Tissue-specific promoters regulate aromatase cytochrome P450 gene expression in human ovary and fetal tissues.

The formation of estrogens from C19 steroids is catalyzed by a specific form of cytochrome P450, aromatase cytochrome P450 (P450AROM; the product of the CYP19 gene). Previous studies have demonstrated that aromatase activity in human adipose and ovarian granulosa cells is subject to complex multifactorial regulation and that changes in activity are correlated with changes in the levels of mRNA encoding P450AROM. We have previously isolated the human CYP19 gene. Two unique untranslated first exons (exons I.1 and I.2) have been identified in mRNA specific for P450AROM in human placenta. Although the proportion of transcripts encoding exon I.2 is very small, genomic clones encoding the sequences of both exons I.1 and I.2 have recently been isolated. The corpus luteum of human ovary differs in that promoters I.1 and I.2 are completely inactive. Sequence analysis of the DNA immediately 5' of exon II (which contains the start site of translation) demonstrates the presence of a TATAA sequence beginning 149 basepairs 5' of the ATG initiation codon identified in placental exon II. Using a combination of primer extension and S1 nuclease protection analysis, it appears that the initiation site of ovarian P450AROM transcripts aligns 26 basepairs down-stream of the sequence TATAA. It appears, therefore, that the expression of P450AROM-specific mRNA in corpus luteum is regulated by an additional promoter (promoter II), which is located just 5' of exon II. Consistent with these observations, Northern analysis of poly(A)+ RNA isolated from placenta and corpus luteum demonstrates that the major promoter of placental P450AROM is promoter I.1, while the major promoter in the corpus luteum is promoter II.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the gene encoding aromatase cytochrome P450 (CYP19) in fetal tissues.

The formation of estrogens from C19 steroids is catalyzed by a specific form of cytochrome P450, aromatase cytochrome P450 (P450arom; the product of the CYP19 gene). In previous studies we have demonstrated that in adult human tissues and placenta, expression of the CYP19 gene is regulated in part by means of tissue-specific promoters through the use of alternative splicing mechanisms. In addition to placenta, a number of fetal tissues express aromatase, including liver, intestine, skin, and brain. To characterize the CYP19 transcripts present in these and other fetal tissues, we have used reverse transcription and polymerase chain reaction to amplify sequences corresponding to the various untranslated exons from RNA extracted from these tissues. In addition, we have prepared cDNA libraries using RNA from these tissues by the method of rapid amplification of cDNA ends. Sequencing of clones derived from these libraries has been employed to confirm the presence of sequence corresponding to untranslated exons at the 5'-ends of P450arom transcripts. Based on these findings, we conclude that in fetal tissues other than placenta, transcripts containing sequence found in the exon we have previously named I.4 appear to be the most common. Such sequences have been found in cells in which P450arom expression is stimulated by glucocorticoids. Thus, the presence of such transcripts in fetal liver RNA is consistent with our previous observations that aromatase activity in fetal hepatocytes is stimulated by glucocorticoids. Secondly, transcripts are present in the fetal adrenal, although no aromatase activity has ever been detected in this tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Initial characterization of a subclone of the MA-10 mouse Leydig tumor cell line.

Cloned cell lines have proven to be useful models in understanding the regulation of endocrine cells and steroid synthesis. In this study we report the isolation and characterization of a subclone of the MA-10 Leydig tumor cell line. Whereas there was no difference in basal steroid production between the clone (MA-10 LP) and the parent stock (MA-10), MA-10 LP produces very low levels of progesterone after stimulation by hCG or (Bu)2cAMP. In both cell populations, hCG stimulation resulted in the accumulation of comparable amounts of cAMP in the presence of a phosphodiesterase inhibitor, and similar levels of cAMP were measured at 30 min without inhibitor. Measurement of cholesterol side-chain cleavage activity using two separate methods demonstrated that the low steroid production in MA-10 LP could not be accounted for by a decrease in the activity of this enzyme complex. Additionally, no difference in 3 beta-hydroxysteroid dehydrogenase activity could be demonstrated between the two cell populations. Since the lesion that attenuates the ability of MA-10 to synthesize progesterone is somewhere after the production of cAMP and before cholesterol side-chain cleavage activity, this system may provide a useful model for understanding the regulatory mechanisms controlling steroid biosynthesis in Leydig cells.

An endogenous digitalis-factor derived from the adrenal gland: studies of adrenocortical tumor cells.

The present studies demonstrate that the murine adrenocortical tumor cell line Y-1 releases a digoxin-like immunoreactive material into both serum-supplemented nutrient medium and minimal Krebs-Ringer bicarbonate medium. Release of pregnenolone into minimal medium from these cells was consistently inhibited by addition of the cholesterol side-chain cleavage inhibitor aminoglutethimide. However, release of digoxin-like immunoreactivity (DLI) was not similarly affected. To exclude the possibility that DLI could be accounted for by cross-reaction with another known adrenal steroid, aminoglutethimide inhibition was accompanied by inhibition of 17 alpha-hydroxylase with SU-10603 and inhibition of 3 beta-hydroxysteroid dehydrogenase with cyanoketone. Once again, pregnenolone release was effectively inhibited, but no similar pattern of inhibition of DLI release was observed. Increasing the time of the incubation periods from 1 to 2 h did not change the pattern of secretion of pregnenolone or DLI. HPLC analysis of DLI released over prolonged culture periods into serum-supplemented nutrient medium showed high levels of DLI in a single major and several adjacent peaks. Analysis of the ability of extracts of Y-1-conditioned medium to compete with tritiated ouabain for binding to erythrocytes indicates that conditioned medium contained highly enriched levels of ouabain-like activity. On HPLC analysis, the distribution of this activity showed partial correlation with the distribution of DLI. These observations indicate that Y-1 cells produce and release significant quantities of a material with cardiac glycoside-like properties reflected in the cross-reactivity with antidigoxin antibodies and the ability to compete with ouabain for binding to erythrocytes. In substantiation of previous findings in chopped adrenal cultures, the cardiac glycoside-like activity does not appear to result from cholesterol side-chain cleavage or pregnenolone production, since inhibition of side-chain cleavage as well as subsequent 17 alpha-hydroxylation and 3 beta-dehydrogenation did not result in consistent inhibition of DLI release.

Use of tissue-specific promoters in the regulation of aromatase cytochrome P450 gene expression in human testicular and ovarian sex cord tumors, as well as in normal fetal and adult gonads.

We have previously demonstrated that the tissue-specific regulation of human aromatase cytochrome P450 (P450arom) gene expression is, in part, the consequence of the use of tissue-specific promoters. Promoter I.1 (PI.1) and PI.2-specific transcripts are expressed in the placenta, whereas promoter II (PII) appears to be the only active promoter in the corpus luteum. Testicular and ovarian sex cord tumors with annular tubules (SCTATs) associated with gynecomastia in prepubertal boys and isosexual precocity in girls with Peutz-Jeghers syndrome (P-JS) have been previously reported. In the present study, we investigated the regulatory elements directing P450arom gene transcription in samples of SCTAT from three prepubertal boys and a girl with P-JS and an ovarian granulosa cell tumor from an adult woman, as well as in healthy fetal and adult testicular and ovarian tissues. Placental tissue was used as a control. Using polymerase chain reaction linked to reverse transcription and northern blotting, we determined the tissue-specific use of various P450arom promoters by analyzing specific 5'-termini from messenger RNA templates. Results indicate a universal gonadal promoter (PII) directs P450arom gene expression in healthy fetal and adult ovaries and testes, as well as in SCTAT of the P-JS and an adult ovarian granulosa cell tumor. These results are interpreted to mean that use of PII in human ovary and testis is preserved from the fetal period into adult life as well as in transformed neoplastic Sertoli and granulosa cells. On the other hand, transcripts from placenta are specific for PI.1 (and to a much lesser extent, PI.2). In SCTAT, immunoreactive P450arom is detected only in the cytoplasm of neoplastic cells, whereas the normal-appearing sex cords do not contain any immunoreactive P450arom. These results further suggest that the markedly increased aromatase expression of these transformed neoplastic cells is not a consequence of using different tissue-specific promoters. Rather it appears to involve activation (or failure of inhibition) of the upstream regulatory elements of the same promoter, which is normally functional in all gonadal tissues, namely the proximal PII.

Use of tissue-specific promoters in the regulation of aromatase cytochrome P450 gene expression in human testicular and ovarian sex cord tumors, as well as in normal fetal and adult gonads.

We have previously demonstrated that the tissue-specific regulation of human aromatase cytochrome P450 (P450arom) gene expression is, in part, the consequence of the use of tissue-specific promoters. Promoter I.1 (PI.1) and PI.2-specific transcripts are expressed in the placenta, whereas promoter II (PII) appears to be the only active promoter in the corpus luteum. Testicular and ovarian sex cord tumors with annular tubules (SCTATs) associated with gynecomastia in prepubertal boys and isosexual precocity in girls with Peutz-Jeghers syndrome (P-JS) have been previously reported. In the present study, we investigated the regulatory elements directing P450arom gene transcription in samples of SCTAT from three prepubertal boys and a girl with P-JS and an ovarian granulosa cell tumor from an adult woman, as well as in healthy fetal and adult testicular and ovarian tissues. Placental tissue was used as a control. Using polymerase chain reaction linked to reverse transcription and northern blotting, we determined the tissue-specific use of various P450arom promoters by analyzing specific 5'-termini from messenger RNA templates. Results indicate a universal gonadal promoter (PII) directs P450arom gene expression in healthy fetal and adult ovaries and testes, as well as in SCTAT of the P-JS and an adult ovarian granulosa cell tumor. These results are interpreted to mean that use of PII in human ovary and testis is preserved from the fetal period into adult life as well as in transformed neoplastic Sertoli and granulosa cells. On the other hand, transcripts from placenta are specific for PI.1 (and to a much lesser extent, PI.2). In SCTAT, immunoreactive P450arom is detected only in the cytoplasm of neoplastic cells, whereas the normal-appearing sex cords do not contain any immunoreactive P450arom. These results further suggest that the markedly increased aromatase expression of these transformed neoplastic cells is not a consequence of using different tissue-specific promoters. Rather it appears to involve activation (or failure of inhibition) of the upstream regulatory elements of the same promoter, which is normally functional in all gonadal tissues, namely the proximal PII.

MCF-7 and T47D human breast cancer cells contain a functional peroxisomal response.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors that regulate transcription of target genes. Since attempts have been made to correlate the ingestion of high-fat diets, itself a peroxisome proliferator, with the occurrence of breast cancer, we set about to determine if human breast cancer cells contained a functional PPAR. In this report we demonstrate the presence of an mRNA in two breast cancer cell lines (MCF-7 and T47D) which is specifically recognized by a mouse PPARgamma2 probe. Furthermore, in gel shift assays a consensus PPAR response element (PPRE) was specifically bound by nuclear extracts from MCF-7 cells and was further retarded by antibodies raised to mouse PPARgamma. Finally, when transfected with a PPRE-luciferase transcriptional reporter construct, transcription was increased in response to activators of PPAR and its dimmeric partner the retinoic acid X receptor (RXR). These data indicate that peroxisomal proliferators are capable of mediating transcription in human breast cells and suggest the possibility of a physiological role in the breast.

Alternative promotion of aromatase P-450 expression in the human placenta.

We have previously reported the isolation and characterization of the human gene encoding aromatase cytochrome P-450 (P-450AROM). The gene had been demonstrated to span at least 52 kb and contain ten exons, the first of which, exon I.1, is untranslated. Here we report the isolation and characterization of a P-450AROM cDNA from a human placental primer-extended cDNA library which contains a unique 5' sequence. This cDNA has been isolated and sequences used to screen a human placental genomic library for the presence of a unique first exon. The exon (exon I.2) lies 9 kb 5' of the second, ATG-containing exon (exon II) and is spliced onto exon II at the same site as that reported for exon I.1. DNA sequence analysis indicates that exon I.2 has a putative TATA (TAAA) sequence 33 base pairs (bp) upstream from a putative transcription start site and putative CAAT (CATT) binding sequence beginning at 54 bp upstream from this start site. Polymerase chain reaction (PCR) amplification experiments indicate that mRNA containing exon I.2-specific sequences can be demonstrated in tissues of fetal, but not adult, origin. These data have been confirmed by Northern analysis in the placenta. Characterization of this genomic clone containing exons I.2 and II now establishes the P-450AROM gene to be at least 72 kb in length and raises new questions regarding tissue specific and developmental control of aromatase expression in the human.

Differential transcriptional activation of peroxisome proliferator-activated receptor gamma by omega-3 and omega-6 fatty acids in MCF-7 cells.

While the role of dietary fats in breast cancer remains controversial, the recent cloning of peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear hormone receptor, from human breast cancer cells lines provides a potential molecular link. Several fatty acids from four classes of dietary fats were tested for their ability to mediate the transcriptional activity of PPARgamma in MCF-7 and MDA-MB-231 cells using growth media with minimal serum. Whereas omega-3 fatty acids inhibit transactivation of PPARgamma to levels below control, omega-6, monounsaturated and saturated fatty acids stimulate the activity of the transcriptional reporter. These studies indicate that individual fatty acids differentially regulate the transcriptional activity of PPARgamma by selectively acting as agonists or antagonists. Furthermore, the transcriptional activation of PPARgamma correlates with cell proliferation in MCF-7 cells. Understanding the effects of individual fats on breast cancer cells and PPARgamma transactivation could provide important new insights into the epidemiology of breast cancer and the role of dietary fat.

Regulation of steroidogenesis in subclones of the MA-10 mouse Leydig tumor cell line.

We have previously reported the isolation of a subclone of the MA-10 mouse Leydig tumor cell line (MA-10 LP) which secretes less than 10% of the steroid synthesized by the parent, accumulates comparable amounts of cAMP and has equivalent cholesterol side-chain cleavage activity as the parent population (Kilgore and Stocco (1989) Endocrinology 124, 1210-1216). In the present study we show that addition of exogenous sterol carrier protein 2 (SCP2) to isolated mitochondria was not able to overcome the deficient steroid response of MA-10 LP. We have also demonstrated that human chorionic gonadotropin (hCG)-stimulated cellular events which activate steroid production by subsequently isolated mitochondria require ongoing protein synthesis, release of intracellular calcium and are mediated through the calcium-calmodulin complex. Additionally, mitochondrial sonicates from hCG-stimulated parent cells were able to stimulate steroid production by intact mitochondria isolated from unstimulated parent cells, whereas sonicates from similarly treated MA-10 LP had no effect on steroid production in these mitochondria. Together these data suggest that hCG induces changes in the mitochondria of the parent stock which are not induced to the same extent in the mitochondria of MA-10 LP.

Hemiatrophy of the tongue: a rare complication of the hemiplegic migraine.

A rare case of a patient with complicated migraine is presented. The patient had hemiatrophy of the tongue as a residual of a migraine episode. This cleared after several months. The paper goes on to trace the history of complicated migraine and the rather unusual complication of hemiatrophy of the tongue.

Brain aromatase cytochrome P-450 messenger RNA levels and enzyme activity during prenatal and perinatal development in the rat.

Aromatase cytochrome P-450 (P-450AROM) enzyme activity catalyzes the conversion of androgens to estrogens in specific brain areas. During development local estrogen formation is thought to influence the sexual differentiation of neural structures (i.e. increase neurite growth and establish neural circuitry) and modulate reproductive functions. This study was undertaken to investigate the ontogeny of the (P-450AROM) enzyme and its messenger RNA (mRNA) in medial basal hypothalamic (MBH) and preoptic area (POA) tissue during late fetal and perinatal development of the rat. Aromatase activity in the MBH-POA was negligible before gestational day (GD) 16 (< 0.1 pmol/h/mg protein), increased over 10-fold at GD 17 and continued to increase (over 5-fold) to peak levels at GD 19 (> 5.0 pmol/h/mg protein), and then declined to low levels at GD 22 and 2 days post-birth (approximately 1 pmol/h/mg protein). The profile of P-450AROM mRNA in the MBH-POA tissue was characterized by a predominant 2.7 kilobase (kb) mRNA species, similar in size to the largest functional P-450AROM mRNA observed in adult rat ovarian tissue. At GD 15, the P-450AROM mRNA was undetectable; low but detectable levels were seen at GD 17, the abundance increased at later time points and remained at peak levels on GDs 18 through 20, decreased slightly by GD 22, and then declined further by 2 days post-birth. The developmental increase in P-450AROM mRNA levels correlated with the ascending pattern of enzyme activity before GD 19, but the marked decrease in enzyme activity seen after GD 19 was not accompanied by a corresponding decline in mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

17alpha-Hydroxylase gene expression in the bovine ovary: mechanisms regulating expression differ from those in adrenal cells.

17alpha-Hydroxylase cytochrome P450 (P450(17alpha)) is the enzyme which synthesizes C19 steroids in a two-step reaction in which 17alpha-OH pregnenolone is an intermediate. In the bovine and human adult female, 17alpha-hydroxylase is expressed in adrenocortical cells where 17alpha-OH pregnenolone and 17alpha-OH progesterone are precursors of cortisol, and in theca cells of the ovary where these intermediates are precursors of C19 steroids. In both adrenal cortex and theca, 17alpha-hydroxylase gene expression is stimulated by cyclic AMP (cAMP). The aim of this study was to determine the mechanism regulating 17alpha-hydroxylase gene expression in the bovine ovary. Our results indicate that the bovine 17alpha-hydroxylase gene is regulated in a tissue-specific fashion. Primer extension and S1 nuclease protection assays reveal that the start site of transcription in the theca is identical to that in the adrenal. Transfection studies employing beta-globin reporter gene constructs fused to successive deletions of the 5' regulatory region of the bovine 17alpha-hydroxylase gene indicate that sequences between -80 and -37 basepairs (bp) (CRS2) confer cAMP-regulated transcription in bovine theca cells in culture. These results are in contrast to similar studies conducted in bovine adrenocortical cells, which indicate that the major cAMP response element (referred to as CRS1) is located at -243 to -225 bp. The Ad4 element (AGGTCA, -42 to -37 bp) within CRS2, which has been shown to be involved in cAMP responsiveness in other steroidogenic P450 genes, cannot by itself confer cAMP-regulated reporter gene expression in bovine cells. These results indicate that in the cow, 17alpha-hydroxylase gene expression is regulated in a tissue-specific fashion, and that this regulation may be conferred, at least in part, by the use of tissue-specific cis-acting elements in the bovine 17alpha-hydroxylase gene.

Tissue-specific promoters regulate aromatase cytochrome P450 expression.

In the human, estrogen biosynthesis occurs in several tissue sites, including ovary, placenta, adipose, and brain. Recent work from our laboratory has indicated that tissue-specific expression of aromatase cytochrome P450 (P450arom), the enzyme responsible for estrogen biosynthesis, is determined, in part, by the use of tissue-specific promoters. Thus the expression of P450arom in human ovary appears to utilize a promoter proximal to the translation start-site. This promoter is not utilized in placenta but instead, the promoter used to drive aromatase expression in placenta is at least 40 kb upstream from the translational start-site. In addition, there is a minor promoter used in the expression of a small proportion of placental transcripts which is 9 kb upstream from the start of translation. Transcripts from these promoters are also expressed in other fetal tissues including placenta-related cells such as JEG-3 choriocarcinoma cells, hydatidiform moles, and other fetal tissues such as fetal liver. On the other hand, in adipose tissue expression of P450arom may be achieved by yet another, adipose-specific promoter. The various 5'-untranslated exons unique for expression driven by each of these promoters are spliced into a common intron/exon boundary upstream from the translational start-site. This means that the protein expressed in each of the various tissue-specific sites of estrogen biosynthesis is identical.

Brain stem auditory evoked responses in clinical practice.

A relatively new noninvasive technique for the diagnosis of abnormalities affecting the central nervous system is presented. It is especially useful in patients with acoustic neuroma, brain stem disorder such as tumors of the posterior fossa with neuraxis impingement, as well as demyelinating disorders such as multiple sclerosis. It is certainly useful in the verification of neurologic normalcy in patients complaining of dizziness and vertigo.

Oxygen uptake in mice infected with Trichinella spiralis.

Oxygen consumption, rectal temperature, and the level of activity in mice infected with Trichinella spiralis were significantly reduced below that seen in uninfected controls for periods of time during the first 30 days following infection. The differences in oxygen consumption between controls and infected animals were evident throughout the 24-hr period comprising day 7 postinfection. Both oxygen consumption and rectal temperature increased with decreasing level of infection. These changes in oxygen consumption, rectal temperature, and activity are discussed in terms of pathophysiologic and immunopathologic changes known to occur during the course of infection.

Induction of mitochondrial proteins in MA-10 Leydig tumour cells with human choriogonadotropin.

The side-chain cleavage of cholesterol is the rate-limiting enzymic step in steroidogenesis and occurs in the mitochondria of steroid-producing tissues. In these studies, the effect of acute stimulation of MA-10 Leydig-tumour cells on the synthesis of mitochondrial proteins was investigated. Cells were incubated in the presence of stimulating levels of human choriogonadotropin (hCG) and [35S]methionine for 2 h periods. Mitochondria were isolated and their proteins analysed by two-dimensional polyacrylamide-gel electrophoresis and fluorography. At least three mitochondrial-specific proteins were found in cells exposed to gonadotropin, and these proteins were also found in cells treated with dibutyryl cyclic AMP (db cyclic AMP). The appearance of these proteins was prevented by the addition of cycloheximide to the cells, as was the production of progesterone, the major steroid produced in MA-10 cells. In addition, mitochondria isolated from cells stimulated with db cyclic AMP produced progesterone at a rate 3-fold greater than mitochondria isolated from control cells during 3 h of incubation. Lastly, mixing experiments demonstrated that sonicated mitochondria isolated from db cyclic AMP-treated cells stimulated progesterone production in control mitochondria 6-fold. These studies show that hCG and db cyclic AMP stimulation of MA-10 cells results in the rapid induction of cycloheximide-sensitive proteins located in the mitochondria which may be instrumental in the acute regulation of steroidogenesis.

Glucose absorption in vitro by the enteric stages of Trichinella spiralis.

Absorption of glucose in vitro by enteric stages of Trichinella spiralis was examined in worms isolated at different times following infection. Glucose absorption by 6-h-old worms was similar to that seen during moulting (24 h post-infection (p.i.]. As embryogenesis began (48 h p.i.) glucose absorption in vitro by the parasite rose dramatically to a peak in 72-h-old worms. Absorption of glucose in vitro remained high in 96-h-old worms but as larviposition began (120 h p.i.) glucose uptake by T. spiralis dropped significantly. Glucose absorption by worms isolated from the host during the primary immune response was unaffected. Glucose absorption in vitro remained at similar levels in worms isolated between 120 and 312 h p.i. The percentage of absorbed label incorporated into parasite tissues was greatest in 48- and 312-h-old worms. Glucose uptake in vitro by pre-adult and adult worms changed with time and these changes accompanied specific events in the biology of the parasite.