Eric Dupont

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)

Antiangiogenic properties of a novel shark cartilage extract: potential role in the treatment of psoriasis.

Background: A number of inflammatory and immune diseases are associated with vascular changes. Psoriasis, as an example, is a common inflammatory skin disease with dilation of capillaries as an early histological change. In more developed psoriatic lesions there is proliferation of blood vessels and neovascularization. The use of agents that target these vascular changes represents a novel therapeutic strategy in the treatment of inflammatory diseases. Since cartilage is an avascular tissue, it has been hypothesized that there may be factors found in cartilage that inhibit blood vessel formation. Objective: The objectives of this study were 1) to determine whether extracts of cartilage could inhibit angiogenesis, and 2) since altered angiogenesis is associated with certain diseases, including psoriasis, to examine whether inhibition of angiogenesis could potentially contribute to the treatment of psoriasis. Methods: Extracts of shark cartilage were prepared by homogenization and ultrafiltration to derive the active agent termed Æ-941. This agent was tested for antiangiogenesis activity using the embryonic vascularization test, which is a modification of the ex vivo chick embryo culture (CAM). Since one of the first steps in angiogenesis is degradation by metalloproteinases of the basement membrane of capillaries, Æ-941 was tested for collagenase activity using a fluorogenic peptide substrate. Anti-inflammatory properties were tested using a cutaneous irritation model in humans. Results: A dose dependent inhibition in embryonic neovascularization as well as in collagenase activity by Æ-941 was demonstrated. When test compounds were applied on the forearms of test subjects, Æ-941 was shown to have anti-inflammatory properties. Anecdotal data suggested that topical Æ-941 had a beneficial effect in psoriasis. Conclusion: Our results show that Æ-941 has anti-angiogenic and anti-inflammatory properties. Antiangiogenesis agents such as Æ-941 provide an entirely new class of agents to treat cutaneous and systemic diseases associated with altered vascularity.

Effects of human chorionic gonadotropin (hCG) and prolactin (PRL) on 3β-hydroxy-5-ene-steroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) expression and activity in the rat ovary☆

Abstract Using a recently cloned rat ovary 3β-HSD cDNA and antibodies raised against purified human placental 3β-HSD, we have studied the effects of treatment with human chorionic gonadotropin (hCG) and hyperprolactinemia achieved by pituitary implants, alone or in combination, on the expression and activity of ovarian 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) in intact adult rats. 32P-and 35S-labeled cDNA probes were used to evaluate the effects of treatments on 3β-HSD mRNA levels by dot blot and in situ hybridization, respectively, while enzymatic activity was measured by the conversion of [14C]dehydroepiandrosterone into [14C]androstenedione. The present data show that hCG exerts a marked trophic effect on rat corpora lutea with an increase in total ovarian 3β-HSD mRNA levels, 3β-HSD protein content as well as enzymatic activity, resulting in an increase in serum progesterone levels. Prolactin-secreting pituitary implants alone, on the other hand, while exerting small effects on 3β-HSD expression and activity, led to a marked potentiation of the stimulatory effect of hCG on all parameters. The present data show that hCG and PRL act synergistically to stimulate ovarian progesterone secretion via an increase in 3β-HSD mRNA levels, protein content and enzymatic activity.

Cartilage as a Source of Natural Inhibitors of Angiogenesis.

Ever since tumor-induced neovascularization was recognized as one of the key parameters that control tumor growth, considerable effort has been expended to identify ways to inhibit angiogenesis (1–3). Two approaches have been attempted.

Molecular Cloning of Rat 3β-HSD: Structure of Two Types of cDNAs and Differential Expression of Corresponding mRNAs in the Ovary

Following cleavage of the aliphatic side-chain of cholesterol, thus leading to the formation of pregnenolone and related \( \Delta \) 5-3β-hydroxysteroids, the next obligatory step in the formation of all classes of steroid hormones—namely, progesterone, mineralocorticoids, glucocorticoids, androgens and estrogens—requires the oxidation and isomerization of these \( \Delta \) 5-3β-hydroxysteroid precursors into \( \Delta \) 4-3-ketosteroids. This irreversible oxidative conversion is catalyzed by the enzymatic complex \( \Delta \) 5-3β-hydroxysteroid dehydrogenase/\( \Delta \) 5-\( \Delta \) 4 isomerase (3β-HSD). This membrane-bound enzymatic system is found in steroidogenic tissues such as the placenta, adrenal cortex, testis, and ovary (1–5), as well as in several peripheral tissues including the prostate, breast, brain, and liver (6–10).