Hui-Fen Zhao

Structure and tissue-specific expression of 3ß-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase genes in human and rat classical and peripheral steroidogenic tissues

The enzyme 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3 beta-HSD) catalyzes the oxidation and isomerization of 5-ene-3 beta-hydroxypregnene and 5-ene-hydroxyandrostene steroid precursors into the corresponding 4-ene-ketosteroids necessary for the formation of all classes of steroid hormones. We have recently characterized two types of human 3 beta-HSD cDNA clones and the corresponding genes which encode deduced proteins of 371 and 372 amino acids, respectively, and share 93.5% homology. The human 3 beta-HSD genes containing 4 exons were assigned by in situ hybridization to the p11-p13 region of the short arm of chromosome 1. We have also recently elucidated the structure of three types of rat 3 beta-HSD cDNAs as well as that of one type of 3 beta-HSD from bovine and macaque ovary lambda gt11 cDNA libraries which all encode 372 amino acid proteins. The human type I 3 beta-HSD is the almost exclusive mRNA species detected in the placenta and skin, while the human type II is the predominant mRNA species in the adrenals, ovaries and testes. The predicted rat type I and type II 3 beta-HSD proteins expressed in adrenals, gonads and adipose tissue share 94% homology while they share 80% similarity with the liver-specific type III 3 beta-HSD. Transient expression of human type I and type II as well as rat type I and type II 3 beta-HSD cDNAs in HeLa human cervical carcinoma cells reveals that 3 beta-ol dehydrogenase and 5-ene-4-ene isomerase activities reside within a single protein and these cDNAs encode functional 3 beta-HSD proteins that are capable of converting 3 beta-hydroxy-5-ene-steroids into 3-keto-4-ene derivatives as well as the interconversion of 3 beta-hydroxy and 3-keto-5 alpha-androstane steroids. We have found that the rat type III mRNA species was below the detection limit in intact female liver while, following hypophysectomy, its accumulation increased to 55% of the levels measured in intact or HYPOX male rats, an increase which can be blocked by administration of ovine prolactin (oPRL). In addition, in female rats, treatment with oPRL for 10 days starting 15 days after HYPOX, markedly decreased ovarian 3 beta-HSD mRNA accumulation accompanied by a similar decrease in 3 beta-HSD activity and protein levels. Treatment with the gonadotropin hCG reversed the potent inhibitory effect of oPRL on these parameters and stimulated 3 beta-HSD mRNA levels in ovarian interstitial cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular cloning, cDNA structure and predicted amino acid sequence of bovine 3β-hydroxy-5-ene steroid dehydrogenase/Δ5-Δ4 isomerase

We have used our recently characterized human 3β‐hydroxy‐5‐ene steroid dehydrogenase/Δ5‐Δ4‐isomerase (3β‐HSD) cDNA as probe to isolate cDNAs encoding bovine 3β‐HSD from a bovine ovary λgtll cDNA library. Nucleotide sequence analysis of two overlapping cDNA clones of 1362 bp and 1536 bp in length predicts a protein of 372 amino acids with a calculated molecular mass of 42093 (excluding the first Met). The deduced amino acid sequence of bovine 3β‐HSD displays 79% homology with human 3β‐HSD while the nucleotide sequence of the coding region shares 82% interspecies similarity. Hybridization of cloned cDNAs to bovine ovary poly(A)+ RNA shows the presence of an approximately 1.7 kb mRNA species.

Characterization of macaque 3β-hydroxy-5-ene steroid dehydrogenase/Δ5-Δ4 isomerase: structure and expression in steroidogenic and peripheral tissues in primate

Abstract The conversion of 3β-hydroxy-5-ene steroids by the enzyme complex 3β-hydroxysteroid dehydrogenase/Δ 5 -Δ 4 isomerase (3β-HSD) is an obligatory step in the biosynthesis of all classes of hormonal steroids in classical steroidogenic as well as in peripheral tissues. To develop a model more closely related to the human, we have isolated and characterized cDNA clones encoding macaque 3β-HSD by screening a rhesus monkey ovary κgt11 cDNA library using a human 3β-HSD cDNA probe. Nucleotide sequence of 1629 bp from overlapping cDNA clones predicts a protein of 372 amino acids with a calculated molecular mass of 41,874 (excluding the first Met). The deduced amino acid sequence of macaque 3β-HSD displays 79.4% and 93.9% similarity with that of bovine and human 3β-HSD, respectively. RNA blot analysis performed under high stringency conditions of macaque poly(A) + RNA samples using full-length 32 P-labeled macaque 3β-HSD cDNA revealed the presence of an approximately 1.7 kb mRNA species in classical steroidogenic tissues, namely the ovary, testis and adrenal glands as well as in several peripheral tissues including the liver, kidney and epididymis. Computer analysis of the deduced macaque 3/gb-HSD protein sequence predicts the presence of an NH 2 -terminal membrane-associated segment as well as four additional membrane-spanning segments, thus suggesting that 3β-HSD is an integral protein. The availability of macaque cDNA should permit detailed studies concerning the tissue-specific expression as well as the hormonal regulation of 3β-HSD mRNA in classical steroidogenic glands as well as in peripheral tissues which are an important site of Steroidogenesis in primates.

Synthetic progestins stimulate prostatic binding protein messenger RNAs in the rat ventral prostate

In order to assess the intrinsic androgenic activity of the synthetic progestins currently used as antiandrogens for the treatment of prostate cancer and other androgen-sensitive diseases, cyproterone acetate (CPA), medroxyprogesterone acetate (MPA) and megestrol acetate (MEG) were administered for 4 days to adult rats castrated 4 days previously. The effects of these compounds were measured on highly specific and sensitive markers of androgen action in the rat ventral prostate, namely the levels of messenger RNAs encoding the C1 (PBP-C1) and C3 (PBP-C3) components of rat prostatic binding protein (PBP). Steady-state mRNA levels were measured by dot-blot hybridization as well as by in situ hybridization. Treatment with CPA or MEG, at the twice daily dose of 10 mg, caused respective 2- and 4.5-fold increases in the steady-state levels of mRNA encoding PBP-C1. MPA, at the dose of 0.45 mg, twice daily, was approximately 40 times as potent as MEG, leading to an 8-fold increase in PBP-C1 mRNA levels. While the pure nonsteroidal antiandrogen flutamide (10 mg, twice daily) did not cause accumulation of PBP mRNAs when administered to castrated rats, it completely reversed the stimulatory effects of the synthetic progestins CPA, MPA and MEG. The results obtained by in situ hybridization were similar to those obtained by dot-blot analysis. Moreover, the synthetic progestins caused similar androgenic effects on PBP-C3 mRNA levels. The present data indicate that all three synthetic progestins currently used for the treatment of prostate cancer possess significant intrinsic androgenic activity as evidenced by their stimulatory effects on the accumulation of mRNAs sensitive to androgen action. Consequently, as indicated by this sensitive and androgen-specific in vivo rat model, such compounds are not recommended for the treatment of conditions requiring an optimal blockade of androgens, especially prostate cancer.

Potent antagonism between estrogens and androgens on GCDFP-15 expression and cell growth in the ZR-75-1 human breast cancer cells

While the presence of androgen receptors has been documented in normal1* and neoplastic breast tissue, as well as in several established breast cancer cell lines, very little is kcown about their functional significance. Physiological (0.110 nM) concentrations of androgens can counteract induction of the progesterone receptor by 17P-estradiol (E,) in MCF-7 cells through an androgen-receptor-mediated mechan i ~ m . ~ . ~ Moreover, in the T47-D human breast cancer cell line, Sa-dihydrotestosterone (5a-DHT) specifically induces the secretion of specific proteins, an effect that is reversed by the antiandrogen flutamide.6. Androgens have also been found to modulate the number of specific prolactin binding sites in another human breast cancer cell line. Although the above-mentioned studies indicate that breast cancer cells possess functional androgen receptors mediating various biochemical responses, no specific effect of androgens on cell proliferation has yet been reported. Androgens such as

Structure and Control of Expression of the 3βHSD and 17βHSD Genes in Classical Steroidogenic and Peripheral Intracrine Tissues

It is remarkable that humans, in addition to possessing a highly sophisticated endocrine system, have largely vested sex steroid formation in peripheral tissues (1). In fact, while the ovaries and testes are the exclusive sources of androgens and estrogens in the lower mammals, the situation is very different in higher primates, where active sex steroids are in a large part or whole synthesized locally, thus providing autonomous control to target tissues that are thus able to adjust formation and metabolism of sex steroids to local requirements. The situation of a high secretion rate of adrenal precursor sex steroids in men and women is thus completely different from current animal models used in the laboratory; namely rats, mice, guinea pigs, and all others except monkeys, where the secretion of sex steroids takes place exclusively in the gonads (1–3). Primates are thus unique in having adrenals that secrete large amounts of the precursor steroids dehydroepiandrosterone (DHEA) and especially DHEA-sulfate (DHEA-S) that are converted into androstenedione (Δ4-dione) and then into potent androgens and estrogens in peripheral tissues (2, 4).