Mitochondrial Processing of Newly Synthesized Steroidogenic Acute Regulatory Protein (StAR), but Not Total StAR, Mediates Cholesterol Transfer to Cytochrome P450 Side Chain Cleavage Enzyme in Adrenal Cells
2001; Elsevier BV; Volume: 276; Issue: 49 Linguagem: Inglês
10.1074/jbc.m107815200
ISSN1083-351X
AutoresIrina Artemenko, Dong Zhao, Dale B. Hales, Karen H. Hales, Colin R. Jefcoate,
Tópico(s)Hormonal Regulation and Hypertension
ResumoThe metabolism of cholesterol by cytochrome P450 side chain cleavage enzyme is hormonally regulated in steroidogenic tissues via intramitochondrial cholesterol transport. The mediating steroidogenic acute regulatory protein (StAR) is synthesized as a 37-kDa (p37) precursor that is phosphorylated by protein kinase A and cleaved within the mitochondria to generate 30-kDa forms (p30, pp30). The effectiveness of modified recombinant StAR forms in COS-1 cells without mitochondrial import has led to a prevailing view that cholesterol transport is mediated by p37 StAR via activity on the outer mitochondrial membrane. The present study of the activation of cholesterol metabolism by bromo-cAMP in adrenal cells in relation to35S-StAR turnover indicates that targeting of pp30 to the inner membrane provides the dominant cholesterol transport mechanism. We show that 1) only newly synthesized StAR is functional, 2) phosphorylation and processing of p37 to pp30 occurs rapidly and stoichiometrically, 3) both steps are necessary for optimum transport, and 4) newly synthesized pp30 exhibits very high activity (400 molecules of cholesterol/StAR/min). Segregation of cAMP activation and synthesis of StAR from cholesterol metabolism showed that very low levels of newly synthesized StAR (1 fmol/min/106 cells) sustained activated cholesterol metabolism (0.4 pmol/min/106 cells,t 1/2 = 70 min) long after complete removal of p37 (t 1/2 = 5 min). This activity was highly sensitive to inhibition of processing by CCCP only until sufficient pp30 was formed. Maximum activation preceded bromo-cAMP-induced StAR expression, indicating other limiting steps in cholesterol metabolism. The metabolism of cholesterol by cytochrome P450 side chain cleavage enzyme is hormonally regulated in steroidogenic tissues via intramitochondrial cholesterol transport. The mediating steroidogenic acute regulatory protein (StAR) is synthesized as a 37-kDa (p37) precursor that is phosphorylated by protein kinase A and cleaved within the mitochondria to generate 30-kDa forms (p30, pp30). The effectiveness of modified recombinant StAR forms in COS-1 cells without mitochondrial import has led to a prevailing view that cholesterol transport is mediated by p37 StAR via activity on the outer mitochondrial membrane. The present study of the activation of cholesterol metabolism by bromo-cAMP in adrenal cells in relation to35S-StAR turnover indicates that targeting of pp30 to the inner membrane provides the dominant cholesterol transport mechanism. We show that 1) only newly synthesized StAR is functional, 2) phosphorylation and processing of p37 to pp30 occurs rapidly and stoichiometrically, 3) both steps are necessary for optimum transport, and 4) newly synthesized pp30 exhibits very high activity (400 molecules of cholesterol/StAR/min). Segregation of cAMP activation and synthesis of StAR from cholesterol metabolism showed that very low levels of newly synthesized StAR (1 fmol/min/106 cells) sustained activated cholesterol metabolism (0.4 pmol/min/106 cells,t 1/2 = 70 min) long after complete removal of p37 (t 1/2 = 5 min). This activity was highly sensitive to inhibition of processing by CCCP only until sufficient pp30 was formed. Maximum activation preceded bromo-cAMP-induced StAR expression, indicating other limiting steps in cholesterol metabolism. cytochrome P450 side chain cleavage enzyme adrenocorticotropic hormone cycloheximide carbonyl cyanidem-chlorophenylhydrazone glutathioneS-transferase 70-kDa heat shock protein 90-kDa heat shock protein high pressure liquid chromatography 20α-hydroxycholesterol 1,10-phenanthroline polyacrilamide gel electrophoresis radioimmunoassay steroidogenic acute regulatory protein bromo-cyclic AMP The conversion of cholesterol to pregnenolone, the first step in steroid synthesis, is catalyzed by cytochrome P450 side chain cleavage enzyme (P450scc),1 which is localized on the matrix side of the inner mitochondrial membrane (1Miller W.L. Endocr. Rev. 1988; 9: 295-318Crossref PubMed Scopus (1223) Google Scholar,2Churchill P.F. Kimura T. J. Biol. Chem. 1979; 254: 10443-10448Abstract Full Text PDF PubMed Google Scholar). The transfer of cholesterol to P450scc, a limiting step in this conversion (3Simpson E.R. Jefcoate C.R. Brownie A.C. Boyd G.S. Eur. J. Biochem. 1972; 28: 442-450Crossref PubMed Scopus (123) Google Scholar, 4Privalle C.T. Crivello J.F. Jefcoate C.R. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 702-706Crossref PubMed Scopus (334) Google Scholar), requires hormonal activation of cholesterol mobilization to the mitochondria. This mobilization is dependent on the uptake of lipoproteins, lysosomal-cytosolic transfer, and hydrolysis of cholesterol esters as well as on two subsequent transfer steps (5DiBartolomeis M.J. Jefcoate C.R. J. Biol. Chem. 1984; 259: 10159-10167Abstract Full Text PDF PubMed Google Scholar). The first step involves transfer of cholesterol to the mitochondria and is dependent on an intact cytoskeleton but not on protein synthesis (6Hall P.F. Can. J. Biochem. Cell Biol. 1984; 62: 653-665Crossref PubMed Scopus (44) Google Scholar). The second step involves transfer within the mitochondria from outer to inner membrane. This trophic hormone-dependent transport of cholesterol from mitochondrial outer membrane to inner membrane (4Privalle C.T. Crivello J.F. Jefcoate C.R. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 702-706Crossref PubMed Scopus (334) Google Scholar, 7Brownie A.C. Alfano J. Jefcoate C.R. Orme-Johnson W. Beinert H. Simpson E.R. Ann. N. Y. Acad. Sci. 1973; 212: 344-360Crossref PubMed Scopus (68) Google Scholar,8Jefcoate C.R. Simpson E.R. Boyd G.S. Eur. J. Biochem. 1974; 42: 539-551Crossref PubMed Scopus (105) Google Scholar) is blocked in vivo within 10 min by protein synthesis inhibitors, such as cycloheximide (CHX) (9Garren L.D. Gill G.N. Masui H. Walton G.M. Rec. Prog. Horm. Res. 1971; 27: 433-478PubMed Google Scholar, 10Crivello J.F. Jefcoate C.R. Biochim. Biophys. Acta. 1978; 542: 315-329Crossref PubMed Scopus (95) Google Scholar). A series of cAMP-stimulated and CHX-sensitive phosphoproteins (30–37 kDa), which localize to the mitochondria, have been identified in cultured adrenal and testis cells (11Krueger R.J. Orme-Johnson N.R. J. Biol. Chem. 1983; 258: 10159-10167Abstract Full Text PDF PubMed Google Scholar, 12Alberta J.A. Andersson L.A. Dawson J.H. J. Biol. Chem. 1989; 264: 20467-20473Abstract Full Text PDF PubMed Google Scholar, 13Epstein L.F. Orme-Johnson N.R. J. Biol. Chem. 1991; 266: 19739-19745Abstract 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, 15Pon L.A. Hartigan J.A. Orme-Johnson N.R. J. Biol. Chem. 1986; 261: 13309-13316Abstract Full Text PDF PubMed Google Scholar, 16Stocco D.M. Kilgore M.W. Biochem. J. 1988; 249: 95-103Crossref PubMed Scopus (99) Google Scholar, 17Stocco D.M. Chen W. Endocrinology. 1991; 128: 1918-1926Crossref PubMed Scopus (105) Google Scholar). The gene that encodes these proteins has been cloned, and the active form has been named the steroidogenic acute regulatory protein (StAR) (18Clark B.J. Wells J. King S.R. Stocco D.M. J. Biol. Chem. 1994; 269: 28314-28322Abstract Full Text PDF PubMed Google Scholar). The expression of recombinant StAR in modified COS-1 cells enhances cholesterol metabolism (19Sugawara T. Holt J.A. Driscoll D. Strauss Jr., J.F. Lin D. Miller W.L. Patterson D. Clancy K.P. Hart I.M. Clark B.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4778-4782Crossref PubMed Scopus (351) Google Scholar), even when StAR is mutated to prevent the mitochondrial uptake of the protein (20Arakane F. Sugawara T. Nishino H. Liu Z. Holt J.A. Pain D. Stocco D.M. Miller W.L. Strauss Jr., J.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13731-13736Crossref PubMed Scopus (256) Google Scholar). This has raised essential questions concerning the role of N-terminal processing in the activity of StAR. Deletions or mutations of StAR (all C-terminal) have been found in humans born with the steroid deficiency disease, lipoid adrenal congenital hyperplasia (21Lin D. Sugawara T. Strauss Jr., J.F. Clark B.J. Stocco D.M. Saenger P. Rogol A. Miller W.L. Science. 1995; 267: 1828-1831Crossref PubMed Scopus (889) Google Scholar, 22Bose H.S. Sugawara T. Strauss J.F. Miller W.L. N. Engl. J. Med. 1996; 335: 1870-1878Crossref PubMed Scopus (558) Google Scholar, 23Saenger P. Klonari Z. Black S.M. Compagnone N. Mellon S.H. Fleischer A. Abrams C.A. Shackelton C.H. Miller W.L. J. Clin. Endocrinol. Metab. 1995; 80: 200-205Crossref PubMed Google Scholar). Disruption of the StAR gene in mice produces a similar phenotype (24Caron 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 (398) Google Scholar). The effect of these deletions establishes that StAR is necessary for 80–90% of adrenal cholesterol metabolism. StAR is selectively expressed in all steroidogenic tissues, except the placenta (25Clark 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 latter may express another StAR family member, such as has been cloned from mammary cells (26Watari H. Arakane F. Moog-Lutz C. Kallen C.B. Tomasetto C. Gerton G.L. Rio M.C. Baker M.E. Strauss Jr., J.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8462-8467Crossref PubMed Scopus (206) Google Scholar). Before the cloning of StAR, co-translational phosphorylation of the precursor protein (p37), followed by rapid intramitochondrial turnover was suggested as a reason for the rapid effect of protein synthesis inhibitors (11Krueger R.J. Orme-Johnson N.R. J. Biol. Chem. 1983; 258: 10159-10167Abstract Full Text PDF PubMed Google Scholar, 13Epstein L.F. Orme-Johnson N.R. J. Biol. Chem. 1991; 266: 19739-19745Abstract Full Text PDF PubMed Google Scholar). Transfer of the positively charged leader sequence through the import pore requires a mitochondrial membrane potential and is linked to a matrix proteolytic cleavage of the leader sequence (27Pfanner N. Craig E.A. Honlinger A. Annu. Rev. Cell Dev. Biol. 1997; 13: 25-51Crossref PubMed Scopus (147) Google Scholar, 28Brunner M. Neupert W. Methods Enzymol. 1995; 248: 717-728Crossref PubMed Scopus (15) Google Scholar, 29Isaya G. Kalousek F. Methods Enzymol. 1995; 248: 556-567Crossref PubMed Scopus (25) Google Scholar). This is consistent with the predominance of a p37 form of StAR (13Epstein L.F. Orme-Johnson N.R. J. Biol. Chem. 1991; 266: 19739-19745Abstract Full Text PDF PubMed Google Scholar) or retention of intermediate forms, which occur when CCCP, a powerful respiratory uncoupler, prevents entry into the matrix (30Stocco D.M. Sodeman T.C. J. Biol. Chem. 1991; 266: 19731-19738Abstract Full Text PDF PubMed Google Scholar). Inhibition of cholesterol metabolism by o-phenanthroline (o-phen), an inhibitor of mitochondrial proteolysis, and by CCCP (13Epstein L.F. Orme-Johnson N.R. J. Biol. Chem. 1991; 266: 19739-19745Abstract Full Text PDF PubMed Google Scholar, 30Stocco D.M. Sodeman T.C. J. Biol. Chem. 1991; 266: 19731-19738Abstract Full Text PDF PubMed Google Scholar) suggests that this cleavage of StAR is necessary for cholesterol transfer. In isolated mitochondria, cholesterol metabolism, mediated by recombinant StAR, is blocked by CCCP and by inhibition of ATP turnover, each consistent with a need for StAR processing (31King S.R. Liu Z. Soh J. Eimerl S. Orly J. Stocco D.M. J. Steroid Biochem. Mol. Biol. 1999; 69: 143-154Crossref PubMed Scopus (55) Google Scholar). However, the activity of StAR without the targeting sequence in COS-1 cells has led to a broad acceptance that StAR functions on the outer membrane and is than removed from the active site by intramitochondrial transfer (20Arakane F. Sugawara T. Nishino H. Liu Z. Holt J.A. Pain D. Stocco D.M. Miller W.L. Strauss Jr., J.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13731-13736Crossref PubMed Scopus (256) Google Scholar, 30Stocco D.M. Sodeman T.C. J. Biol. Chem. 1991; 266: 19731-19738Abstract Full Text PDF PubMed Google Scholar). In a previous study (32Ariyoshi N. Kim Y-C. Artemenko I. Bhattacharyya K. Jefcoate C.R. J. Biol. Chem. 1998; 273: 7610-7619Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), we showed that ACTH maximally stimulated steroid synthesis in rats in vivo within 10 min, without any measurable change in multiple forms of total StAR. In this paper, we address the question of whether mitochondrial processing is, indeed, essential for activated adrenal cholesterol metabolism, despite the evident possibility of extramitochondrial StAR activity. Here, we resolve why changes in cholesterol metabolism appear to occur without measurable changes in the multiple forms of immunodetectable StAR. We show that these paradoxes are explained by the dependence of activity exclusively on newly synthesized StAR and by the need for extraordinarily little intramitochondrial StAR for maximum activity. The basis for differences between these adrenal studies and experiments with recombinant StAR are discussed. 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. Rats were housed in a stress-minimizing environment. Rats were given multiple injections of ACTH (1–24 fragment) (Sigma) every 30 min during a period of 3 h. A group of rats was injected with CHX (Sigma) (20 mg/rat) 20 min prior to sacrifice, in addition to ACTH treatment. Animals were sacrificed by decapitation, blood was collected to measure steroids, and adrenals were surgically removed, defatted, and rapidly homogenized in TRIzol reagent (Life Technologies, Inc.; 50–100 mg of tissue/1 ml of reagent) for protein and RNA analysis. Antibody was elicited to the full-length mature form of StAR. The StAR cDNA (18Clark B.J. Wells J. King S.R. Stocco D.M. J. Biol. Chem. 1994; 269: 28314-28322Abstract Full Text PDF PubMed Google Scholar) was digested with SmaI and ligated to BamHI linkers. The DNA was digested with BamHI, and the 1273-base pair fragment was cloned into the dephosphorylated BamHI site of GEX2T (Amersham Pharmacia Biotech). The linker sequences (5′-CGCGGATCCGCG-3′) were added to maintain the reading frame and add an alanine residue between the thrombin cleavage site of the vector and amino acid 46 of StAR. This removes all but two amino acid residues of the predicted signal peptide, resulting in a fusion of the mature 30-kDa form of StAR with GST. The protein was expressed in bacteria, purified by glutathione affinity chromatography, and injected into rabbits. A specific, high titer antibody was elicited that was suitable for immunoprecipitation and Western blot analysis. Rat adrenal cells were isolated from inner cortex zone of adrenals (fasciculata cells) that were obtained fresh from Harlan Bioproducts (Madison, WI), subjected to collagenase A (Roche Molecular Biochemicals) digestion, and cultured for 24–48 h as described previously (33Brake P.B. Jefcoate C.R. Endocrinology. 1995; 136: 5034-5041Crossref PubMed Scopus (42) Google Scholar). Cell treatment and labeling were performed in Krebs buffer containing 0.2% glucose and 0.1% bovine serum albumin for 30 min at 37 °C. Cells (2 × 106) were labeled with 1 mCi of [35S]methionine (EXPRE35S35S labeling kit; PerkinElmer Life Sciences). Cells were washed three times with Krebs buffer, scraped from the plates, and pelleted. Cell pellets were used for 35S-protein gel electrophoresis analysis. Y-1 cells (mouse tumor adrenal cells) were obtained from American Type Culture Collection (Manassas, VA) and maintained in Ham's F-10 (Sigma) supplemented with 15% horse serum (Atlanta Biological, Norcross, GA), 2.5% fetal bovine serum (Hi-Clone, Logan, UT), penicillin (50 units/ml; Sigma), and streptomycin (50 mg/ml; Sigma). All cell treatments were performed in six-well plates with 1–2 × 106 cells/well in serum-free medium. In some experiments, cells were prelabeled for 5 min with [35S]methionine (0.4 mCi/ml) in methionine-free Dulbecco's modified Eagle's media. Cells were typically stimulated with 8-Br-cAMP, as designated. Cell medium was collected after each treatment to measure pregnenolone. 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 μl of the methanol and subjected to HPLC 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. Cell medium was collected after each treatment and assayed for pregnenolone by RIA. 10 μmtrilostane (a gift from Dr. R. J. Harmon (University of Kentucky)) and 20 μm SU-10603 (CIBA-GEIGY Corp., Summit, NJ) were added before each treatment to block further pregnenolone metabolism. Since pregnenolone is rapidly removed in the absence of these inhibitors, pregnenolone formation reflects only metabolism after the addition of these inhibitors. Pregnenolone was extracted from cell media with hexane. The organic phase was then evaporated under nitrogen with heating at 37 °C. The residue was reconstituted in RIA buffer, and RIA was performed as described before (34Abrahams G.E. Buster J.E. Kyle F.W. Corrales P.C. Teller R.C. J. Clin. Endocrinol. Metab. 1973; 37: 40-45Crossref PubMed Scopus (73) Google Scholar) using anti-pregnenolone from ICN Biomedicals, (Costa Mesa, CA). Cells were plated in six-well plates at a density of 0.8–1 × 106 cells/well. In phase 1, cells were incubated with 1 mm 8-Br-cAMP (Roche Molecular Biochemicals) with or without one of the following: 2 mm o-phen, 20 μm CCCP, and 0.2 mm CHX (all of the inhibitors were obtained from Sigma). Each phase 1 incubation was carried out at 37 °C for 5–15 min as specified. Medium was collected for pregnenolone measurement. Cells then were washed with 10 °C medium (without inhibitors or Br-cAMP), and new medium was added also without Br-cAMP but with the inclusion of CHX. This incubation was carried out for 15 min at 37 °C (phase 2). Again, medium was collected to determine the level of generated pregnenolone. The pelleted cells were lysed in osmotic lysis buffer (10 mm Tris, pH 7.4, 0.3% SDS, 50 μg/ml RNase, and 100 μg/ml DNase in 5 mmMgCl2, 1% protease inhibitor mixture (Sigma)). The samples were then boiled with SDS boiling buffer (5% SDS, 10% glycerol, and 60 mm Tris, pH 6.8). Aliquots of the samples were removed for protein determination and trichloroacetic acid precipitation for protein-bound 35S counts. The rest of the material was lyophilized and dissolved in SDS-boiling buffer with 5% β-mercaptoethanol to a protein concentration of 5 mg/ml. Two-dimensional electrophoresis was performed using isoelectric focusing followed by SDS-PAGE (10% acrylamide) as described previously (32Ariyoshi N. Kim Y-C. Artemenko I. Bhattacharyya K. Jefcoate C.R. J. Biol. Chem. 1998; 273: 7610-7619Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Protein (200 μg) was loaded on each tube gel for isoelectric focusing. For experiments that required immunoblotting after electrophoresis, the proteins 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 (35Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (46343) 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.) and using polyclonal α-StAR antibody, 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 Biotech). To perform fluorography following second dimension separation, the gels were fixed overnight in 50% methanol, 10% acetic acid; rehydrated in 10% acetic acid; dried onto filter paper; and exposed to x-ray film.14C molecular weight markers (Diversified Biotechnology, Newton Center, MA) were loaded on the basic edge of each gel. Y-1 cells were labeled with [35S]methionine (0.4 mCi/ml) in methionine-free Dulbecco's modified Eagle's medium with or without 1 mmBr-cAMP, 20 μm CCCP, and 2 mm o-phen (phase 1). After washing the cells, the radioactive media was replaced with serum-free culture media containing 0.2 mm CHX. Incubation was continued for 15 or 35 min (phase 2). Then cells were rinsed with phosphate-buffered saline and scraped into 0.6 ml of ice-cold immunoprecipitation buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm Na3VO4, 1% Nonidet P-40, 0.25% deoxycholate, 0.05% SDS, 40 mm NaF, 10 mm NaMo, 1 mmphenylmethylsulfonyl fluoride, 1% protease inhibitor mixture (Sigma)). Cell lysates were repeatedly passed through a 23-gauge needle and centrifuged at 12,000 × g for 5 min in an Eppendorf centrifuge. Equal aliquots of protein from each supernatant were first incubated with 20 μl of protein A-agarose plus (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) to eliminate nonspecifically bound proteins for 30 min at 4 °C. After centrifugation, supernatants were incubated with 1 μl of StAR antibody overnight at 4 °C and then for 3 h with 100 μl of agarose A. Immunocomplexes were washed several times with immunoprecipitation buffer and collected by centrifugation. The final pellets were resuspended in sample buffer, and equal amounts of counts were loaded on SDS-PAGE 10% gels. Gels were fixed for 30 min in 50% methanol, 10% acetic acid fixation solution, soaked for 15 min in Amplify (Amersham Pharmacia Biotech), dried for 90 min at 65 °C, and exposed to Kodak BioMax film (Eastman Kodak Co.). Quantitative analysis of StAR expression was completed by soft laser-scanning densitometry employing a Zeineh model SL-504-XL soft laser-scanning densitometer (Fullerton, CA). Protein was measured using the BCA method (Pierce) according to the manufacturer's instructions. In previous work, we have detected multiple forms of StAR in rat adrenals in vivo (32Ariyoshi N. Kim Y-C. Artemenko I. Bhattacharyya K. Jefcoate C.R. J. Biol. Chem. 1998; 273: 7610-7619Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). CHX (protein synthesis inhibitor) rapidly inhibits steroidogenesis in vivo by limiting access of cholesterol to P450scc (4Privalle C.T. Crivello J.F. Jefcoate C.R. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 702-706Crossref PubMed Scopus (334) Google Scholar, 36Jefcoate C.R. DiBartolomeis M. Williams C.A. McNamara B.C. J. Steroid Biochem. 1987; 27: 721-729Crossref PubMed Scopus (101) Google Scholar). Here, we have tested whether changes in StAR correlate with this inhibition. Fig.1 shows that a 3-h stimulation of female rats by ACTH produces a 4-fold increase in the level of blood corticosterone. Injection of the CHX in the last 20 min of the ACTH treatment lowered blood corticosterone below unstimulated levels. This indicates a rapid and full block of corticosterone secretion within this period. Based on our recent work, a 20-min ACTH stimulation, which is sufficient for the peak of corticosterone levels, did not elevate immunodetectable StAR isoforms (32Ariyoshi N. Kim Y-C. Artemenko I. Bhattacharyya K. Jefcoate C.R. J. Biol. Chem. 1998; 273: 7610-7619Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Analysis of this 3-h ACTH stimulation with a StAR anti-peptide antibody (Fig. 1 B) revealed four StAR forms. Two forms that were observable in unstimulated rats were relatively insensitive to this ACTH treatment. Two additional forms that exhibit more acidic pI values (<6.5) were only evident after this hormonal stimulation. These forms with the more acidic pI values were exactly compatible with previously described phosphorylated forms (13Epstein L.F. Orme-Johnson N.R. J. Biol. Chem. 1991; 266: 19739-19745Abstract Full Text PDF PubMed Google Scholar). This complete inhibition of corticosterone formation by CHX takes place without an affect on any of StAR forms. The lack of correlation of StAR with this activity change remained even when analyzed with a much more sensitive antibody raised against recombinant StAR, which detected at least four additional forms (Fig. 1 C). We conclude that none of these eight detectable StAR forms is either particularly labile or responsible for the rapid loss of steroidogenesis produced by CHX. Like the results with acute ACTH stimulation in vivo, this suggests that total immunodetectable StAR is not a good indicator of StAR activity. Previous work has shown that processing of p37 StAR to mature p30 forms is blocked by o-phen, an inhibitor of mitochondrial proteolysis (13Epstein L.F. Orme-Johnson N.R. J. Biol. Chem. 1991; 266: 19739-19745Abstract Full Text PDF PubMed Google Scholar, 30Stocco D.M. Sodeman T.C. J. Biol. Chem. 1991; 266: 19731-19738Abstract Full Text PDF PubMed Google Scholar). In view of the controversy concerning the role of StAR processing in cholesterol metabolism, we measured the changes in immunodetectable StAR forms in relation to effects of o-phen on cholesterol metabolism. Primary cells, which were isolated from rat adrenals and cultured up to 48 h, were stimulated with Br-cAMP in the presence of o-phen. Pregnenolone production was stimulated 4-fold (from 2 to 8.0 ng/15 min/106 cells), and this increase was, indeed, completely blocked by o-phen (2.0 ng/15 min/106 cells). Two-dimensional immunoblots with α-StAR antibody (Fig. 2 A) showed approximately equal levels of a set of four major spots, in pairs corresponding closely to the pI values previously seen for unphosphorylated forms (1 and 2) and more acidic phosphorylated StAR forms (3 and 4). The separation of major and additional minor spots was highly reproducible but failed to detect changes in any of StAR isoforms in response to either Br-cAMP or o-phen. (Fig. 2 A). This experiment further establishes that expression of total levels of individual forms of StAR is a poor indicator of activity. As an alternative mechanism, we have tested the possibility that only newly synthesized StAR determines cholesterol transfer to P450scc (13Epstein L.F. Orme-Johnson N.R. J. Biol. Chem. 1991; 266: 19739-19745Abstract Full Text PDF PubMed Google Scholar). This is necessarily blocked by CHX and also should be sensitive to the processing inhibitor,o-phen. In parallel with the above immunodetection analysis, a second set of primary cells were stimulated with 8-Br-cAMP for 30 min and labeled concurrently with [35S]methionine. This brief labeling time preferentially detects incorporation of 35S into more the labile proteins. Fig. 2 B shows that Br-cAMP decreased 35S-labeling of two more basic isoforms (1 and 2) in parallel with appearance of two more acidic 30-kDa proteins (3 and 4). These changes and the relative location of acidic spots correspond closely to the previously reported generation of phosphorylated forms (13Epstein L.F. Orme-Johnson N.R. J. Biol. Chem. 1991; 266: 19739-19745Abstract Full Text PDF PubMed Google Scholar, 37Stocco D.M. Clark B.J. J. Steroid Biochem. Mol. Biol. 1993; 46: 337-347Crossref PubMed Scopus (55) Google Scholar).o-Phenanthroline treatment blocked the acute formation of35S-labeled p30 and p32 proteins. An additional p37 protein was detectable in the expected location for the StAR precursor but only in the presence of o-phen (Fig. 2 B,white arrowhead). The failure of 35S labeling in control cells to detect acidic StAR forms that are strongly represented in the equivalent immunoblots could result from a slow accumulation of these forms under basal culture conditions. A 32-kDa protein (Fig. 2 B, 5), which was largely insensitive to Br-cAMP treatment, was also removed byo-phen. No equivalent form was seen in the immunoblots, thus raising doubts about whether this was a StAR isoform. This correlation between disappearance of 35S-StAR forms and loss of stimulated cholesterol metabolism after o-phen treatment and the insensitivity of immunodetectable forms suggests that only newly synthesized and processed StAR mediates cholesterol access to P450scc. The levels of preexisting StAR are evidently too high to allow detection of changes in small amounts of synthesized StAR by immunoblotting. This lack of change in StAR immunoblots also shows that various forms of immunodetectable StAR turn over very little in this 30-min incubation period. We have studied the turnover of the immunoprecipitated35S-StAR in mouse Y-1 adrenal cells, which, unlike primary cells, proliferate in culture. The Y-1 cells respond to Br-cAMP with a similar rapid stimulation of cholesterol metabolism although with diminished activity. o-Phenanthroline and CHX, however, similarly inhibit this stimulation. Cells were prelabeled for 5 min with [35S]methionine, prior to Br-cAMP treatment, followed by immunoprecipitation of StAR. After this 5-min labeling period, we were able to detect p37 at a much higher level than p30 (Fig. 3 B). After a further 7.5 min, the labeling of p37 in control cells doubled to a steady state level, which was maintained in the next 7.5-min period. By contrast, the p30 band in control cells, which was nearly absent after the 5 min, increased linearly in each subsequent 7.5-min period. This labeling sequence indicates that the p37 form is indeed a precursor of the p30 form. The labeling of the p30 form in each of t
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