Molecular pathomechanism of hereditary pancreatitis

 描述(由申请人提供)：这笔赠款的主要目标是确定羧基肽酶A1(CPA1)突变作为人类慢性胰腺炎风险因素的机制。大多数非酒精性慢性胰腺炎病例是在遗传易感性的基础上发展起来的，这些易感基因是由编码消化酶的危险基因突变驱动的，这些消化酶包括阳离子胰蛋白酶原(蛋白酶丝氨酸1，PRSS1)、胰腺分泌胰蛋白酶抑制物(丝氨酸蛋白酶抑制物1，SPINK1)、胰凝乳蛋白酶C(CTRC)或羧基肽酶A1(CPA1)。我们在以前的资助时期的研究清楚地定义了与PRSS1、SPINK1和CTRC突变导致的胰腺内胰酶活性增加相关的病理途径。然而，我们最近关于CPA1突变的结果表明，并不是所有的遗传风险因素都会增加胰酶活性。目前该基金的主要假设是，突变诱导的错误折叠和随之而来的内质网(ER)应激是增加CPA1突变携带者胰腺炎风险的基本机制。为了验证这一假设，我们将系统地研究28个CPA1变体，并证明致病的CPA1变体在细胞内滞留和降解，从而减少分泌和显著增加内质网应激。相反，中性的、非致病的CPA1变异体正常分泌，不会造成显著的内质网应激。此外，为了深入了解错误折叠CPA1突变体在体内的影响，我们将研究一种新的在小鼠Cpa1基因中含有人类突变p.N256K的敲入小鼠系。我们预计，错误折叠的Cpa1突变体的胰腺表达将导致ER应激，细胞凋亡和/或NFκB激活增加，并可能引发急性和/或慢性胰腺炎。最后，我们将证明这种错误折叠的Cpa1突变体在小鼠胰腺中的表达使胰腺对损伤和随之而来的炎症反应敏感，并在急性和慢性胰腺炎的实验模型中增加胰腺炎反应。这些特定目标的成功完成将坚定地确立突变诱导的错误折叠是人类慢性胰腺炎的相关疾病机制，并将在体外和体内提供补充证据，证明内质网应激及其相关信号通路介导了胰腺炎风险的增加。

 DESCRIPTION (provided by applicant): The main objective of this grant is to determine the mechanisms by which carboxypeptidase A1 (CPA1) mutations act as risk factors for chronic pancreatitis in humans. The majority of non-alcoholic cases of chronic pancreatitis develop on the basis of genetic susceptibility, driven by mutations in risk genes that encode digestive enzymes such as cationic trypsinogen (protease serine 1, PRSS1), the pancreatic secretory trypsin inhibitor (serine protease inhibitor Kazal type 1, SPINK1), chymotrypsin C (CTRC) or carboxypeptidase A1 (CPA1). Our studies in previous funding periods clearly defined a pathological pathway associated with increased intra-pancreatic trypsin activity as a result of mutations in PRSS1, SPINK1 and CTRC. Our more recent results on CPA1 mutations, however, indicate that not all genetic risk factors increase trypsin activity. The overarching hypothesis of the current grant is that mutation-induced misfolding and consequent endoplasmic reticulum (ER) stress are the fundamental mechanisms increasing pancreatitis risk in carriers of CPA1 mutations. To test this hypothesis, we will systematically study 28 CPA1 variants and demonstrate that pathogenic CPA1 variants suffer retention and degradation inside the cell with consequently diminished secretion and markedly increased ER stress. In contrast, neutral, non-pathogenic CPA1 variants are secreted normally and do not cause significant ER stress. Furthermore, to obtain insight into the in vivo effects of misfolding CPA1 mutants, we will study a novel knock-in mouse line harboring the human mutation p.N256K in the mouse Cpa1 gene. We expect that pancreatic expression of a misfolding Cpa1 mutant will cause ER stress with increased apoptosis and/or NFκB activation, and may elicit acute and/or chronic pancreatitis. Finally, we will demonstrate that expression of this misfolding Cpa1 mutant in the mouse pancreas sensitizes the pancreas to injury and consequent inflammation and increases pancreatitis responses in experimental models of acute and chronic pancreatitis. Successful completion of these specific aims will firmly establish that mutation-induced misfolding is a relevant disease-mechanism in human chronic pancreatitis and will provide complementary in vitro and in vivo evidence that ER stress and associated signaling pathways mediate increased pancreatitis risk.