Molecular Mechanisms Of Cell Adhesion And Invasion

细胞粘附和侵袭的分子机制

• 批准号: 8553682
• 负责人: Steven Akiyama
• 金额: $ 156.54万
• 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/8553682
• 关键词: 1-Phosphatidylinositol 3-Kinase; Acute; Acute Disease; Adhesions; Adhesives; Affinity Chromatography; Animals; Autoimmunity; Automobile Driving; Basement membrane; Binding; Biological Models; Blood; Blood Platelets; Bone remodeling; CD29 Antigen; Cell Adhesion; Cell Line; Cell Proliferation; Cell surface; Cell to Cell Adhesion Signaling Pathway; Cell-Cell Adhesion; Cell-Matrix Junction; Cells; Chimeric Proteins; Chronic; Chronic Disease; Collaborations; Collagen; Collagen Type IV; Complex; Cues; Cyclin D1; Cytoskeleton; Data; Development; Disease; Disease Progression; E-Cadherin; E-Selectin; Embryo; Embryonic Development; Endogenous Factors; Endothelial Cells; Endothelium; Environmental Risk Factor; Event; Exposure to; Extracellular Matrix; Family; Fibronectins; Gelatinase B; Glycoproteins; Goals; Growth; Human; Immunoglobulin G; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Injection of therapeutic agent; Integrin-mediated Cell Adhesion Pathway; Integrins; Intervention; JUN gene; L-Selectin; Laminin; Lead; Leukocyte Rolling; Leukocyte Trafficking; Leukocytes; Lung; MAP Kinase Gene; MAPK14 gene; MAPK8 gene; Malignant Neoplasms; Mediating; Melanoma Cell; Membrane Glycoproteins; Membrane Proteins; Messenger RNA; Migration Assay; Molecular; Morphogenesis; N-Cadherin; Neoplasm Metastasis; Nodule; Nuclear; P-Selectin; Pathway interactions; Play; Post-Translational Protein Processing; Prevention strategy; Process; Proteomics; Recombinants; Regulation; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Selectins; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Site; Stimulus; System; Tail; Therapeutic; Thrombosis; Tissues; Toxic Environmental Substances; Transcription Factor AP-1; Tumor Necrosis Factor-alpha; Tyrosine Phosphorylation; Universities; Veins; Wound Healing; adhesion receptor; cell motility; design; exposed human population; extracellular; human TNF protein; human disease; implantation; in vivo; loss of function; malignant phenotype; melanoma; member; migration; mitogen-activated protein kinase p38; mutant; neoplastic cell; novel; nucleolin; prevent; receptor; response; restoration; subcutaneous; treatment strategy; tumor; tumor growth

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 transemembrane cell-cell adhesion glycoproteins 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 expressed on activated platelets as well as endothelial cells as a result of a secretory cascade initiated in response to environmental factors that result in inflammation. In some diseases, including certain cancers, elevated levels of P-selectin can be found in the blood. All three members of the selectin family, E-, L-, and P-selectin can bind to human tumor cells and cancer-derived cell lines. Our research has focused recently on the possible role of cues from the tumor microenvironment that can regulate 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 identified nucleolin as a novel cell surface P-selectin receptor on Colo-320 cells using affinity chromatography and a proteomic approach and 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. 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 binds to cultured human A375 melanoma cells in a concentration-dependent and saturable manner via cell surface nucleolin resulting in concommitant stimulation of matrix metalloproteinase-9 (MMP-9) secretion. We are currently investigating the mechanism by which P-selectin binding to A375 melanoma cells stimulates MMP-9 secretion. Our preliminary data indicate that P-selectin stimulates a 3-fold increase of MMP-9 mRNA, as judged by RT-PCR. Activation of p38 MAP kinase may be involved in this process. 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 using two indepedent in vitro migration assays can occur even in the absence of catalytic activity. In collaboration with investigators at Duke University, we have shown that exogenous expression of the tumor supressor CYLD markedly inhibits melanoma cell proliferation and migration in vitro and subcutaneous tumor growth in vivo. In addition, the melanoma cells expressing exogenous CYLD were unable to form pulmonary tumor nodules following tail-vein injection. At the molecular level, CYLD decreased beta-1 integrin expression and inhibited pJNK induction by TNF-alpha or cell-attachment to collagen IV. Moreover, CYLD induced an array of other molecular changes associated with modulation of the malignant phenotype, including a decreased expression of cyclin D1, N-cadherin and nuclear Bcl3, and an increased expression of p53 and E-cadherin. Most interestingly, co-expression of the constitutively active MKK7 or c-Jun mutants with CYLD prevented the above molecular changes, and fully restored melanoma growth and metastatic potential in vivo. Our findings demonstrate that JNK/AP-1 signaling pathway underlies the melanoma growth and metastasis that is associated with CYLD loss-of-function. Thus, restoration of CYLD and inhibition of JNK and beta-1 integrin function represent potential therapeutic strategies for treatment of malignant melanoma.

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。 选择素是跨膜细胞-细胞黏附糖蛋白，参与白细胞转运、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-选择素在活化的血小板和内皮细胞上表达，是对导致炎症的环境因素所启动的分泌级联反应的结果。在一些疾病中，包括某些癌症，可以在血液中发现P-选择素水平升高。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。 我们最近的研究集中在肿瘤微环境中的信号对整合素介导的肿瘤细胞迁移和侵袭的可能作用。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。我们用亲和层析和蛋白质组学方法鉴定了核仁素是一种新的细胞表面P-选择素受体，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素、PI3-K和p38MAPK的信号复合体。 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-选择素通过细胞表面核素以浓度依赖且饱和的方式与培养的人A375黑色素瘤细胞结合，导致基质金属蛋白酶-9(MMP9)的分泌。我们目前正在研究P-选择素与A375黑色素瘤细胞结合刺激基质金属蛋白酶-9分泌的机制。我们的初步数据表明，根据RT-PCR的判断，P-选择素刺激了3倍于基质金属蛋白酶-9mRNA的表达。P38MAP的激活可能参与了这一过程。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，使用两种独立的体外迁移试验，即使在缺乏催化活性的情况下，也可以使用基质金属蛋白酶-9刺激细胞迁移。 与杜克大学的研究人员合作，我们发现外源表达肿瘤抑制因子CyLD在体外显著抑制黑色素瘤细胞的增殖和迁移，并在体内抑制皮下肿瘤生长。此外，尾静脉注射后，表达外源性CyLD的黑色素瘤细胞不能形成肺肿瘤结节。在分子水平上，CyLD降低了β-1整合素的表达，抑制了肿瘤坏死因子-α或细胞对IV型胶原的黏附诱导的pJNK。此外，CyLD还诱导了一系列与调节恶性表型相关的分子变化，包括细胞周期蛋白D1、N-钙粘蛋白和核Bcl3的表达降低，p53和E-钙粘素的表达增加。最有趣的是，MKK7或c-jun突变体与CyLD共表达阻止了上述分子变化，并在体内完全恢复了黑色素瘤的生长和转移潜能。我们的发现表明，JNK/AP-1信号通路是黑色素瘤生长和转移的基础，而黑色素瘤的生长和转移与CyLD功能丧失有关。因此，修复CyLD和抑制JNK和β-1整合素功能是治疗恶性黑色素瘤的潜在治疗策略。