Department of Physiology, The Faculty of Medicine, Jagiellonian University Medical College, Cracow, Poland.
Department of Cell Biology, The Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Cracow, Poland.
Helicobacter. 2019 Oct;24(5):e12653. doi: 10.1111/hel.12653. Epub 2019 Aug 14.
Colonization of the gastric mucosa with Helicobacter pylori (Hp) leads to the cascade of pathologic events including local inflammation, gastric ulceration, and adenocarcinoma formation. Paracrine loops between tissue cells and Hp contribute to the formation of gastric cancerous loci; however, the specific mechanisms underlying existence of these loops remain unknown. We determined the phenotypic properties of gastric fibroblasts exposed to Hp (cagA+vacA+) infection and their influence on normal epithelial RGM-1 cells.
RGM-1 cells were cultured in the media conditioned with Hp-activated gastric fibroblasts. Their morphology and phenotypical changes associated with epithelial-mesenchymal transition (EMT) were assessed by Nomarski and fluorescence microscopy and Western blot analysis. Motility pattern of RGM-1 cells was examined by time-lapse video microscopy and transwell migration assay. The content of TGF-β in Hp-activated fibroblast-conditioned media was determined by ELISA.
The supernatant from Hp-activated gastric fibroblasts caused the EMT-like phenotypic diversification of RGM-1 cells. The formation of fibroblastoid cell sub-populations, the disappearance of their collective migration, an increase in transmigration potential with downregulation of E-cadherin and upregulation of N-cadherin proteins, prominent stress fibers, and decreased proliferation were observed. The fibroblast (CAF)-like transition was manifested by increased secretome TGF-β level, α-SMA protein expression, and its incorporation into stress fibers, and the TGF-βR1 kinase inhibitor reduced the rise in Snail, Twist, and E-cadherin mRNA and increased E-cadherin expression induced by CAFs.
Gastric fibroblasts which are one of the main targets for Hp infection contribute to the paracrine interactions between Hp, gastric fibroblasts, and epithelial cells. TGF-β secreted by Hp-activated gastric fibroblasts prompting their differentiation toward CAF-like phenotype promotes the EMT-related phenotypic shifts in normal gastric epithelial cell populations. This mechanism may serve as the prerequisite for GC development.
幽门螺杆菌（Hp）定植于胃黏膜，引发一连串病理事件，包括局部炎症、胃溃疡和腺癌形成。组织细胞与 Hp 之间的旁分泌环促进胃癌灶的形成；然而，这些环存在的具体机制尚不清楚。我们确定了暴露于 Hp（cagA+vacA+）感染的胃成纤维细胞的表型特性及其对正常上皮 RGM-1 细胞的影响。
将 RGM-1 细胞培养在 Hp 激活的胃成纤维细胞条件培养基中。通过相差和荧光显微镜以及 Western blot 分析评估与上皮-间充质转化（EMT）相关的形态和表型变化。通过延时视频显微镜和 Transwell 迁移测定法检查 RGM-1 细胞的迁移模式。通过 ELISA 测定 Hp 激活的成纤维细胞条件培养基中 TGF-β的含量。
Hp 激活的胃成纤维细胞的上清液引起 RGM-1 细胞发生 EMT 样表型多样化。形成成纤维细胞样细胞亚群，消失其集体迁移，迁移潜能增加，E-钙粘蛋白下调，N-钙粘蛋白蛋白上调，出现明显的应激纤维，增殖减少。成纤维细胞（CAF）样转化表现为分泌的 TGF-β水平增加，α-SMA 蛋白表达增加，并整合到应激纤维中，TGF-βR1 激酶抑制剂减少了 CAFs 诱导的 Snail、Twist 和 E-钙粘蛋白 mRNA 的升高以及 E-钙粘蛋白表达的增加。
Hp 感染的主要靶标之一胃成纤维细胞有助于 Hp、胃成纤维细胞和上皮细胞之间的旁分泌相互作用。Hp 激活的胃成纤维细胞分泌的 TGF-β促使其向 CAF 样表型分化，促进正常胃上皮细胞群体发生 EMT 相关表型改变。这种机制可能是 GC 发展的前提。
