Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390.
Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, Texas 75390.
J Biol Chem. 2018 May 25;293(21):7942-7968. doi: 10.1074/jbc.RA118.002046. Epub 2018 Apr 6.
In aortic vascular smooth muscle (VSM), the canonical Wnt receptor LRP6 inhibits protein arginine (Arg) methylation, a new component of noncanonical Wnt signaling that stimulates nuclear factor of activated T cells ( NFATc4). To better understand how methylation mediates these actions, MS was performed on VSM cell extracts from control and LRP6-deficient mice. LRP6-dependent Arg methylation was regulated on >500 proteins; only 21 exhibited increased monomethylation (MMA) with concomitant reductions in dimethylation. G3BP1, a known regulator of arteriosclerosis, exhibited a >30-fold increase in MMA in its C-terminal domain. Co-transfection studies confirm that G3BP1 (G3BP is Ras-GAP SH3 domain-binding protein) methylation is inhibited by LRP6 and that G3BP1 stimulates NFATc4 transcription. NFATc4 association with VSM osteopontin () and alkaline phosphatase () chromatin was increased with LRP6 deficiency and reduced with G3BP1 deficiency. G3BP1 activation of NFATc4 mapped to G3BP1 domains supporting interactions with RIG-I (retinoic acid inducible gene I), a stimulus for mitochondrial antiviral signaling (MAVS) that drives cardiovascular calcification in humans when mutated in Singleton-Merten syndrome (SGMRT2). Gain-of-function SGMRT2/RIG-I mutants increased G3BP1 methylation and synergized with osteogenic transcription factors (Runx2 and NFATc4). A chemical antagonist of G3BP, C108 (C108 is 2-hydroxybenzoic acid, 2-[1-(2-hydroxyphenyl)ethylidene]hydrazide CAS 15533-09-2), down-regulated RIG-I-stimulated G3BP1 methylation, Wnt/NFAT signaling, VSM TNAP activity, and calcification. deficiency reduced RIG-I protein levels and VSM osteogenic programs. Like and deficiency, MAVS deficiency reduced VSM osteogenic signals, including TNAP activity and Wnt5-dependent nuclear NFATc4 levels. Aortic calcium accumulation is decreased in MAVS-deficient LDLR mice fed arteriosclerotic diets. The G3BP1/RIG-I/MAVS relay is a component of Wnt signaling. Targeting this relay may help mitigate arteriosclerosis.
在主动脉血管平滑肌 (VSM) 中，经典 Wnt 受体 LRP6 抑制蛋白质精氨酸 (Arg) 甲基化，这是非经典 Wnt 信号的一个新组成部分，可刺激活化 T 细胞核因子 (NFATc4)。为了更好地理解甲基化如何介导这些作用，对来自对照和 LRP6 缺陷小鼠的 VSM 细胞提取物进行了 MS。LRP6 依赖性 Arg 甲基化受调节的蛋白超过 500 个；只有 21 个表现出单甲基化 (MMA) 的增加，同时伴有二甲基化的减少。G3BP1 是动脉粥样硬化的已知调节剂，其 C 端结构域的 MMA 增加了 30 多倍。共转染研究证实，LRP6 抑制 G3BP1 甲基化，G3BP1 刺激 NFATc4 转录。LRP6 缺乏时，NFATc4 与 VSM 骨桥蛋白 () 和碱性磷酸酶 () 染色质的结合增加，而 G3BP1 缺乏时则减少。G3BP1 对 NFATc4 的激活映射到支持与 RIG-I（维甲酸诱导基因 I）相互作用的 G3BP1 结构域，RIG-I 是一种刺激物，可在 Singleton-Merten 综合征 (SGMRT2) 中发生突变时驱动人类心血管钙化。功能获得性 SGMRT2/RIG-I 突变体增加了 G3BP1 的甲基化，并与成骨转录因子 (Runx2 和 NFATc4) 协同作用。G3BP 的化学拮抗剂 C108（C108 是 2-羟基苯甲酸，2-[1-(2-羟基苯基)亚乙基]肼 CAS 15533-09-2）下调了 RIG-I 刺激的 G3BP1 甲基化、Wnt/NFAT 信号、VSM TNAP 活性和钙化。LRP6 缺乏减少了 RIG-I 蛋白水平和 VSM 成骨程序。与 和 缺乏一样，MAVS 缺乏也减少了 VSM 成骨信号，包括 TNAP 活性和 Wnt5 依赖性核 NFATc4 水平。动脉粥样硬化饮食喂养的 MAVS 缺陷 LDLR 小鼠主动脉钙积累减少。G3BP1/RIG-I/MAVS 接力是 Wnt 信号的一个组成部分。靶向该接力可能有助于减轻动脉粥样硬化。
