Journal
HYPERTENSION
Volume 37, Issue 1, Pages 99-104Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1161/01.HYP.37.1.99
Keywords
human; renin; gene expression; hormones, thyroid; response elements
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Funding
- NIDDK NIH HHS [R01 DK072408, R01 DK072408-01A1] Funding Source: Medline
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [R01DK072408] Funding Source: NIH RePORTER
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We previously reported that thyroid hormone stimulates renin synthesis in vivo and in vitro. Here, we analyzed the 5'-flanking sequence of the human renin gene for promoter activity responsive to thyroid hormone using Calu-6 cells, which secrete renin endogenously and express thyroid hormone receptor-beta. The luciferase reporter gene was cloned together with 5'-flanking portions of the human renin gene of various lengths into the pGL3-Basic vector. Luciferase activity assays were performed using the Dual Luciferase Reporter Ass ay System. 3,3',5 -Triiodo-L-thyronine stimulated the promoter activity of pGL3-Basic-1111/+12 and pGL3-Basic-1298/+12 by 2.3 +/- 0.1- and 1.7 +/- 0.1-fold, respectively. Shorter constructs (pGL3-Basic-144/+12, pGL3-Basic-226/+12, pGL3-Basic-452/+12, and pGL3-Basic-953/+12) were not stimulated by thyroid hormone. These results suggest that there is a possible thyroid hormone response element (5'-AGG TCA GGT CAc aat GTT CCT-3') between nucleotides -1111 and -953. In 3 constructs with site-directed mutations in this sequence, basal promoter activities were significantly increased, whereas promoter activation by thyroid hormone was abolished. Electrophoretic mobility shift assays showed that the -1111/-953 DNA fragment of the intact human renin gene was bound to nuclear proteins of Calu-6 cells; however, none of the 3 mutant probes were bound to any nuclear proteins. These results suggest that thyroid hormone stimulates the promoter activity of the human renin gene through thyroid hormone response element-dependent mechanisms in Calu-6 cells.
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