Identification and characterization of the minimal androgen-regulated kidney-specific kidney androgen-regulated protein gene promoter.
The kidney androgen-regulated protein (Kap) gene is tissue specific and regulated by androgen in mouse kidney proximal tubule cells (PTCs). In the present study, we aimed to identify the minimal PTC-specific androgen-regulated Kap promoter and analyze its androgen response elements (AREs). A deletion series of the Kap1542 promoter/luciferase constructs were assayed in opossum kidney (OK) PTCs in the presence or absence of 15 nM dihydrotestosterone (DHT). Kap1542 and Kap637 had low activity and no androgen induction; Kap224 had a basal activity that was 4- to 5-fold higher than that of Kap1542, but was only slightly induced by DHT. Kap147 had a basal activity that was 2- to 3-fold higher than that of Kap1542 and was induced by DHT 4- to 6-fold. Kap77 abolished basal promoter activity but was still induced by DHT. Results showed that, in vitro, Kap147 was a minimal androgen-regulated promoter. Transient transfection in different cells demonstrated that Kap147 specifically initiated reporter gene expression in PTCs. Sequence analysis revealed two potential AREs located at positions -124 and -39 of Kap147. Mutational assays showed that only the ARE at -124 was involved in androgen response in OK cells. Electrophoretic mobility shift assay also verified -124 ARE bound specifically to androgen receptor. In conclusion, we defined the minimal Kap147 promoter that may be a good model for the study of kidney PTC-specific expression and molecular mechanisms that lead to an androgen-specific responsiveness in vivo.
Fan
Liqiang
L
The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China. fanglq@ecust.edu.cn
Hardy
Dianne O
DO
Catteral
James F
JF
Zhao
Jian
J
Li
Suxia
S
D000728:Androgens / Q000502:physiology*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics*
D000728:Androgens; D017931:DNA Primers; C067670:Kap protein, mouse; D011506:Proteins