Supplementary Material for: Requirement of Na+/H+ exchanger NHE1 for vasopressin-induced osteogenic signaling and calcification in human aortic smooth muscle cells

Background/Aims: Vasopressin is a powerful stimulator of vascular calcification, augmenting osteogenic signaling in vascular smooth muscle cells (VSMCs) including up-regulation of the transcription factors core-binding factor α-1 (CBFA1), msh homeobox 2 (MSX2), and SRY-Box 9 (SOX9), as well as of tissue-nonspecific alkaline phosphatase (ALPL). Vasopressin-induced osteogenic signaling and calcification require the serum- & glucocorticoid-inducible kinase 1 (SGK1). Known effects of SGK1 include up-regulation of Na+/H+ exchanger 1 (NHE1). NHE1 further participates in the regulation of reactive oxygen species (ROS). NHE1 has been shown to participate in the orchestration of bone mineralization. The present study, thus, explored whether vasopressin modifies NHE1 expression and ROS generation, as well as whether pharmacological inhibition of NHE1 disrupts vasopressin-induced osteogenic signaling and calcification in VSMCs. Methods: Human aortic smooth muscle cells (HAoSMCs) were treated with vasopressin in the absence or presence of SGK1 siRNA, SGK1 inhibitor GSK-650394, and NHE1 blocker cariporide. Transcript levels were determined by using qRT-PCR, protein abundance by western blotting, ROS generation with 2’,7’-dichlorofluorescein diacetate (DCFDA) fluorescence, as well as ALP activity and calcium content by using colorimetric assays. Results: Vasopressin significantly enhanced the NHE1 transcript and protein levels in HAoSMCs, effects significantly blunted by SGK1 inhibition with GSK-650394 or SGK1 siRNA. Vasopressin increased ROS accumulation, an effect significantly blocked by NHE1 inhibitor cariporide. Vasopressin further significantly increased osteogenic markers CBFA1, MSX2, SOX9, and ALPL transcript levels, as well as ALP activity and calcium content in HAoSMCs, all effects significantly blunted by SGK1 silencing or in the presence of GSK-650394 and cariporide. Conclusion: Vasopressin stimulates NHE1 expression and ROS generation, an effect dependent on SGK1 and required for vasopressin-induced stimulation of osteogenic signaling and calcification of VSMCs.

研究背景与目的：血管升压素（vasopressin）是强效的血管钙化刺激因子，可增强血管平滑肌细胞（vascular smooth muscle cells, VSMCs）内的成骨信号通路，具体包括上调转录因子核心结合因子α1（core-binding factor α-1, CBFA1）、msh同源框2（msh homeobox 2, MSX2）、SRY盒转录因子9（SRY-Box 9, SOX9）以及组织非特异性碱性磷酸酶（tissue-nonspecific alkaline phosphatase, ALPL）的表达。血管升压素诱导的成骨信号激活与血管钙化过程依赖于血清与糖皮质激素诱导激酶1（serum- & glucocorticoid-inducible kinase 1, SGK1）。已知SGK1可上调Na+/H+交换体1（Na+/H+ exchanger 1, NHE1）的表达，而NHE1可参与活性氧（reactive oxygen species, ROS）的调控，且已有研究证实NHE1参与骨矿化过程的调控。因此，本研究旨在探讨血管升压素是否会改变NHE1的表达与ROS的生成，以及NHE1的药理学抑制是否会阻断血管升压素诱导的VSMCs内成骨信号通路激活与血管钙化。

研究方法：将人主动脉平滑肌细胞（human aortic smooth muscle cells, HAoSMCs）分为多组，分别仅用血管升压素处理，或联合应用SGK1小干扰RNA（SGK1 siRNA）、SGK1抑制剂GSK-650394、NHE1抑制剂卡立泊来德（cariporide）进行处理。采用实时荧光定量PCR（quantitative real-time PCR, qRT-PCR）检测细胞转录本水平，通过蛋白质印迹法（western blotting）检测蛋白表达丰度，使用2',7'-二氯荧光素二乙酸盐（2’,7’-dichlorofluorescein diacetate, DCFDA）荧光法检测ROS生成量，并通过比色法检测碱性磷酸酶（ALP）活性与细胞钙含量。

研究结果：血管升压素可显著上调HAoSMCs中NHE1的转录与蛋白水平，该效应可被SGK1抑制剂GSK-650394或SGK1 siRNA显著削弱。血管升压素可促进ROS积累，该效应可被NHE1抑制剂卡立泊来德显著阻断。此外，血管升压素可显著上调HAoSMCs中成骨标志物CBFA1、MSX2、SOX9及ALPL的转录水平，同时提升ALP活性与钙含量，上述所有效应均可被SGK1基因沉默或联合应用GSK-650394与卡立泊来德显著抑制。

研究结论：血管升压素可通过SGK1依赖的途径刺激NHE1表达与ROS生成，而该过程是血管升压素诱导VSMCs成骨信号通路激活与血管钙化的必要条件。