Abstract 12123: Novel Biology of Sortilin 1: Posttranslational Modification of Sortilin 1 Regulates Its Trafficking and Causes Matrix Vesicle Calcification
Background: Emerging evidence suggests that microcalcifications lead to plaque rupture. Human genome wide association studies linked the SORT1 gene, encoding sortilin 1, with increased risk of cardiovascular diseases. We recently proposed a direct role for sortilin 1 in vascular calcification; however, the underlying mechanisms remain unknown. We hypothesized that sortilin 1 mediates calcification by its recruitment into matrix vesicles (MVs), precursors to microcalcifications, conferring potency for mineralization of vascular extracellular matrix.
Methods and Results: Sortilin 1 co-immunoprecipitation combined with mass spectrometry (MS) revealed that caveolin-1 (Cav-1) and tissue-non-specific alkaline phosphate (TNAP) interactions are promoted as a function of smooth muscle cell (SMC) calcification. Immunogold labeling showed sortilin 1 in the plasma membrane of human carotid plaque SMCs. In agreement, sortilin 1 was enriched in the lipid raft/caveolae- membrane fractions of calcified SMCs. Cav-1 silencing reduced sortilin 1 expression (-50%, P≤0.05), TNAP activity (-31%, P≤0.01) and matrix mineralization (-47%, P≤0.01). Stimulation of SMCs isolated from Cav-1 deficient mice revealed decreased sortilin 1-mediated induction of TNAP activity (-26%, P≤0.05) and calcification (-37%, P≤0.01) compared to wild-type SMCs. Sortilin 1, Cav-1 and TNAP were present in MVs as shown by Western blot, electron microscopy and MS. Overexpression of sortilin 1 increased its levels in MVs. Silencing of cellular sortilin 1 by siRNA reduced TNAP activity within the MVs (-45%; P≤0.01). Quantitative MS showed that a C-terminal phosphorylation was increased by 3-fold in calcified SMCs as compared to control SMCs. Of note, the peptide harboring this phosphorylation site was absent in the MV isolated from these SMCs, suggesting that sortilin 1 modification directly controls its loading into MV.
Conclusion: We demonstrated a new role of sortilin 1 in MV calcification, and propose that posttranslational modification profiles of the C-terminus regulate the trafficking and packaging properties of sortilin 1 into the MVs. With these novel findings, we can develop strategies aimed at sortilin 1 as a therapeutic target for vascular calcification.
- © 2013 by American Heart Association, Inc.