Inorganic phosphate signaling mechanisms through PiT-1 in VSMCs

VSMC 中通过 PiT-1 的无机磷酸信号传导机制

• 批准号: 8979371
• 负责人: Nicholas W Chavkin
• 金额: $ 3.79万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8979371
• 关键词: Actins; Adaptor Signaling Protein; Age; Binding; Binding Sites; Blood Vessels; C57BL/6 Mouse; Cardiovascular system; Chronic Kidney Failure; Co-Immunoprecipitations; Data; Deposition; Dimerization; Excretory function; Fluorescence Resonance Energy Transfer; Human; Hypertension; Image; In Vitro; Inorganic Phosphate Transporter; Kidney; Lead; MAPK3 gene; MEKs; Minerals; Morbidity - disease rate; Mutate; Mutation; Pathway interactions; Patients; Phenotype; Phosphorylation; Physiologic pulse; Physiological; Play; Population; Prevalence; Property; Protein Binding; Proteins; Receptor Signaling; Renal function; Risk; Role; Serum; Signal Pathway; Signal Transduction; Site; Small Interfering RNA; Smooth Muscle Myocytes; Sodium; Stimulus; Structure-Activity Relationship; Vascular calcification; calcification; calcium phosphate; cardiovascular risk factor; dimer; extracellular; inorganic phosphate; mineralization; monomer; mortality; mutant; normal aging; novel; novel therapeutics; overexpression; patient population; public health relevance; receptor; response; symporter; therapeutic target; uptake

 DESCRIPTION (provided by applicant): Vascular calcification (VC) is the inappropriate deposition of calcium phosphate mineral in the vasculature, and arterial calcification can cause cardiovascular complications through hypertension, decreased vascular compliance, and increased pulse wave velocity. VC occurs in the normal ageing population, but patients with chronic kidney disease (CKD) have a highly increased prevalence of VC compared to age- matched controls. In CKD patients, reduced renal function diminishes the ability to excrete inorganic phosphate (Pi), which leads to hyperphosphatemia. Elevated Pi is a recognized risk factor for cardiovascular morbidity and mortality in the CKD population due to increased calcification, which is caused by active deposition of mineral by vascular smooth muscle cells (VSMCs). Elevated Pi induces VSMCs to undergo an osteochondrogenic phenotype transition, which involves a decrease in SMC markers (SM22a, SMa-actin, SM- MHC) and an increase in osteochondrogenic markers (Runx2, OPN, OCN, ALP). However, the mechanism of Pi-induced VSMC phenotype change and matrix mineralization is unclear. Our lab has previously shown that the type III sodium-dependent phosphate co-transporter, PiT-1, is the main Pi transporter in human VSMCs and is required for human VSMC matrix mineralization and osteochondrogenic differentiation in vitro. Although this suggested PiT-1 promoted mineralization through increased Pi uptake, recent studies have questioned that conclusion. Pi uptake through PiT-1 was shown to be saturated at Pi concentrations below 0.5 mM, which is well below the Pi concentration required to induce mineralization (around 2.4 mM), suggesting Pi uptake is not required for Pi-induced effects. Recently, our lab has observed that elevated Pi (3.0 mM) induces ERK1/2 phosphorylation in VSMCs, and deletion of PiT-1 from VSMCs removed this induction. Furthermore, we could rescue Pi-induced ERK1/2 phosphorylation and osteochondrogenic differentiation of VSMCs by overexpression of either wild-type PiT-1 or a Pi-deficient PiT-1 mutant protein. These results suggest PiT-1 can sense and respond to elevated Pi by a Pi uptake-independent mechanism through ERK1/2 cell signaling. Given preliminary data and previous studies, we hypothesize that elevated Pi induces PiT-1 transition from a dimer to a monomer state, which exposes a cryptic site and allows for protein interactions with the receptor signal initiator protein RAPGEF1 and causes activation of the RAF/MEK/ERK signaling pathway. In this proposal, we aim to elucidate the mechanisms of PiT-1 cell signaling by 1) investigating the importance of RAPGEF1 in Pi-induced ERK1/2 phosphorylation and protein binding between RAPGEF1 and PiT-1 in VSMCs, and 2) investigating the role of PiT-1 dimerization in response to elevated Pi and the functional PiT-1 domains required for dimerization. This novel signaling pathway is the first Pi sensing pathway discovered in VSMCs. We believe that this pathway can provide novel therapeutic strategies that could inhibit the effects of Pi on VSMCs and block hyperphosphatemia-induced VC in CKD patients.

 描述（由申请人提供）：血管钙化（VC）是磷酸钙矿物质在血管系统中的不适当沉积，动脉钙化可通过高血压、血管顺应性降低和脉搏波速度增加引起心血管并发症。VC发生在正常的老龄化人群中，但与年龄匹配的对照组相比，慢性肾脏病（CKD）患者的VC患病率高度增加。在CKD患者中，肾功能下降降低了排泄无机磷酸盐（Pi）的能力，从而导致高磷酸盐血症。由于钙化增加，PI升高是CKD人群中心血管发病率和死亡率的公认风险因素，钙化增加是由血管平滑肌细胞（VSMC）的矿物质活性沉积引起的。升高的Pi诱导VSMC经历骨软骨形成表型转变，其涉及SMC标志物（SM 22 a、SMA-肌动蛋白、SM-MHC）的减少和骨软骨形成标志物（Runx 2、OPN、OCN、ALP）的增加。然而，Pi诱导VSMC表型改变和基质矿化的机制尚不清楚。我们的实验室之前已经证明，III型钠依赖性磷酸盐共转运蛋白PiT-1是人VSMC中主要的Pi转运蛋白，并且是体外人VSMC基质矿化和骨软骨分化所需的。虽然这表明PiT-1通过增加Pi吸收促进矿化，但最近的研究质疑这一结论。通过PiT-1的Pi摄取显示在低于0.5 mM的Pi浓度下饱和，这远低于诱导矿化所需的Pi浓度（约2.4 mM），表明Pi诱导的效应不需要Pi摄取。最近，我们的实验室已经观察到升高的Pi（3.0 mM）诱导VSMCs中的ERK 1/2磷酸化，并且从VSMCs中删除PiT-1消除了这种诱导。此外，我们可以通过过度表达野生型PiT-1或PiT-1缺陷突变蛋白来挽救Pi诱导的ERK 1/2磷酸化和VSMC的骨软骨分化。这些结果表明，PiT-1可以通过ERK 1/2细胞信号转导通过Pi摄取非依赖性机制感知和响应升高的Pi。鉴于初步数据和以前的研究，我们假设，Pi升高诱导PiT-1从二聚体转变为单体状态，这暴露了一个隐蔽位点，并允许与受体信号起始蛋白RAPGEF 1的蛋白质相互作用，并导致RAF/MEK/ERK信号通路的激活。在这个提议中，我们的目标是阐明PiT-1细胞信号转导的机制，1）调查的重要性，在PI诱导的ERK 1/2磷酸化和RAPGEF 1和PiT-1之间的蛋白结合在VSMC，和2）调查的作用PiT-1二聚化响应于升高的Pi和二聚化所需的功能PiT-1结构域。这条新的信号通路是在VSMCs中发现的第一条Pi传感通路。我们认为，这一途径可以提供新的治疗策略，可以抑制Pi对VSMC的影响，并阻断CKD患者高磷血症诱导的VC。