Van Luu-The

Pathways of adipose tissue androgen metabolism in women: depot differences and modulation by adipogenesis

The objective was to examine pathways of androgen metabolism in abdominal adipose tissue in women. Abdominal subcutaneous (SC) and omental (OM) adipose tissue samples were surgically obtained in women. Total RNA was isolated from whole adipose tissue samples and from primary preadipocyte cultures before and after induction of differentiation. Expression levels of several steroid-converting enzyme transcripts were examined by real-time RT-PCR. Androgen conversion rates were also measured. We found higher expression levels in SC compared with OM adipose tissue for type 1 3beta-hydroxysteroid dehydrogenase (3beta-HSD-1; P < 0.05), for aldo-keto reductase 1C3 (AKR1C3; P < 0.0001), for AKR1C2 (P < 0.0001), and for the androgen receptor (P < 0.0001). 17beta-HSD-2 mRNA levels were lower in SC adipose tissue (P < 0.05). Induction of adipocyte differentiation led to significantly increased expression levels in SC cultures for AKR1C3 (4.7-fold, P < 0.01), 11-cis-retinol dehydrogenase (6.9-fold, P < 0.02), AKR1C2 (5.6-fold, P < 0.004), P-450 aromatase (5.7-fold, P < 0.02), steroid sulfatase (3.1-fold, P < 0.02), estrogen receptor-beta (11.8-fold, P < 0.01), and the androgen receptor (4.0-fold, P < 0.0005). Generally similar but nonsignificant trends were obtained in OM cultures. DHT inactivation rates increased with differentiation, this effect being mediated by dexamethasone alone, through a glucocorticoid receptor-dependent mechanism. In conclusion, higher mRNA levels of enzymes synthesizing and inactivating androgens are found in differentiated adipocytes, consistent with higher androgen-processing rates in these cells. Glucocorticoid-induced androgen inactivation may locally modulate the exposure of adipose cells to active androgens.

Human type 3 5α-reductase is expressed in peripheral tissues at higher levels than types 1 and 2 and its activity is potently inhibited by finasteride and dutasteride

Abstract 5α-Reductases are crucial enzymes involved in the biosynthesis of dihydrotestosterone, the most potent natural androgen. To date, three types of 5α-reductases, chronologically named types 1, 2 and 3 5α-reductases (SRD5a-1, 2 and 3) have been described. In the present paper, we characterized the activity and compared the mRNA expression levels of SRD5a-3 with those of SRD5a-1 and 2 in various human tissues, and determined its sensitivity to finasteride and dutasteride. We have established HEK-293 cell line that stably expressed SRD5a-3 for studying its activity and the inhibitory effect of finasteride, using [14C]labeled steroids. mRNA expression levels were quantified using real-time PCR in many male and female human tissues including the prostate, adipose tissue, mammary gland, as well as breast and prostate cancer cell lines. Incubation of HEK-SRD5a-3 cells with [14C]4-androstenedione and [14C]testosterone allowed us to show that SRD5a-3 can catalyze very efficiently both substrates 4-androstenedione and testosterone into 5α-androstanedione and dihydrotestosterone, respectively. We observed that the affinity of the enzyme for 4-androstenedione is higher than for testosterone. The activity of SRD5a-3 and SRD5a-2 are similarly sensitive to finasteride, whereas dutasteride is a much more potent inhibitor of SRD5a-3 than SRD5a-2. Tissue distribution analysis shows that SRD5a-3 mRNA expression levels are higher than those of SRD5a-1 and SRD5a-2 in 20 analyzed tissues. In particular, it is highly expressed in the skin, brain, mammary gland and breast cancer cell lines, thus suggesting that SRD5a-3 could play an important role in the production of androgens in these and other peripheral tissues.

Steroidal lactones as inhibitors of 17β-hydroxysteroid dehydrogenase type 5 : Chemical synthesis, enzyme inhibitory activity, and assessment of estrogenic and androgenic activities

Androgens are well known to play a predominant role in prostate cancer and other androgen-dependent diseases. To decrease the level of androgen testosterone in the prostate, we are interested in developing inhibitors of 17beta-hydroxysteroid dehydrogenase type 5 (17beta-HSD5). This enzyme expressed in the prostate is one of the two enzymes able to convert 4-androstene-3,17-dione into testosterone. From a screening study, it was found that a series of steroid derivatives bearing a lactone on D-ring demonstrated potent inhibition of 17beta-HSD5 over-expressed in HEK-293 cells. The results of enzymatic assays using intact cells indicated that a C18-steroid (estradiol or 3-deoxyestradiol) backbone and a spiro-delta-lactone (six-member ring) are important for a strong inhibitory activity. Moreover, the presence of a dimethyl group at the alpha-position of the lactone carbonyl increases the selectivity of the inhibitor toward 17beta-HSD5. Compound 26, a 3-deoxyestradiol derivative with a dimethylated spiro-delta-lactone at position 17, possesses the most potent inhibitory activity for 17beta-HSD5 (IC(50)=2.9 nM). It showed no binding affinity for estrogen, androgen, progestin and glucocorticoid receptors (ER, AR, PR and GR). A weak proliferative effect was, however, observed on ZR-75-1 (ER+) cells in culture at high concentration (1 microM), but not at 0.03 microM. Interestingly, no significant proliferative effect was detected on Shionogi (AR+) cells in culture in the presence of 0.1 and 1 microM of lactone 26.

Structure-based inhibitor design for an enzyme that binds different steroids: a potent inhibitor for human type 5 17beta-hydroxysteroid dehydrogenase.

Human type 5 17β-hydroxysteroid dehydrogenase plays a crucial role in local androgen formation in prostate tissue. Several chemicals were synthesized and tested for their ability to inhibit this enzyme, and a series of estradiol derivatives bearing a lactone on the D-ring were found to inhibit its activity efficiently. The crystal structure of the type 5 enzyme in complex with NADP and such a novel inhibitor, EM1404, was determined to a resolution of 1.30Å. Significantly more hydrogen bonding and hydrophobic interactions were defined between EM1404 and the enzyme than in the substrate ternary complex. The lactone ring of EM1404 accounts for important interactions with the enzyme, whereas the amide group at the opposite end of the inhibitor contributes to the stability of three protein loops involved in the construction of the substrate binding site. EM1404 has a strong competitive inhibition, with a Ki of 6.9 ± 1.4 nm, demonstrating 40 times higher affinity than that of the best inhibitor previously reported. This is observed despite the fact that the inhibitor occupies only part of the binding cavity. Attempts to soak the inhibitor into crystals of the binary complex with NADP were unsuccessful, yielding a structure with a polyethylene glycol fragment occupying the substrate binding site. The relative crystal packing is discussed. Combined studies of small molecule inhibitor synthesis, x-ray crystallography, enzyme inhibition, and molecular modeling make it possible to analyze the plasticity of the substrate binding site of the enzyme, which is essential for developing more potent and specific inhibitors for hormone-dependent cancer therapy.

Expression of Enzymes Involved in Estrogen Metabolism in Human Prostate

There is evidence that estrogens can directly modulate human prostate cell activity. It has also been shown that cultured human prostate cancer LNCaPcan synthesize the active estrogen estradiol (E2). To elucidate the metabolism of estrogens in the human prostate, we have studied the expression of enzymes involved in the formation and inactivation of estrogens at the cellular level. 17β-Hydroxysteroid dehydrogenase (17β-HSD) types 1, 2, 4, 7, and 12, as well as aromatase mRNA and protein expressions, were studied in benign prostatic hyperplasia (BPH) specimens using in situ hybridization and immunohistochemistry. For 17β-HSD type 4, only in situ hybridization studies were performed. Identical results were obtained with in situ hybridization and immunohistochemistry. All the enzymes studied were shown to be expressed in both epithelial and stromal cells, with the exception of 17β-HSD types 4 and 7, which were detected only in the epithelial cells. On the basis of our previous results, showing that 3β-HSD and 17β-HSD type 5 are expressed in human prostate, and of the present data, it can be concluded that the human prostate expresses all the enzymes involved in the conversion of circulating dehydroepiandrosterone (DHEA) to E2. The local biosynthesis of E2 might be involved in the development and/or progression of prostate pathology such as BPH and prostate cancer through modulation of estrogen receptors, which are also expressed in epithelial and stromal cells. (J Histochem Cytochem 54:911-921, 2006)

The crystal structure of human Delta4-3-ketosteroid 5beta-reductase defines the functional role of the residues of the catalytic tetrad in the steroid double bond reduction mechanism.

The 5beta-reductases (AKR1D1-3) are unique enzymes able to catalyze efficiently and in a stereospecific manner the 5beta-reduction of the C4-C5 double bond found into Delta4-3-ketosteroids, including steroid hormones and bile acids. Multiple-sequence alignments and mutagenic studies have already identified one of the residues presumably located at their active site, Glu 120, as the major molecular determinant for the unique activity displayed by 5beta-reductases. To define the exact role played by this glutamate in the catalytic activity of these enzymes, biochemical and structural studies on human 5beta-reductase (h5beta-red) have been undertaken. The crystal structure of h5beta-red in a ternary complex with NADP (+) and 5beta-dihydroprogesterone (5beta-DHP), the product of the 5beta-reduction of progesterone (Prog), revealed that Glu 120 does not interact directly with the other catalytic residues, as previously hypothesized, thus suggesting that this residue is not directly involved in catalysis but could instead be important for the proper positioning of the steroid substrate in the catalytic site. On the basis of our structural results, we thus propose a realistic scheme for the catalytic mechanism of the C4-C5 double bond reduction. We also propose that bile acid precursors such as 7alpha-hydroxy-4-cholesten-3-one and 7alpha,12alpha-dihydroxy-4-cholesten-3-one, when bound to the active site of h5beta-red, can establish supplementary contacts with Tyr 26 and Tyr 132, two residues delineating the steroid-binding cavity. These additional contacts very likely account for the higher activity of h5beta-red toward the bile acid intermediates versus steroid hormones. Finally, in light of the structural data now available, we attempt to interpret the likely consequences of mutations already identified in the gene encoding the h5beta-red enzyme which lead to a reduction of its enzymatic activity and which can progress to severe liver function failure.

Glucocorticoid-induced androgen inactivation by aldo-keto reductase 1C2 promotes adipogenesis in human preadipocytes

Adipogenesis and lipid storage in human adipose tissue are inhibited by androgens such as DHT. Inactivation of DHT to 3α-diol is stimulated by glucocorticoids in human preadipocytes. We sought to characterize glucocorticoid-induced androgen inactivation in human preadipocytes and to establish its role in the antiadipogenic action of DHT. Subcutaneous and omental primary preadipocyte cultures were established from fat samples obtained in subjects undergoing abdominal surgeries. Inactivation of DHT to 3α/β-diol for 24 h was measured in dexamethasone- or vehicle-treated cells. Specific downregulation of aldo-keto reductase 1C (AKR1C) enzymes in human preadipocytes was achieved using RNA interference. In whole adipose tissue sample, cortisol production was positively correlated with androgen inactivation in both subcutaneous and omental adipose tissue (P < 0.05). Maximal dexamethasone (1 μM) stimulation of DHT inactivation was higher in omental compared with subcutaneous fat from men as well as subcutaneous and omental fat from women (P < 0.05). A significant positive correlation was observed between BMI and maximal dexamethasone-induced DHT inactivation rates in subcutaneous and omental adipose tissue of men and women (r = 0.24, n = 26, P < 0.01). siRNA-induced downregulation of AKR1C2, but not AKR1C1 or AKR1C3, significantly reduced basal and glucocorticoid-induced androgen inactivation rates (P < 0.05). The inhibitory action of DHT on preadipocyte differentiation was potentiated following AKR1C2 but not AKR1C1 or AKR1C3 downregulation. Specifically, lipid accumulation, G3PDH activity, and FABP4 mRNA expression in differentiated preadipocytes exposed to DHT were reduced further upon AKR1C2 siRNA transfection. We conclude that glucocorticoid-induced androgen inactivation is mediated by AKR1C2 and is particularly effective in omental preadipocytes of obese men. The interplay between glucocorticoids and AKR1C2-dependent androgen inactivation may locally modulate adipogenesis and lipid accumulation in a depot-specific manner.

Potent and Selective Steroidal Inhibitors of 17β-Hydroxysteroid Dehydrogenase Type 7, an Enzyme That Catalyzes the Reduction of the Key Hormones Estrone and Dihydrotestosterone

17beta-Hydroxysteroid dehydrogenase type 7 (17beta-HSD7) catalyzes the reduction of estrone (E(1)) into estradiol (E(2)) and of dihydrotestosterone (DHT) into 5alpha-androstane-3beta,17beta-diol (3beta-diol), therefore modulating the level of mitogenic estrogens and androgens in humans. By classical and parallel chemistry, we generated several 4-methyl-4-aza-5alpha-androstane derivatives differing in their C-17 substituent: 17beta-formamide, 17beta-benzamide, and 17beta-tertiary amine. Best candidates in each category had demonstrated good inhibitory potency toward the conversion of E(1) into E(2) (IC(50) = 189-451 nM) and also toward the conversion of DHT into 3beta-diol (69-91% at 3 microM). Inhibition assays with 17beta-HSD1, 17beta-HSD5, 5alpha-reductase (5alpha-R) 1 and 5alpha-R2 revealed that 17beta-HSD7 inhibitors with a 4-methyl-4-aza nucleus were also able to inhibit 5alpha-Rs but not the other enzymes tested. Two 4-aza-5alpha-androstane inhibitors were, however, selective and still showed good inhibition of 17beta-HSD7. First selective and efficient inhibitors of 17beta-HSD7 are now available for additional mechanistic and therapeutic studies.

Updated survey of the steroid-converting enzymes in human adipose tissues

Over the past decade, adipose tissues have been increasingly known for their endocrine properties, that is, their ability to secrete a number of adipocytokines that may exert local and/or systemic effects. In addition, adipose tissues have long been recognized as significant sites for steroid hormone transformation and action. We hereby provide an updated survey of the many steroid-converting enzymes that may be detected in human adipose tissues, their activities and potential roles. In addition to the now well-established role of aromatase and 11β-hydroxysteroid dehydrogenase (HSD) type 1, many enzymes have been reported in adipocyte cell lines, isolated mature cells and/or preadipocytes. These include 11β-HSD type 2, 17β-HSDs, 3β-HSD, 5α-reductases, sulfatases and glucuronosyltransferases. Some of these enzymes are postulated to bear relevance for adipose tissue physiology and perhaps for the pathophysiology of obesity. This elaborate set of steroid-converting enzymes in the cell types of adipose tissue deserves further scientific attention. Our work on 20α-HSD (AKR1C1), 3α-HSD type 3 (AKR1C2) and 17β-HSD type 5 (AKR1C3) allowed us to clarify the relevance of these enzymes for some aspects of adipose tissue function. For example, down-regulation of AKR1C2 expression in preadipocytes seems to potentiate the inhibitory action of dihydrotestosterone on adipogenesis in this model. Many additional studies are warranted to assess the impact of intra-adipose steroid hormone conversions on adipose tissue functions and chronic conditions such as obesity, diabetes and cancer.

Assessment of steroidogenic pathways that do not require testosterone as intermediate

Abstract Traditional literature and textbooks generally describe that estradiol (E2) and dihydrotestosterone (DHT) are synthesized from the aromatization and 5α-reduction of testosterone (T), respectively, following a pathway in which T is an essential intermediate (Tpath). This pathway implies that the steps of aromatization and 5α-reduction follow the reaction of the androgenic 17β-hydroxysteroid dehydrogenase (17β-HSD) that catalyzes the conversion of 4-androstenedione (4-dione) into T, and that estrogenic 17β-HSDs are not required. Contrary to this belief, the cloning of many estrogen-specific 17β-HSDs and the observation of higher affinity of aromatase and 5α-reductase for 4-dione than T are strongly in favor of biosynthetic pathways in which the steps catalyzed by aromatase and 5α-reductase precede that catalyzed by 17β-HSDs. Such pathways do not require T as an intermediate, as demonstrated by experiments using [14C]-labeled DHEA and 4-dione as substrates and incubation with SZ95 sebaceous gland, DU-145 prostate cancer and JEG-3 choriocarcinoma cell lines cultured in the presence of inhibitors of 5α-reductase and aromatase. A review of early literature about patients with testicular 17β-HSD deficiency and of steroid metabolism appears to confirm the physiological functionality of the E2 and DHT biosynthetic pathway not requiring T as intermediate (noTpath).