Role of IKKβ/NFκβ signaling in the regulation of CKD-dependent vascular calcification

 描述（由申请方提供）：本研究计划的长期目标是确定血管钙化的分子机制，以确定治疗慢性肾脏病（CKD）依赖性血管钙化的新靶点。血管钙化与慢性肾病患者的心血管死亡率密切相关。CKD受试者中超过一半的死亡可归因于心血管疾病。我们以前的研究表明，硬脂酸盐来源于从头脂肪生成促进主动脉钙化。最近，我们发现，由于硬脂酰辅酶A去饱和酶（SCD）活性降低，CKD受试者中硬脂酸盐代谢产物之一二硬脂酰磷脂酸（18：0/18：0-PA）的水平显著升高。硬脂酸盐的促钙化和促成骨作用与二硬脂酰磷脂酸（18：0/18：0-PA）和NF-κ B介导的TNFα表达水平高度相关。我们假设血管钙化发病机制的中心事件是通过血管平滑肌细胞（VSMCs）中的PKC β/IKKβ/NF-κB信号传导增加TNFα的表达。我们的假设基于以下证据，这些证据来自于我们实验室的一系列初步结果：1）CKD增加血清和主动脉TNFα 2）通过甘油脂质从头合成的GPAT 4-AGPAT 3途径合成的18：0/18：0-PA是血管钙化发病机制中的关键代谢产物。3)TNFα可增加人和小鼠VSMCs和18：0/18：0-PA的矿化，导致IKKβ/NFκB通路激活。4)CKD激活主动脉PKC β。5)已知PA激活PKC β，PKC β进而磷酸化并激活IKKβ。6)在血管钙化的小鼠模型如具有5/6 nx的DBA 2/J小鼠和VSMC特异性SCD 1/2敲除小鼠的动脉中，活性NF-κB蛋白增加。7)TNFα单克隆抗体（英夫利西单抗）治疗可完全减弱CKD依赖性血管钙化。为了确定PA-PKCζ-IKKβ-NF-κB-TNFα轴在血管钙化发病机制中的关键作用，我们提出了三个具体目标。具体目标1：确定18：0/18：0-PA激活PKC β-IKKβ-NF-κB是否是VSMC成骨分化和矿化所必需的。具体目标二：A）确定重新18：0/18：0-PA合成的调节是否影响体内PKC β-IKKβ-NF-κB - TNFα轴和血管钙化，和B）确定SMC特异性活化和PKC β-IKKβ-NF-κB轴的抑制是否影响体内血管钙化。

 DESCRIPTION (provided by applicant): The long term objective of this research proposal is to determine the molecular mechanisms of vascular calcification in order to identify novel target(s) for the treatment of chronic kidney disease (CKD)-dependent vascular calcification. Vascular calcification is closely associated with cardiovascular mortality in patients with CKD. More than half of all deaths in CKD subjects can be attributed to cardiovascular diseases. Our previous study revealed that stearate derived from de novo lipogenesis promotes aortic calcification. Recently, we found that levels of one of the stearate metabolites, distearoyl-phosphatidic acid (18:0/18:0-PA) are significantly increased in subjects with CKD due to lower stearoyl-CoA desaturase (SCD) activity. The pro-calcific and pro- osteogenic effects of stearate were highly correlated with levels of distearoyl-phosphatidic acid (18:0/18:0-PA) and NF-κΒ-mediated TNFα expression. We hypothesized that a central event in the pathogenesis of vascular calcification is increased expression of TNFα through PKCζ/IKKβ/NF-κB signaling in vascular smooth muscle cells (VSMCs). Our hypothesis is based on the following evidence derived from a series of preliminary results from our lab: 1) CKD increased serum and aortic TNFα 2) 18:0/18:0-PA synthesized through the GPAT4-AGPAT3 pathway of de novo glycerolipid synthesis is a critical metabolite in the pathogenesis of vascular calcification. 3) TNFα increased mineralization of VSMCs and 18:0/18:0-PA in humans and mice, resulting in activation of the IKKβ/NFκB pathway. 4) CKD activated aortic PKCζ. 5) PA is known to activate PKCζ, which in turn phosphorylates and activates IKKβ. 6) Active NF-κB proteins were increased in the aortas of murine models of vascular calcification such as DBA2/J mice with 5/6 nx and VSMC-specific SCD1/2 knockout mice. 7) Treatment with a TNFα monoclonal antibody (Infliximab) completely attenuated CKD-dependent vascular calcification. To determine the pivotal role of the PA-PKCζ-IKKβ-NF-κB-TNFα axis in the pathogenesis of vascular calcification, we propose three specific aims. Specific Aim 1: Determine whether activation of PKCζ-IKKβ-NF-κB by 18:0/18:0-PA is required for osteoblastic differentiation and mineralization of VSMCs. Specific Aim 2: A) Determine whether the modulation of de novo 18:0/18:0-PA synthesis affects the PKCζ-IKKβ-NF-κB - TNFα axis and vascular calcification in vivo and B) determine whether SMC-specific activation and inhibition of the PKCζ-IKKβ-NF-κB axis influences vascular calcification in vivo.