Proc. Natl. Acad. Sci. USA Vol. 92, pp. 4778-4782, May 1995 Biochemistry Human steroidogenic acute regulatory protein: Functional activity in COS-1 cells, tissue-specffic expression, and mapping of the structural gene to 8pll.2 and a pseudogene to chromosome 13 (steroidogenesis/cAMP/pregnenolone/cholesterol side-chain cleavage) TERUO SUGAWARA*, JOHN A. HOLT*, DEBORAH DRISCOLL*, JEROME F. STRAUSS III*t, DONG LINt, WALTER L. MILLERt, DAVID PATTERSON§, KEVIN P. CLANCY§, IRIS M. HART§, BARBARA J. CLARK%, AND DOUGLAS M. STOCCOV *Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104; *Department of Pediatrics, University of California, San Francisco, CA 94143; §The Eleanor Roosevelt Institute, Denver, CO 80206; and 1Department of Cell Biology and Biochemistry, Texas Tech University Health Science Center, Lubbock, TX 79430 Communicated by Seymour Lieberman, The St. Luke's-Roosevelt Institute for Health Sciences, New York, NY, February 24, 1995 (received for review November 28, 1994) ABSTRACT Steroidogenic acute regulatory protein kindly provided by Andre Lacroix, Alain Belanger, and Yves (StAR) appears to mediate the rapid increase in pregnenolone Tremblay (University of Laval, Quebec). The library was synthesis stimulated by tropic hormones. cDNAs encoding screened with a partial-length mouse StAR cDNA (11). More StAR were isolated from a human adrenal cortex library. than 50 positive clones were detected in the screening of Human StAR, coexpressed in COS-1 cells with cytochrome 600,000 plaques. Two plaque-purified phage clones were se- P450scc and adrenodoxin, increased pregnenolone synthesis lected for sequence analysis. Each contained an insert of -1.6 >4-fold. A major StAR transcript of 1.6 kb and less abundant kb. Both inserts were subcloned into pSPORT transcripts of 4.4 and 7.5 kb were detected in ovary and testis. (GIBCO/BRL) Kidney had a lower amount of the 1.6-kb message. StAR and sequenced utilizing an automated DNA sequencer (Ap- mRNA was not detected in other tissues including placenta. plied Biosystems) employing Taq DyeDeoxy sequencing re- Treatment of granulosa cells with 8-bromo-adenosine 3',5'- agents. Ambiguities were corrected by manual sequencing. cyclic monophosphate for 24 hr increased StAR mRNA 3-fold Expression of StAR cDNA in COS-1 Cells. COS-1 cells were or more. The structural gene encoding StAR was mapped transfected with various expression vectors with Lipo- using somatic cell hybrid mapping panels to chromosome 8p. fectamine (GIBCO/BRL) using 10 ,lA per dish (12). The Fluorescence in situ hybridization placed the StAR locus in the vectors included pSPORTwithout cDNA insert, pSPORTwith region 8pll.2. A StAR pseudogene was mapped to chromo- the 1.6-kb StAR cDNA (pStAR), and expression vectors for some 13. We conclude that StAR expression is restricted to bovine P450scc (pCDP450scc) and adrenodoxin (pCDADX), tissues that carry out mitochondrial sterol oxidations subject kindly provided by Michael Waterman (Vanderbilt University, to acute regulation by cAMP and that StAR mRNA levels are Nashville, TN). Forty-eight hours after transfection, medium regulated by cAMP. was collected for radioimmunoassay of pregnenolone as de- scribed (12). In one experiment, the hydroxysterol, 20a-hy- Cholesterol side-chain cleavage in the adrenals and gonads and droxycholesterol, was added (5 ,g/ml) to the incubation la-hydroxylation of vitamin D in the kidney are acutely medium. This hydroxysterol is a more soluble pregnenolone stimulated by tropic hormones that act through the interme- and an diacy of cAMP (1, 2). It has been recognized that proteins are precursor intermediate in the cholesterol side-chain required for the translocation of sterol substrates to the inner cleavage reaction. Hydroxysterols bypass the regulated trans- mitochondrial membrane where the P450 enzymes that cata- location mechanism of cholesterol movement and, therefore, lyze these reactions reside (3-5). A 30-kDa phosphorylated generally provide an index of maximal cholesterol side-chain protein rapidly appears in mitochondria of steroidogenic cells cleavage activity (13). following tropic stimulation (6-10). This protein, named ste- Expression of StAR mRNA. Northern blots containing 2 ,g roidogenic acute regulatory protein (StAR), has recently been of poly(A)+ RNA from various human tissues were purchased purified from a murine Leydig cell tumor line (MA-10 cells) from Clontech and probed with the 1.6-kb StAR cDNA and a and its cDNA has been cloned (11). The cDNA encodes a 3-actin cDNA according to the supplier's protocol. protein with an N-terminal mitochondrial targeting sequence. Total RNA was also isolated from cultures of human Transient expression of the StAR cDNA in MA-10 cells granulosa cells obtained from women undergoing in vitro resulted in enhanced steroidogenesis. These findings are fertilization/embryo transfer or from purified human cytotro- strong evidence that StAR is the factor responsible for the phoblast cells. The human granulosa cells were cultured for 4 rapid movement of substrate from the outer to the inner mitochondrial membranes. days and then treated with 1.5 mM 8-bromoadenosine 3',5'- To explore the role of StAR in human tissues and its cyclic monophosphate (8-Br-cAMP) for 24 hr. The cytotro- potential involvement in disorders of steroidogenesis, we iso- phoblast cells were cultured for 24 hr in the absence or lated cDNAs encoding human StAR, examined the pattern of presence of 1.5 mM 8-Br-cAMP. Detailed protocols for the StAR expression, and mapped the StAR structural gene. preparation, culture, and isolation of total RNA from the granulosa cells and trophoblast cells have been described (14, MATERIALS AND METHODS Abbreviations: StAR, steroidogenic acute regulatory protein; 8-Br- Isolation of Human StAR cDNA Clones and DNA Sequence cAMP, 8-bromoadenosine 3',5'-cyclic monophosphate; FISH, fluo- Analysis. A human adrenal cortex cDNA library in Agt22A was rescence in situ hybridization; YAC, yeast artificial chromosome. tTo whom reprint requests should be addressed at: Department of Obstetrics and Gynecology, 778 Clinical Research Building, 415 The publication costs of this article were defrayed in part by page charge Curie Boulevard, Philadelphia, PA 19104. payment. This article must therefore be hereby marked "advertisement" in IThe sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession No. U17280). 4778 Downloaded by guest on September 23, 2021 Biochemistry: Sugawara et al Proc. NatL Acad Sci USA 92 (1995) 4779 15). Northern blots were probed with the StAR cDNA and a sponding to the 3' untranslated sequences. The sense primer cDNA encoding human 28S rRNA. was 5'-CCTACTGGAAGCCTGCAAGTCTAAG-3'. The an- Mapping ofthe StAR Structural Gene and Pseudogene. The tisense primer was 5'-TGGTTTTAGGTGGGTACATA- StAR gene and its pseudogene were mapped by hybridization AGGG-3'. StAR sequences in YAC DNA were amplified in a to Southern blots of DNA from somatic cell hybrids (16) and standard PCR. The reaction products were analyzed for the by polymerase chain reaction (PCR) analyses using primers presence of the expected 240-nt amplification product. specific for the structural gene or pseudogene. Genomic DNAs YAC FISH was performed as described (27, 28) with the from human x hamster and human x mouse somatic cell following modifications. The biotin-labeled probe was dena- hybrid lines were obtained from the NIGMS Human Genetic tured at 75°C for 5 min, preannealed with human Cot-1 DNA Mutant Cell Repository (1992/1993 Catalog of Cell Lines, for 1 hr at 37°C, and applied to human chromosome slide National Institutes of Health), and DNAs from human x preparations that had been previously denatured and dehydrated. hamster somatic cell hybrids were purchased from Bios (New In some experiments, a chromosome 8 centromere-specific probe Haven, CT). (D8Z2; Oncor) was added to the hybridization mixture. Detec- Regional mapping of the StAR structural gene was accom- tion was by avidin-fluorescein isothiocyanate, with one amplifi- plished with a chromosome 8 regional mapping panel consist- cation by the manufacturer's directions (Oncor). ing of hybrids 9HL10, ISHL27, and 20XP0435-2, generously Twelve metaphase spreads were G-banded by trypsin supplied by M. Wagner (17), 8q-, 21q+, and clone 17 (17-19), (GTG) and photographed prior to FISH, which was performed and VErec8-7al, which is a hybrid produced by the fusion of after destaining and dehydration of the slides. Metaphase the GlyB CHO-Ki mutant with cells from a patient suffering spreads were relocated and banding patterns were compared from recombinant 8 syndrome (20). with probe signal to determine chromosomal location. Frac- Southern Blotting. Ten to 12 ,g of genomic DNAs from 24 tional length measurements confirmed the assignment (27). somatic cell hybrids, human, hamster (RJK88), and mouse (GM Cl 1-D) was digested with HindIII and subjected to RESULTS AND DISCUSSION Southern blotting (16). PCR Analyses. The StAR structural gene and pseudogene Human StAR cDNA. The two human StAR cDNAs that were were mapped by PCR of characterized by DNA sequence analysis had identical analysis somatic cell hybrid DNA with 126-nt 5' untranslated sequence-specific primers. For the structural gene the forward regions. Both clones contained an 855-nt open frame a primer used was 5'-GTGAGCAAAGTCCAGGTGCG-3' and reading encoding 285-amino acid protein and a 623-nt 3' untranslated sequence that ended in a the reverse primer was 5'-TGTGGCCATGCCAGCCAGCA- poly(A)+ tail preceded 23 nt upstream by an AATAAA sequence. 3'. These sequences span a small intron and yield an product The deduced human StAR amino acid sequence is 87% of 300 nt. Primers derived from the DNA sequence of the identical to that of mouse StAR (11). It contains an N-terminal PCR-amplified pseudogene, the sequence of which will be sequence that is composed of basic and hydrophobic amino reported elsewhere, were used to determine the pseudogene acids that are characteristic of mitochondrial targeting se- location.