Functional role of a novel cis-acting element (GAGA box) in human type-1 angiotensin II receptor gene transcription.
GH/growth factors have been shown to increase angiotensin type 1 receptor expression. In the present study we determined the cis-acting regulatory region controlling GH-induced transcription of the human type-1 angiotensin receptor (hAT(1)). In human proximal tubule cells transfected with a chloramphenicol acetyl transferase (CAT) reporter plasmid under the control of the hAT(1) promoter, GH induced CAT activity. Serial deletions of the hAT(1) promoter region indicated that an area between -314 bp and -70 bp upstream of the 5'-end of the cDNA sequence was essential for this activation to occur. Although sequence analysis identified putative multiple nuclear protein binding sites in this region, we determined that a 12 bp sequence (5'-GAGAGGGAGGAG-3', GAGA box) located between -161 bp and -149 bp was important for GH-mediated activation. Using mobility shift assays we demonstrated increased DNA binding activity to the labeled GAGA box in nuclear extracts treated with GH, suggesting this sequence is a GH response element. Southwestern analysis identified an 18 kDa GAGA box-binding protein (GAGA-BP). GH-induced activity of the GAGA-BP occurred within 2.5 min and reached a maximum at 5 min. Activation did not require de novo protein synthesis. Removal of the GAGA box abolished GH-induced transcription as well as basal transcription of the hAT(1) gene. Additional studies demonstrated that epidermal growth factor, platelet-derived growth factor and insulin activate the GAGA-BP, suggesting these growth factors can also regulate the transcription of the hAT(1) gene through the GAGA box. Our data show that the GAGA-BP acts as a trans-acting factor binding to the cis-acting regulatory element in the hAT(1) promoter, which is necessary for the basal and growth factor(s)-mediated transcriptional activation of the hAT(1) gene.
Wyse
B D
BD
Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Linas
S L
SL
Thekkumkara
T J
TJ
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D017930:Genes, Reporter; D006133:Growth Substances / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D017931:DNA Primers; D006133:Growth Substances; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D015500:Chloramphenicol O-Acetyltransferase