Ethanol Regulates Vacuolar ATPase & PEDF

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 以促进胰腺癌的发展。在这两种疾病中，基质内的纤维化改变了功能。在……里面 慢性胰腺炎，纤维化会导致疼痛、胰腺功能不全并增加癌症风险。结缔组织增殖物 胰腺癌的反应增加了转移性疾病的可能性。在这两家公司中，矩阵更替 导致色素上皮衍生因子(PEDF)等起作用的蛋白质的丢失 细胞增殖的内源性障碍。因此，靶向对基质很重要的酶 周转可能是治疗的新靶点。 一个潜在的靶点是空泡ATPase(v-ATPase)。V-ATPase是一种高度受调控的 质子泵，其功能是将质子移入细胞器并移出到细胞外空间。它 在调节pH和酸化隔间方面起着重要的动态平衡作用 含有能在低pH值下发挥最佳作用的酶。这发生在破骨细胞等细胞中，其中 V-ATPase基因突变会导致严重的骨骼异常和死亡 老鼠模型。质膜上有v-ATPase的癌细胞最近也被报道 比没有质膜v-ATPase的癌细胞表现得更具侵略性。这表明 质膜上的V-ATPase有助于基质降解和细胞侵袭。我们的 初步研究发现，v-ATPase存在于星状细胞和 胰腺癌细胞，其激活调节基质金属蛋白酶(MMPs)的活性。 由于MMPs能降解PEDF，因此抑制v-ATPase活性可以维持PEDF水平。 这一建议检验了v-ATPase介导的质子通量导致 基质金属蛋白酶的激活和蛋白质的周转，如维持细胞静止的PEDF。 此外，乙醇等刺激物会增加v-ATPase向血浆的转位 膜。我们的初步研究支持这一假设，因为它表明： 1)小鼠体内缺乏PEDF可导致PSC的激活。 2)PEDF基因缺失小鼠多种促纤维化/炎性细胞因子表达增强 和参与基质周转的蛋白酶。 3)蓝豆素诱导的胰腺炎导致体重增加和胶原沉积增加。 在PEDF基因缺失的小鼠中与WT动物进行比较。 4)乙醇刺激导致依赖于v-ATPase的PEDF的丢失。 5)乙醇或瘦素刺激导致可溶性v-ATPase(V1E)从胞浆移位到 星状细胞的质膜。 6)乙醇/瘦素刺激导致V1E与特定的v-ATPase亚型共定位。 7)胰腺癌细胞表现出v-ATPase依赖性的基质金属蛋白酶原激活。 8)shRNA介导的v-ATPase亚单位V1E基因敲除的胰腺癌细胞株 升高PEDF水平，降低基质金属蛋白酶-2/9活性。 9)在人胰腺组织中，v-ATPase在Panin病变中的极化分布转变为 癌细胞中的扩散模式，并与细胞侵袭潜能相关。 为了测试v-ATPase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-ATPase功能。我们将选择性地以v-ATPase为目标 使用分子方法确定这是否会抑制胰腺星状细胞和 胰腺细胞在体内的生长。

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.