Expression of the angiotensinogen gene is synergistically stimulated by 8-BrcAMP and Dex in opossum kidney cells
Ming, M.; Wang, T.T.; Lachance, S.; Delalandre, A.; Carrière, S.; Chan, J.S.
American Journal of Physiology 268(1 Pt 2): R105-R111
1995
ISSN/ISBN: 0002-9513 PMID: 7840309 Document Number: 445096
We transiently transfected fusion genes with the 5'-flanking region of the angiotensinogen gene linked to a bacterial chloramphenicol acetyltransferase (CAT) coding sequence as a reporter into opossum kidney (OK) cells. The addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) (10-3-10-7 M) or forskolin (10-9-10-5 M) stimulated the expression of the plasmid pOCAT (angiotensinogen nucleotide (N) -1498/+18) fusion gene in OK cells in a dose-dependent manner. The addition of dexamethasone (Dex) (10-6 M) further enhanced the stimulatory effect of 8-BrcAMP or forskolin, whereas the addition of (R)-p-adenosine 3',5'-cyclic monophosphorothioate ((Rp)-cAMP(S), an inhibitor of cAMP-dependent protein kinase A, I and III blocked the stimulatory effect of 8-BrcAMP. Furthermore, the addition of 8-BrcAMP (10-3 M) or Dex (10-6 M) or a combination of both stimulated the expression of pOCAT (angiotensinogen N -1138/+18), pOCAT (angiotensinogen N -960/+18), pOCAT (angiotensinogen N -814/+18), and pOCAT (angiotensinogen N -688/+18), but had no effect on the expression of pOCAT (angiotensinogen N -280/+18), pOCAT (angiotensinogen N -198/+18), pOCAT (angiotensinogen N -110/+18), pOCAT (angiotensinogen N -53/+18), and pOCAT (angiotensinogen N-35/+18). To further localize the putative cAMP-responsive element (CRE) in the angiotensinogen gene, we constructed fusion genes by inserting the DNA fragments angiotensinogen N -814 to N -689, angiotensinogen N -814 to N -761, and angiotensinogen N -760 to N -689 of the 5'-flanking region of the angiotensinogen gene upstream of the thymidine kinase (TK) promoter fused to a CAT gene and introduced them into OK cells. The addition of 8-BrcAMP (10-3 M) stimulated the transcriptional activity of the pTKCAT (angiotensinogen N -814/-689), pTKCAT (angiotensinogen N -814/-761), and pTKCAT (angiotensinogen N -760/-689) by 1.3-, 2.9-, and 1.2-fold, compared with controls (without the addition of 8-BrcAMP). Similar responses were also observed with the addition of forskolin. These studies demonstrated that 8-BrcAMP and Dex act synergistically to stimulate the expression of the angiotensinogen gene in OK cells, and we provided evidence for the presence of a putative CRE in the 5'-flanking region of the rat angiotensinogen gene.