This project is designed to examine, at the level of molecular regulation, the function of extracellular matrix (ECM) remodeling in the loss and maintenance of tissue-specific function and gene expression. The hypothesis to be tested is that the balance between proteinases and their inhibitors regulate the interaction between mammary epithelial cells and ECM. Thus the loss of ECM through proteolysis may be the key factor in the loss of differentiated function of mammary gland during involution. Initial studies have indicated that a critical balance exists between proteinases and inhibitors in maintaining differentiated tissue-specific function in mammary gland. The approach uses adult mouse mammary gland during pregnancy, lactation and involution for experiments in in vivo and in culture to study the normal expression and function of metalloproteinases and the tissue inhibitor of metalloproteinases (TIMP). It also exploits transgenic methods to achieve overexpression or inappropriate expression of metalloproteinases and their inhibitors in vivo and in culture to analyze the function of ECM. The expression of such transgenes can be targeted with high fidelity and specificity to mammary gland through the use of tissue-specific promoters. Metalloproteinases and TIMP will be targeted to mammary gland or mammary cell lines for inappropriate expression, and thus either produce phenotypes of excessive ECM degradation or inhibition of ECM degradation. For transgenic mice the whey acidic protein (WAP) promoter will be used attached to cDNA sequences of TIMP or metalloproteinases, because it is expressed in mammary gland from day 10-14 of pregnancy through lactation, declining abruptly during involution. Animals expressing the transgenes will be analyzed for the effect of these proteins on gland morphology and function. Therefore, if ECM is required for growth and differentiation, overexpression of these genes may result in abnormal gland morphogenesis including premature involution of failure to develop. Mammary cell strains transfected with cDNA constructs to produce inappropriate expression of metalloproteinases and TIMP will be cultured on ECM or introduced in vivo into cleared mammary fat pads to study how these genes affect structure and mammary gland expression of differentiated gene products. These approaches will help elucidate whether ECM regulates tissue-specific function in vivo, how ECM is remodeled in normal mammary development, and how proteinases and their inhibitors play an essential regulatory role. These studies are related to an important aspect of women's health, that of regulation of normal and abnormal mammary gland function, and, in the long term can be used as a data base to determine whether overexpression or inhibition of proteinases alters the gland's susceptibility to mammary tumorigenesis and the subsequent tumor metastasis.
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