Homeostasis of the prostate gland and mammary gland is maintained through an intricate balance between cell survival and cell death regulated by steroid hormones. Depletion of these hormones results in apoptotic cell death and tissue involution. Although well studied, the initiating mechanism of involution remains vague. A potential target of hormone action during involution may be the intercellular junction of adjacent secretory epithelium. This is supported by the long-standing observation that one of the first visible stages of prostate and mammary involution is the disruption of interepithelial adhesion prior to the onset of apoptosis. In addressing this specific aspect of hormone-regulated involution, my laboratory has acquired compelling evidence indicating that the disruption of E-cadherin-dependent adhesion induces epithelial apoptosis during prostate and mammary involution. In cultured prostate and mammary epithelial cells, inhibition of E-cadherin-dependent aggregation resulted in synergistic cell death following apoptotic stimuli. Loss of cell-cell adhesion in the non-aggregated population appeared to result from rapid proteolytic cleavage of the mature l20kDa species of E-cadherin (E- cad120). Since our original submission, we have mapped the cleavage site to a concise, 22 amino acid region spanning residues 792 to 814. These conclusions are drawn from recent experiments from our laboratory in which we have fine mapped the cleavage region employing 6 different region- or peptide-specific antibodies. Co-immunoprecipitations from cell culture and involuting mammary gland demonstrated that this cleavage event removed the beta-catenin binding domain from the cytoplasmic tail of E-cadherin resulting in a non beta-catenin binding, membrane-bound 97kDa species (E-cad97) and a free cytoplasmic 35kDa form (E-cad?5) that binds strongly to beta-catenin. Examination of E-cadherin expression and cellular distribution in the involuting prostate gland and mammary gland revealed that the loss of junctional membrane immunoreactivity and E-cadherin proteolysis preceded apoptosis. The observation that E-cad97 and E-cad35 accumulated during mammary involution suggested that hormone depletion signals the same apoptotic pathway in vivo as observed in vitro . The intent of this revised proposal will be to elucidate the functional role of E-cadherin cleavage in the disruption of intercellular adhesion and induction of cell death. Our experimental approach will be to engineer an inducible expression system in prostate and mammary epithelial cells to determine if expression of E-cad35 or E-cad can differentially initiate cellular dissociation and apoptosis. We will utilize immuno-affinity purification and Edman degradation amino-terminal peptide sequencing to determine the precise proteolytic site. If a known proteolytic site is identified, we will determine if the purified protease or the corresponding inhibitors recapitulate or inhibit the proteolytic event respectively. Additionally, we will engineer a truncation-resistant mutant of E-cad120 to be expressed in prostate and mammary epithelial cells to determine if cellular dissociation and apoptosis are inhibited. We anticipate results from these studies will support our central hypothesis: That rapid proteolytic processing occurs in the cytoplasmic domain of the mature E-cadherin protein resulting in the loss of beta-catenin binding and cellular dissociation that signals epithelial apoptosis during prostate and mammary involution.
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