Trefoil factor family (TFF)は消化管粘膜防御において重要なペプチドであるが,胃粘膜で高レベルに発現しているTFF1 (pS2)の作用機構については不明の部分が多い.本研究では胃粘膜上皮細胞のTFF1発現に対するcAMP/PKA経路の影響を,ヒト胃癌由来細胞株MKN45及びラット正常胃粘膜上皮由来のRGM-1を用いて検討した.定量的RT-PCRにより内因性TFF1 mRNAの発現を測定し,ヒトTFF1遺伝子のプロモーター領域(-953から+34)をpGL3-basicベクターに組み込みんだTFF1レポーター遺伝子を用いてTFF1プロモーター活性を評価した. Forskolin, DbcAMP刺激によりMKN45細胞において内因性TFF1 mRNA発現及びTFF1プロモーター活性は増強した.さらに段階的にTFF1プロモーター領域を欠失させたレポーターを作製し検討するとTFF1遺伝子プロモーター上でcAMP/PKA経路の活性化に対する応答領域は-456から-327にマップされた. MKN45細胞において,消化管防御機能を有するPGE_2の受容体であるEP2とEP4の発現が認められ,さらにPGE_2によりTFF1の発現レベルが上昇することも確認された. Forskolin, PGE_2によるTFF1発現の増強はRGM-1細胞でも認められた.これらの結果より, cAMP/PKA経路は胃粘膜上皮細胞においてTFF1の発現調節に関与し, PGE_2はcAMP/PKA経路を介してTFF1発現を増強していると考えられた.Trefoil factor family (TFF) is a group of small peptides that play important roles in the defense of the gastrointestinal mucosa. Among TFF subtypes, TFF1 is expressed at high levels in gastric epithelial cells. However, the regulatory mechanisms of gastric TFF1 expression are not fully understood. In this study, we examined whether cAMP/PKA pathway modulates the expression of TFF1 in gastric epithelial cells. MKN45 gastric cells were used. RGM-1, a cell line derived from normal rat gastrcinc mucosa, was also used in some experiments. Endogenous EP1-EP4 receptor expression was examined by conventional RT-PCR. Endogenous TFF1 mRNA expression was analyzed by real-time quantitative RAT PER. The promotor sequence of the human TFF1 gene (-956 to +36) was cloned into pGL3-basic vector to make a TFF1 reporter gene (TFF1-Luc) and various mutant reporters were also made. In each reporter gene assay, phRL-TK vector was co-transfected for standardization. Forskolin or DbcAMP signifcanlty up-regulated the expression of endogenous TFF1 mRNA and TFF1 reporter genes in MKN45 cells. cAMP/PKA responsive element was mapped between -456 and -327 of the TFF1 gene promoter. EP2 and EP4 receptors, which link to Gs, were expressed in MKN45 cells, and PGE_2 was found to up-regulate TFF1 expression. In RGM-1cells, forskolin, and PGE_2 also increased the expression of endogenous TFF1 mRNA. These results suggest that cAMP/PKA pathway is involved in the regulation of TFF1 expression in gastric epithelial cells, and that PGE_2, a gastroprotective prostaglandin, up-reglates TFF1 expression through cAMP/PKA pathway
