The broad goal of this research is to investigate the hypothesis that extracellular matrix (ECM) exerts regulatory effects which determine the differentiated status and physiological function of type II pulmonary epithelial cells (T2P). There is substantial evidence to support this premise. In other tissues and in the lung, the ECM exerts regulatory effects on nearby cells. Recent studies suggest that T2P-matrix interactions are complex and bidirectional: not only does the ECM Influence the cells, but also the cells synthesize matrix components and actively degrade ECM from the culture surface. This suggests that synthesis and turnover determine final ECM composition and thereby the extent of T2P differentiation. We propose to investigate synthesis and turnover of ECM component molecules by T2P, to define regulation of these pathways, and to relate these observations to changes in type 11 cell phenotype during lung growth and repair. The approach anticipated Involves the following specific aims: (1) to define and quantitate synthesis and deposition of specific ECM components by T2P In primary culture; (2) define the qualitative and quantitative aspects of ECM degradation by T2P, thereby to determine how the pathways of matrix synthesis, deposition and turnover contribute to net matrix production; (3) to identify physiologically relevant regulators of ECM synthesis and turnover by the T2P and to investigate the specific regulatory effects and mechanisms of action of these agents; (4) to address the physiological significance of ECM synthesis and turnover as a component of the role of the T2P in lung growth and repair. T2P differentiation will be characterized based on specific phenotypic markers. ECM synthesis and turnover by T2P will be studied in primary culture using radiolabeling, immunologic, biochemical and molecular techniques. Expression of ECM molecules by T2P will be verified using specific immunologic markers and in situ hybridization. ECM turnover will be studied based on loss of radiolabeled molecules from prelabeled matrix, using identification and separation techniques as above. Both the molecular specificity and the pathway of matrix degradation will be defined. The role of external factors to control ECM synthesis and degradation will be defined and localized to specific steps in the turnover pathways. A long term goal is to relate observations in cultured cells to similar conditions in situ in order to identify factors which regulate synthesis and turnover of ECM components by T2P, and to place the role of these pathways and their regulation in a physiological and/or pathophysiological context. The results will address the role of the pulmonary epithelium to establish normal cell-ECM interactions and to modulate the effect of the ECM on cellular differentiation; they also will help to define the role of the T2P in integration of lung structure and function under both physiological and pathophysiological conditions.
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