CBP磷酸化缺失造成大腸幹性與再生失調之研究

Abstract

CREB-binding protein (CBP) 為具HAT活性之轉錄輔助因子，利於基因轉錄，參與調控多種細胞功能。我們已證明CBP Serine 1383/1387位點磷酸化缺失之CbpS1383/1387Am/m knock-in小鼠 (AA小鼠) 會產生自發性腸炎，有腸道屏障失衡及表皮細胞自我更新延遲之現象。腸道幹細胞可維持腸道屏障之完整性，本研究利用腸道表皮細胞培養成球狀聚落 (spheres)，探討CBP磷酸化缺失對幹性之影響。過量表現人類CBP AA磷酸化缺失之突變型蛋白於HCT116腸道上皮細胞，培養之spheres直徑較小、球體生成效率較低、細胞存活率較低，CD44+/CD166+ 與ALDH+ 細胞亦較少，顯示其幹性降低。為了進一步探討CBP磷酸化於近生理條件中之角色，使用小鼠結腸crypts培養初代organoids。AA小鼠之organoids生成效率較低、直徑較小，幹性相關基因如Lgr5、Ascl2、Mmp7之表現量亦較低。以RNA sequencing探討CBP磷酸化缺失導致腸道幹性失調之可能機制，顯示WT和AA小鼠結腸organoids基因表現確實有差異；Gene Set Enrichment Analysis (GSEA)分析顯示，與維持幹性之訊息傳遞路徑如stem cell proliferation、Wnt/β-catenin及Notch signaling於四週齡AA小鼠之表現較低。 為了解腸道表皮細胞幹性失調是否影響腸道損傷之修復，以DSS誘發小鼠腸炎。在損傷及修復期，AA小鼠之體重、潛血反應、軟便症狀及腸組織結構有延遲復原之現象，修復所需之Ki-67+ proliferating cells亦延緩出現。WT小鼠於損傷期， organoids數量增加，但復原期減少；然而AA小鼠於損傷與復原階段，organoids之數量並無變化，但於復原期organoids較WT多但直徑較小。 因此，CBP磷酸化缺失可能影響spheres及結腸organoids之幹性，阻礙腸道之損傷修復。

CREB-binding protein (CBP) is a transcriptional coactivator involving in the regulation of DNA accessibility to affect a variety of cellular functions. We have previously demonstrated that impaired phosphorylation of CBP at Ser1383/1387 in mice (CbpS1383/1387Am/m knock-in mice, AA mice) caused spontaneous colitis instigated by dysregulation of colonic epithelial homeostasis. The integrity of intestinal epithelial barrier is maintained by the self-renew and continuous replacement of intestinal stem cells; therefore, whether impaired phosphorylation of CBP affected stemness was assessed by culturing spheres derived from intestinal epithelial cell (IEC) enriched in stem cell population. The stemness of spheres from human HCT116 IECs overexpressing CBP AA mutant (Ser1382/1386Ala) was decreased, characterized by smaller size of spheres, lower sphere-forming efficiency, a decrease in cell viability, lower CD44+/CD166+ and ALDH+ population. To further investigate the role of CBP in a near-physiological environment, primary colonic epithelium-derived 3D organoid culture system was employed. Decreased organoid-forming efficiency and size were also observed in organoids generated from AA mice. The mRNA levels of stemness-related genes, such as Lgr5, Ascl2 and Mmp7 were decreased in organoids from AA mice. The possible mechanisms of dysregulation of stemness in AA mice were investigated by RNA sequencing. Heat map showed a reverse expression pattern of genes between WT and AA organoids. A decrease in stem cell related signaling such as stem cell proliferation, Wnt/β-catenin, and Notch signaling, as well as downregulation of NF-kB signaling by Gene Set Enrichment Analysis (GSEA) analysis was observed in 4-week-old AA mice. To examine whether dysregulation of IEC stemness delays epithelial regeneration upon intestinal injury, chemical (DSS)-induced model was performed, symptoms were assessed and organoids were generated. After acute DSS treatment, a delay in weight restoration and histopathological recovery in both injury and recovery phases was seen in AA mice. The number of organoids derived from WT mice in injury phase was increased, followed by a decrease in recovery phase. However, those derived from AA mice during injury and recovery phases had no alteration; besides, more and smaller organoids were seen in AA mice compared to WT mice in recovery phase. These findings reveal that impaired phosphorylation of CBP might affect the stemness of both spheres and colonic organoids, impeding intestinal regeneration.