Molecular Mechanisms Of Cell Adhesion And Invasion

细胞粘附和侵袭的分子机制

• 批准号: 8148988
• 负责人: Steven Akiyama
• 金额: $ 185.17万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8148988
• 关键词: Cell Adhesion; Molecular

Cell adhesion and migration contribute to normal processes such as differentiation, embryonic development, and wound healing as well as to the progression of diseases and pathological conditions that can result from either acute or chronic exposure to environmental toxicants, such as cancer and inflammatory responses. Key mechanistic steps in these processes involve the interactions of extracellular glycoproteins--such as fibronectin, laminin, and collagens--with specific adhesive receptors, the best characterized of which are the integrins, a family of heterodimeric complexes consisting of an alpha subunit and a beta subunit. Integrins are highly regulated receptors that can exist in either an active or inactive state. Selectins are vascular cell-cell adhesion molecules involved in leukocyte trafficking, inflammation, thrombosis, autoimmunity and cancer. Accumulation of leukocytes at sites of inflammation is initiated by selectins that mediate the capturing and rolling of leukocytes on endothelium. Three major members of the selectin family have been identified: L-selectin, E-selectin and P-selectin. L-Selectin is constitutively expressed on leukocytes. P-and E-selectins are expressed on activated endothelial cells in response to microenvirnomental stimuli. P-selectin is also expressed on thrombin-activated platelets. All three members of the selectin family, E-, L-, and P-selectin can bind to human tumor cells and cancer-derived cell line. Our research has focused recently on the possible role of inflammatory cues in activation of integrin-mediated tumor cell migration and invasion. As a model system, we are examining the ability of one selectin, P-selectin, to trigger integrin-mediated adhesion and migration of cultured human tumor cells. We focus on two closely related aspects of this project: to characterize the mechanisms of P-selectin-induced activation of integrin-mediated cell adhesion and cell migration. We have previously shown that binding of soluble, recombinant P-selectin-IgG Fc chimeric protein to Colo-320 cells stimulates cell adhesion to fibronectin through the specific activation of the alpha5-beta1 integrin by means of the p38 MAP kinase and PI-3 kinase (PI3-k) signaling pathways. We have now identified nucleolin as a novel cell surface P-selectin receptor on Colo-320 cells using affinity chromatography and a proteomic approach. This finding was validated by showing that an anti-nucleolin mAb D3 inhibits P-selectin interactions with living Colo-320 cells and that nucleolin becomes clustered at the external side of the plasma membrane of living, intact cells when bound to cross-linked P-selectin-IgG-Fc chimeric protein. We have also found that P-selectin binding to Colo-320 cells induces tyrosine phosphorylation specifically of cell-surface nucleolin and formation of a signaling complex containing cell surface nucleolin, PI 3-K, and p38 MAPK. Using siRNA approaches, we showed that both P-selectin binding to Colo-320 cells and formation of the P-selectin-mediated p38 MAPK/ PI 3-K signaling complex require nucleolin expression. Thus, we have characterized nucleolin (or a nucleolin-like protein) as a novel, signaling cell-surface receptor for P-selectin on Colo-320 cells and suggest a mechanism for linkage of nucleolin to P-selectin-induced signal transduction pathways that regulate the adhesion of Colo-320 on fibronectin substrates. We have recently been characterizing the stimulation of integrin-mediated tumor cell migration by the binding of soluble, recombinant P-selectin-IgG Fc chimeric protein. We have found that P-selectin stimulates the secretion of matrix metalloproteinase-9 (MMP-9) by A375 human melanoma cells and that direct addition of exogenous MMP-9 to A-375 cells can stimulate the migratory phenotype. The current paradigm in the field states that MMP-9 stimulates migration by digesting basement membrane proteins, allowing tumor cells to clear a migratory pathway. However, we have preliminary data that show that stimulation of cell migration by MMP-9 can occur even in the absence of catalytic activity.

细胞黏附和迁移有助于正常的过程，如分化、胚胎发育和伤口愈合，以及疾病和病理条件的进展，这些疾病和病理条件可能是由于急性或长期暴露于环境毒物，如癌症和炎症反应造成的。这些过程中的关键机制步骤涉及细胞外糖蛋白--如纤维连接蛋白、层粘连蛋白和胶原蛋白--与特定的黏附受体的相互作用，其中最典型的是整合素，这是一个由α亚基和β亚基组成的异二聚体复合体家族。整合素是高度受调控的受体，可以存在于活性或非活性状态。 选择素是一种血管细胞-细胞黏附分子，参与白细胞运输、炎症、血栓形成、自身免疫和癌症。白细胞在炎症部位的聚集是由选择素启动的，它介导了白细胞在内皮细胞上的捕获和滚动。目前已发现选择素家族的三个主要成员：L-选择素、E-选择素和P-选择素。L-选择素在白细胞上有结构性表达。P-选择素和E-选择素在激活的内皮细胞上表达，以响应微环境刺激。P-选择素也表达在凝血酶激活的血小板上。选择素家族的三个成员，E-、L-和P-选择素都能与人类肿瘤细胞和肿瘤衍生细胞系结合。 我们的研究最近集中在炎症信号在整合素介导的肿瘤细胞迁移和侵袭激活中的可能作用。作为一个模型系统，我们正在检测一种选择素，P-选择素，触发整合素介导的黏附和迁移培养的人类肿瘤细胞的能力。本研究主要集中在两个密切相关的方面：研究P-选择素激活整合素介导的细胞黏附和细胞迁移的机制。 我们先前已经证明，可溶性的重组P-选择素-Ig G Fc嵌合蛋白与Colo-320细胞的结合通过p38 MAP和PI-3激酶(PI3-k)信号通路特异性地激活α5-β1整合素来刺激细胞与纤维连接蛋白的黏附。我们现在已经用亲和层析和蛋白质组学的方法鉴定了核仁素是一种新的细胞表面P-选择素受体。这一发现得到了证实，表明抗核仁单抗D3抑制P-选择素与活的COLO-320细胞的相互作用，并且当与交联的P-选择素-免疫球蛋白-Fc嵌合蛋白结合时，核仁会聚集在活的完整细胞的质膜外侧。我们还发现，P-选择素与COLO-320细胞结合可诱导细胞表面核仁特异性酪氨酸磷酸化，并形成包含细胞表面核仁蛋白、PI3-K和p38MAPK的信号复合体。使用siRNA方法，我们发现P-选择素与COLO-320细胞的结合和P-选择素介导的p38MAPK/PI 3-K信号复合体的形成都需要核仁素的表达。因此，我们将核仁(或一种核仁样蛋白)描述为一种新型的细胞表面P-选择素受体，并提出了一种将核仁链接到P-选择素诱导的信号转导通路的机制，该信号转导通路调节了COLO-320在纤维连接蛋白底物上的黏附。 最近，我们一直在研究通过结合可溶性重组P-选择素-Ig G Fc嵌合蛋白来刺激整合素介导的肿瘤细胞迁移。我们发现P-选择素能刺激A375人黑色素瘤细胞分泌基质金属蛋白酶-9(MMP9)，并且将外源性MMP9直接加入A375细胞可以刺激其迁移表型。目前该领域的范例表明，基质金属蛋白酶-9通过消化基底膜蛋白刺激迁移，使肿瘤细胞清除迁移途径。然而，我们有初步数据表明，即使在缺乏催化活性的情况下，基质金属蛋白酶-9也可以刺激细胞迁移。