Q-PCR validation of the Wnt/β-catenin signaling pathway alterations in <i>Iqgap2^−/−</i> livers.

<p>The same liver RNA samples used for microarray (24-month-old WT, N  =  3, and 24-month-old KO, N  =  3) were analyzed using the Mouse Wnt Signaling Pathway RT² Profiler PCR Array. 11 genes belonging to the pathway and found to be differentially expressed in <i>Iqgap2^−/−</i> livers are shown.</p

Hepatic RNA microarray analysis of <i>Iqgap2^−/−</i> mice.

<p><b>A</b>: Schema of a four-way microarray analysis of <i>Iqgap2^−/−</i> knockout (KO) and wild-type (WT) mouse livers at 6 months and 24 months of age. Four-way comparison using SAM identified 554 genes that were differentially expressed among four groups (cutoff fold change ≥3 and FDR≤0.05). <b>B</b>: Unsupervised hierarchical cluster analysis identified 11 subsets of genes (clusters) within 554 genes that change expression in similar pattern across four groups; <b>C</b>: Top canonical biological pathways for genes from the cluster # 9 as identified by Ingenuity Pathway Analysis (IPA).</p

Unsupervised hierarchical clustering analysis of the 399 genes subset representing the <i>Iqgap2^−/−</i> HCC transcript signature.

<p>The two-dimension hierarchical clustering procedure was performed based on the 1- Pearson correlation distance and the average linkage method. All data were centered by rows to mean 0 and standard deviation of 1, meanwhile the data range was confined to −3 to 3 for a more comparable scale. The data are presented in a matrix format with columns representing individual samples and rows representing genes, thereby each cell in the matrix represents the expression level of a gene feature in an individual sample. The red and green colors in cells reflect high and low expression levels, respectively, as indicated in the fold-change scale bar. Tumor-free samples have blue font labels and HCC samples have maroon font labels.WT-1, WT-2, WT-3 – wild-type liver samples from 6-month-old mice; KO-1, KO-2, KO-3 - <i>Iqgap2^−/−</i> liver samples from 6-month-old mice; WT-4, WT-5, WT-6 - wild-type liver samples from 24-month-old mice; and KO-4, KO-5, KO-6 - <i>Iqgap2^−/−</i> liver tumor samples from 24-month-old mice. Note that the three <i>Iqgap2^−/−</i> HCC tumor samples from 24-month-old mice show a distinct pattern and form a separate cluster. The rest of the samples have more similar transcript profiles with the highest similarity found between livers from the younger (6-month-old) mice irrespectively of genotype.</p

Comparison of HCC transcript profiles of <i>Iqgap2^−/−</i> mouse model and human HCC.

<p>Cross-species clustering profiles were obtained using comparative functional genomics approach. The clustering dendrogram was generated based on 1 - Pearson correlation and an average distance linkage. The data scale was also confined to a range of −3 to 3 for a more comparable heat map. Hierarchical clustering analysis was performed on 151 ortholog genes shared between 24-month-old wild-type and the age-matched <i>Iqgap2^−/−</i> mice (N = 3 in each group, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071826#pone-0071826-g003" target="_blank"><b>Figure 3</b></a> legend), and the human GSE6222 integrated microarray data set. This data set included four T1 HCC tumors (early stage HCC), six T3 HCC tumors (advanced HCC), and 2 normal liver controls. Mouse and human tumor-free samples have blue font labels; human T1 HCC samples have red font labels; and <i>Iqgap2^−/−</i> HCC and human T3 HCC samples have maroon font labels. All three <i>Iqgap2^−/−</i> HCC samples (KO.4 through KO.6) co-clustered with four out of six human T3 HCC samples. Genes differentially expressed between mouse and human livers are enclosed in a box at the top of the heat diagram.</p

Ingenuity Pathway Analysis of biological functions of 371 genes that change expression with age in <i>Iqgap2^−/−</i> but not in WT livers, leading to the development of HCC.

<p>The top five canonic biological pathways (<b>A</b>), top five associated networks (<b>B</b>) and top five Tox functions (<b>C</b>) are shown.</p

<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

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 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