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Biochemical factors governing the steady-state estrone/estradiol ratios catalyzed by human 17beta-hydroxysteroid dehydrogenases types 1 and 2 in HEK-293 cells.

ABSTRACT: Human 17beta-hydroxysteroid dehydrogenase types 1 and 2 (17betaHSD1 and 17betaHSD2) regulate estrogen potency by catalyzing the interconversion of estrone (E1) and estradiol (E2) using nicotinamide adenine dinucleotide (phosphate) cofactors NAD(P)(H). In intact cells, 17betaHSD1 and 17betaHSD2 establish pseudo-equilibria favoring E1 reduction or E2 oxidation, respectively. The vulnerability of these equilibrium steroid distributions to mutations and to altered intracellular cofactor abundance and redox state, however, is not known. We demonstrate that the equilibrium E2/E1 ratio achieved by 17betaHSD1 in intact HEK-293 cell lines is progressively reduced from 94:6 to 10:90 after mutagenesis of R38, which interacts with the 2'-phosphate of NADP(H), and by glucose deprivation, which lowers the NADPH/NADP(+) ratio. The shift to E2 oxidation parallels changes in apparent K(m) values for purified 17betaHSD1 proteins to favor NAD(H) over NADP(H). In contrast, mutagenesis of E116 (corresponding to R38 in 17betaHSD1) and changes in intracellular cofactor ratios do not alter the greater than 90:10 E1/E2 ratio catalyzed by 17betaHSD2, and these mutations lower the apparent K(m) of recombinant 17betaHSD2 for NADP(H) only less than 3-fold. We conclude that the equilibrium E1/E2 ratio maintained by human 17betaHSD1 in intact cells is governed by NADPH saturation, which is strongly dependent on both R38 and high intracellular NADPH/NADP(+) ratios. In contrast, the preference of 17betaHSD2 for E2 oxidation strongly resists alteration by genetic and metabolic manipulations. These findings suggest that additional structural features, beyond the lack of a specific arginine residue, disfavor NADPH binding and thus support E2 oxidation by 17betaHSD2 in intact cells.

SUBMITTER: Sherbet DP

PROVIDER: S-EPMC2736091 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Biochemical factors governing the steady-state estrone/estradiol ratios catalyzed by human 17beta-hydroxysteroid dehydrogenases types 1 and 2 in HEK-293 cells.

Sherbet Daniel P DP Guryev Oleg L OL Papari-Zareei Mahboubeh M Mizrachi Dario D Rambally Siayareh S Akbar Sharareh S Auchus Richard J RJ

Endocrinology 20090625 9

Human 17beta-hydroxysteroid dehydrogenase types 1 and 2 (17betaHSD1 and 17betaHSD2) regulate estrogen potency by catalyzing the interconversion of estrone (E1) and estradiol (E2) using nicotinamide adenine dinucleotide (phosphate) cofactors NAD(P)(H). In intact cells, 17betaHSD1 and 17betaHSD2 establish pseudo-equilibria favoring E1 reduction or E2 oxidation, respectively. The vulnerability of these equilibrium steroid distributions to mutations and to altered intracellular cofactor abundance an ...[more]

PMID: 19556422

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Project description:BackgroundPituitary tumor transforming gene (PTTG), also known as securin, is highly expressed in various tumors including pituitary, thyroid, colon, ovary, testis, lung, and breast. An overexpression of PTTG enhances cell proliferation, induces cellular transformation in vitro, and promotes tumor development in nude mice. PTTG also inhibits separation of sister chromatids leading to aneuploidy and genetic instability. A great amount of work has been undertaken to understand the biology of PTTG and its expression in various tumors. However, mechanisms by which PTTG mediates its tumorigenic function are not fully understood. To utilize this gene for cancer therapy, identification of the downstream signaling genes regulated by PTTG in mediation of its tumorigenic function is necessary. For this purpose, we expressed PTTG in human embryonic kidney (HEK293) cells that do not express PTTG and analyzed the downstream genes using microarray analysis.ResultsA total of 22,277 genes printed on an Affymetrix HG-U133A 2.0 GeneChip array were screened with labeled cRNA prepared from HEK293 cells infected with adenovirus vector expressing PTTG cDNA (AdPTTG cDNA) and compared with labeled cRNA prepared from HEK293 cells infected with control adenovirus (control Ad) or adenovirus vector expressing GFP (AdGFP). Out of 22,277 genes, 71 genes were down-regulated and 35 genes were up-regulated with an FDR corrected p-value of < or = 0.05 and a fold change of > or =2. Most of the altered genes identified are involved in the cell cycle and cell apoptosis; a few are involved in mRNA processing and nitrogen metabolism. Most of the up-regulated genes belong to the histone protein family.ConclusionPTTG is a well-studied oncogene for its role in tumorigenesis. In addition to its importance in regulation of the cell cycle, this gene has also been recently shown to play a role in the induction of cell apoptosis. The microarray analysis in the present study demonstrated that PTTG may induce apoptosis by down-regulation of oncogenes such as v-Jun and v-maf and up-regulation of the histone family of genes.

Dataset Information

Biochemical factors governing the steady-state estrone/estradiol ratios catalyzed by human 17beta-hydroxysteroid dehydrogenases types 1 and 2 in HEK-293 cells.

Publications

Biochemical factors governing the steady-state estrone/estradiol ratios catalyzed by human 17beta-hydroxysteroid dehydrogenases types 1 and 2 in HEK-293 cells.

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