Binding conformations of top-scored compounds from natural products library in the SH2 domain of hSTAT1 and hSTAT3.

<p><b>(A)</b> Binding pose variation of the top-scored hSTAT1-specific inhibitor in SH2 domain of hSTAT1 and hSTAT3. <b>(B)</b> Binding pose variation of the top-scored hSTAT3-specific inhibitor in SH2 domain of hSTAT1 and hSTAT3. The binding pose variations are shown in line representation, colored in blue and violet. Results were obtained using Surflex-Dock 2.6 program.</p

STAT3-CBAVs of STAT3-specific inhibitors.

<p>Graph presents comparative binding affinity values of a selection of STAT3-specific inhibitors docked to models of all hSTAT monomers.</p

Binding conformations of top-scored compounds from clean leads library in the SH2 domain of hSTAT1 and hSTAT3.

<p>(A) Binding pose variation of the top-scored hSTAT1-specific inhibitor in SH2 domain of hSTAT1 and hSTAT3. (B) Binding pose variation of the top-scored hSTAT3-specific inhibitor in SH2 domain of hSTAT1 and hSTAT3. The binding pose variations are shown in line representation, colored in yellow and green. Results were obtained using Surflex-Dock 2.6 program.</p

Top-scored binding conformation of stattic in the SH2 domain of all hSTATs.

<p>Stattic is shown in stick representation, pTyr-linker is presented as green colored lines with pTyr residue in pink. Results were obtained using Surflex-Dock 2.6 program.</p

Structural models and phylogenetic comparison of hSTAT monomers (1, 2, 3, 4, 5A, 5B and 6) with their specific pTyr-linkers.

<p><b>(A)</b> Phylogenetic distribution of hSTATs in form of a simplified phylogenetic tree. <b>(B)</b> Models of the monomers are shown in the cartoon representation with pTyr-peptides in the stick representation. Specific domains are positioned as follows: N-domain on the top-left, coiled-coiled domain on the bottom-center, C-domain on the top-right and SH2 domain on the top-center, to facilitate visual analysis of phosphotyrosine (pTyr)-linker and the SH2 interactions. Monomers are colored according to the predicted local deviation from the real structure (the predicted error of the model), as calculated by MetaMQAP. Blue indicates low predicted deviation of Cα atoms down to 0Å, red indicates unreliable regions with deviation > 5Å, green to orange indicate intermediate values. pTyr-peptides are colored in violet, while pTyr residue is colored in pink. <b>(C)</b> Models of hSTAT dimers with the linker of monomer I in the SH2 domain of monomer II. pTyr-peptides are presented in stick representation, pY+0—pTyr binding pocket, pY-X—hydrophobic side-pocket. SH2 domains are in the surface representation, colored according to the distribution of the electrostatic surface potential, calculated with APBS. Blue indicates positively charged regions, red indicates negatively charged regions.</p

STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production.

<p>A, <i>WT</i> and <i>STAT1^−/− VSMCs</i> were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113318#pone-0113318-g001" target="_blank">Fig. 1</a>. RNA was isolated and qRT-PCR for <i>Nos2</i> using <i>Gapdh</i> as internal control was performed (upper panel) B, After stimulation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113318#pone-0113318-g001" target="_blank">Fig. 1</a>, medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p<0.05 was considered as significant. Data were tested for significance by one-way ANOVA followed by post-hoc Tukey or unpaired two-tailed student T-test when appropriate. C, D Isolated aortic rings from WT and STAT^−/− mice were incubated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. Next, response to norepinephrine and sodium nitroprusside was tested on the wire myograph. C, Response to noradrenaline in WT and STAT1-deficient aortic rings presented as a percentage of maximal constriction to KPSS.*p<0.001 vs. WT control; •p<0.001 vs. WT LPS; ○p<0.001 vs. STAT1^−/− control. D, Response to stepwise increased concentration of sodium nitroprusside. xp<0.05 vs. WT control; ∞p<0.01 vs. WT LPS; □p<0.05 STAT1^−/−control. Aortas isolated from 3–4 animals per group were taken. Two-way ANOVA test with Bonferroni post hoc test was used. Statistical significance for the highest concentration is given.</p

STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis

<div><p>Signal integration between IFNγ and TLRs in immune cells has been associated with the host defense against pathogens and injury, with a predominant role of STAT1. We hypothesize that STAT1-dependent transcriptional changes in vascular cells involved in cross-talk between IFNγ and TLR4, reflect pro-atherogenic responses in human atherosclerosis. Genome-wide investigation identified a set of STAT1-dependent genes that were synergistically affected by interactions between IFNγ and TLR4 in VSMCs. These included the chemokines <i>Cxcl9</i>, <i>Ccl12</i>, <i>Ccl8</i>, <i>Ccrl2</i>, <i>Cxcl10</i> and <i>Ccl5</i>, adhesion molecules <i>Cd40</i>, <i>Cd74</i>, and antiviral and antibacterial genes <i>Rsad2</i>, <i>Mx1</i>, <i>Oasl1</i>, <i>Gbp5</i>, <i>Nos2</i>, <i>Batf2</i> and <i>Tnfrsf11a</i>. Among the amplified genes was also <i>Irf8</i>, of which <i>Ccl5</i> was subsequently identified as a new pro-inflammatory target in VSMCs and ECs. Promoter analysis predicted transcriptional cooperation between STAT1, IRF1, IRF8 and NFκB, with the novel role of IRF8 providing an additional layer to the overall complexity. The synergistic interactions between IFNγ and TLR4 also resulted in increased T-cell migration and impaired aortic contractility in a STAT1-dependent manner. Expression of the chemokines CXCL9 and CXCL10 correlated with STAT1 phosphorylation in vascular cells in plaques from human carotid arteries. Moreover, using data mining of human plaque transcriptomes, expression of a selection of these STAT1-dependent pro-atherogenic genes was found to be increased in coronary artery disease (CAD) and carotid atherosclerosis. Our study provides evidence to suggest that in ECs and VSMCs STAT1 orchestrates a platform for cross-talk between IFNγ and TLR4, and identifies a STAT1-dependent gene signature that reflects a pro-atherogenic state in human atherosclerosis.</p></div

Effect of STAT1 dependent signal integration on chemokine expression.

<p><i>WT</i> and <i>STAT1^−/− VSMCs</i>, HMECs or <i>WT</i> aortic ring segments were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113318#pone-0113318-g001" target="_blank">Fig. 1</a>. A, RNA from VSMCs was isolated and qRT-PCR for <i>Ccl5</i>, <i>Cxcl9</i> using <i>Gapdh</i> as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of <i>CXCL10, CXCL9 and CCL5</i> upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for <i>Cxcl10</i>, <i>Cxcl9</i> using <i>Gapdh</i> as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p<0.05 was considered as significant. Data were tested for significance by one-way ANOVA followed by post-hoc Tukey or unpaired two-tailed student T-test when appropriate. E, ChIP-qPCR analysis of the <i>Cxcl10</i> promoter region containing NFκB and ISRE binding sites show the enrichment with STAT1, NFκB and IRF1 antibodies compared with IgG control in an IFNγ, LPS or IFNy+LPS-dependent manner in <i>WT VSMCs</i>. Immunoprecipitated DNA was quantified by qPCR and normalized to values obtained after amplification of unprecipitated (input) DNA. A representative experiment is shown.</p

Expression of synergistically amplified genes in atherosclerotic vessels.

<p>A, Venn diagram with analysis of microarray datasets obtained from human coronary plaques and human carotid plaques. B, Promoter analysis of the differentially expressed genes in carotid (left panel) and coronary plaques (right panel). For details see text.</p

Genes prone to synergistic amplification upon stimulation with IFNγ and/or LPS and their promoter analysis.

<p>The table introduces genes that expression is at least 2-fold higher upon stimulation with IFNγ+LPS as compared to the sum of the treatments with both factors alone (see column “Signal integration”). Other numbers represent fold changes compared to control. Cross indicates presence of specific sequence in the promoter regions.</p><p>Genes prone to synergistic amplification upon stimulation with IFNγ and/or LPS and their promoter analysis.</p