Steroidogenic acute regulatory protein (StAR) plays a critical role Xin regulating the rate-limiting step in steroid hormone synthesis, cholesterol side-chain cleavage. StAR gene expression is transcriptionally controlled in the gonads by gonadotropic hormones via a cAMP second message. We have begun to analyze factors responsible for the transcriptional activation of the StAR gene. The human StAR gene promoter has at least two cis elements that govern basal and cAMP-regulated gene expression. One of these elements (the distal element) is a consensus binding sequence for the orphan nuclear receptor transcription factor, steroidogenic factor 1 (Sf-1); the other (the proximal element) is a related motif. The human StAR promoter is not active in BeWo choriocarcinoma cells, but is functional and cAMP-responsive in murine Y1 adrenal cortical tumor cells. Cotransfection of a plasmid expressing SF-1 allows a StAR promoter construct to function in BeWo cells. Other orphan nuclear transcription factors do not support StAR promoter function in BeWo cell hosts. Deletion or mutation of the distal and proximal cis elements individually substantially reduces SF-1-supported StAR promoter activity. The distal site binds SF-1 with high affinity, whereas the proximal site binds SF-1 with lower affinities. These findings demonstrate a requirement for SF-1 for human StAR gene expression.

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroid hormone biosynthesis, presumably by facilitating the delivery of cholesterol to P450scc in the inner mitochondrial membranes. StAR is synthesized as a 37-kDa preprotein that is processed to a 30-kDa mature form by cleavage of an N-terminal mitochondrial import sequence. To identify structural features required for StAR biological activity, we mutated the human StAR cDNA, including the deletion of N- and C-terminal sequences, and examined the ability of the mutants to promote steroidogenesis and enter the mitochondria of transfected COS-1 cells. Deletion of up to 62 residues from the N terminus (N-62) did not significantly affect steroidogenesis-enhancing activity. The N-terminal deletion mutants were associated with mitochondria-enriched fractions, but import and processing were progressively impaired with increasing length of the deletion. Immunogold electron microscopy and in vitro import assays showed that the active N-62 mutant was not imported into the mitochondria. Removal of the 28 C-terminal amino acids (C-28) inactivated StAR. Deletion of the C-terminal 10 amino acids (C-10) reduced steroidogenic activity by 53%, while truncation of the last 4 amino acids had no effect. The C-28 mutant StAR was not efficiently imported into mitochondria or processed, whereas some of the C-10 mutant was processed, indicating that import had occurred. We conclude that in the COS-1 cell system used, StAR does not need to enter into mitochondria to stimulate steroidogenesis and that residues in the C terminus are essential for steroidogenesis-enhancing activity. These findings imply that StAR acts via C-terminal domains on the outside of the mitochondria.

Steroidogenic acute regulatory protein (StAR) is required for efficient adrenal cortical and gonadal but not trophoblast steroid hormone synthesis. StAR gene expression in gonadal cells is stimulated by tropic hormones acting through the intermediacy of cAMP. DNA sequence analysis of the human StAR gene promoter revealed two motifs resembling binding sites for steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor transcription factor family that controls expression of steroidogenic hydroxylases. The 5'-most sequence (distal site) is a consensus SF-1 binding site. The proximal site is a consensus estrogen receptor binding half-site. The StAR gene promoter is not active in BeWo choriocarcinoma cells, COS-1 cells, HeLa cells, or SK-OV-3 ovarian adenocarcinoma cells, all of which do not express significant levels of SF-1 mRNA. Introduction of SF-1 into these cells stimulated StAR promoter activity, particularly in response to cAMP. Two orphan nuclear transcription factors that bind to sequences similar to SF-1 sites, NGFI-B/Nur77 and RNR-1, did not support cAMP-stimulated StAR promoter activity in BeWo cells. Mutation of the distal putative SF-1 binding site reduced basal and cAMP-stimulated promoter activity in BeWo cells by 82% and 71%, respectively. Mutation of the proximal putative SF-1 binding site reduced basal and cAMP-stimulated promoter activity by 89% and 96%, respectively. Mutations in both sites reduced basal promoter activity to 7% of wild type promoter activity and cAMP-stimulated promoter activity to less than 5% of the wild type. Deletion analyses of promoter activity were consistent with the mutation studies. Electrophoretic mobility shift assays (EMSAs) demonstrated that the distal site binds to SF-1 expressed in COS-1 cells and to an SF-1-GST fusion protein with high affinity, but that the mutated distal sequence does not. An anti-SF-1 antibody ablated the characteristic SF-1-DNA complex with the distal sequence. The proximal site formed a number of protein-DNA complexes with COS-1 cell extracts, but appeared to have at best only very modest affinity for SF-1. Collectively, our findings demonstrate that SF-1 plays a key role in controlling the basal and cAMP-stimulated expression of the StAR gene. SF-1 can function at two distinct sites in the human StAR gene promoter, apparently by two different types of interaction, to control transcription.

Purpose: Our study determines and compares how the major organs of large animals handle exogenous halogenated bioactive sex steroids within the first minutes after their i.v. or i.a. injection, The rationale is that an understanding is needed of the acute physiological events because they affect decisions for how to optimize delivery of radiohalogenated sex steroid receptor ligands for purposes of medical imaging and modes of radiotherapy.Methods and Materials: We used an indicator dilution technique that allows monitoring of blood-tissue exchange of radioactivity in a continuous manner in anesthetized surgically prepared swine.Results: In swine, with by-passed liver circulation, the lungs allow the vast majority of [I-125]-16alpha-iodo-17beta-estradiol ([I-125]E) to be extracted from the blood perfusing the lung in the initial transit after i.v. injection in vivo. Similar outcome was observed for most major organs, including the CNS, intestines, spleen, peripheral appendages, and kidneys after i.a. injection of [I-125]E in vivo. However, within minutes the organs released the [I-125]E in its original chemical form back into the vascular system, with the exception of estrogen receptor (ER) rich tissues and the kidneys that retained the [I-125]E in its original form, although in the kidneys a nonpolar metabolite also accumulated.Conclusion: Our experiments confirm in a large animal model that radioiodoestradiol can be sequestered or concentrated in ER-rich sites. The liver and sex steroid receptor-rich organs modify considerably, by metabolism and sequestration, respectively, the acute distribution of bioactive steroids, Our data indicate potential for detection of ER in vivo in hormone-sensitive tumors, that is, in breast and endometrial cancers, and offer improved understanding of the recent studies in subjects with breast cancer that demonstrated that receptor imaging in vivo of steroid receptors with high-affinity radiolabeled ligands is possible in a clinical setting.

Congenial lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH. Affected individuals can make no adrenal or gonadal steroids. All affected individuals are phenotypic females irrespective of gonadal sex, and frequently die in infancy if mineralocorticoid and glucocorticoid replacements are not instituted. Recent data implicate the steroidogenic acute regulatory (StAR) protein in this disorder. We now describe a 46,XY patient of Vietnamese ancestry with lipoid CAH who had a somewhat milder form of the disease. Diagnosis was at 10 weeks of age, and low levels of plasma progesterone, corticosterone, 180H-corticosterone and androstenedione were detectable. Testicular RNA for StAR was reverse transcribed, amplified, cloned and sequenced, revealing a 185 bp deletion corresponding to all of exon 5. The corresponding mRNA did not encode active protein in transfected cells. Cloned genomic DNA from the patient revealed only a T-->A transversion in intron 4,11 bp from the splice acceptor site of exon 5. This transversion destroys an NcoI site; digestion of PCR-amplified genomic DNA from the patient and both parents confirmed that the patient was homozygous and the parents were heterozygous. Expression vectors for StAR minigenes were constructed containing all StAR exons plus introns 4, 5 and 6 either with or without the T-->A mutation in intron 4. RNase protection assays showed that expression of the vector with normal intron 4 yielded correctly spliced StAR mRNA in transfected COS-1 cells, while most, but not all StAR mRNA from the vector with the T-->A transversion in intron 4 was abnormally spliced. RNase protection of the patient's testicular RNA confirmed that most, but not all StAR mRNA was similarly spliced abnormally. Splicing errors appear to be a rare cause of genetic diseases, but subtle intronic mutations may be missed when genomic DNA is the only material available for study. The low level of normal StAR mRNA produced may account for the later clinical presentation and low levels of steroid hormones detected in this patient.

Sterol carrier protein X (SCPx) is a 58-kDa protein that is localized to peroxisomes. The amino acid sequence of the protein suggests that SCPx may function as a thiolase. The gene encoding SCPx also codes for a 15.3-kDa protein called sterol carrier protein 2 (SCP2). Here we report the structure of this gene (SCP2), which spans approximately 80 kb and consists of 16 exons and 15 introns. Multiple transcription start sites were identified. The 5′ flanking region has characteristics of other peroxisomal protein promoters, which include the absence of a TATA box and a G+C-enriched region containing several reverse GC boxes.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTHuman Sterol Carrier Protein x/Sterol Carrier Protein 2 Gene Has Two PromotersTakashi Ohba, John A. Holt, Jeffrey T. Billheimer, and Jerome F. Strauss, IIICite this: Biochemistry 1995, 34, 33, 10660–10668Publication Date (Print):August 22, 1995Publication History Published online1 May 2002Published inissue 22 August 1995https://pubs.acs.org/doi/10.1021/bi00033a042https://doi.org/10.1021/bi00033a042research-articleACS PublicationsRequest reuse permissionsArticle Views98Altmetric-Citations51LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

Steroidogenic acute regulatory protein (StAR) plays a key role in steroid hormone synthesis by enhancing the metabolism of cholesterol into pregnenolone. We determined the organization of the StAR structural gene, mapped to 8p11.2. The gene spans 8 kb and consists of seven exons interrupted by six introns. The 1.3 kb of DNA upstream from the transcription start site directed expression of a luciferase reporter gene in mouse Y-1 adrenal cortical tumor cells but not in BeWo choriocarcinoma cells. Reporter gene expression in the Y-1 cells was increased more than 2-fold by 8-Br-cAMP, indicating that the 1.3 kb DNA fragment contains sequences that confer tissue-specific expression and cAMP regulation. The sequence of a related StAR pseudogene, mapped to chromosome 13, lacks introns and has an insertion, numerous substitutions, and deletions. Expression of StAR in COS-1 cells cotransfected with cholesterol 27-hydroxylase (P450c27) and adrenodoxin resulted in a 6-fold increase in formation of 3 beta-hydroxy-5-cholestenoic acid, demonstrating that StAR's actions are not specific to steroidogenesis but extend to other mitochondrial cholesterol-metabolizing enzymes.

Steroidogenic acute regulatory protein (StAR) appears to mediate the rapid increase in pregnenolone synthesis stimulated by tropic hormones. cDNAs encoding StAR were isolated from a human adrenal cortex library. Human StAR, coexpressed in COS-1 cells with cytochrome P450scc and adrenodoxin, increased pregnenolone synthesis > 4-fold. A major StAR transcript of 1.6 kb and less abundant transcripts of 4.4 and 7.5 kb were detected in ovary and testis. Kidney had a lower amount of the 1.6-kb message. StAR mRNA was not detected in other tissues including placenta. Treatment of granulosa cells with 8-bromo-adenosine 3',5'-cyclic monophosphate for 24 hr increased StAR mRNA 3-fold or more. The structural gene encoding StAR was mapped using somatic cell hybrid mapping panels to chromosome 8p. Fluorescence in situ hybridization placed the StAR locus in the region 8p11.2. A StAR pseudogene was mapped to chromosome 13. We conclude that StAR expression is restricted to tissues that carry out mitochondrial sterol oxidations subject to acute regulation by cAMP and that StAR mRNA levels are regulated by cAMP.

Schlufolgerung  - Erhhte Serumspiegel des p185erbB-2 Onkoproteins knnen mit einem EIA bei einzelnen Ovarialkarzinompatientinnen gemessen werden. - Wegen des seltenen Vorkommens versprechen p185erbB-2 Serumkonzentrationen keinen klinischen Nutzen bei der Identifikation oder Verlaufskontrolle von high-risk Patientinnen.

PURPOSE:We undertook this study because confirmation of a rapid vascular escape and slow release back into the circulatory system suggests that arterial injection of radiohalogenated steroid receptor ligands might provide an efficacious route of administration for imaging or treatment of receptor-rich malignant tumors in peripheral tissues. METHODS AND MATERIALS:We injected radiolabeled 16 alpha-iodo,17 beta-estradiol ([I]-E), an estrogen receptor ligand, into the femoral artery of swine in a solution that contained [125I]-E in a known ratio to [99mTc]-labeled red blood cells. Fractions of femoral venous blood were collected at short intervals during 10 min. We looked for changes in the ratio of the radiolabels. [99mTc]-labeled red blood cells are known to remain in the vascular system for an hour or more. RESULTS:After passage of the injectate through the capillary bed of the swine leg, a dramatic decrease of the initial 125I:99mTc ratio to only 10% was observed in the femoral venous blood. This ratio increased gradually during the next 10 min to approximately 30% of that in the injectate, indicating that a significant portion (approximately 90%) of the [125I]-E was initially trapped in the limb and then slowly re-entered the vascular system. To obtain visual confirmation of the rapid vascular escape of iodo-estrogen, we injected either an imageable form of [I]-E (123I]-E) or [99mTc]-labeled red blood cells into the dorsal aorta of superovulated rabbits, whose smaller size allowed whole-body imaging. The biodistributions of these radiopharmaceuticals were surveyed continuously by real-time planar gamma imaging. Within 2 min after the injection of [123I]-E, the outlines of the circulatory system were entirely lost; however, some estrogen receptor-rich tissues (the ovaries) as well as some non-target tissues, for example, the lower leg extremities, yielded well-defined images. In contrast, after intra-arterial injection of [99mTc]-labeled red blood cells, the circulatory system remained sharply defined for the duration of the study (40 min). CONCLUSION:A large fraction of [I]-E escapes from the vascular system during the first pass through an organ or limb, without regard to the estrogen receptor content of the tissue.

Strategies for diagnosis and therapy in which sex steroid receptor ligands serve as carriers for radionuclides are attractive because a high incidence of carcinomas of the female genital tract and the breast that are seen clinically have an abundant expression of one or more of the receptor proteins. A radiohalogenated estrogen receptor (ER) ligand, 16 alpha-[123I]iodo-17 beta-estradiol [123I]E, has met clinical criteria for receptor-mediated diagnostic imaging. Its [125]I-labeled sister nuclide derivative [125I]E decays by orbital electron capture with emission of very-low-energy (Auger) electrons, which gives this latter radiohalogen the potential to serve in pharmaceuticals for radiotherapy; as examples, [125I]deoxyuridine, when incorporated into the DNA molecule, or [125I]E, when bound to the receptor within ER-rich tumor cells, are both cytotoxic in vitro. Whereas the mechanisms and subcellular changes that accompany the cytotoxicity from [125I]deoxyuridine are well documented in the form of aberrations and breaks in the cellular DNA, the effects at the subcellular level causing the cytotoxicity of the sex steroid receptor ligand [125I]E have not been characterized and are the focus of our study. We found that in a standard colony-forming assay the addition of [125I]E to the cultures decreased the survival rate of ER-positive MCF-7 cells in a dose-dependent manner. The decreased survival rate was prevented by the addition of competing excess radioinert ER ligand (diethylstilbestrol); [125I]E did not reduce survival in ER-negative MCF-7 cells. The [125I]E-induced and ER-mediated cytotoxicity was accompanied by aberrations in the DNA components of the nuclei of the cells. These included chromatid and chromosome breaks, gaps, and tri-radial chromosome formation. Our findings add plausibility and credence to the notion that the cytotoxicity imparted by Auger-electron-emitting radioligands for sex steroid receptors is in part attributable to radiodecay that causes double-stranded breakage of DNA.

The binding of von Willebrand factor (vWF) to platelet glycoprotein (GP) lb receptor is one of the initial events in thrombus formation. Previous studies have shown that RG12986, a reduced and alkylated recombinant fragment of vWF (Ser445-Val733), can inhibit binding of native vWF to GP lb and offers potential as an anti-thrombotic agent. We have now evaluated a series of deletion mutants of RG12986 and found that reduced and alkylated rvWF508-704 is close to the minimal sequence with optimal RG12986-like activity (IC50 for inhibition of GP Ib-dependent platelet aggregation in the absence of modulators: 0.022 muM +/- 0.01, n = 3) and that it too binds directly to GP Ib. Under in vitro conditions, with no exogenous modulators present and in the absence of shear stress, oxidized rvWF508-704 (containing a disulfide bond between Cys508 and Cys659) is approximately 5-fold less active than reduced and alkylated rvWF508-704; the two fragments, however, display comparable activity in the presence of the modulator botrocetin. The smaller rvWF508-704 fragment offers distinct advantages over RG12986. In particular, removal of non-active NH2 and COOH terminal sequences may reduce the risk of antigenicity and may contribute to rendering the molecule mostly monomeric in solution, as opposed to the monomer-dimer equilibrium previously described for RG12986.

Der Östrogenrezeptor (ER)-Status von Mamma- und Endometriumkarzinom ist wichtig für die Prognose und Therapie (1). Die Notwendigkeit zur Entnahme von Biopsien zur ER-Bestimmung limitiert die Zahl von Tumorlokalisationen, die simultan untersucht werden können, und sequentielle Bestimmungen bei Metastasierung und Rezidiv. Heterogenität von Primärtumor und Metastasen und mögliche Änderungen des ER-Phänotyps während der Therapie werden daher unzureichend erfaßt. Durch Tumorexzision und Aufarbeitung des Gewebes können Artefakte auftreten, so daß der vom Labor ermittelte ER-Status eventuell nicht mehr mit der Situation in-vivo entspricht. Das Verständnis der Rezeptorbiologie und die Behandlung der Patienten würden von Techniken profitieren, welche die Bestimmung des ER-Status in vivo gestattet. Wir testeten, ob ein Gamma-emittierender, hochaffiner ER-Ligand (16α-[123J]Jod-Östradiol-17β, [123J]E, spez. Aktivität > 10.000 Ci/mmol) (2) in ER-reichen Geweben für eine szintigraphische Darstellung in-vivo hinreichen spezifisch konzentriert wird.

During the normal menstrual cycle the volume of cervicovaginal fluid (CVF), as determined by the patient at home using a simple volumetric aspirating pipette, increases significantly over several days prior to the luteinizing hormone (LH) surge and decreases characteristically shortly after ovulation. The present study was undertaken to test the hypothesis that self-determined measurements of CVF volume would correlate positively with serum estradiol (E2)levels and with total follicular volume (TFV) in cycles stimulated with exogenous gonadotropins. Consequently, 20 infertility patients, undergoing human menopausal gonadotropin (hMG)-stimulated cycles, were asked to measure daily CVF. Routine serum E2determinations and vaginal follicular ultrasound studies were performed up to and including the day of human chorionic gonadotropin (hCG) administration (designated day 0). The mean daily CVF volume (±SD) increased from 0.1 ± 0.01 ml on day −6 to 0.7 ± 0.40 ml on day — 1 and then decreased to 0.6 ± 0.40 ml on day 0. On day 0, 7 of 20 cycles (35%) demonstrated a decrease in CVF, which ranged from 0.1 to 1.0 ml (mean, 0.42 ml). The mean daily CVF correlated positively (correlation coefficient = r) with the mean daily serum E2 (r = 0.89) and with the mean daily TFV (r =0.88). The mean daily correlation of TFV for E2 was r =0.98. The correlation between CVF and E2of individuals ranged from r =0.38 to r =0.99 and the correlation between CVF and TFV ranged from r =0.12 to r =1.0, while the individual correlations ofE2 to TFV ranged from r =0.60 to r =0.99. These early data suggest that, although mean daily levels of CVF increase positively over time, individual CVF levels are not sensitive enough, alone, to replace the traditional methods of monitoring hMG-stimulated cycles.

Conversion of cholesterol to pregnenolone is the most important and, at the same time, the rate-limiting step of steroidogenesis. Sterol carrier protein-2 (SCP2) or nonspecific lipid transfer protein (nsL-TP) is an intracellular protein, which plays an important role in the pre- and transmitochondrial transport of cholesterol and for the mitochondrial synthesis of pregnenolone. Synthesis of pregnenolone in rat Leydig cells can be increased by LH and LH-RH; however, only LH leads to characteristic changes in intracellular concentrations of SCP2. This means that synthesis of pregnenolone is regulated in two different ways. In this study we aimed to find out whether such a 'second way' of steroidogenesis is also demonstrable for the human corpus luteum (i.e. human luteal cells). Human luteal cells were collected during follicle punctures and were cultured as described previously. We demonstrate that (human) LH/hCG are able to enhance pregnenolone synthesis; this process is accompagnied by typical changes of SCP2 and an increase in activity of 7-dehydrocholesterol reductase, which is a marker enzyme for SCP2. LH-RH was shown to exert no effect. Thus, we conclude that a second way of steroidogenesis (i.e. synthesis of pregnenolone) cannot be proved for the human corpus luteum.

The biological activities of steroid hormones are effected via intracellular receptors. The receptors are part of a ligand-activated family of transcription regulator proteins that are critical for steroid-regulated cell differentiation. With recombinant cDNA technology, yeast and cultured animal cells can be made to express mammalian cDNA steroid receptors from cDNA clones that contain deletions and substitutions. Among the leading problems addressed in these models is the characterization of sequences that promote association or interaction with other transcription regulating molecules, including oncogene products. Recently it has been found that heat shock proteins may serve not only to stabilize the receptor proteins but also to precondition the activation imparted by ligand binding. Aberrant receptor proteins can be found in ovarian cancer. Whether aberrant receptor proteins are associated with transformation in general or with a variable clinical response to steroidal or anti-steroidal therapy is not known. Even after chemotherapy, steroid receptors are expressed in the metastases of ovarian cancers seen clinically, and they may have potential uses for localization and treatment of receptor-rich cancers. Radioligand pharmaceuticals appropriate for imaging or for site-directed radiocytotoxicity can be sequestered to the nuclei of receptor-rich cancers. Initial clinical imaging and therapy trials with such pharmaceuticals have been approved and begun. In the use of halogenated estrogen radiopharmaceuticals, liver metabolism and enterohepatic recirculation are important considerations. Ascites prolongs retention of a radiohalogenated estrogen in the abdominal cavity. Distant metastases have been localized with [123I]-estrogen in breast cancer patients in pre-operative procedures. Receptor-mediated cytotoxicity occurs when estrogen receptor radioligand pharmaceuticals that are Auger electron emitters are used in vitro.