Expression of the angiotensinogen gene is synergistically stimulated by 8-BrcAMP and Dex in opossum kidney cells.
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 II] 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Ming
M
M
University of Montreal, Maisonneuve-Rosemont Hospital, Research Center, Montreal, Quebec, Canada.
Wang
T T
TT
Lachance
S
S
Delalandre
A
A
Carrière
S
S
Chan
J S
JS
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D000808:Angiotensinogen / Q000096:biosynthesis*; D000818:Animals; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D015870:Gene Expression / Q000187:drug effects*; D007687:Kidney Tubules, Proximal; D009893:Opossums; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection
D011993:Recombinant Fusion Proteins; D013873:Thionucleotides; D000808:Angiotensinogen; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; C016957:adenosine-3',5'-cyclic phosphorothioate; D003907:Dexamethasone; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase