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

 描述(申请人提供)：本研究方案的长期目标是确定血管钙化的分子机制，以寻找治疗慢性肾脏疾病依赖性血管钙化的新靶点(S)。血管钙化与慢性肾脏病患者的心血管死亡率密切相关。在慢性肾脏病患者中，超过一半的死亡可归因于心血管疾病。我们先前的研究表明，来自新生脂肪生成的硬脂酸盐促进了主动脉钙化。最近，我们发现，由于硬脂酰辅酶A脱饱和酶(SCD)活性降低，CKD患者硬脂酰磷脂酸(18：0/18：0-PA)水平显著升高。硬脂酸盐的促钙化和促成骨作用与双硬脂酰磷脂酸(18：0/18：0-PA)水平和NF-κΒ介导的肿瘤坏死因子α表达高度相关。我们推测在血管钙化发病机制中的一个中心事件是血管平滑肌细胞(VSMC)中通过PKCα/IKKζ/NF-βB信号通路增加了肿瘤坏死因子α的表达。我们的假说基于我们实验室的一系列初步结果：1)慢性肾脏病增加血清和主动脉肿瘤坏死因子α2)18：0/18：0-PA是通过GAPAT4-AGPAT3从头合成甘油脂途径合成的，是血管钙化发病机制中的关键代谢产物。3)肿瘤坏死因子α增加人和小鼠血管平滑肌细胞和18：0/18：0-PA的矿化，从而激活IKKβ/NFκB通路。(4)CKD激活主动脉PKCζ。5)已知PA能激活蛋白激酶Cζ，进而使IKKβ磷酸化并激活。6)血管钙化模型小鼠，如5/6NX基因敲除的DBA2/J小鼠和血管平滑肌细胞特异的κ/2基因敲除小鼠的主动脉中，活化的NF-SCD1B蛋白表达增加。7)用肿瘤坏死因子α单抗(英夫利昔单抗)治疗完全减轻慢性肾脏病依赖的血管钙化。为了确定PA-PKCζ-IKKβ-NF-κB-α轴在血管钙化发病机制中的关键作用，我们提出了三个特定的目标。特异目的1：确定18：0/18：0-PA激活PKCζ-IKKβ-NF-κB是否是VSMC成骨分化和矿化所必需的。具体目的2：A)确定18：0/18：0-PA合成的调节是否影响PKCζ-IKKβ-NF-κB-α轴和体内血管钙化；B)确定SMC特异性激活和抑制PKCζ-IKKβ-NF-κB轴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.