Project Summary Arterial medial calcification (AMC) and arterial stiffening are a prevalent pathological process in different pathological conditions or diseases such as hypertension, aging, atherosclerosis, diabetes and chronic kidney disease. Enhanced exosome secretion by smooth muscle cells (SMCs) has been reported to be an essential mechanism for calcifying nidus formation and extracellular matrix mineralization in the arterial wall to result in AMC. Recent studies have also shown that lysosome function plays a critical role in controlling multivesicular body (MVB) fate and enhancing exosome secretion and thereby in the development of arterial calcification. However, it remains poorly understood how lysosome function is controlled to determine exosome secretion and thereby lead to AMC. This proposal seeks to explore a novel epigenetic mechanism that regulates lysosome trafficking and exosome secretion, which may contribute to the development of AMC. This epigenetic regulation of lysosome function may be associated with the lysine methyltransferase Kmt6- mediated repression of gene transcription of Smpd1, a lysosome enzyme that hydrolyzes sphingomyelin into ceramide. Kmt6 is considered as a crucial epigenetic regulator that represses the target gene expression by methylation of lysine residue in histone proteins. In preliminary studies, we demonstrated that SMC-specific Kmt6 gene deletion exacerbated AMC and arterial stiffening, which were associated with increased Smpd1 expression and ceramide production, reduced lysosome TRPML1 channel activity, and lysosome trafficking dysfunction. These observations led us to hypothesize that Kmt6 is an essential epigenetic regulatory enzyme that controls lysosomal Smpd1-mediated sphingolipid metabolism and thereby regulates lysosome trafficking or its fusion to MVBs and subsequent exosome secretion in SMCs. Kmt6 gene defect or functional deficiency may disturb lysosome-mediated degradation of MVBs leading to increased exosome secretion, calcifying nidus formation, osteogenic transition, and ultimately AMC in face of different pathological challenges. To test this hypothesis, the following Specific Aims are proposed. Aim 1 will determine loss of Kmt6 contributes to osteogenic transition and AMC in SMC-specific Kmt6 knockout mice with analysis of SMC phenotypes and calcification. Aim 2 attempts to test whether Kmt6-mediated epigenetic regulation of Smpd1 critically controls lysosome trafficking and exosome secretion by increasing TRPML1 channel activity and associated Ca2+ release using patch clamping of isolated lysosomes and lysosome-specific Ca2+ imaging. Aim 3 will explore the molecular mechanisms how Smpd1 gene is epigenetically regulated by Kmt6 with a focus on its action on histone and DNA methylation in cultured arterial SMCs. Our findings will for the first time define an epigenetic mechanism controlling Smpd1 expression and activity via Kmt6 in SMCs and reveal a novel role of epigenetic dysregulat...