An epigenetic regulatory loop controls pro-osteogenic activation by TGF-β1 or bone morphogenetic protein 2 in human aortic valve interstitial cells

Abstract

Calcific aortic valve disease (CAVD) is common in the elderly population, but pharmacological interventions for managing valvular calcification are unavailable. Transforming growth factor β1 (TGF-β1) and bone morphogenetic protein 2 (BMP-2) induce pro-osteogenic activation of human aortic valve interstitial cells (AVICs) that play an important role in valvular calcification. However, the molecular mechanism underlying pro-osteogenic activation in AVICs is incompletely understood. Here, we investigated an epigenetic regulatory mechanism in human AVIC pro-osteogenic activation induced by TGF-β1 and BMP-2. Microarray and real-time PCR analyses revealed that microRNA (miR)-486 up-regulation and miR-204 down-regulation were characteristic changes in TGF-β1- and BMP-2-stimulated normal AVICs and in AVICs from calcified valves. Both TGF-β1 and BMP-2 down-regulated miR-204 through Smad pathways. Interestingly, an miR-486 antagomir diminished the effect of TGF-β1 and BMP-2 on miR-204 levels and calcium deposit formation. Furthermore, the miR-486 antagomir increased the expression of Smurf2, a Smad inhibitor, in the presence or absence of TGF-β1 or BMP-2 stimulation, whereas a miR-486 mimic reduced Smurf2 expression. Smurf2 knockdown augmented TGF-β1- or BMP-2-induced miR-204 down-regulation and resulted in increased expression of the osteoblastic biomarkers Osx and Runx2. In summary, we found that TGF-β1 and BMP-2 up-regulate miR-486 and down-regulate miR-204 in human AVICs to promote pro-osteogenic activity and that miR-486 inhibits Smurf2 expression to augment the miR-204 down-regulation. We conclude that the miR-486-Smurf2-Smad loop plays an important role in regulating AVIC pro-osteogenic activation in response to TGF-β1 or BMP-2. Targeting this regulatory loop may have therapeutic potential for suppressing aortic valve calcification.