Contribution of Ceramide Signaling to Activation of the mTORC1 Pathway and Calcification Nidus Formation in Coronary Arterial Smooth Muscle Cells

Abstract

The mammalian target of rapamycin complex 1 (mTORC1) signaling on late endosomes (LE)/lysosomes may control cargo selection, membrane biogenesis, exosome secretion and lysosome trafficking, which is fine controlled by lysosomal sphingolipids such as ceramide. This lysosomal sphingolipid‐regulated mTOR signaling may be a crucial molecular mechanism responsible for development of the arterial medial calcification (AMC) given the crucial role of exosome secretion from vascular smooth muscle cells (SMCs). In the present study, we used coronary artery SMCs (CASMCs) from Asah1fl/fl/SMcre mice with SMC‐specific deletion of lysosomal acid ceramidase (mouse gene code: Asah1) to demonstrate the role of lysosomal ceramide in mTOR signaling and arterial calcification. First, we demonstrated that mTORC1, a 289 kD cytoplasm protein is enriched in CASMCs by Western blot analysis and treatment of CASMCs isolated from Asah1fl/fl/SMcre mice with high Pi increased more calcium deposition compared to WT/WT cells as shown by the presence of Alizarin Red‐stained nodules. This calcium deposition was significantly decreased by specific mTOR inhibitor (Torin‐1) and by acid sphingomyelinase (Asm) inhibitor (Imipramine). Correspondingly, Asah1 gene deletion induced phenotypic change in Pi‐treated CASMCs, as depicted by decreased SM22‐α (SMC marker) expression in Western blot analysis, which was reversed by Torin‐1 or Imipramine. Using confocal microscopy, we further found that Pi markedly increased colocalization of mTORC1 with Lamp‐1, a lysosome targeting of mTORC1, but decreased colocalization of VPS16 with Lamp‐1, an indicative of multivesicular bodies (MVBs) interaction with lysosomes in CASMCs from Asah1fl/fl/SMcre mice, suggesting that activation of mTOR signaling inhibits lysosome trafficking or fusion with MVBs if AC was deficient. However, mTOR and Asm inhibition produced opposite effects on lysosome trafficking and interaction with MVBs compared to CASMCs treated with AC deficiency, further suggesting the role of ceramide‐mTOR signaling in lysosome trafficking or interactions with MVBs. Using nanoparticle tracking analysis, Pi‐induced excretion of exosomes (100–150 nm in size) were found to be significantly increased in CASMCs of Asah1fl/fl/SMcre mice compared to CASMCs from their control littermates. These results suggest that lysosomal ceramide‐mTOR signaling critically controls MVB fate and exosome release and that AC deficiency may lead to increased mTOR activation, thereby increasing exosome release and leading to calcification nidus formation in CASMCs.Support or Funding Information(Supported by NIH grants HL057244, HL075316 and DK120491)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.