Vol. 62, Issue 6, 1344-1355, December 2002

Transcriptional Regulation of the Rat Bradykinin B2 Receptor Gene: Identification of a Silencer Element

Heloisa A. Baptista, Maria C. W. Avellar, Ronaldo C. Araujo, Jorge L. Pesquero, Joost P. Schanstra, Jean L. Bascands, Jean P. Esteve, Antonio C. M. Paiva, Michael Bader, and João B. Pesquero

Center for Development of Experimental Models in Medicine and Biology (H.A.B), Interdisciplinary Center for Biochemical Research (R.C.A.), and Departments of Biophysics (A.C.M.P., J.B.P.) and Pharmacology (M.C.W.A.), Universidade Federal de São Paulo, São Paulo, Brazil; Universidade Mogi das Cruzes, Mogi das Cruzes, Brazil; Department of Physiology and Biophysics, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (J.L.P.); Institut Louis Bugnard, Institut National de la Santé et de la Recherche Médicale U388 (J.P.S., J.L.B.) and U531 (J.P.E.), Toulouse, France; and Max-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany (M.B.)

Kinins are involved in a variety of physiological and pathophysiological processes related to cardiovascular homeostasis, inflammation, blood flow, and nociception. Under physiological conditions, the bradykinin B2 (BKB2) receptor is constitutively expressed and mediates most of kinins' actions. However, the mechanisms regulating BKB2 receptor gene expression are still poorly understood. In this study, 4.6 kilobases of the 5'-flanking region from the rat BKB2 receptor gene were sequenced, and computer analysis revealed several sites for transcriptional factors. Nine promoter mutants were cloned in luciferase reporter gene vectors and transfected in NG108-15 cells and rat aorta vascular smooth muscle cells (VSMCs), showing several positive and negative regulatory elements. A classical silencer with 56 base pairs (bp) caused a decrease in reporter gene activity in NG108-15 cells and VSMCs and was able to inhibit the thymidine kinase promoter. Using electrophoretic mobility shift assay and surface plasmon resonance assay, protein-DNA interactions in the silencer region were determined and specific sets of protein-silencer complexes were detected in both cell types. More intense complexes were observed in the central 21 bp of the silencer and mutation in a putative SRE-1 site strongly impaired the protein-DNA binding. Down-regulation of the BKB2 receptor population in NG108-15 cells promoted by N⁶, 2'-O-dibutyryladenosine 3':5'-cyclic monophosphate was paralleled by an increase in the amount of nuclear proteins bound to the silencer sequence showing an inverse relationship between protein-silencer complexes and the transcription of the BKB2 receptor gene. In summary, these data highlight the cell-specific regulation of the BKB2 receptor and the importance of a silencer element present in the regulatory region of the gene.