Inorganic phosphate signaling mechanisms through PiT-1 in VSMCs

 描述(申请人提供)：血管钙化(VC)是钙磷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而导致心血管并发症。VC发生在正常老龄化人群中，但与年龄匹配的对照组相比，慢性肾脏疾病(CKD)患者VC的患病率大大增加。在CKD患者中，肾功能下降会降低排泄无机磷(PI)的能力，从而导致高磷血症。在CKD人群中，PI升高是公认的心血管发病率和死亡率的危险因素，原因是钙化增加，这是由血管平滑肌细胞(VSMCs)活跃的矿物质沉积引起的。PI升高诱导VSMCs发生成骨表型转换，包括SMC标志物(SM22a、SMA-Actin、SM-MHC)减少和骨软骨形成标志物(Runx2、OPN、OCN、ALP)增加。然而，PI诱导VSMC表型改变和基质矿化的机制尚不清楚。本实验室以前的研究表明，III型钠依赖的磷酸共转运体PIT-1是人VSMC中主要的PI转运体，在体外对人VSMC基质的矿化和成骨分化是必需的。虽然这表明PIT-1通过增加磷吸收促进矿化，但最近的研究对这一结论提出了质疑。当PI浓度低于0.5 mM时，PIT-1对PI的吸收达到饱和，远低于诱导矿化所需的PI浓度(约2.4 mM)，这表明PI诱导的效果不需要PI吸收。最近，我们实验室观察到，PI升高(3.0 mM)诱导VSMC ERK1/2磷酸化，而VSMC中PIT-1的缺失消除了这种诱导。此外，我们可以通过过表达野生型PIT-1或PI缺陷型PIT-1突变蛋白来挽救PI诱导的ERK1/2磷酸化和VSMCs的成骨分化。这些结果表明，PIT-1可以通过ERK1/2细胞信号通路，通过不依赖于PI摄取的机制来感知和响应PI升高。根据初步数据和以前的研究，我们假设PI升高导致PIT-1从二聚体到单体状态的转变，这暴露了一个隐藏的位点，允许蛋白质与受体信号起始蛋白RAPGEF1相互作用，并导致RAF/MEK/ERK信号通路的激活。在这项研究中，我们的目的是通过1)研究RAPGEF1在PI诱导的ERK1/2磷酸化以及RAPGEF1与PIT-1之间的蛋白结合中的重要性，以及2)研究PIT-1二聚化在PI升高以及PIT-1二聚化所需的功能结构域中的作用，来阐明PIT-1细胞信号转导的机制。这一新的信号通路是在VSMC中发现的第一个PI传感通路。我们相信，这一通路可以提供新的治疗策略，可以抑制PI对CKD患者VSMCs的影响，并阻断高磷血症诱导的VC。

 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.