Antagonism of Forkhead Box Subclass O Transcription Factors Elicits Loss of Soluble Guanylyl Cyclase Expression.

Nitric oxide (NO) stimulates soluble guanylyl cyclase (sGC) activity, leading to elevated intracellular cyclic guanosine 3',5'-monophosphate (cGMP) and subsequent vascular smooth muscle relaxation. It is known that 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. Here, we demonstrate that pharmacological inhibition of Forkhead box subclass O (FoxO) transcription factors using the small-molecule inhibitor AS1842856 significantly blunts sGC and mRNA expression by more than 90%. These effects are concentration-dependent and concomitant with greater than 90% reduced expression of the known FoxO transcriptional targets, glucose-6-phosphatase and growth arrest and DNA damage protein 45 (Gadd45). Similarly, sGC and sGC protein expression showed a concentration-dependent downregulation. Consistent with the loss of sGC and mRNA and protein expression, pretreatment of vascular smooth muscle cells with the FoxO inhibitor decreased sGC activity measured by cGMP production following stimulation with an NO donor. To determine if FoxO inhibition resulted in a functional impairment in vascular relaxation, we cultured mouse thoracic aortas with the FoxO inhibitor and conducted ex vivo two-pin myography studies. Results showed that aortas have significantly blunted sodium nitroprusside-induced (NO-dependent) vasorelaxation and a 42% decrease in sGC expression after 48-hour FoxO inhibitor treatment. Taken together, these data are the first to identify that FoxO transcription factor activity is necessary for sGC expression and NO-dependent relaxation.