DNA METHYLATION OF AN INTRONIC ENHANCER DYSREGULATES PHOSPHOLIPID PHOSPHATASE 3 AND PROMOTES OSTEOGENESIS IN THE AORTIC VALVE

Abstract

Calcific aortic valve disease (CAVD) is characterized by the transition of valve interstitial cells (VICs) into osteoblast-like cells. Lysophosphatidic acid (LPA), which is produced in mineralized aortic valves (AVs), is a potent driver of osteogenic reprograming. LPA is degraded by the integral membrane protein phospholipid phosphatase 3, encoded by the PLPP3 gene. We examined the expression of PLPP3 in surgically explanted CAVD and in control nonmineralized AVs. The epigenetic regulation of PLPP3 was investigated in control and CAVD. The impact of PLPP3 on the osteogenic transition was examined in human VICs. The mRNA and enzymatic activity of PLPP3 were significantly decreased in CAVD compared to control AVs. Multidimensional gene profiling showed that intronic (intron 1 of PLPP3) DNA methylation was associated with PLPP3 expression. Bisulfite pyrosequencing in 52 AVs showed that intronic methylation of a CpG site was increased by 2.3-fold in CAVD compared to control AVs. In AVs, intronic CpG methylation level was inversely related to PLPP3 expression. The intronic site is highly conserved and showed histone marks of enhancer (H3K4me1). In reporter assay, the cloned intronic sequence with a PLPP3 minimal promoter induced a high level of activity in an orientation-independent manner. Co-transfection of ubiquitous transcription factors increased by several-fold the reporter activity. Valves with lower mRNA level of PLPP3 had higher content of LPA. In VICs treated with small interfering RNA (siRNA) for PLPP3, we observed that LPA-induced osteogenic transition was exacerbated as shown by higher expression of RUNX2, BGLAP and BMP2 as well as elevated alkaline phosphatase (ALP) activity. DNA methylation of an intronic enhancer decreases the expression of PLPP3 during CAVD. A lower expression of PLPP3 promotes the osteogenic transition of VICs.