Epithelial Signaling and Transport Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland.
Genetics and Pathogenesis of Allergy Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.
Gastroenterology. 2019 Dec;157(6):1660-1672.e2. doi: 10.1053/j.gastro.2019.08.042. Epub 2019 Sep 4.
BACKGROUND & AIMS: Pancreatitis is characterized by increased influx of Ca into acinar cells, by unknown mechanisms. Inhibitors of Ca influx channels could be effective in treating acute pancreatitis, but these have deleterious side effects that can result in death. We investigated the expression patterns and functions of acinar cell Ca channels and factors that regulate them during development of acute pancreatitis, along with changes in the channel inactivator store-operated calcium entry-associated regulatory factor (SARAF). We investigated whether SARAF is a target for treatment of acute pancreatitis and its status in human with pancreatitis.
We generated mice that expressed SARAF tagged with hemagglutinin, using CRISPR/Cas9 gene editing, and isolated acinar cells. We also performed studies with Saraf mice, Saraf mice, mice without disruption of Saraf (control mice), and mice that overexpress fluorescently labeled SARAF in acinar cells. We analyzed interactions between stromal interaction molecule 1 (STIM1) and SARAF in HEK cells stimulated with carbachol using fluorescence resonance energy transfer microscopy and immunoprecipitation. Mice were given injections of caerulein or L-arginine to induce pancreatitis. Pancreatic tissues and blood samples were collected and levels of serum amylase, trypsin, tissue damage, inflammatory mediators, and inflammatory cells were measured. We performed quantitative polymerase chain reaction analyses of pancreatic tissues from 6 organ donors without pancreatic disease (controls) and 8 patients with alcohol-associated pancreatitis.
Pancreatic levels of Ca influx channels or STIM1 did not differ significantly between acinar cells from mice with vs. without pancreatitis. By contrast, pancreatic levels of Saraf messenger RNA and SARAF protein initially markedly increased but then decreased during cell stimulation or injection of mice with caerulein, resulting in excessive Ca influx. STIM1 interacted stably with SARAF following stimulation of HEK or mouse acinar cells with physiologic levels of carbachol, but only transiently following stimulation with pathologic levels of carbachol, leading to excessive Ca influx. We observed reduced levels of SARAF messenger RNA in pancreatic tissues from patients with pancreatitis, compared with controls. SARAF knockout mice developed more severe pancreatitis than control mice after administration of caerulein or L-arginine, and pancreatic acinar cells from these mice had significant increases in Ca influx. Conversely, overexpression of SARAF in acini reduced Ca influx, eliminated inflammation, and reduced severity of acute pancreatitis.
In mice with pancreatitis, SARAF initially increases but is then degraded, resulting in excessive, pathological Ca influx by acinar cells. SARAF knockout mice develop more severe pancreatitis than control mice, whereas mice that express SARAF from a transgene in acinar cells develop less-severe pancreatitis. SARAF therefore appears to prevent pancreatic damage during development of acute pancreatitis. Strategies to stabilize or restore SARAF to acinar cells might be developed for treatment of pancreatitis.
胰腺炎的特征是细胞溶质 Ca2+ 流入增加，但其机制尚不清楚。Ca2+ 流入通道抑制剂在治疗急性胰腺炎方面可能是有效的，但它们具有有害的副作用，可能导致死亡。我们研究了在急性胰腺炎发展过程中，细胞溶质 Ca2+ 通道的表达模式和功能，以及通道失活剂储存操作钙进入相关调节因子 (SARAF) 的变化。我们研究了 SARAF 是否是治疗急性胰腺炎的靶点，以及其在胰腺炎患者中的状态。
我们使用 CRISPR/Cas9 基因编辑生成了带有血球凝集素标签的 SARAF 表达的小鼠，并分离了胰腺细胞。我们还对 Saraf 小鼠、Saraf 缺失小鼠（对照小鼠）和在胰腺细胞中过表达荧光标记 SARAF 的小鼠进行了研究。我们使用荧光共振能量转移显微镜和免疫沉淀分析了在细胞溶质 Ca2+ 刺激下 HEK 细胞中基质相互作用分子 1 (STIM1) 和 SARAF 之间的相互作用。用蛙皮素或 L-精氨酸给小鼠注射诱导胰腺炎。收集胰腺组织和血液样本，测量血清淀粉酶、胰蛋白酶、组织损伤、炎症介质和炎症细胞的水平。我们对 6 名无胰腺疾病的器官捐献者（对照）和 8 名酒精相关性胰腺炎患者的胰腺组织进行了定量聚合酶链反应分析。
与无胰腺炎的小鼠相比，胰腺细胞溶质 Ca2+ 通道或 STIM1 的水平在有胰腺炎的小鼠中没有显著差异。相比之下，Saraf 信使 RNA 和 SARAF 蛋白的胰腺水平在细胞刺激或注射蛙皮素的小鼠中最初显著增加，但随后减少，导致 Ca2+ 流入过多。在生理水平的蛙皮素刺激 HEK 或小鼠胰腺细胞后，STIM1 与 SARAF 稳定相互作用，但在病理水平的蛙皮素刺激后仅短暂相互作用，导致 Ca2+ 流入过多。我们观察到与对照组相比，胰腺炎患者的胰腺组织中 SARAF 信使 RNA 水平降低。在给予蛙皮素或 L-精氨酸后，SARAF 敲除小鼠比对照小鼠发生更严重的胰腺炎，而这些小鼠的胰腺腺泡细胞中 Ca2+ 流入显著增加。相反，在胰腺细胞中过表达 SARAF 可减少 Ca2+ 流入、消除炎症并减轻急性胰腺炎的严重程度。
在胰腺炎小鼠中，SARAF 最初增加，但随后降解，导致胰腺细胞发生过度的、病理性的 Ca2+ 流入。SARAF 敲除小鼠比对照小鼠发生更严重的胰腺炎，而在胰腺细胞中表达来自转基因的 SARAF 的小鼠发生的胰腺炎较轻。因此，SARAF 似乎在急性胰腺炎发展过程中防止胰腺损伤。稳定或恢复 SARAF 到胰腺细胞的策略可能被开发用于治疗胰腺炎。
