内质网 (ER) 伴侣 GRP78 在 ER 应激期间分泌并减轻内皮细胞炎症,European Heart Journal

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内质网 (ER) 伴侣 GRP78 在 ER 应激期间分泌并减轻内皮细胞炎症
European Heart Journal ( IF 37.6 ) Pub Date : 2023-11-11 , DOI: 10.1093/eurheartj/ehad655.3290
E Repges ₁ , M A Z Al Zaidi ₁ , F J Jansen ₁ , S Z Zimmer ₁ , V T Tiyerlili ₂ , G N Nickenig ₁ , A A Aksoy ₁

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简介葡萄糖调节蛋白 78kD (GRP78) 是一种分子伴侣，也是 ER 应激反应的主要调节因子，该反应由多种干扰 ER 中蛋白质折叠的条件触发。在内质网应激时，GRP78 会激活未折叠蛋白反应 (UPR)，其目的是清除未折叠蛋白并恢复内质网稳态。最近，描述了 GRP78 的细胞外分泌。然而，分泌型 GRP78 在动脉粥样硬化和内皮细胞炎症中的病理生理学相关性仍有待阐明。本研究的目的是探讨 GRP78 分泌在内皮细胞中的作用。方法和结果首先，我们试图研究血管细胞在 ER 应激期间是否分泌 GRP78。用 ER 应激诱导剂衣霉素处理人冠状动脉内皮细胞 (HCAEC) 长达 48 小时。ER 应激诱导后，Western Blot 和 ELISA 实验检测到细胞内 GRP78 表达增加。有趣的是，长时间的内质网应激也促进了 GRP78 的细胞外分泌。HCAEC 分泌组的无偏蛋白质组学分析证实，在 ER 应激诱导后，GRP78 是上调程度最高的胞外蛋白之一（2.43 倍）。与内质网-高尔基体蛋白转运抑制剂 Brefeldin A 共孵育可消除细胞外分泌（图 1）。因此，ER 应激诱导的 GRP78 分泌是一个主动调节的过程。接下来，分析含有GRP78的条件培养基(CM)对HCAEC的影响。作为对照，HCAEC 与 BFA 共孵育，或通过 siRNA 进行 GRP78 敲低。用含有CM的GRP78处理降低了靶细胞中的GRP78 mRNA表达(与对照[+BFA]相比0.35倍，p＜0.0001)。此外，它还增加了活力（BFA：93.0% vs. 83%，p=0.002；GRP78-siRNA：94% vs. 85%，p=0.01）并降低了细胞凋亡率（例如 BFA：1.7 倍 vs. 5.1 倍），p=0.02）。此外，与不含GRP78的CM相比，血管炎症和ER应激标志物（例如NF-κB和CHOP）的表达降低（图2）。此外，测量了增加的H 2 O 2 产生(BFA：1.4倍，p＞0.001，GRP78-siRNA：1.3倍，p＝0.004)(图2)。为了确认 CM 的有益作用实际上是由 GRP78 引起的，我们用重组 GRP78 处理 HCAEC，并观察到细胞活力以剂量依赖性方式增加（1000 ng/ml：与对照相比，p=0.02）。CRIPTO 是生长因子的膜结合共受体，之前与 GRP78 信号传导有关。无论是否进行 GRP78 处理，CRIPTO 的 siRNA 敲低都会降低活力并导致细胞凋亡。因此，GRP78 介导的作用可能是由 CRIPTO 介导的。结论内皮ER应激促进GRP78分泌。条件培养基中 GRP78 的存在可改善随后的 ER 应激和内皮炎症，这在动脉粥样硬化形成中发挥着关键作用。进一步的研究对于阐明释放机制和对靶细胞的影响非常重要。

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The endoplasmic reticulum (ER) chaperone GRP78 is secreted during ER Stress and alleviates endothelial cell inflammation

Introduction Glucose-Regulated Protein 78kD (GRP78) is a chaperone and the main regulator of the ER-stress response which is triggered by a variety of conditions that disturb folding of proteins in the ER. Upon ER Stress, GRP78 activates the unfolded protein response (UPR), which aims to clear unfolded proteins and restore ER homeostasis. Recently, extracellular secretion of GRP78 was described. However, the pathophysiological relevance of secreted GRP78 in atherosclerosis and endothelial cell inflammation remains to be elucidated. Aim of this study is to investigate the role of GRP78 secretion in endothelial cells. Methods and Results First, we sought to investigate if vascular cells secrete GRP78 during ER Stress. Human coronary artery endothelial cells (HCAEC) were treated with the ER stress inductor tunicamycin for up to 48h. After ER Stress induction, Western Blot and ELISA experiments detected an increased intracellular GRP78 expression. Intriguingly, prolonged ER Stress also promoted extracellular secretion of GRP78. Unbiased proteomic analysis of the HCAEC secretome confirmed that after ER-Stress induction, GRP78 is one of the most highly upregulated extracellular proteins (2.43-fold). Co-incubation with Brefeldin A, an inhibitor of ER-Golgi protein transport, abolished extracellular secretion (Fig.1). Hence, ER-Stress-induced GRP78 secretion is an actively regulated process. Next, the effect of GRP78 containing conditioned medium (CM) on HCAEC was analyzed. For control, HCAEC were co-incubated with BFA, or GRP78-knockdown was performed by siRNA. Treatment with GRP78 containing CM decreased GRP78 mRNA expression in target cells (0.35-fold vs. control [+BFA], p<0.0001). Furthermore, it increased viability ( BFA: 93.0 % vs. 83%, p=0.002; GRP78-siRNA: 94% vs. 85%, p=0.01) and decreased the rate of apoptosis (e.g. BFA: 1.7-fold vs. 5.1, p=0.02). Moreover, expression of markers of vascular inflammation and ER Stress (e.g., NF-κB and CHOP) were decreased when compared to CM without GRP78 (Fig.2). Furthermore, increased H2O2-generation was measured (BFA: 1.4-fold, p>0.001, GRP78-siRNA:1.3-fold, p=0.004) (Fig. 2). To confirm that the beneficial effects of the CM are in fact caused by GRP78, we treated HCAEC with recombinant GRP78 and observed an increased cell viability in a dose-dependent manner (1000 ng/ml: p=0.02 vs. control). CRIPTO is a membrane-bound co-receptor for growth factors and was previously linked to GRP78-signalling. siRNA-Knockdown of CRIPTO reduces viability and leads to cell apoptosis, irrespective of GRP78-treatment. Thus, GRP78-mediated effects may be mediated by CRIPTO. Conclusion Endothelial ER Stress promotes GRP78 secretion. Presence of GRP78 in conditioned medium ameliorates subsequent ER Stress and endothelial inflammation, which play a critical role in atherogenesis. Further investigations are important to shed light on the mechanism of release and the effect on target cells.

更新日期：2023-11-11

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