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(Hypertension. 2004;43:1270.)
© 2004 American Heart Association, Inc.

Scientific Contributions

Characterization of a cGMP-Response Element in the Guanylyl Cyclase/Natriuretic Peptide Receptor A Gene Promoter

David Hum; Sandrine Besnard; Rocio Sanchez; Dominic Devost; Francis Gossard; Pavel Hamet; Johanne Tremblay

From the Centre de recherche, Centre hospitalier de l’Université de Montréal (CHUM)—Hôtel-Dieu, Montréal, Québec, Canada.

Correspondence to Dr Johanne Tremblay, Centre de recherche, CHUM—Hôtel-Dieu, 3850 St. Urbain St., Montréal, Québec H2W 1T7, Canada. E-mail johanne.tremblay{at}umontreal.ca

Previous studies have shown that atrial natriuretic peptide (ANP) can inhibit transcription of its receptor, guanylyl cyclase A, by a mechanism dependent on cGMP and have suggested the presence of a putative cGMP-response element (cGMP-RE) in the Npr1 gene promoter. To localize and characterize the putative cis-acting element, we have subcloned a 1520-bp fragment of the rat Npr1 promoter in an expression vector containing the luciferase reporter gene. Several fragments, generated by exonuclease III-directed deletions, were transiently transfected into cells to measure their promoter activity. Deletion from –1520 to –1396 of a 1520-bp-long Npr1 promoter led to a 5-fold increase in luciferase activity. Subsequent deletion to the position –1307 resulted in a decrease of luciferase activity by 90%. Preincubation of cells with 100 nM of ANP or 100 µM 8-bromo-cGMP inhibited luciferase activity of the 1520-bp and 1396-bp-long fragments, but not the activity of the 1307-bp fragment, suggesting that the cGMP-RE is localized between positions –1396 and –1307. The cGMP regulatory region was narrowed by gel shift assays and footprinting to position –1372 to –1354 from the transcription start site of Npr1 and indicated its interaction with transcriptional factor(s). Cross-competition experiments with mutated oligonucleotides led to the definition of a consensus sequence (–1372 AaAtRKaNTTCaAcAKTY –1354) for the novel cGMP-RE, which is conserved in the human (75% identity) and mouse (95% identity) Npr1 promoters.

Key Words: cyclic GMP • natriuretic peptides • receptors, atrial natriuretic factor

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