sheppard MC 1988 Calcium-dependence of prolactin mRNA accumulation in GH3 rat pituitary tumor cells. J Mol Endocrinol 1:111-116 12. White BA, Power E, Fay FS 1989 Calcium regulation of prolactin gene expression: opposing effects of extracellular CaCI2 and Ca2+ ionophores. Mol Endocrinol 3:17571764 13. Enyeart JJ, Biagi B, Day RN 1990 Opposing actions of Bay K 8644 enantiomers on calcium current, prolactin secretion, and synthesis in pituitary cells. Mol Endocrinol 4:727-735 14. Jackson AE, Bancroft C 1988 Proximal upstream flanking sequences direct calcium regulation of the rat prolactin gene. Mol Endocrinol 2:1139-1144 15. Murdoch GH, Waterman M, Evans RM, Rosenfeld MG 1985 Molecular mechanisms of phorbol ester, thyrotropinreleasing hormone, and growth factor stimulation of prolactin gene-transcription. J Biol Chem 260:11852-11858 16. White BA 1985 Evidence for a role of calmodulin in the regulation of prolactin gene expression. J Biol Chem 260:1213-1217 17. MacNeil S, Griffin M, Cooke AM, Pettett NJ, Dawson RA, Owen R, Blackburn GM 1988 Calmodulin antagonists of improved potency and specificity for use in the study of calmodulin biochemistry. Biochem Pharmacol 37:17171723 18. Day RN, Maurer RA 1990 Pituitary calcium channel modulation and regulation of prolactin gene expression. Mol Endocrinol 4:736-742 19. Elsholtz HP, Mangalam HJ, Potter E, Albert V, Supowit
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S, Evans RM, Rosenfeld MG 1986 Two different cisactive elements transfer the transcriptional effects of both EGF and phorbol esters. Science 234:1552-1557 Bandyopadhyay SK, Bancroft C 1989 Calcium induction of the mRNAs for prolactin and c-fos is independent of protein kinase C activity. J Biol Chem 264:14216-14219 Bancroft C, Gick GG, Johnson ME, White BA 1985 Regulation of growth hormone and prolactin gene expression by hormones and calcium. Biochem Actions of Horm Xll:173-213 Davis JRE, Belayew A, Sheppard MC 1989 Prolactin and growth hormone. In: Sheppard MC (ed) Molecular Biology of Endocrinology. Bailliere's Clinical Endocrinology and Metabolism. Bailliere Tindall, London, vol 2: 797-834 Day RN, Maurer RA 1989 The distal enhancer region of the rat prolactin gene contains elements conferring response to multiple hormones. Mol Endocrinol 3:3-9 Nelson C, Albert VR, Elsholtz HP, Lu Ll-P, Rosenfeld MG 1988 Activation of cell-specific expression of rat growth hormone and prolactin genes by a common transcription factor. Science 239:1400-1405 Gutierrez-Hartmann A, Siddiqui S, Loukin S 1987 Selective transcription and DNase I protection of the rat prolactin gene by GH3 pituitary cell-free extracts. Proc Natl Acad Sci USA 84:5211-5215 Cao Z, Barron EA, Sharp ZD 1988 Prolactin upstream factor 1 (PUF-1) mediates cell-specific transcription. Mol Cell Biol 8:5432-5438 Lufkin T, Jackson AE, Pan WT, Bancroft C 1989 Proximal rat prolactin promoter sequences direct optimal, pituitary cell-specific transcription. Mol Endocrinol 3:559-566 White BA, Preston GM 1988 Evidence for a role of topoisomerase II in the Ca2+-dependent basal level expression of the rat prolactin gene. Mol Endocrinol 2:40-46 White BA, Bancroft FC 1982 Cytoplasmic dot hybridization: simple analysis of relative mRNA levels in multiple small cell or tissue samples. J Biol Chem 257:8569-8572 Davis JRE, Lynam TC, Franklyn JA, Docherty K, Sheppard MC 1986 Triiodothyronine and phenytoin reduce prolactin messenger RNA levels in cultured rat pituitary cells. J Endocrinol 109:359-364 Lufkin T, Bancroft FC 1987 Identification by cell fusion of gene sequences that interact with positive trans-acting factors. Science 237:283-286 Luckow B, Schutz G 1987 CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res 15:5490 Gorman CM, Moffat L, Howard BH 1982 Recombinant genomes which express chloramphenicol acetyltransferase activity in mammalian cells. Mol Cell Biol 2:10441051
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Julian R. E. Davis, Nigel Hoggard, Elizabeth M. Wilson, Maria E. Vidal, and Michael C. Sheppard Department of Medicine (J.R.E.D., N.H.) Manchester University Manchester M13 9PT, United Kingdom Department of Medicine Queen Elizabeth Hospital Edgbaston, Birmingham B15 2TH, United Kingdon
The effects of the calcium/calmodulin signaling system on expression of the rat PRL gene were studied in rat pituitary GH3 cells using two specific naphthalene sulfonamide calmodulin (CaM) antagonist drugs, W7 and a more potent and more highly specific iodo-derivative, 5-iodo-1-C8. PRL (but not GH) mRNA accumulation was markedly inhibited by W7, which in coincubations abolished the stimulation normally seen with TRH. Transient transfection assays showed that expression of the reporter gene chloramphenicol acetyl transferase (CAT) linked to 5'-flanking sequences from the PRL gene was inhibited by the calcium-channel blocker verapamil and by the two CaM antagonists. The calcium effects showed partial promoter specificity, in that transcription of PRL-CAT constructs was markedly inhibited by verapamil, but the Rous sarcoma virus-CAT construct also showed significant inhibition, whereas the pBL-CAT2 construct was unaffected. Three hundred ninety five base pairs were sufficient to confer the full inhibitory effect of calcium channel blockade or CaM antagonist seen with longer constructs. The data indicate that CaM is important for PRL gene transcription, and that the effects of CaM are exerted on DNA sequences within the proximal 395bp of prolactin 5'-flanking DNA. (Molecular Endocrinology 5: 8-12, 1991)
intracellular calcium stores and influx of extracellular calcium through voltage-gated L-type channels are involved (12, 13). The proximal 172 base pairs of 5'flanking DNA from the PRL gene are able to confer calcium-responsiveness onto a reporter gene (14). The effects of calcium on the PRL gene may be mediated either by protein kinase C or by CaM. Earlier work has suggested that CaM is involved also in regulating PRL mRNA accumulation and gene transcriptional rate (15, 16), but many of the drugs used, notably the phenothiazines, are recognized to have effects on other intracellular signaling systems including protein kinase C itself. In order to address the question of the role of CaM in PRL gene regulation we have employed two specific CaM antagonist agents: the highly specific naphthalene sulfonamide CaM antagonist W7 (A/-(6-aminohexyl)-1 naphthalene sulfonamide; IC50 ~30 ^mol/liter) which has only very slight effects on protein kinase C, and a more potent and more specific iodo-derivative of W7, 5-iodo-1-C8 (abbreviated W8 in this report, IC50 ~3 /iimol/liter) which has no detectable effect on protein kinase C (17). We have demonstrated CaM dependence of PRL mRNA levels (to a much greater extent than GH mRNA levels) in GH3 cells. Secondly, we have demonstrated that the proximal 395 bp of upstream flanking DNA from the rat PRL gene confers calcium and CaM dependence on a bacterial reporter gene, expressed in transfected GH3 cells.
INTRODUCTION PRL secretion is dependent upon calcium and calmodulin (CaM; 1-6), and the calcium-CaM signaling system is thought to mediate in part the effects of TRH (7). PRL mRNA accumulation (much more than GH mRNA) is critically dependent upon calcium (8-11), and both 0888-8809/91/0008-0012$02.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
RESULTS Hybridization Experiments The CaM antagonist W7 reduced PRL secretion, and PRL mRNA levels fell to 26% of control after 24-h incubation with 100 HM W7 (Fig. 1a). W7 (100 ^mol/ liter) reduced both PRL secretion and PRL mRNA levels
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Calmodulin Regulation of PRL Gene
NS
mRNA 0.5 (O.D. u n i t s )
lull
lOOuM Control D vi lOugnQ
release
0
lun
irxii tw7]
mRNA O.S (0.0. units)
ng/ml
lun
iooun ( W 7 1
Fig. 1. Effects of CaM antagonist W7 on GH3 cells A, Effects of treatment with 1 or 100 HM W7 for 24 h on PRL mRNA accumulation and PRL secretion, n = 6 from a single experiment. B, Effects of coincubation with 100 MM W7 and 100 nM TRH for 24 h (n = 12 replicates per group pooled from three similar experiments, P < 0.01 compared with control in each case). C, Effects of W7 on GH mRNA in the same experiments; n = 6 from the experiment shown in A, and n = 12 from the experiments shown in B. *, P < 0.03 compared to respective controls.
and prevented any stimulation by 100 nM TRH (Fig. 1 b). GH mRNA levels in these experiments showed slight reductions (to 77-92% of control) in the presence of 100 fiM W7, but these were much less marked than for PRL mRNA (Fig. 1c). High doses of the CaM antagonists caused rounding of GH3 cells and reduced adherence to the culture dishes, but did not affect cell viability as determined by trypan blue exclusion.
% OF CONTROL ACETYLATION
r395CAT
Transfection Experiments
r958CAT
RSVCAT
pBLCAT2
Fig. 2. Effects of verapamil (50 HM for 24 h) on acetylation of C-chloramphenicol by cytoplasmic extracts of GH3 cells transfected with rPRL(-395)-CAT, rPRL(-958)-CAT, RSVCAT, or pBL-CAT2 The inhibition due to verapamil is shown as a percentage of control values for each construct. Values shown are means ± SEM of triplicate determinations, and similar results were obtained in two or three independent experiments in each case. *, P < 0.01 compared to respective controls. 14
GH3 cells showed no endogenous CAT activity, while cells transfected with the Rous sarcoma virus (RSV> chloramphenicol acetyl transferase (CAT) construct had high levels of activity, with up to 90% acetylation of chloramphenicol per 100 ng protein. Transfection efficiency differed somewhat between different experiments, but in general transfection with the PRL-CAT constructs resulted in acetylation of approximately 15% of that seen with the RSV-CAT construct. In experiments to validate calcium dependence of the transfected constructs, GH3 cells were transfected with different constructs and treated with control medium or 50 nM verapamil for 24 h. Cells were transfected before being divided into treatment groups to avoid possible differences due to different transfection efficiency. Verapamil treatment reduced CAT activity from the PRL(-395)-CAT and PRL(-958)-CAT constructs to 12.5 ± 3.4% and 16.3 ± 2.1 % of control levels, respectively, but had less effect on RSV-CAT (30.1 ± 3.3% of control) and none on pBL-CAT2 (Fig. 2). On the other hand, treatment with the calcium channel agonist Bay K 8644 increased CAT activity from the PRL-CAT con-
structs but not from the other promoters, indicating a degree of promoter specificity of calcium channel blockade or activation, as has been reported previously (14, 18) (data not shown). The two CaM antagonists W7 and W8 both markedly reduced CAT activity in GH3 cells transfected with PRL(-395)-CAT (Fig. 3). W8 at 3 fiM reduced CAT activity to 73% of control levels (mean of two similar experiments), and to 14 ± 6% of control at 10 ^ M concentration (mean ± SEM from four separate experiments). W7 reduced CAT activity to 6% and 5% of control at 10 and 50 HM concentrations, respectively (mean of two similar experiments). The degree of inhi-
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RSVCAT
50 nM W7
PRL(-395)-CAT
Fig. 3. Effects of two CaM antagonists (W7 and W8) on acetylation of 14C-chloramphenicol by cytoplasmic extracts of GH3 cells transfected with rPRL-(-395)-CAT The first lane shows cells transfected with RSV-CAT plasmid. A representative experiment is shown (see text), and cells were treated with CaM antagonists for 24 h before assay for CAT activity.
bition of CAT activity by W8 was similar using both PRL-CAT constructs: using the PRL(-395)-CAT construct activity was reduced by 10 HM W8 to 14% of control levels, and with the PRL(-958)-CAT construct to 22% of control (means of four and two independent experiments, respectively).
DISCUSSION
The data presented in this report indicate that highly specific CaM antagonists have major effects on rat PRL gene transcription in GH3 cells, and show that the mechanism for calcium-CaM action may involve the proximal enhancer region from the 5'-flanking DNA within the gene. The precise role of calcium in basal and stimulated PRL gene transcription has been a subject of some controversy. Calcium is an important cofactor for protein kinase C, which is an established stimulator of PRL gene transcription (15, 19), but it also activates the calcium-binding protein CaM. Recent evidence has shown that down-regulation of protein kinase C by chronic exposure of GH3 cells to phorboi esters does not affect the increase in PRL mRNA levels induced by calcium (20), implying that this kinase is not essential for this effect. Previous studies have suggested that CaM is important in PRL gene transcription (15, 16, 21), but much previous work has relied upon CaM antagonists whose specificity is relatively poor as they also inhibit a number of other intracellular enzymes including protein kinase C itself (17). The present results, obtained using two highly specific CaM antagonists which have only minimal effect on protein kinase
C, confirm that PRL gene expression is indeed CaM dependent, in contrast to GH mRNA. The effects of calcium and CaM appear to be mediated by effects on the 5'-flanking DNA in the PRL gene. The PRL gene contains a proximal enhancer region (up to 400 bp upstream from the cap site) and a distal region (up to 1700 bp upstream); response elements have recently been defined for several hormones and intracellular regulators within these enhancer regions (22). For example, the transcriptional response to both epidermal growth factor (EGF) and protein kinase C activation by phorboi ester is conferred by DNA sequences between positions - 3 5 and - 7 8 bp upstream from the cap site (19). A precise response element for calcium itself has not yet been identified, but the information required for a full transcriptional response to calcium is contained within the first 174 bp of DNA upstream from the cap site (14), and our results with Bay K 8644 and verapamil using the PRL(-395)CAT construct confirm these findings. The present data show that the CaM-sensitivity of the endogenous PRL gene shown by the hybridization studies is, like the calcium responsiveness, conferred onto the CAT reporter gene by the first 395 bp of the proximal enhancer region of the gene. Pharmacological manipulations of the calcium/CaM signaling system are likely to have wide-spread effects on cellular metabolism and on a variety of different genes, and indeed our data show that the transcription of CAT from the RSV promoter sequence is also substantially reduced by verapamil, whereas that with the pBL-CAT2 construct was unaffected. Likewise, three control promoter-CAT constructs tested by Jackson and Bancroft (14) in fact showed some degree of calcium induction. However these effects were much different in magnitude compared to the effects on the PRL gene, implying some degree of promoter specificity. The proximal enhancer region of the PRL gene therefore appears to contain specific CaM response elements) which are important in basal and TRH-stimulated gene transcriptional control. However the rPRL gene appears to contain multiple potential response elements, and TRH, EGF, and cAMP effects have recently been shown to be conferred onto a reporter gene by the distal as well as the proximal enhancer regions of the gene (23), and Day and Maurer (18) have recently shown that the distal enhancer also confers a transcriptional response to a pure L-type calcium channel agonist. The 174 bp calcium-responsive stretch of the PRL 5'-flanking DNA contains three of the four proximal binding sites for a pituitary-specific protein factor (termed Pit-1, PUF-1, or LSF-1) (24-27). The first such site coincides with a response region for EGF and phorboi ester (19) and also with a possible recognition sequence for the CaM-dependent enzyme DNA topoisomerase (28), inviting the speculation that these intracellular signals may exert their effect by modifying the binding of tissue-specific proteins to DNA or to other DNA-binding factors, or alternatively by modifying al-
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Calmodulin Regulation of PRL Gene
ready-bound trans factors to affect their interaction with the transcription initiation complex. So far there are few data available that address this question, but preliminary data suggest that the binding of pituitary-specific nuclear protein(s) to putative Pit-1 binding sites within the proximal enhancer region is affected by CaM antagonists (Wilson, J., I. D. Phillips, and J. R. E. Davis, manuscript in preparation). Further studies will be required to investigate in detail the mechanisms of calcium-CaM effects on expression of the PRL gene.
11
natants from the cell lysate were assayed for protein content, and stored for assay of CAT activity. Assay for CAT Activity CAT activity was assayed as described (33) in a 100 n\ reaction volume containing 100 ^g cell lysate protein, 12 mM acetyl Coenzyme A, and 0.2 nC\ 14C-labeled chloramphenicol. Reactions were incubated for 16 h at 37 C, and samples were then extracted with ethyl acetate before acetylation products were separated by TLC, and quantified by scintillation counting. Results are expressed as percent acetylation of total chloramphenicol/100 M9 protein. Reagents
MATERIALS AND METHODS GH3 Cell Culture and RIAs GH3 cells obtained from the European Collection of Animal and Cell Cultures, were grown in continuous monolayer culture in Ham's F-10 medium supplemented with 5% fetal calf serum (GIBCO, Paisley, Strathclyde, UK) and 10% horse serum (GIBCO). Cells were subcultured from single donor cultures 2 days before experiments, and then washed with serum-free medium before treatment with test substances in serum-free F-10 medium as described. PRL secretion into culture fluid was measured by RIA using reagents supplied by the National Hormone and Pituitary Program, NIADDK (Bethesda, MD); results are expressed in terms of the standard rPRL-RP3. Hybridization Studies: mRNA Measurements Relative cytoplasmic concentrations of PRL and GH mRNA were measured by cytoplasmic dot hybridization analysis (29) as described previously (30). Briefly, plasmid probes containing complementary DNA inserts specific for rat PRL or rat GH were labeled with 32P-dCTP by nick translation to a specific activity of 1-2 x 108 dpm/^g DNA. After filter hybridization and autoradiography, mRNA data were quantified by scanning densitometry and data expressed in arbitrary units of optical density (O.D.) of the developed autoradiographs. Transfection Studies GH3 cells were transfected with synthetic DNA constructs consisting of 395 bp or 958 bp of the rat PRL promoter (31) linked to the bacterial CAT gene, or as a control RSV-CAT or pBL-CAT2 (32). Transfection was carried out either by the diethylaminoethyl (DEAE)-Dextran method (CaM antagonist data) or by electroporation (verapamil data). Two days before transfection, GH3 cells were subcultured onto 100-mm Petri dishes at 4 x 106 cells per dish. For the DEAE-Dextran method, cells were washed with serum-free medium, before the addition to each dish of 10 ng plasmid DNA in 5 ml medium containing 50 ITIM Tris (pH 7.5) and 100 M9/ml DEAE dextran. After 30-60 min this medium was removed, and the cells were washed before addition of fresh medium or test substances for 24 h. For the electroporation technique, approximately 50 x 106 cells in 0.8 ml culture medium were transferred to an electroporation cuvette containing 20 ^9 plasmid DNA and then transfected by a 275 V pulse at a total capacitance of 960 nf (Electropore apparatus, SEDD, Liege, Belgium). The cells were immediately diluted in culture medium and then divided into a series of 35-mm multiwell culture dishes for treatment with test substances. At the end of the incubation period, medium was removed from the culture dishes, and the cells were washed in ice-cold PBS. Cells were then harvested by trypsinization, washed, and lysed in 0.5% Triton X-100 in 0.25 mui Tris (pH 7.8). Cell lysates were centrifuged at 15,000 x g for 4 min, and super-
Restriction enzymes and kinases were purchased from Boehringer Mannheim (Lewes, East Sussex, UK). W7 (Sigma, Poole, Dorset, UK) and W8 (17) were initially dissolved in dimethylsulfoxide and freshly diluted in F-10 culture fluid before experiments. Verapamil was purchased from Abbott Laboratories Ltd. (Queensborough, Kent, UK), and Bay K 8644 was a generous gift from Bayer Pharmaceuticals Ltd. Radiolabeled chloramphenicol was purchased from Amersham International PLC (Amersham, Bucks, UK), and DEAE-dextran from Sigma.
Acknowledgments The authors are very grateful to Prof. F. C. Bancroft, New York, New York for generous provision of the PRL(-395)CAT, PRL(-958)-CAT clones, and for the rGH cDNA probe, and to Dr. R. A. Maurer, Iowa City, Iowa, for the rPRL cDNA probe. Dr. G. M. Blackburn, Department of Chemistry, Sheffield University, UK, kindly donated the W7 derivative 5-iodo1-C8 (termed W8 in this paper). Received December 19, 1989. Revision received October 5,1990. Accepted October 5,1990 Address requests for reprints to: Dr. J. R. E. Davis, Department of Medicine, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom. These studies were supported by grants from the Central Birmingham Health Authority Endowment Fund, the University of Birmingham Faculty of Medicine Scientific Projects Committee, and the Wellcome Trust. Portions of this work were presented at the 71st meeting of The Endocrine Society, Seattle, WA, 1989 (Abstract 64).
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Vol 5 No. 1
MOL ENDO-1991 12
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