緊密連接蛋白Occludin調控上皮細胞凋亡以參與黏膜穩態之機制探討

Abstract

Occludin 是第一個被發現調節上皮細胞屏障通透性的跨膜緊密連接蛋白。臨床數據顯示，在發炎性腸道疾病 (IBD) 患者中，潰瘍組織有Occludin表現量下降現象。我們先前發現，缺乏Occludin可透過降低caspase-3的mRNA和蛋白質表現來減少細胞死亡，這個過程對於維持黏膜平衡至關重要，但其中調節的分子機制尚不清楚。Occludin與TGFβR結合促進調節TGFβ引起的緊密連接溶解現象。 Occludin可能抑制依賴TGFβ的SMAD3訊息途徑，對調控caspase-3在維持黏膜穩態中有著重要作用。 目標：探討OCLN/TGFβR訊號如何調控上皮細胞凋亡caspase-3的分子機制。 材料與方法：我們使用RT-qPCR、西方墨點法和免疫螢光染色技術在腸道上皮WT、OCLN KD和OCLN KO Caco-2細胞中檢測包括OCLN、TGFβR、SMAD3和CASP3在內的分子訊號，使用鄰近連接測定法 (PLA) 檢測蛋白質交互作用。TGFβR抑制劑SB-525334和LY2109761用於研究OCLN/TGFβR訊號在調控caspase-3表現的特異性。透過腸道上皮細胞特異性Occludin敲除小鼠 (Ocln KOIEC) 和WT小鼠給予硫酸葡聚醣 (DSS) 誘發結腸炎模型，並分析結腸組織中Ocln、Tgfbr/Smad和凋亡訊號的轉錄體表現。最後，我們利用基因表現綜合資料集 (GEO) 分析健康和IBD患者的上述分子訊號表現以確認體外細胞研究發現。 結果：首先確認OCLN KD和OCLN KO Caco-2細胞具備Occludin缺乏現象，這些細胞的跨膜屏障通透性遠高於WT Caco-2細胞。缺乏Occludin導致caspase-3的轉錄和蛋白質表現量降低，而TGFβR和SMAD3表現量保持不變。在基底外側給予TGFβ不會改變TGFβR的表現量。PLA顯示，TGFβR與Occludin的交互作用會減少Occludin與早期內吞標記物EEA1的內吞。Occludin缺乏細胞中的SMAD3表現不受TGFβ處理影響。然而，它增加了SMAD3的核轉位並減少了caspase-3的生成。這些數據顯示，Occludin與TGFβR相互作用可能透過阻礙Occludin內化來抑制TGFβ/SMAD3訊息途徑，進而影響caspase-3的生成。TGFβR激酶活性的藥理抑制劑SB-525334和LY2109761不會影響TGFβR與Occludin的相互作用。在TGFβR抑制後，與TGFβ處理相比，Occludin與EEA1的交互作用增加，SMAD3的核轉位減少。這些結果顯示Occludin與TGFβR的相互作用發生在TGFβR磷酸化之前，SMAD3訊號在調節caspase-3訊號中扮演著重要角色，這對於維持黏膜穩態至關重要。在DSS誘導的結腸炎模型中，Ocln KOIEC小鼠的Casp3表現量降低，Tgfb/Smad3訊號沒有變化，與體外細胞研究結果一致。最後，IBD患者的GEO資料集顯示OCLN表現量降低，CASP3升高，伴隨著上皮細胞標記KRT8表現量降低，顯示在IBD患者需要進一步針對上皮細胞的研究。 結論：這項研究發現Occludin缺乏對凋亡調節的可能新機制，它還顯示Occludin表現量下降如何透過減少caspase-3合成從而降低凋亡。Occludin的內吞現象和TGFβ/SMAD3訊號在這複雜機制中扮演重要角色，這一途徑可能是一種在病理情況下減少上皮組織損傷的適應性策略。

Occludin is the first transmembrane tight junction protein found to regulate epithelial cell barrier permeability. Clinical evidence of inflammatory illness (IBD) shows destructured Occludin expression in ulcer tissue. We previously found that the absence of Occludin reduces cellular death via reducing mRNA and protein expression of caspase-3. This procedure is essential for mucosal lining balance. The mechanism underlying this regulation is unclear. Occludin binding to TGFβR facilitates the dissolution of tight junctions. Occludin may inhibit the TGFβ-dependent SMAD3 signaling pathway, which controls the role of caspase-3 role in maintaining mucosal homeostasis. Objective: To explore the mechanism by which OCLN/TGFβR signaling governs epithelial apoptotic caspase-3. Materials and Methods: We examined molecular signals including OCLN, TGFβR, SMAD3, and CASP3 in intestinal epithelial WT, OCLN KD, and OCLN KO Caco-2 cells using RT-qPCR, Western blotting, and immunostaining. The protein interactions was measured by proximity ligation assay (PLA). TGFβR inhibitors SB-525334 and LY2109761 were used to study the specificity of OCLN/TGFβR signaling in controlling caspase-3 expression. Dextran sulfate sodium (DSS) was given to intestinal-specific Occludin knockout (Ocln KOIEC) and WT littermates to create a colitis model and analyze transcription of Ocln, Tgfbr/Smad, and apoptotic signals in colonic tissues. The Gene Expression Omnibus (GEO) dataset of healthy and IBD patients was also investigated to confirm in vitro findings. Results: OCLN KD and OCLN KO Caco-2 cells showed Occludin deficient efficacy. These cells had far higher barrier permeability than WT. Lack of Occludin resulted in lower caspase-3 transcript and protein levels, while TGFβR and SMAD3 levels remained unchanged. Applying TGFβ to the basolateral side did not change the quantity of TGFβR. PLA showed that TGFβR contact with Occludin decreased its internalization with early endosomal EEA1, as indicated by the interaction. SMAD3 expression was unaffected by TGFβ treatment in Occludin-deficient cells. However, it increased SMAD3 nucleus translocation and decreased caspase-3 production. These data suggest that Occludin-TGFβR interaction may inhibit the TGFβ/SMAD3 pathway by hindering Occludin internalization, which affects caspase-3 production. Pharmacological inhibitors SB-525334 and LY2109761, targeting TGFβR kinase activity, did not affect the interaction between TGFβR and Occludin. After TGFβR inhibition, Occludin interaction with EEA1 increased and SMAD3 nuclear translocation decreased compared to TGFβ treatment. Occludin and TGFβR interact before phosphorylation, suggesting that SMAD3 signals play a vital role in regulating caspase-3 signals, which maintain mucosal homeostasis. In the DSS-induced colitis paradigm, Ocln KOIEC mice had downregulated Casp3 and unaltered Tgfb/Smad3 signals, confirming in vitro studies. Finally, the GEO dataset of IBD patients showed downregulation of OCLN, elevated CASP3, and low epithelial marker KRT8, suggesting that further investigation of IBD epithelia needs to be conducted. Conclusions: This study discovers new Occludin deficient effects on apoptosis regulation. It also shows how dropped Occludin levels reduce caspase-3 synthesis in turn lowering apoptosis. Occludin endocytosis and TGFβ/SMAD3 signaling play a significant role in the complicated mechanism. This pathway may be an adaptive strategy to reduce epithelial tissue damage under pathogenic situations.