Basement Membranes and Associated Protein Factors In Development and Disease

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Basement membranes (BMs) are thin extracellular matrices (ECMs) that separate epithelial and mesenchymal cells and surround other cells, such as endothelial, muscular, and neural cells. BMs consist of a unique set of proteins, such as laminins, perlecan, and collagen IV. They are also associated with other ECM proteins, such as fibulin and fibronectin, in various tissues. BMs are the first ECMs to appear during development and are critical for organ development and tissue repair. They provide the scaffold for cells and cell layers and play essential roles in cell adhesion, migration, proliferation, and differentiation during morphogenesis. We have studied the specific functions of basement membranes and their associated proteins to delineate their structural and functional relationships, to elucidate their regulatory mechanisms, and to describe related protein interactions that occur during development and disease. (A) A C-terminal fibulin-7 fragment (Fbln7-C) shows anti-angiogenesis activity Fibulins comprise a family of seven secreted glycoproteins that are associated with basement membranes, elastic fibers, and other matrices. Fibulins mediate cell-to-cell and cell-to-matrix communication, as well as stabilize the ECM during organogenesis and vasculogenesis. Fibulins have been implicated in the modulation of cell morphology, growth, adhesion, and motility. Fibulins also act as both tumor-suppressors and oncogenic factors. We previously identified fibulin-7 (Fbln7)/TM14, the newest member of the fibulin family, as being expressed by odontoblasts as a cell adhesion molecule for dental mesenchyme cells and odontoblasts. We also found that Fbln7 interacts with ECM proteins and growth factors. ECM proteins and their fragments play an important role in angiogenesis through either the promotion or suppression of angiogenic and inflammatory processes. We showed that Fbln7 was expressed in avascular and immunotolerant tissues, such as cartilage, the eye, and the placenta. We found that recombinant Fbln7 proteins inhibited angiogenesis by blocking tube formation in human umbilical vein endothelial cells (HUVECs). A C-terminal Fbln7 fragment (Fbln7-C) had the strongest inhibitory effect on HUVEC tube formation and on vessel sprouting in aortic ring assays in vitro. Fbln7-C bound to HUVECs through alpha5beta1 integrin, but it did not promote cell spreading or migration. We showed that Fbln7-C inhibited actin stress fiber formation and induced alpha5beta1 integrin clustering at cell adhesion sites with other focal adhesion molecules, and it promoted sustained activation of FAK, p130Cas, and Rac1. However, RhoA activation was decreased, thereby inhibiting HUVEC spreading and migration. Our findings suggest that Fibln7 fragments such as Fbln7-C may prevent vascular invasion into avascular tissues by inhibiting endothelial cell spreading and migration. (B) Critical role of laminin alpha1 in kidney function Laminins, components of basement membranes, comprise a family of heteromeric ECM proteins consisting of alpha, beta, and gamma chains. Laminin alpha1 (Lama1), a subunit of laminin-111 (Lm-111), is the first laminin to be expressed during mouse embryogenesis. Mouse embryos that are deficient in Lama1 lack Reicherts membrane and die by embryonic day 7. Although Lama1 is expressed by mesangial cells in the kidneys, its roles in the kidneys are unknown because of early embryonic lethality. In collaboration with Dr. Eri Arikawa-Hirasawa, a former lab member, and Dr. Jeffrey Miner, we used a viable conditional KO mouse model with a specific deletion of Lama1 in the epiblast lineage (Lama1CKO) using Sox-2 Cre mice. This enabled us to study the role of Lama1 in kidney development and function. Lama1CKO mice showed delayed glomeruli development. Adult Lama1CKO mice developed focal glomerulosclerosis and proteinuria with age. In addition, mesangial cell proliferation was increased, and the mesangial matrix, which normally contains laminin-111, was greatly expanded. In vitro, mesangial cells from Lama1CKO mice exhibited significantly increased proliferation compared with those from controls. This increased proliferation was inhibited by the addition of exogenous Lama1-containing laminin-111, but not by laminin-211 or laminin-511, suggesting Lama1s specific role in regulating mesangial cell behavior. Moreover, the absence of Lama1 increased TGF-beta1 and induced Smad2 phosphorylation, and inhibitors of TGF-beta1 receptor I kinase blocked Smad2 phosphorylation in both control and Lama1CKO mesangial cells, indicating that the increased Smad2 phosphorylation occurred in the absence of Lama1 via the TGF-beta1 receptor. These findings suggest that Lama1 plays a critical role in kidney function and kidney aging by regulating the mesangial cell population and mesangial matrix deposition through TGF- beta1/Smad signaling. (C) Perlecan is required for FGF-2-dependent neurogenesis We previously showed that perlecan, a major heparan sulfate proteoglycan (HSPG) in basement membranes, is present in both blood vessel walls and fractones, extracellular matrix-containing structures, in the neurogenic niche. Perlecan interacts with extracellular molecules, growth factors, and cell surface receptors, and is implicated in many biological functions in tissue development, homeostasis, and diseases. Perlecan deficiency causes perinatal lethal chondrodysplasia in mice and in humans. We previously created perinatal lethality rescued (Hspg2-/-;Tg) mice by expressing recombinant perlecan specifically in the cartilage of perlecan-null (HSPG2-/-) genetic background mice to study the role of perlecan in tissue homeostasis in adult mice. The subventricular zone (SVZ) is one of the main locations where neurogenesis occurs in the adult brain. Recent studies suggest that blood vessels and fractones play important roles in neurogenesis. Perlecan is expressed in both fractones and adult subventricular zone (SVZ) blood vessels. In collaboration with Dr. Eri Arikawa-Hirasawa, we showed that perlecan deficiency reduced the number of neural stem cells in the SVZ in Hspg2-/-;Tg mice. We also showed that FGF-2 treatment induced the expression of cyclin D2 through the activation of Akt and Erk1/2 pathways and promoted neurosphere formation in vitro. However, in the absence of perlecan, FGF-2 failed to promote neurosphere formation. These results suggest that perlecan is a critical component of the neurogenic niche that regulates FGF-2 signaling and acts by promoting neural stem cell self-renewal and neurogenesis.