Regulation of human renin gene promoter activity: a new negative regulatory region determines the responsiveness to TNF alpha.
BACKGROUND
The renin-angiotensin system has been known to regulate blood pressure and body fluid homeostasis. Several lines of evidence have shown that renin gene expression and release are up-regulated by beta-adrenergic stimulation, sodium depletion, and angiotensin converting enzyme inhibition, but down-regulated by cytokines. To further characterize the human renin gene (hREN) promoter structure, its regulation, and to identify an appropriate cell system for study, we examined five cell lines and investigated drug effects on the hREN promoter expression.


METHODS
Using the hREN-luciferase reporter gene constructs in the DNA transfection assays, approximately 5 kb of the hREN 5' flanking region was assessed for promoter activity in five different cell lines. Regulation of the hREN promoter activity was investigated using Y-1 adrenal cells that were transfected with the hREN-luciferase DNA and were treated with forskolin, calcium ionophore A23187, phorbol ester, angiotensin II (Ang II), or cytokines.


RESULTS
Transient transfection analysis showed that the 5 kb hREN 5' flanking DNA alone was able to confer significant promoter activity in Y-1 adrenal cells. In transfected Y-1 cells, luciferase reporter expression was induced by forskolin, suppressed by the calcium ionophore A23187, and phorbol ester in a dose-dependent manner, but was unaffected by angiotensin II (Ang II). However, when Y-1 reporter cells were transfected with human angiotensin II receptor type 1 (AT1) cDNA, hREN promoter activity was dose-dependently down-regulated by Ang II, which was blockable by losartan, an AT1-selective antagonist. Further studies also showed that hREN promoter activity in Y-1 cells was selectively down-regulated by TNF alpha. Deletion of the hREN promoter sequences between position -3916 and -2822 not only enhanced hREN promoter activity by approximately tenfold, but also caused a failure of down-regulation by TNF alpha. In contrast, neither interleukin (IL)-1 alpha, IL-1 beta, IL-2, nor IL-6 exerted any significant effect.


CONCLUSIONS
Together the results suggest that TNF alpha is a negative regulator of the hREN expression in the adrenal cells, and that the TNF alpha responsiveness may be controlled by elements located between the positions -3916 and -2822 of the hREN promoter. Moreover, the Y-1 cell line may provide a valuable model system for studying renin gene regulation.
Chen
L S
LS
Division of Cardiovascular & Metabolic Diseases, Wyeth-Ayerst Research, Princeton, New Jersey, USA. 870400@ucl.itri.org.tw
Cuddy
M P
MP
LaVallette
L A
LA
D000311:Adrenal Glands / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D016207:Cytokines / Q000494:pharmacology; D004247:DNA / Q000235:genetics; D000882:Haplorhini; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D051379:Mice; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology*; D012083:Renin / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology / Q000502:physiology*
D016207:Cytokines; D007476:Ionophores; D014409:Tumor Necrosis Factor-alpha; D000804:Angiotensin II; D005576:Colforsin; D000001:Calcimycin; D004247:DNA; D012083:Renin; D013755:Tetradecanoylphorbol Acetate