optimal-lentivirus-production-cell-culture-conditions-necessary-to

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

University of Miami, Miller School of Medicine

Forskolin and heregulin synergistically drive human Schwann cell (HSC) proliferation in vitro, but the role of forskolin is not completely understood. To learn how forskolin might affect receptor levels in HSC cultured from adult nerve roots, we first studied expression and localization of HER2 and HER3 in intact roots, using Western blotting and light and electron microscopic immunocytochemistry. We then determined the effect of forskolin and heregulin on receptor expression in HSC cultured from nerve roots using Western blotting and RNase protection assays. HER2 and HER3 were expressed in nonmyelinating Schwann cells in roots and in cultured HSCs before exposure to forskolin. HER2, but not HER3, was also expressed in endoneurial fibroblasts and in cultured nerve root-derived fibroblasts. Treatment with forskolin for 24 h consistently increased HER2 and HER3 protein levels in HSCs but did not alter HER2 and HER3 mRNA levels. In addition, 24-h treatment with heregulin alone decreased HER2 and HER3 protein levels, an effect not previously described. When both heregulin and forskolin were present, HER2 and HER3 protein levels were similar to initial control values. The effect of forskolin on receptor levels was mimicked by dibutyryl-cAMP and receptor levels in both untreated and forskolin treated HSCs were decreased by treatment with the protein kinase A inhibitor H-89. Following pretreatment of HSCs with forskolin, increased receptor levels were correlated with increased rates of thymidine incorporation into HSCs. These results suggest that forskolin/heregulin synergy might derive, at least in part, from post-transcriptional effects leading to increased steady-state receptor levels.

Forskolin increases neuregulin receptors in human Schwann cells without increasing receptor mRNA.

Atypical protein kinase iota (PKCiota) is a key organizer of the apical domain in epithelial cells. Ezrin is a cytosolic protein that, upon activation by phosphorylation of T567, is localized under the apical membrane where it connects actin filaments to membrane proteins and recruits protein kinase A (PKA). To identify the kinase that phosphorylates ezrin T567 in simple epithelia, we analyzed the expression of active PKC and the appearance of T567-P during enterocyte differentiation in vivo. PKCiota phosphorylated ezrin on T567 in vitro, and in Sf9 cells that do not activate human ezrin. In CACO-2 human intestinal cells in culture, PKCiota co-immunoprecipitated with ezrin and was knocked down by shRNA expression. The resulting phenotype showed a modest decrease in total ezrin, but a steep decrease in T567 phosphorylation. The PKCiota-depleted cells showed fewer and shorter microvilli and redistribution of the PKA regulatory subunit. Expression of a dominant-negative form of PKCiota also decreased T567-P signal, and expression of a constitutively active PKCiota mutant showed depolarized distribution of T567-P. We conclude that, although other molecular mechanisms contribute to ezrin activation, apically localized phosphorylation by PKCiota is essential for the activation and normal distribution of ezrin at the early stages of intestinal epithelial cell differentiation.

Atypical protein kinase C (iota) activates ezrin in the apical domain of intestinal epithelial cells.

Los corticoesteroides inhiben transportadores de cationes org&aacute;nicos (PTU) que juegan un papel importante en la absorci&oacute;n de la droga, distribuci&oacute;n tisular y eliminaci&oacute;n. Sensibilidad del corticoesteroide de broncodilatador trata el tejido de la v&iacute;a a&eacute;rea, sin embargo, es mal entendida. Evaluar los efectos de los corticosteroides inhalados en los mecanismos de absorci&oacute;n y eliminaci&oacute;n de las v&iacute;as respiratorias de &beta; 2-agonistas, humana v&iacute;a a&eacute;rea epitelial y captaci&oacute;n celular de m&uacute;sculo liso de captaci&oacute;n formoterol y salmeterol se midi&oacute; in vitro. Los corticosteroides causaron una inhibici&oacute;n r&aacute;pida, dependiente de la concentraci&oacute;n de la absorci&oacute;n de los cati&oacute;nico formoterol por las c&eacute;lulas de m&uacute;sculo liso de las v&iacute;as respiratorias, pero no las c&eacute;lulas epiteliales de las v&iacute;as respiratorias. Absorci&oacute;n del salmeterol lipof&iacute;lico no cargado era del corticoesteroide-insensible en ambos tipos celulares. En las c&eacute;lulas musculares lisas, inhala absorci&oacute;n de formoterol inhibido de corticosteroides con un orden de potencia de novela: des-ciclesonida &gt; budesonida &gt; beclometasona 17-monopropionate &gt; dipropionato de beclometasona &gt; ciclesonida &gt; fluticasona. La acci&oacute;n inhibitoria fue r&aacute;pidamente reversible y no fue mejorada por la exposici&oacute;n prolongada del corticoesteroide o sensibles a un inhibidor de la transcripci&oacute;n. Supresi&oacute;n de expresi&oacute;n OCT3 utilizando la producci&oacute;n mediada por lentivirus de shRNA reduce la sensibilidad del corticoesteroide de formoterol absorci&oacute;n por las c&eacute;lulas musculares lisas. Nuestros datos apoyan una absorci&oacute;n insensible de corticosteroide y un mecanismo sensible del corticoesteroide disposici&oacute;n para broncodilatadores de 2 agonistas &beta; cati&oacute;nico de acci&oacute;n prolongada en las v&iacute;as respiratorias. Orden de rango de potencia y otras caracter&iacute;sticas &#39;cl&aacute;sicas&#39; de efectos antiinflamatorios no se aplican a las medidas de transporte de drogas r&aacute;pida de los corticosteroides inhalados.

Acciones de nongenomic r&aacute;pida de los corticosteroides inhalados en transporte 2-agonista de acci&oacute;n prolongada &beta; en la v&iacute;a a&eacute;rea.

&#x7CD6;&#x76AE;&#x8D28;&#x6FC0;&#x7D20;&#x6291;&#x5236;&#x836F;&#x7269;&#x7684;&#x5438;&#x6536;&#x3001; &#x7EC4;&#x7EC7;&#x5206;&#x5E03;&#x548C;&#x6D88;&#x9664;&#x4E2D;&#x5177;&#x6709;&#x91CD;&#x8981;&#x4F5C;&#x7528;&#x7684;&#x6709;&#x673A;&#x9633;&#x79BB;&#x5B50;&#x8F6C;&#x8FD0; (Oct)&#x3002;&#x7CD6;&#x76AE;&#x8D28;&#x6FC0;&#x7D20;&#x654F;&#x611F;&#x6027;&#x7684;&#x5E73;&#x5598;&#x836F;&#x7269;&#x8D29;&#x8FD0;&#x6C14;&#x9053;&#x7EC4;&#x7EC7;&#xFF0C;&#x4E0D;&#x8FC7;&#xFF0C;&#x662F;&#x751A;&#x5C11;&#x3002;&#x4E0A;&#x6C14;&#x9053;&#x5438;&#x6536;&#x548C;&#x5904;&#x7F6E;&#x673A;&#x5236;&#x957F;&#x6548; &beta; 2 &#x53D7;&#x4F53;&#x6FC0;&#x52A8;&#x5242;&#x3001; &#x4EBA;&#x6C14;&#x9053;&#x4E0A;&#x76AE;&#x548C;&#x5E73;&#x6ED1;&#x808C;&#x7EC6;&#x80DE;&#x5438;&#x6536;&#x7684;&#x6C1A;&#x798F;&#x83AB;&#x7279;&#x7F57;&#x3001; &#x6C99;&#x7F8E;&#x7279;&#x7F57;&#x5438;&#x5165;&#x7CD6;&#x76AE;&#x8D28;&#x6FC0;&#x7D20;&#x7684;&#x5F71;&#x54CD;&#x8FDB;&#x884C;&#x8BC4;&#x4F30;&#x7684;&#x8861;&#x91CF;&#x662F;&#x4F53;&#x5916;&#x3002;&#x7CD6;&#x76AE;&#x8D28;&#x6FC0;&#x7D20;&#x5F15;&#x8D77;&#x7684;&#x9633;&#x79BB;&#x5B50;&#x798F;&#x7684;&#x6C14;&#x9053;&#x5E73;&#x6ED1;&#x808C;&#x7EC6;&#x80DE;&#xFF0C;&#x4F46;&#x4E0D;&#x662F;&#x6C14;&#x9053;&#x4E0A;&#x76AE;&#x7EC6;&#x80DE;&#x5438;&#x6536;&#x8FC5;&#x901F;&#x3001; &#x6D53;&#x5EA6;&#x4F9D;&#x8D56;&#x6027;&#x6291;&#x5236;&#x3002;&#x5438;&#x6536;&#x975E;&#x88AB;&#x63A7;&#x7684;&#x4EB2;&#x8102;&#x6027;&#x6C99;&#x7F8E;&#x7279;&#x7F57;&#x662F;&#x76AE;&#x8D28;&#x7C7B;&#x56FA;&#x9187;&#x533A;&#x5206;&#x8FD9;&#x4E24;&#x79CD;&#x5355;&#x5143;&#x683C;&#x7C7B;&#x578B;&#x3002;&#x5728;&#x5E73;&#x6ED1;&#x808C;&#x7EC6;&#x80DE;&#x4E2D;&#x5438;&#x5165;&#x7CD6;&#x76AE;&#x8D28;&#x6FC0;&#x7D20;&#x6291;&#x5236;&#x798F;&#x83AB;&#x7279;&#x7F57;&#x5438;&#x6536;&#x4E0E;&#x65B0;&#x578B;&#x6548;&#x80FD;&#x6392;&#x540D;&#x987A;&#x5E8F;&#xFF1A; des &#x5948;&#x5FB7; &gt; &#x5948;&#x5FB7; &gt; &#x4E19;&#x9178;&#x500D;&#x6C2F; 17 monopropionate &gt; &#x4E19;&#x9178;&#x500D;&#x6C2F;&#x7C73;&#x677E; &gt; &#x5948;&#x5FB7; &gt; &#x6C1F;&#x66FF;&#x5361;&#x677E;&#x3002;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x662F;&#x5FEB;&#x901F;&#x53EF;&#x9006;&#xFF0C;&#x4EA6;&#x6CA1;&#x6709;&#x957F;&#x671F;&#x7CD6;&#x76AE;&#x8D28;&#x6FC0;&#x7D20;&#x66B4;&#x9732;&#xFF0C;&#x4ECE;&#x800C;&#x589E;&#x5F3A;&#x6216;&#x654F;&#x611F;&#x5BF9;&#x8F6C;&#x5F55;&#x6291;&#x5236;&#x56E0;&#x5B50;&#x3002;&#x6291;&#x5236; OCT3 &#x8868;&#x8FBE;&#x5F0F;&#x4F7F;&#x7528;&#x7684; shRNA &#x6162;&#x75C5;&#x6BD2;&#x4ECB;&#x5BFC;&#x751F;&#x4EA7;&#x51CF;&#x5C11;&#x7CD6;&#x76AE;&#x8D28;&#x6FC0;&#x7D20;&#x654F;&#x611F;&#x6027;&#x7684;&#x798F;&#x83AB;&#x7279;&#x7F57;&#x5438;&#x6536;&#x7684;&#x5E73;&#x6ED1;&#x808C;&#x7EC6;&#x80DE;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x6570;&#x636E;&#x652F;&#x6301;&#x9633;&#x79BB;&#x5B50;&#x957F;&#x6548; &beta; 2-&#x6FC0;&#x52A8;&#x5242;&#x652F;&#x6C14;&#x7BA1;&#x6269;&#x5F20;&#x5242;&#x5728;&#x6C14;&#x9053;&#x4E2D;&#x7CD6;&#x76AE;&#x8D28;&#x6FC0;&#x7D20;&#x4E0D;&#x654F;&#x611F;&#x5438;&#x6536;&#x548C;&#x7CD6;&#x76AE;&#x8D28;&#x6FC0;&#x7D20;&#x654F;&#x611F;&#x5904;&#x7F6E;&#x673A;&#x5236;&#x3002;&#x6548;&#x80FD;&#x6392;&#x540D;&#x987A;&#x5E8F;&#x548C;&#x6297;&#x708E;&#x4F5C;&#x7528;&#x7684;&#x5176;&#x4ED6; &#39;&#x7ECF;&#x5178;&#39; &#x529F;&#x80FD;&#x4E0D;&#x9002;&#x7528;&#x4E8E;&#x5438;&#x5165;&#x7CD6;&#x76AE;&#x8D28;&#x6FC0;&#x7D20;&#x5FEB;&#x901F;&#x836F;&#x7269;&#x4F20;&#x8F93;&#x64CD;&#x4F5C;&#x3002;

&#x5FEB;&#x901F; nongenomic &#x64CD;&#x4F5C;&#x4E0A;&#x957F;&#x6548; &beta; 2-&#x6FC0;&#x52A8;&#x5242;&#x8FD0;&#x8F93;&#x4E2D;&#x6C14;&#x9053;&#x5438;&#x5165;&#x7CD6;&#x76AE;&#x8D28;&#x6FC0;&#x7D20;&#x3002;

Corticosteroids inhibit organic cation transporters (OCTs) that play an important role in drug absorption, tissue distribution and elimination. Corticosteroid sensitivity of bronchodilator trafficking in the airway tissue, however, is poorly understood. To assess the effects of inhaled corticosteroids on airway absorption and disposal mechanisms of long-acting Î²(2)-agonists, human airway epithelial and smooth muscle cell uptake of tritiated formoterol and salmeterol was measured inÂ vitro. Corticosteroids caused a rapid, concentration-dependent inhibition of uptake of the cationic formoterol by airway smooth muscle cells, but not airway epithelial cells. Uptake of the non-charged lipophilic salmeterol was corticosteroid-insensitive in both cell types. In smooth muscle cells, inhaled corticosteroids inhibited formoterol uptake with a novel potency rank order: des-ciclesonideÂ >Â budesonideÂ >Â beclomethasone 17-monopropionateÂ >Â beclomethasone dipropionateÂ >Â ciclesonideÂ >Â fluticasone. The inhibitory action was rapidly reversible, and was not enhanced by prolonged corticosteroid exposure or sensitive to a transcription inhibitor. Suppression of OCT3 expression using lentivirus-mediated production of shRNA reduced corticosteroid sensitivity of formoterol uptake by smooth muscle cells. Our data support a corticosteroid insensitive absorption and a corticosteroid-sensitive disposition mechanism for cationic long-acting Î²(2)-agonist bronchodilators in the airway. Potency rank order and other 'classical' features of anti-inflammatory effects do not apply to inhaled corticosteroids' rapid drug transport actions.

Rapid nongenomic actions of inhaled corticosteroids on long-acting Î²(2)-agonist transport in the airway.

Les corticost&eacute;ro&iuml;des inhibent les transporteurs de cations organiques (PTOM) qui jouent un r&ocirc;le important dans l&#39;absorption de m&eacute;dicaments, distribution tissulaire et l&#39;&eacute;limination. Sensibilit&eacute; de corticost&eacute;ro&iuml;de de bronchodilatateur traite dans les tissus des voies a&eacute;riennes, cependant, est mal comprise. Pour &eacute;valuer les effets des corticost&eacute;ro&iuml;des inhal&eacute;s sur les m&eacute;canismes d&#39;absorption et d&#39;&eacute;limination des voies a&eacute;riennes de &beta; 2-agonistes &agrave; longue, human airway epithelial et apport des cellules musculaires lisses du formot&eacute;rol triti&eacute;e et salmeterol a &eacute;t&eacute; mesur&eacute;e in vitro. Corticost&eacute;ro&iuml;des caus&eacute; une inhibition rapide, concentration-d&eacute;pendante de la capture de la formot&eacute;rol cationique de cellules musculaires lisses des voies a&eacute;riennes, mais pas des cellules &eacute;pith&eacute;liales des voies respiratoires. Captation du salm&eacute;t&eacute;rol lipophile non charg&eacute;e est ind&eacute;pendante de corticost&eacute;ro&iuml;des dans les deux types de cellules. Dans les cellules musculaires lisses, inhal&eacute; absorption formoterol inhib&eacute; corticost&eacute;ro&iuml;des avec un ordre de puissance roman : des-cicl&eacute;sonide &gt; budesonide &gt; beclomethasone 17-monopropionate &gt; beclomethasone dipropionate &gt; cicl&eacute;sonide &gt; fluticasone. L&#39;action inhibitrice a &eacute;t&eacute; rapidement r&eacute;versible et n&#39;&eacute;tait pas renforc&eacute;e par une exposition prolong&eacute;e de corticost&eacute;ro&iuml;des ou sensible &agrave; un inhibiteur de la transcription. Suppression de l&#39;expression OCT3 utilisant la production induite par le lentivirus de shARN r&eacute;duit sensibilit&eacute; corticost&eacute;ro&iuml;de de formot&eacute;rol absorption par les cellules musculaires lisses. Nos donn&eacute;es supportent une absorption insensible de corticost&eacute;ro&iuml;des et d&#39;un m&eacute;canisme de corticost&eacute;ro&iuml;de-sensible &agrave; la disposition des bronchodilatateurs 2-agoniste &beta; de long-acting cationiques dans les voies respiratoires. Classement par ordre de puissance et d&#39;autres fonctions &laquo; classiques &raquo; des effets anti-inflammatoires ne s&#39;appliquent pas aux actions de transport rapide de m&eacute;dicaments corticost&eacute;ro&iuml;des en inhalation.

Nongenomic rapide des actions de corticost&eacute;ro&iuml;des en inhalation sur transport 2-agoniste &beta; de long-action dans les voies respiratoires.

Kortikosteroide hemmen organische kation-Transporter (&Uuml;LG), die eine wichtige bei der Droge Absorption, Verteilung von Gewebe und Beseitigung Rolle. Kortikosteroid-Empfindlichkeit von Bronchodilatator Menschenhandel die Atemwege Gewebe, ist jedoch oft missverstanden. Um die Auswirkungen der inhalativen Kortikoiden Airway Absorption und Beseitigung Mechanismen der langwirksamen &beta; 2-Agonisten, epitheliale menschlichen Atemwege und glatten Muskel Zelle Aufnahme tritiiertem Formoterol und Salmeterol f&uuml;r in-vitro gemessen wurde. Kortikosteroiden verursacht eine schnelle, Konzentration abh&auml;ngige Hemmung der Aufnahme von den kationischen Formoterol von Airway glatten Muskelzellen, aber nicht die Epithelzellen der Atemwege. Aufnahme von der nicht aufgeladenen lipophile Salmeterol war Kortikosteroid-unempfindlich in beiden Zelltypen. In glatten Muskelzellen, inhalative Kortikosteroide gehemmter Formoterol Aufnahme mit einer neuartigen Potenz-Rangfolge: des-Ciclesonide &gt; Budesonid &gt; Beclometason-17-Monopropionate &gt; Beclomethasondipropionat &gt; Ciclesonide &gt; Fluticason. Die hemmende Aktion war schnell reversibel, und nicht durch l&auml;ngere Kortikosteroid-Exposition oder empfindlich auf eine Transkription-Inhibitor. Unterdr&uuml;ckung von mit OCT3 Ausdruck mit Lentiviren-vermittelte Produktion von ShRNA reduziert Kortikosteroid-Empfindlichkeit der Formoterol-Aufnahme von glatten Muskelzellen. Unsere Daten unterst&uuml;tzen eine Kortikosteroid-unempfindlich-Absorption und ein kortikosteroid-Sensitive Disposition-Mechanismus f&uuml;r kationische langwirksamen &beta; 2-Agonisten Bronchodilatatoren in die Atemwege. Potenz Rangordnung und andere &#39;klassischen&#39; entz&uuml;ndungshemmende Wirkung gelten nicht f&uuml;r Aktionen von inhalativen Kortikoiden rapid Droge Verkehrs.

Schnelle Nongenomic Aktionen von inhalativen Kortikoiden langwirksamen &beta; 2-Agonisten Verkehr in die Atemwege.

I corticosteroidi inibiscono i trasportatori di cationi organici (PTOM) che svolgono un ruolo importante nell&#39;assorbimento del farmaco, distribuzione tissutale e l&#39;eliminazione. Sensibilit&agrave; corticosteroide di broncodilatatore traffico nel tessuto delle vie aeree, tuttavia, &egrave; scarsamente compreso. Per valutare gli effetti dei corticosteroidi su meccanismi di assorbimento e smaltimento delle vie aeree di lunga durata d&#39;azione &beta; (2) per via inalatoria-agonisti, vie respiratorie umane epiteliali e assorbimento cellule muscolari lisce di trizio formoterolo e Salmeterolo &egrave; stata misurata in vitro. Corticosteroidi ha causato una rapida, concentrazione-dipendente inibizione dell&#39;assorbimento del formoterolo cationico di cellule muscolari lisce delle vie aeree, ma non nelle cellule epiteliali delle vie aeree. Assorbimento del Salmeterolo lipofilica non caricati era insensibile di corticosteroidi in entrambi i tipi di cella. Nelle cellule muscolari lisce, inalato assorbimento formoterolo inibito corticosteroidi con un ordine di rango romanzo potenza: des-ciclesonide &amp;gt; budesonide &amp;gt; Beclometasone 17-monopropionate &amp;gt; beclometasone dipropionato &amp;gt; ciclesonide &amp;gt; fluticasone. L&#39;azione inibitoria &egrave; stato rapidamente reversibile e non &egrave; stata avanzata da esposizione prolungata di corticosteroidi o sensibili a un inibitore della trascrizione. Soppressione dell&#39;espressione OCT3 usando produzione lentivirus-mediata di shRNA ridotta sensibilit&agrave; corticosteroide di formoterolo assorbimento da parte delle cellule muscolari lisce. I nostri dati supportano un assorbimento insensibili corticosteroide e un meccanismo di corticosteroidi sensibili disposizione per cationici long-acting &beta; (2)-broncodilatatori agonisti nelle vie aeree. Ordine di rango di potenza ed altre caratteristiche &#39;classiche&#39; di effetti anti-infiammatori non si applicano alle azioni di trasporto rapido di droga dei corticosteroidi per via inalatoria.

Azioni rapide nongenomic di corticosteroidi per via inalatoria a lunga durata d&#39;azione &beta; (2)-trasporto agonista nelle vie aeree.

&#x30B3;&#x30EB;&#x30C1;&#x30B3;&#x30B9;&#x30C6;&#x30ED;&#x30A4;&#x30C9;&#x306F;&#x3001;&#x85AC;&#x7269;&#x306E;&#x5438;&#x53CE;&#x3001;&#x7D44;&#x7E54;&#x5206;&#x5E03;&#x304A;&#x3088;&#x3073;&#x9664;&#x53BB;&#x3067;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3059;&#x6709;&#x6A5F;&#x30AB;&#x30C1;&#x30AA;&#x30F3;&#x8F38;&#x9001;&#x4F53; (&#x8D70;&#x67FB;&#x653E;&#x5C04;&#x8A08;) &#x3092;&#x6291;&#x5236;&#x3057;&#x307E;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x6C17;&#x9053;&#x306E;&#x7D44;&#x7E54;&#x3067;&#x306F;&#x3001;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x306E;&#x6C17;&#x7BA1;&#x652F;&#x62E1;&#x5F35;&#x5264;&#x306E;&#x30B3;&#x30EB;&#x30C1;&#x30B3;&#x30B9;&#x30C6;&#x30ED;&#x30A4;&#x30C9;&#x611F;&#x5EA6;&#x306F;&#x3088;&#x304F;&#x308F;&#x304B;&#x3063;&#x3066;&#x3044;&#x307E;&#x305B;&#x3093;&#x3002;&#x5438;&#x5165;&#x30B9;&#x30C6;&#x30ED;&#x30A4;&#x30C9;&#x6C17;&#x9053;&#x5438;&#x53CE;&#x3068;&#x5EC3;&#x68C4;&#x6A5F;&#x69CB;&#x306E;&#x9577;&#x6642;&#x9593;&#x4F5C;&#x7528;&#x578B; &beta; (2) &#x306E;&#x52B9;&#x679C;&#x3092;&#x8A55;&#x4FA1;&#x3059;&#x308B;-&#x30A2;&#x30B4;&#x30CB;&#x30B9;&#x30C8;&#x3001;&#x3072;&#x3068;&#x6C17;&#x9053;&#x4E0A;&#x76AE;&#x7D30;&#x80DE;&#x304A;&#x3088;&#x3073;&#x5E73;&#x6ED1;&#x7B4B;&#x7D30;&#x80DE;&#x53D6;&#x308A;&#x8FBC;&#x307F;&#x30C8;&#x30EA;&#x30C1;&#x30A6;&#x30E0; &#x30DB;&#x30EB;&#x30E2;&#x30C6; &#x30ED;&#x30FC;&#x30EB;&#x304A;&#x3088;&#x3073;&#x30B5;&#x30EB;&#x30E1;&#x30C6; &#x30ED;&#x30FC;&#x30EB;&#x306E;&#x751F;&#x4F53;&#x5916;&#x3067;&#x6E2C;&#x5B9A;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x30B3;&#x30EB;&#x30C1;&#x30B3;&#x30B9;&#x30C6;&#x30ED;&#x30A4;&#x30C9;&#x306F;&#x6025;&#x901F;&#x306B;&#x3001;&#x6FC3;&#x5EA6;&#x4F9D;&#x5B58;&#x306E;&#x963B;&#x5BB3;&#x30AB;&#x30C1;&#x30AA;&#x30F3; &#x30DB;&#x30EB;&#x30E2;&#x30C6; &#x30ED;&#x30FC;&#x30EB;&#x306B;&#x3088;&#x308B;&#x6C17;&#x9053;&#x5E73;&#x6ED1;&#x7B4B;&#x7D30;&#x80DE;&#x304C;&#x306A;&#x3044;&#x6C17;&#x9053;&#x4E0A;&#x76AE;&#x7D30;&#x80DE;&#x306E;&#x5438;&#x53CE;&#x306E;&#x539F;&#x56E0;&#x3002;&#x8FFD;&#x52A0;&#x6599;&#x91D1;&#x3067;&#x306F;&#x975E;&#x8102;&#x6EB6;&#x6027;&#x30B5;&#x30EB;&#x30E1;&#x30C6; &#x30ED;&#x30FC;&#x30EB;&#x306E;&#x53D6;&#x308A;&#x8FBC;&#x307F;&#x306F;&#x3001;&#x30B3;&#x30EB;&#x30C1;&#x30B3;&#x30B9;&#x30C6;&#x30ED;&#x30A4;&#x30C9;&#x3092;&#x4E21;&#x65B9;&#x306E;&#x30BB;&#x30EB;&#x30BF;&#x30A4;&#x30D7;&#x306B;&#x533A;&#x5225;&#x3057;&#x306A;&#x3044;&#x3060;&#x3063;&#x305F;&#x3002;&#x5E73;&#x6ED1;&#x7B4B;&#x7D30;&#x80DE;&#x306E;&#x5438;&#x5165;&#x30B3;&#x30EB;&#x30C1;&#x30B3;&#x30B9;&#x30C6;&#x30ED;&#x30A4;&#x30C9;&#x6291;&#x5236;&#x30DB;&#x30EB;&#x30E2;&#x30C6; &#x30ED;&#x30FC;&#x30EB;&#x53D6;&#x308A;&#x8FBC;&#x307F;&#x5C0F;&#x8AAC;&#x30DD;&#x30FC;&#x30C6;&#x30F3;&#x30B7;&#x30FC;&#x306E;&#x9806;&#x4F4D;&#x3068;: des &#x30B7;&#x30AF;&#x30EC;&#x30BD;&#x30CB;&#x30C9;&#x76EE;&#x30D6;&#x30C7;&#x30BD;&#x30CB;&#x30C9;&#x76EE;&#x30D9;&#x30AF;&#x30ED;&#x30E1;&#x30BF;&#x30BE;&#x30F3; 17 monopropionate &#x76EE;&#x30B8;&#x30D7;&#x30ED;&#x30D4;&#x30AA;&#x30F3;&#x9178;&#x30D9;&#x30AF;&#x30ED;&#x30E1;&#x30BF;&#x30BE;&#x30F3;&#x76EE;&#x30B7;&#x30AF;&#x30EC;&#x30BD;&#x30CB;&#x30C9;&#x76EE;&#x30D5;&#x30EB;&#x30C1;&#x30AB;&#x30BE;&#x30F3;&#x3002;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x306F;&#x6025;&#x901F;&#x306B;&#x4E0D;&#x53EF;&#x9006;&#x7684;&#x3060;&#x3063;&#x305F;&#x3057;&#x3001;&#x9577;&#x671F;&#x30B9;&#x30C6;&#x30ED;&#x30A4;&#x30C9;&#x306E;&#x66B4;&#x9732;&#x306B;&#x3088;&#x3063;&#x3066;&#x5F37;&#x5316;&#x3055;&#x308C;&#x305F;&#x307E;&#x305F;&#x306F;&#x8EE2;&#x5199;&#x963B;&#x5BB3;&#x5264;&#x306B;&#x654F;&#x611F;&#x3067;&#x306F;&#x306A;&#x304B;&#x3063;&#x305F;&#x3002;ShRNA &#x30EC;&#x30F3;&#x30C1; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x5A92;&#x4ECB;&#x751F;&#x7523;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066; OCT3 &#x767A;&#x73FE;&#x306E;&#x6291;&#x5236;&#x306F;&#x3001;&#x30B3;&#x30EB;&#x30C1;&#x30B3;&#x30B9;&#x30C6;&#x30ED;&#x30A4;&#x30C9;&#x611F;&#x5EA6;&#x30DB;&#x30EB;&#x30E2;&#x30C6; &#x30ED;&#x30FC;&#x30EB;&#x53D6;&#x308A;&#x8FBC;&#x307F;&#x306B;&#x3088;&#x308B;&#x5E73;&#x6ED1;&#x7B4B;&#x7D30;&#x80DE;&#x306E;&#x6E1B;&#x5C11;&#x3002;&#x79C1;&#x305F;&#x3061;&#x306E;&#x30C7;&#x30FC;&#x30BF; &#x30B3;&#x30EB;&#x30C1;&#x30B3;&#x30B9;&#x30C6;&#x30ED;&#x30A4;&#x30C9;&#x3092;&#x533A;&#x5225;&#x3057;&#x306A;&#x3044;&#x5438;&#x53CE;&#x3068;&#x30B3;&#x30EB;&#x30C1;&#x30B3;&#x30B9;&#x30C6;&#x30ED;&#x30A4;&#x30C9;&#x654F;&#x611F;&#x306A;&#x51E6;&#x5206;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0; &#x30B5;&#x30DD;&#x30FC;&#x30C8; &#x30AB;&#x30C1;&#x30AA;&#x30F3;&#x9577;&#x6642;&#x9593;&#x4F5C;&#x7528;&#x578B; &beta; (2)-&#x6C17;&#x9053;&#x30A2;&#x30B4;&#x30CB;&#x30B9;&#x30C8;&#x6C17;&#x7BA1;&#x652F;&#x62E1;&#x5F35;&#x3002;&#x52B9;&#x529B;&#x9806;&#x4F4D;&#x3068;&#x6297;&#x708E;&#x75C7;&#x4F5C;&#x7528;&#x306E;&#x4ED6;&#x306E; &#39;&#x30AF;&#x30E9;&#x30B7;&#x30C3;&#x30AF;&#39; &#x6A5F;&#x80FD;&#x5438;&#x5165;&#x30B9;&#x30C6;&#x30ED;&#x30A4;&#x30C9;&#x6025;&#x901F;&#x306A;&#x85AC;&#x7269;&#x8F38;&#x9001;&#x30A2;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x306B;&#x306F;&#x9069;&#x7528;&#x3055;&#x308C;&#x307E;&#x305B;&#x3093;&#x3002;

&#x9577;&#x6642;&#x9593;&#x4F5C;&#x7528;&#x578B; &beta; (2) &#x5438;&#x5165;&#x30B9;&#x30C6;&#x30ED;&#x30A4;&#x30C9;&#x85AC;&#x306E;&#x6025;&#x901F;&#x306A;&#x975E;&#x30B2;&#x30CE;&#x30E0;&#x7684;&#x30A2;&#x30AF;&#x30B7;&#x30E7;&#x30F3;-&#x6C17;&#x9053;&#x306B;&#x304A;&#x3051;&#x308B;&#x30A2;&#x30B4;&#x30CB;&#x30B9;&#x30C8;&#x8F38;&#x9001;&#x3002;

&#xCF54;&#xB974; &#xD2F0; &#xCF54; &#xC2A4;&#xD14C;&#xB85C;&#xC774;&#xB4DC; &#xC57D;&#xBB3C; &#xD761;&#xC218;, &#xC870;&#xC9C1; &#xBD84;&#xD3EC; &#xBC0F; &#xC81C;&#xAC70;&#xC5D0; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xD558;&#xB294; &#xC720;&#xAE30; &#xC591;&#xC774;&#xC628; &#xC804;&#xC1A1;&#xAE30; (OCTs)&#xB97C; &#xC5B5;&#xC81C; &#xD569;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098; &#xCF54;&#xB974; &#xD2F0; &#xCF54; &#xC2A4;&#xD14C;&#xB85C;&#xC774;&#xB4DC; &#xAC10;&#xB3C4; bronchodilator &#xAE30;&#xB3C4; &#xC870;&#xC9C1;&#xC5D0; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;&#xC758;, &#xC11C; &#xD22C;&#xB974; &#xAC8C; &#xC774;&#xD574;. &#xAE34;-&#xC5F0;&#xAE30; &beta; (2)-agonists, &#xC778;&#xAC04;&#xC758; &#xAE30;&#xB3C4; &#xC0C1;&#xD53C; &#xBC0F; tritiated formoterol&#xC640; &#xC0B4; &#xBA54; &#xD14C; &#xB864;&#xC758; &#xBD80;&#xB4DC;&#xB7EC;&#xC6B4; &#xADFC;&#xC721; &#xC138;&#xD3EC; &#xD1B5;&#xD48D; &#xAD00;&#xC758; &#xAE30;&#xB3C4; &#xD761;&#xC218; &#xBC0F; &#xCC98;&#xB9AC; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC5D0; &#xD761;&#xC785;&#xB41C; &#xCF54;&#xB974; &#xD2F0; &#xCF54; &#xC2A4;&#xD14C;&#xB85C;&#xC774;&#xB4DC;&#xC758; &#xD6A8;&#xACFC; &#xD3C9;&#xAC00; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC0DD;&#xCCB4; &#xC678;&#xC5D0;&#xC11C; &#xCE21;&#xC815; &#xD588;&#xB2E4;. &#xCF54;&#xB974; &#xD2F0; &#xCF54; &#xC2A4;&#xD14C;&#xB85C;&#xC774;&#xB4DC;&#xB294; &#xAE30;&#xB3C4; &#xD3C9;&#xD65C; &#xADFC; &#xC138;&#xD3EC;, &#xD558;&#xC9C0;&#xB9CC; &#xD558;&#xC9C0; &#xAE30;&#xB3C4; &#xC0C1;&#xD53C; &#xC138;&#xD3EC;&#xC5D0; &#xC758;&#xD574; &#xC591;&#xC774;&#xC628; formoterol&#xC758; &#xD1B5;&#xD48D; &#xAD00;&#xC758; &#xAE09;&#xC18D; &#xD55C;, &#xB18D;&#xB3C4; &#xC758;&#xC874; &#xC800;&#xD574;&#xB97C; &#xBC1C;&#xC0DD;&#xD569;&#xB2C8;&#xB2E4;. -&#xCDA9;&#xC804; &#xBE14;&#xB808;&#xBBF8;&#xC26C; &#xC0B4; &#xBA54; &#xD14C; &#xB864;&#xC758; &#xD638;&#xC751;&#xC744; &#xC5BB; &#xC5C8;&#xC740; &#xB450; &#xC140; &#xD615;&#xC2DD;&#xC5D0;&#xC11C; &#xCF54;&#xB974; &#xD2F0; &#xCF54; &#xC2A4;&#xD14C;&#xB85C;&#xC774;&#xB4DC; &#xAD6C;&#xBD84; &#xD558;&#xC9C0;. &#xBD80;&#xB4DC;&#xB7EC;&#xC6B4; &#xADFC;&#xC721; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xCF54;&#xB974; &#xD2F0; &#xCF54; &#xC2A4;&#xD14C;&#xB85C;&#xC774;&#xB4DC; &#xC800;&#xD574; formoterol &#xAE00;&#xADC0; &#xC18C;&#xC124; &#xD798; &#xB4F1;&#xAE09; &#xC21C;&#xC11C;&#xC640; &#xD761;&#xC785;: des ciclesonide &amp;gt; &#xB4DC; &amp;gt; &#xB514; 17 monopropionate &amp;gt; &#xB514; &#xD504;&#xB85C; &amp;gt; ciclesonide &amp;gt; &#xD30C;&#xB178;. &#xAE08;&#xC9C0; &#xC870;&#xCE58;&#xB294; &#xC2E0;&#xC18D; &#xD558; &#xAC8C; &#xB418;&#xB3CC;&#xB9B4; &#xC218; &#xADF8;&#xB9AC;&#xACE0; &#xC7A5;&#xAE30;&#xAC04;&#xB41C; &#xCF54;&#xB974; &#xD2F0; &#xCF54; &#xC2A4;&#xD14C;&#xB85C;&#xC774;&#xB4DC; &#xB178;&#xCD9C;&#xC5D0; &#xC758;&#xD574; &#xD5A5;&#xC0C1; &#xB41C; &#xB610;&#xB294; &#xC804;&#xC0AC; &#xC5B5;&#xC81C; &#xBB3C;&#xC5D0; &#xBBFC;&#xAC10;&#xD55C; &#xC544;&#xB2C8;&#xC5C8;&#xB2E4;. ShRNA &#xC0DD;&#xC0B0; lentivirus &#xC911;&#xC7AC;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; OCT3 &#xC2DD;&#xC758; &#xBD80;&#xB4DC;&#xB7EC;&#xC6B4; &#xADFC;&#xC721; &#xC138;&#xD3EC;&#xC5D0; &#xC758;&#xD574; formoterol &#xD1B5;&#xD48D; &#xAD00;&#xC758; &#xCF54;&#xB974; &#xD2F0; &#xCF54; &#xC2A4;&#xD14C;&#xB85C;&#xC774;&#xB4DC; &#xAC10;&#xB3C4; &#xAC10;&#xC18C;. &#xC6B0;&#xB9AC;&#xC758; &#xB370;&#xC774;&#xD130;&#xB294; &#xC591;&#xC774;&#xC628; &#xAE34; &#xC5F0;&#xAE30; &beta; &#xAE38; &#xD56D; (2)-&#xC81C; bronchodilators &#xAE30;&#xB3C4;&#xC5D0;&#xC11C; &#xCF54;&#xB974; &#xD2F0; &#xCF54; &#xC2A4;&#xD14C;&#xB85C;&#xC774;&#xB4DC;&#xB97C; &#xAD6C;&#xBD84; &#xD558;&#xC9C0; &#xC54A;&#xB294; &#xD761;&#xC218; &#xBC0F; &#xCF54;&#xB974; &#xD2F0; &#xCF54; &#xC2A4;&#xD14C;&#xB85C;&#xC774;&#xB4DC;&#xC5D0; &#xBBFC;&#xAC10;&#xD55C; &#xCC98;&#xB9AC; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xC9C0;&#xC6D0;&#xD569;&#xB2C8;&#xB2E4;. &#xD798; &#xB4F1;&#xAE09; &#xC21C;&#xC11C; &#xBC0F; &#xD56D; &#xC5FC;&#xC99D; &#xD6A8;&#xACFC;&#xC758; &#39;&#xD074;&#xB798;&#xC2DD;&#39; &#xAE30;&#xB2A5; &#xB2E4;&#xB978; &#xD761;&#xC785;&#xB41C; corticosteroids &#xC2E0;&#xC18D;&#xD55C; &#xC57D;&#xBB3C; &#xC804;&#xC1A1; &#xC791;&#xC5C5;&#xC5D0; &#xC801;&#xC6A9; &#xB418;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xAE34;-&#xC5F0;&#xAE30; &beta; &#xAE38; &#xD56D; (2)-&#xC81C; &#xAD50;&#xD1B5; &#xAE30;&#xB3C4;&#xC5D0; &#xD761;&#xC785;&#xB41C; &#xCF54;&#xB974; &#xD2F0; &#xCF54; &#xC2A4;&#xD14C;&#xB85C;&#xC774;&#xB4DC;&#xC758; &#xC2E0;&#xC18D;&#xD55C; nongenomic &#xC791;&#xC5C5;.

The epithelium that lines the conducting airways is composed of several distinct cell types that differentiate from common progenitor cells. The signals that control fate selection and differentiation of ciliated cells, a major component of the epithelium, are not completely understood. Ciliated cell differentiation can be accomplished in vitro when primary normal human bronchial epithelial (NHBE) cells are cultured at an air-liquid interface, but is inhibited when NHBE cells are cultured under submerged conditions. The mechanism by which submersion prevents ciliogenesis is not understood, but may provide clues to in vivo regulation of ciliated cell differentiation. We hypothesized that submersion creates a hypoxic environment that prevents ciliated cell differentiation by blocking the gene expression program required for ciliogenesis. This was confirmed by showing that expression of multicilin and Forkhead box J1, key factors needed for ciliated cell differentiation, was inhibited when NHBE cells were cultured in submerged and hypoxic conditions. Multicilin and Forkhead box J1 expression and ciliated cell differentiation were restored in submerged and hypoxic cells upon treatment with the γ-secretase inhibitor, N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT), which suggested that Notch signaling was involved. Overexpression of Notch intracellular domain inhibited differentiation in the presence of DAPT, confirming the role of Notch signaling. These results indicate that submersion and hypoxia prevent ciliated cell differentiation by maintaining Notch signaling, which represses genes necessary for ciliogenesis. These data provide new insights into the molecular mechanisms that control human bronchial differentiation.

Submersion and hypoxia inhibit ciliated cell differentiation in a notch-dependent manner.

Loss of ciliated cells and increases in goblet cells are seen in respiratory diseases such as asthma. These changes result in part from reduced differentiation of basal progenitor cells to ciliated cells during injury and repair. The T helper 2 cytokine, IL-13, has been shown to inhibit ciliated cell differentiation, but the mechanism is not clearly understood. We recently showed that Notch signaling inhibits ciliated cell differentiation in submerged culture by repressing multicilin and forkhead box J1 (FOXJ1) expression, genes required for ciliogenesis. Using a novel method to study ciliated cell differentiation, we investigated the relationship between IL-13 and Notch signaling pathways. We found that IL-13 inhibits ciliated cell differentiation by repressing multicilin and FOXJ1 expression but does so independent of Notch signaling. In addition, we show that pharmacological inhibition of Janus kinase/signal transducer and activator of transcription, but not mitogen activated protein kinase kinase, signaling rescues multicilin and FOXJ1 expression and ciliated cell differentiation in the presence of IL-13. These findings indicate that regulation of multicilin expression by two distinct signaling pathways affects ciliated cell differentiation. In addition, the requirement for Janus kinase activation in IL-13-induced inhibition of ciliogenesis provides a potential therapeutic target for the treatment of respiratory disease.

IL-13 Inhibits Multicilin Expression and Ciliogenesis via Janus Kinase/Signal Transducer and Activator of Transcription Independently of Notch Cleavage.

Nevis L. Fregien

Department of Cell Biology

University of Miami, Miller School of Medicine

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells