The akr1b7 gene encodes an aldose reductase-like protein that is responsible for detoxifying isocaproaldehyde generated by the conversion of cholesterol to pregnenolone. The regulation of gene expression by human chorionic gonadotropin (hCG) was first investigated in the MA-10 Leydig tumor cell line. The akr1b7 gene was constitutively expressed and accumulation of its mRNA was increased in a dose- and time-dependent manner by treatment with hCG. akr1b7 mRNA accumulation was sharply increased in the presence of 0.25 nM hCG and it reached a fivefold increase within 2 h. AKR1B7 protein accumulation was delayed compared with that of the corresponding mRNA. In agreement, hCG significantly increased the levels of mRNA and protein of akr1b7 in primary cultures of adult mouse Leydig cells, thus suggesting that LH potentially regulates akr1b7 gene expression in vivo. Expression of akr1b7 was developmentally regulated in the testis. Unexpectedly, levels of akr1b7 mRNA increased from embryonic day 15 to the day of birth and declined until adulthood while AKR1B7 protein levels followed an inverse pattern, suggesting an important role for translational mechanisms.
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S Baron, M Manin, C Aigueperse, M Berger, C Jean, G Veyssiere, and L Morel
A Dassouli, Ch Darne, S Fabre, M Manin, G Veyssière, and Cl Jean
The understanding of androgen-regulated gene expression requires a cell culture system that mimics the functions of cells in vivo. In the present paper we have examined a vas deferens epithelial cell subculture system. Cultured vas deferens epithelial cells have been shown to exhibit polarized properties characteristic of functioning epithelia and to display a high level of androgen receptors. Incubation of cells with androgen caused a decrease in cellular androgen receptor mRNA that was time-dependent. Total suppression was observed after 24 h of exposure to androgen. By contrast, incubation of vas deferens epithelial cells with androgen resulted in a threefold increase in the cellular content of androgen receptor protein, as assayed by ligand binding. In response to androgens, vas deferens epithelial cells expressed mouse vas deferens protein mRNA (MVDP mRNA). Maximum expression of the MVDP gene, at both mRNA and protein levels, was observed after 24 h of androgen induction.
DEAE-dextran transfection conditions were defined using the MMTV-CAT vector. Dihydrotestosterone stimulated the transcription activation of MMTV-CAT gene in vas deferens epithelial cells in a dose- and time-dependent manner. No induction was seen when fragments of the MVDP promoter region were cloned directly in front of the CAT gene and transiently transfected into vas deferens epithelial cells. It was found that cotransfection of cells with MVDP-CAT constructs and with an androgen receptor expression vector resulted in a small but consistent androgen-dependent increase in reporter gene activity. Transiently transfected vas deferens epithelial cells are a suitable model with which to study the effect of androgen on gene regulatory elements.