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.

Molecular genetics of steroid 5 alpha-reductase 2 deficiency.

Two isozymes of steroid 5 alpha-reductase encoded by separate loci catalyze the conversion of testosterone to dihydrotestosterone. Inherited defects in the type 2 isozyme lead to male pseudohermaphroditism in which affected males have a normal internal urogenital tract but external genitalia resembling those of a female. The 5 alpha-reductase type 2 gene (gene symbol SRD5A2) was cloned and shown to contain five exons and four introns. The gene was localized to chromosome 2 band p23 by somatic cell hybrid mapping and chromosomal in situ hybridization. Molecular analysis of the SRD5A2 gene resulted in the identification of 18 mutations in 11 homozygotes, 6 compound heterozygotes, and 4 inferred compound heterozygotes from 23 families with 5 alpha-reductase deficiency. 6 apparent recurrent mutations were detected in 19 different ethnic backgrounds. In two patients, the catalytic efficiency of the mutant enzymes correlated with the severity of the disease. The high proportion of compound heterozygotes suggests that the carrier frequency of mutations in the 5 alpha-reductase type 2 gene may be higher than previously thought.

Four-amino acid segment in steroid 5 alpha-reductase 1 confers sensitivity to finasteride, a competitive inhibitor.

The 4-azasteroid 17 beta-(N-t-butyl)carbamoyl-4-aza-5 alpha-androst-1-en-3-one (finasteride) is 100-fold more potent as a competitive inhibitor of the rat NADPH:delta 4-3-oxosteroid-5-alpha- oxidoreductase (steroid 5 alpha-reductase) type 1 enzyme (Ki = 3-5 nM) than of the human type 1 enzyme (Ki greater than or equal to 300 nM). In this study, we exploit this differential sensitivity to map a major determinant of finasteride sensitivity in steroid 5 alpha-reductase. Chimeric steroid 5 alpha-reductase cDNAs composed of different combinations of rat and human exon sequences were created by genetic engineering, expressed in human embryonic kidney 293 cells, and assayed for their sensitivity to finasteride. Hybrid proteins containing sequences encoded by rat exon 1 were found to be as sensitive to finasteride as the parental enzyme. The exchange of progressively smaller protein segments encoded within exon 1 identified a tetrapeptide sequence (Val-Ser-Ile-Val) in the rat enzyme that conferred sensitivity to finasteride. The analogous sequence in the human enzyme (Ala-Val-Phe-Ala) conferred partial resistance to the drug. Finasteride was a competitive inhibitor of the native and all chimeric enzymes tested, suggesting that the tetrapeptide segments form a portion of the substrate-binding domain of steroid 5 alpha-reductase.

Rat C1-tetrahydrofolate synthase. cDNA isolation, tissue-specific levels of the mRNA, and expression of the protein in yeast.

We have isolated and characterized cDNA clones encoding rat cytoplasmic C1-tetrahydrofolate (H4folate) synthase. In eukaryotes, this enzyme is trifunctional and contains the activities of 10-formyl-H4folate synthetase, 5,10-methenyl-H4folate cyclohydrolase, and 5,10-methylene-H4folate dehydrogenase. The deduced sequence of the 935-amino acid open reading frame contained exact matches to NH2-terminal (15 residues) and internal (residues 436-450) peptide sequences obtained from the purified enzyme. The amino acid sequence derived from the rat cDNA shows extensive homology to analogous proteins from bacterial, yeast, and mammalian sources. We have used the cDNA to determine the steady-state levels of the mRNA in various rat tissues and have found that gene expression is regulated in a tissue-specific manner. Transcript levels are highest in kidney and liver with liver transcripts reduced about 30% relative to those found in kidney. Brain, heart, testis, lung and skeletal muscle display even lower transcript levels; reductions range from 70 to 80% of transcript levels found in kidney. Comparison to the levels of enzyme in these tissues allows us to conclude that pretranslational events predominate in the tissue-specific expression. The rat enzyme has been functionally expressed in Saccharomyces cerevisiae as evidenced by its capacity to complement a chromosomal deletion of ADE3, the yeast gene encoding cytoplasmic C1-H4folate synthase.

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.

Cell type specific expression of steroid 5 alpha-reductase 2.

Isozymes of steroid 5 alpha-reductase (5 alpha-reductase) have crucial roles in androgen physiology by synthesizing the potent hormone dihydrotestosterone. The expression pattern of the 5 alpha-reductase type 2 isozyme was determined in genital and extragenital tissues by developing an immunohistochemical assay using formalin-fixed tissue and affinity purified polyclonal antibodies that specifically recognize this isozyme. Expression was detected in basal epithelial and stromal cells of the normal prostate but not in luminal epithelial cells. Stromal cells of the seminal vesicle also expressed the type 2 isozyme. In contrast, staining was detected in epithelial cells of the epididymis but not in the surrounding stroma. Myofibroblasts in foreskin samples of normal and hypospadiac individuals expressed antigen and were distributed in bands throughout the prepuce, suggesting a clonal origin. In most cells the type 2 isozyme exhibited a perinuclear subcellular distribution. However, in liver hepatocytes the protein was distributed throughout the intracellular membrane compartment.

Increased expression of early growth response-1 messenger ribonucleic acid in prostatic adenocarcinoma.

PURPOSE: We isolated genes whose expression differs between normal and malignant prostate. MATERIALS AND METHODS: Differential display polymerase chain reactions revealed a messenger ribonucleic acid (mRNA) with higher expression in prostatic adenocarcinoma versus age matched normal tissue. Deoxyribonucleic acid sequencing identified this mRNA as the product of the early growth response-1 gene. RESULTS: Early growth response-1 mRNA levels were elevated in 12 of 12 intraprostatic adenocarcinomas but not in breast or ovarian cancers, or in rapidly dividing rat ventral prostate cells. Early growth response-1 mRNA was detected in epithelial and stromal cells at tumor margins but not in lymph node metastases. CONCLUSIONS: Early growth response-1, a nuclear transcription factor, is implicated in the growth and invasion of intraprostatic cancers.

Expression and regulation of steroid 5 alpha-reductase 2 in prostate disease.

The androgen dihydrotestosterone is synthesized by the enzyme steroid 5 alpha-reductase, and it is required for growth and development of the prostate. We used immunohistochemistry to examine the expression of the type 2 isozyme of 5 alpha-reductase in benign prostatic hyperplasia and prostate cancer. The type 2 isozyme is highly expressed within stromal cells in both disease states. No type 2 isozyme is detectable in a lymph node metastasis. Immunoblotting studies show that androgen ablation therapies substantially decrease isozyme expression in the epididymis but have a lesser effect on expression in the prostate. Finasteride therapy (2 weeks to 3 years) did not abolish expression of the prostatic type 2 isozyme nor did this drug treatment induce expression of the type 1 isozyme.