緊密連接蛋白ZO-1調節有絲分裂紡錘體方向性促進上皮細胞修復的機制探討

Abstract

免疫誘發的腸道和全身性疾病（包括黏膜損傷）會導致腸道屏障功能降低。為恢復平衡，黏膜迅速癒合至關重要。緊密連接蛋白ZO-1除了已知的屏障功能外，目前研究也發現其可透過有絲分裂紡錘體的排列方向發揮關鍵作用來促進上皮細胞生長，然而，ZO-1調節細胞分裂的分子機制尚未明瞭。 目的：確定ZO-1在黏膜癒合的關鍵過程中調節有絲分裂紡錘體方向的機制。 結果：應用CRISPR/Cas9技術產生ZO-1缺陷的腸上皮Caco-2細胞，與正常Caco-2細胞相比，這些細胞中缺乏ZO-1表達，且屏障功能下降。ZO-1缺失的細胞表現出傷口癒合受損和細胞遷移不良的現象。缺乏ZO-1蛋白的細胞在有絲分裂的Sub-G1期出現程序性細胞凋亡的細胞數量較多，且細胞分裂方向不規則。透過使用三維細胞培養，結果顯示在給予paclitaxel或nocodazole處理增加有絲分裂細胞後，ZO-1的缺失會導致有絲分裂紡錘體的排列方向錯誤。先前的研究顯示，抑制ZO-1的表達會對微絨毛的排列和上皮細胞頂部表面的結構產生負面影響。透過使用抑制和穩定肌動蛋白聚合的latrunculin A、cytochalasin B和jasplakinolide結果證明，ZO-1- cortical actin相互作用的存在不會影響Caco-2細胞中有絲分裂紡錘體的方向。值得一提的是，YBX3和VCL轉錄子的含量增加，這些基因參與細胞分裂方向的調節並與ZO-1交互作用。相反地，缺乏ZO-1的Caco-2細胞，DNMBP和CTTN表達減少。此外，ZO-1的缺失會降低有絲分裂機制和訊號分子的水平，包括pericentrin 和aurora A。同樣地，這些發現也出現在被診斷患有發炎性腸道疾病(IBD)的個體中，這些人的ZO-1表現量降低。ZO-1蛋白透過與細胞骨架結合蛋白（而不是皮質肌動蛋白）相互作用來幫助有絲分裂的有效執行。 結論：這項研究顯示ZO-1在有絲分裂紡錘體的排列方向方面具有還未被探索的非典型功能，這與其參與維持屏障功能無關。根據我們的觀點，ZO-1相關的細胞骨架結合蛋白和轉錄因子YBX3在調節有絲分裂方向上具有重要功能。IBD患者可能由於潛在的根本原因而導致黏膜癒合受損。

Immune-mediated intestinal and systemic illnesses that include mucosal injury result in a reduced intestinal barrier function. It is crucial that the mucosa be promptly healed in order to restore balance. Zonula occludens-1 (ZO-1) facilitates the growth of epithelial cells by playing a crucial role in the orientation of the mitotic spindle, irrespective of its barrier function. However, the mechanism by which ZO-1 drives cell division has not yet been established. Objective: To determine the mechanism by which ZO-1 regulates the orientation of the mitotic spindle, a crucial process for mucosal healing. Results: Applying CRISPR/Cas9 technology to generate ZO-1-deficient intestinal epithelial Caco-2 cells demonstrated the absence of ZO-1 expression in these cells, leading to a decreased barrier function compared to cells that do express ZO-1. Cells missing ZO-1 exhibited impaired healing of purse-string wounds and poor cell migration. Cells lacking the ZO-1 protein show a higher number of cells undergoing programmed cell death in the sub-G1 phase of mitosis, as well as irregular cellular orientation. Through the use of three-dimensional cell culture, it was shown that the absence of ZO-1 led to a misorientation of the mitotic spindle after exposure to paclitaxel or nocodazole. Previous studies have shown that suppressing the expression of ZO-1 has negative consequences on the arrangement of microvilli and the structure at the top surface of epithelial cells. By using latrunculin A, cytochalasin B, and jasplakinolide, which inhibit and stabilize actin polymerization, the presence of ZO-1-cortical actin interaction did not influence the orientation of the mitotic spindle in Caco-2 cells. It is important to mention that the levels of YBX3 and VCL transcripts were increased. These genes are involved in the regulation of cell division direction and interact with ZO-1. Conversely, cells lacking ZO-1, DNMBP and CTTN exhibited a decrease in expression. In addition, the absence of ZO-1 reduces the levels of mitotic machinery and signaling molecules, including pericentrin and aurora A. Similarly, the findings were seen in individuals diagnosed with inflammatory bowel disease (IBD) who have decreased levels of ZO-1 expression. The ZO-1 protein aids in the effective execution of mitosis by interacting with cytoskeletal binding proteins, rather than cortical actin. Conclusion: This work reveals that ZO-1 has an unexplored, unconventional function in the orientation of the mitotic spindle, which is not related to its involvement in maintaining barriers. According to our perspective, ZO-1-associated cytoskeletal binding proteins and transcription factor YBX3 have significant functions in regulating mitotic direction. Patients with IBD may have impaired mucosal healing due to a potential underlying cause.