Estrogen-producing steroidogenic pathways in parietal cells of the rat gastric mucosa

Recently we demonstrated the presence of aromatase (P450(arom)), estrogen synthetase, and the active production of estrogen in parietal cells of the rat stomach. We therefore investigated the steroidogenic pathways of estrogen and also other steroid metabolites in the gastric mucosa of male rats, by showing the mRNA expression of steroidogenic enzymes using RT-PCR and in situ hybridization histochemistry, and by measuring the blood concentration of steroids in the artery and the portal vein. RT-PCR analysis showed the strong mRNA expression of 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), 17beta-hydroxysteroid dehydrogenase (HSD) type III and P450(arom), and the weak mRNA expression of 17beta-HSD type II, 5alpha-reductase type I and 3alpha-HSD. The other mRNAs of steroidogenic enzymes examined were not detected. In situ hybridization histochemistry demonstrated the localization of mRNAs for P450(17alpha), 17beta-HSD type III and P450(arom) in the parietal cells. Higher levels of progesterone and testosterone were found in the artery compared with the portal vein. Higher amounts of estrone and 17beta-estradiol, by contrast, were present in the portal vein compared with the artery. These results indicate that parietal cells of rat stomach convert circulating progesterone and/through androstenedione and testosterone to synthesize both estrone and 17beta-estradiol, which then enter the liver via the portal vein.     

Structure and tissue-specific expression of 3ß-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase genes in human and rat classical and peripheral steroidogenic tissues

The enzyme 3ß-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3ß-HSD) catalyzes the oxidation and isomerization of 5-ene-3ß-hydroxypregnene and 5-ene-hydroxyandrostene steroid precursors into the corresponding 4-ene-ketosteroids necessary for the formation of all classes of steroid hormones. We have recently characterized two types of human 3ß-HSD cDNA clones and the corresponding genes which encode deduced proteins of 371 and 372 amino acids, respectively, and share 93.5% homology. The human 3ß-HSD genes containing 4 exons were assigned by in situ hybridization to the p11–p13 region of the short arm of chromosome 1. We have also recently elucidated the structure of three types of rat 3ß-HSD cDNAs as well as that of one type of 3ß-HSD from bovine and macaque ovary λgt11 cDNA libraries which all encode 372 amino acid proteins. The human type I 3ß-HSD is the almost exclusive mRNA species detected in the placenta and skin, while the human type II is the predominant mRNA species in the adrenals, ovaries and testes. The predicted rat type I and type II 3ß-HSD proteins expressed in adrenals, gonads and adipose tissue share 94% homology while they share 80% similarity with the liver-specific type III 3ß-HSD. Transient expression of human type I and type II as well as rat type I and type II 3ß-HSD cDNAs in Hela human cervical carcinoma cells reveals that 3ß-ol dehydrogenase and 5-ene-4-ene isomerase activities reside within a single protein and these cDNAs encode functional 3ß-HSD proteins that are capable of converting 3ß-hydroxy-5-ene-steroids into 3-keto-4-ene derivatives as well as the interconversion of 3ß-hydroxy and 3-keto-5-androstane steroids. We have found that the rat type III mRNA species was below the detection limit in intact female liver while, following hypophysectomy, its accumulation increased to 55% of the levels measured in intact or HYPOX male rats, an increase which can be blocked by administration of ovine prolactin (oPRL). In addition, in female rats, treatment with oPRL for 10 days starting 15 days after HYPOX, markedly decreased ovarian 3ß-HSD mRNA accumulation accompanied by a similar decrease in 3ß-HSD activity and protein levels. Treatment with the gonadotropin hCG reversed the potent inhibitory effect of oPRL on these parameters and stimulated 3ß-HSD mRNA levels in ovarian interstitial cells. In intact females, hCG exerted marked trophic effects on rat corpora lutea with an increase in total ovarian 3ß-HSD expression and activity. We have also shown that treatment with hCG for 15 days in intact male rats caused a marked increase in testicular 3ß-HSD expression and activity while glucocorticoids exerted inhibitory effects on these parameters. We have also observed that the ontogeny of 3ß-HSD expression in human and rat adrenal gland, testis and ovary is closely correlated with steroid hormone biosynthesis, thus suggesting that regulation of the expression of 3ß-HSD is a limiting step in the biosynthesis of steroids in these tissues.     

Androgen and estrogen treatments alter steady state messengers RNA (mRNA) levels of testicular steroidogenic enzymes in the rainbow trout, Oncorhynchus mykiss

Recent investigations have shown that estrogens have profound inhibitory effects on steroidogenic enzyme gene expressions before and after testicular differentiation in the rainbow trout, Oncorhynchus mykiss. This present study bring new data on juvenile rainbow trout treated with estrogens and androgens. Following a 8 days oral treatment of juvenile male with 17alpha-ethynyl-estradiol (EE2, 20 mg/kg diet) or 11beta-hydroxyandrostenedione (11betaOHDelta4, 10 mg/kg diet), we observed a fast and marked decrease of steady-state mRNA levels for 3betaHSD, P450scc, P450c17, and P450c11 enzymes in the testis. After completion of these treatments, mRNA levels of these enzymes remained low in EE2 treated males whereas in 11betaOHDelta4 treated males they recovered their initial levels in 8 days. This demonstrate that both androgen and estrogen treatments have profound effects on testicular steroidogenesis by decreasing steroid enzymes steady-state mRNA. After in vitro incubation of testicular explants with 17beta-estradiol (E2, 600 ng/ml of medium), we also observed a decrease of mRNA levels for 3betaHSD and P450c11. This suggest that estrogens effects could be triggered, at least to some extend, directly on the testis. We also investigated the hypothesis of a negative feedback of steroids on follicle stimulating hormone (FSH) secretion, but FSH plasmatic levels in treated fish did not showed any significant decrease. This demonstrate that FSH is not implied in this steroids inhibition of steroidogenic enzymes gene expression.     

Expression of steroidogenic factors 1 and 2 in normal human pancreas

Orphan nuclear receptor steroidogenic factor-1 (SF-1) is crucial for development and function of steroidogenic organs. The steroidogenic factor-2 (SF-2) is an essential factor involved in cholesterol transfer and activation of promoters of steroidogenic enzymes CYP11A1, CYP17 and Steroidogenic Acute Regulatory Protein (StAR). We have previously demonstrated steroidogenic activity in pancreatic tissue. The aim of this study was to investigate the presence of SF-1 and SF-2 in human pancreas. Total RNA was extracted from normal male (five) and female (five) samples, obtained from the organs donor program. RT-PCR approach was used to analyze the expression of SF-1 and SF-2. Immunohistochemical analysis was performed for SF-1. The bands of expression were present in both male and female samples, although differential expression was observed. For both factors, the signal detected was more evident in males than in females. A similar pattern was present in the immunohistochemical study. Normal human pancreas expresses SF-1 and SF-2 factors similarly to ovary and adrenals. A distinctive characteristic is the sexually dimorphic expression of these factors. Our data provide evidence suggesting that the pancreas achieves steroidogenic activity supporting the presence of gender- and location-related differences in the expression of these steroidogenic factors.     

Sex differences, developmental changes, response to injury and cAMP regulation of the mRNA levels of steroidogenic acute regulatory protein, cytochrome p450scc, and aromatase in the olivocerebellar system

Compelling evidence has now demonstrated direct biological actions of sex steroids at the cerebellum. Likewise, the expression of key steroidogenic factors, such as the steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (P450scc), and aromatase, at this neural site has been reported. Little is known, however, about the regulation of their genes in the cerebellum. Assessment of StAR, P450scc, and aromatase mRNAs in the cerebellum of male and female rats revealed that the expression of these genes is developmentally regulated, with the highest levels at early postnatal ages in both sexes and with significantly higher mRNA levels in postnatal males. Expression of these genes in the female remained unaltered after perinatal androgenization and along the estrous cycle. In contrast, damage of cerebellar afferent neurons of the inferior olivary nucleus evoked a significant increase in StAR, P450scc, and aromatase mRNA levels at this site, as well as a transient elevation in StAR mRNA at the cerebellum. Finally, enhancement of cAMP levels in cultured cerebellar neurons induced a significant increase in StAR and aromatase mRNA levels. In summary, we present herein novel evidence for the developmentally regulated and partially sexually dimorphic pattern of expression of StAR, P450scc, and aromatase genes in the rat cerebellum. These observations, together with the finding that the mRNA levels of these steroidogenic molecules are sensitive to injury and are regulated by intracellular cAMP, strongly suggest that local steroidogenesis is likely to play an important role during development and adaptation to neurodegenerative processes in the olivocerebellar system.     

Cellular localization of mRNA expression of enzymes involved in the formation and inactivation of hormonal steroids in the mouse prostate.

It is well documented that several tissues, including the prostate, are actively involved in the local formation and inactivation of hormonal steroids. To identify the cell types involved in the formation and inactivation of androgens and estrogens in the ventral lobe prostate, we have localized by in situ hybridization (ISH) a large number of steroidogenic as well as steroid-inactivating enzyme mRNAs in the adult mouse prostate. In parallel studies, we also measured enzyme mRNA levels by quantitative real-time PCR (RT-PCR) in ventral lobe prostates. From the results obtained with quantitative RT-PCR, it appears that, with a few exceptions, the enzyme with low mRNA expression could not be detected by ISH. The following enzymes have been localized by ISH: 17beta-hydroxysteroid dehydrogenase (17beta-HSD) types 1, 2, 3, 4, 7, 8, 9, 10, and 11; 5alpha-reductase type 2; 5beta reductase type 1; P450 7alpha hydroxylase; estrogen sulfotransferase type 1; 11beta-HSD types 1 and 2; and UDP-glucuronosyltransferase 1A6. All of these mRNAs are expressed in the epithelial cells of prostatic acini. Several enzyme mRNAs were also localized in stromal cells. Types 1, 7, and 10 17beta-HSD, estrogen sulfotransferase type 1, and 11beta-HSD types 1 and 2 were found only in epithelial cells. The present results indicate that both epithelial and stromal cells in the mouse prostate play a role in local formation and inactivation of hormonal steroids.     

Estrogen sulfotransferase of the rat liver: complementary DNA cloning and age- and sex-specific regulation of messenger RNA.

Mammalian estrogen sulfotransferase (EST; EC 2.8.2.4) sulfurylates the hydroxyl group of estrogenic steroids by transferring the sulfate from a cosubstrate adenosine 3'-phosphate-5'-phosphosulfate. Sulfurylated steroids do not bind to the estrogen receptor with high affinity and, therefore, are hormonally inactive. We have purified rat liver EST and developed monoclonal antibody to this enzyme. By immunoscreening a lambda gt-11 expression library constructed from male rat liver cDNAs, the cDNA clone corresponding to EST was identified and isolated. A recombinant expression plasmid (pCMV5) containing this cDNA insert when transfected into COS-7 cells generated both immunologically and enzymatically active EST. With the help of this cDNA probe, we have explored the regulation of the EST mRNA in the liver and the possible role of this enzyme in sex hormone action. During the lifespan of male rats, only the young adult animals show hepatic androgen responsiveness. Also, estrogenic hormones strongly antagonize androgen action in the rat liver. Northern blot analysis of liver RNA derived from male rats of different ages shows that the androgen sensitivity of young adult animals is associated with a high expression of EST mRNA. During the same period, mRNA corresponding to dehydroepiandrosterone sulfotransferase is markedly (approximately 10-fold) down-regulated. Such a correlation is in concordance with the role of these enzymes in the maintenance of hepatic androgen sensitivity during young adult life by inactivating the estrogenic and sparing the androgenic steroids. Furthermore, the increase in the hepatic androgen sensitivity of androgen-treated female rats is also associated with the induction of EST.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure, function and tissue-specific gene expression of 3β-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase enzymes in classical and peripheral intracrine steroidogenic tissues

The membrane-bound enzyme 3β-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3β-HSD) catalyses an essential step in the transformation of all 5-pregnen-3β-ol and 5-androsten-3β-ol steroids into the corresponding 3-keto-4-ene-steroids, namely progesterone as well as all the precursors of androgens, estrogens, glucocorticoids and mineralocorticoids. We have recently characterized two types of human 3β-HSD cDNA clones and the corresponding genes which encode type I and II 3β-HSD isoenzymes of 372 and 371 amino acids, respectively, and share 93.5% homology. The human 3β-HSD genes containing 4 exons were assigned by in situ hybridization to the p11-p13 region of the short arm of chromosome 1. Human type I 3β-HSD is the almost exclusive mRNA species present in the placenta and skin while the human type II is the predominant mRNA species in the adrenals, ovaries and testes. The type I protein possesses higher 3β-HSD activity than type II. We elucidated the structures of three types of rat 3β-HSD cDNAs as well that of one type of 3β-HSD from bovine and macaque ovary λgt11 cDNA libraries, which all encode a 372 amino acid protein. The rat type I and II 3β-HSD proteins expressed in the adrenals, gonads and adipose tissue share 93.8% homology. Transient expression of human type I and II as well as rat type I and II 3β-HSD cDNAs in HeLa human cervical carcinoma cells reveals that 3β-ol dehydrogenase and 5-ene-4-ene isomerase activities reside within a single protein. These expressed 3β-HSD proteins convert 3β-hydroxy-5-ene-steroids into 3-keto-4-ene derivatives and catalyze the interconversion of 3β-hydroxy and 3-keto-5α-androstane steroids. By site-directed mutagenesis, we demonstrated that the lower activity of expressed rat type II compared to rat type I 3β-HSD is due to a change of four residues probably involved in a membrane-spanning domain. When homogenates from cells transfected with a plasmid vector containing rat type I 3β-HSD is incubated in the presence of dihydrotestosterone (DHT) using NAD? as co-factor, 5α-androstanedione was formed (A-dione), indicating an intrinsic androgenic 17β-hydroxysteroid dehydrogenase (17β-HSD) activity of this 3β-HSD. We cloned a third type of rat cDNA encoding a predicted type III 3β-HSD specifically expressed in the rat liver, which shares 80% similarity with the two other isoenzymes. Transient expression in human HeLa cells reveals that the type III isoenzyme does not display oxidative activity for the classical substrates of 3β-HSD. However, in common with the type I enzyme, it converts A-dione and DHT to the corresponding 3β-hydroxysteroids, thus showing an exclusive 3-ketosteroid reductase activity. When NADPH is used as co-factor, the affinity for DHT of the type III enzyme becomes 10-fold higher than that of the type I. Rat type III mRNA was below the detection limit in intact female liver. Following hypophysectomy, its concentration increased to 55% of the values measured in intact or hypophysectomized male rats, an increase which can be blocked by administration of ovine prolactin (oPRL). Treatment with oPRL for 10 days starting 15 days after hypophysectomy markedly decreased ovarian 3β-HSD mRNA accumulation accompanied by a similar decrease in 3β-HSD activity and protein levels. Treatment with the gonadotropin hCG reversed the potent inhibitory effect of oPRL on these parameters and stimulated 3β-HSD mRNA levels in ovarian interstitial cells. These data indicate that the presence of multiple 3β-HSD isoenzymes offers the possibility of tissue-specific expression and regulation of this enzymatic activity that plays an essential role in the biosynthesis of all hormonal steroids in classical as well as peripheral intracrine steroidogenic tissues.     

The role of phosphoenolpyruvate carboxykinase in neuronal steroidogenesis under acute inflammation

Phosphoenolpyruvate carboxykinase (PEPCK) is a key gluconeogenic enzyme found in many tissues throughout the body including brain. In the present study, we have investigated the effect of bacterial lipopolysaccharide (LPS) on PEPCK and its role in neuronal steroidogenesis. Adult female albino rats were administered LPS (5 mg/kg body weight) to induce acute inflammation. LPS administration resulted in a significant increase of PEPCK mRNA expression with concomitant increase in mRNA levels of steroidogenic acute regulatory (StAR) protein and other steroidogenic enzymes including 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD) and aromatase in brain tissue. Further, the inhibition of PEPCK expression by glipizide significantly decreased the mRNA expression of steroidogenic proteins and concurrently increased the mRNA levels of proinflammatory cytokines under LPS administration. The results of this study suggest a novel finding that PEPCK may have an important role in neuronal steroidogenesis; which serves as an adaptive response under inflammation.     

StAR protein and steroidogenic enzyme expressions in the rat Harderian gland

The Harderian gland (HG) of the rat (Rattus norvegicus) secretes copious amounts of lipids, such as cholesterol. Here we report a study of the expressions of the StAR protein and key steroidogenic enzymes in the HG of male and female rats. The objective of the present investigation was to ascertain (a) whether the rat HG is involved in steroid production starting with cholesterol, and (b) whether the pattern of gene and protein expressions together with the enzymatic activities display sexual dimorphism. The results demonstrate, for the first time, the expression of StAR gene and protein, and Cyp11a1, Hsd3b1, Hsd17b3, Srd5a1, Srd5a2 and Cyp19a1 genes in the rat HG. StAR mRNA and protein expressions were much greater in males than in females. Immunohistochemical analysis demonstrated a non-homogeneous StAR distribution among glandular cells. Hsd17b3 and Cyp19a1 mRNA levels were higher in males than in females, whereas Srd5a1 mRNA levels were higher in females than in males. No significant differences were observed in mRNA levels of Cyp11a1, Hsd3b1 and Srd5a2 between sexes. Furthermore, the in vitro experiments demonstrated a higher 5α-reductase activity in the female as compared to the male HG vice versa a higher P450 aro activity in males as compared to females. These results suggest that the Harderian gland can be classified as a steroidogenic tissue because it synthesizes cholesterol, expresses StAR and steroidogenic enzymes involved in both androgen and estrogen synthesis. The dimorphic expression and activity of the steroidogenic enzymes may suggest sex-specific hormonal effects into the HG physiology.     

Adrenal glands of mouse and rat do not synthesize androgens.

Human adrenal glands produce considerable amounts of the C-19 steroids dehydroepiandrosterone (DHEA) and androstenedione. To investigate the capability of rodent adrenals to produce these steroids, cell suspensions of mouse and rat adrenal glands were incubated in the absence and presence of adrenocorticotropic hormone (ACTH). Corticosterone levels in the incubation medium increased dramatically in the presence of ACTH, but no significant amounts of 17-hydroxyprogesterone or androstenedione could be detected. This indicates that the adrenals of rat and mouse lack the enzyme 17 alpha-hydroxylase. Absence of plasma cortisol in the presence of high levels of corticosterone confirmed these data. Plasma levels of androstenedione were significantly decreased in castrated male rats as compared to levels observed in intact males, showing the contribution of the testes to the plasma content of androstenedione. Very low levels of androstenedione were observed in female, male and castrated male mice. Plasma concentrations of DHEA were not detectable in intact and castrated male mice and rats. It is concluded that rat and mouse lack the enzyme necessary to synthesize adrenal C-19 steroids and that the adrenals in these animals, therefore, do not contribute to plasma levels of androstenedione and DHEA.     

Quantitative analysis of messenger RNA expression of matrix metalloproteinases (MMP-2 and MMP-9), tissue inhibitor-2 of matrix metalloproteinases (TIMP-2), and steroidogenic enzymes in bovine placentomes during gestation and postpartum

The relationship between the mRNA expression of proteolytic and steroidogenic enzymes in bovine placentomes was examined. Caruncle and cotyledon tissues were collected every 6 hr after spontaneous parturition until the fetal membranes were released. Based on the time of fetal membrane release after parturition, the specimens were classified as follows: (1) the early group, in which the fetal membranes were released within 6 hr after parturition; and (2) the late group, in which the fetal membranes were released 6-12 hr after parturition. The placentomes from a slaughterhouse were additionally collected as samples for the examination of enzymes during the gestation period. The mRNA expression of steroidogenic enzymes in the cotyledon was observed to be higher than that in caruncle tissues; however, the mRNA expression patterns of P450scc and StAR tended to be similar in both placental tissues. On the other hand, although the expression levels of TIMP-2 mRNA in both caruncle and cotyledon tissues were similar, during gestation and postpartum the expression levels of MMP-2 and MMP-9 mRNA were approximately 10 times higher in caruncle than in cotyledon tissue. Marked contrasting changes in mRNA expression patterns between pre- and postpartum periods were observed for MMP-2 and MMP-9 in caruncle tissues and for MMP-9 and TIMP-2 in cotyledon tissues. The present study provides the first evidence that MMP-2, MMP-9, and TIMP-2 mRNAs are expressed in bovine placentomes during the gestational and postpartum periods and suggests that these enzymes, in conjunction with steroidogenic enzymes, mediate fetal membrane detachment after parturition.     

Expression level of enzymes related to in situ estrogen synthesis and clinicopathological parameters in breast cancer patients

In order to evaluate the importance of estrogen production in tumor and surrounding tissues, we measured mRNA expression levels of 5 enzymes participating to estrogen synthesis in situ and 4 breast cancer-related proteins in 27 pairs of tumor and non-malignant tissues. Steroid sulfatase (STS) mRNA was more frequently detected in tumor tissues rather than in their non-malignant counterparts. Estrogen sulfotransferase (EST) was constantly expressed with high level not only in tumor tissues but also in their surrounding non-malignant counterparts. In contrast, mRNA expression levels of aromatase, and 17β-hydroxysteroid dehydrogenase type I and II were relatively low and detected only in small proportion of the patients. We also measured the mRNA expression levels of the same nine genes in tumor tissues of 197 breast cancer patients, and analyzed relationship between the mRNA expression level and the clinicopathological parameters. The mRNA expression levels of STS, aromatase and erbB2 in tumor tissues increased as breast cancer progressed. The tumoral mRNA expression levels of STS, estrogen receptor β, and erbB2 in patients with recurrence were higher than those in patients without recurrence. Upregulation of STS expression plays an important role in tumor progression of human breast cancer and is considered to be responsible for estrogen production in tumor and surrounding tissues.