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• W2005930762 abstract "The steroidogenic acute regulatory protein (STAR) participates in steroidogenesis through the mitochondrial transfer of cholesterol to cytochrome P450scc. The rat adrenal Star gene is transcribed as a 3.5-kilobase pair (kb) and 1.6-kb mRNA with the larger mRNA predominating (∼85% of total) in vivo. Hypophysectomy (HPX) produced a 3–5-fold decrease in Star mRNA along with a loss of adrenal steroids, whereas P450scc mRNA decreased by less than 2-fold. Adrenocorticotropic hormone (ACTH) treatment of HPX rats maximally stimulated steroidogenesis rates within 5 min with over 10-fold elevation of steady state blood levels occurring within 10 min. For intact rats there was a 5–10-fold larger increase, paralleling previously observed elevations of cholesterol-cytochrome P450scc association and metabolism in subsequently isolated adrenal mitochondria. ACTH did not increase either total STAR protein or a group of modified forms until at least 30 min after completion of acute stimulation, indicating that elevated translation of STAR protein cannot alone mediate this acute stimulation. Parallel slow changes in STAR protein and corticosterone formation after ACTH treatment are consistent with participation of STAR forms as co-regulators of these hormonal responses. ACTH stimulation of HPX rats increasedStar mRNA by 2.5-fold within 20 min and by 4.5-fold after 1 h, thus preceding the rise in the STAR protein. A 3.5-kbStar cDNA clone isolated from a rat adrenal cDNA library exhibited a 0.9-kb open reading frame and a 2.5-kb 3′-untranslated region (3′-UTR). The open reading frame sequence differed at only 12 amino acids from that of the mouseStar. The rat Star gene seven exons with exon 7 encoding the entire 2.5 kb of 3′-UTR of the 3.5-kb mRNA. The 3′-UTR sequence suggests that 1.6- and 3.5-kb mRNA are formed by an alternative usage of different polyadenylation signals. Multiple UUAUUUA(U/A)(U/A) motifs also suggest additional regulation through this extended 3′-UTR. Although elevation of STAR protein by ACTH does not cause the acute increase in adrenal cholesterol metabolism, changes in the turnover or distribution of an active STAR subfraction cannot be excluded. The steroidogenic acute regulatory protein (STAR) participates in steroidogenesis through the mitochondrial transfer of cholesterol to cytochrome P450scc. The rat adrenal Star gene is transcribed as a 3.5-kilobase pair (kb) and 1.6-kb mRNA with the larger mRNA predominating (∼85% of total) in vivo. Hypophysectomy (HPX) produced a 3–5-fold decrease in Star mRNA along with a loss of adrenal steroids, whereas P450scc mRNA decreased by less than 2-fold. Adrenocorticotropic hormone (ACTH) treatment of HPX rats maximally stimulated steroidogenesis rates within 5 min with over 10-fold elevation of steady state blood levels occurring within 10 min. For intact rats there was a 5–10-fold larger increase, paralleling previously observed elevations of cholesterol-cytochrome P450scc association and metabolism in subsequently isolated adrenal mitochondria. ACTH did not increase either total STAR protein or a group of modified forms until at least 30 min after completion of acute stimulation, indicating that elevated translation of STAR protein cannot alone mediate this acute stimulation. Parallel slow changes in STAR protein and corticosterone formation after ACTH treatment are consistent with participation of STAR forms as co-regulators of these hormonal responses. ACTH stimulation of HPX rats increasedStar mRNA by 2.5-fold within 20 min and by 4.5-fold after 1 h, thus preceding the rise in the STAR protein. A 3.5-kbStar cDNA clone isolated from a rat adrenal cDNA library exhibited a 0.9-kb open reading frame and a 2.5-kb 3′-untranslated region (3′-UTR). The open reading frame sequence differed at only 12 amino acids from that of the mouseStar. The rat Star gene seven exons with exon 7 encoding the entire 2.5 kb of 3′-UTR of the 3.5-kb mRNA. The 3′-UTR sequence suggests that 1.6- and 3.5-kb mRNA are formed by an alternative usage of different polyadenylation signals. Multiple UUAUUUA(U/A)(U/A) motifs also suggest additional regulation through this extended 3′-UTR. Although elevation of STAR protein by ACTH does not cause the acute increase in adrenal cholesterol metabolism, changes in the turnover or distribution of an active STAR subfraction cannot be excluded. The conversion of cholesterol to pregnenolone is the first step of steroid synthesis and is catalyzed by cytochrome P450 side chain cleavage enzyme (P450scc) 1The abbreviations used are: P450scc, P450 side chain cleavage enzyme; STAR, steroidogenic acute regulatory protein; RT-PCR, reverse transcription and polymerase chain reaction; HPX, hypophysectomy; ACTH, adrenocorticotropic hormone; kb, kilobase pair(s); bp, base pair(s); UTR, untranslated region; ORF, open reading frame. 1The abbreviations used are: P450scc, P450 side chain cleavage enzyme; STAR, steroidogenic acute regulatory protein; RT-PCR, reverse transcription and polymerase chain reaction; HPX, hypophysectomy; ACTH, adrenocorticotropic hormone; kb, kilobase pair(s); bp, base pair(s); UTR, untranslated region; ORF, open reading frame.localized on the matrix side of the inner mitochondrial membrane (1Miller W.L. Endocr. Rev. 1988; 9: 295-318Crossref PubMed Scopus (1183) Google Scholar,2Churchill P.F. Kimura T. J. Biol. Chem. 1979; 254: 10443-10448Abstract Full Text PDF PubMed Google Scholar). This conversion is the rate-limiting step in steroidogenesis and is hormonally activated (3Stone D. Hechter O. Arch. Biochem. Biophys. 1954; 51: 457-469Crossref PubMed Scopus (282) Google Scholar). The availability of cholesterol to P450scc which limits this conversion (4Simpson E.R. Jefcoate C.R. Brownie A.C. Boyd G.S. Eur. J. Biochem. 1972; 28: 443-450Crossref Scopus (122) Google Scholar, 5Privalle C.T. Crivello J.F. Jefcoate C.R. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 702-706Crossref PubMed Scopus (333) Google Scholar) requires hormonal activation of cholesterol mobilization to the mitochondria and then trophic hormone-dependent transport of cholesterol from mitochondrial outer membrane to inner membrane (6Brownie A.C. Alfano J. Jefcoate C.R. Beinert H. Orme-Johnson W.H. Ann. N. Y. Acad. Sci. 1973; 212: 344-360Crossref PubMed Scopus (68) Google Scholar, 7Jefcoate C.R. Simpson E.R. Boyd G.S. Eur. J. Biochem. 1974; 42: 539-551Crossref PubMed Scopus (103) Google Scholar). This latter process is blocked within 10 min by protein synthesis inhibitors, such as cycloheximide, which also correlate with the loss of steroid synthesis after removal of ACTH (8Garren L.D. Gill G.N. Masui H. Walton G.M. Recent Prog. Horm. Res. 1971; 27: 433-478PubMed Google Scholar, 9Crivello J.F. Jefcoate C.R. Biochim. Biophys. Acta. 1978; 542: 315-329Crossref PubMed Scopus (93) Google Scholar). A series of phosphoproteins (30–37 kDa) have been identified in cultured adrenal cells that localize to the mitochondria and increase in response to hormone stimulation (10Krueger R.J. Orme-Johnson N.R. J. Biol. Chem. 1983; 258: 10159-10167Abstract Full Text PDF PubMed Google Scholar, 11Alberta J.A. Epstein L.F. Pon L.A. Orme-Johnson N.R. J. Biol. Chem. 1989; 264: 2368-2372Abstract Full Text PDF PubMed Google Scholar). The larger precursor forms are rapidly removed by cycloheximide concomitant with mitochondrial proteolysis (10Krueger R.J. Orme-Johnson N.R. J. Biol. Chem. 1983; 258: 10159-10167Abstract Full Text PDF PubMed Google Scholar, 11Alberta J.A. Epstein L.F. Pon L.A. Orme-Johnson N.R. J. Biol. Chem. 1989; 264: 2368-2372Abstract Full Text PDF PubMed Google Scholar). It has been proposed by Epstein and Orme-Johnson (12Epstein L.F. Orme-Johnson N.R. J. Biol. Chem. 1991; 266: 19739-19745Abstract Full Text PDF PubMed Google Scholar) that the effects of these proteins account for the cycloheximide sensitivity and hormonal stimulation in intermembrane cholesterol transport. Subsequent studies from several laboratories have shown that these changes in 30-kDa proteins occur in adrenal, testis, and ovarian cells of several species (13Pon L.A. Orme-Johnson N.R. J. Biol. Chem. 1986; 261: 6594-6599Abstract Full Text PDF PubMed Google Scholar, 14Pon L.A. Epstein L.F. Orme-Johnson N.R. Endocr. Res. 1986; 12: 429-446Crossref PubMed Scopus (66) Google Scholar, 15Juengel J.L. Meberg B.M. Turzillo A.M. Net T.M. Niswender G.D. Endocrinology. 1995; 136: 5423-5429Crossref PubMed Google Scholar, 16Hartung S. Rust W. Balvers M. Ivell R. Biochem. Biophys. Res. Commun. 1995; 215: 646-653Crossref PubMed Scopus (82) Google Scholar, 21Sugawara T. Holt J.A. Driscoll D. Strauss III, J.F. Lin D. Miller W.L. Patterson D. Clancy K.P. Hart I.M. Clark B.J. Stocco D.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4778-4782Crossref PubMed Scopus (349) Google Scholar). The peripheral benzodiazepine receptor that is localized in the adrenal mitochondrial outer membrane is also essential for this step, suggesting the involvement of a complex multistep process (17Papadopoulos V. Brown A.S. J. Steroid Biochem. Mol. Biol. 1995; 53: 103-110Crossref PubMed Scopus (101) Google Scholar,18Boujrad N. Gaillard J.L. Garnier M. Papadopoulos V. Endocrinology. 1994; 135: 1576-1583Crossref PubMed Scopus (56) Google Scholar). Recently, a cDNA encoding the 30-kDa protein has been cloned and renamed the Steroidogenic acuteregulatory protein (STAR) (19Clark B.J. Wells J. King S.R. Stocco D.M. J. Biol. Chem. 1994; 269: 28314-28322Abstract Full Text PDF PubMed Google Scholar). Several sites for phosphorylation by cAMP-dependent protein kinase have been recognized in the sequence which otherwise shows no similarity to other proteins (20Stocco D.M. Clark B.J. Endocr. Rev. 1996; 17: 221-224Crossref PubMed Scopus (934) Google Scholar). Star expression in COS-1 cells that have been previously transfected with expression vectors encoding P450scc and adrenodoxin further enhances cholesterol metabolism (21Sugawara T. Holt J.A. Driscoll D. Strauss III, J.F. Lin D. Miller W.L. Patterson D. Clancy K.P. Hart I.M. Clark B.J. Stocco D.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4778-4782Crossref PubMed Scopus (349) Google Scholar). Recent work shows that this STAR activity in COS-1 cells is independent of the mitochondrial uptake that is seen in steroidogenic cells (22Arakane R. Sugawara T. Nishino H. Liu Z. Holt J.A. Pain E. Stocco D.M. Miller W.L. Strauss III, J.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13731-13736Crossref PubMed Scopus (253) Google Scholar). MutatedSTAR has been found in humans born with the steroid deficiency disease, congenital lipoid adrenal hyperplasia (23Lin D. Sugawara T. Strauss III, J.F. Clark B.J. Stocco D.M. Saenger P. Rogol R. Miller W.L. Science. 1995; 267: 1828-1831Crossref PubMed Scopus (864) Google Scholar, 24Bose H.S. Sugawara T. Strauss III, J.F. Miller W.L. N. Engl. J. Med. 1996; 335: 1870-1878Crossref PubMed Scopus (525) Google Scholar, 25Saenger P. Klonari Z. Black S.M. Compagnone N. Mellon S.H. Fleischer A. Abrams C.A.L. Shackelton C.H.L. Miller W.L. J. Clin. Endocrinol. & Metab. 1995; 80: 200-205Crossref PubMed Google Scholar). In these cases C-terminal truncation of the protein removes steroidogenic activity. Disruption of the Star gene in mice produces a similar phenotype (58Caron K.M. Soo S.C. Wetsel W.C. Stocco D.M. Clark B.J. Parker K.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11540-11545Crossref PubMed Scopus (382) Google Scholar). Star has been detected in all steroidogenic tissues except for brain and placenta (26Clark B.J. Soo S.-C. Caron K.M. Ikeda Y. Parker K.L. Stocco D.M. Mol. Endocrinol. 1995; 9: 1346-1355Crossref PubMed Google Scholar). Two species of Star mRNA (1.6 and 3.4 kb) are stimulated by cAMP in cultured mouse Leydig tumor cells (26Clark B.J. Soo S.-C. Caron K.M. Ikeda Y. Parker K.L. Stocco D.M. Mol. Endocrinol. 1995; 9: 1346-1355Crossref PubMed Google Scholar). The regulation of the relative proportions of these Star mRNA species may be important in determining the post-transcriptional activation that has been implicated in the rapid hormone stimulation of steroidogenesis (19Clark B.J. Wells J. King S.R. Stocco D.M. J. Biol. Chem. 1994; 269: 28314-28322Abstract Full Text PDF PubMed Google Scholar, 20Stocco D.M. Clark B.J. Endocr. Rev. 1996; 17: 221-224Crossref PubMed Scopus (934) Google Scholar, 21Sugawara T. Holt J.A. Driscoll D. Strauss III, J.F. Lin D. Miller W.L. Patterson D. Clancy K.P. Hart I.M. Clark B.J. Stocco D.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4778-4782Crossref PubMed Scopus (349) Google Scholar). Here we have investigated acute effects of ACTH on levels of Star mRNA and protein in the rat adrenal gland in vivo under conditions that exhibit ACTH-enhanced transfer of cholesterol to P450scc within adrenal mitochondria (4Simpson E.R. Jefcoate C.R. Brownie A.C. Boyd G.S. Eur. J. Biochem. 1972; 28: 443-450Crossref Scopus (122) Google Scholar, 5Privalle C.T. Crivello J.F. Jefcoate C.R. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 702-706Crossref PubMed Scopus (333) Google Scholar, 6Brownie A.C. Alfano J. Jefcoate C.R. Beinert H. Orme-Johnson W.H. Ann. N. Y. Acad. Sci. 1973; 212: 344-360Crossref PubMed Scopus (68) Google Scholar). We have particularly addressed the kinetics of the activation of steroidogenesis in relation to changes in Star mRNA and protein, in particular whether changes in STAR are sufficiently fast to account for the acute response. Although the gene encoding STAR has been cloned from both mouse (19Clark B.J. Wells J. King S.R. Stocco D.M. J. Biol. Chem. 1994; 269: 28314-28322Abstract Full Text PDF PubMed Google Scholar) and human cells (21Sugawara T. Holt J.A. Driscoll D. Strauss III, J.F. Lin D. Miller W.L. Patterson D. Clancy K.P. Hart I.M. Clark B.J. Stocco D.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4778-4782Crossref PubMed Scopus (349) Google Scholar), we know little about the mechanism of transcriptional and translational regulation. The present structures of the mouse and human STAR gene only account for the expression of a 1.6-kb Star mRNA even though steroidogenic cells show extensive expression of a 3.5-kb mRNA. Here we show that this longer mRNA is the predominant species in rat adrenals in vivo, thus raising questions about this extended sequence. Several elements in the 3′-UTRs are targets for RNA-binding proteins which regulate their processing, translational efficiency, and stability (27Jackson R.J. Cell. 1993; 74: 9-14Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 28Jacobson A. Peltz S.W. Annu. Rev. Biochem. 1996; 65: 693-739Crossref PubMed Scopus (576) Google Scholar). Here we describe an extended structure for the Star gene encoding the complete rat 3.5-kb mRNA that is a key step toward characterizing the acute control of the activation of this protein. We show that the extended sequence indeed contains motifs that may affect regulation of thisStar mRNA. Intact and hypophysectomized female Sprague-Dawley rats (175–200 g) were obtained from Harlan Sprague-Dawley (Madison, WI). All animal treatments were performed at 10:00 a.m. Animals were kept in stress minimizing environment. Each rat was then injected with ACTH. Intact rats were treated intraperitoneally with 4 units/rat of Cortrosyn (Organon, West Orange, NJ) up to 20 min prior to sacrifice (20-min group), or subcutaneously with 4 units/rat of Acthar gel (Rhone-Pouleuc Rorer Pharmaceuticals Inc., Collegeville, PA) 24 h prior to sacrifice (24-h group), or subcutaneously (4 units of Acthar gel/rat/day) for 3 consecutive days (3-day group). Hypophysectomized rats were allowed to recover after surgery for 3 days when they were maintained on Purina Lab Chow diets supplemented with 5% sucrose solution. A group of rats was given 4 units of Cortrosyn intraperitoneally for stimulations of up to 20 min prior to sacrifice. For 1 h stimulation, a second injection was given after 30 min to compensate for in vivo degradation of ACTH. For prolonged administrations ACTH was injected subcutaneously as Acthar gel (4 units) 24 h prior to sacrifice. In 24-h groups a second subcutaneous injection of 4 units of Acthar gel was given 3 h prior to sacrifice. All animals were sacrificed by decapitation, and blood was collected into vacutainers containing 15% EDTA, and adrenals were surgically removed, defatted, and rapidly homogenized in TRIzol reagent (Life Technologies, Inc.) Adrenal protein and RNA fractions from this treatment were analyzed for STAR protein and mRNA (see below). Steroids were extracted from plasma with ethyl acetate/acetone (2:1). Cortisone was used during extraction as an internal standard. The organic phase was then evaporated under nitrogen with heating at 37 °C. The residue was dissolved in 100 ml of the methanol and subjected to high pressure liquid chromatography analysis using a reverse-phase C18 column (Beckman, 5 m, 4.6 mm × 25 cm) with a linear gradient of methanol (50–100%). Detection was carried out at 254 nm by a Beckman System Gold programmable Detector Module 166, and the data were analyzed by System Gold Software via a Beckman System Gold Analog Interface Module 406. Two-dimensional gel electrophoresis was performed as described previously (29Elliot M.E. Goodfriend T.L. Jefcoate C.R. Endocrinology. 1993; 133: 1669-1677Crossref PubMed Scopus (54) Google Scholar) using the Millipore Investigator Two-dimensional Electrophoresis System. In brief, tube gels (200 × 1 mm) were prepared with isoelectrofocusing gel solution (ESA, Inc., Chelmsford, MA), broad range ampholytes (Millipore, pH 3–10), and 10% ammonium persulfate. The protein fraction from rat adrenal tissue homogenized in TRIzol reagent (50–100 mg of tissue/1 ml of reagent; Life Technologies, Inc.) was obtained according to the protocol from Life Technologies, Inc. The protein pellet was washed with ethanol and then solubilized with 1% SDS solution. Protein concentration was determined by BCA method, according to the manufacturer's instructions (Pierce) using bovine serum albumin as a standard. Protein samples were mixed with an equal volume of a buffer containing 10 mm Tris, 120 mm dithiothreitol, 0.06% SDS, 3.2% (v/v) Nonidet P-40, 1.8% ampholytes, and 8 m urea (all reagents from ESA, Inc.). 80 μg of protein was loaded on the tube gel and subjected to electrofocusing at 1000 V for 18 h. Gels were extruded from the tubes and equilibrated for 2 min in a 373 mm Tris (pH 8.6), 50 mm dithiothreitol, 3% SDS, and 0.02% bromphenol blue buffer. Tube gels were placed on top of slab gels, consisting of 375 mm Tris (pH 8.8), 0.1% SDS, and 12.5% Duracryl. Molecular weight standards, prepared in 1% agarose, were applied on the top of the slab gel at either end of the tube gel. Gels were run at a constant power of 16 watts/gel for 5 h. After electrophoresis, the proteins from the gel were transferred to the nitrocellulose membrane. The position of the molecular weight markers was determined by staining the membrane with Ponceau S. Western immunoblot analysis (30Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44915) Google Scholar) was completed, using a rabbit antibody (1:6000) raised against a peptide sequence of mouse Star, kindly provided by Drs. N. Boujrad and V. Papadopoulos (Georgetown University, Washington, D. C.), followed by the secondary horseradish peroxidase-conjugated goat anti-rabbit antibody (Promega, Madison, WI). Proteins were visualized by exposing the nitrocellulose membranes following treatment with the ECL detection reagent (Amersham Pharmacia Biotech), to the ECL x-ray film (Amersham Pharmacia). Construction of rat cDNA library in λZAPII was performed as described previously (31Bhattacharyya K.K. Brake P.B. Eltom S.E. Otto S.A. Jefcoate C.R. J. Biol. Chem. 1995; 270: 11595-11602Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). The coding region of rat Star cDNA was cloned by PCR usingpfu DNA polymerase (Stratagene, La Jolla, CA) with primers designed against mouse Star cDNA sequences 15–34 and 973–992 (19Clark B.J. Wells J. King S.R. Stocco D.M. J. Biol. Chem. 1994; 269: 28314-28322Abstract Full Text PDF PubMed Google Scholar). The 0.9-kb amplified product was then subcloned into the SmaI site of the pBluescript SK(+) plasmid (Stratagene, La Jolla, CA). The nucleotide sequence was analyzed by the dideoxy method using Sequenase Version II kit (U. S. Biochemical Corp.), and sequence comparison with reported mammalian Star genes was completed using the GCG program (Genetics Computer Group, Inc.). 5 × 105 plaques of the cDNA library were screened using the 0.9-kb rat Star cDNA as a probe. The probe was labeled by the random primer method (Prime-It kit II, Stratagene) using [α-32P]dCTP (3000 Ci/mmol) (NEN Life Science Products) and nitrocellulose (Schleicher & Schuell) filter replicas for hybridization. From more than 100 positive clones, 18 were randomly selected and excised into the pBluescript vector and then characterized. cDNA clones were isolated and purified using the Qiagen plasmid kit (Qiagen Inc.). The dye terminator cycle sequencing reaction was carried out for both strands using GeneAmp PCR System 9600 (Perkin-Elmer). 5 × 105plaques of the WiStar Furth rat genomic DNA library were screened using a 0.9-kb rat Star cDNA as described above. From 10 positives, 5 clones were selected and characterized. Five clones were amplified by the standard plate lysate and liquid culture method, prepared by the polyethylene glycol method (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 1.21-3.51Google Scholar), and then purified by Qiagen plasmid kit. The gene structure was verified by PCR using a set of specific primers derived from the 0.9-kb cDNA sequence (Table I). For determination of genomic sequence and, specifically, the sequences for introns and exon-intron junctions, primers were designed against sequences from the ends of each exon. We assumed a distribution of ORF sequences between exons equivalent to the mouse sequence (see Table I and Fig. 6). PCR products were purified using a QIAEX-gel extraction kit and then directly sequenced using GeneAmp PCR System 9600. The gene sequences from within each exon were compared with those of rat Star cDNA.Table IPrimers used for the generation of PCR productsPCR productsPrimersForwardReversePCR 15′-AGC TTC TAC AGA CAT ATG CG-3′ (1F)5′-GCT CTC CGG TTC AGC TCT TG-3′ (1R)PCR 25′-AGC TTC TAC AGA CAT ATG CG-3′ (1F)5′-TCC TGG TCA CTG TAG AGT GT-3′ (2R)PCR 35′-CAG GAA GGC TGG AAG AAG GA-3′ (2F)5′-AAC ACC TTG CCC ACA CCT GG-3′ (3R)PCR 45′-CAG GAA GGC TGG AAG AAG GA-3′ (2F)5′-TTT CCA ATC TTC TTC AGG AC-3′ (4R)PCR 55′-CTG TGT GCT GGC AGG CAT GG-3′ (3F)5′-ACC ATG CAG GTG GGA CCG TG-3′ (5R)PCR 65′-CTG TGT GCT GGC AGG CAT GG-3′ (3F)5′-ATG GTC TTT GGC AGC CAC CC-3′ (6R)PCR 75′-TGA GGC AAT CAT TCC ATC CT-3′ (4F)5′-ATG GTC TTT GGC AGC CAC CC-3′ (6R)PCR 85′-CGA GGC CTG TAC ATG CTG AC-3′ (5F)5′-GTG TTC CCG TTA CAG CCA CA-3′ (7R) Open table in a new tab Restriction digestion of the five genomic clones was carried out with a set of restriction enzymes, followed by Southern blotting using a probe synthesized against a partial 5′-flanking region (∼150 bp) of the rat Star gene. A 4.4-kb DNA fragment produced bySacI digestion of one genomic clone was recognized by the 5′-flanking region cDNA probe, was subcloned, and was partially sequenced. A dye terminator cycle sequencing reaction was carried out for both strands of the construct using the GeneAmp PCR System 9600. Rats were injected with long acting ACTH 24 h prior to sacrifice. Total RNA (20 μg) was then prepared from the adrenal glands with TRIzol reagent. A reverse primer [5′-CCC AGC ACA CAG CTT GAA TGT AGC TAG TAA-3] was synthesized against bases 4–33 downstream to the translation initiation codon, ATG. The extended product was extracted with phenol/chloroform and precipitated with 100% ethanol. The pellet was washed with 70% ethanol and briefly dried for a few minutes and then dissolved in 5 ml of TE buffer (pH 7.4) with 5 ml of formamide loading buffer and kept at −20 °C until use. The sample was heated with boiling water for 3 min just before loading and analyzed on a 6% sequencing gel together with the samples for dideoxynucleotide sequencing using32P-end-labeled primer. 5′-End labeling of the primer with [γ-32P]ATP was conducted with T4 polynucleotide kinase, and the labeled primer (5 × 104 cpm) was mixed with RNA. Hybridization was performed at 30 °C overnight, and primer extension reaction was performed at 42 °C for 90 min with 100 units of reverse transcriptase. The same labeled primer (10 ng) was used for the dideoxynucleotide sequencing reaction using a construct containing rat Star 5′-flanking region as a template. Primer extension product was then loaded on a denaturing sequencing gel in parallel to the DNA sequencing ladder. Northern hybridization was conducted with either the 32P-labeled 0.9-kbStar ORF cDNA after agarose-formaldehyde gel (1%) electrophoresis. Quantitation of Northern hybridization was performed by laser densitometry using the NIH image program. Rat Star mRNA species containing poly(A)+tails were identified by using a nested RT-PCR according to the method reported previously (33Toriello H.V. Glover T.W. Takahara K. Byers P.H. Miller D.E. Higgins J.V. Greenspan D.S. Nat. Genet. 1996; 13: 361-365Crossref PubMed Scopus (109) Google Scholar). Briefly, cDNA was synthesized using Superscript reverse transcriptase (Life Technologies, Inc.), 2 μg of total RNA isolated from untreated rat adrenal gland, and an adapter primer, 5′-TAC GCC AAG CTC GAA ATT AAC CCT CAC TAA AGG G(T)16-3′. Long distance PCR was then performed usingTaq polymerase under previously published conditions (34Barnes W.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2216-2220Crossref PubMed Scopus (980) Google Scholar,35Cheng S. Fockler C. Barnes W.M. Higuchi R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5695-5699Crossref PubMed Scopus (574) Google Scholar). We used a set of 4 primers as follows: the Star exon 1-specific primer 5′-CTG CAG CAC TAC CAC AGA AAG CAT-3′ and adapter-specific primer 5′-TAC GCC AAG CTC GAA ATT AAC CCT C-3′, followed by a second nested PCR with Star exon 1-specific primer 5′-CTA CAT TCA AGC TGT GTG CTG GGA-3′ and adapter-specific primer 5′-TCG AAA TTA ACC CTC ACT AAA GGG-3′ in the presence of RNasin. After incubation at 37 °C for 1 h, reverse transcriptase was inactivated by boiling for 5 min followed by vigorous shaking to inactivate RNasin. Single strand cDNA was then obtained by digestion of template mRNA with RNase A. The product was dissolved in 50 ml of TE buffer (pH 7.4). The first PCR reaction was conducted using one-tenth of the product as a template. PCR was performed according to the automatic program designed as follows: denaturation, 94 °C for 3.5 min; cycle denaturation, 98 °C for 7 s; annealing, 65 °C for 1 min; autosegment extension, 72 °C, 4 min + 10 s/cycle; 30 cycles. At the end of the last cycle, the tubes were held at 70 °C for 10 min for elongation reaction and then cooled down to 4 °C. The second nested PCR reaction was performed with a 100 × diluted sample of the first PCR product as a template, using the conditions and primers described above. We have characterized the expression of immunodetectable STAR protein in relation to the time course of steroid synthesis following ACTH stimulation of rats in vivo. Fig. 1 shows the time course of the increase in corticosterone synthesis in hypophysectomized (HPX) and intact rats. Similar to previous studies, we show that removal of ACTH through HPX lowers circulating adrenal steroid levels by over 10-fold. Administration of ACTH to the HPX rats produced a biphasic steroidogenic response, including an acute phase which is complete in 20 min and a chronic phase which occurs between 1 and 24 h post-treatment. In the acute phase there is a short lag (3–5 min) followed by a rapid increase to a steady state that is reached in 10 min and is maintained at least until 1 h after stimulation. The chronic phase corresponds to a further 3.5-fold increase in serum glucocorticoids, which occurs between 1 and 24 h after stimulation. In intact rats the acute phase kinetics are retained but with much higher amplitude of the response. The change to a new steady state at 20 min corresponds to a 3-fold increase in the rate of secretion of glucocorticoids relative to the basal secretion rate. We analyzed the effect of ACTH treatment on STAR protein by carrying out immunoblotting from two-dimensional electrophoresis gels of total adrenal protein. This approach has been used previously to resolve modified forms of rat STAR, notably phosphorylated forms, which locate at a more acidic pH on these gels (52Jefcoate C.R. McNamara B.C. Artemenko I. Yamazaki T. J. Steroid Biochem. Mol. Biol. 1992; 43: 751-767Crossref PubMed Scopus (152) Google Scholar). Prior to ACTH treatment of HPX and intact rats, we detected two proteins whose pI (pI 6.5 and 6.6) and size (30 kDa) are fully compatible with previous determinations for rat STAR in unstimulated cultured adrenal cells (Fig. 2) (13Pon L.A. Orme-Johnson N.R. J. Biol. Chem. 1986; 261: 6594-6599Abstract Full Text PDF PubMed Google Scholar, 52Jefcoate C.R. McNamara B.C. Artemenko I. Yamazaki T. J. Steroid Biochem. Mol. Biol. 1992; 43: 751-767Crossref PubMed Scopus (152) Google Scholar). In the same animals that were used for these steroid measurements, the STAR protein levels were 2–4 times lower in HPX rats but showed absolutely no response to ACTH within the 20-min period of the acute phase response (Fig. 2 B). Increases in total STAR were only apparent 60 min after stimulation (data not shown) and rose 3–4-fold by 24 h corresponding to the period of chronic changes in steroidogenesis. During this period, initial pair o" @default.
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