Abstract 673: KLF10 is a Critical Mediator of Wnt Signaling in Aortic Valve Interstitial Cells

Abstract

We have previously demonstrated that β-catenin plays important roles in valve calcification with a specific osteogenic phenotype defined by increased bone mineral content and overall valve thickening. Recent studies indicate that KLF10 may be involved in mediating the Wnt signaling pathway in bone, which is known to play critical roles in osteoblast differentiation and mineralization. Therefore, we sought to test the role of KLF10 in mediating Wnt signaling, as well as differentiation and mineralization, in valve interstitial cells (VICs) isolated from porcine valves. Exposure of VICs to differentiation media led to increased expression of Runx2, Sox9 and osteocalcin. Differentiated cells also stained positive with Von Kossa while undifferentiated cells stained negative confirming the induction of an osteogenic phenotype. As expected, over-expression of both Lef1 and β-catenin led to activation of the top-flash reporter when transfected into VICs. Interestingly, over-expression of KLF10 also significantly up-regulated the top-flash reporter alone and further enhanced the activity of both Lef1 and β-catenin when co-transfected. We further confirmed the role of TIEG in an atherosclerotic mouse model ApoE/LRP5 double KO and found a two-fold increase in KLF10, Lrp6, and Runx2 expression in the cholesterol treated aortic valves as compared to controls. These data suggested that KLF10, Lef1 and β-catenin interact with each other to form a transcriptionally active protein complex leading to enhanced Wnt signaling in VICs. This possibility was further confirmed by the observation that KLF10 and β-catenin co-localize with one another in the nucleus of VICs following stimulation with LiCl and/or TGF-β. Taken together, these data implicate an important role for KLF10 in mediating Wnt signaling and Lef1 transcriptional activity in VICs, and implicate a potential role for canonical Wnt signaling in the observed osteogenic bone phenotype of cardiac aortic valves.