Department of Medicine A, University Medicine Greifswald, Ferdinand-Sauerbruchstrasse, Greifswald, 17475, Germany.
Veterans Affairs Greater Los Angeles Healthcare System, David Geffen School of Medicine, Southern California Research Center for Alcoholic Liver and Pancreatic Disease and Cirrhosis, University of California at Los Angeles, Los Angeles, CA, 90073, USA.
Cell Mol Life Sci. 2020 May;77(9):1811-1825. doi: 10.1007/s00018-019-03254-7. Epub 2019 Jul 30.
Premature intrapancreatic trypsinogen activation is widely regarded as an initiating event for acute pancreatitis. Previous studies have alternatively implicated secretory vesicles, endosomes, lysosomes, or autophagosomes/autophagolysosomes as the primary site of trypsinogen activation, from which a cell-damaging proteolytic cascade originates. To identify the subcellular compartment of initial trypsinogen activation we performed a time-resolution analysis of the first 12 h of caerulein-induced pancreatitis in transgenic light chain 3 (LC3)-GFP autophagy reporter mice. Intrapancreatic trypsin activity increased within 60 min and serum amylase within 2 h, but fluorescent autophagosome formation only by 4 h of pancreatitis in parallel with a shift from cytosolic LC3-I to membranous LC3-II on Western blots. At 60 min, activated trypsin in heavier subcellular fractions was co-distributed with cathepsin B, but not with the autophagy markers LC3 or autophagy protein 16 (ATG16). Supramaximal caerulein stimulation of primary pancreatic acini derived from LC3-GFP mice revealed that trypsinogen activation is independent of autophagolysosome formation already during the first 15 min of exposure to caerulein. Co-localization studies (with GFP-LC3 autophagosomes versus Ile-Pro-Arg-AMC trypsin activity and immunogold-labelling of lysosomal-associated membrane protein 2 [LAMP-2] versus trypsinogen activation peptide [TAP]) indicated active trypsin in autophagolysosomes only at the later timepoints. In conclusion, during the initiating phase of caerulein-induced pancreatitis, premature protease activation develops independently of autophagolysosome formation and in vesicles arising from the secretory pathway. However, autophagy is likely to regulate overall intracellular trypsin activity during the later stages of this disease.
胰蛋白酶原提前在胰腺内激活被广泛认为是急性胰腺炎的起始事件。之前的研究提示，在细胞损伤的蛋白水解级联反应起始之前，分泌小泡、内体、溶酶体或自噬体/自噬溶酶体可能是胰蛋白酶原激活的主要部位。为了鉴定最初胰蛋白酶原激活的亚细胞区室，我们在转 Lc3-GFP 自噬报告基因的小鼠中进行了蓝斑肽诱导胰腺炎的前 12 小时的时间分辨分析。胰蛋白酶活性在 60 分钟内增加，血清淀粉酶在 2 小时内增加，但荧光自噬体形成仅在胰腺炎 4 小时时出现，同时 Western blot 显示细胞质 LC3-I 向膜结合 LC3-II 转移。在 60 分钟时，较重亚细胞级分中的激活胰蛋白酶与组织蛋白酶 B 共分布，但与自噬标记物 LC3 或自噬蛋白 16 (ATG16) 不共分布。来自 LC3-GFP 小鼠的原代胰腺腺泡的超量蓝斑肽刺激显示，在暴露于蓝斑肽的前 15 分钟内，胰蛋白酶原激活就独立于自噬溶酶体形成。共定位研究（用 GFP-LC3 自噬体与 Ile-Pro-Arg-AMC 胰蛋白酶活性，以及免疫金标记溶酶体相关膜蛋白 2 [LAMP-2] 与胰蛋白酶原激活肽 [TAP]）表明，只有在后期时间点才有活性胰蛋白酶存在于自噬溶酶体中。总之，在蓝斑肽诱导的胰腺炎的起始阶段，过早的蛋白酶激活独立于自噬溶酶体形成和分泌途径中的小泡发生。然而，自噬可能在该疾病的后期阶段调节细胞内总体胰蛋白酶活性。
