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J. Biol. Chem., Vol. 281, Issue 27, 18560-18568, July 7, 2006
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1
From the
Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215 and
INCELL Corporation, San Antonio, Texas 79249
Received for publication, December 29, 2005 , and in revised form, April 12, 2006.
| ABSTRACT |
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| INTRODUCTION |
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B and AP-1 and leading to up-regulation of proinflammatory cytokine gene expression such as IL-8 and MIP3
in nontransformed human colonic epithelial cells, NCM460 (4). More recently, we also characterized the pathophysiological role of flagellin in an experimental colitis model using dextran sulfate sodium (DSS) (5). According to our recent results, only injured, not intact colonic mucosa is highly vulnerable to intracolonic flagellin, causing proinflammatory gene expression and histologically evident colonic inflammation. These data strongly indicate that bacterial flagellin in the gut participates in the development and progress of colonic inflammation.
Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase that phosphorylates phosphoinositides at position 3 of the inositol ring, leading to activation of the well characterized serine/threonine kinase Akt/protein kinase B (6). Class 1 PI3Ks are composed of a p85 regulatory subunit interacting with an upstream signaling molecule, generally a membrane receptor, and a p110 catalytic subunit responsible for their enzymatic activity (6). This class of PI3Ks can be activated by various receptor-mediated extracellular stimuli, including TLR2, TLR3, TLR4, IL-1R, and platelet-derived growth factor receptor (712).
The human gut harbors a large collection of commensal microbes, releasing various pathogen-associated molecular patterns (2, 13). These microbial ligands are able to initiate TLR engagement and trigger PI3K activation. Sheng et al. (14) recently showed that PI3K activation is essential for intestinal epithelial cell proliferation in vitro and in vivo by inducing cyclin D1 expression. Moreover, the up-regulation of PI3K has been suggested to cause a hyperproliferative epithelium (15). Indeed, increased PI3K activity was observed in a majority of colon cancers (16). Therefore, inappropriate TLR engagement probably results in an aberrant PI3K activation leading to a hyperproliferative epithelium. Nevertheless, the molecular mechanisms by which TLR5 activates PI3K have not been studied.
In this study, we demonstrate that in nontransformed human colonic epithelial cells, the adaptor molecule MyD88 binds to TLR5 in response to flagellin, and MyD88 recruits the p85 regulatory subunit of PI3K, implying that MyD88 plays a bridging role between TLR5 and the p85 regulatory subunit. These results provide a molecular mechanism whereby TLR5 engagement by bacterial flagellin leads to activation of PI3K in human colonic epithelial cells.
| EXPERIMENTAL PROCEDURES |
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B-luciferase reporter, TLR5, and TLR5 (
TIR) were described previously (4). The dominant negative plasmid of the PI3K regulatory subunit (p85-DN, lacks the binding site for the catalytic subunit p110) and wild type p85 expression plasmid were kindly provided by Dr. Julian Downward (Imperial Cancer Research Fund, London, UK) (17). p85
-FLAG, a generous gift from Dr. David Fruman (University of California, Irvine), was subcloned into pcDNA3.1/zeo (). Akt wild type and dominant negative constructs (Akt-DN) were from Upstate (Charlottesville, VA). All plasmids were prepared using the EndoFree plasmid purification kit (Qiagen, Valencia, CA) as recommended by the manufacturer. Monoclonal antibodies against FLAG and HA were from Sigma and Roche Applied Science, respectively. The nontransformed human colonic epithelial cells, NCM460, and the culture medium M3D were obtained from INCELL Corp. (San Antonio, TX) (4). NCM460 cells and human embryonic kidney (HEK) 293 cells were cultivated as described in our previous studies (4, 18).
8-Week-old male mice (C3H/HeJ) were from The Jackson Laboratories (Bar Harbor, ME) and housed in a pathogen-free facility. To induce colitis, mice were fed dextran sulfate sodium (DSS, 2.5%) (MP Biomedicals, Irvine, CA) dissolved in regular tap water during the entire experimental period, as described previously (5). The Institutional Animal Care and Use Committee of Beth Israel Deaconess Medical Center approved all animal procedures.
Generating the Various Mutant Constructs of TLR5 and MyD88TLR5 (Y798F)-HA, TLR5 (Y815F)-HA, TLR5 (Y798F/Y815F)-HA, and TLR5 (Y691F/Y693F/Y696F)-HA were generated by PCR-based mutagenesis using Pfu enzyme (Stratagene, La Jolla, CA) and appropriate primers. To generate TLR5 (Y798F)-HA, we performed the first PCR using TLR5 WT-HA as template and primers F-1 harboring Y798F substitution and R-2. Then, by using the first PCR product as reverse primer and F-2 primer, we performed the second PCR with TLR5 WT as a template. The second PCR product was inserted into XbaI and BamHI sites in the pUNO-TLR5-HA construct, resulting in the TLR5 (Y798F)-HA construct. To generate TLR5 (Y815F)-HA and TLR5 (Y798F/Y815F)-HA, the first PCR was done with primers F-3 containing the Y815F mutation and R-1 and TLR5 WT-HA as a template. The second PCR was performed with the first PCR product and F-2 as primers, and TLR5 WT-HA or TLR5 (Y798F)-HA was used as a template. The PCR product from the second PCR was inserted into XbaI and BamHI sites in the pUNO-TLR5-HA construct, giving rise to TLR5 (Y815F)-HA and TLR5 (Y798F/Y815F)-HA constructs, respectively. To generate TLR5 (Y691F/Y693F/Y696F)-HA, the primers R-1 and F-4 harboring the Y691F mutation were used for the first PCR with a template TLR5 WT-HA. The primer F-2 and the first PCR product were used for the second PCR with TLR5 WT-HA to generate TLR5 (Y691F)-HA. PCR was again performed with primers R-1 and F-5 possessing Y693F/Y696F mutations and a template TLR5 (Y691F)-HA. The PCR product was used as primers along with F-2 and TLR5 (Y691F)-HA as a template. The PCR product was used to generate TLR5 (Y691F/Y693F/Y696F)-HA construct.
To generate human MyD88 (Y257F)-HA expressing construct, the first PCR was performed with an F-6 primer containing the Y257F mutation and an R-2 primer along with MyD88 WT-HA as a template. The MyD88 (Y257F)-HA fragment was amplified through PCR using the first PCR product and F-7 as primers and the pUNO-MyD88 WT-HA construct as a template. The integrity of the whole sequence was confirmed by sequencing all DNA constructs. Primers used for PCR were as described below, and substitution of tyrosine with phenylalanine is marked in boldface with an underline. The primers used are as follows: F-1, 5'-GGT GGT GGTT GGG TCC TTG TCC CAG TTC CAG TTG ATG AAA CAT CAA TCC-3'; F-2, 5'-CC TTACA GCG AAC CTC ATC CAC TTA TCAG-3'; F-3, 5'-GGC TTT GTA CAG AAA CAG CAG TTT TTG AGG TGGC-3'; F-4, 5'-CCT GAT ATG TTC AAA TAT GAT GCC TAT TTG TGC-3'; F-5, 5'-CCT GAT ATG TTC AAA TTT GAT GCC TTT TTG TGC TTC AGC AGC-3'; F-6, 5'-CCCC ATC AAG TTC AAG GCA ATG AAG AAA GAG TTC CCC-3'; F-7, 5'-CCC TGC TTG CTC AAC TCT ACG TC-3'; R-1, 5'-GGC GTA GTC TGG CAC ATC ATA GGG GTA GG-3'; and R-2, 5'-GGC ATA GTC TGG CAC ATC ATA AGG GTA TCC-3'.
Immunofluorescence StainingCells grown on a chamber slide system (Nalge Nunc, Rochester, NY) were fixed in 10% formalin and permeabilized using 0.5% Triton X-100. Slides were washed with PBS and blocked with normal goat serum (1%) plus 1% free bovine serum albumin (1%) in PBS for 20 min at room temperature. Slides were then incubated for 1 h with primary antibodies (1:50 dilution) against FOXO1 (Santa Cruz Biotechnology, Santa Cruz, CA) in bovine serum albumin (1%), PBS. Samples were then washed with PBS and incubated with Cy3-conjugated anti-rabbit secondary antibody (1:200) for 1 h. The slides were then rinsed and mounted with DAPI mounting solution (Vector Laboratories, Burlingame, CA). Images were analyzed with a Zeiss Axioskop-2 microscope.
Generating TLR5 or MyD88 Silenced CellsThe silencing vector expressing siRNA targeting the human TLR5 or MyD88 were obtained from InvivoGen (San Diego) (19). NCM460 or HEK293 cells were transfected with the silencing vector and an empty control vector. Stably transfected cells were isolated in selection media containing Zeocin. Silencing of the target molecule was confirmed in several isolated colonies.
Immunoblotting AnalysisCell lysates were prepared in lysis buffer (150 mM NaCl, 50 mM Tris-Cl (pH 8.0), 5 mM EDTA, 1% Nonidet P-40) containing a protease inhibitor mixture (Roche Applied Science) and a phosphatase inhibitor mixture (Sigma). The equal amount of total protein was subjected to SDS-PAGE analysis, and immunoblotting with the appropriate antibodies was performed as described previously (4).
Immunoprecipitation AssayTotal cell lysates were prepared in lysis buffer as described above. Equal amounts of total protein were mixed with a primary antibody or control IgG and incubated at 4 °C for 45 h. The protein-A or -G beads (Pierce) were then added and incubated overnight. The precipitants were washed three times with lysis buffer and fractionated on SDS-PAGE, followed by the immunoblotting procedure described above.
Luciferase Reporter AssaysCells were plated in 6-well plates (0.5 x 106 cells/well) and transfected with the appropriate plasmid DNA, including a
-galactosidase expression plasmid (HSP70-
-gal) as an internal control, using SuperFect transfect reagent (Qiagen, Valencia, CA) according to the manufacturer's instructions. One day after transfection, cells were stimulated with flagellin (100 ng/ml), LPS (100 ng/ml), IL-1 (50 ng/ml), or tumor necrosis factor-
(50 ng/ml) for 6 h, and the relative luciferase activity was determined by normalization with
-galactosidase activity as described previously (4). The total amount of plasmid DNA was kept consistent by adding the empty vector for each transfection. All assays were performed in triplicate, and a single representative experiment is shown. Data are expressed as mean values ± S.E.
IL-8 and MIP3
MeasurementAn enzyme-linked immunosorbent assay (ELISA) was performed to measure the level of human IL-8 and MIP3
using the appropriate kits from BIOSOURCE and R & D Systems (Minneapolis, MN), respectively, by following the manufacturer's instructions. All assays were performed in triplicate, and data were expressed as mean ± S.E.
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Infection of AdenovirusesThe recombinant adenoviruses, Adeno-p85-DN and control Adeno-lacZ, were generous gifts from Dr. Masato Kasuga, Kobe University Graduate School of Medicine (Japan) (20). For infection, NCM460 cells (2.0 x 106 cells/60-mm dish) were cultivated for a day and incubated with the adenoviruses at the same multiplicity of infection (plaque-forming units per cell) in culture media for 6 h at 37 °C. The virus-containing medium was replaced with fresh medium and cultivated for 1 day. Cells were stimulated with flagellin (100 ng/ml) overnight, and IL-8 was measured in the culture media. The expression of p85-DN was also confirmed by immunoblotting analysis in the cell lysates.
| RESULTS |
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Smythies et al. (21) showed that human intestinal macrophages are anergic to various inflammatory stimuli, including LPS. In addition, TLR5, a specific receptor for bacterial flagellin, is highly expressed in intestinal epithelial cells such as the adenocarcinoma cell lines HT29 and T84, as well as in nontransformed human NCM460 colonocytes, as we described previously (4). Therefore, we investigated if flagellin/TLR5 engagement induces PI3K activation in NCM460 cells. As shown in Fig. 1B, flagellin/TLR5 engagement transiently phosphorylates Akt within 10 min, and this effect is evident for up to 40 min after stimulation. Flagellin-stimulated Akt phosphorylation is blocked by the PI3K-specific inhibitor LY294002 (Fig. 1C), indicating that Akt phosphorylation is mediated by PI3K activated in response to flagellin and suggesting that Akt phosphorylation can be used as a readout for PI3K activation in NCM460 cells. Moreover, because activation of Akt results in the phosphorylation of the forkhead transcription factor Foxo1, followed by the inhibition of translocation of Foxo1 to the nucleus (22), another readout of PI3K activation is the expected localization of Foxo1 that should be expelled from the nucleus by PI3K activation. Thus, we examined if flagellin stimulation to NCM460 cells inhibits FOXO1 translocation to the nucleus by immunofluorescence staining with an antibody recognizing FOXO1. As shown in Fig. 1D, control cells show the presence of FOXO1 (in red) at the nucleus (counterstained by DAPI, in blue). In contrast, in cells stimulated with flagellin FOXO1 is localized outside the nuclei (Fig. 1D). The merged image of FOXO1 and DAPI nuclei counterstaining confirmed that FOXO1 is re-localized to the cytoplasmic region following flagellin exposure (Fig. 1D). Together, these data demonstrate that flagellin/TLR5 engagement induces PI3K activation in nontransformed human colonocytes.
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B activation is indeed knocked down in TLR5-silenced cell in response to flagellin. To address this, because NF
B activation is a canonical outcome of TLR engagement, TLR5-silenced NCM460 cells were transiently transfected with an NF
B-luciferase reporter plasmid followed by measuring NF
B reporter activity in response to flagellin. As shown in Fig. 2B, TLR5-silenced NCM460 cells were not responsive to flagellin stimulation, but they responded to IL-1 that utilizes a conserved molecular signaling pathway downstream of the TLR5 and IL-1 receptor, implying the specific inhibition of TLR5-mediated signaling at the receptor level (1). In contrast, NCM460 cells transfected with control vector were responsive to flagellin, as measured by NF
B reporter activity (Fig. 2B). Furthermore, because flagellin stimulation of NCM460 cells leads to IL-8 production (4), knockdown of TLR5 expression should inhibit flagellin-induced IL-8 production in TLR5-silenced NCM460 cells. To test this, we treated TLR5-silenced and control NCM460 cells with flagellin overnight and measured the level of IL-8 protein production in cell supernatants by using ELISA. We found that flagellin-induced IL-8 production was blocked in TLR5-silenced cells, whereas control cells produced large amounts of IL-8 in response to flagellin (Fig. 2C). In contrast, TLR5-silenced NCM460 cells had increased IL-8 production in response to IL-1 (Fig. 2C), indicating that TLR5 expression is selectively silenced in NCM460 cells. Next, we stimulated TLR5-silenced NCM460 cells with flagellin and examined PI3K activation by immunoblotting analysis. As shown in Fig. 2D, flagellin exposure stimulated Akt phosphorylation only in control, but not in TLR5-silenced NCM460 cells. Flagellin stimulation also failed to change the localization of FOXO1 in TLR5-silenced NCM460 cells (data not shown). Together, these data clearly indicate that PI3K activation is a specific outcome achieved from TLR5 engagement in response to flagellin in nontransformed human colonocytes.
TLR5 Associates with p85 Regulatory Subunit of PI3K, as Mediated by Its Cytoplasmic TIR DomainBecause TLR2 and TLR3 have been associated with the p85 regulatory subunit of PI3K (7, 8), we next investigated whether engagement of TLR5 by flagellin recruits the p85 regulatory subunit of PI3K in NCM460 cells. To study this, total cell lysates from NCM460 cells stimulated with flagellin, as indicated in Fig. 3A, were co-immunoprecipitated with an antibody against p85. The immunoprecipitant was next fractionated in SDS-PAGE followed by immunoblotting with an antibody against TLR5. As shown in Fig. 3A, the co-immunoprecipitation assay demonstrated that TLR5 associates with p85 in response to flagellin in nontransformed colonic epithelial cells. Moreover, HEK293 cells were transiently co-transfected with an HA-tagged TLR5 wild type (TLR5 WT-HA) or an HA-tagged TLR5 (
TIR) (cytoplasmic TIR domain deleted TLR5, TLR5 (
TIR)-HA) construct along with a FLAG-tagged p85 (p85-FLAG) construct as indicated in Fig. 3B. Two days after transfection, total cell lysates were prepared and subjected to co-immunoprecipitation with FLAG antibody followed by immunoblot analysis with HA antibody, as described under "Experimental Procedures." As shown in Fig. 3B, TLR5 was able to co-immunoprecipitate with the p85 regulatory subunit of PI3K, whereas TLR5 without the cytoplasmic TIR domain failed to associate with p85, implying that the cytoplasmic TIR domain of TLR5 mediates the association of TLR5 with the p85 regulatory subunit of PI3K.
The SH2 Domain Binding Motif (YXXM) in the Cytoplasmic TIR Domain of TLR5 Is Not Involved in Recruiting the p85 Subunit of PI3K to TLR5PI3K is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit. There are two SH2 domains (N-SH2 and C-SH2) in the p85 regulatory subunit that allow direct binding to the "YXXM" motif present in membrane-bound proteins, such as TLR2, IL-1 receptor, or platelet-derived growth factor receptor (7, 1012). Previous studies showed that TLR2 directly associates with the p85 regulatory subunit of PI3K via the YXXM motif in the cytoplasmic TIR domain of TLR2 (7). Along these lines, we analyzed the amino acid sequence of the TLR5 and identified the presence of a YXXM motif at amino acid residues 798801 YXXM within the cytoplasmic TIR domain of TLR5. Thus, we hypothesized that the YXXM motif in the cytoplasmic region of TLR5 is responsible for recruiting p85 protein, thereby resulting in TLR5-induced PI3K activation. To verify this hypothesis, we replaced the tyrosine residue in the YXXM motif within the cytoplasmic TIR domain of TLR5 with phenylalanine in order to disrupt the YXXM motif (TLR5 (Y798F)). TLR5 (Y798F)-HA expression construct was prepared as described under "Experimental Procedures." The HEK293 cells were then co-transfected with TLR5 (Y798F)-HA or TLR5 WT-HA expression construct and a p85-FLAG construct in the indicated combinations shown in Fig. 4B. Total cell lysates were prepared and subjected to co-immunoprecipitation with a FLAG antibody followed by immunoblot analysis using HA antibody to examine whether the disruption of YXXM motif prevents TLR5 (Y798F) from associating with p85. As presented in Fig. 4B, like TLR5 WT, TLR5 (Y798F) was still able to interact with p85. In addition, an adjacent tyrosine residue (Tyr-815) to the YXXM motif was also mutated to generate TLR5 (Y815F)-HA and co-transfected with p85-FLAG followed by co-immunoprecipitation assays. As shown in Fig. 4C, a point mutation of Y815F in the TIR domain of TLR5 is still capable of recruiting p85 protein. Furthermore, a double mutation of Y798F in the YXXM motif and Y815F in the TIR domain of TLR5 still enables p85 regulatory protein to co-immunoprecipitate with TLR5 (Fig. 4D). We also substituted various tyrosine residues (tyrosine 691, 693, and 696) at the tyrosine-rich region within the TIR domain of TLR5, and co-immunoprecipitation assays together with p85 were performed as described above. As shown in Fig. 4E, the triple mutation of tyrosine at 691, 693, and 696 amino acid residues did not alter the association of p85 protein with TLR5. Together, these data implied that the SH2 binding YXXM motif in the cytoplasmic TIR domain of TLR5 is not involved in recruiting the p85 regulatory subunit of PI3K. Furthermore, our data obtained from the various tyrosine mutants of TLR5 suggested that tyrosine residues at the TIR domain of TLR5 do not appear to be involved in p85 recruitment.
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B reporter construct followed by flagellin stimulation. As shown in Fig. 6B, NF
B reporter activity was not induced in MyD88-silenced HEK293 cells in response to flagellin stimulation, whereas strong NF
B reporter activity was evident in flagellin-exposed control HEK293 cells. In addition, because HEK293 cells do not express TLR4 to which MyD88 is a critical adaptor molecule, we transiently co-transfected TLR4 and NF
B reporter constructs in these cells followed by LPS stimulation. As shown in Fig. 6B, silencing MyD88 completely blocked LPS/TLR4-induced NF
B reporter activity, whereas LPS strongly induced NF
B reporter activity in control HEK293 cells. These data confirmed that MyD88 is biochemically and functionally silenced in HEK293 cells. To test whether the association between TLR5 and the p85 regulatory subunit of PI3K is MyD88-dependent, we next transiently co-transfected TLR5-HA and p85-FLAG constructs into both MyD88-silenced and control HEK293 cells in the indicated combination in Fig. 6C, followed by co-immunoprecipitation with a FLAG antibody. The immune complex was fractionated by SDS-PAGE followed by immunoblotting analysis using an HA antibody. As presented in Fig. 6C, TLR5 failed to co-immunoprecipitate with the p85 regulatory subunit of PI3K in MyD88-silenced cells, whereas TLR5 associates with p85 in control cells, implying that MyD88 is required for the association between TLR5 and p85.
MyD88 Mediates Flagellin/TLR5-induced PI3K Activation in Human ColonocytesNext, we investigated if the adaptor molecule MyD88 is required to mediate PI3K activation triggered by flagellin/TLR5 engagement in nontransformed human colonic epithelial cells (NCM460). To address this, we first generated MyD88-silenced NCM460 cells by stably transfecting MyD88 siRNA-expressing construct into NCM460 cells, as described under "Experimental Procedures." We confirmed the silenced MyD88 expression by immunoblotting analysis (Fig. 7A). Moreover, we demonstrated that flagellin-induced NF
B reporter activity was blocked in MyD88-silenced cells, in contrast to increased NF
B activation in control cells. However, tumor necrosis factor-
that mediates NF
B activation via MyD88-independent pathways was able to induce NF
B activation in both MyD88-silenced and control NCM460 cells (Fig. 7B), indicating that MyD88-dependent signaling was specifically blocked. Furthermore, we also evaluated if silencing MyD88 is able to inhibit flagellin-induced IL-8 production. As shown in Fig. 7C, flagellin-stimulated IL-8 production was completely blocked in MyD88-silenced NCM460 cells, whereas control NCM460 cells rendered substantially increased IL-8 production in response to flagellin. Together, these data present strong evidence indicating that the endogenous MyD88 expression was specifically and effectively silenced in these cells.
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YXXM Motif in MyD88 Is Responsible for Associating with the p85 Regulatory Subunit of PI3KOjaniemi et al. (23) showed that MyD88 associates with the p85 regulatory subunit of PI3K following LPS/TLR4 engagement in murine macrophages. Moreover, Aksoy et al. (9) suggested that the adaptor molecule TRIF downstream of TLR3 and -4 mediates PI3K activation. Because our data implied that MyD88 mediates TLR5-induced PI3K activation (Fig. 6), we investigated if flagellin/TLR5 engagement enables MyD88 to associate with the p85 regulatory subunit in nontransformed human colonocytes. To test this, NCM460 cells were stimulated with flagellin as indicated in Fig. 8A, and co-immunoprecipitation assays were performed using an antibody against the p85 regulatory subunit of PI3K, followed by immunoblot analysis with an antibody recognizing MyD88. As shown in Fig. 8A, MyD88 and the p85 regulatory subunit of PI3K are associated following TLR5 engagement by flagellin.
Because there is an SH2 domain-binding YXXM motif ("YKAM" at 257260 amino acids) in the C terminus of MyD88, we next investigated if disruption of this motif within MyD88 alters the association between MyD88 and the p85 regulatory subunit of PI3K. To assess this, we first generated an HA-tagged MyD88 (Y257F) mutant construct in which the tyrosine residue in the YKAM motif was replaced with phenylalanine, as described under "Experimental Procedures." As indicated in Fig. 8B, we transfected the MyD88 WT-HA or the MyD88 (Y257F)-HA expression construct into HEK293 cells along with a p85-FLAG expression construct. A co-immunoprecipitation assay was then performed with the FLAG antibody followed by immunoblot analysis with the HA antibody, as described under "Experimental Procedures." As presented in Fig. 8B, our co-immunoprecipitation results demonstrate that disruption of the YXXM motif in MyD88 interferes with the association between MyD88 and the p85 regulatory subunit of PI3K, whereas wild type MyD88 is able to associate with p85. Together with the results presented in Figs. 6 and 7, these data indicate that MyD88 is responsible for recruiting the p85 regulatory subunit of PI3K, thereby mediating flagellin/TLR5-induced PI3K activation.
PI3K Activation Induced by Flagellin/TLR5 Engagement Is Involved in IL-8 Expression in Nontransformed Human ColonocytesFukao et al. (24) suggested a negative regulation of PI3K in IL-12 production by TLR ligands such as LPS, peptidoglycan, or CpG DNA in mouse dendritic cells. To study the biological consequence of PI3K activation in TLR5-mediated signal transduction pathways triggered by flagellin, we performed cytokine micro-array, as described in our previous study (4), using total cell lysates from NCM460 cells stimulated with flagellin with or without the PI3K-specific inhibitor LY294002. Flagellin stimulation leads to increased production of IL-8 (red arrow) and MIP3
(blue arrow) (Fig. 9A), confirming our prior observations (4). Moreover, LY294002 significantly inhibited flagellin-induced IL-8 production, whereas MIP3
production was not significantly altered (Fig. 9A). Consistent with these results, the PI3K-specific inhibitor, LY294002, blocked flagellin-induced IL-8 but not MIP3
secretion measured by ELISA (Fig. 9B). We next transiently co-transfected NCM460 cells with a dominant negative mutant of p85 (p85-DN) or a wild type p85 expressing construct along with an IL-8 promoter reporter construct, as described under "Experimental Procedures" and evaluated flagellin-stimulated IL-8 promoter activity. As shown in Fig. 9C, overexpression of the p85 dominant negative mutant inhibited IL-8 promoter activity in response to flagellin. In addition, inhibition of the PI3K downstream signaling molecule Akt by overexpressing a dominant negative Akt mutant (Akt-DN) substantially reduced IL-8 promoter activity stimulated by flagellin in NCM460 cells. To evaluate if overexpressing p85-DN is able to inhibit flagellin-induced IL-8 production, NCM460 cells were infected with p85-DN expressing adenoviruses (Adeno-p85-DN) or control viruses (Adeno-lacZ), as described elsewhere (20). Cells were next stimulated with flagellin overnight, and IL-8 levels were measured by ELISA in the cell supernatants. As shown in Fig. 9D, overexpressing p85-DN reduced flagellin-induced IL-8 protein production in NCM460 cells. Together, these data demonstrate that TLR5-linked PI3K activation is at least in part involved in IL-8 protein production in human colonocytes.
| DISCUSSION |
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Meanwhile, Sarkar et al. (8) recently showed that tyrosine phosphorylation of TLR3 is involved in PI3K activation, indicating that tyrosine phosphorylation of TLR may be an important biochemical event in TLR-associated signaling. In the case of TLR5, however, mutating the various tyrosine residues with phenylalanine in the cytoplasmic TIR domain did not appear to disrupt TLR5-mediated NF
B activation in NCM460 cells (data not shown). Further studies are needed to determine the biological significance of the tyrosine residues in the cytoplasmic TLR domain of TLR5.
PI3K activation mediates several critical cellular responses, including cell differentiation, proliferation, survival, and shape (6, 25). Of greater interest, Fukao and Koyasu (24) showed that blocking PI3K activation resulted in enhanced IL-12 production by several TLR ligands in mouse dendritic cells. However, using nontransformed human colonic epithelial cells stimulated with flagellin, our cytokine micro-array data did not show any enhanced cytokine production by blocking PI3K activation in flagellin/TLR5-induced signaling (Fig. 9). In contrast, pharmacologic antagonism or overexpression of a dominant negative PI3K mutant substantially reduced IL-8 production, but not MIP3
production in response to flagellin in colonocytes (Fig. 9). These results indicate that in human colonocytes, the IL-8 gene expression in response to flagellin is at least in part PI3K-dependent. In general, NF
B activation is a common regulator, critical for proinflammatory cytokine expression such as IL-8 and MIP3
(4). Interestingly, we determined that blocking PI3K activation by its specific inhibitor LY294002 substantially reduces flagellin/TLR5-derived NF
B reporter activity in colonic epithelial cells,3 although LY294002 significantly inhibits IL-8 but not MIP3
production (Fig. 8A). Although the mechanism of these two different responses is not clear, we hypothesize that, other than the canonical NF
B (p65/p50) transcription factor activation (26), TLR5-derived PI3K activation might be able to mediate the activation of the noncanonical transcription factors of the Rel family, such as RelB/p52. As a result PI3K activation might differentially influence expression of IL-8 and MIP3
in flagellin-stimulated colonic epithelial cells.
In conclusion, our results provide important information relevant to the molecular mechanism by which PI3K is activated in response to bacterial flagellin. These results might be critical to our understanding of the mechanism(s) by which TLR5/PI3K interactions participate in the pathophysiology of intestinal inflammation in response to bacteria, including inflammatory bowel disease.
| FOOTNOTES |
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1 To whom correspondence should be addressed: Beth Israel Deaconess Medical Center, Harvard Medical School, Gastrointestinal Neuropeptide Center, Division of Gastroenterology, Dana 601, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-1259; Fax: 617-667-2767; E-mail: cpothoul{at}bidmc.harvard.edu.
2 The abbreviations used are: TLR, Toll-like receptor; DSS, dextran sulfate sodium; FOXO, Forkhead transcription factors; LTA, lipoteichoic acid; MyD88, myeloid differentiation factor 88; PI3K, phosphatidylinositol 3-kinase; TIR, Toll/IL-1 receptor; TRIF, Toll/IL-1R domain-containing adaptor inducing IFN-
; HA, hemagglutinin; WT, wild type; PBS, phosphate-buffered saline; DAPI, 4,6-diamidino-2-phenylindole; siRNA, small interfering RNA; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; HEK, human embryonic kidney; SH, Src homology; LPS, lipopolysaccharide; DN, dominant negative. ![]()
3 S. H. Rhee, H. Kim, M. P. Moyer, and C. Pothoulakis, unpublished data. ![]()
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