Uwada Junsuke, Yazawa Takashi, Islam Md Tariqul, Khan Md Rafiqul Islam, Krug Susanne M, Fromm Michael, Karaki Shin-Ichiro, Suzuki Yuichi, Kuwahara Atsukazu, Yoshiki Hatsumi, Sada Kiyonao, Muramatsu Ikunobu, Taniguchi Takanobu
Division of Cellular Signal Transduction, Department of Biochemistry, Asahikawa Medical University, Asahikawa, Japan.
Division of Cellular Signal Transduction, Department of Biochemistry, Asahikawa Medical University, Asahikawa, Japan; Department of Pharmacy, University of Rajshahi, Rajshahi, Bangladesh.
Cell Signal. 2017 Jul;35:188-196. doi: 10.1016/j.cellsig.2017.04.007. Epub 2017 Apr 12.
Intestinal epithelial cells form a tight barrier to act as selective physical barriers, repelling hostile substances. Tumor necrosis factor-α (TNF-α) is a well characterized pro-inflammatory cytokine which can compromise intestinal barrier function and the suppression of TNF-α function is important for treatment of inflammatory bowel disease (IBD). In this study, we investigated the contribution of G-protein-coupled receptor (GPCR)-induced signalling pathways to the maintenance of epithelial barrier function. We first demonstrated the existence of functional muscarinic M3 and histamine H1 receptors in colonic epithelial cell HT-29/B6. As we previously reported, muscarinic M3 receptor prevented TNF-α-induced barrier disruption through acceleration of TNF receptor (TNFR) shedding which is carried out by TNF-α converting enzyme (TACE). M3 receptor-mediated suppression of TNF-α function depends on Gα protein, however, histamine H1 receptor could not ameliorate TNF-α function, while which could induce Gα dependent intracellular Ca mobilization. We found that p38 MAPK was predominantly phosphorylated by M3 receptor through Gα protein, whereas H1 receptor barely upregulated the phosphorylation. Inhibition of p38 MAPK abolished M3 receptor-mediated TNFR shedding and suppression of TNF-α-induced NF-κB signalling. The p38 MAPK was also involved in TACE- mediated EGFR transactivation followed by ERK1/2 phosphorylation. These results indicate that not H1 but M3 receptor-induced activation of p38 MAPK might contribute to the maintenance of epithelial barrier function through down-regulation of TNF-α signalling and activation of EGFR.
肠上皮细胞形成紧密屏障，作为选择性物理屏障，排斥有害物质。肿瘤坏死因子-α（TNF-α）是一种特征明确的促炎细胞因子，它会损害肠道屏障功能，抑制TNF-α功能对于炎症性肠病（IBD）的治疗很重要。在本研究中，我们研究了G蛋白偶联受体（GPCR）诱导的信号通路对上皮屏障功能维持的作用。我们首先证明了结肠上皮细胞HT-29/B6中存在功能性毒蕈碱M3和组胺H1受体。正如我们之前报道的，毒蕈碱M3受体通过加速由TNF-α转换酶（TACE）进行的TNF受体（TNFR）脱落来防止TNF-α诱导的屏障破坏。M3受体介导的TNF-α功能抑制依赖于Gα蛋白，然而，组胺H1受体不能改善TNF-α功能，而它可以诱导Gα依赖性细胞内钙动员。我们发现p38丝裂原活化蛋白激酶（p38 MAPK）主要通过Gα蛋白被M3受体磷酸化，而H1受体几乎不上调磷酸化。抑制p38 MAPK消除了M3受体介导的TNFR脱落和对TNF-α诱导的核因子κB（NF-κB）信号传导的抑制。p38 MAPK还参与了TACE介导的表皮生长因子受体（EGFR）反式激活，随后是细胞外信号调节激酶1/2（ERK1/2）磷酸化。这些结果表明，不是H1受体而是M3受体诱导的p38 MAPK激活可能通过下调TNF-α信号传导和激活EGFR来维持上皮屏障功能。
