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• W2011721908 abstract "Thyrotropin (TSH) increases 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene transcription in FRTL-5 rat thyroid cells, and the effect of TSH can be mimicked by cAMP.Sequence analysis of the rat reductase promoter has revealed a hitherto unnoticed cAMP-responsive element (CRE)-like octamer. This octamer is located between 53 and 60 nucleotides downstream of the sterol regulatory element 1; its first 6 nucleotides are identical to the consensus somatostatin CRE, and the entire octamer is identical to the fos CRE. A synthetic oligonucleotide containing the HMG-CoA reductase CRE-like octamer (RED CRE) formed protein-DNA complexes with nuclear extracts from FRTL-5 cells, which could be prevented by unlabeled CRE-containing oligonucleotides whose flanking sequences were otherwise nonidentical. The complexes were specifically supershifted by anti-CREB antibodies. FRTL-5 cells transfected with a fusion plasmid carrying the bacterial chloramphenicol acetyl transferase (CAT) under the control of the HMG-CoA reductase promoter displayed CAT activity, which was specifically stimulated by TSH. In contrast, CAT activity in FRTL-5 cells transfected with similar constructs carrying mutations in the reductase CRE was significantly lower and did not increase after TSH challenge.We suggest that the HMG-CoA reductase gene contains a functional CRE, important for TSH regulation of transcription. The data presented provide the molecular basis for a novel regulatory mechanism for HMG-CoA reductase gene expression in rat thyroid cells, which involves the direct effect of cAMP. Thyrotropin (TSH) increases 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene transcription in FRTL-5 rat thyroid cells, and the effect of TSH can be mimicked by cAMP. Sequence analysis of the rat reductase promoter has revealed a hitherto unnoticed cAMP-responsive element (CRE)-like octamer. This octamer is located between 53 and 60 nucleotides downstream of the sterol regulatory element 1; its first 6 nucleotides are identical to the consensus somatostatin CRE, and the entire octamer is identical to the fos CRE. A synthetic oligonucleotide containing the HMG-CoA reductase CRE-like octamer (RED CRE) formed protein-DNA complexes with nuclear extracts from FRTL-5 cells, which could be prevented by unlabeled CRE-containing oligonucleotides whose flanking sequences were otherwise nonidentical. The complexes were specifically supershifted by anti-CREB antibodies. FRTL-5 cells transfected with a fusion plasmid carrying the bacterial chloramphenicol acetyl transferase (CAT) under the control of the HMG-CoA reductase promoter displayed CAT activity, which was specifically stimulated by TSH. In contrast, CAT activity in FRTL-5 cells transfected with similar constructs carrying mutations in the reductase CRE was significantly lower and did not increase after TSH challenge. We suggest that the HMG-CoA reductase gene contains a functional CRE, important for TSH regulation of transcription. The data presented provide the molecular basis for a novel regulatory mechanism for HMG-CoA reductase gene expression in rat thyroid cells, which involves the direct effect of cAMP. In mammalian cells, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)1( 1The abbreviations used are: HMG-CoA3-hydroxy-3-methylglutaryl coenzyme ATSHthyroid-stimulating hormone, thyrotropinCREcAMP-responsive elementCREBCRE binding proteinCATchloramphenicol acetyl transferasePKAprotein kinase AATFactivating transcription factorbpbase pair(s)MHCmajor histocompatibility complex.) reductase (mevalonate:NADP+ oxidoreductase CoA-acylating, EC 1.1.1.34) is the rate-limiting enzyme for the synthesis of mevalonic acid, the precursor common to many isoprenoids, including cholesterol (1). Because of its involvement in cholesterol biosynthesis, HMG-CoA reductase has become the target of many drugs aimed at lowering threatening levels of serum cholesterol(2Alberts A.W. Am. J. Cardiol. 1988; 62: 10J-15JAbstract Full Text PDF PubMed Scopus (314) Google Scholar). An additional interest in this enzyme has mounted in recent years, since mevalonate is also the precursor of the donor isoprenoids, farnesyl and geranyl-geranyl diphosphate, for a growing class of biologically relevant proteins, which include monomeric G proteins, most notably the product of the ras gene, p21, a key transducer of mitogenic signals(3Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3767) Google Scholar). As the rate-limiting enzyme for synthesis of mevalonate, the precursor of isoprenes, HMG-CoA reductase is, therefore, a critical regulator of cell proliferation in normal as well as in tumor cells(3Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3767) Google Scholar, 4Grieco D. Beg Z.H. Romano A. Bifulco M. Aloj S.M. J. Biol. Chem. 1990; 265: 19343-19350Abstract Full Text PDF PubMed Google Scholar). 3-hydroxy-3-methylglutaryl coenzyme A thyroid-stimulating hormone, thyrotropin cAMP-responsive element CRE binding protein chloramphenicol acetyl transferase protein kinase A activating transcription factor base pair(s) major histocompatibility complex. So far it has been firmly believed that, at the transcriptional level, HMG-CoA reductase is negatively controlled by end-product (cholesterol) since the enzyme is expressed at a relatively high rate when cells are starved for mevalonate(5Reynolds G.A. Basu S.K. Osborne T.F. Chin D.J. Gil G. Brown M.S. Goldstein J.L. Luskey K.L. Cell. 1984; 38: 275-285Abstract Full Text PDF PubMed Scopus (266) Google Scholar, 6Osborne T.F. Gil G. Brown M.S. Kowal R.C. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3614-3618Crossref PubMed Scopus (50) Google Scholar, 7Osborne T.F. Goldstein J.L. Brown M.S. Cell. 1985; 42: 203-212Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 8Sudhof T.C. Russell D.W. Brown M.S. Goldstein J.L. Cell. 1987; 48: 1061-1069Abstract Full Text PDF PubMed Scopus (135) Google Scholar, 9Dawson P.A. Hofmann S.L. van der Westhuyzen D.R. Sudhof T.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 1988; 263: 3372-3379Abstract Full Text PDF PubMed Google Scholar, 10Osborne T.F. Gil G. Goldstein J.L. Brown M.S. J. Biol. Chem. 1988; 263: 3380-3387Abstract Full Text PDF PubMed Google Scholar, 11Osborne T.F. J. Biol. Chem. 1991; 266: 13947-13951Abstract Full Text PDF PubMed Google Scholar, 12Osborne T.F. Bennett M. Rhee K. J. Biol. Chem. 1992; 267: 18973-18982Abstract Full Text PDF PubMed Google Scholar). However, we have reported that in FRTL-5 rat thyroid cells, HMG-CoA reductase is transcriptionally induced by thyrotropin (TSH), via cAMP(4Grieco D. Beg Z.H. Romano A. Bifulco M. Aloj S.M. J. Biol. Chem. 1990; 265: 19343-19350Abstract Full Text PDF PubMed Google Scholar). TSH is the physiologic mitogen in FRTL-5 cells(13Ambesi-Impiombato, F. S., (August 26, 1986) U. S. Patent 4,608,341.Google Scholar, 14Ambesi-Impiombato F.S. Parks L.A.M. Coon H.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3455-3459Crossref PubMed Scopus (974) Google Scholar). When starved of TSH, these cells become quiescent and develop a greater than 2-fold increase in total cholesterol, caused by the doubling of available low density lipoprotein receptors(15Bifulco M. Santillo M. Tedesco I. Zarrilli R. Laezza C. Aloj S.M. J. Biol. Chem. 1990; 265: 19336-19342Abstract Full Text PDF PubMed Google Scholar). TSH challenge increases HMG-CoA reductase gene expression, reduces low density lipoprotein receptor activity, decreases total cholesterol, and increases cholesterol biosynthesis(4Grieco D. Beg Z.H. Romano A. Bifulco M. Aloj S.M. J. Biol. Chem. 1990; 265: 19343-19350Abstract Full Text PDF PubMed Google Scholar, 15Bifulco M. Santillo M. Tedesco I. Zarrilli R. Laezza C. Aloj S.M. J. Biol. Chem. 1990; 265: 19336-19342Abstract Full Text PDF PubMed Google Scholar). The TSH-induced increase in HMG-CoA reductase and cholesterol biosynthesis precedes the TSH-induced increase in thymidine incorporation into DNA and cell doubling(4Grieco D. Beg Z.H. Romano A. Bifulco M. Aloj S.M. J. Biol. Chem. 1990; 265: 19343-19350Abstract Full Text PDF PubMed Google Scholar). The apparent paradox of a lower cholesterol content in an actively dividing cell population has been explained as follows. In order for FRTL-5 cells to proceed through the cell cycle, active isoprenoid synthesis is a mandatory requirement; the TSH-induced reduction in cholesterol content would maintain a high rate of HMG-CoA reductase synthesis(4Grieco D. Beg Z.H. Romano A. Bifulco M. Aloj S.M. J. Biol. Chem. 1990; 265: 19343-19350Abstract Full Text PDF PubMed Google Scholar, 15Bifulco M. Santillo M. Tedesco I. Zarrilli R. Laezza C. Aloj S.M. J. Biol. Chem. 1990; 265: 19336-19342Abstract Full Text PDF PubMed Google Scholar). We have cloned and sequenced the promoter region of the rat gene in an effort to elucidate the mechanism by which TSH/cAMP act. Analysis of the 5′-flanking region of the HMG-CoA reductase gene revealed the octamer, 5′-TGACGTAG-3′, which is closely homologous to a consensus cAMP-responsive element (CRE)(16Montminy M.R. Sevarino K.A. Wagner J.A. Mandel G. Goodman R.H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6682-6686Crossref PubMed Scopus (1056) Google Scholar). In this study, we investigate the role of this CRE-like sequence and show that it is involved in both basal and TSH-induced HMG-CoA reductase gene expression in thyroid cells. We also show that the CRE-binding protein, CREB(17Habener J.F. Mol. Endocrinol. 1990; 4: 1087-1094Crossref PubMed Scopus (324) Google Scholar), modulates the reductase CRE. The data presented strongly support a direct cAMP regulation of HMG-CoA reductase gene transcription in thyroid cells, involving cis-acting CRE. TSH was a highly purified preparation from bovine pituitary extracts(18Kohn L.D. Winand R.J. J. Biol. Chem. 1975; 250: 6503-6508Abstract Full Text PDF PubMed Google Scholar). Insulin, cristalline bovine serum albumin, phenylmethylsulfonyl fluoride, leupeptin, pepstatin A, and other reagents were obtained from Sigma. Trypticase peptone and yeast extract were from Beckton Dickinson. M13-mp18 DNA was purchased from Life Technologies, Inc. Restriction enzymes, T4 DNA ligase, DNA polymerase I (Klenow fragment), T4 polynucleotide kinase, and Random primed DNA labeling kits were obtained from Boehringer Mannheim. [α32P]dATP (3,000 Ci/mmol), [α32P]dGTP (3,000 Ci/mmol), [g32P]dATP (6,000 Ci/mmol), and nylon filters were from Amity. Sephadex nick columns and DEAE-dextran were obtained from Pharmacia Biotech Inc. BA-85 nitrocellulose filters were purchased from Schleicher & Schuell. For autoradiography, Kodak XAR-5 films have been used with DuPont Cronex intensifying screens (Du Pont). All plasmids have been purified by Qiagen plasmid kits purchased from Qiagen Inc. Synthetic oligonucleotides were purchased from Operon Inc. For sequencing, the Sequenase kit “from U. S. Biochemical Corp. has been used. Plasmid pRED-227 was supplied by the American Type Culture Collection, Rockville, MD. The pCAT-promoter plasmid was purchased from Promega Corp. Antiserum to CREB or activating transcription factor-2 (ATF-2), each with its respective control preimmune counterpart, were kindly provided by Dr. J. P. Hoeffler, University of Colorado Health Center, Denver, CO. pEMBL-8-CAT vector was kindly provided by Dr. R. Cortese, EMBL, Heidelberg, Germany. Luciferase expression vector (pRSV-Luc) was kindly provided by Dr. S. Subramani, Stanford University, Stanford, CA. FRTL-5 cells (ATCC CRL 8305) are a strain of rat thyroid cells whose characteristics and culture conditions have been extensively described(13Ambesi-Impiombato, F. S., (August 26, 1986) U. S. Patent 4,608,341.Google Scholar, 14Ambesi-Impiombato F.S. Parks L.A.M. Coon H.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3455-3459Crossref PubMed Scopus (974) Google Scholar, 19Valente W.A. Vitti P. Rotella C.M. Vaughan M.M. Aloj S.M. Grollman E.V. Ambesi-Impiombato F.S. Kohn L.D. N. Engl. J. Med. 1983; 309: 1028-1034Crossref PubMed Scopus (142) Google Scholar). They were grown at 37°C in a humidified atmosphere composed of 95% air and 5% CO2, in Coon's modified F-12 medium supplemented with 5% calf serum and a six-hormone mixture of TSH, insulin, hydrocortisone, transferrin, somatostatin, and glycil-L-histidyl-L-lysine acetate. The medium containing the complete mixture will be referred to as 6H. Depending on experimental needs, a similar mixture, but lacking TSH (referred to as 5H), was used. Unless otherwise stated, cells were provided fresh medium every 3 days. A FRTL-5 genomic library was screened with the XhoI-XbaI fragment (referred to as probe 5′) and the BstEII-XbaI fragment (referred to as probe 3′) of plasmid pRED-227 containing the full-length hamster HMG-CoA reductase cDNA(20Chin D.J. Gil G. Russell D.W. Liscum L. Luskey K.L. Basu S.K. Okayama H. Berg P. Goldstein J.L. Brown M.S. Nature. 1984; 308: 613-617Crossref PubMed Scopus (155) Google Scholar). The screening was accomplished using in situ plaque hybridization according to Maniatis(21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). For labeling of cDNA probes, random primed DNA labeling kits were used. The clone that hybridized only with probe 5′ was further characterized. Digestion of this clone with BamHI-EcoRI gave rise to three fragments that were transferred to nylon filters. The fragment that still hybridized with probe 5′, approximately 4.5 kilobase pairs, was further analyzed by restriction enzymes. This analysis revealed the presence of five PstI sites. The one positive PstI fragment was subcloned into M13-mp18 bacteriophage or pCAT plasmid for sequencing. Digestions of DNA with restriction endonucleases, electrophoresis on agarose gels, transfer of DNA fragments from gels to nylon filters, and hybridizations were performed using standard procedures(21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). DNA restriction fragments, cloned into M13-mp18 bacteriophage or pCAT-promoter plasmid, were sequenced by the method of Sanger(22Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52505) Google Scholar). Nuclear extracts from quiescent or TSH-challenged FRTL-5 cells were prepared as described previously(23Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9149) Google Scholar, 24Bunce C.M. Thick J.A. Lord J.M. Mills D. Brown G. Anal. Biochem. 1988; 75: 67-73Crossref Scopus (37) Google Scholar). Cells were washed with Dulbecco's modified phosphate-buffered saline without Mg2+ and Ca2+ at pH 7.4, harvested by scraping, and, after centrifugation at 500 ´ g, suspended in 5-pellet volumes of 0.3 M sucrose and 2% Tween 40 in buffer A (10 mM HEPES-KOH, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, and 2 μg/ml pepstatin A). After freezing in liquid nitrogen, the cells were thawed and gently homogenized using hand homogenizer pestel B; the suspension was layered onto 1.5 M sucrose in buffer A and centrifuged at 25,000 ´ g in a swinging bucket rotor. Nuclei were washed with 0.3 M sucrose in buffer A, and nuclear proteins were extracted with 2.5 volumes of buffer B (10 mM HEPES-KOH, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EGTA, 10% glycerol, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, and 2 μg/ml pepstatin A). Extracts were centrifuged at 100,000 ´ g for 1 h; the supernatant was dialyzed for 4 h at 4°C against buffer C (20 mM HEPES-KOH, 100 mM KCl, 0.1 mM EDTA, and 30% glycerol), and precipitated materials were discarded by centrifigation at 25,000 ´ g for 15 min prior to use in electrophoretic mobility shift assays. Electrophoretic mobility shift assays were performed as described previously(25Hennighausen L. Lubon H. Methods Enzymol. 1987; 152: 721-735Crossref PubMed Scopus (165) Google Scholar). The 18-base pair (bp) RED CRE synthetic double-stranded oligonucleotide was labeled with [g-32P]ATP and T4 polynucleotide kinase and then purified on an 8% native polyacrylamide gel. The nuclear extract (3 μg) was incubated for 20 min on ice in a 20-μl reaction volume containing 10 mM Tris-HCl, pH 7.6, 50 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 12.5% glycerol, 0.1% Triton X-100, and 0.5 μg of poly(dI-dC). Approximately 1.5 fmol of 32P-labeled probe (approximately 50,000 cpm) were added, and the incubation was continued for an additional 20 min at room temperature. Where indicated, unlabeled double-stranded oligonucleotides were also added to the binding reaction as competitors and incubated with the extract for 20 min, prior the addition of labeled DNA. Protein-DNA complexes were subjected to electrophoresis on 5% native polyacrylamide gels at 160 V in 1 ´ Tris borate/EDTA buffer at 4°C. In experiments using antiserum to CREB or ATF-2, nuclear extracts were incubated with the antiserum or its control counterpart in the same buffer for 20 min on ice before adding the labeled probe and processing as above. Gels were dried and autoradiographed. The 765-nucleotide PstI fragment (from −323 to +442, see Fig. 1) (10Osborne T.F. Gil G. Goldstein J.L. Brown M.S. J. Biol. Chem. 1988; 263: 3380-3387Abstract Full Text PDF PubMed Google Scholar) spanning the entire reductase promoter was cloned into the pEMBL-8-CAT vector (26) upstream to the bacterial gene for CAT. Mutations of the HMG-CoA reductase promoter sequence were created by two-step, recombinant polymerase chain reaction methods(27Higuchi R. Krummel B. Saiki R.K. Nucleic Acids Res. 1988; 16: 7351-7367Crossref PubMed Scopus (2094) Google Scholar). In the first step, two polymerase chain reaction products were generated using the 5′ or 3′ end of the final products and overlapping sequence containing the same mutation. The second step was performed using the overlapping polymerase chain reaction products as templates, and DNA sequences of the 5′ or 3′ end of the final products as primers. Amplified fragments were ligated to pCAT-promoter plasmid and sequenced to verify the predicted sequence. Similar to the wild-type HMG-CoA reductase promoter, the CRE-mutated reductase fragment (765 bp) was cloned into the pEMBL-8-CAT vector. pRSV-Luc was used as positive control to evaluate transfection efficiency(28de Wet J.R. Wood K.V. DeLuca M. Helinski D.R. Subramani S. Mol. Cell. Biol. 1987; 7: 725-737Crossref PubMed Scopus (2474) Google Scholar). FRTL-5 cells were grown to 50-60% confluence and then shifted to 5H medium (without TSH) for 3-5 days. Transfection used a DEAE-dextran protocol(21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), with minor modifications. Four hours before transfection, the 5H medium was changed. Then, medium was removed, and cells were washed once with phosphate-buffered saline and incubated at 37°C with 0.25 ml of DEAE-dextran (2 mg/ml) containing 20 μg of pEMBL-8-CAT under the control of wild-type or mutated HMG-CoA reductase promoter, and 2 μg of pRSV-Luc/dish was added to 4.75 ml of 5H medium containing no calf serum. After a 1-h incubation, medium was aspirated, and the cells were treated for 3 min with 2.5 ml of 10% dimethyl sulfoxide in phosphate-buffered saline, washed once with phosphate-buffered saline, and then incubated in 5H medium. After 24 h, medium was changed, and cells were exposed to either 5H medium (control) or 6H medium (TSH challenge), both in the presence or in the absence of 25-hydroxycholesterol (25 μM) and mevalonate (0.8 mM). After 16-18 h, cells were harvested. Where indicated, forskolin (10 μM) was added to the cells during the last 6 h of incubation. CAT assays were performed as described previously(29Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5292) Google Scholar). Luciferase activity was measured using a luminometer. Protein concentration was determined using a Bio-Rad kit; recrystallized bovine serum albumin was the standard. A PstI fragment of 765 bp, a candidate to contain the complete rat reductase promoter, was sequenced. The fragment spans 323 nucleotides upstream and 442 nucleotides downstream of the major transcription start site, which is identical to the hamster promoter (Fig. 1). Comparison with the hamster sequence revealed nearly 90% homology in the region −283 to +14 (data not shown). Long stretches of identity included the sterol regulatory element 1 and the major transcription start site. Ninety-five nucleotides upstream of the major transcription start site we noticed an 8-nucleotide sequence closely homologous to a CRE. The first 6 nucleotides of this octamer are identical to a consensus CRE (Fig. 2). We questioned whether this element, if a functionally active CRE, could be involved in the direct cAMP regulation of HMG-CoA reductase gene transcription. We synthesized an 18-bp double-stranded oligonucleotide that includes the octamer identified as a potential CRE in the HMG-CoA reductase promoter (RED CRE). The CRE octamer was flanked 5′ by 6 bp and 3′ by 4 bp (Fig. 2). The oligonucleotide was end-labeled and used as a probe to detect protein-DNA complexes with FRTL-5 cell nuclear extracts. When reacted with extracts from either quiescent (5H) or chronically TSH-stimulated (6H) FRTL-5 cells, protein-DNA complexes were formed that gave rise to a broad retarded band in both cases (Fig. 2). We performed competition experiments using several synthetic oligonucleotides (Fig. 3A), which included wild-type or mutated CRE sequences. When we used the oligonucleotide containing the reductase CRE (self) as competitor, the retarded band was significantly reduced by a 50-fold excess, and abolished by a 500-fold excess of the unlabeled probe (Fig. 3B, lanes3-5, and Fig. 3C, lanes2-4). Oligonucleotides containing the major histocompatibility complex (MHC) Class I CRE (Fig. 3B, lanes6-8) and oligonucleotides containing either somatostatin CRE (Fig. 3C, lanes5-7) or TSH receptor CRE (Fig. 3C, lanes8-10) were as effective as the homologous RED CRE in preventing complex formation, whereas oligonucleotides with the CRE octamer deleted (ΔCRE) (Fig. 3B, lanes9-11) or mutated to a nonpalindromic sequence (NPCRE) (Fig. 3Clanes12-14) did not behave as competitors, even at a 500-fold excess concentration. The addition of an antibody to CREB, to the incubation mixture containing the end-labeled synthetic RED CRE and FRTL-5 nuclear extracts resulted in a supershift of the protein-DNA complex (Fig. 4, lane3). In contrast, antisera to the trans-ATF-2 did not cause supershift (Fig. 4, lane4), nor did the preimmune serum (Fig. 4, lane5). The specificity of the reaction was further indicated by the ability of a 500-fold excess of the unlabeled RED CRE to eliminate the supershifted complex (Fig. 4, lane6) but not of an unlabeled oligonucleotide containing identical flanking regions and a mutated CRE resembling the MHC Class I nonpalindromic CRE (data not shown). To address the question as to whether the stimulatory effect of TSH on HMG-CoA reductase gene expression involves CRE transactivation, we transfected FRTL-5 cells with a chimeric plasmid containing the coding sequence of the bacterial enzyme CAT linked to the rat PstI fragment (765 bp, between nucleotides −323 and +442, see Fig. 1), which comprised the HMG-CoA reductase promoter. Assays for CAT activity were performed in cells maintained in the absence or in the presence of TSH (1 ´ 10-9M) for 16-18 h, i.e. the time required for the maximal hormone-induced HMG-CoA reductase gene transcription, or treated with forskolin (10 μM) for 6 h. Separate sets of cells were also exposed to 25-hydroxycholesterol (25 μM) and mevalonate (0.8 mM). Fig. 5 shows that TSH exposure increased CAT activity and that forskolin (10 μM) mimicked the TSH effect; it also shows that the effects of TSH and forskolin were decreased, but not abated, when cells were exposed to 25-hydroxycholesterol and mevalonate. These results indicate that the rat HMG-CoA reductase promoter-CAT construct is sensitive to sterol feedback regulation and that the TSH/cAMP effect is measurable independent of sterol regulatory element-mediated repression. In order to prove that the reductase CRE octamer was involved in cAMP-mediated TSH or forskolin effects, experiments were performed using the same PstI fragment of the reductase promoter linked 5′ to the CAT gene but carrying the CRE octamer mutated from 5′-TGACGTAG-3′ to the nonpalindromic sequence, 5′-CGACACGA-3′ (Fig. 6). TSH or forskolin failed to increase CAT activity in FRTL-5 cells transfected with the CAT gene under the control of the mutated HMG-CoA reductase promoter. It should be noted that in cells maintained in the absence of TSH (control), CAT activity was still measurable (Figure 5:, Figure 6:); this activity is likely to be sustained by residual cAMP in TSH-starved cells. These findings indicate that the CRE plays a functional role in the TSH/cAMP regulation of HMG-CoA reductase transcription in FRTL-5 cells. In a previous report (4Grieco D. Beg Z.H. Romano A. Bifulco M. Aloj S.M. J. Biol. Chem. 1990; 265: 19343-19350Abstract Full Text PDF PubMed Google Scholar) we showed that TSH, a specific mitogen and physiologic regulator for thyroid cells, induces HMG-CoA reductase and cholesterol synthesis in FRTL-5 rat thyroid cells. In these cells, TSH induction of reductase gene transcription precedes the increase of DNA synthesis and cell proliferation and is mediated by cAMP(4Grieco D. Beg Z.H. Romano A. Bifulco M. Aloj S.M. J. Biol. Chem. 1990; 265: 19343-19350Abstract Full Text PDF PubMed Google Scholar). In the current study, we have investigated the molecular basis for cAMP regulation of HMG-CoA reductase gene expression, and show that an 8-nucleotide sequence within the reductase promoter is structurally and functionally homologous to a consensus CRE(16Montminy M.R. Sevarino K.A. Wagner J.A. Mandel G. Goodman R.H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6682-6686Crossref PubMed Scopus (1056) Google Scholar). The rat reductase promoter is nearly 90% homologous with the hamster promoter between nucleotides −283 and +14 (see Fig. 1) and identical in long stretches of nucleotides, most notably around the major transcription start site and the sequence known as footprint 4, which is required for reductase gene transcription(6Osborne T.F. Gil G. Brown M.S. Kowal R.C. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3614-3618Crossref PubMed Scopus (50) Google Scholar). This particular region includes the CRE-like octamer 5′-TGACGTAG-3′. We have focused on this sequence as a potential target for cAMP regulation and provide evidences indicating that it is indeed a functional CRE involved in TSH regulation of reductase gene expression. These can be summarized as follows. Oligonucleotides containing the CRE-like sequence and nuclear extracts from FRTL-5 cells formed specific protein-DNA complexes that reacted with antibodies to CREB but failed to react with antibodies to ATF-2 or preimmune serum(16Montminy M.R. Sevarino K.A. Wagner J.A. Mandel G. Goodman R.H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6682-6686Crossref PubMed Scopus (1056) Google Scholar, 17Habener J.F. Mol. Endocrinol. 1990; 4: 1087-1094Crossref PubMed Scopus (324) Google Scholar, 30Saji M. Moriarty J. Ban T. Kohn L.D. Singer D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1944-1948Crossref PubMed Scopus (51) Google Scholar, 31Shimura H. Ikuyama S. Shimura Y. Kohn L.D. J. Biol. Chem. 1993; 268: 24125-24137Abstract Full Text PDF PubMed Google Scholar). TSH or forskolin could stimulate CAT activity in FRTL-5 cells in which the reporter gene was under the control of the reductase promoter. Stimulation was significant also under conditions of cholesterol/mevalonate down-regulation. In FRTL-5 cells transfected with a construct in which the reductase CRE was mutated to nonpalindromic sequence but was otherwise identical, TSH or forskolin failed to affect the activity of the reporter gene. Thus, in FRTL-5 cells, the HMG-CoA reductase gene is positively regulated by cAMP through modulation of a cis-acting element on its promoter, a process that has not been reported so far. The role of cAMP regulation of the reductase gene expression has never been addressed in detail. We have previously reported that in rat thyroid cells, cAMP has a key role in regulating sterol metabolism by down-regulating LDL receptor activity and inducing HMG-CoA reductase gene transcription. Studies on different cell lines should tell us whether this is a general phenomenon rather than a unique feature of thyroid epithelial cells; we suggest, at this point, that it may well be applicable to all cells where the cAMP signal plays a role in the growth process. The identification of a CRE-like sequence as a functional CRE in the reductase promoter indicates direct transcriptional activation through cAMP signaling; however, we are still unable to resolve quantitatively the contribution of cAMP-mediated cholesterol depletion(4Grieco D. Beg Z.H. Romano A. Bifulco M. Aloj S.M. J. Biol. Chem. 1990; 265: 19343-19350Abstract Full Text PDF PubMed Google Scholar, 15Bifulco M. Santillo M. Tedesco I. Zarrilli R. Laezza C. Aloj S.M. J. Biol. Chem. 1990; 265: 19336-19342Abstract Full Text PDF PubMed Google Scholar), since CAT assays on cells transfected with chimeric constructs of the reporter gene and the reductase promoter are affected by cholesterol and mevalonate incubation under conditions of high intracellular cAMP (see Fig. 5). In summary, this study has identified and characterized a CRE in the HMG-CoA reductase promoter and in FRTL-5 cells and has demonstrated that, at least in this particular cell line, the gene can be positively regulated by cAMP. The relevance of this regulatory mechanism, which has been ignored so far, is still a matter of investigation. We thank M. Berardone, F. D'Agnello, and F. Moscato for the art work." @default.
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