HORMONE REGULATED INVOLUTION SIGNALED THROUGH E CADHERIN

前列腺和乳腺的内稳态是通过类固醇激素调节的细胞生存和细胞死亡之间的复杂平衡来维持的。这些激素的耗竭导致细胞凋亡和组织退化。虽然研究得很好，但对合的启动机制仍然模糊。在复旧过程中，激素作用的一个潜在目标可能是相邻分泌上皮的细胞间连接处。长期以来的观察支持了这一点，即前列腺和乳腺复旧的第一个可见阶段是细胞凋亡发生之前上皮间粘连的破坏。在解决激素调节的复旧的这一特定方面时，我的实验室已经获得了令人信服的证据，表明在前列腺和乳腺复旧过程中，e-钙粘蛋白依赖性粘附的破坏会诱导上皮细胞凋亡。在培养的前列腺和乳腺上皮细胞中，抑制e -钙粘蛋白依赖性聚集导致凋亡刺激后的协同细胞死亡。在非聚集群体中，细胞-细胞粘附的丧失似乎是由于成熟的l20kDa E-cadherin （E- cad120）的快速蛋白水解裂解造成的。自我们最初的提交以来，我们已经将裂解位点映射到一个简洁的22个氨基酸区域，跨越残基792到814。这些结论是从我们实验室最近的实验中得出的，我们利用6种不同的区域或肽特异性抗体绘制了切割区域的精细图谱。细胞培养和乳腺的共免疫沉淀表明，这一切割事件从E-cadherin的细胞质尾部去除了β -连环蛋白结合域，导致非β -连环蛋白结合，膜结合的97kDa物种（E-cad97）和自由的细胞质35kDa形式(E-cad？5)与-连环蛋白紧密结合。检查E-cadherin在纠缠性前列腺和乳腺中的表达和细胞分布，发现连接膜免疫反应性的丧失和E-cadherin蛋白水解发生在细胞凋亡之前。E-cad97和E-cad35在乳腺复旧过程中积累的结果表明，激素消耗在体内与在体外观察到的凋亡途径相同。这一修订建议的目的是阐明e -钙粘蛋白切割在破坏细胞间粘附和诱导细胞死亡中的功能作用。我们的实验方法是在前列腺和乳腺上皮细胞中设计一个诱导表达系统，以确定E-cad35或E-cad的表达是否能不同地启动细胞解离和凋亡。我们将利用免疫亲和纯化和Edman降解氨基末端肽测序来确定精确的蛋白水解位点。如果确定了已知的蛋白水解位点，我们将确定纯化的蛋白酶或相应的抑制剂是否分别概括或抑制蛋白水解事件。此外，我们将设计一个抗截断的E-cad120突变体，在前列腺和乳腺上皮细胞中表达，以确定细胞解离和凋亡是否被抑制。我们预计这些研究的结果将支持我们的中心假设：成熟的E-cadherin蛋白的细胞质区域发生快速的蛋白水解过程，导致β -连环蛋白结合和细胞解离的丧失，这是前列腺和乳腺退化过程中上皮细胞凋亡的信号。

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.