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Characterization of homogeneous recombinant rat ovarian 20alpha-hydroxysteroid dehydrogenase: fluorescent properties and inhibition profile.

ABSTRACT: In rat ovary, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, converts progesterone into the inactive progestin 20alpha-hydroxyprogesterone and has been implicated in the termination of pregnancy. Here we report a convenient overexpression system that permits the purification of milligram quantities of homogeneous recombinant 20alpha-HSD with wild-type enzyme activity. The availability of this enzyme has permitted detailed kinetic, inhibition and fluorescence analyses. The enzyme exhibited narrow steroid specificity, catalysing reactions only at C-20; it reduced progesterone and 17alpha-hydroxyprogesterone and oxidized 20alpha-hydroxypregnanes. It also turned over common AKR substrates, such as 9, 10-phenanthrenequinone and 4-nitrobenzaldehyde. The intrinsic fluorescence spectrum of 20alpha-HSD was characterized and was quenched on the binding of NADP(H), yielding a KNADPd of 0.36 microM and a KNADPHd of 0.64 microM. NADP(H) binding generated an energy transfer band that could not be quenched by steroids. Inhibition studies conducted with non-steroidal and steroidal anti-inflammatory drugs and synthetic oestrogens indicated that even though rat ovarian 20alpha-HSD and rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) share more than 67% amino acid identity, their inhibition profiles are markedly different. Unlike 3alpha-HSD, most of these compounds did not inhibit 20alpha-HSD. Only meclofenamic acid and hexoestrol were potent competitive inhibitors for 20alpha-HSD, yielding K(i) values of 18.9 and 14.3 microM respectively. These studies suggest that selective non-steroidal AKR inhibitors could be developed for 20alpha-HSD that might be useful in maintaining pregnancy and that specific inhibitors might be developed from either N-phenylanthranilates or biphenols.

SUBMITTER: Ma H

PROVIDER: S-EPMC1220427 | biostudies-other | 1999 Aug

REPOSITORIES: biostudies-other

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Project description:Human 17beta-hydroxysteroid dehydrogenase type 1 (17?-HSD1) is a steroid-converting enzyme that has long been known to play critical roles in estradiol synthesis and more recently in dihydrotestosterone (DHT) inactivation, showing a dual function that promotes breast cancer cell proliferation. Previously, we reported the first observation of the influence of the enzyme on endogenous estrogen-responsive gene expression. Here, we demonstrate the impact of 17?-HSD1 expression on the breast cancer cell proteome and investigate its role in cell migration.17?-HSD1 was stably transfected in MCF7 cells and the proteome of the generated cells overexpressing 17?-HSD1 (MCF7-17?HSD1 cells) was compared to that of the wild type MCF7 cells. Proteomics study was performed using two-dimensional gel electrophoresis followed by mass spectrometry analysis of differentially expressed protein spots. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to investigate the transcription of individual gene. The effect of 17?-HSD1 on MCF7 cell migration was verified by a wound-healing assay.Proteomic data demonstrate that the expression of more than 59 proteins is modulated following 17?-HSD1 overexpression. 17?-HSD1 regulates the expression of important genes and proteins that are relevant to cell growth control, such as BRCA2 and CDKN1A interacting protein (BCCIP) and proliferating cell nuclear antigen (PCNA) which are down- and upregulated in MCF7-17?HSD1 cells, respectively. RT-qPCR data reveal that 17?-HSD1 increases the mRNA levels of estrogen receptors (ER) alpha and beta by 171 and 120%, respectively, while decreasing that of the androgen receptor by 64%. Interestingly, 17?-HSD1 increases the mRNA transcript (by 3.6 times) and the protein expression of the metastasis suppressor gene nm23-H1 and the expression of the two enzymes are closely correlated. We have further shown that 17?-HSD1 expression is associated with an increase of MCF7 cell migration.In addition to the regulation of important genes, we have demonstrated for the first time that 17?-HSD1 increases breast cancer cell migration, in spite of its positive regulation of the antimetastatic gene NM23. This is also correlated to its stimulation of breast cancer cell growth, further confirming its targeting in ER positive breast cancer. The novel findings in this study suggest several directions for future research on the contribution of 17?-HSD1 to breast cancer progression and related treatment.

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Characterization of homogeneous recombinant rat ovarian 20alpha-hydroxysteroid dehydrogenase: fluorescent properties and inhibition profile.

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