The kidney androgen-regulated protein (KAP) is specifically expressed and differentially regulated by androgens and tri-iodothyronine (T3) in intact mouse early (PCT) and late (PR) proximal-tubule cells. Until now, detailed characterization of the molecular elements mediating androgen-responsive gene expression in the kidney has been hampered by the lack of appropriate cultured cell systems suitable for DNA transfection studies. In the present study we have analysed the hormone-dependent transactivation of the KAP gene promoter in immortalized differentiated PCT and PR proximal-tubule cells derived from L-PK/Tag1 transgenic mice. Transient transfection studies with different KAP promoter constructs indicated that a 224bp-truncated fragment was sufficient to mediate cell-specific expression of the KAP promoter. Dihydrotestosterone (DHT) stimulated in an androgen-dependent manner the transactivation of KAP in PCT and PR cells, while mutation of a putative androgen-response element (ARE) sequence located at −39bp from the transcription initiation site abolished the transactivation induced by DHT. Furthermore, insulin-like growth factor 1 (IGF-1), but not T3, enhanced the androgen-dependent transactivation of KAP in cultured PCT cells. These results demonstrate that the short 224bp fragment of the KAP promoter is sufficient to drive the proximal-tubule androgen-specific regulated expression of KAP and reveal synergistic interactions between IGF-1 and androgens for KAP regulation in PCT cells.
- gene expression
- promoter assays
- steroid hormones
Abbreviations used: AR, androgen receptor; ARE, androgen-response element; CAT, chloramphenicol acetyltransferase; CsA, cyclosporin A; CyPB, the CsA-binding protein cyclophilin B; DHT, dihydrotestosterone; FCS, fetal-calf serum; gal, galactosidase; GH, growth hormone; β-gluc, β-glucuronidase; GR, glucocorticoid receptor; HAGT, human angiotensinogen gene; IGF-1, insulin-like growth factor 1; KAP, kidney androgen-regulated protein; LUC, luciferase; MMTV, murine-mammary-tumour virus; ODC, ornithine decarboxylase; PCT, intact mouse early proximal-tubule cells; PR, intact mouse late proximal-tubule cells; RT-PCR, reverse-transcription PCR; RXR, retinoid X receptor; SAGE, serial analysis of gene expression; SV40, simian virus 40; T3, tri-iodothyronine; THR, thyroid-hormone receptor.
- The Biochemical Society, London ©2002