Investigation of the effects of a panel of IGFBP-5 mutants on apoptosis in a 3D mammary co-culture system

在青春期前和青春期后，乳腺在乳房脂肪垫中发育为一个基本的导管结构。在怀孕期间，它经历了大规模的发展，导致了分化的上皮细胞，最终形成了分泌肺泡的结构，在哺乳期产生乳汁。在哺乳期结束时，绝大多数上皮细胞通过一种称为细胞凋亡的程序性细胞死亡过程而死亡。乳腺的发育和存活依赖于多种激素和生长因子，但已有研究表明，胰岛素样生长因子-I(IGF-I)的缺失会导致乳腺发育的严重障碍。IGF-I现在被认为是体内许多不同类型细胞的重要生存因素。令人惊讶的是，哺乳结束时细胞死亡的过程并没有伴随着血液中IGF-I浓度的下降。相反，我们已经证明，上皮细胞产生一种自杀蛋白，IGF结合蛋白-5(IGFBP-5)，它与IGF-I结合并抑制IGF-I的作用。情况实际上更为复杂，因为我们和其他人已经表明，IGFBP-5可以独立于IGF-I发挥作用，但它这样做的方式尚不清楚。例如，IGFBP-5可以独立于IGF-I激活参与降解细胞外环境的蛋白水解酶(这是哺乳期结束时乳腺重建的关键部分)。我们已经成功地产生了IGFBP-5的突变形式，其中IGF依赖和/或独立的作用已经分离。这些分子已被初步研究，并显示出新的性质，当前提议的一个主要目标是利用这些突变形式的IGFBP-5来更清楚地了解IGFBP-5在诱导乳腺细胞凋亡和组织重塑中的作用机制。这一提议的优势之一在于使用了复杂的3D模型，与典型的2D细胞培养相比，这种模型更接近体内情况。我们的方法包括在以胶原/层粘连蛋白为基础的细胞外环境中将乳腺上皮细胞与脂肪细胞(脂肪细胞)共培养。我们认为这种共培养是至关重要的，因为首先，乳腺上皮发育成3D导管和肺泡结构，它们是极化的，有一个内腔(这是大多数细胞系所不能实现的过程)。其次，乳腺上皮与乳腺脂肪细胞广泛相互作用，这些脂肪细胞分泌影响和“指导”上皮细胞形态发生和分化的因素。我们对这些共培养物以及其中IGF-I和IGFBP-5突变体的影响的评估将涉及最先进的技术，包括共聚焦显微镜、显性-阴性分子和突变蛋白的腺病毒感染，以及96孔RTPCR筛选方法。我们还将使用更经典的免疫组织化学和免疫印迹技术来检查细胞内信号事件。此外，我们将使用快速筛选技术，利用已建立的细胞系，以重点研究更复杂的3D培养。最后，我们将利用在乳腺中特异表达突变的IGFBP-5分子的转基因动物(由另一个项目提供)来比较它们的表型与非突变的IGFBP-5过度表达所诱导的表型(这会损害体内的乳腺发育)。因此，这些研究将在体内和共培养中确定IGFBP-5依赖于IGF和不依赖于IGF的作用的相对重要性，并探索这是否涉及影响细胞存活和迁移的细胞表面蛋白的变化。我们还旨在确定哪些细胞内信号通路(S)被IGFBP-5激活。

The mammary gland develops as a rudimentary ductal structure in the mammary fat pad, during pre- and post-pubertal priods. During pregnancy it undergoes massive development resulting in differentiated epithelial cells which ultimately form the secretory alveolar structures that make milk during lactation. At the end of lactation the vast majority of the epithelial cells die by a process of programmed cell death known as apoptosis. The development and survival of the mammary gland depends upon a variety of hormones and growth factors, but it has been shown that the absence of insulin-like growth factor-I (IGF-I) leads to a dramatic impairment of mammary development. IGF-I is now known to be an important survival factor for many different cell types in the body. Surprisingly, the process of cell death at the end of lactation is not accompanied by a decrease in the concentration of IGF-I in the blood. Instead, we have demontrated that the epithelial cells produce a suicide protein, IGF-binding protein-5 (IGFBP-5) which binds to and inhibits the actions of IGF-I. The situation is actually more complex in that we, and others, have shown that IGFBP-5 can act independently of IGF-I, but the manner in which it does so is not yet understood. For example, IGFBP-5 can, independently of IGF-I , activate proteases which are involved in degrading the extracellular environment (a crucial part of the re-modeling of the mammary gland which occurs at the end of lactation). We have already succesfully generated mutated forms of IGFBP-5 in which the IGF-dependent and /independent effects have been separated. These molecules have been studied in preliminary fashion and shown to exhibit novel properties and a principal objective of the current proposal is to use these mutated forms of IGFBP-5 to provide a clearer insight into the mechanisms of action of IGFBP-5 in inducing mammary apoptosis and tissue remodelling. One of the strengths of this proposal lies in the use of a complex 3D model which more closely resembles the in vivo situation than do typical 2D cultures of cells. Our approach involves co-culture of mammary epithelial cells with fat cells (adipocytes) in a collagen/laminin-based extracellular environment. We believe this co-culture to be crucial as, firstly, the mammary epithelium develops as 3D ducts and alveolar structures which are polarised and have an internal cavity (processes which are not achieved with most cell lines). Secondly, the mammary epithelium interacts extensively with the mammary adipocytes, which secrete factors that influence and 'instruct' epithelial cell morphogenesis and differentiation. Our assesment of these co-cultures, and the effects of IGF-I and IGFBP-5 mutants therein, will involve state-of-the-art technologies including confocal microscopy, adenoviral infection with dominant-negative molecules and mutated proteins, and 96-well rtPCR screening approaches. We will also use more classical approaches of immnohistochemistry and western-immunoblotting techniques to examine intra-cellular signalling events. In addition, we will use rapid screening techniques, utilising established cell lines, in order to focus our studies of the more complex 3D cultures. Finally, we will take advantage of transgenic animals expressing the mutant IGFBP-5 molecules specifically in the mammary gland (provided by a separate project) to compare their phenotype with that induced by over-expression of non-mutated IGFBP-5 (this impairs mammary development in vivo). Thus these studies will identify, in vivo and in co-cultures, the relative importance of the IGF-dependent and IGF-independent effects of IGFBP-5 and explore whether this involves changes in cell surface proteins which influence cell survival and migration. We also aim to determine which intracellular signalling pathway(s) are activated by IGFBP-5.