Merton Bernfield - US grants

Our long-term goal is to understand the function of the basal lamina, a component of the basement membrane, in cellulose physiology and in various pathological states. Our studies have shown that mouse mammary epithelial cells cultured on a collagen gel substratum form a proteoglycan-containing basal lamina. Basal lamina formation correlates with reduced glycosaminoglycan degradation and the apparent conversion of a cellular proteoglycan fraction to a slowly degrading extracellular fraction. Similarly cultured neoplastically transformed cells of the same origin show defective lamina formation and lack of proteoglycan processing. Based upon the hypothesis that the presence and interactions of the proteoglycans in the basal lamina are crucial to its structure and function, we will continue these studies. Using mouse mammary epithelial cells in culture, the focus will be on: (1) isolating and purifying the various proteoglycans and determining the structural relationships between them by analyzing their protein and glycosaminglycan components; (2) elucidating the macromolecular organization of the proteoglycan-containing basal lamina by analyzing the interactions of its various extracellular matrix constituents; (3) establishing the steps in the processing and degradation of basal laminar proteoglycans to determine the role of the collagen substratum in facilitating lamina formation; and (4) applying this new information to elucidate the basis for the defective lamina formation shown by the malignant cells. The functional role of proteoglycans in the lamina will be further assessed by comparing laminar proteoglycans of cultured mammary and vascular endothelial cells.

The cellular behavior involved in morphogenesis are regulated by molecules at cell surfaces that bind cells to the extracellular matrix and to other cells. Epithelial cells contain a cell surface proteoglycan that is a high affinity receptor specific for interstitial matrix materials. The proteoglycan appears to stabilize epithelial sheets by physically linking the intracellular cytoskeleton with the matrix produced by mesenchymal cells. Recent results lead to the working hypothesis that the core protein of the cell surface proteoglycan, the protein to which the glycosaminoglycan chains are covalently bound, is a developmentally regulated molecule that functions as a matrix receptor or as a cell adhesion molecule depending on its post-translational modifications. As a matrix receptor, it would contain heparan and chondroitin sulfate chains, be expressed late in development and, in mature tissues, be solely on the basolateral surface of simple epithelial cells. As a cell adhesion molecule, it would be less extensively glycosylated, be expressed early in development and, in mature tissues, surround stratified epithelial cells. During embryogenesis, it would be present on both epithelial and mesenchymal cells, but change its expression in association with morphogenetic events. This application is designed to explore the hypothesis that the epithelial cell surface proteoglycan is a developmentally regulated multifunctional adhesion molecule. The specific aims are to: (i) evaluate the regulation of cell surface proteoglycan expression in early mouse embryos and during subsequence morphogenetic events by immunological and molecular assessments of its time of appearance, cell and tissue localization, post- translational modifications and mRNA level, (ii) test whether the cell surface proteoglycan functions as a cell adhesion molecule by attempting to perturb cell aggregation and histogenesis with defined antibodies and ligands, to isolate an endogenous ligand(s) and comparing its modifications in simple and stratified epithelia, (iii) define the matrix receptor role by examining whether it traverses the basal lamina, comparing its function with other receptors, evaluating whether it mediates receptor-specific cellular responses and generating cells that are deficient or enhanced in the cell surface proteoglycan. Knowledge of the mechanisms involved in cell-cell and cell-matrix adhesion is critically important to an understanding of cell behavior during development and neoplastic invasion. This research will provide new insight into these mechanisms, potentially leading to diagnosis, treatment and ultimately, prevention of birth defects and metastases.