Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.
Invest Ophthalmol Vis Sci. 2011 Jul 29;52(8):5641-6. doi: 10.1167/iovs.11-7196.
Notch proteins are a family of transmembrane receptors that coordinate binary cell fate decisions and differentiation in wet-surfaced epithelia. We sought to determine whether Notch signaling contributes to maintaining mucosal homeostasis by modulating the biosynthesis of cell surface-associated mucins in an in vitro model of human corneal (HCLE) and conjunctival (HCjE) epithelial cell differentiation.
HCLE and HCjE cells were grown at different stages of differentiation, representing nondifferentiated (preconfluent and confluent) and differentiated (stratified) epithelial cultures. Notch signaling was blocked with the γ-secretase inhibitor dibenzazepine (DBZ). The presence of Notch intracellular domains (Notch1 to Notch3) and mucin protein (MUC1, -4, -16) was evaluated by electrophoresis and Western blot analysis. Mucin gene expression was determined by TaqMan real-time polymerase chain reaction.
Here we demonstrate that Notch3 is highly expressed in undifferentiated and differentiated HCLE and HCjE cells, and that Notch1 and Notch2 biosynthesis is enhanced by induction of differentiation with serum-containing media. Inhibition of Notch signaling with DBZ impaired MUC16 biosynthesis in a concentration-dependent manner in undifferentiated cells at both preconfluent and confluent stages, but not in postmitotic stratified cells. In contrast to protein levels, the amount of MUC16 transcripts were not significantly reduced after DBZ treatment, suggesting that Notch regulates MUC16 posttranscriptionally. Immunoblots of DBZ-treated epithelial cells grown at different stages of differentiation revealed no differences in the levels of MUC1 and MUC4.
These results indicate that MUC16 biosynthesis is posttranscriptionally regulated by Notch signaling at early stages of epithelial cell differentiation, and suggest that Notch activation contributes to maintaining a mucosal phenotype at the ocular surface.
Notch 蛋白是一类跨膜受体，可协调湿表面上皮细胞的二元细胞命运决定和分化。我们试图通过在体外人角膜 (HCLE) 和结膜 (HCjE) 上皮细胞分化模型中调节细胞表面相关粘蛋白的生物合成来确定 Notch 信号是否通过调节细胞表面相关粘蛋白的生物合成来维持粘膜稳态。
HCLE 和 HCjE 细胞在不同的分化阶段生长，代表未分化（预融合和融合）和分化（分层）上皮培养物。用 γ-分泌酶抑制剂二苯并氮杂卓 (DBZ) 阻断 Notch 信号。通过电泳和 Western blot 分析评估 Notch 细胞内结构域（Notch1 至 Notch3）和粘蛋白蛋白（MUC1、-4、-16）的存在。通过 TaqMan 实时聚合酶链反应确定粘蛋白基因表达。
在这里，我们证明 Notch3 在未分化和分化的 HCLE 和 HCjE 细胞中高度表达，并且 Notch1 和 Notch2 的生物合成通过含血清培养基诱导分化而增强。用 DBZ 抑制 Notch 信号会以浓度依赖的方式损害未分化细胞在预融合和融合阶段的 MUC16 生物合成，但不会损害有丝分裂后分层细胞的 MUC16 生物合成。与蛋白水平相反，DBZ 处理后 MUC16 转录物的量没有显着减少，这表明 Notch 在后转录水平调节 MUC16。在不同分化阶段生长的 DBZ 处理上皮细胞的免疫印迹显示 MUC1 和 MUC4 的水平没有差异。
这些结果表明，MUC16 的生物合成在上皮细胞分化的早期阶段通过 Notch 信号的转录后调节，并且表明 Notch 激活有助于维持眼表面的粘膜表型。
