レチノインサン ワ ダイチョウ ジョウヒ サイボウ ニ オイテ NF-k B シグナリング オ カッセイカ スル

All-trans retinoic acid(ATRA)は核内受容体であるretinoic acid receptor(RAR)のリガンドであり,消化管を含む多くの組織で重要な生理作用を有している.最近,臨床的なレチノイン酸の使用と炎症性腸疾患との関連が注目されているが,この問題に関する基礎的な検討は行われていない.今回我々は,大腸癌由来細胞株を用いて,炎症・免疫応答において中心的な役割を果たしているnuclear factor- k B(NF-k B)シグナリングに対するATRA の影響について,レポーター遺伝子解析法,リアルタイム定量的 RT-PCR法にて検討を行った.ATRA および他のレチノイド化合物(9-cis retinoic acid,13-cis retinoic acid,AM580,retinol)は,大腸癌由来細胞株において用量依存性に NF-k B を活性化した.また,ATRA はNF-k B の代表的な標的遺伝子であるIL-8の発現を誘導した.RAR の活性化と比較するとNF-k B 活性化に要するATRA の濃度域は高かった.TNF-a によるNF-k B の活性化,IL-8発現誘導は,ATRA をプレインキュベーションすることにより著明に増大し,相乗的な効果が認められた.ATRA はNF-k B サブユニット(RelA,p50)の発現を増大させ,TNF受容体(TNFR1)の発現レベルも上昇させていた.これらの結果より,レチノイン酸は大腸上皮細胞においてNF-k B シグナリングを活性化し,TNF-a に対する感受性を増大させている可能性が示唆された.All-trans retinoic acid(ATRA)is a ligand for the retinoicacid receptor(RAR), a member of the nuclear receptorsuperfamily, and it is well established that RARs play acritical role in the development and differentiation of variousorgans including gastrointestinal tract. Recently, severalcase reports suggested a possible association between theclinical use of retinoic acid and inflammatory bowel diseases.However, it is not known whether ATRA affects the inflammatoryresponse of colonic epithelial cells. In this study,we examined the effect of ATRA on NF-k B activity andIL-8 expression in colonic epithelial cells in vitro. NF-k Bactivation and RAR activation were assessed by the reportergene assay and IL-8 mRNA expression was assessed bythe real-time quantitative RT-PCR. ATRA and other retinoidcompounds(9-cis retinoic acid, 13-cis retinoic acid,AM580 and retinol)activated NF-k B in a dose- and timedependentmanner in colonic cell lines(HCT116, Caco2,SW480, DLD-1, and LS174T). However, compared to RARactivation, much higher concentrations of ATRA wereneeded to activate NF-k B. ATRA also up-regulated theexpression of IL-8, a target gene of NF-k B. ATRA-inducedNF-k B activation was repressed by a MEK inhibitorand a p38 MAP kinase inhibitor. Preincubation with ATRAsignificantly potentiated TNF-a -induced activation of NFkB and TNF-a -induced expression of IL-8. ATRA wasfound to up-regulate the expression of NF-k B subunits(RelA and p50)and TNF-a receptor 1. These results suggestthat ATRA and other retinoid compounds can activateNF-k B signaling and potentiate the inflammatory responseto TNF-a in colonic epithelial cells

イ ネンマク ジョウヒ サイボウ ノ TFF1 ハツゲン ニ タイスル cAMP/Protein kinase A ケイロ ノ エイキョウ

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

イネンマク ジョウヒ サイボウ ノ trefoil factor family ハツゲン ニ オヨボス indomethacin ノ エイキョウ

Trefoil factor family(TFF)に属するペプチド(TFF1,TFF2,TFF3)は,消化管粘膜の健常性維持,傷害修復過程に重要な役割を果たしているが,その発現調節については不明の部分が多い。本研究では,胃癌由来細胞株であるMKN45細胞を用いて,胃粘膜傷害の起因因子であるNSAIDsのTFF発現に対する影響をリアルタイム定量的RT-PCR法で解析した。MKN45細胞では,TFF1の発現レベルが最も高く,次いでTFF2であり,TFF3の発現レベルは非常に低かった。IndomethacinはTFF1,TFF3の発現に対しては有意な影響を与えず,TFF2の発現は濃度依存性に上昇させた。IndomethacinによるTFF2発現誘導は他の胃癌由来細胞株(AGS,JR)でも認められ,また,他のNSAIDs (aspirin,NS-398)もMKN45細胞においてTFF2発現を上昇させた。このindomethacinの効果は外来性のPGE2の投与では拮抗されなかった。以上の結果から,NSAIDsはそのCOX抑制作用とは別の機構を介して胃粘膜上皮細胞のTFF2発現を誘導していることが示唆された。NSAIDsの投与によるTFF2発現レベルの上昇は,NSAIDs起因性胃粘膜傷害の発生をある程度防御している可能性が考えられるAlthough trefoil factor family (TFF) peptides (TFF1, TFF2, and TFF3) are assumed to play important roles in the protection of gastrointestinal mucosa, regulatory mechanisms of TFF expression are poorly understood at present. In the present study, we examined the effect of indomethacin, a non-selective inhibitor of cyclooxygenase (COX), on the expression of TFF mRNA expression in MKN 45 gastric cells by real-time quantitative RT-PCR method. In MKN 45 cells, relative expression level of TFF1, TFF2, and TFF3 mRNA was 617 : 12 : 1 in the control condition. Indomethacin up-regulated the expression level of TFF 2 in a dose-dependent manner, whereas TFF 1 and TFF 3 expression levels were not significantly affected. Luciferase reporter gene assay confirmed this stimulatory effect of indomethacin on TFF 2 expression. Other COX inhibitors, such as aspirin and NS -398, also up-regulated TFF 2 expression and indomethacin-induced up-regulation of TFF2 expression was also observed in other gatric cell lines, such as AGS and JR. Externally applied PGE2 did not antagonize the effect of indomethacin on TFF2 expression. Since TFF peptides play a critical role in gastric mucosal protection, indomethacin-induced TFF2 may reduce the degree of gastric mucosal damage induced by indomethacin. The present results also suggest that the effect of indomethacin on TFF2 expression is COX-independent

イ ネンマク ジョウヒ サイボウ ニオケル trefoil factor family 1 (TFF1) ハツゲン ニ エイキョウ オ アタエル インシ ニ カンスル ケントウ

乳癌由来細胞株MCF-7でエストロゲン誘導遺伝子として見出されたTFF1は,生理的には胃粘膜に発現し重要な粘膜防御因子として機能している.本研究では,胃癌由来細胞株MKN45を用い,胃におけるTFF1発現調節機構について検討した.MCF-7ではTFF1発現はエストロゲン依存的であったが,MKN45ではTFF1の発現はエストロゲン非感受性であり,TFF1遺伝子プロモーター上のエストロゲン応答配列よりもさらに近位側の配列が,MKN45におけるTFF1発現に大きな影響を与えていた.MKN45にもエストロゲン受容体(ERα,ERβ)の発現は認められたが,MCF-7と比べるとERαの発現レベルが低く,ERαを強制的に発現させるとMKN45でもTFF1発現がエストロゲン感受性となった.胃粘膜でのTFF1発現は粘膜損傷時に上昇することが知られているので,このような誘導性のTFF1発現機構を検討する目的でphorbol ester (TPA),TNF-αの影響をみたところ,TPAはAP-1を介して,TNF-αはNF-κBを介してTFF1の発現を上昇させることが示唆された.これらの結果より,胃粘膜上皮細胞ではTFF1発現はエストロゲン非依存性であり,また,AP-1やNF-κBが誘導性の発現調節に関与していると考えられた.Trefoil factor family 1 (TFF1) is a protease-resistant polypeptide expressed at a high level in gastric epithelial cells and plays a critical role in the defense and repair of gastric mucosa. In the present study, we examined the regulatory mechanisms of gastric TFF1 expression using a gastric cancer cell line, MKN45. Since TFF1 was originally discovered as an estrogen-inducible gene in a breast cancer cell line, MCF-7, MCF-7 was also examined for comparison. The promoter sequence of human TFF1 gene (-953 to +34) was cloned into pGL3 basic vector to make a TFF1 reporter gene and several deletion mutant reporters were also made. Endogenous TFF1 mRNA expression was analyzed by real-time quantitative RT- PCR. In MCF- 7 cells, basal TFF1 expression was dependent on the presence of an estrogen-responsive element (ERE) (-406), while deletion of ERE had no significant effect on the reporter gene expression in MKN45 cells. In MKN45 cells, deletion of more proximal region (-393 to -365) significantly affected the reporter gene expression. Compared to estrogen receptor β (ER β), the expression level of estrogen receptor α (ER α) was low in MKN45 cells and we found that TFF1 expression became estrogen-sensitive when ER a was overexpressed in MKN45 cells. Phorbol-12-miristate-13-acetate (TPA) and tumor necrosis factor-α (TNF-α) up-regulated the expression of endogenous TFF1 mRNA and TFF1 reporter genes. A series of reporter gene experiments suggested that AP-1 site (-338) and NF-κB sites (-251, -230) are involved in the action of TPA and TNF-α, respectively. These results suggest that, compared to MCF-7 cells, the basal TFF1 expression is differently regulated in gastric epithelial cells, and that up-regulation of TFF1 expression, which is often observed at the site of gastric mucosal lesions, may be mediated by AP-1 and/or NF-κB

Surface Potential Studies on Adsorption Processes of Clay Nanosheets onto a Floating Molecular Film of an Amphiphilic Alkylammonium Cation

A floating molecular film was formed when a chloroform solution of dimethyldioctadecylammonium bromide (DMDOA⁺Br^–) was spread on an aqueous dispersion of a clay mineral (sodium montmorillonite). At a low concentration (<50 ppm: ppm = mg dm^–3), a clay mineral was exfoliated into negatively charged layers (denoted by clay nanosheets). Clay nanosheets in a dispersion were adsorbed onto a floating film because of electrostatic interactions. At various clay concentrations (0–50 ppm), surface potential was measured as a function of time to obtain the quantitative information about the adsorption of clay nanosheets on a condensed floating film of DMDOA⁺ ions. It was concluded that the adsorption equilibrium obeyed the Langmuir adsorption isotherm, which was supported by the atomic force microscopy (AFM) observation. The rate constants of adsorption and desorption processes were determined by the fitting analyses based on the Langmuir type kinetics. Interestingly, the delay of the adsorption was observed in the early stage indicating that clay nanosheets were removed from the surface region through the repulsion by a counteranion (Br^–). This explanation was supported by the model simulation using the forward difference method