Calcineurin in Pathologic Zymogen Activation

描述(由申请人提供)： 急性胰腺炎是一种常见的威胁生命的胰腺疾病。虽然有几种已知的病因，但导致这种疾病的机制尚不清楚。然而，我们确实知道，无论病因如何，早期的步骤是胰腺腺泡细胞内消化酶原(酵素)的过早激活。我们以前的研究表明，这种激活需要细胞内钙离子明显的病理性升高。然而，钙离子升高的下游效应尚不清楚。钙离子的一个重要靶点是依赖于钙离子/钙调蛋白的丝氨酸/苏氨酸磷酸酶PP2B，或钙调神经磷酸酶。它与胰腺炎有关，也在胰腺生理学中发挥作用。这一设想的假设是，由于腺泡细胞钙离子升高而引起的钙调神经磷酸酶激活，介导了病理性胰腺酶原的激活。在这项建议中，我们将结合药理学和遗传学策略，(1)鉴定在胰腺腺泡细胞中表达的钙调神经磷酸酶亚型，(2)研究钙调神经磷酸酶在胰腺腺泡细胞过早激活胰酶原中的作用，以及(3)研究体内钙调神经磷酸酶在胰腺炎长期影响中的作用。我们的主要方法将是使用钙调神经磷酸酶抑制剂、CN异构体特异性基因敲除小鼠和siRNA基因敲除。初步结果表明：(1)钙调神经磷酸酶A-α分布于胰腺腺泡细胞的胞浆中；(2)钙调神经磷酸酶抑制可减少酶原的过早激活，但不影响腺泡细胞的酶分泌。预计这些研究将对导致胰腺炎的因素提供新的见解，并可能建议针对胰腺钙调神经磷酸酶的预防性治疗策略。

DESCRIPTION (provided by applicant): Acute pancreatitis is a common life-threatening disorder of the pancreas. Although there are several known etiologies, the mechanisms leading to the disease are unclear. However, we do know that irrespective of etiology an early step is the premature activation of digestive proenzymes (zymogens) within the pancreatic acinar cell. Our previous studies demonstrate that this activation requires a distinct pathologic rise in cytosolic Ca2+. However, the downstream effectors of the Ca2+ rise are not known. An important target of Ca2+ is the Ca2+/calmodulin-dependent serine/threonine phosphatase PP2B, or calcineurin. It has been associated with pancreatitis and also plays a role in pancreatic physiology. The hypothesis of this proposal is that calcineurin activation due to a rise in acinar cell Ca2+ mediates pathologic pancreatic zymogen activation. In this proposal, using a combination of pharmacologic and genetic strategies, we will (1) characterize the calcineurin isoforms expressed in the pancreatic acinar cell, (2) study the role of calcineurin in premature pancreatic zymogen activation in isolated pancreatic acinar cells, and (3) study the role of calcineurin in vivo on long term effects of pancreatitis. Our primary methods will be to use calcineurin inhibitors, CN isoform-specific knockout mice, and siRNA knockdown. In preliminary results, we have shown that (1) calcineurin A-alpha is distributed in the cytoplasm of pancreatic acinar cells and (2) calcineurin inhibition reduces premature zymogen activation without affecting acinar cell enzyme secretion. It is anticipated that these studies will provide new insight into the factors causing pancreatitis and may also suggest prophylactic treatment strategies that target calcineurin in the pancreas.