CagA 轉位及胃上皮細胞緊密接合的損壞：幽門螺旋桿菌可能之致癌機轉

Abstract

雖然世界衛生組織基於流行病學的研究成果，認為幽門螺旋桿菌與人類胃部惡性腫瘤生成有關，特別是與人類胃腺癌形成有著相當密切的關係，已經將幽門螺旋桿菌列為第一類人類致癌因子，但是確實的致病機轉仍尚未被清楚的分析瞭解。由於幽門螺旋桿菌CagA蛋白可以被轉位進入胃上皮細胞，並且在酪氨酸磷酸化模體發生磷酸化，這現象被認為與致癌機轉有關。 所以我們研究CagA酪氨酸磷酸化、酪氨酸磷酸化模體變異性是否與增加致癌風險有關，另外我們建立初次培養人類胃上皮細胞感染幽門螺旋桿菌的模式，希望能比胃癌上皮細胞或非胃上皮細胞的模式，提供更接近人類活體感染的研究方式。 我們研究發現由胃腺癌病人的胃中所分離培養得來的幽門螺旋桿菌菌株發生CagA蛋白酪氨酸磷酸化的比例，比起自一般性胃炎病人的胃中分離得到的菌株，顯著要高了許多（27/29:15/29）。而CagA酪氨酸磷酸化模體變異性與CagA蛋白酪氨酸磷酸化並非絕對相關。所以發生CagA蛋白酪氨酸磷酸化現象與增加致癌風險有關，或者是功能完整的第四型分泌系統與致癌機轉有關。我們也建立初次培養人類胃上皮細胞感染幽門螺旋桿菌的模式，並且發現CagA蛋白酪氨酸磷酸化產生，與細胞形態變化有關；也發現CagA蛋白轉位至胃上皮細胞可以造成細胞緊密接合的破壞。 經由研究顯示能夠被轉位進入胃上皮細胞，並且被磷酸化的CagA蛋白會增加致癌風險，另外由CagA轉位造成細胞緊密接合的破壞而產生的可能致癌機轉，以初次培養上皮細胞的模式來看，是極有可能發生人類活體中。本研究提供對CagA轉位及被磷酸化的致癌機轉更進一步的瞭解。

Background Tyrosine phosphorylation of Helicobacter pylori cytotoxin-associated protein (CagA) of in gastric epithelial cells is reported. Phosphorylated CagA trigger downstream cellular signaling that alters cell morphology and proliferation and induce pro-inflammatory process. Translocated CagA involves epithelial-to-mesenchymal transition and disruption of cellular apical-junction complex in MDCK cells. These events may play important role in the development of malignancy. To clarify these issues, our aims in this study are: 1) to examine the occurrence of CagA tyrosine phosphorylation in H. pylori strains isolated from differnet patients with gastric adenocarcinoma and gastritis, 2) to analyze the relationship between the diversity of tyrosine phosphorylation motifs and the presence of CagA tyrosine phosphorylation, 3) to establish primary human gastric epithelial cell for better understanding effects of CagA protein in non-cancer epithelial cells and 4) to observe cellular junction change in primary human gastric epithelial cell but not non-gastric epithelial cells . Methods Fifty-eight clinical isolates of H. pylori from patients with gastric adenocarcinoma (29 cases) and gastritis (29 cases) were studied for CagA tyrosine phosphorylation by Western blotting. Sequence diversity of tyrosine phosphorylation motifs was analysed among positive- or negative-CagA tyrosine phosphorylation isolates. To elucidate whether events of CagA translocation and phosphorylation take place in normal human gastric epithelium, we infected human primary gastric epithelial cells with H. pylori. Tight junction proteins: ZO-1 and occludin changes were observed by confocal microscopy. Results Positive CagA tyrosine phosphorylation was found in 93.1% (27 of 29) of strains from gastric adenocarcinoma patients and 51.7% (15 of 29) of strains from gastritis patients, (p<0.001). Intact motifs were found in H. pylori isolates with CagA tyrosine phosphorylation. Of the 16 negative CagA tyrosine phosphorylation isolates, intact tyrosine phosphorylation motifs were found in 15 isolates. Our results also demonstrate that CagA protein was translocated into primary gastric epithelial cells and tyrosine phosphorylated. The translocated CagA induces cytoskeleton rearrangement and the disruption of tight junctions in primary gastric epithelial cells. Conclusions CagA tyrosine phosphorylation, which is significantly greater in strains from gastric adenocarcinoma patients, may play a role in gastric carcinogenesis, and could be a better marker of more virulent strains than the cag pathogenicity island in Asia, where the cag pathogenicity island is present in nearly all H. pylori strains. Sequence diversity of tyrosine phosphorylation motifs on CagA was not related to the presence of tyrosine phosphorylation. The absence of tyrosine phosphorylation motif might result in negative tyrosine phosphorylation phenotypes, but such motifs are not the sole factors associated with CagA tyrosine phosphorylation. The establishment of primary culture gastric epithelial cells also provides direct evidence of the modulation of gastric epithelial cells by CagA translocation, and advances our understanding of the carcinogenesis of H. pylori infection.