Stromal markers AKR1C1 and AKR1C2 are prognostic factors in primary human breast cancer.

BACKGROUND: Stromal fibroblasts influence tumor growth and progression. We evaluated two aldo-keto reductases, AKR1C1 and AKR1C2, in stromal fibroblasts and carcinoma cells as prognostic factors in primary human breast cancer. They are involved in intratumoral progesterone metabolism. METHODS: Immunohistochemistry was performed on tissue microarrays from 504 core biopsies from breast cancer patients. Primary endpoints were disease-free (DFS) and overall (OS) survival. RESULTS: AKR1C1 and AKR1C2 expression in fibroblasts and tumor cells correlated with favorable tumor characteristics, such as small tumor size and negative nodal status. In univariate analysis, AKR1C1 expression in carcinoma cells correlated positively with DFS und OS; AKR1C2 expression in both fibroblasts and tumor cells also showed a positive correlation with DFS and OS. In multivariate analysis, AKR1C1 expression in carcinoma cells was an independent prognostic marker. CONCLUSION: It can be assumed that our observations are due to the independent regulatory function of AKR1C1/2 in progesterone metabolism and therefore provide a basis for new hormone-based therapy options for breast cancer patients, independent of classic hormone receptor status.

Organization and activity in the pre- and postovulatory follicle of Necturus maculosus.

The established follicle envelope of Necturus maculosus consists of a layer of follicle cells (granulosa) surrounding the developing oocyte, a layer of theca comprised of connective tissue cells, fibers, and matrix, and a layer of serosal cells. The changes in shape and fine structure of these layers during differentiation accompanying oogenesis are described. The cells and capillaries of the follicle envelope are engaged in an extensive pinocytotic activity, the details of which are described. We used cytochemical techniques to analyze the activity of the follicle envelope with respect to lipid accumulation and alkaline phosphatase activity. Radioautographic results indicate that cells of the follicle envelope are capable of incorporating tritium-labeled uridine and amino acids at certain times during oocyte growth. A comparative analysis was made of the soluble proteins in follicle envelopes isolated from immature oocytes and of those in follicle envelopes isolated from nearly mature oocytes and in postovulatory follicles. After the oocyte is ovulated, the cells of the follicle envelope are converted into a postovulatory follicle. The cells of the postovulatory follicle undergo further differentiation resulting in their becoming actively engaged in the formation of a secretion, the details of which are described at the electron microscope level. Analysis of the postovulatory follicle by thin-layer chromatography and cytochemistry demonstrated the presence of a wide variety of lipid substances and the possible presence of steroid. That the postovulatory follicle may be engaged in steroid biosynthesis is also suggested by studies involving the demonstration of 3 beta-hydroxysteroid dehydrogenase activity with cytochemical techniques applied to frozen sections and to soluble proteins separated by gel electrophoresis.

Mechanism of gonadotropin action in amphibia: involvement of mitochondria.

Luteinizing hormone (a pituitary gonadotropic hormone) stimulates Delta(5)-3beta- hydroxysteroid dehydrogenase activity in the microsomal fraction of frog testes when incubated together with the mitochondria; incubation together with the nuclei instead of the mitochondria does not result in increased, Delta(5)-3beta-hydroxysteroid dehydrogenase activity. The increase is not, induced by adenosine triphosphate, it appears to be hormone-specific, and it is sensitive to puromycin and actinomycin D. These data suggest that the mitochondrial DNA may be involved in mediating the action of luteinizing hormone in amphibian steroidogenesis.

The corticosteroid metabolic profile of the mouse.

Data are presented on the urinary corticosteroid metabolic profile of the mouse strain 129/svJ. Through the use of GC/MS we have characterized, or tentatively identified corticosterone (Kendall's compound B) metabolites of both the 11beta-hydroxy and 11-carbonyl (compound A) series in urine. Full mass spectra of the methyloxime-trimethylether derivatives of 15 metabolites are included in the paper as an aid to other researchers in the field. Metabolites ranged in polarity from tetrahydrocorticosterone (THB) to dihydroxy-corticosterone with dominance of highly polar steroids. We found that prior to excretion corticosterone can undergo oxidation at position 11beta, reduction at position 20 and A-ring reduction. Metabolites retaining the 3-oxo-4-ene structure can be hydroxylated at position 6beta- as well as at an unidentified position, probably 16alpha-. Saturated steroids can be hydroxylated at positions 1beta-, 6alpha-, 15alpha- and 16alpha. A pair of hydroxy-20-dihydro-corticosterone metabolites (OH-DHB) were the most important excretory products accounting for about 40% of the total. One metabolite of this type was identified as 6beta-hydroxy-DHB; the other, of similar quantitative importance was probably 16alpha-hydroxy-DHB. The ratio of metabolites of corticosterone (B) to those of 11-dehydro-corticosterone (A) was greater than 9:1, considerably higher than that for the equivalent "human" ratio of 1:1 for cortisol to cortisone metabolites. Results from this study allowed the evaluation of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) activity in mice with deleted glucose-6-phosphate transporter (G6PT). These mice had attenuated back-conversion of A to B resulting in an increased ratio of A-metabolites to B-metabolites [Walker EA, Ahmed A, Lavery GG, Tomlinson JW, Kim SY, Cooper MS, Stewart PM, 11beta-Hydroxysteroid dehydrogenase type 1 regulation by intracellular glucose-6-phosphate, provides evidence for a novel link between glucose metabolism and HPA axis function. J Biol Chem 2007;282:27030-6]. We believe this study is currently the most comprehensive on the urinary steroid metabolic profile of the mouse. Quantitatively less steroid is excreted in urine than in feces by this species but urine analysis is more straightforward and the hepatic metabolites are less subject to microbial degradation than if feces was analyzed.

[Subcutaneous transplants of juvenile rat testicular tissues continue to develop and secret androgen in adult rats].

Objective To explore the effects of subcutaneous microenvironment of adult rats on survival, development and androgen secretion of Leydig cells of transplanted juvenile rat testis. Methods Healthy adult SD rats were randomly divided into control group, sham group, castrated group and non-castrated group. Rats in the control group were kept intact, no testis was transplanted subcutaneously after adult recipients were castrated in the sham group; 5-7-day juvenile rat testes were transplanted subcutaneously in the castrated group, with one testis per side; Testes resected from juvenile rats were directly transplanted subcutaneously on both sides of the recipients in the non-castrated group. The grafts were obtained and weighed 4 weeks later. Then the histological features of the grafts were examined by HE staining; the expression and distribution of hydroxysteroid 17-beta dehydrogenase 1 (HSD-17ß1) were investigated by immunohistochemistry; and the serum androgen level was determined by ELISA. Results The average mass of grafts obtained from the castrated group was significantly higher than that of the non-castrated group. Immunohistochemistry indicated that Leydig cells were visible in the tissues from both the castrated and non-castrated groups, but the number of HSD-17ß1-posotive cells in the castrated group was larger than that in the non-castrated group. ELISA results showed that the serum androgen level was higher in the control group and non-castrated group than in the sham group and castrated group, and compared with the sham group, the serum androgen level in the castrated group was significantly higher. Conclusion The juvenile rat testis subcutaneously transplanted could further develop under the adult recipient rat skin, and the Leydig cells of grafts harbored the ability to produce and secret androgen.

Modification of steroidogenesis in rat adrenocortical cells transformed by Kirsten murine sarcoma virus.

Adrenal fibroblasts from adult rats acquire some adrenocortical parenchymal characteristics as a consequence of transformation in early passage with Kirsten murine sarcoma virus. To further define the effects of Kirsten murine sarcoma virus-induced transformation on the steroid enzymes of these cells, we investigated the capacity of Kirsten murine sarcoma virus-transformed and untransformed adrenocortical fibroblasts to convert progesterone to C19 and C21 steroid metabolites. Over 95% of metabolites produced were identified and quantitated, and rates of enzyme activities over 24 h were calculated. The transformed and untransformed cells exhibited 5 alpha- and 5 beta-reductase, 3 alpha-, 3 beta-, and 20 alpha-hydroxysteroid dehydrogenase; 11 beta-, 17-, and 21-hydroxylase (HY); and C17-20-lyase activities. Viral transformation resulted in several metabolites not found in untransformed cells, significantly increased 5 beta-reductase, 3 beta-hydroxysteroid dehydrogenase, and C17-20-lyase activities, and significantly decreased 5 alpha-reductase, 3 alpha- and 20 alpha-hydroxysteroid dehydrogenase, and 21-HY activities. The 11 beta-HY and 17-HY activities remained unchanged. The results support previous data suggesting that adrenocortical fibroblasts express some characteristics of adrenocortical parenchymal stem cells. In contrast to other experimental systems, viral transformation of adrenocortical fibroblasts did not cause a generalized reduction of differentiated functions. Instead, specific increases and decreases in individual enzyme activities, with persisting synthesis of fetal and adult adrenocortico-specific steroids, resulted in an altered steroid profile that may have unique effects on the biology of the malignant cells.

Renal epithelial cell lines (BSC-1, MDCK, LLC-PK1) express 11 beta-hydroxysteroid dehydrogenase activity.

Renal tissue of several species has been shown to express considerable 11 beta-hydroxysteroid dehydrogenase (11-HSD, EC 1.1.1.146) activity. However, it is uncertain as to which renal cell types exhibit 11-HSD activity. In the present study, we investigated corticosterone metabolism in BSC-1 cells, a continuous renal epithelial cell line derived from the African green monkey (Cercopithecus aethiops). In incubation experiments using 3H-labelled corticosterone and HPLC, we have demonstrated oxidative 11-HSD activity in intact monolayers of BSC-1 cells as well as in BSC-1 cell homogenates. 11-HSD activity in cell homogenates could be stimulated 7-9-fold by the addition of exogenous NADP+ (1 mM). In contrast, no reductive 11-HSD could be detected either in intact cells or in cell homogenates under various experimental conditions which were designed to favor reductive 11-HSD activity. Pilot experiments were performed in cell homogenates from two other renal epithelial cell lines derived from canine (MDCK) and porcine (LLC-PK1) kidney. They also revealed oxidative but no reductive 11-HSD activity. The data provide evidence for an epithelial localization of renal oxidative 11-HSD activity.

The 11 beta-hydroxysteroid dehydrogenase type 2 activity in human placental microsomes is inactivated by zinc and the sulfhydryl modifying reagent N-ethylmaleimide.

Proper glucocorticoid exposure in utero is vital to normal fetal organ growth and maturation. The human placental 11 beta-hydroxysteroid dehydrogenase type 2 enzyme (11 beta-HSD2) catalyzes the unidirectional conversion of cortisol to its inert metabolite cortisone, thereby controlling fetal exposure to maternal cortisol. The present study examined the effect of zinc and the relatively specific sulfhydryl modifying reagent N-ethylmaleimide (NEM) on the activity of 11 beta-HSD2 in human placental microsomes. Enzyme activity, reflected by the rate of conversion of cortisol to cortisone, was inactivated by NEM (IC(50)=10 microM), while the activity was markedly increased by the sulfhydryl protecting reagent dithiothreitol (DTT; EC(50)=1 mM). Furthermore, DTT blocked the NEM-induced inhibition of 11 beta-HSD2 activity. Taken together, these results suggested that the sulfhydryl (SH) group(s) of the microsomal 11 beta-HSD2 may be critical for enzyme activity. Zn(2+) also inactivated enzyme activity (IC(50)=2.5 microM), but through a novel mechanism not involving the SH groups. In addition, prior incubation of human placental microsomes with NAD(+) (cofactor) but not cortisol (substrate) resulted in a concentration-dependent increase (EC(50)=8 microM) in 11 beta-HSD2 activity, indicating that binding of NAD(+) to the microsomal 11 beta-HSD2 facilitated the conversion of cortisol to cortisone. Thus, this finding substantiates the previously proposed concept that a compulsorily ordered ternary complex mechanism may operate for 11 beta-HSD2, with NAD(+) binding first, followed by a conformational change allowing cortisol binding with high affinity. Collectively, the present results suggest that cellular mechanisms of SH group modification and intracellular levels of Zn(2+) may play an important role in regulation of placental 11 beta-HSD2 activity.

Localization of 11beta-hydroxysteroid dehydrogenase type 2 in rat tissues: in situ studies.

In the rat, the enzyme 11beta-hydroxysteroid dehydrogenase 2 (11betaHSD2) converts the glucocorticoid corticosterone into receptor-inactive 11-dehydrocorticosterone, thereby allowing preferential access of aldosterone to mineralocorticoid receptors (MR). The present study examines the distribution of this enzyme by in situ hybridization, using a homologous complementary RNA probe for 11betaHSD2. 11betaHSD2 messenger RNA was detected in classic epithelial aldosterone target tissues (kidney, salivary glands, and colon), the female reproductive system (ovary, oviduct, uterus, and placenta), and the adrenals; levels in heart, testis, and liver were below the limits of detection. We interpret the finding of 11betaHSD2 expression in both classical MR-containing aldosterone target tissues and a variety of other tissue as evidence that in the rat, the enzyme may play physiological roles in addition to that of excluding glucocorticoids from epithelial MR.

Corticosteroid 11 beta-dehydrogenase in rat testis.

Corticosteroid 11 beta-dehydrogenase, the enzyme that catalyzes the oxidation of the biologically active steroid cortisol to its inactive metabolite cortisone, is present in testis. Since excess cortisol in men and other mammals and excess corticosterone in rodents cause physiological abnormalities including abnormal testicular function, it was pertinent to study the cellular distribution of 11 beta-dehydrogenase in the testis. Purified antiserum directed against homogeneous rat 11 beta-dehydrogenase was used to localize the enzyme in the developing rat testis. With immunofluorescence, the enzyme was not detectable in fetal testis or in the testis of young male rats until the 26th day of development. A few interstitial cells were stained in the testis of 26-day-old animals. In the testis of 31-day-old rats many cells in the interstitium were positive. In adult animals the entire interstitial region displayed bright fluorescence. Depleting animals of germ cells did not abolish the fluorescence. The appearance of this enzyme correlates temporally with the postnatal increase in Leydig cell number and the developmental rise in serum testosterone. We suggest that 11 beta-dehydrogenase of Leydig cells protects the testis from the deleterious effects of cortisol.

Hybridization histochemical localization of 11 beta-hydroxysteroid dehydrogenase type 2 in rat brain.

11 beta-hydroxysteroid dehydrogenase (11-HSD) activity allows aldosterone occupancy of mineralocorticoid receptors by inactivating endogenous glucocorticoids. The expression of the 11-HSD2 gene, a low Km, NAD+ dependent species of 11-HSD, was found in several discrete areas of the rat brain by in situ hybridization. Cells strongly positive for 11-HSD2 mRNA were found in the commissural portion of the nucleus tractus solitarius, subcommissural organ and ventrolateral ventromedial hypothalamus. Scattered labeled cells were also seen in the medial vestibular nucleus. The expression of 11-HSD2 mRNA in the brain is quite distinct from that of 11-HSD1 mRNA and allows for diverse roles in modulating corticosteroid receptor involvement in control of salt appetite, blood pressure and the hypothalamo-pituitary-adrenal axis.

Ontogeny of 11 beta-hydroxysteroid dehydrogenase in rat brain and kidney.

Close regulation of circulating corticosteroid levels during the early postnatal period is crucial for normal development and maturation of the central nervous system. In the first weeks of life cerebral glucocorticoid receptor concentrations are low and the hypothalamic-pituitary-adrenal axis is relatively unresponsive to stress, which might, in part, protect the developing brain from elevated corticosteroid levels. However, central mineralocorticoid receptors are at near adult levels and free glucocorticoid concentrations may approximate adult values as corticosteroid binding globulin is absent. Thus other mechanisms controlling cerebral exposure to corticosteroids may be of importance. 11 beta-Hydroxysteroid dehydrogenase (11 beta-OHSD) determines the access of corticosterone to peripheral mineralocorticoid and glucocorticoid receptors in adults in vivo by metabolizing corticosterone to inactive 11-dehydrocorticosterone. The enzyme has recently been demonstrated in brain subregions and may modulate local corticosteroid-receptor interactions. We therefore examined 11 beta-OHSD bioactivity and messenger RNA (mRNA) expression in the brain, compared with kidney, during the neonatal period. 11 beta-OHSD bioactivity (expressed as the percentage conversion of corticosterone to 11-dehydrocorticosterone) was moderately high in hippocampus and parietal cortex at birth (46 +/- 4% and 48 +/- 5%, respectively), fell significantly to a nadir (32 +/- 1% and 30 +/- 1%, respectively) at postnatal day 10 and then gradually rose to adult values (52 +/- 3% and 58 +/- 3%). By contrast, 11 beta-OHSD activity in cerebellum was high at birth (60 +/- 3%), rose significantly to a peak at postnatal day 10 (74 +/- 3%), and then fell to adult values by postnatal day 15 (64 +/- 3%). Renal 11 beta-OHSD activity was moderately high (69 +/- 3%) at birth and reached adult values (80 +/- 2%) by postnatal day 5. Northern blots showed high and similar expression of a single species of 11 beta-OHSD mRNA from birth to adulthood in the hippocampus. Only low expression of 11 beta-OHSD (two or three separate species) was found in the kidney during the first 2 weeks of life, whereas, in adults high expression of 11 beta-OHSD mRNA was detected in kidney (four species). Using in situ hybridization high 11 beta-OHSD mRNA expression was localized to the neuronal layers of the postnatal hippocampus, neocortex, and cerebellum, and low but detectable expression was found in the neonatal renal cortex. Thus, 11 beta-OHSD is highly expressed in rat brain subregions in the early postnatal period with specific developmental patterns of activity.(ABSTRACT TRUNCATED AT 400 WORDS)

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 11beta-hydroxysteroid dehydrogenase types 1 and 2 in the male reproductive tract.

The action of glucocorticoids in target tissues is dependent on the local expression of glucocorticoid receptors and two 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes, 11beta-HSD1 and 11beta-HSD2, which interconvert active and inactive glucocorticoids. This study examined expression of the 11beta-HSD enzymes in the male reproductive tract of the adult rat. 11beta-HSD1 was immunolocalized to the apical region of principal epithelial cells of the caput epididymis, with the less numerous clear cells devoid of signal. Epididymal 11beta-HSD1 expression was confirmed by Western blot analysis, with immunoreactive species identified at 34 kDa (the expected size for 11beta-HSD1) and at approximately 48 kDa. 11beta-HSD bioactivity was readily detectable in the epididymis, with 11-oxoreductase activity clearly the favored reaction (as observed in liver), consistent with 11beta-HSD1 expression. The epithelium of the vas deferens, seminal vesicle, and penile urethra were also immunopositive for 11beta-HSD1, as were smooth muscle cells of the vas deferens and penile blood vessels. 11beta-HSD2 was also immunolocalized to the epididymal epithelium, but its distribution was complementary to that of 11beta-HSD1 (i.e. clear cells showing intense 11beta-HSD2 staining but principal cells devoid of signal). 11beta-HSD2 was also present in the corpora cavernosa of the penis but not in other tissues. In conclusion, the differential expression of 11beta-HSD1 and 11beta-HSD2 throughout the male reproductive tract suggests that these enzymes locally modulate glucocorticoid and mineralocorticoid actions, particularly in the epididymis and penile vasculature.

Immunolocalization of 11beta-hydroxysteroid dehydrogenase types 1 and 2 in rat uterus: variation across the estrous cycle and regulation by estrogen and progesterone.

Glucocorticoid hormone action in several target tissues is dependent not only on the expression of the glucocorticoid receptor, but also on that of the 11beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes, 11betaHSD-1 and -2. In the uterus, glucocorticoids can exert inhibitory effects on a range of important functions, particularly in relation to the effects of estrogen. Therefore, the present study examined immunolocalization of the two 11betaHSD enzymes in the rat uterus at each stage of the estrous cycle and after ovariectomy with or without estrogen/progesterone replacement. In cycling rats 11betaHSD-1 was localized to luminal and glandular epithelial cells and to eosinophils in both the endometrial stroma and myometrium. In contrast, 11betaHSD-2 immunostaining was localized to endometrial stromal cells and myometrial cells, with no staining evident in epithelial cells or eosinophils. Immunostaining for both enzymes was cycle dependent, being maximal at proestrus and minimal at diestrus. Western blot analysis of whole uterus at proestrus showed the presence of 34- and 40-kDa immunoreactive species for 11betaHSD-1 and -2, respectively. These immunoreactive signals were almost abolished by ovariectomy, but this effect was reversed for both enzymes by estrogen replacement with or without progesterone. These effects of ovariectomy and steroid replacement were confirmed by immunocytochemical analysis, with the exception that progesterone appeared to enhance the stimulatory effects of estrogen on 11betaHSD-2 specifically within the endometrial stroma. In conclusion, these results establish the presence of both 11betaHSD-1 and -2 in the nonpregnant rat uterus and show distinct distributions for the two enzymes and cyclic variation related to positive regulation by ovarian steroids. The physiological implications of these patterns of 11betaHSD expression will ultimately depend on the reaction direction for each enzyme, but 11betaHSD-2 is likely to limit disruptive effects of glucocorticoids on the endometrial stroma, and 11betaHSD-1 may then serve to selectively reactivate glucocorticoids in epithelial cells.

Corticosteroid 11 beta-dehydrogenase of rat tissues: immunological studies.

Monospecific polyclonal antibodies to purified homogeneous rat liver corticosteroid 11 beta-dehydrogenase were generated in rabbits. The antibodies were immunoprecipitins, but enzyme activity was not completely suppressed in the antigen-antibody complex. Two antibody preparations, 56-125 and 56-126, used to detect 11 beta-dehydrogenase antigen in Western blots, generated different staining patterns for kidney, liver, brain, and heart. Using the two antibodies together, the total number of antibody-reacting components in kidney was three, and that in liver was two. Based on rates of digestion with proteases, the two prominent immunoreactive proteins in kidney appeared to be structurally or conformationally different. A prominent immunostaining component was present in stomach. Tissues that showed immunochemical evidence of 11 beta-dehydrogenase antigen showed corresponding levels of 11 beta-dehydrogenase activity. Most active were liver, testis, kidney, and lung. Lower levels of activity were found in prostate and epididymis, brain, and reproductive tract. We conclude that 11 beta-dehydrogenase is widely distributed in rat organs and is present at low levels with significant exceptions. The data indicate that 11 beta-dehydrogenase may occur in several enzyme forms, and that the distribution of these forms is to some extent tissue specific.

Purification and characterization of the corticosteroid 11 beta-dehydrogenase component of the rat liver 11 beta-hydroxysteroid dehydrogenase complex.

We have proposed that 11 beta-hydroxysteroid dehydrogenase is composed of structurally independent units with 11 beta-dehydrogenase and 11-reductase activities. We now report the purification of rat liver 11 beta-dehydrogenase to apparent homogeneity. Starting with microsomes, 800-fold purification was achieved with agarose-NADP affinity chromatography. No 11-reductase accompanied the purification. Homogeneity of 11 beta-dehydrogenase was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid end-group analysis and immunoprecipitation. The terminal amino acid was methionine. Monomer mol wt was 34,000. The enzyme was found to be a glycoprotein. A sequence of 40 amino acid units was identified from the amino end. The amino-terminal region was found to be highly nonpolar. Unlike unpurified microsomal 11 beta-dehydrogenase, which showed curvilinear Eadie plots, homogeneous enzyme gave rectilinear plots. Michaelis constants were 1.83 +/- 0.06 microM for corticosterone and 17.3 +/- 2.24 microM for cortisol. First order rate constants were 10 times greater for corticosterone than cortisol, and maximum velocities were similar.

Tissue-specific distribution of the NAD(+)-dependent isoform of 11 beta-hydroxysteroid dehydrogenase.

11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) converts the active glucocorticoid corticosterone to inactive 11-dehydrocorticosterone in rat (or cortisol to cortisone in man), thereby protecting renal mineralocorticoid receptors from corticosterone or cortisol and allowing preferential access for aldosterone. Recent work suggests that a nicotinamide adenine dinucleotide (NAD+)-dependent 11 beta-OHSD isoform is expressed in distal renal tubule, in contrast with the hepatic isoform which is NAD(+)-phosphate (NADP+)-dependent. To establish the distribution of the NAD(+)-dependent isoform we measured in vitro conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone in homogenized rat tissues in the presence of NADP+ or NAD+. In most tissues (liver, testis, hippocampus, heart, aorta, mesenteric artery) NADP+ increased activity and NAD+ was without effect. However, in whole renal cortex, colon, placenta, and lung both NADP+ and NAD+ increased activity. No difference in cofactor utilization was demonstrated between proximal and distal renal tubules following density gradient separation. This distribution of NAD(+)-dependent activity corresponds with: (i) the distribution of multiple mRNA and/or protein species of 11 beta-OHSD; (ii) the distribution of aldosterone-specific mineralocorticoid receptors; and (iii) the equilibrium between active and inactive glucocorticoids in each tissue. We suggest that the tissue-specific expression of isoforms of 11 beta-OHSD with different kinetic properties confers on them diverse roles in modulating corticosteroid receptor activation.

Localization of renal 11 beta-dehydrogenase by in situ hybridization: autocrine not paracrine protector of the mineralocorticoid receptor.

In the kidney, 11 beta-dehydrogenase (11 beta-DH) converts the active steroid cortisol to inactive cortisone (corticosterone to 11-dehydrocorticosterone in the rat). In man, congenital and acquired deficiency of 11 beta-dehydrogenase are rare causes of hypertension in which cortisol acts as a potent mineralocorticoid. Observations from these clinical studies indicate that 11 beta-DH conveys specificity for the mineralocorticoid receptor in distal tubules and collecting ducts. However, while some studies do indicate 11 beta-DH activity in rat distal tubules and collecting ducts, immunohistochemical studies localize 11 beta-DH only to proximal tubules. to resolve this dilemma, we have performed in situ hybridization localization of 11 beta-DH mRNA in rat kidney tissue using 35S-labeled sense and antisense cRNA probes to rat 11 beta-DH. In contrast to our immunohistochemical studies in which 11 beta-DH protein was localized predominantly to proximal tubules in the inner cortex, 11 beta-DH mRNA was expressed in tubules in both the inner and outer cortex, most probably proximal and distal tubules, and in collecting ducts extending across the corticomedullary junction to the papillary tip. Weak hybridization was also seen in glomeruli, but no hybridization to the sense 11 beta-DH cRNA or to sections pretreated with RNase-A was observed. We conclude that renal 11 beta-DH is suitably located to prevent access of glucocorticoid to the MR in an autocrine and not a paracrine fashion. 11 beta-DH in proximal tubules may protect the glucocorticoid receptor.

Localization and developmental regulation of 11beta-hydroxysteroid dehydrogenase-1 and -2 in the baboon syncytiotrophoblast.

We previously showed that the messenger RNA and protein levels of the 11ss-hydroxysteroid dehydrogenase (11betaHSD) enzymes catalyzing glucocorticoid reduction (11betaHSD-1) and oxidation (11betaHSD-2) increased with advancing baboon gestation and concluded that the estrogen-regulated change in placental cortisol metabolism from reduction at midgestation to oxidation near term is not simply the result of a change in the relative concentrations of these two enzymes. Therefore, in the current study we determined whether 11betaHSD-1 and -2 are located in different regions of the baboon and human syncytiotrophoblast and whether there is a developmental change in their localization with advancing baboon gestation. Western blot analyses, immunofluorescence, and electron microscopic immunocytochemistry indicated that 11betaHSD-1 expression was abundant in microvillus membranes (MVM) juxta the maternal circulation, and their levels are significantly lower, but detectable, in more internal regions of the syncytiotrophoblast, including membranes contiguous with the basal membrane (BM(m)) facing the fetal vasculature in both the human and baboon. In contrast, in both species 11betaHSD-2 expression was limited in the MVM and extensive throughout the remainder of the syncytiotrophoblast, including the BM(m). In the baboon, the relative mean (+/-SE) concentrations (arbitrary densitometric units per microgram protein) of 11betaHSD-1 in the MVM were similar at mid (i.e. day 100; 38,859 +/- 3,484; n = 3) and late (i.e. day 180; 43,561 +/- 1,784; n = 3) gestation (term = day 184) and exceeded (P < 0.01) respective values for 11betaHSD-2 by approximately 16-fold. In contrast, levels of 11betaHSD-1 in the BM(m) declined (P < 0.05) by approximately 50% between mid (7,099 +/- 758) and late (4,013 +/- 738) gestation, whereas levels of 11betaHSD-2 in this fraction increased. Thus, the ratio of 11betaHSD-2 to 11betaHSD-1 in the BM(m) at midgestation (1.22 +/- 0.10) was increased (P < 0.05) 2-fold in late gestation (2.66 +/- 0.05). Collectively, these findings indicate that the 11betaHSD-1 and -2 enzymes are localized to different membrane fractions of the baboon and human placental syncytiotrophoblast. Moreover, we propose that the developmental increase in the ratio of 11betaHSD-2 to 11betaHSD-1 in membranes facing fetal blood near term is consistent with and perhaps the subcellular mechanism responsible for the previously demonstrated switch in transplacental glucocorticoid metabolism from reduction at midgestation to oxidation late in gestation and appears to be responsible for the activation/maturation of the fetal pituitary-adrenocortical axis.