Isoprenylcysteine Carboxyl Methyltransferase and Its Substrate Ras Are Critical Players Regulating TLR-Mediated Inflammatory Responses

In this study, we investigated the functional role of isoprenylcysteine carboxyl methyltransferase (ICMT) and its methylatable substrate Ras in Toll-like receptor (TLR)-activated macrophages and in mouse inflammatory disease conditions. ICMT and RAS expressions were strongly increased in macrophages under the activation conditions of TLRs by lipopolysaccharide (LPS, a TLR4 ligand), pam3CSK (TLR2), or poly(I:C) (TLR3) and in the colons, stomachs, and livers of mice with colitis, gastritis, and hepatitis. The inhibition and activation of ICMT and Ras through genetic and pharmacological approaches significantly affected the activation of interleukin-1 receptor-associated kinase (IRAK)s, tumor necrosis factor receptor associated factor 6 (TRAF6), transforming growth factor-β-activated kinase 1 (TAK1), mitogen-activated protein kinase (MAPK), and MAPK kinases (MAPKKs); translocation of the AP-1 family; and the expressions of inflammation-related genes that depend on both MyD88 and TRIF. Interestingly, the Ras/ICMT-mediated inflammatory reaction critically depends on the TIR domains of myeloid differentiation primary response 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF). Taken together, these results suggest that ICMT and its methylated Ras play important roles in the regulation of inflammatory responses through cooperation with the TIR domain of adaptor molecules.


Mice
Six-week-old ICR, Balb/c, and C57BL/6 male mice (see Table S1 for genetic background information) were purchased from Daehan Biolink (DBL, Chungbuk, Korea) and housed eight mice per group under a 12-h light/dark cycle (lights on at 6 a.m.).

Preparation of cell lysates and nuclear fractions from cells/tissues for immunoblotting analyses
Whole lysates were extracted from animal tissues, RAW264.7 cells, and HEK293 cells. Cells and tissues were washed with PBS, lysis buffer (20 mM Tris-HCl [pH 7.4], 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM ethyleneglycotetraacetic acid, 50 mM glycerophosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol [DTT], 1% Triton X-100, 10% glycerol, 10 g/ml aprotinin, 10 g/ml pepstatin, 1 mM benzamide, and 2 mM phenylmethanesulfonyl fluoride [PMSF]) was added, and samples were lysed at -70°C for 30 minutes. Lysates were clarified by centrifugation at 16,000 rpm for 10 min at 4°C and then stored at -20°C until needed. Extractions of nuclear and membrane fractions proceeded in a similar manner. Tissues or cells were washed with 400 μl of lysis solution A (50 mM KCl, 2 mM MgCl2, 10 mM HEPES [pH 7.8] with KOH, 0.1 mM EDTA, 0.1 mM PMSF, 2 μg/ml leupeptin, 2 μg/ml aprotinin, and 1 mM DTT) and mixed vigorously for 30 seconds with 25 μl of 10% Nonidet P-40. After centrifugation at 14,000 RPM for 30 seconds, the supernatant (cytosolic fraction) and pellet (nuclear fraction) were separated. The supernatant was centrifuged at 14,000 rpm for 1 hour to obtain a membrane fraction. To isolate the nuclear fraction, we added 50 μl of lysis solution B (50 mM KCl, 300 mM NaCl, 10 mM HEPES [pH 7.8] with KOH, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml aprotinin, and 1 mM DTT) to the pellet and incubated it at 4°C for 20 minutes. During this time, vortexing was performed every 5 minutes. We then centrifuged the sample at 14,000 rpm for 5 minutes and collected the nuclear fraction. Proteins were separated by electrophoresis on 10-15% SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane by electroblot. The membrane was blocked by 3% bovine serum albumin (BSA) and dissolved in TBST (Tris-buffered saline containing 3% FBS, 20 mM NaF, 2 mM EDTA, and 0.2% Tween 20), and primary and secondary antibodies were then added. We determined the total and phosphorylated levels of inflammatory proteins using the ECL system (Amersham, Little Chalfont, Buckinghamshire, UK).

Human tissue data
Raw data from inflammatory diseases were assessed from the NCBI Gene Expression Omnibus using the following accession IDs: inflammatory cardiomyopathy, 26583460; rheumatoid arthritis, synovial tissues, 21212124; Crohn's disease and ulcerative colitis comparison, 12532971; inflammatory bowel disease, 4225238; chronic obstructive pulmonary disease, 1638739; and alcoholic hepatitis, 85451358. Informed written consent was obtained from all participants.

Cell viability assay
To determine cell viability in the presence of ICMT inhibitors, we used the MTT assay as reported previoulsy. After pre-incubation of RAW264.7 cells, peritoneal macrophages, and HEK293 cells (1×10 6 cells/ml) for 18 h, ICMT inhibitors (CyM from 20 to 40 M, ICMT-Ph from 1.5 to 200 M) were added to the cells, which were then incubated for 24 h. The cytotoxicity of the ICMT inhibitors was evaluated using a conventional MTT assay, as described previously (Pauwels et al., 1988;Yayeh et al., 2012). Three hours prior to culture termination, 10 l of MTT solution (10 mg/ml in PBS, pH 7.4) was added. The incubation was halted by the addition of 15% sodium dodecyl sulfate (SDS) to each well to solubilize the formazan. The absorbance at 570 nm (OD570-630) was measured using a Spectramax 250 microplate reader (BioTex, Bad Friedrichshall, Germany).

Immunoprecipitation assays
We performed immunoprecipitation assays to determine the binding activities of proteins. First, proteins were extracted using lysis buffer (20 mM Tris-HCl [pH 7.4], 2 mM EDTA, 2 mM ethyleneglycotetraacetic acid, 50 mM -glycerophosphate, 1 mM sodium orthovanadate, 1 mM DTT, 1% Triton X-100, 10% glycerol, 10 g/ml aprotinin, 10 g/ml pepstatin, 1 mM benzamide, and 2 mM PMSF). For immunoprecipitation, we added 5 μl of the primary antibody and 1 μl of the control IgG to each sample group and incubated this mixture at 4°C for 24 hours. We then added protein A-coupled Sepharose beads (50% v/v) (Amersham, Little Chalfont, Buckinghamshire, UK) to the sample and allowed the proteins to bind to the beads in a rotating roller for 4 hours. After five washes with lysis buffer, we performed immunoblotting in the manner described above.

Confocal microscopy
We placed RAW264.7 cells plated at 7 × 10 5 /ml in a 12-well plate and treated the cells 18 h later. After washing the cells three times for 5 min with 1 ml of PBS, we fixed the cells in 1 ml of 3.7% formaldehyde for 10 min. After washing, we permeabilized the cells for 5 min with 500 μl of 1% Triton x-100. After washing, 800 μl of 1% BSA was added, and the sample was blocked for 30 min. We next added the primary antibody (anti-ICMT or Ras) and then the secondary antibody (FITC-antibody: BD Sciences; ER tracker-Red: Invitrogen; Hoechst 33258: Sigma Chemical Co.). After washing, 20 μl of mounting solution (ThermoFisher Scientific) was applied, and the cover glass was placed on the slide, which was then analyzed using confocal microscopy (ZEISS LSM 800).

Microarray analysis
After preparing total RNA from RAW264.7 cells treated and untreated with LPS and from RAW264.7-ICMT -/cells treated and untreated with LPS, we used the GeneChip® Mouse Gene 2.0 ST Array as a platform. cDNA was synthesized using the GeneChip Whole Transcript (WT) Amplification kit. The label protocol was as follows. First, sense cDNA was fragmented, and then biotin labeling of terminal deoxynucleotidyl transferase was performed using the GeneChip WT Terminal Labeling kit. Labeled target DNA was hybridized to the Affymetrix GeneChip Array at 45°C for 16 hours. Hybridized samples were washed, stained with a GeneChip Fluidics Station 450, and scanned with a GCS3000 scanner. Array data were processed using Affymetrix® GeneChip Command Console® software. Analyses were performed using Affymetrix® Expression Console™ software (http://www.affymetrix.com/estore/catalog/131414/AFFY/Expression-Console-Software#1_1) and R 3.1.2 (www.r-project.org). Except for total RNA preparation and data analysis, this process was performed by Macrogen, Inc. (Seoul, Korea).

Statistical analyses
All data presented in this paper are expressed as mean ± SD. For statistical comparisons, results were analyzed using either ANOVA/Scheffe's post hoc test or the Kruskal-Wallis/Mann-Whitney test. A p-value < 0.05 was considered to be statistically significant. All statistical tests were carried out using SPSS (SPSS Inc., Chicago, IL, USA). Similar experimental data were also obtained using an additional independent set of in vivo experiments conducted using the same numbers of mice. Figure S1. Protein level of ICMT under normal culture conditions of RAW264.7 cells. Protein level of ICMT in RAW264.7 cells was determined by immunoblotting analysis. Figure S2. Chemical structure of cysmethynil (CyM). Figure S3. The mRNA expression level of ICMT in various disease conditions. mRNA expression level of ICMT in various human chronic inflammatory diseases was calculated using data from the NCBI Gene Expression Omnibus. *: p < 0.05 and **: p < 0.01 compared to healthy group. NS : not significant, NA : not available, IBD (C) : Inflammatory bowel diseases (Colon), and IBD (I) : Inflammatory bowel diseases (Ileum) Figure S4. Amino acid sequence alignment between the TIR domain and Ras, and constructs of Ras and its mutants.