The role of Helicobacter pylori CagA in regulation of Bcl-2, Bcl-xL and Bcl-6

胃幽門螺旋桿菌（Helicobacter pylori）在分類上屬於革蘭氏陰性菌，病人感染胃幽門螺旋桿菌會造成胃炎、胃潰瘍、十二指腸潰瘍、胃線癌以及胃黏膜相關淋巴組織淋巴瘤。胃幽門螺旋桿菌致病因子CagA是分子量120~145 kD的蛋白質，可經由第四型分泌系統(type IV secretion system)注入宿主細胞內。被送入宿主細胞的CagA會被宿主細胞內的磷酸酶所磷酸化。而磷酸化CagA可與SHP-2結合並活化MAPK訊息傳遞途徑。cagA基因轉殖鼠內，發現除了有胃癌與胃腺癌形成外，有些小鼠會有B淋巴癌生成，但磷酸化耐受性CagA(PR-CagA)的基因轉殖鼠內則觀察不到癌症生成，顯示CagA的磷酸化在B細胞轉型扮演重要角色。為了研究胃幽門螺旋桿菌CagA如何造成B細胞轉型的機制，我們假設胃幽門螺旋桿菌可以直接將CagA送入人類B細胞中，造成B細胞轉型的機率增加。首先我們利用胃幽門螺旋桿菌與BJAB細胞共同培養的系統，發現胃幽門螺旋桿菌能直接將CagA送入B細胞中，且隨著菌量以及時間的增加，可以偵測到磷酸化CagA的增加。為了進一步探討胃幽門螺旋桿菌是否透過CagA影響B細胞的存活，我們觀察CagA對抗細胞凋亡分子Bcl-2與Bcl-xL的影響，結果顯示胃幽門螺旋桿菌可誘導較高的Bcl-2與Bcl-xL表現量，然而在cagA基因剔除菌株中Bcl-2以及Bcl-xL表現量則並不會被誘導起來，因此在胃幽門螺旋桿菌CagA可影響B細胞抗細胞凋亡分子的表現。接著我們探討CagA是否會影響B cell的Bcl-6。我們發現胃幽門螺旋桿菌會抑制Bcl-6蛋白質表現，但是在與cagA基因剔除菌株共同培養後，Bcl-6表現量的抑制則不明顯。利用real-time PCR測量Bcl-2、Bcl-xL以及Bcl-6 mRNA表現，也發現到Bcl-2以及Bcl-xL的 mRNA表現量會被胃幽門螺旋桿菌誘導起來，而Bcl-6的 mRNA表現則會受到抑制，然而在cagA基因剔除菌株與B細胞共同培養的組別並沒有觀察到相同結果。為了觀察SHP-2在胃門幽桿菌調控Bcl-2、Bcl-xL以及Bcl-6的角色，我們利用NSC-87877抑制劑來抑制SHP-2的活性以及利用SHP-2 siRNA抑制SHP-2表現。結果發現Bcl-2, Bcl-xL以及Bcl-6的調控並未受到影響。為了觀察是否CagA單獨存在於B細胞中即會影響B細胞的分子調控，因此我們將野生型CagA以及磷酸化耐受性CagA (PR-CagA)轉染於B細胞中。我們發現在B細胞的Bcl-2以及Bcl-xL表現量在轉染野生型CagA或是磷酸化耐受性CagA的組別皆會被誘導，顯示Bcl-2以及Bcl-xL表現的調控為磷酸化CagA不依賴性。為了探討胃幽門螺旋桿菌是否會增加B細胞的存活，我們給予細胞etoposide測試細胞存活率。我們觀察到B細胞與野生型胃幽門螺旋桿菌共同培養的組別中，細胞的存活率在加入etoposide之後與控制組別差不多，然而在與cagA基因剔除胃幽門螺旋桿菌HM-6共同培養的組別中，細胞死亡情況嚴重。顯示野生型胃幽門螺旋桿菌HM-6可以增加B細胞的存活率。而在轉染CagA到B細胞之後，野生型CagA以及磷酸化耐受性CagA，皆可增加B細胞的存活率。因此抗細胞凋亡分子的表現可能參與胃幽門螺旋桿菌造成胃黏膜相關淋巴組織淋巴瘤的形成機制之一。Helicobacter pylori is a Gram-negative bacteria. Infection with Helicobacter pylori leads to chronic gastrits, peptic ulcer disease, adenocarcinoma and gastric MALT lymphoma. CagA, a virulent factor of Helicobacter pylori, is an 120~145 kD bacteria protein. CagA could be translocated into the host cell via type IV secretion system. Within host cells, CagA is phosphorylated by protein kinase, and the phospho-CagA could bind to SHP-2, and to activate intracellular MAPK kinase signaling pathway. In cagA transgenic mice, gastric cancer and adenocarcinomas, as well as hematological malignancies were observed to develop in the CagA transgenic mice, but not in transgenic mice expressing phosphorylation-resistant CagA, suggesting that phosphorylation of CagA plays an important role in transformation of B cell. To study the mechanism of Helicobacter pylori CagA in B cell transformation, we proposed that Helicobacter pylori CagA could directly translocated into B cell, and increase the potential of B cell transformation. First, we found that H. pylori translocates CagA directly into B cell and undergoes phosphoralytion in H. pylori and BJAB cell co-culture system in a dose- and time-dependent manner. To further examine whether H. pylori CagA could promote B cell survival via CagA, we investigate the effect of CagA on antiapoptotic molecules Bcl-2 and Bcl-xL. We demonstrated that H. pylori induced higher Bcl-2 and Bcl-XL expression, not in cagA knock-out H. pylori strain in human B cell lines, indicating H. pylori CagA may increase B cell survival. Next we investigated the effects of CagA on expression of Bcl-6. We found that H. pylori could repress Bcl-6 expression, but not in cagA knock-out H. pylori strain. Expression of Bcl-2 and Bcl-xL mRNA is up-regulated while expression of Bcl-6 mRNA is inhibited in wild type but not in cagA knock-out H. pylori strain in real-time PCR analysis. To further investigate the role of SHP-2 in the regulation of Bcl-2 Bcl-xL and Bcl-6 expression, we used SHP-2 inhibitor NSC-87877 to inhibit SHP-2 activity and SHP-2 siRNA to knockdown SHP-2 expression. Our results demonstrated the effects on Bcl-2, Bcl-xL and Bcl-6 is not affected by knocking down SHP-2. To further confirm whether CagA may directly regulate Bcl-2, Bcl-xL and Bcl-6, we transfected WT-CagA or PR-CagA into B cell. The expression of Bcl-2 and Bcl-xL are induced in B cell transfected with either WT-CagA or PR-CagA, indicating that the expression of Bcl-2 and Bcl-xL are phospho-CagA independent. In order to examine whether H. pylori promote B cell survival, we treated B cell with etoposide after co-culture with H. pylori. The results showed that the survival of B cell after co-cultured with cagA knock-out strain was significantly decreased; in contrast, B cell transfected with WT-CagA or PR-CagA showed increased survival rate compare to vector control, suggesting CagA can promote B cell survival. Thus anti-apoptotic molecules induced by CagA might play an important role in transformation of B cell, and in the development of MALT lymphoma