ABSTRACT
The understanding of androgen-regulated gene expression requires a cell cultures 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 24h 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 24h of androgen induction. DEAE-dextran transfection conditions were defined using the 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 gene and transfection 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.
Subject(s)
Dihydrotestosterone/pharmacology , Gene Expression Regulation , Receptors, Androgen/biosynthesis , Vas Deferens/metabolism , 3T3 Cells , Animals , Cell Differentiation , Cells, Cultured , Chloramphenicol/pharmacology , Chloramphenicol O-Acetyltransferase/biosynthesis , Cycloheximide/pharmacology , Epithelial Cells , Epithelium/drug effects , Epithelium/metabolism , Gene Expression Regulation/drug effects , Male , Mammary Tumor Virus, Mouse , Metribolone/metabolism , Mice , Mice, Inbred Strains , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , Recombinant Proteins/biosynthesis , Restriction Mapping , Transfection , Vas Deferens/cytologyABSTRACT
Mouse vas deferens protein (MVDP) is a member of the aldo-keto reductase superfamily. The regulation of MVDP gene expression by activators of the protein kinase A signalling pathway was investigated in human (H295-R) and murine (Y1) adrenocortical carcinoma cells. Immunoblotting with polyclonal antibodies showed that MVDP is expressed in adrenal glands from mouse, rat, rabbit and guinea-pig, probably under the control of ACTH. In both adrenocortical cell lines used, MVDP is constitutively synthesized and its accumulation is increased by treatment with cAMP or forskolin. MVDP mRNA steady-state levels were up-regulated by forskolin in adrenocortical cells by a process that does not require de novo protein synthesis. The results suggest that cAMP is at least one of the key regulators of adrenal MVDP expression and that this effect is direct.