TLR2 Mediates Gap Junctional Intercellular Communication through Connexin-43 in Intestinal Epithelial Barrier Injury
2009; Elsevier BV; Volume: 284; Issue: 33 Linguagem: Inglês
10.1074/jbc.m901619200
ISSN1083-351X
AutoresBirgit Ey, Annette Eyking, Guido Gerken, Daniel K. Podolsky, Elke Cario,
Tópico(s)Heat shock proteins research
ResumoGap junctional intercellular communication (GJIC) coordinates cellular functions essential for sustaining tissue homeostasis; yet its regulation in the intestine is not well understood. Here, we identify a novel physiological link between Toll-like receptor (TLR) 2 and GJIC through modulation of Connexin-43 (Cx43) during acute and chronic inflammatory injury of the intestinal epithelial cell (IEC) barrier. Data from in vitro studies reveal that TLR2 activation modulates Cx43 synthesis and increases GJIC via Cx43 during IEC injury. The ulcerative colitis-associated TLR2-R753Q mutant targets Cx43 for increased proteasomal degradation, impairing TLR2-mediated GJIC during intestinal epithelial wounding. In vivo studies using mucosal RNA interference show that TLR2-mediated mucosal healing depends functionally on intestinal epithelial Cx43 during acute inflammatory stress-induced damage. Mice deficient in TLR2 exhibit IEC-specific alterations in Cx43, whereas administration of a TLR2 agonist protects GJIC by blocking accumulation of Cx43 and its hyperphosphorylation at Ser368 to prevent spontaneous chronic colitis in MDR1α-deficient mice. Finally, adding the TLR2 agonist to three-dimensional intestinal mucosa-like cultures of human biopsies preserves intestinal epithelial Cx43 integrity and polarization ex vivo. In conclusion, Cx43 plays an important role in innate immune control of commensal-mediated intestinal epithelial wound repair. Gap junctional intercellular communication (GJIC) coordinates cellular functions essential for sustaining tissue homeostasis; yet its regulation in the intestine is not well understood. Here, we identify a novel physiological link between Toll-like receptor (TLR) 2 and GJIC through modulation of Connexin-43 (Cx43) during acute and chronic inflammatory injury of the intestinal epithelial cell (IEC) barrier. Data from in vitro studies reveal that TLR2 activation modulates Cx43 synthesis and increases GJIC via Cx43 during IEC injury. The ulcerative colitis-associated TLR2-R753Q mutant targets Cx43 for increased proteasomal degradation, impairing TLR2-mediated GJIC during intestinal epithelial wounding. In vivo studies using mucosal RNA interference show that TLR2-mediated mucosal healing depends functionally on intestinal epithelial Cx43 during acute inflammatory stress-induced damage. Mice deficient in TLR2 exhibit IEC-specific alterations in Cx43, whereas administration of a TLR2 agonist protects GJIC by blocking accumulation of Cx43 and its hyperphosphorylation at Ser368 to prevent spontaneous chronic colitis in MDR1α-deficient mice. Finally, adding the TLR2 agonist to three-dimensional intestinal mucosa-like cultures of human biopsies preserves intestinal epithelial Cx43 integrity and polarization ex vivo. In conclusion, Cx43 plays an important role in innate immune control of commensal-mediated intestinal epithelial wound repair. The intestinal epithelial cell (IEC) 3The abbreviations used are: IECintestinal epithelial cell(s)Cx43connexin-43DSSdextran sodium sulfateGJgap junctionGJICgap junctional intercellular communicationMDR1multidrug resistance geneTJtight junctionTLRToll-like receptorUCulcerative colitisWTwild typeZO-1zonula occludens-1PCSKPam3CysSK4HAhemagglutininFLfull-lengthFITCfluorescein isothiocyanateDAPI4′,6-diamidino-2-phenylindolesiRNAsmall interfering RNARTreverse transcriptionGAPDHglyceraldehyde-3-phosphate dehydrogenase. barrier provides the front line of mucosal host defense in the intestine. The IEC barrier confers anatomic integrity and immunologic protection of the intestinal mucosal surface. Because the IEC barrier constantly faces diverse populations of lumenal microbes and other potential threats, it must exert a highly defined process of continuous discrimination: excluding harmful antigens while allowing host-beneficial substances to permeate (1Podolsky D.K. Am. J. Physiol. 1999; 277: G495-G499PubMed Google Scholar, 2Sartor R.B. Gastroenterology. 2008; 134: 577-594Abstract Full Text Full Text PDF PubMed Scopus (1475) Google Scholar). Para- and intercellular transit of molecules is modulated by a complex network of closely arranged tight (TJ) and gap junctions (GJ) between juxtaposed IEC. Gap junctional intercellular communication (GJIC) is an essential, but not well understood, mechanism for cellular and tissue homeostasis that coordinates cell-cell passage of ions and small metabolites ( F7) and appropriate WT controls (FVB/N) were purchased and bred under the Research Agreement from Taconic Farms (Germantown, NY). TLR2−/− mice (Tlr2tm1Kir; >F10 (C57BL6/J); The Jackson Laboratory, Bar Harbor, ME) and their WT controls (C57BL6/J) have previously been described (18Cario E. Gerken G. Podolsky D.K. Gastroenterology. 2007; 132: 1359-1374Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). All of the mice were housed and bred in the same room under strict specific pathogen-free conditions (Helicobacter species, murine norovirus-free) at the Central Animal Facility of University Hospital of Essen (Essen, Germany). Extensive animal health monitoring was conducted routinely on representative mice from this room (Gesellschaft für innovative Mikroökologie mbH, Wildenbruch, Germany). The animals were provided with autoclaved tap water and autoclaved standard laboratory chow ad libitum. All male MDR1α−/− developed loose stools and mucous discharge by ∼8 weeks of age in our specific pathogen-free room, and therefore only male MDR1α−/− were included in this study. Histology scoring was modified according to previous reports (29Mizoguchi E. Mizoguchi A. Takedatsu H. Cario E. de Jong Y.P. Ooi C.J. Xavier R.J. Terhorst C. Podolsky D.K. Bhan A.K. Gastroenterology. 2002; 122: 134-144Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 30Nguyen D.D. Maillard M.H. Cotta-de-Almeida V. Mizoguchi E. Klein C. Fuss I. Nagler C. Mizoguchi A. Bhan A.K. Snapper S.B. Gastroenterology. 2007; 133: 1188-1197Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), based on the specific characteristics of this murine colitis model (supplemental Table S3). Colitis was induced in TLR2−/− or WT by the addition of DSS (molecular weight, 36,000–50,000; Lot 6955H; MP Biomedicals, Irvine, CA) in different concentrations in drinking water for 5 days (18Cario E. Gerken G. Podolsky D.K. Gastroenterology. 2007; 132: 1359-1374Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). Female mice aged 8–11 weeks old were used for DSS studies. All of the protocols were in compliance with German law for use of live animals and approved by the Institutional Animal Care and Use Committee at the University Hospital of Essen and the responsible district government. Caco-2 and IEC-6 cells were purchased from ATCC (Manassas, VA) and maintained as described previously (17Cario E. Gerken G. Podolsky D.K. Gastroenterology. 2004; 127: 224-238Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). HA-tagged TLR2 full-length (TLR2-FL) and HA-tagged TLR4 full-length (TLR4-FL) plasmids in the same backbone vector were obtained from InvivoGen. UC mutant TLR2-R753Q was generated within the TLR2-FL expression construct and confirmed by sequencing (Trenzyme). Plasmids (EndoFree Maxi; Qiagen) were transfected into Caco-2 cells on a 12-well plate using 1 μg/well of TLR2-FL, TLR4-FL, or TLR2-R753Q (Lipofectamine LTX; Invitrogen). Stable transfectants were selected with 1 μg/ml blasticidin and maintained on poly-d-lysine-coated tissue culture plates (BD Biosciences). Transfection efficiency of the different clones was comparable with previous findings from other cell types (31Merx S. Neumaier M. Wagner H. Kirschning C.J. Ahmad-Nejad P. Hum. Mol. Genet. 2007; 16: 1225-1232Crossref PubMed Scopus (43) Google Scholar), as tested by Western blotting and immunohistochemistry with anti-HA (InvivoGen; Cell Signaling). Caco-2 controls (mock) were untransfected cells without any exogenous DNA. Fresh tissue samples from healthy patients undergoing complete colonoscopy for regular colon cancer screening examinations and/or polypectomy were collected at the Endoscopy Unit, Department of Gastroenterology and Metabolic Diseases (Head: M. Rünzi, M.D.), Kliniken Essen-Süd (Essen, Germany). Informed consent was obtained from all patients before the procedure, and the protocol was approved by the Human Studies Committee of Kliniken Essen-Süd. Small cecal specimens were obtained by gently touching down the biopsy forceps onto the mucosal surface. The samples were immediately washed twice in ice-cold Hanks舗 buffered salt solution (PAA) supplemented with 10% (v/v) fetal calf serum, gentamicin, and antibiotic/antimycotic solution; then mixed with or without PCSK (20 μg/ml) into a thin layer of cold liquid Matrigel (BD Biosciences) surrounding the biopsy, which was allowed to polymerize, forming a solid gel at 37 °C within 30 min; then covered with warm full L-15 medium; and cultured for 4 h. Matrigel biopsy blocks were snap frozen in liquid nitrogen, embedded in Tissue-Tek OCT compound (Sakura), and stored at −20 °C until further processing. For assessment of GJ communication by microinjection, 5% Lucifer Yellow CH dye (lithium salt in 100 mm water solution; Invitrogen) was injected into IEC-6 cells, and dye transfer was quantitated by determining the number of directly adjacent cells that received dye (dye coupling). The cells were visualized using an inverted phase contrast/confocal laser microscope (LD-Achroplan 40×/0.6 corr (dry) objective; Zeiss LSM510; Axiovert 100M) and injected using a transinjector with micromanipulator (model 5246/5171; Eppendorf). The frequencies of dye transfer to directly adjacent cells were determined in the presence or absence of PCSK 37s after dye injection (pi = 60 hPa; pc = 20 hPa; ti = 0.2 s) with or without cell injury by acquiring fluorescent images in an automated time series with an interval of 11.671 s, with each confocal scan set to take 3.8 s. Control dye was 10% tetramethylrhodamine isothiocyanate-dextran (molecular weight, 3000) in 100 mm LiCl, 50 mm Tris, pH 7.8. The nuclei were counterstained with cell-permeant SytoGreen fluorescent nucleic acid stain (Invitrogen). For assessment of GJ communication by scrape wound loading, Lucifer Yellow CH (0.05% dye in phosphate-buffered saline (without Ca2+ and Mg2+)) was loaded intracellularly by scraping Caco-2-TLR2-FL and Caco-2-TLR2-R753Q cells with a razor blade. The dye solution was left in the dish for 8 min. After washing, fixation, immunostaining with anti-Lucifer Yellow rabbit polyclonal antibody (Invitrogen) followed by FITC-conjugated donkey anti-rabbit secondary antibody (Jackson Immunoresearch), and mounting with Vectashield/DAPI, the cells were examined for dye transfer using a laser-scanning confocal microscope Zeiss Axiovert 100M-LSM 510. The degree of communication was assessed by determining the extent of Lucifer Yellow transfer into contiguous cells. The mice received 2.3% DSS in drinking water for 5 days, followed by oral PCSK therapy (see Fig. 6A). siRNA targeting murine Cx43 (siGENOME on-Target plus SMART pool; catalog number L-051694-00-0005; accession number NM_010288) and scrambled controls (siGENOME on-Target plus siCONTROL pool; catalog number D-001810-10-05) were obtained from Dharmacon and resuspended in sterile RNase-free water. siRNA was freshly lipoplexed with Lipofectamine 2000 in Opti-MEM (Invitrogen) and administered immediately (5 nmol in 20 μl of solution/mouse) to the rectum of isoflurane-anesthesized WT mice (C57BL6/J) once daily (days 3–5) using a P20 pipettor (Eppendorf), as described previously (32Zhang Y. Cristofaro P. Silbermann R. Pusch O. Boden D. Konkin T. Hovanesian V. Monfils P.R. Resnick M. Moss S.F. Ramratnam B. Mol. Ther. 2006; 14: 336-342Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Five hours after receiving the last siRNA treatment, control groups (n = 5 mice/group; group 1, Cx43siRNA (−PCSK); group 2, scrambled (−PCSK)) were sacrificed on day 5 without PCSK therapy, and the rectum was removed and evaluated for rectal epithelial Cx43 protein expression at the local site of siRNA application by immunohistochemistry, as described below. The rest (n = 4–5 mice/group; group 3, Cx43siRNA (−PCSK); group 4, Cx43siRNA (+PCSK); group 5, scrambled (−PCSK); group 6, scrambled (+PCSK)) were sacrificed on day 9 with or without PCSK therapy, and the entire colon was removed from cecum to anus, and colon length was measured as a marker of inflammation. For histologic examination, frozen cross-sections (7 μm; every 60 μm) throughout the application site of the rectum were obtained and stained with a hematoxylin-eosin kit (Polysciences). The degree of DSS-induced acute inflammation in the rectum was graded blinded, as described previously (18Cario E. Gerken G. Podolsky D.K. Gastroenterology. 2007; 132: 1359-1374Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). Frozen sections were cut (7 μm) and mounted on Superfrost Plus Gold slides (Thermo). The cells were directly grown on coated culture slides. For detection of murine/human Cx43 (clone CX-1B1), ZO-1, and TLR2, sections were fixed in acetone (100%) for 1 min at −20 °C followed by air-drying and washing with phosphate-buffered saline. The sections were then blocked with normal goat serum (1:100 in phosphate-buffered saline) for 60 min at room temperature and incubated with primary antibodies (1:50; for pan-cytokeratin, 1:25) overnight at 4 °C. Alexa Fluor®488-conjugated goat anti-mouse IgG (Molecular Probes-Invitrogen) and/or CY5/FITC-conjugated goat anti-rabbit IgG (Jackson Immunoresearch Laboratories) antibodies were used as secondary antibodies (1:100, 60 min, room temperature). After mounting with Vectashield mounting medium with propidium iodide or DAPI (Vector Laboratories), immunofluorescent sections were assessed using a laser-scanning confocal microscope (Plan-Neofluar 40×/1.3 (oil) or Plan-Apochromat 63×/1.4 (oil) differential interference contrast objectives; Zeiss Axiovert 100M-LSM 510), as described previously (18Cario E. Gerken G. Podolsky D.K. Gastroenterology. 2007; 132: 1359-1374Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 33Cario E. Golenbock D.T. Visintin A. Rünzi M. Gerken G. Podolsky D.K. J. Immunol. 2006; 176: 4258-4266Crossref PubMed Scopus (41) Google Scholar). Briefly, the multitrack option of the microscope and sequential scanning for each channel were used to eliminate any cross-talk. All of the images were captured under identical laser settings. The results were only considered significant if more than 80% of the scanned sections/field exhibited the observed effect. Standardized three-dimensional reconstructions and extractions of co-localized areas were generated using LSM 510 v.3.2 software. Control experiments were performed with isotype control IgG (Santa Cruz or Ebioscience) or omitting the primary antibody. All of the experiments were repeated at least twice; representative results are shown for each experiment and subgroup as indicated. Proteins from whole colon samples of distal parts were isolated using the T-PER tissue protein extraction reagent (Thermo) supplemented with complete mini protease ± PhosSTOP phosphatase inhibitor mixture tablets and 2 mm phenylmethylsulfonyl fluoride Plus (Roche Applied Science). Dephosphorylation reaction of Cx43 was carried out by incubating tissue lysates for 1 h at 37 °C in the presence (1 unit/μg) of calf intestinal alkaline phosphatase (Promega). ZO-1 was detected in whole colonic samples as described previously (18Cario E. Gerken G. Podolsky D.K. Gastroenterology. 2007; 132: 1359-1374Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). The lysates were mechanically disrupted by a tissue disruptor homogenizer (Qiagen). For preparation of subcellular fractions, the ProteoExtract subcellular proteome extraction kit from Merck was used according to the manufacturer舗s instructions. Immunoprecipitation/immunoblotting and mouse 62-cyto/chemokine antibody array III (RayBiotech) were performed as described previously (18Cario E. Gerken G. Podolsky D.K. Gastroenterology. 2007; 132: 1359-1374Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 33Cario E. Golenbock D.T. Visintin A. Rünzi M. Gerken G. Podolsky D.K. J. Immunol. 2006; 176: 4258-4266Crossref PubMed Scopus (41) Google Scholar). All of the experiments were repeated at least twice; representative results are shown for each experiment. Total RNA samples from cells and murine colons were extracted using the RiboPure RNA Isolation kit from Ambion and purified using the RNeasy mini kit with RNase-free DNase from Qiagen, following the manufacturers舗 instructions. For conventional RT-PCR analysis, reverse transcription of 50 ng of each RNA sample was performed with Sensiscript RT (Qiagen). 2 μl of the RT reaction was used for subsequent PCR (HotStar Taq DNA polymerase; Qiagen) in duplicate. PCR primers (Primer-BLAST, NCBI) were as follows: human Cx43, 5′-GGA CAT GCA CTT GAA GCA GA and 5′-CAG GAG GAG ACA TAG GCG AG (37 cycles); and human ZO-1, 5′-GTC TGC CAT TAC ACG GTC CT and 5′-GGT CTC TGC TGG CTT GTT TC (32 cycles). Species nonspecific GAPDH primers have previously been described (13Cario E. Rosenberg I.M. Brandwein S.L. Beck P.L. Reinecker H.C. Podolsky D.K. J. Immunol. 2000; 164: 966-972Crossref PubMed Scopus (644) Google Scholar). Negative controls included water or RNA sample as template. All of the PCR products were resolved by 1–2% agarose gel electrophoresis, and DNA bands were visualized by staining the gel with ethidium bromide. For real time PCR analysis, QuantiTect Primer Assays (Qiagen) were used as the gene-specific primer pairs for human Cx43 (QT00012684), human ZO-1 (QT00077308), human GAPDH (QT01192646), mouse Cx43 (QT00173635), mouse ZO-1 (QT00493899), mouse TFF3 (QT00108857), mouse interferon γ (QT01038821), mouse IL-2 (QT00112315), mouse IL-12p40 (QT00153643), mouse IL-23R (QT00138719), mouse IL-4 (QT00160678), mouse IL-6 (QT00098875), mouse IL-10 (QT00106169), mouse IL-13 (QT00099554), and mouse GAPDH (QT00309099). Quantitative real time RT-PCR was performed at least in duplicate using the one-step QuantiTect SYBR Green RT-PCR kit (Qiagen) on the Rotor-Gene 2000 real time amplification system (Corbett Research), according to the manufacturer舗s protocols. Copy numbers of individual transcripts were normalized in parallel against GAPDH as endogenous control (X/100,000 copies of GAPDH). The mRNA level in untreated cells or tissues (as indicated under "Results") was defined as 1 arbitrary unit. The data are expressed as the means ± S.E. of two or more independent experiments, as indicated. The differences between means were evaluated using the two-tailed, unpaired t test (GraphPad Prism version 4.03, GraphPad Software, San Diego, CA), where appropriate. p values of <0.05 were considered as significant. To determine whether ZO-1 complexes with cellular Cx43 in IEC, we performed co-immunolabeling using confocal laser microscopy. ZO-1 and Cx43 were predominantly localized at the apical and lateral plasma membrane in the human model IEC line Caco-2 (Fig. 1A) and were frequently but not exclusively co-localized at distinct cell-cell contacts (overlay panel) in typical punctuate staining patterns (extracted panel). Constitutive interaction between ZO-1 and Cx43 in Caco-2 cells was confirmed by co-immunoprecipitation (data not shown). We next studied the effects of TLR2 in regulating synthesis of the ZO-1-Cx43 complex in IEC. Stimulation of Caco-2 cells with the synthetic TLR2 ligand PCSK increased Cx43 mRNA within 3 h, with a maximum of 3-fold activation after 6 h (Fig. 1B). After 12 h, Cx43 mRNA expression returned to low control levels. Subcellular fractions of intestinal epithelial Cx43 protein electrophoresed as typical multiple isoforms when analyzed by SDS-PAGE (Fig. 1C), including a faster migrating, nonphosphorylated form (P0; ∼42 kDa), and two slower migrating, phosphorylated forms, termed P1 (∼44 kDa) and P2 (∼46 kDa), consistent with previous reports from other cell types (5Musil L.S. Cunningham B.A. Edelman G.M. Goodenough D.A. J. Cell Biol. 1990; 111: 2077-2088Crossref PubMed Scopus (593) Google Scholar, 26Cruciani V. Mikalsen S.O. Exp. Cell Res. 1999; 251: 285-298Crossref PubMed Scopus (43) Google Scholar, 34Lampe P.D. Lau A.F. Arch Biochem. Biophys. 2000; 384: 205-215Crossref PubMed Scopus (471) Google Scholar). PCSK stimulation for 3 h led to redistribution of Cx43-P2 from the cytoskeleton to the plasma membrane. In contrast, TLR2 stimulation did not modulate ZO-1 gene transcription or protein expression in IEC in vitro. To analyze intestinal epithelial GJIC functionality in response to TLR2 activation, we next performed microinjection studies to determine the incidence and extent of dye coupling between IEC with or without PCSK treatment. For optimal microinjection conditions, we used IEC-6 cells, which form a simple, confluent monolayer of polyglonal epithelial cells in culture and express both TLR2 (18Cario E. Gerken G. Podolsky D.K. Gastroenterology. 2007; 132: 1359-1374Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar) and Cx43 (11Leaphart C.L. Qureshi F. Cetin S. Li J. Dubowski T. Baty C. Beer-Stolz D. Guo F. Murray S.A. Hackam D.J. Gastroenterology. 2007; 132: 2395-2411Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). IEC-6 cells were microinjected with Lucifer Yellow, which readily passes through GJ (Fig. 2A). Rhodamine-dextran (not transferable through GJ) and SytoGreen (cell-permeant nuclear stain) were used as control markers of cell and nucleus intactness, respectively (Fig. 2B). Under basal conditions, most IEC-6 cells were not dye-coupled (Fig. 2A, upper panel). The presence of PCSK did not increase the efficacy of dye spreading. However, when the IEC-6 cell was injured by microinjection, the incidence of intercellular diffusion was increased, with most cells showing dye coupling with four or five neighboring cells (Fig. 2A, lower panel), suggesting that cellular damage is required to activate GJIC between otherwise quiescent IEC-6 cells. PCSK rapidly increased the extent of dye transfer around single cell injuries, showing 10–11 coupled cells/injection. In addition, GJ-mediated intercellular diffusion was sustained longer in PCSK-treated c
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