β5インテグリン分子の多様性の病的意義

金沢大学医学部前年度の研究より、一般人の10%に存在することが明らかになった変異型(FNK欠損)のβ5インテグリン機能を知るために、αvを発現しているがβ5を発現しないCHO-K1細胞への変異型cDNAの遺伝子導入の準備を開始始した。まず、発現ベクターに組み込まれた通常型β5インテグリンcDNA(β5pcDNA1/Neo)は米国、Scripps研究所のCheresh博士から分与された。変異型β5インテグリンcDNAについては、変異型5\u27末端に近い9bp欠損部分の短い領域をPCR法を用いて増幅し、制限酵素を用いて変異型の同部分と置き換える方法をとった。適切な制限酵素部位は欠損部より5\u27側に一カ所(BspHI)あったが、欠損部の3\u27側にはなかったので、3\u27側の切断はベクターのクローニングサイト(Xbal)を利用した。変異型9bp欠損部は、通常型β5と変異型β5のヘテロ接合体であるKATO-III細胞のcDNAのPCR産物からTAクローニング法でサブクローニングした。しかしながら、通常型はベクター内に正方向または逆方向にそれぞれ同じ確立(6/12)で挿入されたにも関わらず、変異型は常に逆方向に挿入された(0/11)。従って、逆方向の挿入片の目的部分をBspHIとBamHIで切り出し、BamHI切断部を平滑末端とし、さらにXbaIを有するリンカーを付加した。同様に正常型β5cDNAを有するβ5pcDNA1/Neoから変異部に相当する部分をBspHIとXbaI同じ制限酵素を用いて除去し、変異末端フラグメントを挿入した。この後にCHO-K1細胞に正常型または変異型のβ5cDNAを遺伝子導入する予定である。研究課題/領域番号:09877044, 研究期間(年度):1997 – 1998出典：研究課題「β5インテグリン分子の多様性の病的意義 」課題番号09877044（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09877044/）を加工して作

Protein Phosphatase 2A Regulatory Subunit B Promotes MAP Kinase-mediated Migration of A431 Cells

金沢大学大学院医学系研究科保健学専攻Background/Aims: Phosphatases are involved in regulation of MAP kinase (MAPK). A431 cells migrate on collagen after EGF stimulation using MAPK. To clarify the involvement of PP2A in this MAPK-dependent migration, the expression of an isoform of the B regulatory subunit was inhibited. Methods: An antisense sequence corresponding to B cDNA was transfected into A431 cells. Their migratory activity on collagen was examined using Transwell, and MAPK phosphorylation and phosphatase activity were measured, and the results were compared with those obtained with mock-transfected cells. Results: Antisense-transfected cells showed less B protein and phosphatase activity than mock-transfected controls. Migration of antisense-transfected cells showed a low response to EGF. The response of MAPK phosphorylation of antisense-transfected cells to EGF stimulation and adhesion to collagen in the presence or absence of EGF were markedly decreased. Phosphatase activity of PP2A-B also did not respond to EGF, collagen or EGF plus collagen, and remained at low levels. Conclusion: These results suggested that PP2A-B promotes cell migration through the MAPK cascade

Migratory phenotypes of HSC-3 squamous carcinoma cell line induced by EGF and PMA: Relevance to migration of loosening of adhesion and vinculin-associated focal contacts with prominent filopodia

金沢大学大学院医学系研究科保健学専攻Cell migration is involved in carcinoma cell invasion and wound healing. We examined motogenic cytokines that potentiated migration of human HSC-3 carcinoma cells. To assess migratory activity, modified Boyden chambers were used. Among a variety of potential motogenic cytokines, epidermal growth factor (EGF) enhanced migration of HSC-3 cells both on collagen and fibronectin. Phorbol myristate acetate (PMA) also enhanced migration. Inhibitors of protein kinase C completely inhibited PMA-induced migration, but only partly inhibited EGF-induced migration. Protein kinase A was also involved in the EGF-induced signaling pathway for migration. Although the signaling pathways were independent, and the cell shape on collagen was different from that on fibronectin, migratory cells stimulated by EGF or PMA showed common morphology on different ligands. The cells were polygonal or round in shape and the loss of long cytoplasmic extensions was noted. Migratory HSC-3 cells stimulated by EGF or PMA became less adhesive to collagen and fibronectin. Since both EGF- and PMA-stimulated migration did not require de novo protein synthesis, the signaling pathways possibly lead to assembly and disassembly of an actin cytoskeleton. Immunofluorescence for vinculin was concentrated into focal contacts in EGF- and PMA-stimulated HSC-3 cells, whereas the fluorescence signal was hardly detected in non-stimulated cells. Talin and β1 integrin were immunolocalized at focal contacts in non-stimulated cells, and it remained unchanged in stimulated cells. Numerous filopodia visualized with actin immunofluorescence were formed around stimulated HSC-3 cells, whereas filopodia were short and sparse around elongated cytoplasms in non-stimulated cells. Thus, shortening of cytoplasmic extensions with numerous filopodia, loosening of adhesion, and vinculin-associated focal contacts were regarded as migratory phenotypes

ヘムオキシゲナーゼ1によるクルクミン誘導末梢血単球炎症性サイトカイン産生の制御

クルクミン(diferuloyl methane)は香辛料ターメリックの活性成分であり、試験管内あるいは生体内において強い抗酸化あるいは抗炎症作用などを示すとされているが、このような生物活性の機序としてheme oxygenase-1 (HO-1)の誘導を介することが知られている。本研究では、末梢血単球の炎症性サイトカイン産生におけるクルクミンの関与を明らかにすることを目的として行った。その結果、クルクミンは lipopolysaccharide により末梢血単核球に誘導されたTNF-alpha や IL-6の産生を抑制した一方で、IL-10やHO-1活性を有意に増強した。また、本研究で用いたクルクミンの濃度範囲において、アポトーシスや細胞死はほとんど認めなかったことから、クルクミンによる炎症性サイトカインの抑制効果はクルクミンの細胞毒性に起因したものではないといえた。さらに、HO活性抑制物質SnPP添加により単核球の培養後期においてクルクミンの抑制効果は部分的に解除された、このことから、クルクミンはHO-1産生を介した炎症性サイトカインの抑制を部分的に誘導することが示唆された一方で、他機序を介した単球への関与についても示唆された。クルクミンのような非細胞毒性を示す薬剤を利用することによる単球機能の調節は、さまざまな炎 症性疾患の新しい抗炎症治療への可能性が期待される。Curcumin (diferuloyl methane) is the active component of the spice turmeric and it exerts potent antioxidant and anti-inflammatory functions both in vitro and in vivo. The mechanism through which curcumin exhibits its biological functions is through induction of heme oxygenase-1 (HO-1). In the present study, we aimed to elucidate the direct effect of curcumin on inflammatory cytokine production by irculating monocytes. Curcumin inhibited lipopolysaccharide induced production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) by peripheral blood mononuclear cells, whereas it induced significant levels of interleukin-10 (IL-10) and HO-1. The inhibitory effect of curcumin was not via cytotoxicity of the reagent because there was no significant apoptosis or cell death induced over the range of concentrations used for the assay. The inhibitory effect of curcumin was partially abrogated by adding HO inhibitor SnPP at the late phase of the culture, indicating that the curcumin induced suppression of inflammatory cytokine is partly through production of HO-1. In addition, curcumin may act on monocytes through multiple mechanisms to regulate its inflammatory cytokine production. Modulation of monocyte functions by non-cytotoxic reagent such as curcumin may offer a novel anti-inflammatory therapeutics for the treatment of various inflammatory disorders

Dynamic Regulation of Extracellular Signal-Regulated Kinase (ERK) by Protein Phosphatase 2A Regulatory Subunit B56γ1 in Nuclei Induces Cell Migration

Extracellular signal-regulated kinase (ERK) signalling plays a central role in various biological processes, including cell migration, but it remains unknown what factors directly regulate the strength and duration of ERK activation. We found that, among the B56 family of protein phosphatase 2A (PP2A) regulatory subunits, B56γ1 suppressed EGF-induced cell migration on collagen, bound to phosphorylated-ERK, and dephosphorylated ERK, whereas B56α1 and B56β1 did not. B56γ1 was immunolocalized in nuclei. The IER3 protein was immediately highly expressed in response to costimulation of cells with EGF and collagen. Knockdown of IER3 inhibited cell migration and enhanced dephosphorylation of ERK. Analysis of the time course of PP2A-B56γ1 activity following the costimulation showed an immediate loss of phosphatase activity, followed by a rapid increase in activity, and this activity then remained at a stable level that was lower than the original level. Our results indicate that the strength and duration of the nuclear ERK activation signal that is initially induced by ERK kinase (MEK) are determined at least in part by modulation of the phosphatase activity of PP2A-B56γ1 through two independent pathways. © 2013 Kawahara et al

EGF and β1 Integrin Convergently Regulate Migration of A431 Carcinoma

金沢大学大学院医学系研究科We found that the convergently epidermal growth factor (EGF)-induced signal and the collagen-induced signal activate mitogen-activated protein kinase (MAPK), which induces migration. We examined the signaling mechanisms of EGF-induced cell migration on collagen using the A431 carcinoma cell. EGF (10 ng/ml) induced migration on collagen, but inhibited proliferation. Using a MAPK cascade inhibitor, PD98059, it was shown that EGF-induced migration on collagen was mediated by MAPK whereas EGF-induced migration on fibronectin and vitronectin was not. PD98059 also showed that activation of MAPK induced by EGF enhanced the adhesiveness of A431 cells to collagen. By Western blotting analysis, the kinetics of MAPK phosphorylation induced by EGF and collagen was examined separately, and convergently. First of all, EGF without collagen caused transient MAPK phosphorylation. Collagen without EGF caused MAPK to be immediately and transiently dephosphorylated, and rephosphorylated followed by sustained hyperphosphorylation. EGF together with collagen caused an immediate, and sustained, hyperphosphorylation. These facts suggest that the transient MAPK dephosphorylation induced by collagen is required for migration in order to maintain an appropriate level of sustained phosphorylation. Furthermore, we found that adhesion of A431 cells to collagen was blocked by the anti-β1 integrin antibody or by the mixed antibodies composed of anti-α1, -α2, and -α3 antibodies, indicating that collagen-induced MAPK phosphorylation was mediated through α1β1, α2β1, and α3β1 integrins. © 2002 Elsevier Science

Inhibitory effects of adhesion oligopeptides on the invasion of squamous carcinoma cells with special reference to implication of αv integrins

金沢大学大学院医学系研究科保健学専攻We studied invasion-related adhesion events in vitro using three squamous carcinoma cell lines (HSC-3, poorly differentiated type; OSC-19, well-differentiated type; and KB cells, undifferentiated type). An in vitro invasion assay through matrigel in the transwell chamber revealed that HSC-3 cells were most invasive, OSC-19 cells moderately invasive and KB cells least invasive. Inhibition assay of invasion using synthetic peptides RGD, RGDV, RGDS, RGDT, IKVAV and YIGSR, showed that invasion of the three cell lines was significantly inhibited by RGDV. There were other peptides that inhibited invasion significantly including IKVAV for HSC-3, and RGDS and YIGSR for OSC-19. HSC-3 cells and OSC-19 cells adhered to fibronectin, laminin, vitronectin, and type IV collagen, and KB cells did not adhere to laminin but did to fibronectin, vitronectin and collagen type IV. Pretreatment of cells with RGDV peptide in the attachment assay reduced the ability of these cells to bind to vitronectin and fibronectin more efficiently than pretreatment with RGDS. Anti-αv antibodies inhibited adhesion of HSC-3, OSC-19 and KB cells to vitronectin, but anti-β1 antibodies did not inhibit adhesion. Immunofluorescent microscopic examinations showed that all cell lines were positive for anti-β5 and anti-αv antibodies, and only HSC-β3 cells were positive for antiβ-3 antibody. α5β1 was not clearly demonstrated in any of the cell lines. RGDV was the most effective inhibitor of squamous cell carcinoma invasion among the synthetic oligopeptides used in this experiment, and it is suggested that it affects αvβ3-and/or αvβ5-mediated carcinoma cell invasion

Overexpression of E2F1 associated with LOH at RB locus and hyperphosphorylation of RB in non-small cell lung carcinoma

金沢大学大学院医学系研究科保健学専攻Purpose E2F1 plays a critical role in cell proliferation, and its function is controlled by the retinoblastoma (RB) protein. We examined the expression of E2F1 and the aberration of RB gene and protein to elucidate what factors contribute to the overexpression of E2F1 in non-small cell lung carcinomas. Methods The expression level of E2F1 in tissues of non-small cell lung carcinomas was measured by means of quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. For RB, we examined loss of heterozygosity (LOH) by PCR-restriction fragment length polymorphism and a variable number of tandem repeats, and protein expression by immunohistochemistry. Results Fifteen cases of carcinoma (46%) showed high transcription levels of E2F1 gene. Immunohistochemically, almost all (14 of 15) cases overexpressing E2F1 mRNA were positive for E2F1 protein. LOH at the RB locus was found in 13 of 30 informative cases. In 13 cases with LOH, ten showed overexpression of E2F1 mRNA and protein. Immunohistochemical positivity for phosphorylated RB protein was also closely correlated with overexpression of E2F1. Conclusions Our results suggest that overexpression of E2F1, induced both by LOH at the RB locus and anomalous phosphorylation of the RB protein, is involved in the development of non-small cell lung carcinoma

In situハイブリダイゼーションによる子宮頸部上皮性病変への16, 18ヒト乳頭腫ウイルスの組み込みの検出

高感度 in situ ハイブリゼーション（ISH）法と高感度 PCR 法を用いて子宮頸部扁平上皮病変と腺病変での16型と18型のヒト乳頭種ウイルス感染頻度検索し、核への組み込みの頻度を ISH 法で決定した。16, 18型は PCR と ISH でともに、扁平上皮系病変のグレードが高まるにつれて感染頻度が上がった。浸潤扁平上皮癌のすべての例で ISH で組み込み型を示し、CIN1 の全てでエピソーム型を示した。腺系病変では PCR でも ISH でも16型より18型が多く検出され、そのほぼすべてが組み込み型だった。CIN2ではエピソーム型が多くCIN3では組み込み型が多かった。これらから16, 18型の核への組み込みは扁平上皮病変でも腺病変でも癌の初期に現れる変化、あるいは癌への進展を強く示唆する所見と考えられた。Highly sensitive in situ hybridization (ISH) and highly sensitive PCR methods were used to determine the infection of HPV type 16 (HPV 16) and type 18 (HPV 18) and the integration of them into the cells of squamous and glandular lesions of the uterine cervix. The frequency of HPV infection detected by ISH and PCR increased with progression of squamous lesions. All cases of ICC showed the integrated pattern of positive signals with ISH and all positive cases in CIN1 showed the episomal pattern. In CIN2 episomal patterns were more frequent than integrated pattern and in CIN3 integrated pattern were more frequent. Glandular lesions showed a greater frequency of HPV 18 than HPV 16 on nested PCR and ISH, and almost all of ISH-positive cases showed the integrated pattern. These data suggested that integration of HPV 16 and 18 is required for carcinogenesis in both squamous cell carcinoma and adenocarcinoma of the uterine cervix

Adhesiveness of β5 integrin variant lacking FNK767–769 is similar to that of the prototype containing FNKFNK764–769

金沢大学大学院医学系研究科保健学専攻Little is known about the functions of two different β5 integrins: repeated-FNK (FNKFNK764–769) and single-FNK (FNK764–766) amino acid sequences in the cytoplasmic domain. We examined whether they occurred as germ line mutations or somatic mutations associated with neoplastic transformation, and whether there were functional alterations. Out of six cultured cell lines, only KATO-III cells had the single-FNK β5 sequence. The single-FNK β5 was found in 9 out of 79 patients with colon carcinoma, but no somatic mutations were detected in cancerous tissues. CHO cells were transformed with expression vectors containing single-FNK or repeated-FNK β5 cDNA, which were derived from KATO-III cells. CHO cells transfected with single-FNK and repeated-FNK showed similar adhesiveness to, and proliferative activity on, vitronectin substrates