Human 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase from placenta: expression in nonsteroidogenic cells of a protein that catalyzes the dehydrogenation/isomerization of C21 and C19 steroids

The isolation, cloning, and expression of a cDNA insert complementary to mRNA encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase is reported. The insert contains an open reading frame encoding a protein of 372 amino acids, the initial 29 amino acids corresponding to the N-terminal sequence identified from the purified human placental microsomal enzyme. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental microsomal 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase, as detected by immunoblot analysis, and catalyzed the conversion of 17 alpha-hydroxypregnenolone to 17 alpha-hydroxyprogesterone, pregnenolone to progesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, very efficiently oxidized 5 alpha-androstan-3 beta,17 beta-diol to 5 alpha-dihydrotestosterone and, upon addition of NADH, reduced 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol. Thus, the dehydrogenation/isomerization steps of steroid biosynthesis can be catalyzed by a single polypeptide chain, which can metabolize all of the major physiological substrates.     

Cloning and expression of cDNA encoding human placental estrogen sulfotransferase

Using two oligoprimers derived from the bovine placental estrogen sulfotransferase sequence, we amplified a probe for human placental estrogen sulfotransferase. Using this probe to screen a human placental cDNA library constructed in λgt11, we isolated a cDNA clone of 1.3 kb encoding human estrogen sulfotransferase. DNA analysis predicts a protein of 295 amino acids with a calculated molecular weight of 34199. Alignment of the amino acid sequence with other sulfotransferases indicates that human placental estrogen sulfotransferase shares 68.6, 68.2 and 65.9% similarity with bovine placental, guinea pig adrenocortical, and rat liver estrogen sulfotransferase, respectively. It shows also 95.6, 57.6, 85.3, and 54.2% similarity to human phenol, human DHEA, rat phenol, and rat hydroxysteroid sulfotransferase, respectively. Transfection of expression vectors encoding human estrogen sulfotransferase and dehydroepiandrosterone (DHEA) sulfotransferase in human adrenal adenocarcinoma SW-13 cells indicates that estrogen sulfotransferase transforms estrone more specifically, whereas DHEA sulfotransferase is more specific for DHEA and pregnenolone.     

A working hypothesis for the regulation of steroidogenesis and germ cell development in the gonads by glucocorticoids and 11β-hydroxysteroid dehydrogenase (11βHSD)

The relationship between glucocorticoid secretion from the adrenal gland and gonadal function has previously been attributed to central inhibition by the adrenal steroids of pituitary gonadotropin output. This review focuses on the direct actions of glucocorticoids within the gonads, including positive effects on germ cell maturation and both positive and negative effects on the stimulation of gonadal steroidogenesis by LH and FSH. In addition, we address the role in the gonads of 11β-hydroxysteroid dehydrogenase (11βHSD), which interconverts the glucocorticoids with their inactive 11-ketosteroid derivatives. To date, two isoforms of 11βHSD have been described. 11βHSD1, purified and cloned from the liver, has a relatively low affinity for glucocorticoids and acts instead as an 11-oxoreductase, whereas the high affinity 11βHSD2, first identified in the kidney, acts as an efficient 11β-dehydrogenase to inactivate physiological concentrations of glucocorticoid. We propose that in the gonads, 11βHSD1 promotes the positive effects of glucocorticoids on germ cell maturation (by increasing the local concentration of active glucocorticoids), whereas a high affinity 11β-dehydrogenase activity, consistent with that of 11βHSD2, inactivates glucocorticoids and so protects luteal cells from the inhibitory effects of these steroids during the luteal phase of the ovarian cycle.     

Human cholesterol side-chain cleavage enzyme, P450scc: cDNA cloning, assignment of the gene to chromosome 15, and expression in the placenta.

Conversion of cholesterol to pregnenolone is mediated by P450scc [cholesterol, reduced-adrenal-ferrodoxin: oxygen oxidoreductase (side-chain-cleaving), EC 1.14.15.67]. RNA from several human adrenal samples was translated in vitro and immunoprecipitated with anti-bovine P450scc, indicating that P450scc mRNA represents about 0.5% of human adrenal mRNA in normal, hypertrophied, and malignant adrenals. A 1626-base-pair human adrenal P450scc cDNA was cloned in bacteriophage lambda gt10. Primer extension data indicated P450scc mRNA is about 1850 bases long and that all adrenal P450scc mRNA has the same 5' end. A full-length clone containing 1821 bases was obtained from a human testis cDNA library to yield the complete sequence. The encoded human preP450scc contains 521 amino acids with a molecular weight of 60189.65. The testis and adrenal sequences were identical; the human cDNA and amino acid sequences are 82% and 72% homologous, respectively, with the bovine sequences. P450scc cDNA was used to probe DNA from a panel of mouse-human somatic cell hybrids, showing that the single human P450scc gene lies on chromosome 15. The human P450scc gene is expressed in the placenta in early and midgestation; primary cultures of placental tissue indicate P450scc mRNA accumulates in response to cyclic AMP.     

Immunolocalization of murine type VI 3β-hydroxysteroid dehydrogenase in the adrenal gland,testis, skin,and placenta

The enzyme 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) is essential for the biosynthesis of all active steroid hormones, such as those secreted from the adrenal gland, testis, ovary, skin and placenta. The 3β-HSD enzymes exist in multiple isoforms in humans and rodents. To date, six different isoforms have been identified in the mouse, and these isoforms are speculated to play different roles in different tissues. We previously showed that the murine type VI 3β-HSD isoform (Hsd3b6) is expressed specifically in the aldosterone-producing zona glomerulosa cells within the adrenal gland and that its overexpression causes abnormally increased aldosterone synthesis, revealing a crucial (or rate-limiting) role of this enzyme in steroidogenesis. However, potential contributions of this enzyme to the steroid hormone synthesis outside the adrenal glands are poorly understood. This paucity of knowledge is partly because of the lack of isoform-specific antibody that can be used for immunohistochemistry. Here, we report the development and characterization of specific antibody to Hsd3b6 and show the results of immunohistochemistry for the adrenal gland, testis, ovary, skin and placenta. As expected, Hsd3b6 immunoreactivities within the adrenal gland were essentially confined to the zona glomerulosa cells, where aldosterone is produced. By contrast, no immunopositive cells were observed in the zona fasciculata, which is where corticosterone is produced. In the gonads, while the ovaries did not show any detectable immunoreactivity to Hsd3b6, the testes displayed intense immunoreactivities within the interstitial Leydig cells, where testosterone is produced. In the skin, positive immunoreactivities to Hsd3b6 were only seen in the sebaceous glands, suggesting a specific role of this enzyme in sebaceous function. Moreover, in the placenta, Hsd3b6 was specifically found in the giant trophoblast cells surrounding the embryonic cavity, which suggests a role for this enzyme in local progesterone production that is required for proper embryonic implantation and/or maintenance of pregnancy. Taken together, our data revealed that Hsd3b6 is localized in multiple specific tissues and cell types, perhaps thereby involved in biosynthesis of a number of tissue-specific steroid hormones with different physiological roles.     

Low expression of 3 beta-hydroxy-5-ene steroid dehydrogenase gene in human fetal adrenals in vivo; adrenocorticotropin and protein kinase C-dependent regulation in adrenocortical cultures.

Human fetal adrenals are very active in steroid production. They make large amounts of dehydroepiandrosterone sulfate which is further converted to estrogens in placenta. Fetal adrenals cannot make cortisol efficiently from cholesterol or pregnenolone, but they can convert progesterone to cortisol. To clarify the molecular basis of the very low activity of 3 beta-hydroxy-5-ene steroid dehydrogenase (3 beta HSD) in human fetal adrenals we studied the expression of 3 beta HSD gene in fetal adrenals in vivo and in culture conditions. Human adult adrenals, placenta and a testicular Leydig cell tumor clearly expressed 3 beta HSD gene when studied by Northern blotting, but fetal adrenals and ovaries had no detectable 3 beta HSD mRNA by this method. Polymerase chain reaction analysis of cDNA samples derived from different human tissues revealed 3 beta HSD gene expression in placenta, adult adrenal and adult ovarian granulosa cells after 25 cycles of amplification. Fetal adrenal samples became positive only after additional amplification cycles, which verifies the very low expression of 3 beta HSD gene in fetal adrenals. In cell culture conditions both ACTH and a protein kinase C regulator 12-O-tetradecanoyl phorbol-13-acetate induced 3 beta HSD gene expression. We conclude: 1) the very low activity of 3 beta HSD in human fetal adrenals is due to the low expression of this gene; 2) both cAMP and protein kinase C-dependent mechanisms regulate 3 beta HSD gene expression in adrenocortical cells.     

Expression of 11beta-hydroxylase in rat Leydig cells.

11Beta-hydroxy (11beta-OH) derivatives of certain steroids function as inhibitors of 11beta-hydroxysteroid dehydrogenase isoform 1 (11betaHSD1), an enzyme expressed in Leydig cells that catalyzes the reversible oxidation of biologically active glucocorticoids to inactive 11-dehydro metabolites. 11beta-Hydroxylase is an adrenal enzyme responsible for glucocorticoid biosynthesis, catalyzing 11beta-hydroxylation of steroids and thus producing 11beta-OH-steroid derivatives. The aims of the present study were 1) to examine whether 11beta-hydroxylase is expressed in testis, 2) to define the biochemical characteristics of the testicular form of this enzyme, and 3) to establish whether 11beta-hydroxylated steroids inhibit Leydig cell 11betaHSD1 activities. 11beta-Hydroxylase mRNA was detected in purified rat Leydig cells by RT-PCR. Sequencing confirmed that the PCR products had 100% identity with the published rat adrenal enzyme cDNA sequence. Immunohistochemistry and Western blot analysis using a mouse monoclonal antibody confirmed the expression of 11beta-hydroxylase protein in Leydig cells. Moreover, 11beta-hydroxylase activity, synthesis of corticosterone from 11-deoxycorticosterone, was measurable in Leydig cells, and the K(m) and maximum velocity values were 7.28 +/- 0. 92 microM and 1.13 +/- 0.04 micromol/10(6) cell x h, respectively. When assayed in Leydig cells, several 11beta-hydroxylated steroids were efficient inhibitors of 11betaHSD1 dehydrogenase activity, whereas other 11-keto compounds were effective as inhibitors of oxidoreductase activity. These results provide the first direct evidence that rat Leydig cells express 11beta-hydroxylase, which may be involved in the regulation of glucocorticoid metabolism within the testis through local biosynthesis of endogenous inhibitors of 11betaHSD1.     

Regulation of adrenal 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase expression and activity by adrenocorticotropin and corticosterone in the rat.

This study describes the regulation of adrenal 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) expression and activity by ACTH and corticosterone, alone or in combination, in intact male and female rats as well as the effect of ACTH on 3 beta HSD expression and activity in the adrenals of hypophysectomized female animals. The effect of treatment on total 3 beta HSD mRNA levels was measured by dot blot hybridization using rat 3 beta HSD cDNA, while the specific regulation of type I and type II 3 beta HSD mRNAs was analyzed by ribonuclease protection assay. The concentration of 3 beta HSD protein was measured by Western blot, using cross-reacting antibodies raised against purified human placental 3 beta HSD, while 3 beta HSD enzymatic activity was measured by the conversion of [14C]dehydroepiandrosterone into [14C]androstenedione. The present data show that the trophic effect of ACTH on male and female rat adrenals is accompanied by increases in total 3 beta HSD mRNA, enzymatic activity, and protein content. Hypophysectomy, on the other hand, causes a marked decrease in 3 beta HSD mRNA levels and enzymatic activity, which is completely reversed by administration of ACTH. On the other hand, corticosterone treatment results in a marked inhibition of 3 beta HSD mRNA levels, enzymatic activity, and protein content in intact animals; this effect is probably mediated by a decrease in ACTH secretion. The present data show that ACTH and corticosterone, via its inhibitory action on ACTH secretion, have potent and opposite effects on the expression of two 3 beta HSD genes in the rat adrenal; a parallel effect is observed on both type I and II 3 beta HSD. Such data suggest that 3 beta HSD could well play a major role in the regulation of steroid formation in the adrenal cortex.     

Influence of placental 11β-hydroxysteroid dehydrogenase (11β-HSD) inhibition on glucose metabolism and 11β-HSD regulation in adult offspring of rats

Placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) converts glucocorticoids to 11-keto-products and is believed to play an important role in protecting fetuses from higher maternal glucocorticoid levels. Recent reports have speculated that prenatal glucocorticoid exposure leads to fetal growth retardation and adult offspring hypertension and hyperglycemia. To investigate the effects of placental 11β-HSD2 inhibition on glucose metabolism and the 11β-HSD system in adult offspring, pregnant rats were treated with daily injections of carbenoxolone (CBX), an inhibitor of 11β-HSD. The offspring of the maternal CBX treatment group showed reduced birth weight (treated v control, 5.6 ± 0.5 v 6.4 ± 0.4 g, P < .0001). In adult offspring of the maternal CBX treatment group, plasma hemoglobin A_1c was significantly increased (7.3% ± 1.8% v 4.8% ± 0.3%, P < .01) and glucose intolerance was shown on the oral glucose tolerance test. The gene expression of hepatic 11β-HSD1 and renal 11β-HSD2 was decreased 87.6% (P < .05) and 52.3% (P < .01) in adult offspring of the maternal CBX treatment group, whereas renal 11β-HSD1 was not significantly altered. The change in 11β-HSD activity corresponded to the change in the gene expression. These results suggest that inhibition of placental 11β-HSD2 causes growth retardation, glucose intolerance, and partial suppression of the 11β-HSD system in the offpsring.     

Immunoelectron microscopic localization of three key steroidogenic enzymes (cytochrome P450(scc), 3 beta-hydroxysteroid dehydrogenase and cytochrome P450(c17)) in rat adrenal cortex and gonads.

The biosynthesis of steroid hormones in endocrine steroid-secreting glands results from a series of successive steps involving both cytochrome P450 enzymes, which are mixed-function oxidases, and steroid dehydrogenases. So far, the subcellular distribution of steroidogenic enzymes has been mostly studied following subcellular fractionation, performed in placenta and adrenal cortex. In order to determine in situ the intracellular distribution of some steroidogenic enzymes, we have investigated the ultrastructural localization of the three key enzymes: P450 side chain cleavage (scc) which converts cholesterol to pregnenolone; 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) which catalyzes the conversion of 3 beta-hydroxy-5-ene steroids to 3-oxo-4-ene steroids (progesterone and androstenedione); and P450(c17) which is responsible for the transformation of C(21) into C(19) steroids (dehydroepiandrosterone and androstenedione). Immunogold labeling was used to localize the enzymes in rat adrenal cortex and gonads. The tissues were fixed in 1% glutaraldehyde and 3% paraformaldehyde and included in LR gold resin. In the adrenal cortex, both P450(scc) and 3 beta-HSD immunoreactivities were detected in the reticular, fascicular and glomerular zones. P450(scc) was exclusively found in large mitochondria. In contrast, 3 beta-HSD antigenic sites were mostly observed in the endoplasmic reticulum (ER) with some gold particles overlying crista and outer membranes of the mitochondria. P450(c17) could not be detected in adrenocortical cells. In the testis, the three enzymes were only found in Leydig cells. Immunolabeling for P450(scc) and 3 beta-HSD was restricted to mitochondria, while P450(c17) immunoreactivity was exclusively observed in ER. In the ovary, P450(scc) and 3 beta-HSD immunoreactivities were found in granulosa, theca interna and corpus luteum cells. The subcellular localization of the two enzymes was very similar to that observed in adrenocortical cells. P450(c17) could also be detected in theca interna cells of large developing and mature follicles. As observed in Leydig cells, P450(c17) immunolabeling could only be found in the ER. These results indicate that in different endocrine steroid-secreting cells P450(scc), 3 beta-HSD and P450(c17) have the same association with cytoplasmic organelles (with the exception of 3 beta-HSD in Leydig cells), suggesting similar intracellular pathways for biosynthesis of steroid hormones.     

Isolation of a full-length cDNA insert encoding human aromatase system cytochrome P-450 and its expression in nonsteroidogenic cells.

The isolation and cloning of a full-length cDNA insert complementary to mRNA encoding human aromatase system cytochrome P-450 is reported. The insert contains an open reading frame encoding a protein of 503 amino acids. This gene is clearly a member of the cytochrome P-450 gene superfamily, because the sequence contains regions of marked homology to those of other members, notably a putative membrane-spanning region, I helix, Ozols, and heme-binding regions. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental aromatase system cytochrome P-450, as detected by immunoblot analysis, and catalyzed the aromatization of androstenedione, testosterone, and 16 alpha-hydroxyandrostenedione. This activity was inhibited by the known aromatase inhibitors, 4-hydroxyandrostenedione and econazole. Thus the several steps involved in the aromatization reaction appear to be catalyzed by a single polypeptide chain, which can metabolize the three major physiological substrates.     

A new inherited variant of the 3 beta-hydroxysteroid dehydrogenase-isomerase deficiency syndrome: evidence for the existence of two isoenzymes

The clinical and endocrine features of a unique form of adrenal insufficiency secondary to an inherited deficiency of 3 beta-hydroxysteroid dehydrogenase-isomerase (3-HSD) were studied. The propositus was a 19-yr-old man with a history of repeated episodes of acute adrenal crisis. Family study disclosed that a 6-yr-old female sibling also was affected, and a third sibling had died during the course of an adrenal crisis. The diagnosis of adrenal insufficiency was established on the basis of extremely low serum cortisol levels and urinary 17-hydroxycorticosteroid excretion with concomitantly elevated serum ACTH levels and lack of cortisol response to ACTH administration. Impairment of C-21 steroid 3-HSD activity was strongly suggested by persistency elevated serum 17-hydroxypregnenolone to 17-hydroxyprogesterone and pregnenolone to progesterone ratios, their significant increase after ACTH administration, and their return to normal during cortisol therapy in both patients. Nevertheless, the serum dehydroepiandrosterone to androstenedione ratio, both basally and after ACTH and/or hCG stimulation, was normal. These findings coupled with the normal phenotypic development and onset of puberty in the two patients indicated intact C-19 steroid 3-HSD activity. The overall results indicate an inherited impairment of 3-HSD activity confined only to C-21 steroid substrates and, thus, suggest the existence of at least two 3-HSD isoenzymes under independent genetic regulation.     

Cellular localization of 11β-hydroxysteroid dehydrogenase 2 gene expression in the ovine adrenal gland

The cellular localization of 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) gene expression in the ovine adrenal gland was determined by in situ hybridization histochemistry. 11β-HSD2 mRNA was localized exclusively to the adrenal cortex of the adult sheep, and within the cortex the mRNA was highly expressed in the zona fasciculata and zona reticularis with relatively low expression in the zona glomerulosa. Radiometric conversion assay using adrenal cortical tissues revealed extremely high levels of 11β-HSD activity which was characteristic of 11β-HSD2 in that it was NAD-dependent and displayed a K_m for cortisol of 41 ± 4 nM. This indicates that 11β-HSD2 mRNA within the ovine adrenal gland is translated and functional with respect to enzymatic activity. In marked contrast, 11β-HSD1 mRNA was undetectable in either the cortex or medulla of adult sheep adrenal glands. In conclusion, we have demonstrated, for the first time, the zonal localization of 11β-HSD2 mRNA and the presence of 11β-HSD2 activity in the adult sheep adrenal cortex. The adrenal 11β-HSD2 may function to (1) regulate the rate of cortisol secretion by adrenocortical cells; (2) protect these cells from high levels of locally produced glucocorticoids; and/or (3) provide an important source of circulating cortisone, which can be activated by the action of 11β-HSD1 reductase in organs such as the liver.     

Isolation of a rat liver delta-aminolevulinate dehydrase (ALAD) cDNA clone: evidence for unequal ALAD gene dosage among inbred mouse strains.

We have isolated several cDNA clones encoding delta-aminolevulinate dehydrase [ALAD; porphobilinogen synthase; 5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing), EC 4.2.1.24], the second enzyme in the heme biosynthetic pathway. We used a rabbit polyclonal antibody developed against the purified 35-kDa subunit of rat liver ALAD to screen a lambda gt11 cDNA expression library constructed from rat liver mRNA. A prototype clone (ALAD-1) contained a 680-base-pair insert and expressed a 140-kDa beta-galactosidase gene cDNA insert fusion protein immunoreactive with both polyclonal and monoclonal anti-ALAD. Identity of ALAD-1 was verified by hydridization to ALAD mRNA that had been enriched via immunopurification of rat liver polysomes with anti-ALAD. Intensity of such hybridization to a 1500-nucleotide-long mRNA was approximately equal to 200-fold greater than that realized with whole liver mRNA, a result consistent with the degree of immunoenrichment of ALAD mRNA found independently by analysis of cell-free translation products. A second ALAD cDNA clone (ALAD-3) was isolated when the rat liver cDNA expression library was rescreened with ALAD-1. The identity of both ALAD cDNA clones was established by correspondence between their nucleotide sequence and the reported amino-terminal protein sequence of bovine ALAD. Hybridization of ALAD cDNA to mouse genomic DNA indicates that the previously unexplained incremental differences in ALAD enzymatic activity among inbred mouse strains has arisen through alterations in ALAD gene dose.     

Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7α-hydroxy dehydroepiandrosterone and 7α-hydroxy pregnenolone

Steroids produced locally in brain (neurosteroids), including dehydroepiandrosterone (DHEA), influence cognition and behavior. We previously described a novel cytochrome P450, Cyp7b, strongly expressed in rat and mouse brain, particularly in hippocampus. Cyp7b is most similar to steroidogenic P450s and potentially could play a role in neurosteroid metabolism. To examine the catalytic activity of the enzyme mouse Cyp7b cDNA was introduced into a vaccinia virus vector. Extracts from cells infected with the recombinant showed NADPH-dependent conversion of DHEA (K_m, 13.6 μM) and pregnenolone (K_m, 4.0 μM) to slower migrating forms on thin layer chromatography. The expressed enzyme was less active against 25-hydroxycholesterol, 17β-estradiol and 5α-androstane-3β,17β-diol, with low to undetectable activity against progesterone, corticosterone, and testosterone. On gas chromatography and mass spectrometry of the Cyp7b metabolite of DHEA the retention time and fragmentation patterns were identical to those obtained with authentic 7α-hydroxy DHEA. The reaction product also comigrated on thin layer chromatography with 7α-hydroxy DHEA but not with 7β-hydroxy DHEA; when [7α-³H]pregnenolone was incubated with Cyp7b extracts the extent of release of radioactivity into the medium suggested that hydroxylation was preferentially at the 7α position. Brain extracts also efficiently liberated tritium from [7α-³H]pregnenolone and converted DHEA to a product with a chromatographic mobility indistinguishable from 7α-hydroxy DHEA. We conclude that Cyp7b is a 7α-hydroxylase participating in the synthesis, in brain, of neurosteroids 7α-hydroxy DHEA, and 7α-hydroxy pregnenolone.