Abstract 465: G3BP1 Supports Osteogenic Mitochondrial Antiviral Signaling - NFAT Transcriptional Relays in Aortic Vascular Smooth Muscle Cells

In vascular smooth muscle (VSM), LRP6, a Wnt co-receptor, inhibits arteriosclerotic noncanonical Wnt signals that activate protein arginine (Arg) methylation relays, converging on nuclear factor of activated T cells (NFAT). LRP6 deletion in VSM increases arteriosclerotic calcification and stiffness in low density lipoprotein receptor (LDLR)-deficient mice fed diabetogenic diets. To study how Arg methylation mediates LRP6 actions, immunoaffinity mass spectrometry was performed on aortic VSM cell extracts from SM22-Cre;LRP6(fl/fl);LDLR-/- and LRP6(fl/fl);LDLR-/- mice. LRP6 deficiency altered Arg methylation of over 490 proteins, but only 22 exhibited increased monomethylation (MMA) with reduced dimethylation. One protein involved in atherosclerosis, G3BP1, exhibited a >30-fold increase in MMA of its C-terminal domain with LRP6 deficiency. Co-transfection confirmed that protein Arg methyltransferase 1 modified G3BP1 and was inhibited by LRP6. G3BP1 stimulated Frizzled-dependent NFAT transcription, again inhibited by LRP6. Nuclear NFATc4 and NFATc4 association with osteopontin (OPN) and alkaline phosphatase (TNAP) chromatin increased in LRP6-deficient VSM, and was reduced with G3BP1 deficiency. G3BP1 activation of NFAT required its nuclear transport domain and the MMA modified C-terminal domain to respond to Ddx58 - a modulator of mitochondrial antiviral signaling (MAVS) linked to aortic calcification. Ddx58 colocalized and synergized with G3BP1 in VSM to upregulate targets of noncanonical signaling, including OPN and TNAP. While VSM LRP6 deficiency increased Ddx58 levels, G3BP1 insufficiency reduces Ddx58 and osteogenic expression. RNAi targeting G3BP1, Ddx58 or NFATc4 reduced VSM osteogenic programs; VSM from MAVS-deficient mice exhibited reduced TNAP activity and osteogenic gene expression. Mass spectrometry data indicates that G3BP1 and Ddx58 form complexes with specific nucleoporins regulating nucleocytoplasmic transport. Thus, G3BP1 is a target of MMA regulated by LRP6. G3BP1 promotes osteogenic signals in VSM, conveyed by a MAVS relay to regulate osteogenic transcription. Targeting G3BP1 and protein Arg methyltransferases may mitigate arteriosclerotic calcification in type 2 diabetes.