Hydroxysteroid Dehydrogenase Activity Research Articles

Modulation of oestrone sulphatase activity in breast cancer cell lines by growth factors

Currently there is much interest in the role that growth factors may play in the development of human breast tumours. We have shown previously that growth factors secreted by breast tumours may influence the activity of oestradiol hydroxysteroid dehydrogenase, the enzyme which catalyses the interconversion of oestrone (E 1) and oestradiol. As the formation of E 1 from its sulphate (E 1S) by oestrone sulphatase may be quantitatively more important than production from androstenedione via aromatase, we have studied the effect of insulin-like growth factor-1 (IGF-I) and basic fibroblast growth factor (bFGF) on oestrone sulphatase activity in the hormone-dependent MCF-7 and the hormone-independent MDA-MB-231 breast cancer cell lines. In both these cell types, bFGF (1–200 ng/ml) and IGF-I (25–200 ng/ml) significantly stimulated oestrone sulphatase activity in a dose-dependent manner (by 8–60%) after 48 h. Additionally, cycloheximide significantly inhibited (by 90–120%) this stimulation of oestrone sulphatase activity by the two growth factors in both MCF-7 and MDA-MB-231 cells. Basal oestrone sulphatase activity was higher in the oestrogen receptor, ER - ve MDA-MB-231 cells than in the ER + ve MCF-7 breast cancer cells. We conclude that these growth factors, believed to be secreted by breast tumours, may induce enzymes of oestrogen synthesis and hence increase local production of oestrogens.

Characterization of multiple forms of carbonyl reductase from chicken liver.

Three enzyme forms (CR1, CR2 and CR3) of carbonyl reductase were purified from chicken liver with using 4-benzoylpyridine as a substrate. CR1 was a dimeric enzyme composed of two identical 25-kD subunits. CR2 and CR3 were monomeric enzymes whose molecular weights were both 32 kD. CR1 exhibited 17 beta-hydroxysteroid dehydrogenase activity as well as carbonyl reductase activity in the presence of both NADP(H) and NAD(H). CR2 and CR3 had similar properties with regard to substrate specificity and inhibitor sensitivity. They could exhibit the activity only with NADPH and had no hydroxysteroid dehydrogenase activity. CR2 and CR3 cross-reacted with anti-chicken kidney carbonyl reductase antibody, though CR1 did not. The results suggest that CR1 is a hydroxysteroid dehydrogenase, and CR2 and CR3 are similar to each other and to the kidney enzymes.

Localization of an 11 beta hydroxysteroid dehydrogenase activity to the distal nephron. Evidence for the existence of two species of dehydrogenase in the rat kidney.

An 11 beta hydroxysteroid dehydrogenase (11 beta HSD) activity has been localized in the rat kidney by a histochemical technique which links steroid metabolism with the production of a color reaction. Oxidation of 11 beta-hydroxyandrostenedione was observed in cortical distal convoluted tubules and in medullary collecting ducts. Carbenoxolone abolished staining, no reaction was obtained with androstenedione hydroxylated at the 17 or 19 position, and oxidation of 11 beta-hydroxyandrostenedione was nicotinamide-adenine dinucleotide (NAD) dependent. These results demonstrate the presence of a dehydrogenase activity separate from the nicotinamide-adenine dinucleotide phosphate (NADP)-dependent 11 beta hydroxysteroid dehydrogenase recently purified and cloned from rat liver. We have named this activity 11 beta HSD2 to distinguish it from the NADP-dependent 11 beta HSD. Histological studies showed that 11 beta HSD2 activity does not correlate with the immunocytochemical localization of the previously defined 11 beta HSD enzyme, but rather the 11 beta HSD2 activity is localized in the distal tubules of the rat kidney. In this respect 11 beta HSD2 colocalizes with the mineralocorticoid receptor. No reaction product was obtained using cortisol or corticosterone as substrate with either NAD or NADP as cofactor. Furthermore incubation of tissue sections with 11 beta androstenedione in the presence of deoxycorticosterone completely inhibited cytochemical staining. We interpret these results as evidence of 20 reductase activity which uses the reduced cofactor at the expense of the color reaction. These results support the crucial role played by an 11 beta hydroxysteroid dehydrogenase in the local protection of type I receptors in mineralocorticoid selective tissues.

Localization of an 11 beta hydroxysteroid dehydrogenase activity to the distal nephron. Evidence for the existence of two species of dehydrogenase in the rat kidney

An 11 beta hydroxysteroid dehydrogenase (11 beta HSD) activity has been localized in the rat kidney by a histochemical technique which links steroid metabolism with the production of a color reaction. Oxidation of 11 beta-hydroxyandrostenedione was observed in cortical distal convoluted tubules and in medullary collecting ducts. Carbenoxolone abolished staining, no reaction was obtained with androstenedione hydroxylated at the 17 or 19 position, and oxidation of 11 beta-hydroxyandrostenedione was nicotinamide-adenine dinucleotide (NAD) dependent. These results demonstrate the presence of a dehydrogenase activity separate from the nicotinamide-adenine dinucleotide phosphate (NADP)-dependent 11 beta hydroxysteroid dehydrogenase recently purified and cloned from rat liver. We have named this activity 11 beta HSD2 to distinguish it from the NADP-dependent 11 beta HSD. Histological studies showed that 11 beta HSD2 activity does not correlate with the immunocytochemical localization of the previously defined 11 beta HSD enzyme, but rather the 11 beta HSD2 activity is localized in the distal tubules of the rat kidney. In this respect 11 beta HSD2 colocalizes with the mineralocorticoid receptor. No reaction product was obtained using cortisol or corticosterone as substrate with either NAD or NADP as cofactor. Furthermore incubation of tissue sections with 11 beta androstenedione in the presence of deoxycorticosterone completely inhibited cytochemical staining. We interpret these results as evidence of 20 reductase activity which uses the reduced cofactor at the expense of the color reaction. These results support the crucial role played by an 11 beta hydroxysteroid dehydrogenase in the local protection of type I receptors in mineralocorticoid selective tissues.

Hydroxysteroid dehydrogenase activity associated with sexual differentiation in embryos of the turtle Trachemys scripta.

Many turtles exhibit temperature-dependent sex determination. We have examined the hypothesis that incubation temperature causes a differential expression of steroidogenic enzymes in embryonic turtles. The activities of three steroidogenic enzymes were studied histochemically in turtle (Trachemys scripta) embryos at different developmental stages: sexually undifferentiated (Stage 15), differentiating (Stage 17), and differentiated (Stage 26; i.e., hatchling). Steroidogenic enzymes were detected in several tissues prior to, during, and after gonadal differentiation in embryos incubated at both male-producing and female-producing temperatures. In all embryos, ene-5-3 beta-hydroxysteroid dehydrogenase (HSDH) was detected only in adrenal tissue. 3 alpha-HSDH was localized in adrenal tissue, as well as in the mesonephros and liver. 17 beta-HSDH was evident in mesonephric, hepatic, and gut tissues. In hatchlings, ene-5-3 beta-HSDH and 3 alpha-HSDH were evident in the adrenal gland, whereas 3 alpha-HSDH and 17 beta-HSDH were present in the mesonephros and liver. While there was some variation in the activities of these enzymes during development, no temperature-specific pattern was apparent. At no stage were the enzymes observed in genital ridge/gonad. Our results show that T. scripta embryos possess enzymes necessary for steroid hormone synthesis. The segregated distributions of the enzymes suggest that a multi-organ regulatory system may mediate embryonic steroidogenesis. Our results do not indicate the genital ridge/gonad the principle site of steroid synthesis, although it may possess other enzymes that influence steroidogenesis.

Regulation of 1lβ-Hydroxysteroid Dehydrogenase Activity in Human Skin Fibroblasts: Enzymatic Modulation of Glucocorticoid Action*

The regulation of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) was studied in cultured human skin fibroblasts. 11-Oxo-reductase activity was 5- to 10-fold higher than 11 beta-dehydrogenase activity. Cells treated with 100 nM dexamethasone (Dex) showed a 3-fold increase in the maximum velocity of both activities without a change in the Km values. Dex induction of 11 beta HSD was half-maximal at 48 h and was blocked by glucocorticoid receptor antagonists. Nonglucocorticoid steroids were ineffective. Removal of serum from the culture medium increased maximum velocity values up to 6-fold. Treatment of cells grown in the absence of serum with 8-bromo-cAMP, phorbol esters, or insulin decreased both 11 beta HSD activities. The effects of Dex treatment and serum removal were additive and were blocked by cycloheximide and actinomycin-D. In all experiments both 11 beta HSD activities were modulated in parallel. Both cortisone (200 nM) and cortisol increased the aromatase activity of fibroblasts in the presence of serum. Prior induction of 11 beta HSD by serum removal increased the potency of cortisone from 10-15% to 50% that of cortisol. We conclude that 1) in human fibroblasts 11 beta HSD appears to be a single protein that is under multifactorial regulation; 2) 11 beta HSD may increase or decrease cortisol availability to glucocorticoid receptors; and 3) plasma cortisone levels may be important in assessing glucocorticoid status.

Synthesis and metabolism of vertebrate-type steroids by tissues of insects: A critical evaluation

This review covers the synthesis and the metabolism of vertebrate-type steroids (progesterone, testosterone, estradiol, corticosteroids) by insect tissues and discusses the significance of the reactions for insect physiology. Biosynthesis of vertebrate-type steroids from cholesterol hitherto has been demonstrated in only two insect species, i.e. the water beetle Acilius sulcatus (Coleoptera) and the tobacco hornworm Manduca sexta (Lepidoptera). In Acilius, steroid synthesis is associated with exosecretion (chemical defense). Nothing, however, is known about a physiological role of the C21 steroid conjugate present in ovaries and eggs of Manduca. No synthesis of vertebrate-type steroids was observed in any other insect investigated to date. Most metabolic conversions of steroids by insects concerned oxidoreduction of oxygen groups (hydroxysteroid dehydrogenase activity) and (polar and apolar) conjugate formation. All important enzymatic steps involved in synthesis and catabolism, as known from studies with tissues of vertebrates, were not, or hardly observed. The conclusion is drawn that typical vertebrate-type (C21, C19 and C18) steroids probably do not act as physiologically active substances in insects.

Naltrexone Normalizes the Suppression but not the Surge of Δ5-3β-Hydroxysteroid Dehydrogenase Activity in Leydig Cells of Stressed Rat Fetuses*

Rat fetuses from mothers stressed chronically by immobilization and high intensity illumination beginning on day 14 of gestation have higher than normal levels of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity in Leydig cells on day 17 of gestation and lower than normal levels on days 18 and 19. Plasma testosterone titers in normal and stressed male fetuses closely parallel the activity of 3 beta HSD in fetal Leydig cells. In the present study quantitative cytochemistry was used to determine whether the stress-induced alterations in 3 beta HSD activity could be prevented by treating the mother with naltrexone, an opioid receptor blocker, before each stress session. Naltrexone normalized 3 beta HSD activity on days 18 and 19 of gestation, suggesting that the stress-induced suppression involves the endogenous opioid system. In contrast, naltrexone did not prevent the elevation in enzyme activity seen on day 17 in stressed fetuses. The persistence of a stress-induced surge on day 17, in spite of naltrexone therapy, suggests that some nonopioid mechanism is operational at that time.

Hydroxysteroid dehydrogenase activity in tissues of two insect species

1. 1. The metabolism of two tritium labelled vertebrate-type steroids was studied in two insect species, i.e. the fleshfly, Sarcophaga bullata, and the Colorado potato beetle, Leptinotarsa decemlineata. 2. 2. After injection of [ 3H]androstenedione into Sarcophaga bullata pharate adults, testosterone (both as free steroid and as conjugate) could be identified as a metabolic product. This indicates the presence of the 17β-hydroxysteroid dehydrogenase (HSD) enzyme in the fleshfly. 3. 3. Injection of 17α-hydroxy[ 3H]progesterone into Leptinotarsa decemlineata last instar larvae resulted in the formation of 17α-hydroxy-20α-dihydroprogesterone, 17α-hydroxy-20β-dihydroprogesterone and their conjugates. This indicates the presence of both the 20α-HSD and the 20β-HSD enzyme in Leptinotarsa. 4. 4. Important conversions in the biosynthetic pathway of steroids in vertebrates, such as the conversion of 17α-hydroxyprogesterone to androgens ( Leptinotarsa) and the aromatization of androgens to estrogens ( Sarcophaga), were not demonstrated in the metabolic studies.

Mineralocorticoid activity of carbenoxolone: contrasting effects of carbenoxolone and liquorice on 11β-hydroxysteroid dehydrogenase activity in man

1. 11-beta-Hydroxysteroid dehydrogenase is an enzyme complex consisting of 11 beta-dehydrogenase and 11-oxoreductase responsible for the interconversion of cortisol to cortisone in man. Inhibition of 11 beta-dehydrogenase (e.g. after liquorice ingestion) results in cortisol acting as a potent mineralocorticoid. We have evaluated the effect of the synthetic liquorice derivative, carbenoxolone, on this enzyme complex. 2. Carbenoxolone given to six volunteers in metabolic balance produced sodium retention with suppression of the renin-angiotensin-aldosterone system. Plasma potassium fell, although there was no kaliuresis. This was associated with inhibition of 11 beta-dehydrogenase (as measured by a rise in the plasma half-life of [11 alpha-3H]cortisol). Unlike liquorice, however, carbenoxolone also inhibited 11-oxoreductase (as measured by the generation of cortisol after oral cortisone acetate). 3. The mineralocorticoid activity of carbenoxolone, like liquorice, is mediated via cortisol by inhibition of 11 beta-dehydrogenase. Carbenoxolone, however, also inhibits 11-oxoreductase activity and this may relate to its effect on renal potassium excretion.

3(20)α-Hydroxysteroid Dehydrogenase Activity of Monkey Liver Indanol Dehydrogenase1

Homogeneous indanol dehydrogenase from monkey liver catalyzed the reversible conversion of 3 alpha- or 20 alpha-hydroxy groups of several bile acids and 5 beta-pregnanes to the corresponding 3- or 20-ketosteroids. The kcat values for the steroids determined at pH 7.4 were low, but the kcat/Km values for the 3-ketosteroids were comparable to or exceeded those for 1-indanol and xenobiotic carbonyl substrates. The enzyme transferred the 4-pro-R-hydrogen atom of NADPH to the 3 beta- or 20 beta-face of the ketosteroid substrate. Competitive inhibition of the hydroxysteroid dehydrogenase activity of the enzyme by medroxyprogesterone acetate, hexestrol, and 1,10-phenanthroline suggests that both 1-indanol and hydroxysteroid are oxidized at the same active site on the enzyme. The specific inhibitor of the enzyme, 1,10-phenanthroline, suppressed the 3 alpha-hydroxysteroid dehydrogenase activity in the crude extract of monkey liver by 50%. The results strongly suggest that indanol dehydrogenase acts as a 3(20)alpha-hydroxysteroid dehydrogenase in the metabolism of certain steroid hormones and bile acids.

An electron microscopic study of cells with steroid-secreting morphology in the paraaortic lymph node of the hamster.

Clustered cells with steroid-secreting morphology (SH), located within the paraaortic lymph node (PLN) capsules of normal and pregnant female golden hamsters, were examined by light and electron microscopy. Hydroxysteroid dehydrogenase (HD) activity of the SH cell cluster also was examined by histochemical techniques. The cluster was composed of mostly packed SH cells and surrounding mesenchymal cells. Individual SH cells possessed prominent smooth endoplasmic reticulum, well-developed Golgi complexes, some lipid droplets, and numerous mitochondria containing tubulovesicular cristae common to mammalian SH cells. Intermediate-type junctions were often observed between SH cells. The same SH cells were rarely detected in the subcapsular sinus and in the cortical parenchyma of the PLN. Early oophorectomy of the hamster resulted in cytoplasmic degeneration of the SH cell at day 5 after the operation. On the other hand, normal adult male hamsters possessed similar PLNs, but no SH cells were recognized in the nodes. The present histochemical preparation of normal female PLNs revealed moderate-to-strong HD activity, probably associated with SH cell clusters, in the limited regions of the capsules. Based on the present ultrastructural and histochemical findings, it is proposed that the SH cell cluster consists of steroid hormone producing cells.

Hydroxysteroid dehydrogenase of Cochliobolus lunatus

Cochliobolus lunatus is known to be able to hydroxylate steroids at position 11β. Besides this inducible enzyme, we found a constitutive hydroxysteroid dehydrogenase activity which is strongly regioselective with the highest activity at position 17, and the best substrate was found to be androstenedione. Using different substrates, no such activities were observed at positions 3 or 11. The enzyme is membrane bound and NADH or NADPH dependent. The protoplasts of Cochliobolus lunatus show the same activity as intact cells, which means that the cell wall does not influence the reaction.

Effects of follicle regulatory protein on thecal aromatase and 3 beta-hydroxysteroid dehydrogenase activity in medium- and large-sized pig follicles.

Theca was excised from large (greater than 8 mm) and medium-sized (3-6 mm) pig follicles and prepared as monolayer cultures in serum-free media. After 24 h cells were treated with (1) M199 (control), (2) 5 i.u. hCG, (3) 100 micrograms or 100 ng FRP or (4) hCG (5 i.u.) + FRP (100 micrograms or 100 ng). At 3, 6, 12, 24 and 48 h after treatment, progesterone, oestradiol, androstenedione and testosterone were measured in media. Formation of progesterone by microsomal fractions incubated (37 degrees C) with 1 microM-pregnenolone + 5-microM-NAD+ for 1 h was used as a measure of 3 beta-HSD activity. Aromatase activity was determined by incubating cells with [3H]testosterone for 3 h (37 degrees C) and measuring 3H2O release. In theca from large follicles, hCG enhanced 3 beta-HSD activity after 48 h (P less than 0.05) and secretion of progesterone after 36 h. FRP alone inhibited 3 beta-HSD activity at 36 and 72 h, but had little effect on progesterone secretion. FRP inhibited (P less than 0.05) the hCG-induced increase in 3 beta-HSD activity at 36, 48 and 72 h. HCG enhanced aromatase activity after 48 h while FRP prevented (P less than 0.05) the hCG-induced increase in aromatase activity at 48 and 72 h. Secretion of oestradiol was enhanced (P less than 0.05) at 48 h but inhibited at 72 h by hCG. FRP alone had little effect on secretion of oestradiol but hCG + FRP was inhibitory at 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative determination of oestradiol-17 beta hydroxysteroid dehydrogenase: increased sensitivity by HPLC separation of the hormones permits the measurement of enzyme activity in cryostat sections.

The activity of the oestradiol-17 beta hydroxysteroid dehydrogenase in human endometrial and breast cancer specimens was determined by the NAD-dependent conversion of oestradiol-17 beta to oestrone. The sensitivity of the determination was improved by the separation of the hormones by HPLC. We are now able to determine oestradiol-17 beta hydroxysteroid dehydrogenase quantitatively in cryostat sections. A clear correlation of serum progesterone levels and oestradiol-17 beta hydroxysteroid dehydrogenase activity in the endometrium was demonstrated, and we found a more than 30-fold increase in enzyme activity after the progesterone surge. In contrast, in breast cancer samples, we found no correlation between oestradiol-17 beta hydroxysteroid dehydrogenase and the measured serum parameters.

Delta (5)3 beta hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate-binding co-operativity: a continuous monitoring microdensitometric study with unfixed ovarian tissue sections.

delta (5)3 beta hydroxysteroid dehydrogenase activity transforms biologically inactive delta (5)3 beta hydroxy steroids into the active delta (4)3-keto products (e.g. pregnenolone to progesterone). Using a cytochemical procedure which allows for the continuous microdensitometric monitoring of an enzyme reaction as it proceeds and a well described cytochemical assay for delta (5)3 beta HSD we have analysed the initial velocity rates (Vo) for dehydroepiandrosterone (DHEA) binding to this enzyme in regressing (i.e. 20 alpha hydroxy steroid dehydrogenase positive) corpus luteum (CL) cells in unfixed tissue sections (5 micron) of the dioestrous and proestrous rat ovary. The results are mean +/- S.E.M. The relationship between DHEA concentration (0 to 50 microM) and delta (5)3 beta HSD activity in the dioestrous corpora lutea was sigmoidal and had an atypical 1/Vo versus 1/S plot, the x intercept being positive. Using a 1/Vo versus 1/S2 plot the Vmax was determined to be 1.0 +/- 0.08 mumol min-1 mg-1 CL (n = 6). The Hill constant was 2.7 +/- 0.02 (n = 6) suggesting a high degree of positive co-operativity for DHEA binding. The S concentration for half maximal activity was 17 +/- 1 mumoles (n = 6). In the corpora lutea cells of the proestrous ovary, the Vmax for DHEA transformation was unchanged (0.95 +/- 0.04 mumol min-1 mg-1, n = 3) whilst the S0.5 was significantly increased to 27 +/- 0.1 (p less than 0.01, n = 3). The Hill constant remained positive being 2.9 +/- 0.2 (n = 3).(ABSTRACT TRUNCATED AT 250 WORDS)

Thecal cell 3-beta hydroxysteroid dehydrogenase activity: Modulation by human chorionic gonadotropin, progesterone, estradiol-17 beta and dihydrotestosterone

Thecal cell steroidogenesis plays a major role in folliculogenesis within the porcine ovary. Accorclingly, the effects of physiological concentrations of steroids on 3β-hydroxysteroid dehydrogenase activity (3β-HSD) were determined. Theca was excised from large porcine follicles and prepared in a monolayer culture in 1 ml of serum-free media. Cells were treated 24 h after culture as follows: (1) control, (2) hCG (5IU); (3) progesterone (P, 3μg); estradiol-17β (E, 4μg); 5β-dihydrotestosterone (DHT, 1 μg); (4) hCG + P or E or DHT. At 3, 6, 12, 24 and 48 h after treatment, media were assessed for P levels. For 3β-HSD activity, P formation by microsomal fractions incubated with 1 μM pregnenolone + 5 μM NAD+ for l h (37°C) was monitored. Thecal cell P secretion increased from 27 to 72 h. hCG significantly ( P < 0.05) increased P levels after 36 h compared to controls. E or E + hCG decreased P levels at 36, 48, and 72 h and DHT prevented the hCG-induced increase in P secretion. 3β-HSD activity in thecal microsomes increased significantly from 27 to 72 h. hCG had little effect on 3β-HSD activity compared with controls from 27 to 36 h, but significantly ( P < 0.05) decreased 3β-HSD activity at 48 and 72 h. However, P or P + hCG significantly ( P < 0.05) decreased 3β-HSD activity at all times. In addition, E or E + hCG significantly ( P < 0.05) decreased 3β-HSD activity at 48 and 72 h. DHT prevented the hCG-induced decrease in 3β-HSD activity. In conclusion, porcine thecal secretion of P and microsomal 3β-HSD activity increased during 72 h of culture. Paradoxically, the addition of hCG to cultures enhanced media P concentrations but inhibited 3β-HSD activity. Further, the addition of E to cultures decreased media concentrations of P while P or E decreased 3β-HSD activity. Therefore, paracrine/autocrine effects of locally produced steroids may play a role in modulating thecal cell steroidogenesis.

Inhibition of delta 5-3 beta hydroxysteroid dehydrogenase activity in pre-implantation mouse embryos and their implantation by sheep ovarian follicular fluid peptide.

The activity of delta 5-3 beta hydroxysteroid dehydrogenase (delta 5-3 beta HSD) in pre-implantation mouse embryos was inhibited following their prior incubation with partially purified sheep ovarian follicular fluid peptide (OFFP). OFFP, 5 microliter, injected into the uterine lumen of day 4 pregnant mice impaired the ensuing implantation of embryos. Our observations suggest that OFFP may inhibit steroid metabolizing activity in pre-implantation embryos and thereby interfere with the process of implantation.

Cytochemical demonstration of hydroxysteroid dehydrogenase activity in mouse oocytes and preimplantation embryos : G.G. De Schepper and C.J.F. Van Noorden. Academic Medical Centre, University of Amsterdam, The Netherlands

Cytochemical demonstration of hydroxysteroid dehydrogenase activity in mouse oocytes and preimplantation embryos : G.G. De Schepper and C.J.F. Van Noorden. Academic Medical Centre, University of Amsterdam, The Netherlands

17β Hydroxysteroid dehydrogenase activity in breast adipose tissue from breast cancer patients

17β Hydroxysteroid dehydrogenase activity in breast adipose tissue from breast cancer patients