Abstract P114: Foxo4 Regulates Vascular Smooth Muscle Soluble Guanylyl Cyclase Expression Expression

Soluble guanylyl cyclase (sGC) modulator drugs may offer a new approach to attenuate hypertension by enhancing vasorelaxation through production of cGMP. Loss of sGC leads to impaired vasorelaxation and hypertension, however the mechanisms that regulate sGC transcription are not fully understood. We recently published that Forkhead box subclass O (FoxO) transcription factor inhibition led to decreased sGC mRNA and protein expression and subsequent loss of downstream enzymatic sGC function and vasoreactivity in the aorta. We conducted luciferase experiments in HEK cells which identified several regions which were sensitive to FoxO inhibition and may be responsible for the regulation of sGCβ protein expression (n=4).The objective of this study was to identify which FoxO transcription factor(s) regulates sGC transcription in vascular aortic smooth muscle cells (SMCs) and at what location on the promoter. FoxO1 shRNA produced a 3-fold increase in sGCα and sGCβ mRNA expression (n=3) and a 27% elevation in sGCβ protein expression (n=6). FoxO3a shRNA produced a similar 2-fold increase in sGCα and sGCβ mRNA (n=3) and a 41% increase in sGCβ protein expression (n=6). FoxO4 shRNA, however, produced approximately 55% decrease in sGCα and sGCβ mRNA (n=3) and a similar 52% decrease in sGCβ protein expression (n=6). FoxO1 and FoxO3a shRNA treatment caused no significant change in pVASP expression at baseline or following stimulation with the nitric oxide donor molecule DEA NONOate (n=3). FoxO4 shRNA treatment resulted in a 73% decrease in pVASP expression at baseline and blunted the DEA NONOate-mediated increase in phosphorylation by 33% (n=3). Preliminary ChIP experiments in human aortic SMCs and have identified 4 sites of enriched Foxo4 binding on the human sGCβ promoter consistent with locations previously identified as sensitive to FoxO inhibitor. These data indicate FoxO4 plays a critical role in the maintenance of sGC transcription, protein expression, and downstream enzymatic function of sGC in vascular smooth muscle. Additionally, our data demonstrate that human sGCβ promoter expression requires functional FoxO activity and FoxO4 promoter binding occurs in promoter regions of human smooth muscle found to be important for promoter-luciferase expression.