The goal of this research proposal is to establish a model system in which ectopic gene expression in transgenic mice can be targeted to renal proximal tubule cells under the control of a conditionally regulated promoter. The proposal is based on previous experiments primarily from this laboratory that have characterized the regulation of Kap, the gene for Kidney Androgen Regulated Protein (KAP) in the mouse kidney. Kap is expressed exclusively in the kidney in males and is dramatically induced by androgen. However, the gene is under multihormonal control and, in order to establish androgen regulated expression of transgenes, we propose to examine the promoter elements involved in the hormonal regulation of Kap. The goal of these experiments is to identify the minimal androgen regulated promoter to drive expression of transgenes. A primarily in vivo approach will be taken to the identification and characterization of promoter elements that regulate the response of Kap to hormonal stimulation. Promoter analysis in cultured cells will be performed to confirm and refine the data obtained in vivo. To accomplish the goals of the project, the following Specific Aims will be undertaken. First, Kap promoter function will be examined in vivo by the mapping of Dnase I hypersensitive sites and by in vivo footprinting to identify HREs that regulate the gene. Second, transgenic mice carrying a transgene comprising various lengths of the Kap promoter fused to a reporter gene will be examined for their ability to express the gene in a tissue-specific, hormonally regulated manner to identify the minimal androgen regulated promoter. The cellular localization of mRNA and protein products of the transgenes and of the endogenous Kap gene will be determined. Third, the Kap promoter will be fused to the gene for SV40 T antigen to target tumorigenesis to renal proximal tubule cells. Resulting tumors will be characterized and dispersed as a source of cells for the derivation of stable cells lines exhibiting the selected phenotype, i.e., androgen regulated expression of the transgene. Fourth, the immortalized cells will be used to establish an in vitro transfection system in which data from earlier in vivo experiments will be confirmed and refined. Nuclear extracts of these cells will provide a source of proteins that mediate the regulation of the promoter. Finally, KAP will be characterized to facilitate the identification and cloning of the human homolog of the gene. The stringent regulation of this gene suggests an important role of KAP in the kidney and will provide the first gene targeting capability to the renal proximal tubule.
| Soler, Montse; Tornavaca, Olga; Sole, Esther et al. (2002) Hormone-specific regulation of the kidney androgen-regulated gene promoter in cultured mouse renal proximal-tubule cells. Biochem J 366:757-66 |
| Hardy, D O; Ge, R S; Catterall, J F et al. (2000) Identification of the oxidative 3alpha-hydroxysteroid dehydrogenase activity of rat Leydig cells as type II retinol dehydrogenase. Endocrinology 141:1608-17 |
| Kumar, N; Crozat, A; Li, F et al. (1999) 7alpha-methyl-19-nortestosterone, a synthetic androgen with high potency: structure-activity comparisons with other androgens. J Steroid Biochem Mol Biol 71:213-22 |
