Suppression of Cholesterol 7α-Hydroxylase Transcription and Bile Acid Synthesis by an α1-Antitrypsin Peptide via Interaction with α1-Fetoprotein Transcription Factor
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m205089200
ISSN1083-351X
AutoresMarie-Christine Gerbod-Giannone, Antonio del Castillo‐Olivares, Sabina Janciauskiene, Gregorio Gil, Phillip B. Hylemon,
Tópico(s)Peroxisome Proliferator-Activated Receptors
Resumoα1-Antitrypsin (α1-AT) is a serum protease inhibitor that is synthesized mainly in the liver, and its rate of synthesis markedly increases in response to inflammation. This increase in α1-AT synthesis results in an increase in peptides, like its carboxyl-terminal C-36 peptide (C-36), resulting from α1-AT cleavage by proteases. Atherosclerosis is a form of chronic inflammation, and one of the risk factors is elevated plasma cholesterol levels. Because of the correlation between atherosclerosis, plasma cholesterol content, inflammation, and α1-AT rate of synthesis, we investigated the effect of the C-36 serpin peptide on hepatic bile acid biosynthesis. We discovered that C-36 is a powerful and specific transcriptional down-regulator of bile acid synthesis in primary rat hepatocytes, through inhibition of the cholesterol 7α-hydroxylase/CYP7A1 (7α-hydroxylase) promoter. Mice injected with the C-36 peptide also showed a decrease in 7α-hydroxylase mRNA. A mutated but very similar peptide did not have any effect on 7α-hydroxylase mRNA or its promoter. The sterol 12α-hydroxylase/CYP8B1 (12α-hydroxylase) promoter is also down-regulated by the C-36 peptide in HepG2 cells but not by the mutated peptide. The DNA element involved in the C-36-mediated regulation of 7α- and 12α-hydroxylase promoters mapped to the α1-fetoprotein transcription factor (FTF) site in both promoters. The C-36 peptide prevented binding of FTF to its target DNA recognition site by direct interaction with FTF. We hypothesize that the C-36 peptide specifically interacts with FTF and induces a conformational change that results in loss of its DNA binding ability, which results in suppression of 7α- and 12α-hydroxylase transcription. These results suggest that peptides derived from specific serum proteins may alter hepatic gene expression in a highly specific manner. α1-Antitrypsin (α1-AT) is a serum protease inhibitor that is synthesized mainly in the liver, and its rate of synthesis markedly increases in response to inflammation. This increase in α1-AT synthesis results in an increase in peptides, like its carboxyl-terminal C-36 peptide (C-36), resulting from α1-AT cleavage by proteases. Atherosclerosis is a form of chronic inflammation, and one of the risk factors is elevated plasma cholesterol levels. Because of the correlation between atherosclerosis, plasma cholesterol content, inflammation, and α1-AT rate of synthesis, we investigated the effect of the C-36 serpin peptide on hepatic bile acid biosynthesis. We discovered that C-36 is a powerful and specific transcriptional down-regulator of bile acid synthesis in primary rat hepatocytes, through inhibition of the cholesterol 7α-hydroxylase/CYP7A1 (7α-hydroxylase) promoter. Mice injected with the C-36 peptide also showed a decrease in 7α-hydroxylase mRNA. A mutated but very similar peptide did not have any effect on 7α-hydroxylase mRNA or its promoter. The sterol 12α-hydroxylase/CYP8B1 (12α-hydroxylase) promoter is also down-regulated by the C-36 peptide in HepG2 cells but not by the mutated peptide. The DNA element involved in the C-36-mediated regulation of 7α- and 12α-hydroxylase promoters mapped to the α1-fetoprotein transcription factor (FTF) site in both promoters. The C-36 peptide prevented binding of FTF to its target DNA recognition site by direct interaction with FTF. We hypothesize that the C-36 peptide specifically interacts with FTF and induces a conformational change that results in loss of its DNA binding ability, which results in suppression of 7α- and 12α-hydroxylase transcription. These results suggest that peptides derived from specific serum proteins may alter hepatic gene expression in a highly specific manner. α1-antitrypsin sterol 12α-hydroxylase/CYP8B1 cholesterol 7α-hydroxylase/CYP7A1 α1-fetoprotein bovine serum albumin direct repeat fatty acid synthase α1-fetoprotein transcription factor glutathioneS-transferase 3-hydroxy-3-methyl-glutaryl coenzyme A hepatocyte nuclear factor low density lipoprotein ribonuclease protection assay small heterodimer partner-1 serine protease inhibitor cytomegalovirus Serine protease inhibitors (serpins) are the most common protease inhibitors in mammals and are part of the acute phase response. At sites of inflammation, proteolytic enzymes are released by neutrophils, platelets, mast cells, macrophages, or by any invading microorganisms. Because an uncontrolled proteolytic activity would result in serious unwanted destruction of surrounding tissues, the synthesis of serpins (i.e. α1-antichymotrypsin, α1-antitrypsin (α1-AT),1 and plasminogen activator inhibitor I) is markedly increased (1Rubin H. Nat. Med. 1996; 2: 632-633Crossref PubMed Scopus (51) Google Scholar) to restore homeostasis. Inhibitory serpins interact with their target proteases at a reactive site located within a loop structure of 30–40 amino acid residues from the carboxyl-terminal end (2Zhou A. Carrell R.W. Huntington J.A. J. Biol. Chem. 2001; 276: 27541-27547Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Formation of a stable complex between α1-AT and human leukocyte elastase results from the cleavage of the P1-P′1 bond in the reactive site loop, generating a 4-kDa carboxyl-terminal fragment of 36 amino acids corresponding to residues 359–394 (C-36), that remains non-covalently bound to the cleaved α1-AT in complex with the protease (3Travis J. Salvesen G.S. Annu. Rev. Biochem. 1983; 52: 655-709Crossref PubMed Scopus (1532) Google Scholar). This reactive site loop is also susceptible to proteolysis by non-target proteases (4Desrochers P.E. Mookhtiar K. Van Wart H.E. Hasty K.A. Weiss S.J. J. Biol. Chem. 1992; 267: 5005-5012Abstract Full Text PDF PubMed Google Scholar), which can give rise to the carboxyl-terminal peptide fragments of the serpin. Both of these reactions cause a loss of inhibitor activity and, in the case of α1-AT, it results in a α1-AT molecule composed of two associated chains, a long amino-terminal fragment and a 36-residue carboxyl-terminal fragment, C-36 (5Whisstock J.C. Skinner R. Carrell R.W. Lesk A.M. J. Mol. Biol. 2000; 295: 651-665Crossref PubMed Scopus (48) Google Scholar). This cleaved form of α1-AT has been shown to have biological activities, such as chemotatic activity (6Joslin G. Griffin G.L. August A.M. Adams S. Fallon R.J. Senior R.M. Perlmutter D.H. J. Clin. Invest. 1992; 90: 1150-1154Crossref PubMed Scopus (68) Google Scholar) and regulation of native α1-AT synthesis (7Joslin G. Fallon R.J. Bullock J. Adams S.P. Perlmutter D.H. J. Biol. Chem. 1991; 266: 11282-11288Abstract Full Text PDF PubMed Google Scholar). These observations support the notion that α1-AT may also act as a reservoir of these biological activities that are released upon cleavage of the native form. α1-AT is the archetype of the serpin superfamily and the most abundant serpin in plasma (20–53 μm) and is mainly synthesized by hepatocytes. Its hepatic synthesis increases during acute phase response due to stimulation by the release of interleukin 6 (8Perlmutter D.H. May L.T. Sehgal P.B. J. Clin. Invest. 1989; 84: 138-144Crossref PubMed Scopus (109) Google Scholar). α1-AT concentration in serum can increase up to 8-fold during inflammatory events (9Galloway M.J. Mackie M.J. McVerry B.A. Thromb. Res. 1985; 38: 311-320Abstract Full Text PDF PubMed Scopus (21) Google Scholar). The α1-AT-elastase complexes are cleared from the circulation by the low density lipoprotein (LDL)-related protein receptor or the serpin-enzyme complex receptor, located mainly on the surface of the hepatocytes (7Joslin G. Fallon R.J. Bullock J. Adams S.P. Perlmutter D.H. J. Biol. Chem. 1991; 266: 11282-11288Abstract Full Text PDF PubMed Google Scholar, 10Poller W. Willnow T.E. Hilpert J. Herz J. J. Biol. Chem. 1995; 270: 2841-2845Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). A 5-amino acid peptide within C-36 is actually recognized by the receptor (7Joslin G. Fallon R.J. Bullock J. Adams S.P. Perlmutter D.H. J. Biol. Chem. 1991; 266: 11282-11288Abstract Full Text PDF PubMed Google Scholar). It should be mentioned that free C-36 carboxyl-terminal fragment and other cleavage products have been isolated from plasma, bile, and the spleen (11Johansson J. Grondal S. Sjovall J. Jornvall H. Curstedt T. FEBS Lett. 1992; 299: 146-148Crossref PubMed Scopus (37) Google Scholar). The acute phase response that is triggered by infection, inflammation, and trauma is associated with changes in plasma lipid and lipoprotein levels (12Hardardottir I. Grunfeld C. Feingold K.R. Curr. Opin. Lipidol. 1994; 5: 207-215Crossref PubMed Scopus (269) Google Scholar) as well as in a variety of proteins, such as α1-AT (13Mackiewicz A. Kushner I. Aldred A. G. S. Mackiewicz A. Kushner I. Baumann H. Acute Phase Proteins: Molecular Biology, Biochemistry, and Clinical Applications. CRC Press, Boca Raton, FL1993: 3-37Google Scholar). Plasma cholesterol also increases albeit more moderately than plasma triglycerides (14Cabana V.G. Siegel J.N. Sabesin S.M. J. Lipid Res. 1989; 30: 39-49Abstract Full Text PDF PubMed Google Scholar). Interestingly, however, in experimentally induced acute phase in rabbits, the lipoprotein profile distribution shifts dramatically toward a more atherogenetic profile (14Cabana V.G. Siegel J.N. Sabesin S.M. J. Lipid Res. 1989; 30: 39-49Abstract Full Text PDF PubMed Google Scholar). These changes in cholesterol metabolism can also be induced by the administration of lipopolysaccharide, which mimics Gram-negative infections (12Hardardottir I. Grunfeld C. Feingold K.R. Curr. Opin. Lipidol. 1994; 5: 207-215Crossref PubMed Scopus (269) Google Scholar). Administration of lipopolysaccharide to rodents increases plasma cholesterol levels resulting in an increase in LDL (15Feingold K.R. Hardardottir I. Memon R. Krul E.J. Moser A.H. Taylor J.M. Grunfeld C. J. Lipid Res. 1993; 34: 2147-2158Abstract Full Text PDF PubMed Google Scholar). Atherosclerosis is a form of chronic inflammation resulting from interaction between modified lipoproteins, macrophages, and other cells (for a review see Ref. 16Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). High plasma cholesterol levels are unique in being sufficient to drive the development of atherosclerosis in humans and experimental animals, even in the absence of other known risk factors, and thus cholesterol homeostasis is important. One of the pathways that play a critical role in maintaining cholesterol homeostasis is the bile acid biosynthetic pathway, because nearly 50% of the body cholesterol is catabolized to bile acids. Cholesterol conversion to bile acids occurs via the "classic" (neutral) or the "alternative" (acidic) bile acid biosynthesis pathways (17Javitt N.B. FASEB J. 1994; 8: 1308-1311Crossref PubMed Scopus (150) Google Scholar). Cholic acid and chenodeoxycholic acid are the end products of these pathways and the major primary bile acids found in most vertebrates. Cholic acid is hydroxylated at position 12α, whereas chenodeoxycholic acid is not. There are two enzymes that play major regulatory roles in these two pathways. Cholesterol 7α-hydroxylase/CYP7A1 (7α-hydroxylase) is the rate-limiting enzyme in the classic pathway. Sterol 12α-hydroxylase/CYP8B1 (12α-hydroxylase) is the specific enzyme for cholic acid synthesis and determines the ratio of cholic acid to chenodeoxycholic acid and thus the hydrophobicity of the circulating pool. Because chenodeoxycholic acid is a more potent suppressor of HMG-CoA reductase and 7α-hydroxylase than cholic acid (18Shaw R. Elliot W.H. J. Biol. Chem. 1979; 254: 7177-7182Abstract Full Text PDF PubMed Google Scholar, 19Heuman D.M. Hylemon P.B. Vlahcevic Z.R. J. Lipid Res. 1989; 30: 1161-1171Abstract Full Text PDF PubMed Google Scholar), the relative activity of 12α-hydroxylase may play an important role in the regulation of hepatic cholesterol homeostasis. The alteration of cholic/chenodeoxycholic acid ratio affects biliary cholesterol and phospholipid secretion, thus altering intestinal cholesterol absorption and receptor-mediated lipoprotein uptake by the hepatocyte (19Heuman D.M. Hylemon P.B. Vlahcevic Z.R. J. Lipid Res. 1989; 30: 1161-1171Abstract Full Text PDF PubMed Google Scholar). Recent studies have delineated many of the factors involved in the expression and regulation of these two bile acid biosynthetic enzymes. Thus, liver receptor homolog-1, also known as CYP7A promoter-binding factor, NR5A2 (20Committee N.R.N. Cell. 1999; 97: 161-163Abstract Full Text Full Text PDF PubMed Scopus (963) Google Scholar), or α1-fetoprotein transcription factor (FTF) (Genome Data base Nomenclature Committee) (21Galarneau L. Pare J.F. Allard D. Hamel D. Levesque L. Tugwood J.D. Green S. Belanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar), has been proposed to be required for the transcription of the 7α-hydroxylase gene (22Nitta M., Ku, S. Brown C. Okamoto A.Y. Shan B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6660-6665Crossref PubMed Scopus (249) Google Scholar, 23del Castillo-Olivares A. Gil G. Nucleic Acids Res. 2000; 28: 3587-3593Crossref PubMed Google Scholar). Bile acids activate transcription of the small heterodimer partner 1 (SHP) via binding of the hormone receptor farnesoid X receptor to its binding site in the SHP promoter. In turn, it has been proposed that SHP dimerizes with FTF and diminishes its activity on the 7α-hydroxylase promoter by mechanisms not well understood yet (24Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1569) Google Scholar, 25Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1253) Google Scholar). FTF also plays a key role in the expression and in the regulation of 12α-hydroxylase (26del Castillo-Olivares A. Gil G. J. Biol. Chem. 2000; 275: 17793-17799Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). FTF binds to two sites within the rat 12α-hydroxylase promoter and both sites are required for both promoter activity and bile acid-mediated regulation. We have also shown that SHP is involved in the down-regulation of the 12α-hydroxylase promoter (27del Castillo-Olivares A. Gil G. Nucleic Acids Res. 2001; 29: 4035-4042Crossref PubMed Scopus (81) Google Scholar). Overexpression of SHP in HepG2 cells suppresses 12α-hydroxylase promoter activity. We also showed that SHP prevents binding of FTF to its binding sites within the 12α-hydroxylase promoter providing a mechanism of action for the SHP-mediated suppression of 12α-hydroxylase transcription (27del Castillo-Olivares A. Gil G. Nucleic Acids Res. 2001; 29: 4035-4042Crossref PubMed Scopus (81) Google Scholar). FTF is also the target for a specific sterol regulatory binding protein 2-mediated suppression of the 12α-hydroxylase promoter (28del Castillo-Olivares A. Gil G. J. Biol. Chem. 2002; 277: 6750-6757Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Because of the well documented dramatic increase in α1-AT levels in response to inflammation, which in turns gives rise to an increase in peptides resulting from α1-AT cleavage by proteases and the alteration in cholesterol metabolism during the acute phase response, we have investigated whether there is a connection between the inflammatory response and cholesterol homeostasis through a specific suppression of bile acid biosynthesis by the α1-AT-derived peptide C-36. We report here that the α1-AT C-36 peptide is a powerful inhibitor of the 7α- and 12α-hydroxylase promoters by specifically interacting with FTF, a positive-acting transcription factor. Common laboratory chemicals were obtained from Sigma, Invitrogen, New England BioLabs (Beverly, MA), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and Bio-Rad. Synthetic carboxyl-terminal peptide (C-36 peptide) of α1-antitrypsin (corresponding to residues 359–394; NH2-SIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK) of more than 95% purity was purchased from Saveen Biotech (Denmark). A negative control peptide named C-35 (F → A) (NH2-SIPPEVKFNKPFVALMIEQNTKSPLMGKVVNPTQK) was a generous gift from Dr. Tim Coleman. Plasmids, pGL3-R12α-865, pGL3-R7α-342, and the mutant constructs used for transfections have been previously described (23del Castillo-Olivares A. Gil G. Nucleic Acids Res. 2000; 28: 3587-3593Crossref PubMed Google Scholar, 26del Castillo-Olivares A. Gil G. J. Biol. Chem. 2000; 275: 17793-17799Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). In brief, pGL3-R7α-342 contains a 342-nucleotide fragment of the promoter of the rat 7α-hydroxylase gene (−342 to +59) linked upstream of the luciferase gene, and pGL3-R12α-865 contains a 865-nucleotide fragment of the promoter of the rat 12α-hydroxylase gene (−865 to +37) linked upstream of the luciferase gene. Plasmids, pcDNA3-hHNF-4α carrying the human hepatocyte nuclear factor-4α (HNF-4α) cDNA, and pCMX-hFTF, used for TnT in vitro expression of proteins, have been described previously (27del Castillo-Olivares A. Gil G. Nucleic Acids Res. 2001; 29: 4035-4042Crossref PubMed Scopus (81) Google Scholar). pCMV-βGalactosidase and pBluescript were obtained from Invitrogen. Radioisotopes [γ-32P]ATP, [α-32P]dCTP, [α-32P]UTP, and [14C]cholesterol were purchased from ICN Pharmaceuticals (Costa Mesa, CA). Plasmid constructs containing cDNA fragments encoding antisense RNA used for synthesis of ribonuclease protection assay (RPA) probes have been previously described (29Stravitz R.T. Rao Y.P. Vlahcevic Z.R. Gurley E.C. Jarvis W.D. Hylemon P.B. Am. J. Physiol. 1996; 271: G293-G303PubMed Google Scholar). DNA oligonucleotides used for electrophoretic mobility shift analysis were obtained from Genosys (The Woodlands, TX). Hepatocytes from adult male Sprague-Dawley rats (200–300 g) were isolated according to the method of Bissell and Guzelian (30Bissell D.M. Guzelian P.S. Ann. N. Y. Acad. Sci. 1980; 349: 85-98Crossref PubMed Scopus (286) Google Scholar). Parenchymal hepatocytes (3.5 × 106) were plated onto 60-mm Falcon culture dishes coated with rat tail collagen (Vitrogen 100, Cohesion, Palo Alto, CA). Prior to plating, cells were judged to be >90% viable using trypan blue exclusion (0.04%). Cells were incubated in 3 ml of serum-free William's E medium supplemented with 1.0 μm thyroxine, 0.1 μm dexamethasone (31Hylemon P.B. Gurley E.C. Stravitz R.T. Litz J.S. Pandak W.M. Chiang J.Y. Vlahcevic Z.R. J. Biol. Chem. 1992; 267: 16866-16871Abstract Full Text PDF PubMed Google Scholar), 0.25 unit/ml insulin, and 100 units/ml penicillin in a 5% CO2 atmosphere at 37 °C. Culture medium was changed daily. Peptides were added 48 h after plating, unless otherwise indicated. Hepatocytes were harvested at the indicated times. The conversion of [14C]cholesterol into methanol-water-soluble material was determined as previously described (31Hylemon P.B. Gurley E.C. Stravitz R.T. Litz J.S. Pandak W.M. Chiang J.Y. Vlahcevic Z.R. J. Biol. Chem. 1992; 267: 16866-16871Abstract Full Text PDF PubMed Google Scholar) according to the Folch technique. Adult male C57BBL mice (22–25 g) were injected through the tail vain twice, at 9:00 a.m. and 4:00 p.m., with 2 mg of the C-36 or the C-35 (F → A) peptides dissolved in 150 μl of phosphate-buffered saline (PBS) solution. Control mice were injected with PBS alone. Mice were killed 24 h after the first injection, and liver RNA was extracted by standard procedures (32Hylemon P.B. Gurley E.C. Kubaska W.M. Whitehead T.R. Guzelian P.S. Vlahcevic Z.R. J. Biol. Chem. 1985; 260: 1015-1019Abstract Full Text PDF PubMed Google Scholar). Total RNA was isolated from primary hepatocytes using the guanidine thiocyanate cesium chloride centrifugation method (32Hylemon P.B. Gurley E.C. Kubaska W.M. Whitehead T.R. Guzelian P.S. Vlahcevic Z.R. J. Biol. Chem. 1985; 260: 1015-1019Abstract Full Text PDF PubMed Google Scholar). 7α-Hydroxylase, HMG-CoA reductase, CYP27, actin, glyceraldehyde-3-phosphate dehydrogenase, and cyclophilin mRNAs were quantified by RPA as already described (29Stravitz R.T. Rao Y.P. Vlahcevic Z.R. Gurley E.C. Jarvis W.D. Hylemon P.B. Am. J. Physiol. 1996; 271: G293-G303PubMed Google Scholar) according to the manufacturer's protocol (Ambion, Austin, TX). Fatty acid synthase (FAS), HMG-CoA synthase, and LDL receptor mRNAs were analyzed by Northern blot as previously described (33Pandak W.M., Li, Y.C. Chiang J.Y. Studer E.J. Gurley E.C. Heuman D.M. Vlahcevic Z.R. Hylemon P.B. J. Biol. Chem. 1991; 266: 3416-3421Abstract Full Text PDF PubMed Google Scholar). In brief, total RNA was size-fractionated by electrophoresis on 1% agarose gel containing 7% formaldehyde and then transferred to nitrocellulose membranes (Bio-Rad). To standardize loaded mRNAs, a cyclophilin cDNA fragment was labeled by the same method and used to probe the same membranes under modified hybridization conditions. HepG2 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Rat primary hepatocytes and HepG2 cells were both transfected with Effectene Transfection Reagent from Qiagen (Valencia, CA) according to the manufacturer's instructions. Rat primary hepatocytes were plated in William's E medium containing 10% fetal calf serum, 1.0 μmthyroxine, 0.1 μm dexamethasone, and 100 units/ml penicillin in six-well Primaria tissue culture plates (Becton Dickinson, Franklin Lakes, NJ). Rat primary hepatocytes were transfected with 500 ng of test plasmid, 5 ng of pCMV-βGalactosidase and pBluescript (used as a carrier DNA to adjust the total DNA amount to 1.5 μg) in serum-free, penicillin and hormone-containing (dexamethasone, thyroxine) William's E medium. HepG2 cells were plated on 12-well plates in supplemented minimal essential medium containing 10% fetal calf serum, 0.125 unit/ml insulin, and 0.1 μm dexamethasone. Twenty-four hours later, cells were transfected with 10 ng of test plasmid and 0.2 ng of pCMVsportβGalactosidase and pBluescript (used as a carrier DNA to adjust the total DNA amount to 400 ng). After 16 h, the DNA complexed to Effectene reagent was removed and fresh medium was added with or without the C-36 peptide. Rat primary hepatocytes and HepG2 cells were harvested 24 and 48 h later, respectively, using the Dual-Light Kit (Tropix, Bedford, MA) according to the manufacturer's instructions. Luciferase activity was measured using a Monolight 2010 luminometer (Pharmingen, San Diego, CA), and background activity (pGL3-basic) was subtracted. Luciferase activity was normalized to β-galactosidase activity for transfection efficiency. DNA oligonucleotides used as EMSA probes were labeled using [γ-32P]ATP and T4 polynucleotide kinase (New England BioLabs) according to the manufacturer's instructions. In vitrotranslation/transcription of cDNAs encoding human FTF and human HNF-4α, was performed using the TnT T7-coupled Rabbit Reticulocyte Lysate system from Promega (Madison, WI), according to the manufacturer's protocol. DNA binding reactions were performed in 50 mm KCl, 20 mm Tris-HCl (pH 8.0), 0.2 mm EDTA, 4% Ficoll, 1.0 μg of poly(dI-dC), 1000-fold molar excess of an irrelevant single-stranded DNA, 4 μl of thein vitro translated protein, and the indicated amounts of peptide in a final volume of 20 μl. After 15-min incubation on ice the indicated 32P-labeled DNA probe was added to the reaction. After 20-min incubation on ice, samples were loaded onto a 4% polyacrylamide gel and subjected to electrophoresis at 4 °C. Gels were dried, and exposures were made to XAR film (Kodak). Ninety-six well microtiter plates (Dynatech Laboratories) were coated with either C-36, C-35 (F → A) peptides, or bovine serum albumin (BSA) by incubating the plates overnight at 37 °C with either peptide or BSA at 2.5 μg/ml in phosphate-buffered saline solution, 0.05% NaN3. Plates were washed and treated with blocking buffer (0.017 mNa2B4O7, 0.12 NaCl, 0.05% Tween 20, 1 mm EDTA, 0.25% BSA, 0.05% NaN3). FTF, in the form of a recombinant glutathione S-transferase (GST) fusion protein containing amino acids 141–501 of the human FTF, was then added. After 1-h incubation at 37 °C, plates were washed, blocked, and incubated with goat anti-GST (Amersham Biosciences) for 1 h at 37 °C. After washing and reblocking, a secondary rabbit anti-goat IgG conjugated to horseradish peroxidase was added and incubated for 1 h at 37 °C. A Dako TMB One-Step Substrate system (Dako Corp., Carpenteria, CA) was used to develop the peroxidase activity, the reaction was terminated by adding 0.18 mH2SO4, and absorbance was measured at 450 nm. Specific binding was defined by subtracting the absorbance of the BSA wells from the corresponding peptide wells, and the number was multiplied by 10. For the competition experiments, the indicated molar excess of either C-36 or C-35 (F → A) peptides was included during the incubation with FTF. Results are expressed as the means ± S.E. Statistical significance between two groups was determined by using the Student's t test. A p value of <0.05 was considered significant. We first investigated whether the C-36 peptide, resulting from cleavage of α1-AT by neutrophil elastase, could alter the rates of cholesterol conversion into bile acids in primary rat hepatocytes. Rates of bile acid biosynthesis were measured by the conversion of [14C]cholesterol into methanol-water-soluble material (14C-bile acids). As shown in Fig.1, the C-36 peptide significantly decreased bile acid biosynthesis rates by about 3-fold. No evidence of C-36 peptide toxicity was apparent from lactate dehydrogenase assay (data not shown) or from microscopic examination of the treated hepatocytes. Cholesterol 7α-hydroxylase is the rate-limiting step in the conversion of cholesterol into bile acids via the neutral pathway, so we next determined if the C-36 peptide could regulate 7α-hydroxylase mRNA levels. Forty-eight hours after plating, the C-36 peptide was added to primary rat hepatocytes and total RNA was isolated after 24 h. Fig. 2 shows that the C-36 peptide down-regulated 7α-hydroxylase mRNA levels in a concentrationdependent manner up to 10-fold. Fig.3 shows that the C-36-mediated suppression of 7α-hydroxylase mRNA is time-dependent, and it reaches its peak at 24 h. C-36 had no effect on 7α-hydroxylase mRNA half-life based on actinomycin D experiments (data not shown).Figure 3Time course of the effect of the C-36 serpin peptide on 7α-hydroxylase mRNA levels in primary rat hepatocytes. C-36 (10 μm) was added to the medium 48 h after plating, and total RNA was isolated from cells at indicated times. The ratio of 7α-hydroxylase to cyclophilin (CYC) was expressed as a percentage of untreated controls. Values represent the average of three experiments ± S.E. (p < 0.005). The inset shows a representative experiment.View Large Image Figure ViewerDownload (PPT) To demonstrate the specificity of the C-36 peptide on 7α-hydroxylase mRNA levels, we used a mutated peptide, C-35 (F → A), that has the same amino acid sequence as the C-36 peptide, except it has a mutation at residue 372 (F → A) and a deletion at residue 384 (Fig. 4). This mutation should prevent interaction of C-36 with the serpin-enzyme complex receptor, because the penta-amino acid peptide FVYLI is required for the binding (34Joslin G. Krause J.E. Hershey A.D. Adams S.P. Fallon R.J. Perlmutter D.H. J. Biol. Chem. 1991; 266: 21897-21902Abstract Full Text PDF PubMed Google Scholar). This peptide was tested for its ability to down-regulate CYP7A1 mRNA levels in primary rat hepatocytes as compared with C-36. As shown in Fig. 4, the mutated peptide C-35 (F → A) had no significant effect on 7α-hydroxylase mRNA levels, as compared with the C-36 peptide. Because the regulation of a number of genes involved in cholesterol metabolism and, to a larger extent, in lipid metabolism depends on the size of the free cholesterol pool, we measured mRNA levels of HMG-CoA reductase, HMG-CoA synthase, and FAS by RPA or Northern blot analyses. Following the addition of 10 μm C-36 peptide to primary rat hepatocytes, HMG-CoA reductase, HMG-CoA synthase, and FAS mRNA were suppressed between 2- and 3-fold (Fig.5). Moreover, there was a smaller effect of the C-36 peptide on LDL receptor mRNA levels. Expression of housekeeping genes, such as actin and glyceraldehyde-3-phosphate dehydrogenase was not regulated (Fig. 5). To investigate whether the C-36-mediated suppression of 7α-hydroxylase mRNA that we observed in tissue culture cells also occurs in vivo, mice were injected twice with 2 mg of the C-36 peptide each time. As controls, two other sets of mice were injected with either vehicle alone (PBS) or the C-35 (F → A) control peptide. Fig. 6 shows that injection of the C-36 peptide reduced 7α-hydroxylase expression by more than 2-fold. Control C-35 (F → A) peptide had no significant effect. To characterize the molecular mechanisms involved in the regulation of 7α-hydroxylase expression by the C-36 peptide, we investigated whether the C-36 peptide could regulate 7α-hydroxylase promoter activity. Primary rat hepatocytes were transfected with a chimeric gene containing 342 nucleotides of the rat 7α-hydroxylase promoter in front of the luciferase gene (designated pGL3-R7α-342, Fig.7). This promoter fragment has been shown to contain all the necessary elements for transcription and regulation by bile acids (23del Castillo-Olivares A. Gil G. Nucleic Acids Res. 2000; 28: 3587-3593Crossref PubMed Google Scholar). As shown in Fig. 7, the C-36 peptide suppressed 7α-hydroxylase promoter activity by about 2.5-fold. The C-35 (F → A) mutant peptide did not have any effect (data not shown). Another similar construct that contained only 143 nuc
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