An epigenetic regulatory loop controls pro-osteogenic activation by TGF-β1 or bone morphogenetic protein 2 in human...

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.