Down-regulation by cAMP of angiotensin II type 2 receptor gene expression in PC12 cells.
The rat angiotensin II type 2 receptor (AT2-R) gene was isolated, and cis-regulatory regions in its 5'-flanking area were analyzed. Primer extension and RNase protection analyses revealed a single transcriptional initiation site at the position 24 bp downstream of the TATA box. The 5'-flanking region of AT2-R contained several cis-regulatory elements, such as AP-1, AP-2, C/EBP, NF-1, NF-IL6, NF-kappa B, and glucocorticoid- and cAMP-responsive elements (CRE). The treatment of PC12 cells with dibutyryl cAMP caused a marked decrease (90%) in the AT2-R mRNA level, which was blocked by the inhibitor of protein kinase A and did not require new protein synthesis. The protein level was also reduced 84% after a 24-h exposure to cAMP and the binding affinity was unchanged. The half-life of the AT2-R mRNA decreased -66% by cAMP as compared with control (18.4 +/- 0.4 h). Deletion and mutation analyses of the 5'-flanking region (1.2 Kb) revealed that there were one negative (-1,199 to -739) and two positive cis-regulatory regions (-739 to -436 and -59 to +45), and that the CRE motif located at -426 repressed (-23%) the promoter activity of the rat AT2-R gene. The region between -59 and +45 containing TATA box and AP-2 site accounted for 70% of the promoter activity. These findings indicate that the promoter activity of the rat AT2-R gene is modulated by several cis-regulatory regions and that cAMP markedly downregulates the expression of the AT2-R mainly by inducing AT2-R mRNA destabilization rather than CRE-mediated inhibition of the gene transcription. Thus, humoral factors that transduce cAMP as an intracellular signal may modulate AT2-R-mediated function of Ang II by reducing AT2-R expression.
Murasawa
S
S
Department of Medicine II, Kansai Medical University, Osaka, Japan.
Matsubara
H
H
Kijima
K
K
Maruyama
K
K
Ohkubo
N
N
Mori
Y
Y
Iwasaka
T
T
Inada
M
M
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003994:Bucladesine / Q000494:pharmacology; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000494:pharmacology*; D003609:Dactinomycin / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D008156:Luciferases; D008969:Molecular Sequence Data; D016716:PC12 Cells / Q000187:drug effects / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics
D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D003609:Dactinomycin; D003994:Bucladesine; D000242:Cyclic AMP; D008156:Luciferases