Ethanol Regulates Vacuolar ATPase & PEDF

乙醇调节液泡 ATP 酶

• 批准号: 8245395
• 负责人: CHUHAN CHUNG
• 金额: --
• 依托单位: VA CONNECTICUT HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8245395
• 关键词: Acids; Affect; Alcohol abuse; Animals; Architecture; Cancer cell line; Cancerous; Cell Proliferation; Cell membrane; Cell physiology; Cells; Cessation of life; Chronic; Cicatrix; Collagen; Cytosol; Data; Deposition; Desmoplastic; Development; Diffuse Pattern; Disease; Enzyme Activation; Enzyme Precursors; Enzymes; Ethanol; Exocrine pancreatic insufficiency; Extracellular Matrix; Extracellular Space; Fibroblasts; Fibrosis; Gelatinase A; Gene Mutation; Goals; Growth; Health; Hepatic; Human; In Vitro; Inflammatory; Injury; Knockout Mice; Leptin; Lesion; Malignant Neoplasms; Malignant neoplasm of pancreas; Matrix Metalloproteinases; Mediating; Methods; Modeling; Molecular; Morbidity - disease rate; Movement; Mus; Normal tissue morphology; Organelles; Osteoclasts; Pain; Pancreas; Pancreatic Injury; Pancreatic Intraepithelial Neoplasia; Pancreatitis; Pathogenesis; Peptide Hydrolases; Play; Population; Process; Protein Isoforms; Proteins; Proteolytic Processing; Proton Pump; Protons; Regulation; Reporting; Risk; Risk Factors; Rodent Model; Role; Site; Source; Stimulus; Testing; Tissues; Tumor Suppressor Proteins; Weight Gain; Work; alcohol effect; alcohol exposure; alcohol response; cancer cell; cancer risk; cell growth; cell motility; chronic pancreatitis; clinically relevant; collagenase 3; cytokine; enzyme activity; feeding; human tissue; in vivo; mortality; mouse model; novel; pancreatic cancer cells; pigment epithelium-derived factor; prevent; protein degradation; research study; response; skeletal abnormality; small hairpin RNA; stellate cell; tumor; vacuolar H+-ATPase

DESCRIPTION (provided by applicant): Chronic pancreatitis (CP) is a major cause of morbidity and mortality. CP is also a major risk factor for the development of pancreatic cancer. In both diseases, fibrosis within the matrix alters function. In CP, fibrosis results in pain, pancreatic insufficiency and increases cancer risk. The desmoplastic response in pancreatic cancer increases the likelihood of metastatic disease. In both, matrix turnover leads to the loss of proteins such as pigment epithelium-derived factor (PEDF) that act as endogenous barriers to cellular proliferation. Thus, targeting enzymes that are important for matrix turnover may represent a novel target for treatment. One potential target is the vacuolar-ATPase (v-ATPase). The v-ATPase is a highly regulated proton pump that functions to move protons into organelles and out into the extracellular space. It plays an important homeostatic function in terms of pH regulation and acidifies compartments containing enzymes that work optimally at low pH. This occurs in cells such as osteoclasts where genetic mutations in v-ATPase result in severe skeletal abnormalities and death in humans and in mouse models. Cancer cells with v-ATPase on plasma membranes were also recently reported to behave more aggressively than cancer cells without plasma membrane v-ATPase. This suggests that v-ATPase on plasma membranes contributes to matrix degradation and cellular invasion. Our preliminary work has found that v-ATPase is present on the plasma membranes of stellate cells and pancreatic cancer cells, and its activation modulates matrix metalloproteinase (MMP) activities. Because MMPs degrade PEDF, inhibiting v-ATPase activity can preserve PEDF levels. This proposal examines the hypothesis that proton flux mediated by the v-ATPase results in MMP activation and turnover of proteins such as PEDF that maintain cellular quiescence. Moreover, stimuli such as ethanol and others increase v-ATPase translocation to plasma membranes. Our preliminary studies support this hypothesis by showing that: 1) Absence of PEDF in mice results in PSC activation. 2) PEDF null mice display enhanced expression of multiple pro-fibrogenic/inflammatory cytokines and proteases involved in matrix turnover. 3) Cerulein-induced pancreatitis results in impaired weight gain and increased collagen deposition in PEDF null mice compared to WT animals. 4) Ethanol challenge results in the loss of PEDF that is v-ATPase-dependent. 5) Ethanol or leptin challenge results in translocation of soluble v-ATPase (V1E) from cytosol to plasma membranes in stellate cells. 6) Ethanol/leptin challenge results in V1E co-localization with specific v-ATPase isoforms. 7) Pancreatic cancer cells display MMP zymogen activation that is v-ATPase dependent. 8) Pancreatic cancer cell lines with shRNA-mediated knockdown of v-ATPase subunit V1E increases PEDF levels and demonstrate decreased MMP-2/9 activities. 9) In human pancreatic tissue, a polarized v-ATPase distribution in PanIN lesions changes to a diffuse pattern in cancerous cells and correlates with cellular invasive potential. To test the role of v-ATPase activation in pancreatic fibrosis, in vitro and in vivo experiments will be performed that inhibit v-ATPase function in multiple ways. We will selectively target v-ATPase subunits using molecular methods to determine whether this will inhibit pancreatic stellate cell and pancreatic cell growth in vivo. PUBLIC HEALTH RELEVANCE: Ethanol abuse is a major health problem affecting the VA population and the leading cause of chronic pancreatitis (CP), increased scar tissue within the pancreas. CP results in the loss of pancreatic function and increases the risk of pancreatic cancer. How pancreatic scar tissue develops is incompletely understood. We have identified the activation of an enzyme called vacuolar-ATPase (v-ATPase) in certain cells of the pancreas in response to ethanol. V-ATPase activation increases the activity of enzymes that digest the normal tissue architecture in diseases such as CP. These enzymes also digest pigment epithelium-derived factor (PEDF), a protein that keeps the pancreas in its normal condition. The goal of this proposal is to test whether blocking v-ATPase activity prevents PEDF loss and inhibits pancreatic scar formation.

描述（由申请人提供）： 慢性胰腺炎（CP）是发病和死亡的主要原因。 CP也是一个主要危险因素 对于胰腺癌的发展。在这两种疾病中，基质内的纤维化都会改变功能。在 CP、纤维化会导致疼痛、胰腺功能不全并增加癌症风险。促纤维化剂 胰腺癌的反应增加了转移性疾病的可能性。在这两种情况下，矩阵周转 导致蛋白质的损失，例如色素上皮衍生因子（PEDF），其作用 细胞增殖的内源性障碍。因此，针对基质重要的酶 营业额可能代表一个新的治疗目标。 一个潜在的靶点是液泡-ATP 酶（v-ATP 酶）。 v-ATP酶是一种高度调控的 质子泵，其功能是将质子移入细胞器并移至细胞外空间。它 在 pH 调节和酸化隔室方面发挥重要的稳态功能 含有在低 pH 值下发挥最佳作用的酶。这发生在破骨细胞等细胞中 v-ATP酶的基因突变会导致人类和其他国家的严重骨骼异常和死亡 鼠标模型。最近还报道，质膜上带有 v-ATP 酶的癌细胞 比没有质膜 v-ATP 酶的癌细胞表现得更具攻击性。这表明 质膜上的 v-ATP 酶有助于基质降解和细胞侵袭。我们的 初步工作发现，v-ATP酶存在于星状细胞的质膜上，并且 胰腺癌细胞，其激活可调节基质金属蛋白酶 (MMP) 活性。 由于 MMP 会降解 PEDF，因此抑制 v-ATP 酶活性可以保持 PEDF 水平。 该提案检验了由 v-ATP 酶介导的质子流导致的假设 MMP 激活和维持细胞静止的 PEDF 等蛋白质的周转。 此外，乙醇等刺激物会增加 v-ATP 酶向血浆的易位 膜。我们的初步研究表明： 1) 小鼠中缺乏 PEDF 会导致 PSC 激活。 2) PEDF缺失小鼠表现出多种促纤维化/炎症细胞因子的表达增强 和参与基质周转的蛋白酶。 3) 雨蛙蛋白诱发的胰腺炎导致体重增加受损和胶原蛋白沉积增加 PEDF 缺失小鼠与 WT 动物相比。 4) 乙醇攻击导致 v-ATPase 依赖性 PEDF 损失。 5) 乙醇或瘦素攻击导致可溶性 v-ATP 酶 (V1E) 从细胞质转移到 星状细胞中的质膜。 6) 乙醇/瘦素攻击导致 V1E 与特定 v-ATP 酶亚型共定位。 7) 胰腺癌细胞表现出依赖 v-ATP 酶的 MMP 酶原激活。 8) shRNA介导的v-ATP酶亚基V1E敲低的胰腺癌细胞系 增加 PEDF 水平并降低 MMP-2/9 活性。 9) 在人胰腺组织中，PanIN 病变中的极化 v-ATPase 分布变为 癌细胞中的扩散模式并与细胞侵袭潜力相关。 为了测试 v-ATP 酶激活在胰腺纤维化中的作用，体外和体内实验将 以多种方式抑制v-ATP酶功能。我们将选择性地靶向 v-ATPase 使用分子方法确定亚基是否会抑制胰腺星状细胞和 胰腺细胞在体内的生长。 公共卫生相关性： 乙醇滥用是影响退伍军人事务部人口的一个主要健康问题，也是造成这一问题的主要原因 慢性胰腺炎（CP），胰腺内疤痕组织增加。 CP导致损失 胰腺功能并增加患胰腺癌的风险。胰腺疤痕组织如何形成 的发展还不完全了解。我们已经确定了一种酶的激活作用 胰腺某些细胞中的液泡 ATP 酶（v-ATP 酶）对乙醇有反应。 V-ATP酶 激活增加了消化正常组织结构的酶的活性 CP等疾病。这些酶还消化色素上皮衍生因子 (PEDF)，这是一种 使胰腺保持正常状态的蛋白质。该提案的目标是测试 阻断 v-ATP 酶活性是否可以防止 PEDF 损失并抑制胰腺疤痕形成。