isolation-of-primary-myofibroblasts-from-mouse-and-human-colon-tissue

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

UCLA

The transport and metabolism of organic cationic endobiotics, nutrients, and drugs are essential hepatic functions. Slc22A1 is the basolateral liver transporter mediating the uptake of organic cations; however, little is known about the regulation of this transport protein. Peroxisome proliferator agonist receptor (PPAR)-alpha and -gamma agonists are commonly used agents that regulate many hepatocellular transport functions. Thus the purpose of this study was to examine the effects of PPAR agonists on the hepatic regulation and function of Slc22a1. Mice and H35 cells were administered PPAR-alpha and -gamma agonists, and the effect on Slc22a1 gene expression was measured. We subsequently cloned the Slc22a1 promoter and employed chimeric constructs to assay Slc22a1 gene transcription. The effects of PPAR-agonist treatment on organic cation uptake was also assayed. Slc22a1 expression was increased by PPAR-alpha and -gamma agonist treatment in both murine livers and H35 cells. Gene expression in H35 cells was further increased following transfection with expression vectors of PPAR transcription factors and PPAR agonist treatment. We cloned the promoter region of Slc22a1 and identified a PPAR-response element, and transfections with chimeric Slc22a1; promoter-reporter gene constructs demonstrate that the increased gene expression was transcriptionally regulated. Functional assays confirmed that cells treated with PPAR agonists displayed significant increases in organic cation uptake. PPAR-alpha and -gamma agonists transcriptionally increase Slc22a1 gene expression, and the increased Slc22a1 expression results in enhanced cellular organic cation uptake. These studies may have implication for the uptake of organic cationic drugs and for lipid metabolism.

Transcriptional regulation of murine Slc22a1 (Oct1) by peroxisome proliferator agonist receptor-alpha and -gamma.

The phage T4 HOC, SOC bipartite display system is attractive for the expression of cDNA and display of peptides or proteins at high copy numbers on the phage capsid surface. Until recently, using T4 phage vector to display large foreign molecular immunogens resulted only from either an SOC or HOC single site. In this report, the main advantages of the phage T4 system over other display technologies are substantiated by using the phage T4 SOC, HOC dual site display vector T4-Zh(-) to express: (1) on the SOC site, the classical swine fever virus (CSFV) major antigenic determinant cluster mE2 (123 amino acid, aa) through gene fusion to the SOC gene C-terminus of T4 genome, and (2) on the HOC site, full-length CSFV primary antigen E2 (371 aa) through a co-transformed plasmid, hence leading to a simultaneous display of both proteins on the T4 capsid surface. The immunogenicities of these constructs were measured by ID-ELISA, dot-ELISA, Western blotting, and immunogenic response in mice including humoral and cellular immunity tests. The immunological efficiencies both in vitro and in mice of phage T4 with both single site and dual site displays, as well as conventional Escherichia coli plasmid expression, were evaluated. The animal immune response data showed that the antibody titers elicited by the T4 phage-CSFV recombinants were significantly higher than those obtained by E. coli plasmid expression, and the unpurified double site display T4 phage particles were around two times higher than either single site display or plasmid expression while being at lower phage concentrations than the single site phages. The immunogens were effective in the absence of eukaryotic protein modifications. Therefore, the phage T4 dual site display emerges as a powerful method with an enhanced immune response in animals for research and development of immunological products.

Bacteriophage T4 nanoparticle capsid surface SOC and HOC bipartite display with enhanced classical swine fever virus immunogenicity: a powerful immunological approach.

The myofibroblast (MFB) has recently been identified as an important mediator of tumor necrosis factor-Î± (TNF-Î±)-associated colitis and cancer, but the mechanism(s) involved remains incompletely understood. Here, we show that treatment of 18Co cells, a model of human colonic MFBs, with TNF-Î± and lysophosphatidic acid (LPA) induced striking synergistic cyclooxygenase-2 (COX-2) protein expression and production of PGE(2). This effect was prevented by the LPA(1) receptor antagonist Ki16425, the G(iÎ±)-specific inhibitor pertussis toxin, and by the preferential protein kinase (PK) C inhibitors GF109203X and Go6983. As a known downstream target of LPA and PKC, we tested whether PKD, recently implicated in the regulation of COX-2 expression in MFB, was involved in this response. TNF-Î±, while having no detectable effect on the activation of PKD when added alone, augmented PKD activation stimulated by LPA, as measured by PKD autophosphorylation at Ser(910). LPA-induced PKD activation was also inhibited by Ki16425, pertussis toxin, GF109203X, and Go6983. Transfection of 18Co cells with short interfering RNA targeting PKD completely inhibited the synergistic increase in COX-2 protein, demonstrating a critical role of PKD in this response. Our results imply that cross talk between TNF-Î± and LPA results in the amplification of COX-2 protein expression via a conserved PKD-dependent signaling pathway that appears to involve the LPA(1) receptor and the G protein G(iÎ±). PKD plays a critical role in the expression of COX-2 in human colonic MFBs and may contribute to an inflammatory microenvironment that promotes tumor growth.

TNF-Î± potentiates lysophosphatidic acid-induced COX-2 expression via PKD in human colonic myofibroblasts.

Stromal myofibroblasts regulate extracellular matrix components through the secretion of matrix metalloproteinases such as MMP-3. Both myofibroblasts and MMP-3 have been implicated in colonic inflammation and cancer but the regulatory signaling mechanism(s) are unknown. Exposure of the human colonic myofibroblast cell line 18Co to TNF-Î± and bradykinin induced synergistic MMP-3 mRNA and protein expression, which were blocked by the preferential PKC inhibitors GF109203X and Go6983 and by the MEK inhibitor U0126. Transfection with siRNA targeting PKD1, a known downstream target of both bradykinin and PKC, completely inhibited MMP-3 mRNA and protein expression. Our results imply that TNF-Î± and bradykinin amplify MMP-3 expression at a transcriptional level through a signaling cascade involving PKC, PKD1, and MEK. PKD1 plays a critical role in the expression of MMP-3 in human colonic myofibroblasts, and may contribute to the pathophysiology underlying colitis-associated cancer.

Protein kinase D1 mediates synergistic MMP-3 expression induced by TNF-Î± and bradykinin in human colonic myofibroblasts.

&#x808C;&#x7EA4;&#x7EF4;&#x6BCD;&#x7EC6;&#x80DE;&#x6700;&#x8FD1;&#x5DF2;&#x88AB;&#x786E;&#x5B9A;&#x4E3A;&#x91CD;&#x8981;&#x7684;&#x8C03;&#x89E3;&#x4EBA;&#x7684;&#x80BF;&#x7624;&#x574F;&#x6B7B;&#x56E0;&#x5B50;-&alpha; (TNF-&alpha;)-&#x5173;&#x8054;&#x7684;&#x7ED3;&#x80A0;&#x708E;&#x548C;&#x764C;&#x75C7;&#xFF0C;&#x4F46;&#x673A;&#x5236;&#x6D89;&#x53CA;&#x4ECD;&#x6CA1;&#x6709;&#x5B8C;&#x5168;&#x641E;&#x6E05;&#x695A;&#x3002;&#x6700;&#x8FD1;&#x7684;&#x8BC1;&#x636E;&#x8868;&#x660E;&#x80BF;&#x7624;&#x574F;&#x6B7B;&#x56E0;&#x5B50;-&alpha; &#x662F;&#x591A;&#x79CD;&#x708E;&#x75C7;&#x4FE1;&#x53F7;&#x6805;&#x4E2D;&#x592E;&#x8C03;&#x8282;&#x5668;&#x3002;&#x80BF;&#x7624;&#x574F;&#x6B7B;&#x56E0;&#x5B50;-&alpha; &#x7684;&#x4E00;&#x4E2A;&#x91CD;&#x8981;&#x7684;&#x76EE;&#x6807;&#x53EF;&#x80FD;&#x662F;&#x4FE1;&#x53F7;&#x901A;&#x8DEF;&#x4E0B;&#x6E38;&#x7684;&#x8868;&#x76AE;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x53D7;&#x4F53; (EGFR)&#xFF0C;&#x4E00;&#x76F4;&#x4E0E;&#x8BB8;&#x591A;&#x4EBA;&#x7C7B;&#x7684;&#x764C;&#x75C7;&#x76F8;&#x5173;&#x8054;&#x7684;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x4EBA;&#x7C7B;&#x7ED3;&#x80A0;&#x7684;&#x6210;&#x7EA4;&#x7EF4;&#x7EC6;&#x80DE;&#xFF0C;&#x4E0E;&#x80BF;&#x7624;&#x574F;&#x6B7B;&#x56E0;&#x5B50;-&alpha; &#x7684;&#x6A21;&#x578B;&#x6211;&#x4EEC;&#x663E;&#x793A; 18Co &#x7684;&#x5355;&#x5143;&#x683C;&#xFF0C;&#x8BE5;&#x957F;&#x671F;&#x63A5;&#x89E6;&#x5BFC;&#x81F4;&#x663E;&#x8457;&#x589E;&#x52A0;&#xFF0C;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x7684;&#x8868;&#x76AE;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x8868;&#x8FBE;&#xFF0C;&#x5B8C;&#x5168;&#x7531;&#x653E;&#x7EBF;&#x83CC;&#x916E;&#x6291;&#x5236;&#x7684;&#x6548;&#x679C;&#x3002;&#x8868;&#x76AE;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x548C; HB EGF &#x8868;&#x76AE;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x88C5;&#x8BA2;&#x662F;&#x5173;&#x8054;&#x589E;&#x5F3A;&#x7684; EGFR &#x916A;&#x6C28;&#x9178;&#x6FC0;&#x9176;&#x6D3B;&#x6027;&#x3001; &#x957F;&#x671F;&#x7684; ERK &#x7684;&#x6FC0;&#x6D3B;&#x548C;&#x73AF;&#x6C27;&#x5408;&#x9176;-2 &#x8868;&#x8FBE; 18Co &#x7EC6;&#x80DE;&#x6CBB;&#x7597;&#x8868;&#x76AE;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x548C;&#x8868;&#x76AE;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x7684; HB &#x72EC;&#x81EA;&#x76F8;&#x6BD4;&#x5927;&#x5E45;&#x589E;&#x52A0;&#x3002;&#x80BF;&#x7624;&#x574F;&#x6B7B;&#x56E0;&#x5B50;-&alpha; &#x4E5F;&#x589E;&#x52A0;&#x8868;&#x76AE;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x548C;&#x4FE1;&#x53F7;&#x5728;&#x4E3B;&#x6210;&#x7EA4;&#x7EF4;&#x7EC6;&#x80DE;&#x5206;&#x79BB;&#x4EBA;&#x4F53;&#x7ED3;&#x80A0;&#x7EC4;&#x7EC7;&#x4E2D;&#x3002;&#x8868;&#x76AE;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x80BF;&#x7624;&#x574F;&#x6B7B;&#x56E0;&#x5B50;-&alpha; &#x8BF1;&#x5BFC;&#x4E0A;&#x8C03;&#x53EF;&#x80FD;&#x662F;&#x53EF;&#x4FE1;&#x7684;&#x673A;&#x5236;&#x6765;&#x89E3;&#x91CA;&#x5938;&#x5F20;&#x7684;&#x7EC6;&#x80DE;&#x53CD;&#x5E94;&#x80FD;&#x529B;&#xFF0C;&#x7279;&#x70B9;&#x662F;&#x708E;&#x75C7;&#x6027;&#x80A0;&#x75C5;&#xFF0C;&#x5E76;&#x53EF;&#x5E2E;&#x52A9;&#x4E8E;&#x5FAE;&#x73AF;&#x5883;&#x7ED3;&#x80A0;&#x708E;&#x76F8;&#x5173;&#x764C;&#x75C7;&#x7684;&#x7F6A;&#x9B41;&#x7978;&#x9996;&#x3002;

&#x80BF;&#x7624;&#x574F;&#x6B7B;&#x56E0;&#x5B50;-&alpha; &#x8BF1;&#x5BFC;&#x8868;&#x76AE;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x548C;&#x4EBA;&#x7C7B;&#x7ED3;&#x80A0;&#x808C;&#x6210;&#x7EA4;&#x7EF4;&#x7EC6;&#x80DE;&#x4FE1;&#x53F7;&#x4F20;&#x5BFC;&#x7684;&#x8C03;&#x63A7;&#x3002;

The myofibroblast has recently been identified as an important mediator of tumor necrosis factor-Î± (TNF-Î±)-associated colitis and cancer but the mechanism(s) involved remains incompletely understood. Recent evidence suggests that TNF-Î± is a central regulator of multiple inflammatory signaling cascades. One important target of TNF-Î± may be the signaling pathway downstream of the epidermal growth factor receptor (EGFR), which has been associated with many human cancers. Here, we show that long-term exposure of 18Co cells, a model of human colonic myofibroblasts, with TNF-Î± led to a striking increase in cell surface EGFR expression, an effect that was completely inhibited by cycloheximide. Subsequent EGFR binding by EGF and HB-EGF was associated with enhanced EGFR tyrosine kinase activity, prolonged ERK activation, and a significant increase in COX-2 expression compared to 18Co cells treated with EGF and HB-EGF alone. TNF-Î± also increased EGFR expression and signaling in primary myofibroblasts isolated from human colon tissue. TNF-Î±-induced up-regulation of EGFR may be a plausible mechanism to explain the exaggerated cellular responsiveness that characterizes IBD, and which may contribute to a microenvironment that predisposes to colitis-associated cancer.

TNF-Î± Induces Up-Regulation of EGFR Expression and Signaling in Human Colonic Myofibroblasts.

Le myofibroblastes a r&#xE9;cemment &#xE9;t&#xE9; identifi&#xE9; comme un m&#xE9;diateur important de facteur de n&#xE9;crose tumorale &#x3B1;-(TNF-&#x3B1;) associ&#xE9;e &#xE0; la colite et le cancer, mais le m&#xE9;canisme (s) impliqu&#xE9; demeure incompl&#xE8;tement compris. Des donn&#xE9;es r&#xE9;centes sugg&#xE8;rent que le TNF-&#x3B1; est un r&#xE9;gulateur central de plusieurs cascades de signalisation inflammatoires. Un objectif important du TNF-&#x3B1; peut &#xEA;tre en aval des voies de signalisation du r&#xE9;cepteur du facteur de croissance &#xE9;pidermique (EGFR), qui a &#xE9;t&#xE9; associ&#xE9; &#xE0; de nombreux cancers humains. Ici, nous montrons que l&#x26;#39;exposition &#xE0; long terme de cellules 18Co, un mod&#xE8;le de l&#x26;#39;homme myofibroblastes coliques, avec le TNF-&#x3B1; a conduit &#xE0; une augmentation frappante de l&#x26;#39;expression d&#x26;#39;EGFR de surface cellulaire, un effet qui a &#xE9;t&#xE9; compl&#xE8;tement inhib&#xE9;e par le cycloheximide. Apr&#xE8;s EGFR obligatoire par l&#x26;#39;EGF et l&#x26;#39;HB-EGF a &#xE9;t&#xE9; associ&#xE9;e &#xE0; une activit&#xE9; kinase EGFR tyrosine am&#xE9;lior&#xE9;e, activation de ERK prolong&#xE9;e, et une augmentation significative de la COX-2 par rapport &#xE0; 18Co cellules trait&#xE9;es avec de l&#x26;#39;EGF et l&#x26;#39;HB-EGF seul. TNF-&#x3B1; a &#xE9;galement augment&#xE9; l&#x26;#39;expression d&#x26;#39;EGFR et la signalisation dans les myofibroblastes primaires isol&#xE9;es &#xE0; partir de tissus du colon humain. TNF-&#x3B1; induit une r&#xE9;gulation positive de l&#x26;#39;EGFR peut &#xEA;tre un m&#xE9;canisme plausible pour expliquer la r&#xE9;activit&#xE9; exag&#xE9;r&#xE9;e cellulaire qui caract&#xE9;rise les MII, et qui peut contribuer &#xE0; un micro-environnement qui pr&#xE9;dispose &#xE0; la colite associ&#xE9;e au cancer.

TNF-&#x3B1; induit Up-r&#xE9;gulation de l&#x26;#39;expression de l&#x26;#39;EGFR et de signalisation dans l&#x26;#39;homme myofibroblastes coliques

Vor kurzem wurde der Myofibroblast als ein wichtiger Vermittler von Tumor-Nekrose-Faktor-&alpha; (TNF-&alpha;) ermittelt-assoziierten Kolitis und Krebs, aber die Mechanismen beteiligt bleibt unvollst&auml;ndig verstanden. J&uuml;ngste Hinweise, dass TNF-&alpha; ein zentraler Regulator mehrere entz&uuml;ndliche Signalling Kaskaden ist. Ein wichtiges Ziel des TNF-&alpha; m&ouml;glicherweise den Signalweg stromabw&auml;rts von den epidermalen Wachstumsfaktor-Rezeptor (EGFR), die vielen menschlichen Krebsarten zugeordnet wurde. Hier zeigen wir, dass die langfristigen Exposition 18Co Zellen, f&uuml;hrte ein Modell des menschlichen Kolon Myofibroblasts mit TNF-&alpha; zu einem markanten Anstieg der Zelle Oberfl&auml;che EGFR Ausdruck, ein Effekt, der durch Cycloheximid v&ouml;llig gehemmt wurde. Nachfolgende EGFR-Bindung von EGF und HB-EGF war verbunden mit verbesserten EGFR Tyrosinkinase-Aktivit&auml;t, lang andauernde ERK-Aktivierung und eine deutliche Zunahme der COX-2-Ausdruck im Vergleich zu 18Co Zellen mit EGF und HB-EGF allein behandelt. TNF-&alpha; erh&ouml;ht auch EGFR-Ausdruck und K&ouml;rpersprache prim&auml;re Myofibroblasts aus menschlichem Doppelpunkt Gewebe isoliert. TNF-&alpha; induziert bis-Regelung der EGFR m&ouml;glicherweise einen plausiblen Mechanismus, die &uuml;bertriebene zellul&auml;re Reaktionsf&auml;higkeit zu erkl&auml;ren, die IBD, charakterisiert und k&ouml;nnen beitragen zu einem Untersuchungsverfahren, die Kolitis-assoziierte Krebs pr&auml;disponiert.

TNF-&alpha; induziert Up-Regulierung des EGFR Ausdruck und K&ouml;rpersprache menschlichen Kolon Myofibroblasts.

Il myofibroblast recentemente &egrave; stato identificato come un mediatore importante di fattore di necrosi tumorale-&alpha; (TNF-&alpha;)-colite associata e cancro, ma i meccanismi comprendono coinvolto resta inteso in modo incompleto. Recenti evidenze suggeriscono che il TNF-&alpha; &egrave; un regolatore centrale di cascate di segnalazione infiammatorie pi&ugrave;. Un obiettivo importante di TNF-&alpha; pu&ograve; essere la via di segnalazione a valle del recettore dell&#39;epidermal growth factor (EGFR), che &egrave; stato associato con molti tumori umani. Qui, ci mostrano che l&#39;esposizione a lungo termine delle cellule 18Co, un modello di miofibroblasti del colon umano, con TNF-&alpha; ha portato ad un sorprendente aumento nell&#39;espressione di EGFR superficie delle cellule, un effetto che &egrave; stato completamente inibito dalla cicloeximide. Successiva associazione di EGFR di EGF e HB-EGF &egrave; stato associato con un significativo aumento nell&#39;espressione di COX-2 rispetto a 18Co cellule trattate con EGF e HB-EGF solo rafforzata l&#39;attivit&agrave; della chinasi tirosina EGFR e prolungata attivazione di ERK. TNF-&alpha; aumenta anche l&#39;espressione di EGFR e segnalazione in miofibroblasti primario isolato dal tessuto del colon umano. Indotta da TNF-&alpha; up-regulation di EGFR pu&ograve; essere un meccanismo plausibile per spiegare l&#39;esagerata reattivit&agrave; cellulare che caratterizza IBD, e che possono contribuire ad un microambiente che predispone al cancro colite associate.

TNF-&alpha; induce Up-regolazione di espressione di EGFR e segnalazione in miofibroblasti del colon umano.

&#x7B4B;&#x7DDA;&#x7DAD;&#x82BD;&#x7D30;&#x80DE;&#x306F;&#x6700;&#x8FD1;&#x3001;&#x816B;&#x760D;&#x58CA;&#x6B7B;&#x56E0;&#x5B50;-&#x3B1;&#xFF08;TNF-&#x3B1;&#xFF09; - &#x95A2;&#x9023;&#x5927;&#x8178;&#x708E;&#x3068;&#x764C;&#x304C;&#x3001;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#xFF08;s&#xFF09;&#x306F;&#x5B8C;&#x5168;&#x306B;&#x306F;&#x7406;&#x89E3;&#x306E;&#x307E;&#x307E;&#x95A2;&#x4E0E;&#x306E;&#x91CD;&#x8981;&#x306A;&#x30E1;&#x30C7;&#x30A3;&#x30A8;&#x30FC;&#x30BF;&#x30FC;&#x3068;&#x3057;&#x3066;&#x540C;&#x5B9A;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3002;&#x6700;&#x8FD1;&#x306E;&#x8A3C;&#x62E0;&#x306F;&#x3001;TNF-&#x3B1;&#x306F;&#x8907;&#x6570;&#x306E;&#x708E;&#x75C7;&#x6027;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x30AB;&#x30B9;&#x30B1;&#x30FC;&#x30C9;&#x306E;&#x4E2D;&#x5FC3;&#x7684;&#x8ABF;&#x7BC0;&#x56E0;&#x5B50;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002; TNF-&#x3B1;&#x306E;&#x4E00;&#x3064;&#x306E;&#x91CD;&#x8981;&#x306A;&#x76EE;&#x6A19;&#x306F;&#x3001;&#x591A;&#x304F;&#x306E;&#x30D2;&#x30C8;&#x764C;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x4E0A;&#x76AE;&#x6210;&#x9577;&#x56E0;&#x5B50;&#x53D7;&#x5BB9;&#x4F53;&#xFF08;EGFR&#xFF09;&#x306E;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x7D4C;&#x8DEF;&#x306E;&#x4E0B;&#x6D41;&#x306E;&#x304B;&#x3082;&#x3057;&#x308C;&#x307E;&#x305B;&#x3093;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;TNF-&#x3B1;&#x306F;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x306E;EGFR&#x767A;&#x73FE;&#x306E;&#x8457;&#x3057;&#x3044;&#x5897;&#x52A0;&#x306B;&#x3064;&#x306A;&#x304C;&#x3063;&#x305F;&#x3068;18Co&#x7D30;&#x80DE;&#x306E;&#x9577;&#x671F;&#x66B4;&#x9732;&#x3001;&#x30D2;&#x30C8;&#x5927;&#x8178;&#x7B4B;&#x7DDA;&#x7DAD;&#x82BD;&#x7D30;&#x80DE;&#x306E;&#x30E2;&#x30C7;&#x30EB;&#x306F;&#x3001;&#x5B8C;&#x5168;&#x306B;&#x30B7;&#x30AF;&#x30ED;&#x30D8;&#x30AD;&#x30B7;&#x30DF;&#x30C9;&#x306B;&#x3088;&#x308A;&#x963B;&#x5BB3;&#x3055;&#x308C;&#x305F;&#x52B9;&#x679C;&#x3092;&#x793A;&#x3059;&#x3002;&#x305D;&#x306E;&#x5F8C;&#x306E;EGFR&#x306F;&#x3001;EGF&#x3068;HB-EGF&#x306B;&#x3088;&#x308B;&#x7D50;&#x5408;&#x304C;&#x5F37;&#x5316;&#x3055;&#x308C;&#x305F;EGFR&#x30C1;&#x30ED;&#x30B7;&#x30F3;&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x6D3B;&#x6027;&#x3001;&#x9577;&#x6642;&#x9593;&#x306E;ERK&#x306E;&#x6D3B;&#x6027;&#x5316;&#x3001;&#x5358;&#x72EC;&#x3067;EGF&#x3068;HB-EGF&#x3067;&#x51E6;&#x7406;&#x3055;&#x308C;&#x305F;18Co&#x7D30;&#x80DE;&#x3068;&#x6BD4;&#x8F03;&#x3057;&#x3066;COX-2&#x767A;&#x73FE;&#x306E;&#x6709;&#x610F;&#x306A;&#x5897;&#x52A0;&#x3068;&#x95A2;&#x9023;&#x3057;&#x3066;&#x3044;&#x305F;&#x3002; TNF-&#x3B1;&#x306F;&#x307E;&#x305F;&#x3001;EGFR&#x306E;&#x767A;&#x73FE;&#x3068;&#x30D2;&#x30C8;&#x7D50;&#x8178;&#x7D44;&#x7E54;&#x304B;&#x3089;&#x5206;&#x96E2;&#x3057;&#x305F;&#x521D;&#x4EE3;&#x7DDA;&#x7DAD;&#x82BD;&#x7D30;&#x80DE;&#x306E;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x3092;&#x5897;&#x52A0;&#x3055;&#x305B;&#x305F;&#x3002; EGFR&#x306E;TNF-&#x3B1;&#x8A98;&#x767A;&#x30A2;&#x30C3;&#x30D7;&#x30EC;&#x30AE;&#x30E5;&#x30EC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306F;&#x3001;IBD&#x306E;&#x7279;&#x5FB4;&#x3092;&#x8A87;&#x5F35;&#x3055;&#x308C;&#x305F;&#x7D30;&#x80DE;&#x306E;&#x5FDC;&#x7B54;&#x6027;&#x3092;&#x8AAC;&#x660E;&#x3059;&#x308B;&#x3082;&#x3063;&#x3068;&#x3082;&#x3089;&#x3057;&#x3044;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3067;&#x3042;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x3001;&#x5927;&#x8178;&#x708E;&#x95A2;&#x9023;&#x764C;&#x306E;&#x7D20;&#x56E0;&#x3068;&#x306A;&#x308B;&#x5FAE;&#x5C0F;&#x74B0;&#x5883;&#x306B;&#x8CA2;&#x732E;&#x3059;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308B;&#x3002;

TNF-&#x3B1;&#x306F;&#x3001;EGFR&#x767A;&#x73FE;&#x306E;&#x30A2;&#x30C3;&#x30D7;&#x30EC;&#x30AE;&#x30E5;&#x30EC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3092;&#x8A98;&#x5C0E;&#x3057;&#x3001;&#x30D2;&#x30C8;&#x5927;&#x8178;&#x7B4B;&#x7DDA;&#x7DAD;&#x82BD;&#x7D30;&#x80DE;&#x306E;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;

Myofibroblast &#xCD5C;&#xADFC;&#xC5D0; &#xC885;&#xC591; &#xAD34; &#xC0AC; &#xC778;&#xC790;-&alpha; (TNF-&alpha;)&#xC758; &#xC911;&#xC694; &#xD55C; &#xC911;&#xC7AC;&#xC790;&#xAC00; &#xBC1C;&#xACAC; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;-&#xAD00;&#xB828;&#xB41C; &#xB300; &#xC7A5; &#xC5FC; &#xBC0F; &#xC554; &#xD558;&#xC9C0;&#xB9CC; &#xC7A5;&#xCE58;&#xB97C; &#xB9C8;&#xB354;&#xBCF4;&#xB4DC;&#xC5D0; &#xCC38;&#xC5EC; &#xB0A8;&#xC544; &#xBD88;&#xC644;&#xC804; &#xD558; &#xAC8C; &#xC774;&#xD574;. &#xCD5C;&#xADFC; &#xC99D;&#xAC70; TNF-&alpha;&#xC758; &#xC5EC;&#xB7EC; &#xC5FC;&#xC99D; &#xC2E0;&#xD638; &#xACC4;&#xACE1; &#xC911;&#xC559; &#xB808; &#xADE4; &#xB808;&#xC774; &#xD130; &#xB098;&#xC654;&#xB2E4;. TNF-&alpha;&#xC758; &#xD558;&#xB098;&#xC758; &#xC911;&#xC694; &#xD55C; &#xBAA9;&#xD45C;&#xB294; &#xB9CE;&#xC740; &#xC778;&#xAC04;&#xC758; &#xC554;&#xACFC; &#xC5F0;&#xACB0; &#xB418;&#xC5B4; &#xD45C; &#xD53C; &#xC131;&#xC7A5; &#xC778;&#xC790; &#xC218;&#xC6A9; &#xCCB4; (EGFR)&#xC758; &#xC2E0;&#xD638; &#xD1B5;&#xB85C; &#xD558;&#xB958; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;, TNF-&alpha;&#xC640; &#xC778;&#xAC04;&#xC758; &#xC800; &#xC2B9; myofibroblasts &#xBAA8;&#xB378; &#xC140; &#xD45C;&#xBA74; EGFR &#xC2DD; cycloheximide&#xC5D0; &#xC758;&#xD574; &#xC644;&#xC804;&#xD788; &#xC800;&#xD574; &#xB418;&#xC5C8;&#xB2E4; &#xD6A8;&#xACFC;&#xC5D0;&#xC11C; &#xB208;&#xC5D0; &#xB744;&#xB294; &#xC99D;&#xAC00;&#xB97C; &#xC8FC;&#xB3C4; &#xC6B0;&#xB9AC; 18Co &#xC140;&#xC758; &#xC7A5;&#xAE30; &#xB178;&#xCD9C; &#xD45C;&#xC2DC;. &#xC774;&#xD6C4; EGFR &#xBC14;&#xC778;&#xB529; EGF&#xC640; HB EGF&#xC5D0; &#xC758;&#xD574; &#xD5A5;&#xC0C1; &#xB41C; EGFR &#xD2F0;&#xB85C;&#xC2E0; &#xD0A4; &#xB2C8; &#xC544; &#xC81C; &#xD65C;&#xB3D9;, &#xC7A5;&#xAE30;&#xAC04;&#xB41C; ERK &#xD65C;&#xC131;&#xD654; &#xBC0F; 18Co &#xC140; EGF&#xC640; HB EGF &#xD63C;&#xC790; &#xCE58;&#xB8CC;&#xC5D0; &#xBE44;&#xD574; c o X-2 &#xC2DD;&#xC5D0; &#xC0C1;&#xB2F9;&#xD55C; &#xC99D;&#xAC00;&#xC640; &#xAD00;&#xB828;&#xC774; &#xC788;&#xC5C8;&#xB2E4;. TNF-&alpha;&#xB294; EGFR &#xD45C;&#xD604;&#xACFC; &#xAE30;&#xBCF8; myofibroblasts &#xC778;&#xCCB4; &#xB300; &#xC7A5; &#xC870;&#xC9C1;&#xC5D0;&#xC11C; &#xBD84;&#xB9AC; &#xB41C; &#xC2E0;&#xD638;&#xB3C4; &#xC99D;&#xAC00;. EGFR&#xC758; &#xC5C5; &#xADDC;&#xC81C; TNF-&alpha; &#xC720;&#xB3C4; &#xACFC;&#xC7A5; &#xB41C; &#xC140;&#xB8F0;&#xB7EC; &#xC751;&#xB2F5; IBD, &#xD2B9;&#xC9D5;&#xC744; &#xC124;&#xBA85; &#xD558;&#xAE30; &#xC704;&#xD574; &#xADF8;&#xB7F4;&#xB4EF;&#xD55C; &#xBA54;&#xCEE4;&#xB2C8;&#xC998; &#xC218; &#xC788;&#xB294; colitis &#xAD00;&#xB828; &#xC554;&#xC5D0; &#xAC78;&#xB9AC;&#xAE30; microenvironment&#xC5D0; &#xAE30;&#xC5EC;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

TNF-&alpha; &#xC5C5;-&#xB808; &#xADE4; &#xB808;&#xC774; &#xC158; EGFR &#xD45C;&#xD604;&#xACFC; &#xC778;&#xAC04;&#xC758; &#xC800; &#xC2B9; Myofibroblasts&#xC5D0;&#xC11C; &#xC2DC;&#xADF8;&#xB110;&#xB9C1;&#xC744; &#xC720;&#xB3C4; &#xD569;&#xB2C8;&#xB2E4;.

El miofibroblastos ha sido recientemente identificado como un importante mediador de factor de necrosis tumoral &#x3B1; (TNF-&#x3B1;) asociado a la colitis y el c&#xE1;ncer, pero el mecanismo (s) en cuesti&#xF3;n sigue siendo no del todo comprendidas. La evidencia reciente sugiere que el TNF-&#x3B1; es un regulador central de m&#xFA;ltiples cascadas de se&#xF1;alizaci&#xF3;n inflamatorias. Un objetivo importante de TNF-&#x3B1; puede ser aguas abajo v&#xED;a de se&#xF1;alizaci&#xF3;n del receptor del factor de crecimiento epid&#xE9;rmico (EGFR), que se ha asociado con muchos c&#xE1;nceres humanos. En este sentido, muestran que la exposici&#xF3;n a largo plazo de las c&#xE9;lulas de 18Co, un modelo de humanos miofibroblastos de colon, con el TNF-&#x3B1; dado lugar a un notable aumento en la expresi&#xF3;n de EGFR en la superficie celular, un efecto que fue completamente inhibida por la cicloheximida. EGFR posterior uni&#xF3;n por EGF y HB-EGF se asoci&#xF3; con una mayor actividad de la quinasa de EGFR tirosina, la activaci&#xF3;n de ERK prolongado, y un aumento significativo de la expresi&#xF3;n COX-2 en comparaci&#xF3;n con 18Co c&#xE9;lulas tratadas con EGF y HB-EGF solo. TNF-&#x3B1; tambi&#xE9;n aument&#xF3; la expresi&#xF3;n de EGFR y la se&#xF1;alizaci&#xF3;n en miofibroblastos primarias aisladas de tejido del colon humano. TNF-&#x3B1; inducida por la regulaci&#xF3;n del EGFR puede ser un mecanismo plausible para explicar la capacidad de respuesta exagerada celular que caracteriza a la EII, y que puede contribuir a un microambiente que predispone a la colitis asociada a c&#xE1;ncer.

TNF-&#x3B1; induce Up-regulaci&#xF3;n de la expresi&#xF3;n del EGFR y la se&#xF1;alizaci&#xF3;n en humanos miofibroblastos de colon

BACKGROUND: Enhanced EGF receptor (EGFR) signaling is a hallmark of many human cancers, though the role of enhanced EGFR signaling within the surrounding tumor stroma has not been well studied. The myofibroblast is an important stromal cell that demonstrates enhanced EGFR expression in the setting of inflammation, though the functional relevance is not known. We recently reported that TNF-alpha and the G protein-coupled receptor (GPCR) agonist lysophosphatidic acid (LPA) lead to synergistic cyclo-oxygenase-2 (COX-2) expression, an enzyme strongly associated with the development of colitis-associated cancer. Here, we investigate whether EGFR signaling plays a role in the synergistic COX-2 expression induced by LPA and TNF-alpha. METHODS: 18Co cells, a model of human colonic myofibroblasts, were grown to confluence on 35 x 10mm cell culture dishes and were used from passages 10--14. 18Co cells were treated with TNF-alpha (8.3 ng/ml) and LPA (10 muM). EGFR and COX-2 protein expression, Y1068 phosphorylation, and p42/44 MAPK phosphorylation were assessed by Western Blot, in the presence and absence of various inhibitors. RESULTS: Exposure of 18Co cells to either TNF-alpha or LPA alone had no effect on EGFR autophosphorylation at Y1068. However, chronic exposure to TNF-alpha led to upregulation of EGFR in association with sustained LPA-mediated EGFR phosphorylation at Y1068. TNF-alpha and LPA also led to sustained p42/44 MAPK phosphorylation and synergistic COX-2 expression, effects that were partially inhibited by the EGFR tyrosine kinase inhibitor AG1478. p42/44 MAPK phosphorylation and COX-2 expression were inhibited to the same degree by the MMP inhibitors GM6001 and BB-94, suggesting that LPA-mediated EGFR transactivation involved MMP-mediated release of EGFR ligands from the cell surface. The Src inhibitor SU6556 inhibited TNF-alpha/LPA-mediated EGFR phosphorylation at Y1068, p42/44 MAPK phosphorylation, and COX-2 expression in a dose-dependent fashion, suggesting an upstream role of Src in the transactivation of EGFR. CONCLUSION: Synergistic COX-2 expression induced by TNF-alpha and LPA involves Src/MMP-mediated transactivation of EGFR and downstream p42/44 MAPK activation in human colonic myofibroblasts. Enhanced EGFR expression induced by TNF-alpha promotes GPCR-mediated EGFR transactivation in colonic myofibroblasts, providing an important mechanism for stromal COX-2 over-expression that may predispose to the development of colitis-associated cancer.

Wenxian Nie

Department of Surgery

UCLA

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

Wenxian Nie

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Surgery

UCLA

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

Department of Surgery