Division of Gastroenterology & Hepatology, Department of Medicine, School of Medicine, The Johns Hopkins University, 720 Rutland Avenue, 933 Ross Research Building, Baltimore, MD 21205, USA.
Department of Cellular and Molecular Physiology, School of Medicine, The Johns Hopkins University, Baltimore, MD 21205, USA.
Int J Mol Sci. 2023 May 5;24(9):8273. doi: 10.3390/ijms24098273.
Cholesterol-rich membrane domains, also called lipid rafts (LRs), are specialized membrane domains that provide a platform for intracellular signal transduction. Membrane proteins often cluster in LRs that further aggregate into larger platform-like structures that are enriched in ceramides and are called ceramide-rich platforms (CRPs). The role of CRPs in the regulation of intestinal epithelial functions remains unknown. Down-regulated in adenoma (DRA) is an intestinal Cl/HCO antiporter that is enriched in LRs. However, little is known regarding the mechanisms involved in the regulation of DRA activity. The air-liquid interface (ALI) was created by removing apical media for a specified number of days; from 12-14 days post-confluency, Caco-2/BBe cells or a colonoid monolayer were grown as submerged cultures. Confocal imaging was used to examine the dimensions of membrane microdomains that contained DRA. DRA expression and activity were enhanced in Caco-2/BBe cells and human colonoids using an ALI culture method. ALI causes an increase in acid sphingomyelinase (ASMase) activity, an enzyme responsible for enhancing ceramide content in the plasma membrane. ALI cultures expressed a larger number of DRA-containing platforms with dimensions >2 µm compared to cells grown as submerged cultures. ASMase inhibitor, desipramine, disrupted CRPs and reduced the ALI-induced increase in DRA expression in the apical membrane. Exposing normal human colonoid monolayers to ALI increased the ASMase activity and enhanced the differentiation of colonoids along with basal and forskolin-stimulated DRA activities. ALI increases DRA activity and expression by increasing ASMase activity and platform formation in Caco-2/BBe cells and by enhancing the differentiation of colonoids.
富含胆固醇的膜域，也称为脂筏（LRs），是一种特殊的膜域，为细胞内信号转导提供平台。膜蛋白经常聚集在 LRs 中，这些 LRs 进一步聚集形成更大的平台样结构，富含神经酰胺，称为富含神经酰胺的平台（CRPs）。CRPs 在调节肠道上皮功能中的作用尚不清楚。腺瘤下调物（DRA）是一种富含 LRs 的肠道 Cl/HCO 反向转运蛋白。然而，对于调节 DRA 活性的机制知之甚少。气液界面（ALI）是通过去除特定天数的顶端培养基而创建的；在细胞融合后 12-14 天，Caco-2/BBe 细胞或结肠类器官单层作为浸没培养物生长。共聚焦成像用于检查包含 DRA 的膜微域的尺寸。使用 ALI 培养方法，Caco-2/BBe 细胞和人结肠类器官中 DRA 的表达和活性增强。ALI 导致酸性鞘磷脂酶（ASMase）活性增加，ASMase 是一种负责增加质膜中神经酰胺含量的酶。与作为浸没培养物生长的细胞相比，ALI 培养物表达了更多尺寸 >2 µm 的包含 DRA 的平台。ASMase 抑制剂去甲丙咪嗪破坏了 CRPs，并减少了 ALI 诱导的顶膜中 DRA 表达的增加。将正常的人结肠类器官暴露于 ALI 会增加 ASMase 活性，并增强结肠类器官的分化，以及基础和 forskolin 刺激的 DRA 活性。ALI 通过增加 ASMase 活性和平台形成来增加 Caco-2/BBe 细胞中的 DRA 活性和表达，并通过增强结肠类器官的分化来增强 DRA 活性和表达。
