During involution of the mammary gland epithelial structures are replaced by adipose tissue. This requires the differentiation of preadipocytes into functional adipocytes and involves a change in cell shape from a fibroblast-like (preadipocyte) to a rounded, lipid-filled (adipocyte) morphology, as well as a change in the expression of specific extracellular matrix proteins. We have observed that the serine protease urokinase plasminogen activator (uPA) is expressed in 3T3-L1 preadipocytes but is downregulated when they differentiate to adipocytes. To determine whether serine proteases are involved in adipocyte differentiation in the mammary gland, we developed potent inhibitors of specific serine proteases including uPA by protein engineering of the Eschericia coli serine protease inhibitor ecotin. Treating 3T3-L1 cells with ecotin during differentiation led to a decrease in adipogenic conversion. We also demonstrated that inhibition of serine proteases during involution in vivo blocks adipocyte differentiation and leads to an increase in collagen deposition characteristic of fibroblasts. The data suggests that serine proteases play a major role in adipogenic differentiation, possibly through their action on cytokines and extracellular matrix components. Several recent studies have indicated a link between dietary fat and breast cancer incidence, and our research provides the opportunity to investigate the role that adipose tissue may play in mammary development and carcinogenesis. We use the resources available at the Computer Graphics Laboratory to model interactions between ecotin and target serine proteases to help in designing more specific inhibitors. We are interested in identifying novel serine proteases expressed during involution.
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