Steroid-converting enzymes in human adipose tissues and fat deposition with a focus on AKR1C enzymes.

Adipocytes express various enzymes, such as aldo-keto reductases (AKR1C), 11ß-hydroxysteroid dehydrogenase (11ß-HSD), aromatase, 5α-reductases, 3ß-HSD, and 17ß-HSDs involved in steroid hormone metabolism in adipose tissues. Increased activity of AKR1C enzymes and their expression in mature adipocytes might indicate the association of these enzymes with subcutaneous adipose tissue deposition. The inactivation of androgens by AKR1C enzymes increases adipogenesis and fat mass, particularly subcutaneous fat. AKR1C also causes reduction of estrone, a weak estrogen, to produce 17ß-estradiol, a potent estrogen and, in addition, it plays a role in progesterone metabolism. Functional impairments of adipose tissue and imbalance of steroid biosynthesis could lead to metabolic disturbances. In this review, we will focus on the enzymes involved in steroid metabolism and fat tissue deposition.

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.

Progesterone inhibits 20alpha-hydroxysteroid dehydrogenase expression in the rat corpus luteum through the glucocorticoid receptor.

In this study, we investigated whether progesterone exerts an intraluteal action despite the lack of progesterone receptors (PR) in the rat corpus luteum and whether progesterone acts through the glucocorticoid receptor (GR) to enhance its own levels by down-regulating the expression of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD). We first established that the corpus luteum constitutively expresses the GR throughout pregnancy and after parturition. We also generated a temperature sensitive SV-40 transformed luteal cell line (GG-CL) that expresses the GR and 20alpha-HSD but lacks the PR. Treatment with different doses of either progesterone or dexamethasone caused a dose-related decrease in 20alpha-HSD mRNA in both cultured corpora lutea and in the luteal cell line. RU486, a PR/GR antagonist, completely blocked both the progesterone and the dexamethasone mediated inhibition of 20alpha-HSD expression in GG-CL cells. In summary, this report provides the first evidence that despite the absence of the PR in the rat corpus luteum, progesterone can act through the GR to down-regulate the expression of 20alpha-HSD, an enzyme that catabolizes progesterone and reduces progesterone secretion by the corpus luteum.

Pituitary control of proliferation and differentiation of Leydig cells and their putative precursors in immature hypophysectomized rat testis.

The objective of this study was to determine the effects of pituitary hormones (luteinizing hormone [LH], follicle-stimulating hormone [FSH], growth hormone [GH], and prolactin [PRL]) on interstitial cell proliferation and differentiation in the testis of immature hypophysectomized rats. Macrophages, Leydig cells, precursor mesenchymal cells, endothelial lymphatic cells, and myoid cells were studied. Our experimental approach was aimed at determining whether changes in a cellular subpopulation observed after pituitary hormone treatments were the result of division of existing cells in the population, of differentiation of interstitial precursor cells, or both. In this context, it must be stressed that our data reflected the effects of hormones to prevent the decline of cells due to hypophysectomy rather than their recovery. Macrophage proliferation was taken into account because macrophages closely resemble Leydig cells and are known to proliferate after hormonal treatment. A double-labeling procedure (acid phosphatase and anti-bromodeoxyuridine [anti-BUdR]) revealed that LH, FSH, and PRL increased the number of testicular macrophages 105-, 104-, and 103-fold, respectively, in hypophysectomized rats compared to hypophysectomized control animals. BUdR incorporation in testicular macrophages was greater after PRL treatment than after LH and FSH supplementation. In contrast, we were unable to demonstrate any effect of rat GH on the macrophage population. Light microscopic analysis of plastic embedded sections of treated rat testis revealed that LH increased the numbers of Leydig, precursor mesenchymal, and myoid cells 6-, 4-, and 1.3-fold, respectively. LH also stimulated BUdR incorporation into all interstitial cell types. PRL administration increased both the number of Leydig and precursor mesenchymal cells (each 3-fold) but decreased the number of endothelial lymphatic cells (1.5-fold) when compared to the control animals. In contrast, FSH did not increase the number and proliferation of Leydig cells but exerted a slight proliferative effect on the other interstitial cell populations. In GH-treated rats, the number of precursor mesenchymal cells increased two fold above the control rats. GH also exerted slight proliferative effects on both precursor mesenchymal and myoid cells. Immunohistochemical studies of steroidogenic enzymes in the testicular interstitium of treated rats demonstrated the presence of steroidogenic enzymes, not only in Leydig and precursor mesenchymal cells, but also in some (1%-2%) endothelial lymphatic cells and myoid cells. This may indicate that both of these cell types are also constitutively equipped to perform steroidogenesis or that they are precursor cells undergoing differentiation. Taken together, changes in the number of Leydig cells in our animal model appeared more likely to be dependent on the transformation of precursor cells than on division of preexisting mature Leydig cells.

20 alpha-hydroxysteroid dehydrogenase activity in canine spontaneous neoplasms.

20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activities in 57 neoplastic tissues surgically removed from dogs were measured. Forty-eight of 57 tumor samples were shown to possess 20 alpha-HSD activity. These tissues were histopathologically classified into 22 benign and 26 malignant tumors. Among these tumors, mixed tumor types demonstrated the higher 20 alpha-HSD activity than epithelial and non-epithelial types, and malignant tumors of each tissue type showed slightly but not significantly higher 20 alpha-HSD activities comparing with the corresponding benign ones. Comparing with the activity of normal tissues examined, the corresponding tumor tissues showed significantly higher 20 alpha-HSD activities. Thus, 20 alpha-HSD activity was found in neoplastic tissues at a considerable high rate and the activity seemed to be higher in pathologically malignant tumors.

20 alpha-Hydroxysteroid dehydrogenase activity in rat placenta.

20 alpha-Hydroxysteroid dehydrogenase (20 alpha-HSD) (EC.1.1.1.149) is the enzyme which catabolizes progesterone to 20 alpha-dihydroprogesterone (20 alpha-OHP), a biologically inactive steroid, and is distributed in a variety of tissues including non-steroid-producing tissues. In the present study, changes in cytosolic 20 alpha-HSD activity were investigated in rat placental tissues and its relationship to embryonic mortality was considered; the mesometrial endometrium (including the ectoplacental cone) on days 8-11 of pregnancy (day 0 = estrus), and the chorioallantoic placenta and visceral yolk sac (vitelline membrane) on days 12-21 were separately subjected to measurement of the enzyme activity. 20 alpha-HSD activity was not detected in the chorioallantoic placenta until day 20 and then increased dramatically on day 21. Interestingly, considerable activity of the enzyme was found in the visceral yolk sac from days 14 to 21 and in the mesometrial endometrium from days 8 to 10, whereas it was undetectable in these tissues on days 11 and 12. Analysis of DEAE column chromatography revealed that these tissues contain two different types of 20 alpha-HSD (HSD-1 and HSD-2). By an immunohistochemical method, with polyclonal antiserum to rat 20 alpha-HSD, decidual cells and trophoblastic giant cells adjacent to the ectoplacental cone (day 10), spongiotrophoblasts and visceral yolk sac cells (days 21) were positively stained. The number of fetuses on day 10 of pregnancy was 15.4 and decreased significantly to 12.9 on day 12.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytosolic 20 alpha-hydroxysteroid dehydrogenase activity in spontaneous neoplasms in the dog and cat.

Steroid-producing tissues such as ovary, placenta, testis and adrenal gland and non-steroid-producing cells including lymphocytes are known to contain 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD). In the present study, we measured and partially characterized the 20 alpha-HSD in 11 neoplastic tissues surgically removed from dogs and a cat. The tissues were pathologically classified as 4 benign and 7 malignant. The enzyme activities in the cytoplasmic fraction were positive in 6 of the 7 malignant tissues and 2 of the 4 benign ones. Following DEAE chromatography analysis of 2 malignant tumour samples, multiple forms of enzyme activities with different electric charges were detected. Furthermore, 20 alpha-HSD activity in the cytosol fraction from malignant tumours was increased dramatically by passage through the DEAE column, suggesting that the enzyme is present in the cytosol as an inactive or sequestered form.

Prokaryotic 20 beta-hydroxysteroid dehydrogenase is an enzyme of the 'short-chain, non-metalloenzyme' alcohol dehydrogenase type.

The primary structure of 20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans was determined after FPLC purification of a commercial preparation. Peptides obtained from different proteolytic cleavages were purified by reverse phase HPLC. The 255-residue structure deduced was found to be distantly homologous to those of Drosophila alcohol dehydrogenase and several other dehydrogenases, establishing that prokaryotic 20 beta-hydroxysteroid dehydrogenase as a member of the 'short-chain alcohol dehydrogenase family'. With the enzymes characterized, the identity is greatest (31-34%) towards 4 other prokaryotic dehydrogenases, but the family also includes mammalian steroid and prostaglandin dehydrogenases. These enzymes are low in Cys and have a strictly conserved Tyr residue that appears to be important.

Denaturation by urea and renaturation of 20 beta-hydroxysteroid dehydrogenase studied by high-performance size exclusion chromatography.

The denaturation by urea and renaturation of 20 beta-hydroxysteroid dehydrogenase, a tetrameric enzyme consisting of four identical subunits, were followed by high-performance size exclusion chromatography to detect intermediates in the processes. During the denaturation process no intermediate form (structured monomers or dimers) between the tetramer and the denatured monomer was observed. During the renaturation process, carried out either with or without NADH, high molecular weight aggregates, native tetramers, and low molecular weight intermediates were evidenced and quantified. The contemporaneous measurement of recovery of activity unambiguously demonstrated that the tetrameric structure is essential for enzymatic activity.

Mast cells induced in vitro by interleukin 3 from native murine thymus cells.

To shed further light on the induction and characterization of thymus-derived mast cells, we cultured a variety of cell populations from murine thymus tissues (Balb/c) in the presence or absence of interleukin 3 (IL-3). The whole cell population and the non-adherent T cell-depleted population developed mast cells. The morphological studies revealed granulated cells; the granules were stained with toluidine blue, alcian blue (pH 3.0), and metachromatic dyes. Electron microscopy revealed altered mast cell granules. These cells contained relatively low amounts of histamine (approximately 1700 ng/10(6) cells), were IL-3 (but not IL-2)-dependent, and did not possess T-cell, B-cell, or macrophage markers. No phagocytosis was observed. The cells also had 20 alpha-hydroxysteroid dehydrogenase, and both IL-3 and IgE (145,600/cell) high affinity receptors. The frequency analysis showed 17 precursor cells per 10(6) thymic cells. The results indicate that the thymus indeed contains progenitors of mast cells responsive to IL-3, and that the mast cells are derived from non-T, non-phagocytic, and non-adherent cells of the thymus. Their T-lymphocyte product (IL-3) dependency, ultrastructural appearance, granular stainability, and low content of histamine may support the view that the mast cells originating from the thymus probably belong to a mucosal mast cell lineage.

BALB/c and CBA mice differ in the subtype of T cell involved in Moloney murine leukemia virus-induced lymphomas.

Subsets of T cells can be recognized by presence or absence of terminal deoxynucleotidyl transferase (TdT) or 20 alpha-hydroxysteriod dehydrogenase (20 alpha SDH) activity. By use of these enzyme markers Moloney murine leukemia virus (M-MuLV)-induced lymphomas were shown to involve different T-cell subtypes. In the present paper we show that the genotype of the mouse has a strong influence on the subtype of T cell involved in lymphoma. BALB/c mice preferentially develop 20 alpha SDH-positive lymphomas, whereas CBA lymphomas often have the TdT phenotype. Comparison of the 20 alpha SDH activity of normal bone marrow cells showed that BALB/c mice have higher enzyme levels than CBA mice. The relative availability of a certain type of cell at a critical early step in leukemogenesis may thus influence lymphoma type.

An analysis of factors responsible for resorption of embryos in cisplatin-treated rats.

Pregnant rats were injected ip with 4 or 7 mg cisplatin/kg on gestation day (gd) 6 to study its effect on embryonal resorption. Serum concentrations of prolactin, luteinizing hormone (LH), and progesterone were determined by radioimmunoassay in pregnant rats, and related to the effects of cisplatin on the maintenance of pregnancy. The nocturnal prolactin surge on gd 9 was abolished in cisplatin-treated rats. Within 3 days after drug injection, LH concentrations decreased 39%, while serum progesterone decreased 63% by Day 10. A histochemical study of 20 alpha-hydroxysteroid dehydrogenase activity revealed no enzyme activity by gd 10. It is proposed that the cause of cisplatin-related embryonal resorption in rats may be due to decreases in hormone concentrations observed after drug treatment.

Influence of genotype and the organ of origin on the subtype of T-cell in Moloney lymphomas induced by transfer of preleukemic cells from athymic and thymus-bearing mice.

Thymus, spleen, and bone marrow of 1-month-old neonatally Moloney murine leukemia virus-inoculated mice have been transferred to 400-R-irradiated syngeneic recipients of the opposite sex. The donor or recipient origin of T-cell lymphomas arising in the host animal was identified by the sex chromosome marker. Spleen and bone marrow of athymic BALB-nu/nu mice contain cells with the potential to develop into T-cell lymphomas upon transfer to thymus-bearing BALB/c recipients. Such lymphomas arise from at least two subsets of T-cells, one terminal deoxynucleotidyl transferase (TdT) positive and the other 20 alpha-hydroxysteroid dehydrogenase positive. The enzyme-negative precursor T-cells from the BALB-nu/nu spleen and bone marrow can thus mature to enzyme-positive cells and give rise to lymphoma in the thymus-bearing recipient. Preleukemic spleen and bone marrow, but not thymus, from CBA and BALB/c mice regularly contained cells with the potential to develop lymphoma. The subset of T-cell involved was influenced by the genotype since lymphomas arising after the transfer of CBA and BALB/c spleens were TdT positive and 20 alpha-hydroxysteroid dehydrogenase positive, respectively. In thymus-bearing mice, but not in nude mice, the transfer of preleukemic spleen cells gave lymphomas earlier than did transfer of bone marrow cells. This suggests that the more mature lymphoid cell population in the spleen of thymus-bearing mice may allow leukemic transformation to occur more rapidly than do the less mature cells in the bone marrow. In one-third of the cases, the virus produced by the preleukemic cells transferred induced new lymphomas involving recipient host cells. These de novo-induced lymphomas were all TdT positive. We suggest that leukemic transformation of TdT-positive cells may occur through a different mechanism than does transformation of cells bearing the 20 alpha-hydroxysteroid dehydrogenase marker.

The relative roles of interleukins 1, 2, and 3 in the regulation of T cell differentiation.

The initial description and purification of IL 3 was based on its potential relevance in early T cell differentiation. Although the data do not provide conclusive evidence for a role of IL 3 in T cell differentiation, a number of observations provide some indications that a relationship exists. The most intriguing aspects are concerned with the distribution and regulation of expression of 20 alpha SDH. This particular marker is interesting since it may allow studies of aspects of T cell differentiation that have not been possible with more conventional approaches. The available data are consistent with the hypothesis that in the bone marrow there exists a stem cell that in response to IL 3 is induced to differentiate including the induction of expression of 20 alpha SDH and Thy 1+. Phenotypically this induced cell is similar to a medullary thymocyte, although the cells derived in vitro clearly do not have the functional characteristics of medullary thymocytes. It can be rationalized that a bone marrow-induced prothymocyte may require the thymic microenvironment for continued maturation. Irrespective of this, it now becomes necessary to further explore the possibilities by extending the approaches used to define and study early bone marrow-localized pre-T cell populations as well as continuing to define the necessary components of the thymus in the differentiation pathway for T cells. In these areas of research the experience and systems derived from studies of long-term bone marrow cultures will be of considerable value. In addition to the proposed role that IL 3 plays in early T cell differentiation, it is apparent that the stem cell that is induced to differentiate may have considerably more potentials than the T cell lineage. The ability of IL 3 to induce the differentiation of 20 alpha SDH-positive mastlike cells in vitro is the most demonstrable functional phenotype. The absolute requirement of Il 3 throughout the differentiation of such cells indicates that in vivo, where IL 3 is generally not detectable, the frequency with which such progeny are obtained may be quite low and restricted to unique immunological situations in which high levels of IL 3 are produced. In addition, it appears likely that IL 3 induces the differentiation of a cell that in the presence of erythropoietin can be induced to differentiate along an erythroid pathway. Last, IL 3 may induce the differentiation of promyeloblasts that can differentiate in the presence of CSF-2 [unpublished data].(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of progestin biosynthetic enzymes in cultured rat granulosa cells: effects of prolactin, beta 2-adrenergic agonist, human chorionic gonadotropin and gonadotropin releasing hormone.

Prolactin (Prl), beta 2-adrenergic agents and human chorionic gonadotropin (hCG) are luteotropic in rats, whereas gonadotropin releasing hormone (GnRH) exerts direct inhibitory effects on ovarian steroidogenesis. The present study examined the modulation of the progestin biosynthetic pathway by the luteotropic agents, as well as the actions of GnRH. Rat granulosa cells were primed with follicle-stimulating hormone (FSH) to increase their responsiveness to the luteotropic agents. Subsequent treatment for 2 days with Prl, terbutaline (a beta 2-adrenergic agonist) or hCG stimulated the production of progesterone, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P), pregnenolone and the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). In contrast, treatment with Prl or terbutaline, but not hCG, inhibited 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity by decreasing the apparent maximal velocity of the enzyme with no change in its Km value. Concomitant treatment with GnRH inhibited progesterone, but increased 20 alpha-OH-P production stimulated by Prl or terbutaline. These effects were associated with a stimulation of 20 alpha-HSD activity, while neither 3 beta-HSD activity nor pregnenolone biosynthesis was decreased. In contrast, GnRH inhibited progesterone production in hCG-treated cells without affecting 20 alpha-OH-P production. This was associated with an inhibitory effect of GnRH on pregnenolone biosynthesis with no effect upon 3 beta-HSD activity. Thus, Prl and the beta 2-agonist stimulate progesterone production in granulosa cells by increasing pregnenolone production and 3 beta-HSD activity as well as by decreasing 20 alpha-HSD activity, while hCG stimulates progesterone production by increasing pregnenolone production and 3 beta-HSD activity. The inhibitory effect of GnRH on Prl- or terbutaline-stimulated progesterone production appears to result from a preferential increase in 20 alpha-HSD activity, while the GnRH inhibition of hCG-stimulated progesterone production appears to result from a preferential inhibition of pregnenolone production.

Distribution of progesterone receptor, estradiol dehydrogenase, and 20 alpha-dihydroprogesterone dehydrogenase activities in human endometrial glands and stroma: progestin induction of steroid dehydrogenase activities in vitro is restricted to the glandular epithelium.

Endometrial glands were separated from stromal cells by collagenase digestion of human endometrium, and the distribution of progesterone (P) receptor and the activities of P-regulated enzymes, 17 beta-estradiol dehydrogenase (E2DH) and 20 alpha-dihydroprogesterone dehydrogenase (20 alpha DH), were determined in these preparations. Concentrations of cytosolic P receptor were estimated by Scatchard plot analysis of specific [3H]P binding in intact proliferative endometrium and in glands and stromal cells isolated from this tissue. Epithelial cells were more than 10-fold enriched in high affinity, P-specific binding sites compared to stromal cells. The binding constants (Kd) for [3H]P binding were essentially similar in undissociated endometrium, glandular epithelium, and stroma, ranging from 1--5 nM. The activities of E2DH and 20 alpha DH were also 3-fold higher in the glandular epithelium than in whole tissue or stroma during both proliferative and secretory stages of the menstrual cycle. In addition, the induction of these enzyme activities by progestin in vitro in cultured explants of proliferative endometrium was restricted to the glandular epithelium. Thus, the effects of P on E2DH and 20 alpha DH are expressed in the same cells that contain receptors for P.

Different T-cell subtypes are associated with pathologically distinct forms of Moloney leukemia virus (M-MuLV)-induced lymphoma.

The T-cell enzyme markers, terminal deoxy-nucleotidyltransferase (TDT) and 20 alpha hydroxysteroid dehydrogenase (20 alpha SDH), were used to classify lymphomas induced by the Moloney leukemia virus (M-MuLV). Different subtypes of T cells were shown to be involved in different types of lymphoma. Thymomas were TdT-positive and grew as subcutaneous solid tumors at the site of inoculation. Spleen cells from mice with generalized lymphoma were of two types. In the majority of cases the lymphomas consisted of 20 alpha SDH-positive cells that homed to spleen and lymph nodes upon transplantation. In a few cases the cells of enlarged spleens were TdT-positive and, like the TdT-positive thymomas, could be transplanted as subcutaneous tumors. Thus, TdT-positive and 20 alpha SDH-positive T-cell lymphomas can be distinguished by their homing properties. Preleukemic thymus cells from M-MuLV inoculated mice can, after transfer to 400 -R irradiated syngeneic hosts, induce new lymphomas by virus release or grow in an autonomous fashion in the recipients. Whether of donor or recipient type, these lymphomas are TdT-positive. In contrast, preleukemic bone marrow cells give lymphomas of donor type which are as heterogeneous for T-cell enzymes as are lymphomas induced by neonatal inoculation of M-MuLV.