Abstract P152: Forkhead Box Subclass O Transcription Factors Differentially Regulate Soluble Guanylyl Cyclase Expression in Smooth Muscle

Abstract

Nitric oxide (NO) stimulates soluble guanylyl cyclase (sGC) activity leading to elevated cyclic guanosine 3′, 5′-monophosphate (cGMP) and vascular smooth muscle relaxation. Downregulation of sGC expression attenuates vascular dilation and contributes to the pathogenesis of cardiovascular disease, however, it is not well understood how sGC transcription is regulated. We recently published evidence that inhibition of the Forkhead box subclass O (FoxO) transcription factors resulted in reduced sGC mRNA, protein expression, and cGMP production in cultured smooth muscle. Furthermore, we showed that FoxO inhibitor treatment in ex-vivo aortas decreased sGC β protein expression and impaired NO-dependent vasorelaxation. We now seek to determine which FoxO transcription factor, FoxO1, FoxO3a and/or FoxO4 in vascular smooth muscle cells regulate(s) sGC transcription. In this study, cultured rat aortic smooth muscle cells (RASMC) were treated with adenoviral shRNA constructs targeting FoxO1, FoxO3a, or FoxO4. Western blot analyses measured protein expression and phosphorylation, while RT-qPCR was quantified mRNA expression. Each FoxO shRNA construct produced a variable effects on smooth muscle. FoxO1 shRNA treatment significantly increased both sGC α and β mRNA expression by 3-fold (n=3) and showed a 22% increase in sGC β protein expression (n=3), while FoxO3a shRNA increased both sGC α and β mRNA expression by 2-fold (n=3) and increased sGC β protein expression by 48% (n=3). FoxO4 shRNA treatment, however, decreased sGC α and β mRNA by 50% and 59%, respectively, and also decreased sGC β protein expression by 49% (n=3). FoxO4 shRNA treatment also phosphorylated 38% less vasodilator stimulated protein (pVASP) at its cGMP-dependent phosphorylation site on serine 239 after stimulation with 500 nM treatment with the nitric oxide donor molecule DEA-NONOate (n=4) and 30% less after 1 μM stimulation with DEA-NONOate (n=4) than controls. Taken together, these data suggest a complex FoxO-dependent mechanism wherein FoxO1/3a act antagonistically to FoxO4 regulate sGC gene expression. Future studies will include luciferase promoter-reporter assays and chromatin immunoprecipitation assays to identify integral FoxO binding sites on the sGC promoter.