Two non-reactive ternary complexes of estrogenic 17beta-hydroxysteroid dehydrogenase: crystallization and preliminary structural analysis.

Human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1, EC1.1.1.62) is an important enzyme that catalyses the last step of active estrogen formation. 17β-HSD1 plays a key role in the proliferation of breast cancer cells. The three-dimensional structures of this enzyme and of the enzyme-estradiol complex have been solved (Zhu et al., 1993, J. Mol. Biol. 234:242; Ghosh et al., 1995, Structure 3:503; Azzi et al., 1996, Nature Struct. Biol. 3:665). The determination of the non-reactive ternary complex structure, which could mimic the transition state, constitutes a further critical step toward the rational design of inhibitors for this enzyme (Ghosh et al. 1995, Structure 3:503; Penning, 1996, Endocrine-Related Cancer, 3:41).

To further study the transition state, two non-reactive ternary complexes, 17β-HSD1–EM519-NADP⁺ and 17β-HSD1–EM553-NADP⁺ were crystallized using combined methods of soaking and co-crystallization. Although they belong to the same C2 space group, they have different unit cells, with a=155.59 Å, b=42.82 Å, c=121.15 Å, β=128.5° for 17β-HSD1–EM519-NADP⁺, and a=124.01 Å, b=45.16 Å, c=61.40 Å, β=99.2° for 17β-HSD1–EM553-NADP⁺, respectively. Our preliminary results revealed that the inhibitors interact differently with the enzyme than do the natural substrates.     

The role of 11β-hydroxysteroid dehydrogenase in steroid hormone specificity

11β-hydroxysteroid dehydrogenase (11β-HSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the mineralocorticoid receptor (MR) from occupancy by endogenous glucocorticoids. In aldosterone target cells the type 2 11β-HSD is present, which, in contrast to the type 1 11β-HSD, has very high affinity for its substrate, is unidirectional and prefers NAD as cofactor. cDNAs encoding 11β-HSD2 have been recently cloned from different species, and the cell-specific expression of its mRNA and protein were determined. 11β-HSD2 is expressed in every aldosterone target tissue. Northern analysis revealed that the rabbit 11β-HSD2 is expressed at high levels in the renal collecting duct and at much lower levels in the colon. RT-PCR experiments demonstrated that 11β-HSD2 mRNA is present only in aldosterone target cells within the kidney. We determined the subcellular localization of the rabbit 11β-HSD2 using a chimera encoding 11β-HSD2 and the green fluorescent protein (GFP). This construct was stably transfected into CHO and MDCK cells. The expressed 11β-HSD2/GFP protein retained high enzymatic activity, and its characteristics were undistinguishable from those of the native enzyme. The intracellular localization of this protein was determined by fluorescence microscopy. 11β-HSD2-associated fluorescence was observed as a reticular network over the cytoplasm whereas the plasma membrane and the nucleus were negative, suggesting endoplasmic reticulum (ER) localization. Co-staining with markers for ER proteins, the Golgi membrane, mitochondria and nucleus confirmed that 11β-HSD2 is localized exclusively to the ER. To determine what structural motifs are responsible for the ER localization, we generated deletion mutants missing the C-terminal 42 and 118 amino acids, and fused them to GFP. Similarly as with the intact 11β-HSD2, these mutants localized exclusively to the ER. Both C-terminal deletion mutants completely lost dehydrogenase activity, independently whether activity was determined in intact cells or homogenates. These results indicate that 11β-HSD2 has a novel ER retrieval signal which is not localized to the C-terminal region. In addition, the C-terminal 118 amino acids are essential for NAD-dependent 11β-HSD activity.     

Cortisol as a mineralocorticoid in human disease

The type 2 isozyme of 11β-hydroxysteroid dehydrogenase inactivates cortisol to cortisone and enables aldosterone to bind to the MR. Congenital deficiency of the enzyme results in cortisol-mediated mineralocorticoid excess and arises because of inactivating mutations in the HSD11B2 gene. Inhibition of the enzyme following licorice or carbenoxolone ingestion results in a similar, though milder phenotype and the enzyme is overwhelmed in ectopic ACTH syndrome. Loss of 11β-HSD2 expression may be important in sodium balance and blood pressure control in some patients with renal disease. Finally, while some studies demonstrate impaired 11β-HSD activity in broader populations of patients with hypertension, further studies are required to clarify the role of 11β-HSD2 in ‘essential’ hypertension.     

The type I and type II 11beta-hydroxysteroid dehydrogenase enzymes.

Local tissue concentrations of glucocorticoids are modulated by the enzyme 11β-hydroxysteroid dehydrogenase which interconverts cortisol and the inactive glucocorticoid cortisone in man, and corticosterone and 11-dehydrocorticosterone in rodents. The type I isoform (11β-HSD1) is a bidirectional enzyme but acts predominantly as a oxidoreductase to form the active glucocorticoids cortisol or corticosterone, while the type II enzyme (11β-HSD2) acts unidirectionally producing inactive 11-keto metabolites. There are no known clinical conditions associated with 11β-HSD1 deficiency, but gene deletion experiments in the mouse indicate that this enzyme is important both for the maintenance of normal serum glucocorticoid levels, and in the activation of key hepatic gluconeogenic enzymes. Other important sites of action include omental fat, the ovary, brain and vasculature. Congenital defects in the 11β-HSD2 enzyme have been shown to account for the syndrome of apparent mineralocorticoid excess (AME), a low renin severe form of hypertension resulting from the overstimulation of the non-selective mineralocorticoid receptor by cortisol in the distal tubule of the kidney. Inactivation of the 11β-HSD2 gene in mice results in a phenotype with similar features to AME. In addition, these mice show high neonatal mortality associated with marked colonic distention, and remarkable hypertrophy and hyperplasia of the distal tubule epithelia. 11β-HSD2 also plays an important role in decreasing the exposure of the fetus to the high levels of maternal glucocorticoids. Recent work suggests a role for 11β-HSD2 in non-mineralocorticoid target tissues where it would modulate glucocorticoid access to the glucocorticoid receptor, in invasive breast cancer and as a mechanism providing ligand for the putative 11-dehydrocorticosterone receptor. While previous homologies between members of the SCAD superfamily have been of the order of 20–30% phylogenetic analysis of a new branch of retinol dehydrogenases indicates identities of >60% and overlapping substrate specificities. The availability of crystal structures of family members has allowed the mapping of conserved 11β-HSD domains A–D to a cleft in the protein structure (cofactor binding domain), two parallel β-sheets, and an -helix (active site), respectively.     

3D-structure of human estrogenic 17beta-HSD1: binding with various steroids.

Human estrogenic dehydrogenase (17β-HSD1) catalyses the last step in the biosynthesis of the active estrogens that stimulate the proliferation of breast cancer cells. While the primary substrate for the enzyme is estrone, the enzyme has some activity for the non-estrogenic substrates. To better understand the structure–function relationships of 17β-HSD1 and to provide a better ground for the design of inhibitors, we have determined the crystal structures of 17β-HSD1 in complex with different steroids.

The structure of the complex of estradiol with the enzyme determined previously (Azzi et al., Nature Structural Biology 3, 665–668) showed that the narrow active site was highly complementary to the substrate. The substrate specificity is due to a combination of hydrogen bonding and hydrophobic interactions between the steroid and the enzyme binding pocket. We have now determined structures of 17β-HSD1 in complex with dihydrotestosterone and 20-OH-progesterone. In the case of the C19 androgen, several residues within the enzyme active site make some small adjustments to accommodate the increased bulk of the substrate. In addition, the C19 steroids bind in a slightly different position from estradiol with shifts in positions of up to 1.4 Å. The altered binding position avoids unfavorable steric interactions between Leu 149 and the C19 methyl group (Han et al., unpublished). The known kinetic parameters for these substrates can be rationalized in light of the structures presented. These results give evidence for the structural basis of steroid recognition by 17β-HSD1 and throw light on the design of new inhibitors for this pivotal steroid enzyme.     

Tissue- and temporal-specific regulation of 11beta-hydroxysteroid dehydrogenase type 1 by glucocorticoids in vivo.

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) catalyses the interconversion of active corticosterone and inert 11-dehydrocorticosterone. Short-term glucocorticoid excess upregulates 11β-HSD-1 in liver and hippocampus leading to suggestions that 11β-HSD-1 ameliorates the deleterious effects of glucocorticoid excess by its 11β-dehydrogenase activity. However the predominant activity of 11β-HSD-1 in vivo is 11β-reduction, thus generating active glucocorticoid. We have re-examined the time-course of glucocorticoid regulation of 11β-HSD-1 in the liver, hippocampus and kidney of adult male rats in vivo.

Sham operation markedly reduced 11β-HSD-1 mRNA expression in all tissues, and reduced 11β-HSD bioactivity in liver and hippocampus when compared to untouched controls. Adrenalectomy reduced 11β-HSD-1 expression in all tissues in the short-term (7 days), followed by subsequent recovery of enzyme activity by 21 days in liver and hippocampus. Dexamethasone replacement of adrenalectomised rats attenuated the initial decrease in hepatic 11β-HSD-1 activity, but by 21 days dexamethasone reduced activity compared to control levels.

Thus glucocorticoids regulate 11β-HSD-1 in a complex tissue- and temporal-specific manner. This pattern of regulation suggests glucocorticoids repress 11β-HSD-1 at least in the liver, a pattern of regulation more consistent with the evidence that 11β-HSD-1 is an 11β-reductase in vivo. Operational stress per se down-regulates 11β-HSD-1 which has implications for interpretation and design of in vivo studies of 11β-HSD-1.     

Steroid regulation of progesterone synthesis in a stable porcine granulosa cell line: a role for progestins

The objective of this investigation was to determine the effect of steroid hormones on the synthesis of progesterone in a stable porcine granulosa cell line, JC-410. We also examined the effect of steroid hormones on expression of the genes encoding the steroidogenic enzymes, cytochrome P450-cholesterol side chain cleavage (P450scc) and 3β-hydroxy-5-ene steroid dehydrogenase (3β-HSD). We observed that 48 h exposure of the JC-410 cells to estradiol-17β (estradiol), androstenedione, 5-dihydrotestosterone, levonorgestrel, and 5-cholesten-3β, 25-diol (25-hydroxycholesterol) resulted in stimulation of progesterone synthesis. 25-Hydroxycholesterol augmented progesterone synthesis stimulated by estradiol, 5-dihydrotestosterone, levonorgestrel and 8-bromoadenosine 3′:5′-cyclic monophosphate (8-Br-cAMP). This increase in progesterone synthesis was additive with estradiol, 5-dihydrotestosterone and levonorgestrel, and synergistic with 8-Br-cAMP. Cholera toxin, progesterone, levonorgestrel and androstenedione increased P450scc mRNA levels, whereas estradiol had no effect. Cholera toxin, progesterone and levonorgestrel increased 3β-HSD mRNA levels, but estradiol and androstenedione had no effect. The results were interpreted to mean that estrogens, androgens and progestins regulate progesterone synthesis in the JC-410 cells. The effect of androgens appears to be mediated by stimulation of P450scc gene expression while progestins stimulate both P450scc and 3β-HSD gene expression. Our results support the concept that progesterone is an autocrine regulator of its own synthesis in granulosa cells.     

Effect of nomegestrol acetate on estrone-sulfatase and 17β-hydroxysteroid dehydrogenase activities in human breast cancer cells

It is well recognized that estradiol (E₂) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of the progestin, nomegestrol acetate, on the estrone sulfatase and 17β-hydroxy-steroid dehydrogenase (17β-HSD) activities of MCF-7 and T-47D human breast cancer cells. Using physiological doses of estrone sulfate (E₁S: 5 × 10⁻⁹ M), nomegestrol acetate blocked very significantly the conversion of E₁S to E₂. In the MCF-7 cells, using concentrations of 5 × 10⁻⁶ M and 5 × 10⁻⁵ M of nomegestrol acetate, the decrease of E₁S to E₂ was, respectively, −43% and −77%. The values were, respectively, −60% and −71% for the T-47D cells. Using E₁S at 2 × 10⁻⁶ M and nomegestrol acetate at 10⁻⁵ M, a direct inhibitory effect on the enzyme of −36% and −18% was obtained with the cell homogenate of the MCF-7 and T-47D cells, respectively. In another series of studies, it was observed that after 24 h incubation of a physiological concentration of estrone (E₁: 5 × 10⁻⁹ M) this estrogen is converted in a great proportion to E₂. Nomegestrol acetate inhibits this transformation by −35% and −85% at 5 × 10⁻⁷ M and 5 × 10⁻⁵ M, respectively in T-47D cells; whereas in the MCF-7 cells the inhibitory effect is only significant, −48%, at 5 × 10⁻⁵ M concentration of nomegestrol acetate. It is concluded that nomegestrol acetate in the hormone-dependent MCF-7 and T-47D breast cancer cells significantly inhibits the estrone sulfatase and 17β-HSD activities which converts E₁S to the biologically active estrogen estradiol. This inhibition provoked by this progestin on the enzymes involved in the biosynthesis of E₂ can open new clinical possibilities in breast cancer therapy.     

Progestin regulation of 11beta-hydroxysteroid dehydrogenase expression in T-47D human breast cancer cells

This study examined the enzymatic characteristics and steroid regulation of the glucocorticoid-metabolizing enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) in the human breast cancer cell line T-47D. In cell homogenates, exogenous NAD significantly increased the conversion of corticosterone to 11-dehydrocorticosterone, while NADP was ineffective. There was no conversion of 11-dehydrocorticosterone to corticosterone either with NADH or NADPH demonstrating the lack of reductase activity. In keeping with these results, RT-PCR analysis indicated a mRNA for 11β-HSD2 in T-47D cells, while 11β-HSD1 mRNA levels were undetectable. In T-47D cells treated for 24 h with medroxyprogesterone acetate (MPA), 11β-HSD catalytic activity was elevated 11-fold, while estrone (E₁), estradiol (E₂) and the synthetic glucocorticoid dexamethasone (DEX) were ineffective. The antiprogestin mifepristone (RU486) acted as a pure antagonist of the progestin-enhanced 11β-HSD activity, but did not exert any agonistic effects of its own. In addition, RT-PCR analysis demonstrated that MPA was a potent inducer of 11β-HSD2 gene expression, increasing the steady-state levels of 11β-HSD2 mRNA. Taken together, these results demonstrate that 11β-HSD2 is the 11β-HSD isoform expressed by T-47D cells under steady-state conditions and suggest the existence of a previously undocumented mechanism of action of progestins in breast cancer cells.     

In vitro steroid metabolic studies in human testes II: Metabolism of cholesterol, pregnenolone, progesterone, androstenedione and testosterone by testes of an estrogen-treated man

The effect of long-term in vivo estrogen treatment on in vitro steroidogenesis by the testes of a young man was investigated. In vitro incubation of testicular tissue of this man with ³H-pregnenolone, ³H-progesterone, ³H-androstenedione and ³H-testosterone demonstrated suppression of 17-hydroxylase activity, with little or no effect of the treatment on Δ⁵-3β-hydroxysteroid oxidoreductase, 5a-reductase and aromatase. Increased 20-hydroxysteroid oxidoreductase activity was observed. Determination of intratesticular steroid concentrations led to similar conclusions.     

Two 17beta-hydroxysteroid dehydrogenases (17HSDs) of estradiol biosynthesis: 17HSD type 1 and type 7.

Two 17β-hydroxysteroid dehydrogenases (17HSDs), type 1 and type 7, are enzymes of estradiol biosynthesis, in addition to which rodent type 1 enzymes are also able to catalyze androgens. Both of the 17HSDs are abundantly expressed in ovaries, the type 1 enzyme in granulosa cells and type 7 in luteinized cells. The expression of 17HSD7, which has also been described as a prolactin receptor-associated protein (PRAP), is particularly up-regulated in corpus luteum during the second half of rodent pregnancy. A moderate or slight signal for mouse 17HSD7/PRAP mRNA has also been demonstrated in samples of placenta and mammary gland, for example. Human, but not rodent, 17HSD1 is expressed in placenta, breast epithelium and endometrium in addition to ovaries. A cell-specific enhancer, silencer and promoter in the hHSD17B1 gene participate in the regulation of type 1 enzyme expression. The enhancer consists of several subunits, including a retinoic acid response element, the silencer has a binding motif for GATA factors, and the proximal promoter contains adjacent and competing AP-2 and Sp binding sites.     

Evidence for the DNA binding and adduct formation of estrone and 17β-estradiol after dimethyldioxirane activation

Estrogens, used widely from hormone replacement therapy to cancer treatment, are themselves carcinogenic, causing uterine and breast cancers. However, the mechanism of their carcinogenic action is still not known. Recently, we found that estrone (E₁) and 17β-estradiol (E₂) could be activated by the versatile epoxide-forming oxidant dimethyldioxirane (DMDO), resulting in the inhibition of rat liver nuclear and nucleolar RNA synthesis in a dose-dependent manner in vitro. Since epoxidation is often required for the activation of chemical carcinogens, we proposed that estrogen epoxidation is the underlying mechanism for the initiation of estrogen carcinogenesis (Carcinogenesis 17 (1996) 1957–1961). It is known that initiation requires the binding of a carcinogen to DNA with the formation of DNA adducts. One of the critical tests of our hypothesis is therefore to determine whether E₁ and E₂ after activation are able to bind DNA. This paper reports that after DMDO activation, [³H]E₁ and [³H]E₂ were able to bind to both A-T and G-C containing DNAs. Furthermore, the formation of E₁–DNA and E₂–DNA adducts was detected by ³²P-postlabeling analysis.     

Estrogen-stimulated glucuronidation of dihydrotestosterone in MCF-7 human breast cancer cells

The non-aromatizable androgen dihydrotestosterone (DHT) has been shown to exert a potent inhibitory effect on the proliferation of some human breast cancer cell lines. DHT, however, has little or no significant inhibition on MCF-7 cell proliferation in either the presence or absence of estradiol (E₂). Since the metabolism of DHT into non-active compounds may be responsible for the observed lack of androgenic effect in this cell line, we have investigated the metabolic fate of labeled DHT in MCF-7 cells. A time course incubation was performed with 1 nM [³H]DHT and analysis of the various metabolites formed revealed a time-dependent increase in glucuronidated steroids which was stimulated more than 4-fold by 0.1 nM E₂. The major glucuronidated steroid was androstane-3, 17β-diol in both control and E₂-stimulated cells, comprising 22 ± 1.2% and 30 ± 0.6% of the total radioactivity in the medium, respectively. Other steroid glucuronides observed included DHT, androstane-3β, 17β-diol, and androsterone, all of which were elevated in the E₂-treated cells relative to control values. The present data show that E₂ exerts a stimulatory effect on the glucuronidation of androgens and their metabolites in the estrogen-dependent breast cancer celll line MCF-7. Since glucuronidation is an effective means of cellular elimination of active steroids, such a pathway may be considered as a possible site of regulation of breast cancer cell growth by hormones.     

Some observations on the measurement of estradiol-17β in human plasma by radioimmunoassay

The use of specific and non-specific antisera for estradiol-17β (E₂17β) were compared in the radioimmunoassay of the steroid. The effects of various “blank” mateirials on the standard curve and on the accuracy of recovery of E₂17β added to plasma before and after chromatography on LH-20 Sephadex were examined. It was concluded that the use of the specific antiserum (anti-6-oxoE₂17β -6-(O-carboxymethyl)oxime-bovine serum albumin(antiE₂17β-6-BSA) was an improvement on the non-specific serum anti-E₂17β-17-hemisuccinyl-bovine serum albumin (antiE₂ 17β-17-BSA) following chromatography of extracts. However, although a precise result could be obtained with the anti-E₂17β-6-BSA without the Chromatographic step, recovery of E₂17β added to plasma was only possible if the step was included.

The cross-reactivity of estrone (E₁)with E₂17β using anti-E₂17β-17-BSA as defined by Abraham (J. Clin. Endocr. , 866 (1969) was examined under conditions of constant and of changing E₁:E₂17β ratio.     

Murine NKT cells produce Th17 cytokine interleukin-22

Natural killer T (NKT) cells are known to produce Th17 cytokine IL-17 in addition to Th1/2 cytokines. In this study, the ability of NKT cells to produce IL-22, another Th17 cytokine, was examined in mice. When murine spleen cells were stimulated with α-galactosyl ceramide, a ligand for NKT cells, not only Th1/2 cytokines (IFN-γ, IL-4) but Th17 cytokines (IL-17, IL-22) were produced. NKT cells isolated from splenocytes released IL-17 and IL-22 following CD3, CD3/IL-2 or CD3/CD28 stimulation, in which CD3/CD28 costimulation was most effective. Production of IL-17 and IL-22 in CD4+ and CD8+ T cells from splenocytes was little, if any, even after CD3/CD28 costimulation. Treatment with IL-6/TGF-β decreased CD3/CD28-stimulated production of IL-22, but not that of IL-17, in NKT cells. These findings show for the first time that NKT cells are a cell source of IL-22, and that expression of two Th17 cytokines might be regulated in NKT cells by different mechanisms.     

Kinetic analysis of enzymic activities: Prediction of multiple forms of 17β-hydroxysteroid dehydrogenase

An overview of the application of kinetic methods to the delineation of 17β-hydroxysteroid dehydrogenase (17β-HSD) heterogeneity in mammalian tissues is presented. Early studies of 17β-HSD activity in animal liver and kidney subcellular fractions were suggestive of multiple forms of the enzyme. Subsequently, detailed characterization of activity in cytosol and subcellular membrane fractions of human placenta, with particular emphasis on inhibition kinetics, yielded evidence of two kinetically-differing forms of 17β-HSD in that organ. Gene cloning and transfection experiments have confirmed the identity of these two proteins as products of separate genes. 17β-HSD type 1 is a cytosolic enzyme highly specific for C₁₈ steroids such as 17β-estradiol (E₂) and estrone (E₁). 17β-HSD type 2 is a membrane bound enzyme reactive with testosterone (T) and androstenedione (A), as well as E₂ and E₁. Useful parameters for the detection of multiple forms of 17β-HSD appear to be the E₂/T activity ratio, NAD/NADP activity ratios, steroid inhibitor specificity and inhibition patterns over a wide range of putative inhibitor concentrations. Evaluation of these parameters for microsomes from samples of human breast tissue suggests the presence of 17β-HSD type 2. The 17β-HSD enzymology of human testis microsomes appears to differ from placenta. Analysis of human ovary indicates granulosa cells are particularly enriched in the type 1 enzyme with type 2-like activity in stroma/theca. Mouse ovary appears to contain forms of 17β-HSD which differ from 17β-HSD type 1 and type 2 in their kinetic properties.     

Estrone sulfate-sulfatase and 17β-hydroxysteroid dehydrogenase activities: a hypothesis for their role in the evolution of human breast cancer from hormone-dependence to hormone-independence

The evaluation of estrogens (estrone, estradiol, and their sulfates) in the breast tissue of post-menopausal patients with breast cancer indicates high levels, particularly of estrone sulfate (E₁ S) which is 15–25 times higher than in the plasma. Breast cancer tissue contains the enzymes necessary for local synthesis of estradiol and it was demonstrated that, despite the presence of the sulfatase and its messenger in hormone-dependent and hormone-independent breast cancer cells, this enzyme operates particularly in hormone-dependent cells. Different progestins: Nomegestrol acetate, Promegestone, progesterone, as well as Danazol, can block the conversion of E₁ S to E₂ very strongly in hormone-dependent breast cancer cells. The last step in the formation of estradiol is the conversion of E₁ to this estrogen by the action of 17β-hydroxysteroid dehydrogenase. This activity is preferentially in the reductive direction (formation of E₂) in hormone-dependent cells, but oxidative (E₂ → E₁) in hormone-independent cells. Using intact hormone-dependent cells it was observed that Nomegestrol acetate can block the conversion of E₁ to E₂. It is concluded, firstly, that in addition to ER mutants other factors are involved in the transformation of hormone-dependent breast cancer to hormone-independent, this concerns the enzymatic activity in the formation of E₂; it is suggested that stimulatory or repressive factor(s) involved in the enzyme activity are implicated as the cancer evolves to hormone-independence; secondly, different drugs can block the conversion of E₁ S to E₂. Clinical trials of these “anti-enzyme” substances in breast cancer patients could be the next step to investigate new therapeutic possibilities for this disease.     

Distribution of 17β-hydroxysteroid dehydrogenase gene expression and activity in rat and human tissues

The interconversion of estrone (E₁) and 17β-estradiol (E₂), androstenedione (4-ene-dione) and testosterone (T), as well as dehydroepiandrosterone and androst-5-ene-3β,17β-diol is catalyzed by 17β-hydroxysteroid dehydrogenase (17β-HSD). The enzyme 17β-HSD thus plays an essential role in the formation of all active androgens and estrogens in gonadal as well as extragonadal tissues. The present study investigates the tissue distribution of 17β-HSD activity in the male and female rat as well as in some human tissues and the distribution of 17β-HSD mRNA in some human tissues. Enzymatic activity was measured using ¹⁴C-labeled E₁, E₂, 4-ene-dione and T as substrates. Such enzymatic activity was demonstrated in all 17 rat tissues examined for both androgenic and estrogenic substrates. While the liver had the highestlevel of 17β-HSD activity, low but significant levels of E₂ as well as T formation were found in rat brain, heart, pancreas and thymus. The oxidative pathway (E₂→E₁, T→4-ene-dione) was favored over the reverse reaction in almost all rat tissues while in the human, almost equal rates were found in most of the 15 tissues examined. The widespread distribution of 17β-HSD in rat and human tissues clearly indicates the importance of this enzyme in peripheral sex steroid formation or intracrinology.     

17β-Hydroxysteroid dehydrogenase activity in endometrial cancer cells: Different metabolic pathways of estradiol in hormone-responsive and non-responsive intact cells

In this paper we report that two human long-term endometrial cancer cell lines, Ishikawa and HEC-1A, exhibit quite different abilities in metabolizing estrogens. As a matter of fact, incubation of Ishikawa cells with close-to-physiological concentrations of estradiol (E₂) as precursor resulted in: (1) elevated formation (up to 90%) of E₂-sulphate (E₂-S), using lower precursor concentrations; (2) very limited conversion to estrone (E₁) (< 10% at 24 h incubation), as either free or sulphate; and (3) low but consistent production of other estrogen derivatives, such as 2-hydroxy-estrogens and estriol. Conversely, scant amounts (if any) of E₂-S were found in HEC-1A cells, while no detectable formation of other estrogen metabolites could be observed after 24 h. On the other hand, E₁ production was significantly greater (nearly 60% at 24 h) than in Ishikawa cells, a large proportion of E₁ (over 50% of the total) being formed after only 6 h incubation using time-course experiments. The hypothesis that E₂ metabolism could be minor in Ishikawa cells as a consequence of the high rate of E₂-S formation encountered is contradicted by the evidence that conversion to E₁ also remains limited in the presence of much lower E₂-S amounts, seen using higher molar concentrations of precursor. Overall, we observe that 17β-hydroxysteroid dehydrogenase (17β-HSD) activity diverges significantly in intact Ishikawa and HEC-1A endometrial cancer cells. This difference could not merely be accounted for by the diverse amounts of substrate (E₂) available to the cells, nor may it be imputed to different levels of endogenous estrogens. It should rather be sought in different mechanisms controlling 17β-HSD activity or, alternatively, in the presence of distinct isoenzymes in the two different cell types.