Schoenenberger C A, Zuk A, Groner B, Jones W, Andres A C
Department of Anatomy and Cellular Biology, Harvard Medical School, Boston, Massachusetts 02115.
Dev Biol. 1990 Jun;139(2):327-37. doi: 10.1016/0012-1606(90)90302-y.
In rodents, the whey acidic protein (Wap) is the major whey protein expressed in mammary glands in response to lactogenic hormones. The regulation of the Wap gene differs from that of other milk protein genes, with one consequence being that little or no Wap expression is detectable in cell culture. Here we describe the efficient in vitro induction of the Wap gene in mammary organoids isolated from midpregnant mice. Mammary organoids were isolated as intact epithelial subcomponents which retained the glandular microarchitecture. If organoids were cultured in contact with a monolayer of 3T3-L1 adipocytes, significant levels of Wap mRNA were induced upon hormonal stimulation, with the highest level of Wap mRNA being induced by a combination of hydrocortisone, prolactin, and insulin. Dissociation of the three-dimensional organization abrogated Wap inducibility. Organoids cultured on plastic or hydrated type I collagen did not transcribe Wap mRNA even after hormonal stimulation. Addition of hormones was required to maintain low levels of Wap mRNA in organoids cultured on reconstituted basement membrane, however, Wap mRNA was not induced. Organoid-adipocyte interactions as well as cell-cell interactions inherent in the structure of organoids promote hormone-dependent Wap mRNA expression. In order to study the Wap promoter region in vitro, we cocultured organoids from transgenic mice harboring a chimeric Wap-myc gene with 3T3-L1 adipocytes. Lactogenic hormones induced the Wap-myc transgene in vitro. The kinetics of induction were similar for both the transgene and the endogenous Wap gene indicating that the 2.5-kb regulatory Wap region present in the hybrid gene contains the sequence elements required for hormone-induced gene expression in vitro.
在啮齿动物中，乳清酸性蛋白（Wap）是乳腺中响应泌乳激素而表达的主要乳清蛋白。Wap基因的调控与其他乳蛋白基因不同，其结果之一是在细胞培养中几乎检测不到Wap表达。在此，我们描述了从妊娠中期小鼠分离的乳腺类器官中Wap基因的高效体外诱导。乳腺类器官作为保留腺泡微结构的完整上皮亚组分被分离出来。如果将类器官与3T3-L1脂肪细胞单层接触培养，激素刺激后会诱导出显著水平的Wap mRNA，氢化可的松、催乳素和胰岛素联合使用时诱导出的Wap mRNA水平最高。三维组织结构的解离消除了Wap的诱导能力。在塑料或水合I型胶原上培养的类器官即使在激素刺激后也不转录Wap mRNA。在重组基底膜上培养的类器官中，需要添加激素来维持低水平的Wap mRNA，但不会诱导Wap mRNA。类器官-脂肪细胞相互作用以及类器官结构中固有的细胞-细胞相互作用促进了激素依赖性Wap mRNA的表达。为了在体外研究Wap启动子区域，我们将携带嵌合Wap-myc基因的转基因小鼠的类器官与3T3-L1脂肪细胞共培养。泌乳激素在体外诱导了Wap-myc转基因。转基因和内源性Wap基因的诱导动力学相似，表明杂交基因中存在的2.5 kb Wap调控区域包含体外激素诱导基因表达所需的序列元件。
