Loss of 5α-reductase type 1 accelerates the development of hepatic steatosis but protects against hepatocellular carcinoma in male mice.

Nonalcoholic fatty liver disease (NAFLD) has been associated with glucocorticoid excess and androgen deficiency, yet in the majority of patients with steatohepatitis, circulating cortisol and androgen levels are normal. The enzyme 5α-reductase (5αR) has a critical role in androgen and glucocorticoid action. We hypothesize that 5αR has an important role in the pathogenesis of steatohepatitis through regulation of intracrine/paracrine hormone availability. Human liver samples from patients with NAFLD and normal donor tissue were used for gene expression and immunohistochemical analysis. NAFLD samples were scored using the Kleiner classification. In addition, 5αR1(-/-), 5αR2(-/-), and wild-type (WT) mice were fed normal chow or American lifestyle-induced obesity syndrome (ALIOS) diet for 6 or 12 months. Liver histology was graded and staged. Hepatic and circulating free fatty acid and triglyceride levels were quantified, and gene and protein expression was measured by real-time PCR and immunohistochemistry. 5αR1 and -2 were highly expressed in human liver, and 5αR1 protein expression increased with severity of NAFLD. 5αR1(-/-) (but not 5αR2(-/-)) mice fed an ALIOS diet developed greater hepatic steatosis than WT mice, and hepatic mRNA expression of genes involved in insulin signaling was decreased. Furthermore, 60% of WT mice developed focal hepatocellular lesions consistent with hepatocellular carcinoma after 12 months of the ALIOS diet, compared with 20% of 5αR2(-/-) and 0% of 5αR1(-/-) mice (P < .05). 5αR1 deletion accelerates the development of hepatic steatosis but may protect against the development of NAFLD-related hepatocellular neoplasia and therefore has potential as a therapeutic target.

Brassinosteroid control of sex determination in maize.

Brassinosteroids (BRs) are plant hormones that regulate growth and development. They share structural similarities with animal steroids, which are decisive factors of sex determination. BRs are known to regulate morphogenesis and environmental stress responses, but their involvement in sex determination in plants has been only speculative. We show that BRs control sex determination in maize revealed through characterization of the classical dwarf mutant nana plant1 (na1), which also feminizes male flowers. na1 plants carry a loss-of-function mutation in a DET2 homolog--a gene in the BR biosynthetic pathway. The mutant accumulates the DET2-specific substrate (24R)-24-methylcholest-4-en-3-one with a concomitant decrease of downstream BR metabolites. Treatment of wild-type maize plants with BR biosynthesis inhibitors completely mimicked both dwarf and tasselseed phenotypes of na1 mutants. Tissue-specific na1 expression in anthers throughout their development supports the hypothesis that BRs promote masculinity of the male inflorescence. These findings suggest that, in the monoecious plant maize, BRs have been coopted to perform a sex determination function not found in plants with bisexual flowers.

Methyl esters of N-(dicyclohexyl)acetyl-piperidine-4-(benzylidene-4-carboxylic acids) as drugs and prodrugs: a new strategy for dual inhibition of 5 alpha-reductase type 1 and type 2.

Steroid 5alpha-reductase (5alphaR) inhibitory potency of three N-(dicyclohexyl)acetyl-piperidine-4-(benzylidene-4-carboxylic acids) and their corresponding methyl esters was monitored for type 2 isoenzyme in a benign prostatic hyperplasia cell free preparation and for type 1 isoenzyme in DU145 cells and in a cell free assay. The hydrolytic stability of the esters and their bioconversion to the corresponding acids was assessed in aqueous buffered solution (pH 7.4) and in selected biological media having measurable esterase activities. The carboxylic acids 1, 2, and 3 with high type 2 inhibitory potencies displayed only little type 1 inhibition. The esters 1a, 2a, and 3a, originally designed as prodrugs to enhance cell permeation, proved to be potent type 1 inhibitors and are therefore acting as drugs themselves. They are stable in buffered salt solution (pH 7.4), Caco-2 cells, and human plasma, whereas all esters are cleaved into the corresponding acids in benign prostatic hyperplasia tissue homogenate. Methyl esters, applied as hydrolytically stable precursor drugs to facilitate cell permeation, will yield the corresponding carboxylic acids as type 2 inhibitors after hydrolysis in the target organ. The esters themselves--stable in human plasma and Caco-2 cells--are acting as potent drugs toward 5alphaR type 1. Thus, dual inhibition of 5alphaR type 1 and type 2 can be achieved by applying a single parent compound.

Dutasteride: a potent dual inhibitor of 5-alpha-reductase for benign prostatic hyperplasia.

Dutasteride is a 5alpha-reductase inhibitor that inhibits both types 1 and 2 isozymes of 5alpha-reductase, the enzyme responsible for converting testosterone to dihydrotestosterone in the prostate and other tissues. Dihydrotestosterone is the primary cause of prostate growth and has been proven to play a key role in the development and progression of benign prostatic hyperplasia. Dutasteride has been investigated in three multicenter studies involving 4325 men aged 50 years and above with benign prostatic hyperplasia. Data from these two-year, placebo-controlled studies demonstrated that dutasteride 0.5 mg once daily reduced the risk of both acute urinary retention and the need for benign prostatic hyperplasia-related surgical intervention, improved benign prostatic hyperplasia-related symptoms, decreased prostate volume and increased maximum urinary flow rates with a low incidence of generally mild to moderate adverse events.

Anatomical and cellular localization of neuroactive 5 alpha/3 alpha-reduced steroid-synthesizing enzymes in the spinal cord.

The complementary activities of 5 alpha-reductase (5 alpha-R) and 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) are crucial for the synthesis of neuroactive 5 alpha/3 alpha-reduced steroids, such as 3 alpha-androstanediol, allopregnanolone, and tetrahydrodeoxycorticosterone, which control several important neurophysiological mechanisms through allosteric modulation of gamma-aminobutyric acid type A receptors. Immunocytochemical localization of 3 alpha-HSD in the central nervous system (CNS) has never been determined. The presence and activity of 5 alpha-R have been investigated in the CNS, but only the brain was considered; the spinal cord (SC) received little attention, although this structure is crucial for many sensorimotor activities. We have determined the first cellular distribution of 5 alpha-reductase type 1 (5 alpha-R1) and type 2 (5 alpha-R2) and 3 alpha-HSD immunoreactivities in adult rat SC. 5 alpha-R1 immunostaining was detected mainly in the white matter (Wm). In contrast, intense 5 alpha-R2 labeling was observed in dorsal (DH) and ventral horns of gray matter (Gm). 3 alpha-HSD immunoreactivity was largely distributed in the Wm and Gm, but the highest density was found in sensory areas of the DH. Double-labeling experiments combined with confocal analysis revealed that, in the Wm, 5 alpha-R1 was localized in glial cells, whereas 35% of 5 alpha-R2 and 3 alpha-HSD immunoreactivities were found in neurons. In the DH, 60% of 5 alpha-R2 immunostaining colocalized with oligodendrocyte, 25% with neuron, and 15% with astrocyte markers. Similarly, 45% of 3 alpha-HSD immunoreactivity was found in oligodendrocytes, 35% in neurons, and 20% in astrocytes. These results are the first demonstrating that oligodendrocytes and neurons of the SC possess the key enzymatic complex for synthesizing potent neuroactive steroids that may control spinal sensorimotor processes.

[Expression of type I and type II 5alpha-reductase isoenzymes in prostate cancer tissues].

OBJECTIVE: To study the relative tissue distribution and expression pattern of type I and type type II 5alpha-reductase isozymes in prostate cancer tissues. METHODS: Immunohistochemistry and RT-PCR method were used to investigate qualitatively and semi-quantitatively the expression of type I and type II 5alpha-reductases in prostate tissues from 15 normal persons and 15 patients with prostate cancer. RESULTS: Two isozymes were detected in both normal and cancerous prostate tissues with the two methods. Both 5alpha-reductases were mainly localized in the cytoplasm, and higher degree of staining was observed in epithelial cells than in stroma. Significantly higher level of type I 5alpha-reduetase expression was observed in cancerous tissue than in normal tissue. The expression level of type I 5alpha-reduetase was positively correlated with the tumor stage and grade, and serum PSA concentration. The expression of type II 5alpha-reductase was very weak in prostate cancer tissue. CONCLUSION: The type I 5alpha-reductase, not type II, is involved in the pathogenesis of protate cancer. Selective type I 5alpha-reductase inhibitors or dual inhibitors of both type human 5alpha-reductase isoforms can be used in the treatment of prostate cancer.

Kinetic analysis of LY320236: competitive inhibitor of type I and non-competitive inhibitor of type II human steroid 5alpha-reductase.

Type I and type II steroid 5alpha-reductases (5alpha-R) catalyze the conversion of testosterone (T) to dihydrotestosterone (DHT). LY320236 is a benzoquinolinone (BQ) that inhibits 5alpha-R activity in human scalp skin (Ki(typeI)=28.7+/-1.87 nM) and prostatic homogenates (Ki(typeII)=10.6+/-4.5 nM). Lineweaver-Burk, Dixon, and non-linear analysis methods were used to evaluate the kinetics of 5alpha-R inhibition by LY320236. Non-linear modeling of experimental data evaluated V(max) in the presence or absence of LY320236. Experimental data modeled to the following equation 1v=+ fixing the In0c value equal to 1.0 or 0 are consistent with non-competitive or competitive inhibition, respectively. LY320236 is a competitive inhibitor of type I 5alpha-R (In0c=0, Ki=3.39+/-0.38, RMSE = 1.300) and a non-competitive inhibitor of type II 5alpha-R (In0c=1, Ki=29. 7+/-3.4, RMSE = 0.0592). These data are in agreement with linear transformation of the data using Lineweaver-Burk and Dixon analyses. These enzyme kinetic data support the contention that the BQ LY320236 is a potent dual inhibitor with differing modes of activity against the two known human 5alpha-reductase isozymes. LY320236 represents a class of non-steroidal 5alpha-R inhibitors with potential therapeutic utility in treating a variety of androgen dependent disorders.

Enzyme assay for 5alpha-reductase type 2 activity in the presence of 5alpha-reductase type 1 activity in rat testis.

The relative abundance and physiological role of 5alpha-reductase (5alphaR) isoforms in rat testis, in particular 5alpha-reductase Type 2 (5alphaR2) are poorly understood. Investigation of 5alphaR2 activity using enzyme kinetic studies was hampered by the high concentrations of 5alpha-reductase Type 1 (5alphaR1) in rat testis. Therefore, an assay was developed which exploited the differences in pH optima of the two isoforms. The 5alphaR assays measured the conversion of 3[H]-testosterone to 5alpha-reduced metabolites (dihydrotestosterone+3alpha-Androstanediol) at pH 5.0 and 7.0. To compensate for the overlap of 5alphaR1 activity at pH 5.0, the amount of 5alphaR1 activity at pH 5.0 was determined by measuring recombinant rat 5alphaR1 expressed in COS-7 cells at pH 5.0 and 7.0. The amount of activity at pH 5.0 that was attributed to 5alphaR1 was determined to be 12.4+/-1.4% (mean+/-S.D., n=14). The 5alphaR2 assay was validated by determining recombinant rat 5alphaR2 activity in the presence of recombinant rat 5alphaR1 activity in COS cells. A 99.3+/-14.7% recovery of 5alphaR2 activity was obtained when comparing 5alphaR2 activity recovered versus activity added. 5alphaR1 and 5alphaR2 activities were then assayed in rat testis extracts from 30, 75 and 147 days. Both isoforms markedly declined (50-100-fold) over this age range, with 5alphaR1 as the predominant isoform. In conclusion, an enzymatic assay that detects 5alphaR2 activity in the presence of high concentrations of 5alphaR1 was developed and is applicable in the measurement of 5alphaR2 activity in rat testis.

Expression and regulation of steroid 5 alpha-reductase in the urogenital tract of the fetal rat.

Two androgens, testosterone and dihydrotestosterone, are required for the development of the male urogenital tract in the rat. Testosterone is secreted by the fetal testes and is thought to elicit differentiation of the Wolffian ducts into seminal vesicles, vas deferens, and epididymides. Testosterone is converted into dihydrotestosterone by steroid 5 alpha-reductase in the urogenital tract, and this conversion is necessary for the differentiation of the prostate and external genitalia. Genes encoding two 5 alpha-reductase isozymes, designated type 1 and type 2, have been identified. We examined the expression and regulation of these genes on days 17-21 in the urogenital tracts of male and female fetuses. Expression of the type 1 gene predominated in epithelial cells, whereas type 2 gene expression was limited to mesenchymal cells. Surprisingly, this expression pattern was detected in both testosterone-dependent and dihydrotestosterone-dependent anlagen of the urogenital tract and was the same in both male and female fetuses. Furthermore, transcripts encoding the two isozymes were present in their respective cell types before the overt differentiation of internal genitalia. Androgens stimulated expression of the type 2 gene in the urogenital tracts of both sexes, but did not effect expression of the type 1 gene or the cell type-specific expression patterns of the 5 alpha-reductase genes. In the adult prostate, 5 alpha-reductase gene expression is under feedforward control, in which the product of the enzyme, dihydrotestosterone, stimulates the expression of the gene. However, no evidence for feedforward regulation of either 5 alpha-reductase gene was detected in the fetus.

Cloning, expression and functional characterization of type 1 and type 2 steroid 5 alpha-reductases from Cynomolgus monkey: comparisons with human and rat isoenzymes.

The Cynomolgus monkey may provide an alternative pharmacological model in which to evaluate the efficacy of novel inhibitors of the two known human steroid 5 alpha-reductase (SR) isoenzymes. To evaluate the suitability of this species at the level of the molecular targets, a Cynomolgus monkey prostate cDNA library was prepared and screened using human SR type 1 and 2 cDNAs as hybridization probes. Two distinct cDNA sequences were isolated encoding the monkey type 1 and 2 SR isoenzymes. These sequences share 93 and 95% amino acid sequence identity with their human enzyme counterparts, respectively. Difference in monkey type 1 SR, however, was found within the contiguous four amino acids corresponding to the regions in the human and rat sequences that have been proposed previously to influence steroid and inhibitor affinities. Subsequently, both monkey cDNAs were individually expressed in a mammalian cell (CHO) line. Enzyme activities of both monkey SRs were localized to the membrane fractions of CHO cell extracts. Like the human and rat enzymes, the monkey type 1 and type 2 SRs were most active at neutral and low pH, respectively. The results of inhibition studies with over 30 known SR inhibitors, including epristeride, 4MA, and finasteride, indicate that the monkey SR isoenzymes are functionally more similar to the human than the rat homologues. The results from initial velocity and inhibition studies as functions of pH with the human and monkey type 2 SRs also compare favorably. These results, together, suggest that the monkey SR isoenzymes are structurally and functionally comparable on a molecular level to their respective human counterparts, supporting the relevance and use of the Cynomolgus monkey as a pharmacological model for in vivo evaluation of SR inhibitors.

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.

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.