Alpha-reductase Isozymes Research Articles

Natural mutagenesis study of the human steroid 5 alpha-reductase 2 isozyme.

The enzyme steroid 5 alpha-reductase utilizes NADPH to reduce the double bonds of a variety of steroid substrates with generalized 3-oxo, delta 4,5 structures. One substrate for this membrane-bound enzyme is testosterone, whose reduction to dihydrotestosterone is required for the embryonic differentiation of the external male genitalia and prostate. There are two 5 alpha-reductase isozymes, designated types 1 and 2, which have different biochemical and pharmacological properties. Inherited deficiencies of 5 alpha-reductase type 2 result in a form of male pseudohermaphroditism in which the external genitalia fail to develop normally. Here, nine additional mutations in the 5 alpha-reductase 2 gene in subjects with 5 alpha-reductase deficiency are described. The biochemical consequences of these mutations, as well as 13 previously identified missense mutations, were characterized by recreating the mutations in an expressible cDNA and transfecting into mammalian cells. Twelve of the 22 mutations inactivated the enzyme. The remaining 10 mutations impaired enzyme function by affecting either substrate or cofactor binding. Almost all mutations decreased the half-life of the protein, and all but one of the impaired enzymes had an altered pH optimum. The mutations provide insight into functional domains in the protein as well as an unusual acidic pH optimum characteristic of the 5 alpha-reductase type 2 isozyme.

Cell-type-specific expression of rat steroid 5 alpha-reductase isozymes.

The enzyme steroid 5 alpha-reductase (EC 1.3.99.5) is a component of an intercellular signaling pathway that determines cell fate in the primordium of the mammalian reproductive tract. During male phenotypic sexual differentiation, the dihydrotestosterone product of this enzyme binds to the androgen receptor and initiates development of the external genitalia and prostate. Genes encoding two isozymes of steroid 5 alpha-reductase with different biochemical properties and tissue distributions have recently been isolated. In the current study, we utilize in situ hybridization analysis to determine cell-type-specific expression patterns of the 5 alpha-reductase isozyme mRNAs in two androgen target tissues (regenerating ventral prostate and epididymis) and a peripheral tissue (liver). In regenerating ventral prostate, the type 1 mRNA is expressed in basal epithelial cells whereas expression of the type 2 mRNA is largely confined to stromal cells. These results were confirmed by immunohistochemical analysis and are consistent with distinct roles played by the isozymes in the prostate. In the epididymis, both 5 alpha-reductase isozyme mRNAs are expressed in epithelial cells. Only the type 1 mRNA is present in the liver. This mRNA is distributed in a striking spatial gradient extending from hepatocytes surrounding the portal triad (high expression) to those surrounding the central vein (low to absent expression). These findings demonstrate cell-type-specific expression of the steroid 5 alpha-reductase isozymes and underscore their distinct and overlapping functions in androgen physiology.

Tissue distribution and ontogeny of steroid 5 alpha-reductase isozyme expression.

The synthesis of dihydrotestosterone is catalyzed by steroid 5 alpha-reductase isozymes, designated types 1 and 2. Mutation of type 2 results in male pseudohermaphroditism, in which the external genitalia are phenotypically female at birth. Two striking and unexplained features of this disorder are that external genitalia of affected males undergo virilization during puberty and that these individuals have less temporal hair regression. The tissue-specific and developmental expression patterns of the 5 alpha-reductase isozymes were investigated by immunoblotting. The type 1 isozyme is not detectable in the fetus, is transiently expressed in newborn skin and scalp, and permanently expressed in skin from the time of puberty. There was no qualitative difference in 5 alpha-reductase type 1 expression between adult balding vs. nonbalding scalp. The type 2 isozyme is transiently expressed in skin and scalp of newborns. Type 2 is the predominant isozyme detectable in fetal genital skin, male accessory sex glands, and in the prostate, including benign prostatic hyperplasia and prostate adenocarcinoma tissues. Both isozymes are expressed in the liver, but only after birth. These results are consistent with 5 alpha-reductase type 1 being responsible for virilization in type 2-deficient subjects during puberty, and suggest that the type 2 isozyme may be an initiating factor in development of male pattern baldness.

Characterization of Chinese hamster ovary cell lines expressing human steroid 5 alpha-reductase isozymes

Membrane-bound isozymes of steroid 5 alpha-reductase, designated 1 and 2, synthesize the potent androgen, dihydrotestosterone. Isozyme 1 has an alkaline pH optimum (7.0-8.5), whereas isozyme 2 has an acidic pH optimum (5.0). To gain insight into this enigmatic difference, Chinese hamster ovarian cell lines expressing the human 5 alpha-reductase isozymes were established. The half-lives of both proteins are > 30 h and are not altered by the 4-azasteroid inhibitors finasteride and 17 beta-(N,N,-diethyl)carbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one. Nanomolar concentrations of finasteride block immunoprecipitation of isozyme 2 by antipeptide antibodies, which suggests that drug binding alters protein conformation. In contrast, finasteride (50 microM) has no effect on immunoprecipitation of isozyme 1. Both isozymes are localized to the endoplasmic reticulum by immunocytochemistry and have their carboxyl termini exposed to the cytoplasm. In cell lysates, isozyme 2 exhibits a Vmax at pH 5.0 but has a higher substrate affinity at neutral pH. In intact and permeabilized cells, isozyme 2 has an apparent substrate Km similar to that determined in cell lysates at neutral pH. The results suggest that isozyme 2 is more efficient at neutral pH and that the acidic pH optimum determined in lysates is a consequence of cell lysis.

LY191704: a selective, nonsteroidal inhibitor of human steroid 5 alpha-reductase type 1.

Androgens, in particular dihydrotestosterone (DHT), play a key role in differentiation, growth, and maintenance of the mammalian prostate. Production of DHT from testosterone is catalyzed by two distinct membrane-bound steroid 5 alpha-reductase [5 alpha-reductase; 3-oxo-5 alpha-steroid delta 4-dehydrogenase; 3-oxo-5 alpha-steroid:(acceptor) delta 4-oxidoreductase, EC 1.3.99.5] isozymes designated types 1 and 2. Benign prostatic hyperplasia (BPH), a disease that occurs almost universally in males, is characterized by obstructive and irritative urinary voiding symptoms and has been associated with an overproduction of DHT. Recently, steroidal inhibitors of 5 alpha-reductase type 2 have been used successfully for treatment of BPH. Described here is a nonsteroidal inhibitor of 5 alpha-reductase type 1, LY191704 (8-chloro-4-methyl-1,2,3,4,4a,5,6,10b-octaahydro-benzo[f]quinol in-3(2H)-one). This compound was identified based on its capacity to inhibit 5 alpha-reductase activity in a human genital skin fibroblast cell line (Hs68). Surprisingly, LY191704 is inactive when tested in freshly isolated prostate cells obtained from subjects with BPH, whereas previously described 4-azasteroids are active. LY191704 is, however, a potent inhibitor of the 5 alpha-reductase activity of BPH cells that have been maintained in culture. Analysis of human and rat 5 alpha-reductases expressed from transfected cDNAs in simian COS cells indicates that LY191704 is a specific noncompetitive inhibitor of the human 5 alpha-reductase type 1. Taken together, the results suggest that prostate cells have the capacity to express both 5 alpha-reductase isozymes and that LY191704 may be useful in treatment of human endocrine disorders associated with overproduction of DHT by 5 alpha-reductase type 1.

Tissue distribution and kinetic characteristics of rat steroid 5 alpha-reductase isozymes. Evidence for distinct physiological functions.

The enzyme steroid 5 alpha-reductase (5 alpha-reductase) catalyzes the reduction of delta 4,5 double bonds in a variety of substrates and is thought to play both catabolic and anabolic roles in steroid hormone metabolism. Here, we describe the isolation and characterization of a cDNA encoding the rat type 2 isozyme of 5 alpha-reductase and compare the kinetic properties and tissue-specific expression patterns of this isozyme with those of the type 1 isozyme. The type 2 isozyme has apparent Km values in the nanomolar range for steroid substrates, whereas the type 1 isozyme has micromolar affinities. The isozymes differ in their inhibition by various 4-azasteroids with the type 2 isozyme showing exquisite sensitivity (Ki = 40 pM) to 21,21-pentamethylene-4-aza-5 alpha-pregn-1-ene-3,20-dione. Messenger RNAs encoding the type 2 isozyme are more abundant than type 1 mRNAs in most male reproductive tissues, whereas the type 1 mRNAs predominate in peripheral tissues. Both 5 alpha-reductase mRNAs are more efficiently induced by dihydrotestosterone than by testosterone in the regenerating prostate. The differences in substrate affinities and tissue distributions of the 5 alpha-reductase isozymes suggest that type 2 plays an anabolic role and type 1 a catabolic role in the metabolism of androgens and other steroid hormones.