Basement Membranes and Associated Protein Factors In Development and Disease

基底膜是薄的细胞外基质，分隔上皮细胞和间充质细胞，并包围其他细胞，如内皮细胞、肌肉细胞和神经细胞。基底膜是发育过程中首先出现的细胞外基质，对器官发育和组织修复至关重要。它们为细胞和细胞层提供支架，在形态发生过程中也在细胞黏附、迁移、增殖和分化过程中发挥重要作用。我们最近的研究集中在识别基底膜和相关蛋白的特定功能，以描绘它们的结构/功能关系，阐明它们的调控机制，并描述发生在发育和疾病过程中的相关蛋白质相互作用。 腓骨蛋白由一系列分泌的糖蛋白组成，与基底膜、弹性纤维和其他基质有关。纤维蛋白介导细胞与细胞和细胞与基质之间的通讯，并在器官发生、血管发生和组织内稳态过程中提供细胞外基质(ECM)的稳定。纤维蛋白还参与调节细胞的形态和生长，它们既可以作为肿瘤抑制因子，也可以作为致癌因子。我们先前从牙胚cDNA文库中鉴定出纤毛蛋白-7(Fbln7/TM14)，它是纤毛蛋白家族的最新成员。Fbln7由成牙本质细胞表达，并与牙间充质细胞和成牙本质细胞结合。它还与ECM蛋白相互作用。 细胞外基质(ECM)通过促进或抑制血管生成过程在血管生成中发挥重要作用。我们发现，纤维蛋白-7(Fbln7)在软骨等非血管组织中表达。我们发现，重组Fbln7通过阻断人脐静脉内皮细胞(HUVECs)的管状形成来抑制血管生成。在主动脉环实验中，C端的Fbln7片段(Fbln7-d3)对HUVEC管形成和内皮萌发的抑制作用最强。Fbln7-d3通过整合素与人脐静脉内皮细胞结合，但不促进细胞铺展或迁移，并抑制肌动蛋白应激纤维的形成。Fbln7-d3诱导整合素与其他焦点黏附分子在细胞黏附部位聚集，并持续激活FAK、p130Cas和rac1。我们的发现表明，Fbln7-d3是一种新的抗血管生成因子，可能通过抑制内皮细胞的扩散和迁移来防止血管侵入无血管组织。 免疫细胞通过整合素与基质蛋白及其生物活性片段相互作用，影响其活化和分化。由于Fbln7也在眼和胎盘等免疫耐受组织中表达，我们推测Fbln7或其片段可能作为免疫调节剂发挥作用。我们利用人单核细胞检测了全长(Fbln7-FL)及其C末端片段Fbln7-d3对免疫细胞功能的影响。我们发现单核细胞与Fbln7-FL和Fbln7-d3都有相互作用。我们发现Fbln7-d3抑制细胞扩散和应激纤维的形成，减少炎性细胞因子IL-6和MMP-1/9的产生，而Fbln7-FL减少IL-6的产生，增加IL-10的产生。在体外，Fbln7-d3降低了肿瘤坏死因子激活的单核细胞中ERK1/2的磷酸化。Fbln7-FL和纤连蛋白对活化单核细胞的比较蛋白质组学分析证实，与纤维连接蛋白相比，Fbln7和纤连蛋白对单核细胞功能有抑制作用。我们的结果表明，Fbln7-FL和Fbln7-d3都是炎症的负调控因子。我们正在准备这些结果以供发表。 我们之前发现了一种小鼠突变，命名为FURUE(FURUE在日语中的意思是震颤)，通过插入perlecan转基因导致后肢严重震颤和中枢神经系统髓鞘减退。在皮肤病小鼠的脊髓中，少突胶质细胞的分化受到抑制，少突胶质细胞是中枢神经系统中的髓鞘形成细胞。我们随后确定了Teneurin-4(Ten-4)基因的转基因插入位点，该基因编码一种在中枢神经系统中高表达的跨膜蛋白。我们发现，Ten-4在正常的少突胶质细胞分化过程中被诱导，但在FURUE小鼠中没有表达。在使用少突胶质前体细胞系CG-4的细胞培养中，抑制Ten-4的表达会抑制细胞的分化和突起的形成。Ten-4与磷酸化的FAK和帕西林共定位于细胞突起，尤其是在分化CG-4细胞的突起顶端。免疫沉淀分析表明，Ten-4与FAK、PXLIN、PI3K和CDK5等多种因子形成了一个分子复合体。此外，激活FAK还需要Ten-4。这些发现表明，这对少突胶质细胞的分化至关重要。与东京医科大学的铃木博士和赤泽博士合作，我们证明了Ten-4的表达是在神经母细胞瘤细胞系Neuro-2a的轴突生长过程中诱导的。Ten-4蛋白定位于神经突起生长锥体。Ten-4的过度表达增加了神经突起的长度，而Ten-4的表达下调则减少了丝状突起的形成和神经突起的长度。我们的发现表明，Ten-4对细胞突起的形成和突起的生长具有积极的调节作用。

Basement membranes are thin extracellular matrices that separate epithelial and mesenchymal cells, and surround others such as endothelial, muscular, and neural cells. Basement membranes are the first extracellular matrices to appear during development and are critical for organ development and tissue repair. They provide the scaffold for cells and cell layers and also play essential roles in cell adhesion, migration, proliferation, and differentiation during morphogenesis. Our recent studies have been focused on identifying the specific functions of basement membranes and associated proteins in order to delineate their structure/function relationships, to elucidate their regulatory mechanisms, and to describe the related protein interactions that occur during development and disease. The fibulins comprise a family of secreted glycoproteins associated with basement membranes, elastic fibers, and other matrices. Fibulins mediate cell-to-cell and cell-to-matrix communication and provide stabilization of the extracellular matrix (ECM) during organogenesis, vasculogenesis, and tissue homeostasis. Fibulins are also implicated in the modulation of cell morphology, growth, and they can act as both tumor-suppressors and oncogenic factors. We previously identified fibulin-7 (Fbln7/TM14), the newest member of the fibulin family, from a tooth germ cDNA library. Fbln7 is expressed by odontoblasts and binds to dental mesenchyme cells and odontoblasts. It also interacts with ECM proteins. The extracellular matrix (ECM) plays an important role in angiogenesis through either promotion or suppression of angiogenic processes. We showed that fibulin-7 (Fbln7) is expressed in avascular tissues, such as cartilage. We found that recombinant Fbln7 inhibited angiogenesis by blocking tube formation in human umbilical vein endothelial cells (HUVECs). A C-terminal Fbln7 fragment (Fbln7-d3) had the strongest inhibitory effect on HUVEC tube formation and on endothelial sprouting in aortic ring assays. Fbln7-d3 bound to HUVECs through α5β1 integrin but did not promote cell spreading or migration, and actin stress fiber formation was inhibited. Fbln7-d3 induced α5β1 integrin clustering at cell adhesion sites with other focal adhesion molecules and sustained activation of FAK, p130Cas, and Rac1. Our findings suggest that Fbln7-d3 is a novel anti-angiogenic factor that may prevent vascular invasion into avascular tissues by inhibiting endothelial cell spreading and migration. Interactions of immune cells with matrix proteins and their bioactive fragments via integrins influence their activation and differentiation. Because Fbln7 is also expressed in some immunotolerant tissues such as eye and placenta, we hypothesized that Fbln7 or its fragment may function as an immunomodulator. We have examined the effect of the full-length (Fbln7-FL) and its C-terminal fragment Fbln7-d3 on immune cell functions using human monocytes. We found that monocytes interact with both Fbln7-FL and Fbln7-d3. We showed that Fbln7-d3 inhibits cell spreading and stress fiber formation and reduced the production of inflammatory cytokine IL-6 and MMP-1/9, while Fbln7-FL reduced production of IL-6 and increased the production of IL-10. Fbln7-d3 reduced phosphorylation of Erk1/2 in TNFα-activated monocytes in vitro. Comparative proteomics analysis of activated monocytes cultured on Fbln7-FL and fibulistatin confirmed the inhibitory effect of Fbln7 and fibulistatin on monocyte functions as compared to fibronectin. Our results suggest that both Fbln7-FL and Fbln7-d3 are negative regulators of inflammation. We are preparing these results for publication. We previously identified a mouse mutation, designated furue (furue means tremors in Japanese), by perlecan transgene insertion that causes severe tremors in the hindlimbs and CNS hypomyelination. In the spinal cord of the furue mice, differentiation of oligodendrocytes, the myelin-forming cells in the CNS, is inhibited. We subsequently identified the transgene insertion site into the teneurin-4 (Ten-4) gene encoding a transmembrane protein, which is highly expressed in the central nervous system. We found that Ten-4 is induced during normal oligodendrocyte differentiation, but that in the furue mice its expression is absent. In cell culture using the oligodendrocyte progenitor cell line CG-4, suppression of Ten-4 expression inhibits cell differentiation and process formation. Ten-4 colocalized with phosphorylated FAK and paxillin in cell processes, especially at the tips of the processes in differentiating CG-4 cells. Immunoprecipitation analysis revealed that Ten-4 forms a molecular complex with multiple factors including FAK, paxillin, PI3 kinase, and Cdk5. In addition, Ten-4 is required for the activation of FAK. These findings suggest that is critical for the differentiation of oligodendrocytes. In collaboration with Dr. Suzuki and Dr. Akazawa at Tokyo Medical and Dental University, we showed that Ten-4 expression is induced during neurite outgrowth of the neuroblastoma cell line Neuro-2a. Ten-4 protein is localized at the neurite growth cones. Overexpression of Ten-4 increases neurite length, whereas knockdown of Ten-4 expression decreases the formation of filopodia-like protrusions and the length of neurites. Our findings suggest that Ten-4 is a positive regulator of cellular protrusion formation and neurite outgrowth.