Unraveling the Role of NADPH Oxidase in Inflammation-associated Pancreatic Diseases

摘要 胰腺炎症是胃肠道相关临床发病率和死亡率的主要原因，以及 针对胰腺炎症性疾病的有效治疗策略仍然是医学上尚未满足的需要。 炎症是胰腺炎及其相关的腺泡-导管化生的共同特征。 胰腺癌(ADM)、胰腺上皮内瘤变(Panin)病变和胰腺导管腺癌(PDAC)。 鉴于炎症在胰腺病理中的突出作用，了解胰腺- 特定的炎症机制对相关疾病的预防和治疗至关重要。 众所周知，炎症可诱发活性氧自由基(ROS)。在已知的原因中，ROS既起作用，又起作用 一种信号分子和炎症的中介物。野生型KRAS半胱氨酸受ROS修饰 氧化还原敏感的NKC118D基序导致短暂的过度激活。在~30%的情况下观察到KRAS突变 这表明KRAS突变在胰腺炎症中起着关键作用。 在过去的三十年里，突变的KRAS被认为被锁定在一种结构性活跃的状态中。然而， 最近的研究发现，突变的KRAS在内源水平上并不是结构性活跃的，但可以 被炎性侮辱过度激活，导致持续炎症、不可逆转的ADM和Panin损害。 然而，将炎症与突变的KRAS过度激活和 相关的病理仍然难以捉摸。研究表明，NADPH氧化酶(NOxs)是一种主要的介体 胰腺炎引起的炎症和突变的KRAS激活的产生ROS的主要酶。 然而，突变体KRAS是否受到与野生型KRAS相同的ROS修饰，从而导致 在炎症的背景下观察到的多动是未知的。这项建议的目标是确定 炎症促进突变型KRAS的分子调节和潜在机制 过度活跃与相关的胰腺病理。值得注意的是，通过烧蚀NOx对接亚基p22Phox 在胰腺腺泡细胞表达内源性KrasG12D/+水平的小鼠中，我们证明了NOX 抑制在很大程度上抑制了KrasG12D/+的过度激活，这表明NOX不仅是下游的效应器 也可能是KrasG12D的上游调控因子。因此，NOX和KrasG12D可能形成共同激活 导致慢性炎症、不可逆的ADM和PAIN损害所需的前馈循环。我们 假设在炎症条件下，NOX产生ROS来修改KrasG12D的氧化还原敏感基序， 导致KrasG12D过度激活和相关的病理。我们的假设将通过三个具体的 针对在炎症性侮辱背景下的小鼠模型。目的1是确定NOX在胰腺中的作用 发炎。目标2是确定NOX是否是KrasG12D过度激活、持续炎症、 不可逆的ADM和PANIN病变。目标3是确定氧化还原敏感的NKC118D基序是否需要 KrasG12D过度激活及其相关病理。我们在学习中获得的信息将填补未完成的 关于炎性侮辱如何促进突变的KRAS过度激活和相关病理的知识差距 并指导针对人类这些疾病的新的预防和治疗策略的设计。

Abstract Pancreatic inflammation is the major cause of gastrointestinal-related clinical morbidity and mortality, and effective therapeutic strategies against pancreatic inflammatory diseases remain an unmet medical need. Inflammation is a common feature in the pathogenesis of pancreatitis and associated acinar-to-ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN) lesions, and pancreatic ductal adenocarcinoma (PDAC). Given the prominent role of inflammation in the spectrum of pancreatic pathologies, understanding pancreas- specific inflammatory mechanisms is critical for the prevention and treatment of the associated diseases. Inflammation is known to induce reactive oxygen species (ROS). Among known causal factors, ROS act as both a signaling molecule and a mediator of inflammation. Wild-type KRAS is subject to ROS modification on cysteine of the redox-sensitive NKC118D motif leading to a transient hyperactivation. KRAS mutants are observed in ~30% of patients with chronic pancreatitis, suggesting the critical role of KRAS mutations in pancreatic inflammation. In the past thirty years, mutant KRAS has been viewed as being locked in a constitutively active state. However, recent studies have found that mutant KRAS, at an endogenous level, is not constitutively active but can be hyper-activated by inflammatory insults leading to sustained inflammation, irreversible ADM, and PanIN lesions. However, the molecular mediator and mechanism linking inflammation to mutant KRAS hyperactivation and associated pathologies remain elusive. Studies have shown that NADPH oxidases (NOXs) are a major mediator of pancreatitis-induced inflammation and major enzymes activated by mutant KRAS for the generation of ROS. However, whether mutant KRAS is subject to the same ROS modification as wild-type KRAS leading to the observed hyperactivity in the context of inflammation is unknown. The objective of this proposal is to identify the molecular mediator and unravel the underlying mechanism on how inflammation promotes mutant KRAS hyperactivation and associated pancreatic pathologies. Notably, by ablating the NOX-docking subunit p22phox in mice expressing an endogenous level of KRASG12D/+ in pancreatic acinar cells, we demonstrate that NOX inhibition considerably curbs KRASG12D/+ hyperactivation, suggesting that NOX is not only a downstream effector but also a potential upstream regulator of KRASG12D. Thus, NOX and KRASG12D potentially form a co-activation feed-forward loop necessary to induce chronic inflammation, irreversible ADM, and PanIN lesions. We hypothesize that under inflammation, NOX generates ROS to modify the redox-sensitive motif of KRASG12D, leading to KRASG12D hyperactivation and associated pathologies. Our hypothesis will be tested with three specific aims in mouse models in the context of inflammatory insults. Aim 1 is to determine the role of NOX in pancreatic inflammation. Aim 2 is to determine if NOX is necessary for KRASG12D hyperactivation, sustained inflammation, irreversible ADM, and PanIN lesions. Aim 3 is to determine if the redox-sensitive NKC118D motif is required for KRASG12D hyperactivation and associated pathologies. Information gained in our studies will fill the outstanding knowledge gap on how inflammatory insults promote mutant KRAS hyperactivation and associated pathologies and guide the design of novel preventive and therapeutic strategies against these diseases in humans.