Claude Jean

Androgen receptor expression is regulated by the phosphoinositide 3-kinase/Akt pathway in normal and tumoral epithelial cells

The androgen receptor (AR) is a ligand-responsive transcription factor known to play a central role in the pathogenesis of prostate cancer. However, the regulation of AR gene expression in the normal and pathological prostate remains poorly understood. This study focuses on the effect of the phosphoinositide 3-kinase (PI 3-kinase)/Akt axis on AR expression in vas deferens epithelial cells (VDEC), a suitable model to study androgen regulation of gene expression, and LNCaP cells (derived from a metastasis at the left supraclavicular lymph node from a 50-year-old patient with a confirmed diagnosis of metastatic prostate carcinoma). Taken together, our data show for the first time that the PI 3-kinase/Akt pathway is required for basal and dihydrotestosterone-induced AR protein expression in both VDEC and LNCaP. Inhibition of the PI 3-kinase/Akt pathway reduced AR expression and the decline in AR protein level correlated with a decrease in AR mRNA in VDEC but not in LNCaP. Since PI 3-kinase/Akt axis is active in prostate cancer, cross-talk between PI 3-kinase/Akt and AR signalling pathways may have implications for endocrine therapy.

Product of Side-chain Cleavage of Cholesterol, Isocaproaldehyde, Is an Endogenous Specific Substrate of Mouse Vas Deferens Protein, an Aldose Reductase-like Protein in Adrenocortical Cells

Mouse vas deferens protein (MVDP) is an aldose reductase-like protein that is highly expressed in the vas deferens and adrenal glands and whose physiological functions were unknown. We hereby describe the enzymatic characteristics of MVDP and its role in murine adrenocortical Y1 cells. The murine aldose reductase (AR) and MVDP cDNAs were expressed in bacteria to obtain recombinant proteins and to compare their enzymatic activities. Recombinant MVDP was functional and displayed kinetic properties distinct from those of murine AR toward various substrates, a preference for NADH, and insensitivity to AR inhibitors. For MVDP, isocaproaldehyde, a product of side-chain cleavage of cholesterol generated during steroidogenesis, is the best natural substrate identified so far. In Y1 cells, we found that NADH-linked isocaproaldehyde reductase (ICR) activity was much higher than NADPH-linked ICR activity and was not abolished by AR inhibitors. We demonstrate that in Y1 cells, forskolin-induced MVDP expression enhanced NADH-linked ICR activity by 5–6-fold, whereas no variation in ICR-linked NADPH activity was observed in the same experiment. In cells stably transfected with MVDP antisense cDNA, NADH-linked ICR activity was abolished even in the presence of forskolin, and the isocaproaldehyde toxicity was increased compared with that of intact Y1 cells, as measured by isocaproaldehyde LD50. In Y1 cells transfected with MVDP antisense cDNA, forskolin-induced toxicity was abolished by aminoglutethimide. These results indicate that in adrenocortical cells, MVDP is responsible for detoxifying isocaproaldehyde generated by steroidogenesis.

Physiological functions and hormonal regulation of mouse vas deferens protein (AKR1B7) in steroidogenic tissues.

The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) highly expressed in vas deferens epithelium and zona fasciculata of the adrenal cortex. Recombinant MVDP showed kinetic properties distinct from those of aldose reductase, including its spectrum of substrates, cofactor preference and sensitivity to inhibitors. We demonstrate that in adrenocortical cells, MVDP, rather than aldose reductase, is the principal reductase for isocaproaldehyde (a product of side-chain cleavage of cholesterol) and 4-hydroxynonenal (a lipid peroxidation product). In steroidogenic tissues MVDP expression is regulated by pituitary trophic hormones, namely ACTH in adrenals, FSH in ovaries, and LH in testicular Leydig cells.

Testosterone and dihydrotestosterone in sexual ducts and genital tubercle of rabbit fetuses during sexual organogenesis: effects of fetal decapitation.

Testosterone (T) and dihydrotestosterone (DHT) have been measured in the plasma, sexual ducts and genital tubercle in rabbit fetuses of both sexes during sexual organogenesis. T and DHT were also measured in decapitated male fetuses. In male, T appeared successively in testes (day 19), mesonephros (day 20) and Wolffian ducts (day 22). In sexual ducts, T levels increased from 20 to 25 days, then stabilized until birth. Since DHT was measurable, in sexual ducts of males only from 24 days onwards, T itself is the active hormone on the differentiation of Wolffian ducts. In female fetuses, T and DHT were undetectable, in sexual ducts, at any gestational age. In the genital tubercle T was undetectable in both sexes. In the genital tubercle of males DHT levels increased from 19 to 25 days, then stabilized. In female fetuses, DHT was episodically measurable in the genital tubercle but at levels always lower than in males. DHT is the active hormone on the differentiation of genital tubercle. In male decapitated fetuses T and DHT levels were reduced in sexual ducts and genital tubercle but the differences were not significant.

Isolation of the Mouse Aldose Reductase Promoter and Identification of a Tonicity-responsive Element

Aldose reductase (AR; EC 1.1.1.21) is an oxidoreductase that catalyzes the NADPH-dependent conversion of glucose to sorbitol, the first step of the polyol pathway. AR is of great interest due to its implication in the etiology of diabetic complications. In renal medullary cells, AR also plays an osmoregulatory role by accumulating sorbitol to maintain the intracellular osmotic balance during antidiuresis. We have previously cloned the AR cDNA from mouse kidney, and we report here the isolation of the mouse AR gene promoter. Transient transfection of chloramphenicol acetyltransferase reporter constructs containing various 5′-flanking regions of the mouse AR gene in CV1 cells led to the identification of a sequence spanning base pairs −1053 to −1040, required for an enhancer activity in hypertonic compared with isotonic cell culture conditions. This sequence is similar to the tonicity-responsive element first characterized in the betaine-γ-aminobutyric acid transporter promoter.

Androgens regulate expression of the gene coding for a mouse vas deferens protein related to the aldo-keto reductase superfamily in epithelial cell subcultures.

Mouse vas deferens protein (MVDP), a member of the aldo-keto reductase superfamily, is exclusively produced in the vas deferens. To better understand androgen-regulated MVDP gene expression we have used RNA hybridization to study the effects of androgens on the steady-state levels of MVDP mRNA in vas deferens epithelial cell subcultures. Northern blot analysis revealed that these cells only express MVDP mRNA in the presence of androgens. There was a close relationship between MVDP mRNA levels and dihydrotestosterone concentrations. MVDP mRNA is induced over a period of 24h and maximal induction is about 25-fold. Treatment of cells with cycloheximide completely abolished the observed androgen effect suggesting that the induction of the MVDP gene by androgens depends on continuous protein synthesis. Transient transfection of vas deferens epithelial cells with MMTV-CAT vector showed that these cells contained functional androgen receptors and that they are a suitable system to study androgen effect on MVDP gene regulatory elements.

Adrenal tumorigenesis targeted by the corticotropin-regulated promoter of the aldo-keto reductase AKR1B7 gene in transgenic mice.

Studies of ACTH functions in adrenal steroidogenesis have been facilitated by the availability of immortalized mouse adrenocortical Y1 cells. In order to obtain alternative cell lines with a more differentiated zona fasciculata (ZF) phenotype we used targeted tumorigenesis strategy. We have generated transgenic mice expressing the SV40 T antigen under the control of the ACTH-dependent promoter for the AKR1B7/MVDP gene (aldo-keto reductase 1B7/mouse vas deferens protein), which encodes an enzyme responsible for detoxifying isocaproaldehyde, the product of side-chain cleavage of cholesterol generated by steroidogenesis. Our previous data indicated that in the mouse adrenal, AKR1B7 expression was restricted to the ZF and that a 0.5-kb promoter region was able to target specific adrenal expression in transgenic mice. In situ hybridization analyses indicate that AKR1B7 expression during fetal and post-natal periods paralleled the onset of glucocorticoid synthesis and the development of ZF. In transgenic mice. ACTH control and developmental programming of the CAT gene driven by the 0.5-kb promoter followed endogenous gene regulation. Then transgenic mice harboring the 0.5-kb/SV40 T antigen construct were generated and two founders out of three developed adrenal tumors. Cells derived from the tumor of founder 1 (ATC1) were grown in presence of forskolin to maintain ACTH receptor expression and were tested for ACTH responsiveness by immunocychemistry and northern blot analyses. Even after several passages, the ACTH induced AKR1B7 and P450c11β mRNAs accumulations were similar to that observed in mouse primary adrenocortical cell cultures. Our findings suggest that ATC1 cells have conserved essential features of ZF cells. In order to achieve complete characterization of these cells further analyses are currently performed to investigate their steroidogenic activity.

Age related changes in percent binding of testosterone and dihydrotestosterone and unbound testosterone and dihydrotestesterone in rabbit plasma

Abstract Percent binding of T and DHT to plasma proteins were determined in male and female rabbits from birth to adulthood, using equilibrium dialysis. In males, the percent binding of T and DHT increased from birth (T:87.4 ± 1.2; DHT:92.7 ± 1.2) to 20 days (T:99.5 ± 0.1; DHT: 100 ± 0.1) reaching their maxima. They then decreased progressively until adulthood (T:95.8 ± 0.3; DHT: 96.9 ± 0.4). The B/U ratios of T and DHT followed a similar pattern. These variations were related to the number of specific binding sites: 2.6 × 10 −8 M 1 ± 0.06 at birth; 51.6 × 10 −8 M 1 ± 5.7 at 60 days; 6.4 × 10 −8 M 1 ± 1.3 at 240 days. At 1, 10 and 150 days, but not at 60 days, the percents binding and the B/U ratios of T and DHT were not sex-dependent. Unbound levels of T and DHT were always low (0.1

Androgen dependence during development of the mouse vas deferens protein mRNA

The mRNA encoding a major protein of the mouse vas deferens (MVDP) was first detected in 10-day-old males and its concentration increased sharply between 10 and 20 days, reaching adult levels at 40 days. This increase was not associated with an increase in tissular androgen concentrations. In 30-day-old mice castrated at birth or treated with cyproterone acetate over 29 days, MVDP mRNA levels were not abolished and were similar to those measured in 10- and 20-day-old controls. These results suggest that the neonatal expression of MVDP gene is independent of androgens. In addition, precocious accumulation of MVDP mRNA could be induced by injection of excess amounts of androgens in 20- but not in 10-day-old animals. The prepubertal increase in MVDP mRNA levels is androgen-dependent but other factors may be necessary for MVDP expression.

The Effect of Preweaning Undernutrition upon the Sexual Development of Male Mice

Sexual maturation was evaluated in male mice subjected to preweaning undernutrition by separating pups from their mothers. Underfed and normally fed males were sacrificed at 20, 30, 40, 50 and 60 days of age. From 20 to 60 days, body and organ weights (testes, seminal vesicle) were lower in underfed males. Plasma testosterone levels were lowered in undernourished males at 20 and 30 days of age, and thereafter they were not significantly different from controls. First fertile matings, which occurred between 36 and 46 days (mean age: 40.6 +/- 0.6) in controls, were delayed in underfed males and occurred between 42 and 58 days (mean age: 48 +/- 1.7). The mean body weight, at the time of first fertile matings, was significantly different in controls (29.5 +/- 0.5 g, range 25.9-32.4) and in undernourished males (24.3 +/- 0.3 g, range: 22.0-25.6). Testicular weight and plasma testosterone concentrations were also significantly lower in underfed males, at the time of first fertile matings. The data lead to the conclusion that puberty did not occur at the same body weight in normal and undernourished male mice.