上皮細胞間緊密連接蛋白 Occludin 協調 TGFβ 受體訊息途徑以調控 Caspase-3 依賴性細胞凋亡之機制探討

Abstract

發炎性腸道疾病 (IBD) 的典型病理特徵為黏膜通透性增加與緊密連接蛋白 (如 Occludin, OCLN) 表現量下降。Occludin 最初被歸類為調節上皮屏障的跨膜成分，但近期研究顯示其在黏膜穩態中亦具備非屏障功能。我們先前發現，Occludin 的缺失造成 Caspase-3 的表現量下降來減少上皮細胞凋亡，此抗凋亡表型與 Occludin/TGFβR 的交互作用密切相關，但其下游具體的分子調控機制仍未清楚。 目的： 探討 Occludin 如何透過調控 TGFβ 訊息傳遞路徑與蛋白質空間定位，進而影響腸道上皮細胞中 Caspase-3 依賴性之細胞凋亡。 方法： 以野生型 (WT) 與 OCLN 基因敲除 (KO) 之 Caco-2 腸道上皮細胞為模型，於基底側給予 TGFβ 刺激。透過表現全長及缺乏特定結構域 (ΔN-tail, ΔOCEL, ΔEL1, ΔEL2) 的截短型OCLN 建構體進行結構域功能分析。利用核質分離技術結合西方墨點法與免疫螢光染色，分析典型 (SMAD3, FOXO3A, E2F1) 與非典型 (AKT, ERK1/2, JNK, p-c-Jun) TGFβ 訊息分子之蛋白表現量與細胞空間定位。藉由鄰近連接測定法 (PLA) 與內吞體標記 (EEA1) 檢視受體交互作用及胞內動態。最後，分析 GEO 資料庫中 IBD 患者之臨床轉錄組數據以進行病理與機制驗證。 結果： OCLN 敲除會顯著降低細胞TEER 值，在凋亡調控方面，OCLN KO 細胞中 Caspase-3 的轉錄與蛋白質含量皆顯著降低；然而，在OCLN KO細胞重新表現缺乏 EL2 的截短建構體 (ΔEL2) 能最顯著地將 Caspase-3 表現量恢復至接近 WT。PLA 結果進一步證實，ΔEL2 建構體顯著地增強了 Occludin 與 TGFβR交互作用，證實缺乏 EL2 結構域不僅增加 Occludin 與 TGFβR 之交互作用，更是讓 Caspase-3 表現量得以恢復的關鍵。在 TGFβR 下游訊號傳遞上，OCLN 缺失並未改變 AKT 等分子的整體蛋白質表現量，但顯著降低了 E2F1、ERK 及 TGFβ 長時間刺激下 JNK 的總蛋白表現量，且 OCLN 缺失不僅促進活化後的 SMAD3、FOXO3A、E2F1 及 p-c-Jun 轉移至細胞核內，亦導致了激酶 ERK1/2 與 JNK 的活化。同時，OCLN 缺失改變了激酶的細胞空間分布，使 p-AKT 由表現細胞膜邊緣轉為散布於細胞質，並使 ERK 喪失原有的細胞膜定位而瀰漫於細胞質中。此外，早期內吞體 (EEA1) 與晚期內吞體 (Rab7) 的總量與結構維持穩定。此外，OCLN 缺失亦導致膜上交互作用蛋白 CEACAM1表現量顯著增加，以及促凋亡激酶 SLK 的表現量降低，並使其喪失原本的細胞質邊界定位，基於此二分子皆參與凋亡調控，這些交互作用蛋白在表現量與空間定位上的重整，可能是阻礙下游 Caspase-3 正常表達的機制之一。最後，臨床 GEO 資料庫分析證實，IBD 患者在 OCLN 缺失與 TGFBR 高表現下，其 SMAD/FOXO3 總轉錄量仍維持恆定，並伴隨 AKT 與 CEACAM1 等黏附與存活分子的表現量增加，呼應了體外細胞模型之發現。 結論： Occludin 對 Caspase-3 依賴性凋亡的調控，並非透過改變 TGFβ 下游轉錄因子的總生成量。但是，當 OCLN 缺失時，促使 SMAD3、FOXO3A、E2F1與 p-c-Jun 的核轉位，並連動 CEACAM1 與 SLK 等凋亡調節的交互作用蛋白表現量與空間重整，可能從而影響 Caspase-3 的表現量下降。此非屏障性之 Occludin /TGFβR 空間調控，為腸道上皮細胞在病理發炎中抵抗嚴重組織損傷提供了一項全新的適應性生存策略。

Inflammatory bowel disease (IBD) is pathologically characterized by increased mucosal permeability and downregulated expression of tight junction proteins, such as occludin (OCLN). Initially categorized as a transmembrane component that regulates the epithelial barrier, recent studies have revealed that OCLN also possesses non-barrier functions crucial for mucosal homeostasis. Our previous findings indicated that OCLN deficiency attenuates epithelial apoptosis by downregulating Caspase-3 expression. While this anti-apoptotic phenotype is closely associated with the interaction between OCLN and TGFβR, the specific downstream molecular regulatory mechanisms remain unclear. Objective: To investigate how OCLN modulates the TGFβR signaling pathway and protein spatial localization, thereby influencing Caspase-3-dependent apoptosis in intestinal epithelial cells. Methods: Wild-type (WT) and OCLN knockout (KO) Caco-2 intestinal epithelial cells were employed as in vitromodels and subjected to basolateral TGFβ stimulation. Domain function analysis was conducted by expressing full-length and truncated OCLN constructs lacking specific domains (ΔN-tail, ΔOCEL, ΔEL1, and ΔEL2). Nuclear-cytoplasmic fractionation, Western blotting, and immunofluorescence (IF) staining were utilized to evaluate the protein expression and spatial localization of canonical (SMAD3, FOXO3A, E2F1) and non-canonical (AKT, ERK1/2, JNK, p-c-Jun) TGFβ signaling molecules. Proximity ligation assay (PLA) and endosomal marker (EEA1) tracking were applied to examine receptor interactions and intracellular dynamics. Finally, clinical transcriptomic data of IBD patients from the GEO database were analyzed for pathological and mechanistic validation. Results: OCLN knockout significantly decreased cellular TEER values. Regarding apoptosis regulation, both the transcription and protein levels of Caspase-3 were significantly reduced in OCLN KO cells. However, re-expressing the EL2-lacking truncated construct (ΔEL2) in OCLN KO cells most remarkably restored Caspase-3 expression to near-WT levels. PLA results further demonstrated that the ΔEL2 construct greatly enhanced the interaction between Occludin and TGFβR, confirming that the absence of the EL2 domain not only promotes this interaction but is also the key to restoring Caspase-3 expression. Regarding downstream TGFβR signaling, OCLN deficiency did not alter the overall protein expression of molecules such as AKT, but it significantly decreased the total protein levels of E2F1, ERK, and JNK under prolonged TGFβ stimulation. Furthermore, OCLN deficiency not only promoted the nuclear translocation of activated SMAD3, FOXO3A, E2F1, and p-c-Jun, but also led to the activation of the kinases ERK1/2 and JNK. Concurrently, OCLN depletion altered the spatial distribution of kinases; p-AKT shifted from the cell membrane periphery to become dispersed in the cytoplasm, and ERK lost its original membrane localization, becoming diffusely distributed throughout the cytoplasm. Additionally, the total abundance and structural integrity of early (EEA1) and late (Rab7) endosomes remained stable. Moreover, OCLN deficiency resulted in a significant increase in the expression of the membrane-interacting protein CEACAM1, while decreasing the expression of the pro-apoptotic kinase SLK and disrupting its original cytoplasmic boundary localization. Given that both molecules are involved in apoptosis regulation, this reorganization of interacting proteins in terms of expression and spatial localization may be one of the mechanisms impeding the normal expression of downstream Caspase-3. Finally, clinical GEO database analysis confirmed that in IBD patients presenting with OCLN deficiency and high TGFBR expression, the total transcript levels of SMAD/FOXO3 remained constant, accompanied by increased expression of adhesion and survival molecules such as AKT and CEACAM1, which echoes the findings from the in vitro cell model. Conclusion: Occludin-mediated regulation of Caspase-3-dependent apoptosis does not occur through altering the total production of downstream TGFβ transcription factors. Instead, OCLN deficiency facilitates the nuclear translocation of SMAD3, FOXO3A, E2F1, and p-c-Jun, and drives the reorganization in expression and spatial localization of apoptosis-regulating interacting proteins such as CEACAM1 and SLK, which collectively contributes to the downregulation of Caspase-3 expression. This non-barrier spatial regulation of Occludin/TGFβR provides a novel adaptive survival strategy for intestinal epithelial cells to resist severe tissue damage during pathological inflammation.