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TLR4/Grb2 Mediated N-Ras Activation Contributes to Cholangiocyte Proinflammatory Response Steven P

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TLR4/Grb2 Mediated N-Ras Activation Contributes to Cholangiocyte Proinflammatory Response Steven P TLR4/Grb2 Mediated N-Ras Activation Contributes to Cholangiocyte Proinflammatory Response Steven P. O’Hara, Ph.D., Patrick L. Splinter, Gabriella B. Gajdos, Christy E. Trussoni, Nicholas F. LaRusso, M.D. Center for Basic Research in Digestive Diseases, GI Basic Research Mayo Clinic, Rochester, MN Figure 3 Figure 6 BACKGROUND RESULTS TLR4 and the molecular adaptor, Grb2, are Grb2 depletion diminishes cholangiocyte IL-6 In addition to acting as a physical barrier to pathogen required for LPS-induced N-Ras activation expression dissemination, epithelia participate in host innate immune responses via expression of cytokines, chemokines, antimicrobial scr- TLR4 Grb2 A. siRNA siRNA B. peptides, activation of cell death pathways, and proliferation. Figure 1 H69 Scr siRNA A. 250 B. 800 TLR4 Grb2 Biliary epithelial cells (cholangiocytes) are exposed to microbes 200 IL-6 600 IL-6 LPS activates N-Ras but not K- or H-Ras in RT-PCR or microbe-derived molecules including gram-negative, bacteria- Actin 150 ELISA a dose and time dependent manner Actin 400 derived lipopolysaccharide (LPS). Recent data, including ours, LPS pg/ml LPS 100 indicate that cholangiocytes actively participate in the innate Grb2 200 TLR4 number/ copy 18S 1.0E+06 50 immune response; however, the precise mechanisms regulating LPS (ng/ml) H69 Ctrl Ctrl siRNA Scr Ctrl Ctrl siRNA Scr N-Ras A. 0 10 50 100 200 Lys N-Ras 0 - + - 0 - + - + Grb2 cholangiocyte responses to pathogens and the consequences GST- + Grb2 GST- Media LPS Media LPS siRNA are not clearly defined. K-Ras RBD siRNA RBD Ctrl 3 hour Ctrl 3 hour We have previously demonstrated that: Ha-Ras Neg C. Ctrl Uninf LPS Cp Quantitative PCR and ELISA demonstrate that Grb2 is involved in LPS- -Cholangiocytes express Toll-like receptors. N-Ras MyD88 IP induced IL-6 expression in cholangciocytes A. RT-PCR for IL-6 -LPS induced NFkB activation is TLR4 and MyD88- Grb2 IB demonstrates that this cytokine is upregulated in LPS-treated cells. dependent. GST-RBD Sos1 IP Transfection with the Grb2 siRNA significantly diminishes LPS-induced IL-6 Grb2 IB -Microbial stimuli activates cholangiocyte N-Ras in a LPS (100 ng/ml) expression. B. An ELISA performed on culture media further demonstrates B. TLR4-dependent manner (Program number 2612). 0 5 30 Min An N-Ras activation assay was performed in the context of TLR4 that depletion of Grb2 diminishes cholangiocyte IL-6 secretion following LPS treatment. N-Ras and Grb2 depletion. A. Depletion of TLR4 (top panel) diminishes LPS-induced N-Ras activation (lower panel). B. siRNA depletion GST-RBD of Grb2 (top panel) also blocks LPS-induced N-Ras activation AIM (lower panel). C. Co-immunoprecipitation demonstrates that the To clarify the molecular mechanism and functional consequences P-ERK 1/2 TLR4 signaling complex (MyD88) interacts with Grb2. Also, CONCLUSIONS of microbe induced N-Ras activation. interaction between the guanine exchange factor Sos1 and Grb2 • TLR agonists rapidly activate N-Ras and ERK. ERK 1/2 is induced by microbial stimulus. QUESTION • Microbe-induced N-Ras and ERK activation is Does the adaptor protein Growth Factor receptor-bound protein 2 An N-Ras activation assay was performed following treatment independent of TRAF6. with the TLR4 agonist, lipopolysaccharide (LPS) A. LPS Figure 4 (Grb2) contribute to TLR4-dependent activation of N-Ras and treatment activated N-Ras in a dose dependent manner, while LPS induces the colocalization of Grb2 and • TLR-dependent N-Ras activation requires Grb2. cholangiocyte proinflammatory signaling? activation of the other isoforms was not detected. B. N-Ras was MyD88 Pathogen rapidly activated following LPS treatment. The membrane was • LPS-induced IL-6 expression involves the adaptor recognition TLR4 Plasma membrane stained with ponceau Red to confirm equal loading. molecule Grb2. A. No LPS Grb2-Red B. LPS Grb2-Red Trif/Tram (Grb2/Sos1) MyD88-Green MyD88-Green • LPS induces the interaction between MyD88 and Grb2. ? ACTIVATED ? MyD88 RAS RAF/MEK/ERK Figure 2 MyD88-independent Traf6 The TLR4 Effector, TRAF6, is not required IRF3 activation Proinflammatory for microbe-induced N-Ras activation SUMMARY MyD88-dependent response Confocal microscopy was used to demonstrate the induced NFkB activation interaction between Myd88 and Grb2. A. Unstimulated cells Our results suggest that pathogen induced N-Ras activation contributes Ctrl TRAF6 Ctrl vector B. Ctrl LPS HKLM exhibit minimal co-localization. B. LPS treatment induces the to cholangiocyte cytokine / chemokine expression in a TLR4 / A. vector shRNA N-Ras TRAF6 colocalization of Grb2 and MyD88 (yellow). Grb2-dependent and TRAF6-independent manner. GST-RBD METHODS Actin TRAF6 shRNA NFkB luciferase reporter assay Ctrl LPS HKLM • Cell culture model: Cultured human cholangiocytes (H69 cells) were Figure 5 H69 N-Ras serum starved in EGF-free media and treated with TLR agonists 150 TRAF6 shRNA GST-RBD FRET demonstrates LPS induced interaction between Grb2 and MyD88 including 200 ng/ml E. coli derived LPS. 100 * * Traf6 shRNA •Ras activation assay: Glutathione-S-Transferase Ras-binding domain C. LPS LPS 1.2 50 Ctrl Ctrl A. B. 1.2 LPS Acceptor photobleaching quantitative FRET demonstrates LPS-induced Grb2 of Raf-1 (GST-RBD; Active Motif) was used to pull-down activated # # No LPS Luciferase (ratio to ev) p-ERK 1 1 and MyD88 interaction. A. In the absence of LPS, overexpressed Myc-tagged 0 Ras from total protein extract. Immunoblots were performed to detect CY3 Grb2 and endogenous MyD88 were detected using primary antibodies against Ctrl LPS HKLM Total ERK 0.8 CY3 specific Ras isoforms. CY5 0.8 CY5 Myc and Myd88 followed by secondary antibodies conjugated to Cy3 (donor) and intensity 0.2 0.2 Cy5 (acceptor), respectively. Photobleaching of the acceptor Cy5 (at 16s) did not •Immunoblots: were performed to validate RNAi knockdown of protein Fluorescence 0 0 induce an increase in donor fluorescence. B. LPS-treatment induces an increase expression, and detect phopho-ERK, An N-Ras activation assay was performed in the context of TRAF- 4 12 20 28 36 4 12 20 28 36 6 depletion. A. A stable cell line expressing TRAF6 shRNA was Time (Sec) Time (Sec) in donor Cy3 fluorescence detection following Cy5 (acceptor) photobleaching, demonstrating the induced proximity (≤ 10 nm) of MyD88 and Grb2. C. A positive •RT-PCR and ELISA: were performed to demonstrate IL-6 expression generated. TLR2 (HKLM) and TLR4 (LPS)-induced NFkB 35 C. D. control was performed in which the overexpressed Myc tag was detected by both in the context of Grb2 depletion. activation is blocked in this cell line as demonstrated using a 1.4 Pos Ctrl 25 Cy3 and CY5 conjugated secondary antibodies. D. The FRET efficiency for all NFkB driven luciferase promoter. *, denotes significance (p<0.05) 1 •Forster Resonant Energy Transfer: Grb2-myc wa s 15 conditions (estimate of the fraction of energy transfer to the acceptor per donor compared to control; #, denotes significance compared to same CY3 immunofluorescently labeled with a Cy5 (acceptor). MyD88 was 0.6 excitation event) was calculated using the Donor /Donor method. The LPS- pathogen stimulus without shRNA. B. Both the TLR4 and TLR2 CY5 5 max min immunofleorescently labeled with Cy3 (donor). CY5 was intensity 0.2 0 treated cells demonstrated energy transfer between the fluorophores (~10%), agonists activate N-Ras in control mock transfected cells (ctrl Fluorescence 0 -5 20 28 36 FRET Efficiency (%) photobleached and the fluorescence intensity of CY3 was is 4 12 Pos LPS No while the cells cultured in the absence of LPS did not. vector) and cells depleted of TRAF6. C. TRAF6 depletion does Time (s) measured. FRET is detected when the Cy3 increases in intensity after not diminish the level of LPS-induced ERK phosphorylation. Ctrl LPS acceptor photobleaching. © 2010 Mayo Foundation for Medical Education and Research.
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