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，并在细胞的粘附、迁移、增殖和形态发生过程中发挥重要作用。我们最近的研究主要集中在鉴定基底膜和相关蛋白的特定功能，以描述它们的结构/功能关系，阐明它们的调节机制，并描述在发育和疾病过程中发生的相关蛋白质相互作用。

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.