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Identification of two novel shear stress responsive elements in rat angiotensin I converting enzyme promoter

Laboratory of Genetic and Molecular Cardiology, Heart Institute (InCor) and Internal Medicine Department/LIM13, University of São Paulo Medical School, 05403-901 São Paulo SP, Brazil

Mechanical forces contribute to maintenance of cardiovascular homeostasis via the control of release and production of vasoactive substances. We demonstrated previously that shear stress decreases rat ACE activity and expression. Using a reporter gene approach and mutagenesis, we show now that the classic shear stress responsive element or SSRE (GAGACC) contained within 1,274 bp of this promoter is not functional in response to shear stress (15 dyn/cm², 18 h) [for the wild-type ACE promoter (WLuc), static control (C) = 107 ± 6.5%, shear stress (SS) = 65.9 ± 9.4%, n = 8; for the promoter with the classic SSRE mutated (WSS-mut), C = 100 ± 8.2%, SS = 60.2 ± 5.2%, n = 10, respectively]. Analysis of progressive deletion mutants unraveled a 57-bp fragment, position –251 to –195, from the transcription start site, containing functional SSRE (for WLuc, C = 107 ± 6.5%, SS = 65.9 ± 9.4%, n = 8; for 378, C = 100 ± 6.4%, SS = 60.4 ± 4.3%, n = 11; for 251, C = 99.7 ± 2.6%, SS = 63.2 ± 5.5%, n = 7; for 194, C = 104.6 ± 8.1%, SS = 92.4 ± 6.9%, n = 9). This fragment responded to shear stress even in the context of a heterologous promoter. Finally, functional analysis of mutated candidate regulatory elements identified by gel shift, DNase I footprint, and conservation of aligned sequences revealed that only the double mutant (Barbie/GAGA-mut) but not isolated disruption of the Barbie (WBarbie-mut) or the GAGA (WGAGA-mut) prevented the shear-stress-induced response (for Barbie/GAGA-mut, C = 97.9 ± 5%, SS = 99.4 ± 7.2%, n = 6; for WBarbie-mut, C = 106.1 ± 8.6%, SS = 65.9 ± 9.4%, n = 6; for WGAGA-mut, C = 100.1 ± 2.9%, SS = 66.7 ± 1.6, n = 6;). Taken together, these data provide direct evidence for the new role of Barbie and GAGA boxes in mediating the shear-stress-induced downregulation of rat ACE expression and demonstrate that the classic SSRE (GAGACC) is not functional under the experimental conditions tested.

laminar shear stress; promoter analysis; endothelial cell

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