Ethanol Regulates Vacuolar ATPase & PEDF

乙醇调节液泡 ATP 酶

• 批准号: 8392101
• 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/8392101
• 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.

描述（由申请人提供）： 慢性胰腺炎（CP）是发病率和死亡率的主要原因。CP也是一个主要的风险因素 导致胰腺癌的发生在这两种疾病中，基质内的纤维化改变了功能。在 CP，纤维化导致疼痛，胰腺功能不全，并增加癌症风险。促结缔组织增生 胰腺癌的缓解增加了转移性疾病的可能性。在这两种情况下， 导致色素上皮衍生因子（PEDF）等蛋白质的丢失， 细胞增殖的内源性屏障。因此，靶向对基质重要的酶， 周转率可能是治疗的新靶点。 一个潜在的靶标是液泡-ATP酶（vacuolar-ATPase，v-ATPase）。v-ATP酶是一种高度调节的 质子泵，其功能是将质子移入细胞器并移出细胞外空间。它 在pH调节和酸化隔室方面起着重要的体内平衡功能 含有在低pH下最佳工作的酶。这发生在破骨细胞等细胞中， v-ATP酶的基因突变导致人类严重的骨骼异常和死亡， 小鼠模型。最近还报道了质膜上具有v-ATP酶的癌细胞， 比没有质膜v-ATP酶的癌细胞更具攻击性。这表明 质膜上的v-ATP酶有助于基质降解和细胞侵入。我们 初步研究发现，v-ATP酶存在于星状细胞的质膜上， 胰腺癌细胞，并且其活化调节基质金属蛋白酶（MMP）活性。 由于MMP降解PEDF，抑制v-ATP酶活性可以保持PEDF水平。 该提议检验了由v-ATP酶介导的质子通量导致 MMP激活和蛋白质周转，如维持细胞静止的PEDF。 此外，刺激物如乙醇和其他物质增加了v-ATP酶向血浆的转运 膜。我们的初步研究支持这一假设，表明： 1)小鼠中PEDF的缺乏导致PSC活化。 2)PEDF缺失小鼠显示多种促纤维化/炎性细胞因子的表达增强 和参与基质周转的蛋白酶。 3)蛙皮素诱导的胰腺炎导致体重增加受损和胶原沉积增加 在PEDF敲除小鼠中与WT动物相比。 4)乙醇攻击导致PEDF的损失，这是v-ATP酶依赖性的。 5)乙醇或瘦素刺激导致可溶性v-ATP酶（V1 E）从胞质溶胶易位到 星状细胞的质膜 6)乙醇/瘦素激发导致V1 E与特异性v-ATP酶同工型共定位。 7)胰腺癌细胞显示MMP酶原活化，其是v-ATP酶依赖性的。 8)shRNA介导v-ATP酶亚基V1 E基因敲低的胰腺癌细胞系 增加PEDF水平并显示MMP-2/9活性降低。 9)在人胰腺组织中，PanIN病变中的极化v-ATP酶分布改变为 扩散模式的癌细胞和相关的细胞侵袭潜力。 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 亚基使用分子方法来确定这是否会抑制胰腺星状细胞， 胰腺细胞的体内生长。