Inhibition of KLF5 expression by siRNA abrogates induction of NF-κB subunit levels and binding activity in response to LPS

<p><b>Copyright information:</b></p><p>Taken from "Krüppel-like factor 5 is an important mediator for lipopolysaccharide-induced proinflammatory response in intestinal epithelial cells"</p><p>Nucleic Acids Research 2006;34(4):1216-1223.</p><p>Published online 25 Feb 2006</p><p>PMCID:PMC1383625.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () IEC6 cells were transfected by electroporation with non-specific (NS) siRNA or KLF5-specific siRNA. Mock-transfected cells were used as control. Twenty-four hours following transfection, cells were treated with 5 µg/ml LPS or water control for 2 h, followed by northern blot analysis for the various mRNAs indicated. () Nuclear extracts were prepared from mock-transfected IEC6 cells or cells transfected with non-specific (NS) siRNA or KLF5-specific siRNA that have been treated with 5 µg/ml LPS or water control for 2 h. Electrophoretic mobility shift assay (EMSA) was then performed with a labeled consensus NF-κB binding sequence using 5 µg nuclear extracts per lane. In the last lane (Comp.), 150-fold excess of unlabeled NF-κB probe was included in the reaction containing nuclear extracts from mock-transfected and LPS-treated cells. The same nuclear extracts were also analyzed for the content of KLF5 or actin by western blotting, as shown below the EMSA. () IEC6 cells were pretreated with 10 µM of the NF-κB inhibitor, TLCK, or water control for 1 h and then treated with 5 µg/ml LPS or water control for 2 h before being analyzed for the mRNA levels for KLF5 or GAPDH by northern hybridization

Northern blot analyses of the effects of LPS, PMXB and U0126 on expression of KLF5 and the p65 and p50 subunits of NF-κB in IEC6 cells

<p><b>Copyright information:</b></p><p>Taken from "Krüppel-like factor 5 is an important mediator for lipopolysaccharide-induced proinflammatory response in intestinal epithelial cells"</p><p>Nucleic Acids Research 2006;34(4):1216-1223.</p><p>Published online 25 Feb 2006</p><p>PMCID:PMC1383625.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () IEC6 cells were treated with 5 µg/ml LPS for the time periods specified before extraction of RNA. Twenty micrograms of RNA were loaded in each lane and probed with cDNA encoding KLF5, and the p65 and p50 subunits of NF-κB. GAPDH was used as a loading control. () IEC6 cells were pretreated with 10 µg/ml PMXB for 30 min (lanes 3 and 4) and then treated with 5 µg/ml LPS (lanes 2 and 4) or water control (lanes 1 and 3) for 2 h before being analyzed for the mRNA levels of KLF5, and the p65 and p50 subunits of NF-κB. () IEC6 cells were pretreated with water control (lanes 1 and 2), the vehicle, DMSO (lanes 3 and 4), or the MAPK inhibitor, U0126 (lanes 5 and 6), for 30 min and followed by treatment with 5 µg/ml LPS (lanes 2, 4 and 6) or water control (lanes 1, 3 and 5) for 2 h before northern analyses for the mRNA stated. GAPDH was used as a loading control in both panels

<i>Iqgap2</i>^<i>-/-</i> colons are characterized by diminished production of IL-6 in response to DSS treatment.

<p><b>A.</b> IL-6 (left) and IL-10 (right) mRNA cytokine levels as quantified by qRT-PCR in colons from WT and <i>Iqgap2</i>^<i>-/-</i> mice from the three groups: untreated, treated with DSS, and treated with DSS and allowed to recover for 7 days, N = 3 per group per genotype. The levels of IL-6 mRNA in DSS-treated <i>Iqgap2</i>^<i>-/-</i> colons were beyond the sensitivity of the method used. Data are presented as a transcript fold change relative to actin mRNA transcript levels. <b>B.</b> IF showing reduced IL-6 production (red) in DSS-treated <i>Iqgap2</i>^<i>-/-</i> colons (panel c) compared to DSS-treated WT (panel a). White arrows indicate representative IL-6-positive cells. Panels b and d show corresponding DAPI staining. Magnification is 200 X. Images are representative of N = 3 per genotype. <b>C.</b> Quantification of IF IL-6 positive cells in colons from WT and <i>Iqgap2</i>^<i>-/-</i> mice from the three groups: untreated, treated with DSS, and treated with DSS and allowed to recover for 7 days. Data represent an average of ten randomly selected fields per sample ± SD. N = 3 per genotype/treatment. P-values indicating statistically significant differences are shown. <b>D.</b> IHC for phospho-STAT3(Tyr705) in WT and <i>Iqgap2</i>^<i>-/-</i> colons before and after DSS treatment, N = 5 per group. Representative pSTAT3-positive cells are pointed with black arrows. Magnification is 200 X.</p

Increased secretion of TNF-α and IL-6 in IEC6 cells in response to LPS requires KLF5

<p><b>Copyright information:</b></p><p>Taken from "Krüppel-like factor 5 is an important mediator for lipopolysaccharide-induced proinflammatory response in intestinal epithelial cells"</p><p>Nucleic Acids Research 2006;34(4):1216-1223.</p><p>Published online 25 Feb 2006</p><p>PMCID:PMC1383625.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> IEC6 cells were treated with water control (open bars) or 5 µg/ml LPS (filled bars) for the periods of time specified. The amounts of TNF-α () and IL-6 () in the supernatants were determined by ELISA. = 4 in all experiments. * < 0.05 compared to control. Mock-transfected IEC6 cells or IEC6 cells transfected with non-specific (NS) siRNA or KLF5-specific siRNA were treated with water control (open bars) or 5 µg/ml LPS (filled bars) for 48 h. The amounts of TNF-α () and IL-6 () in the supernatants were determined by ELISA. = 4 in all experiments. * < 0.05 compared to control

IQGAP2 levels in colon specimens of patients with IBD.

<p><b>A.</b> Two cases of ulcerative colitis (UC): panels a and b represent Case #1, and panels c and d–Case #2. <b>B.</b> Two cases of Crohn’s disease (CD), panels are designated as above. Images are representative of the total of 7 IBD patient cases. Magnification is 200 X.</p

IQ Motif-Containing GTPase-Activating Protein 2 (IQGAP2) Is a Novel Regulator of Colonic Inflammation in Mice

<div><p>IQ motif-containing GTPase-activating protein 2 (IQGAP2) is a multidomain scaffolding protein that plays a role in cytoskeleton regulation by juxtaposing Rho GTPase and Ca²⁺/calmodulin signals. While IQGAP2 suppresses tumorigenesis in liver, its role in pathophysiology of the gastrointestinal tract remains unexplored. Here we report that IQGAP2 is required for the inflammatory response in colon. Mice lacking <i>Iqgap2</i> gene (<i>Iqgap2^-/-</i> mice) were resistant to chemically-induced colitis. Unlike wild-type controls, <i>Iqgap2^-/-</i> mice treated with 3% dextran sulfate sodium (DSS) in water for 13 days displayed no injury to colonic epithelium. Mechanistically, resistance to colitis was associated with suppression of colonic NF-κB signaling and IL-6 synthesis, along with diminished neutrophil and macrophage production and recruitment in <i>Iqgap2^-/-</i> mice. Finally, alterations in IQGAP2 expression were found in colons of patients with inflammatory bowel disease (IBD). Our findings indicate that IQGAP2 promotes inflammatory response at two distinct levels; locally, in colonic epithelium through TLR4/NF-κB signaling pathway, and systemically, via control of maturation and recruitment of myeloid immune cells. This work identifies a novel mechanism of colonic inflammation mediated by signal transducing scaffolding protein IQGAP2. IQGAP2 domain-specific blocking agents may represent a conceptually novel strategy for therapy of IBD and other inflammation-associated disorders, including cancer.</p></div

Reduced production of white blood cells (WBC) in <i>Iqgap2</i>^<i>-/-</i> mice.

<p><b>A.</b> Quantification of positive cells as a result of IF shows significantly decreased numbers of infiltrating macrophages (F4/80), neutrophils (Ly-6G) and dendritic cells (CD11c) in <i>Iqgap2</i>^<i>-/-</i> colons compared to WT following DSS treatment. The number of positive fluorescent cells was obtained by counting cells in six randomly selected fields per colon sample from WT and <i>Iqgap2</i>^<i>-/-</i> mice from the three groups: untreated, treated with DSS, and treated with DSS and allowed to recover for 7 days. <b>B.</b> Complete blood count (CBC) confirms reduced numbers of neutrophils, lymphocytes and monocytes in circulation in <i>Iqgap2</i>^<i>-/-</i> mice treated with DSS. Data are presented as the mean ± SEM. N = 3 per genotype/treatment (<b>A</b>), N = 5 per genotype/treatment (<b>B</b>), p-values indicating statistically significant differences are shown.</p

Suppression of NF-κB signaling in <i>Iqgap2</i>^<i>-/-</i> colons.

<p><b>A.</b> IHC shows decreased baseline levels of p65 subunit of NF-κB in <i>Iqgap2</i>^<i>-/-</i> colon compared to WT (panels a, d). While DSS treatment resulted in elevated levels of p65 in WT colon, it failed to elicit the same response in <i>Iqgap2</i>^<i>-/-</i> colon (panel b, e). Termination of DSS treatment results in a restoration of the baseline p65 levels within 7 days in both genotypes (panel c, f). <b>B.</b> IHC of TLR4 in WT and <i>Iqgap2</i>^<i>-/-</i> colons before and after DSS treatment. <b>C.</b> IHC of MyD88 in the same samples. Note low levels of both TLR4 and MyD88 in <i>Iqgap2</i>^<i>-/-</i> colons after DSS exposure (panels d). A representative image of N = 5 per genotype is shown for each IHC. Magnification is 200 X.</p

IQGAP2 expression in organs of the digestive system.

<p><b>A.</b> Immunoblot of digestive tract organs from wild-type (WT) and <i>Iqgap2</i>^<i>-/-</i> mice, probed for IQGAP2 and β-actin as a control for equal protein loading. Representative blots of N = 3 per genotype are shown. <b>B.</b> IHC of IQGAP1 and IQGAP2 in WT (panels a, b) and <i>Iqgap2</i>^<i>-/-</i> (panels c, d) mouse colon. Magnification is 200X.</p

Physiological and histological evidence of resistance to DSS-induced colitis in <i>Iqgap2</i>^<i>-/-</i> mice.

<p><b>A.</b> Schema of a DSS treatment experiment. DSS at the concentration of 3% was administered in drinking water for up to 13 days. A separate group of mice was treated with 3% DSS for 13 days and then allowed to recover on regular water for 7 days. <b>B.</b> Baseline body weight of untreated WT and <i>Iqgap2</i>^<i>-/-</i> mice. The difference between genotypes is not statistically significant (p = 0.1059). <b>C.</b> In contrast to the WT control group, <i>Iqgap2</i>^<i>-/-</i> mice did not lose weight as a result of DSS treatment (left panel); after a 7-day recovery period, WT mice restored 95% of their baseline weight before DSS treatment (right panel). Data are presented as the mean ± SEM. Statistically significant (p < 0.05) differences between genotypes are indicated with asterisks. Resistance of <i>Iqgap2</i>^<i>-/-</i> mice to experimental colitis is also demonstrated by colon length (<b>D</b>) and low colitis disease activity index (DAI) (<b>E</b>). <b>F.</b> Histological evidence (H&E) of the absence of colitis in <i>Iqgap2</i>^<i>-/-</i> mice. While DSS treatment resulted in an expansive colonic epithelium loss (black arrows) in WT colons (panels a, b), <i>Iqgap2</i>^<i>-/-</i> colons were minimally affected (panels c, d). N = 5 per group per experiment; experiment was repeated three times; the results of one representative experiment are shown.</p