ag-490 and stattic

Crohn's disease (CD) has an altered intestinal barrier function, yet the underlying mechanisms remain to be disclosed. The tricellular tight junction protein tricellulin is involved in the maintenance of the paracellular macromolecule barrier and features an unchanged expression level in CD but a shifted localization. As angulins are known to regulate the localization of tricellulin, we hypothesized the involvement of angulins in CD. Using human biopsies, we found angulin-1 was downregulated in active CD compared with both controls and CD in remission. In T84 and Caco-2 monolayers, leptin, a cytokine secreted by fat tissue and affected in CD, decreased angulin-1 expression. This effect was completely blocked by STAT3 inhibitors, Stattic and WP1066, but only partially by JAK2 inhibitor AG490. The effect of leptin was also seen at a functional level as we observed in Caco-2 cells an increased permeability for FITC-dextran 4 kDa indicating an impaired barrier against macromolecule uptake. In conclusion, we were able to show that in active CD angulin-1 expression is downregulated, which leads to increased macromolecule permeability and is inducible by leptin via STAT3. This suggests that angulin-1 and leptin secretion are potential targets for intervention in CD to restore the impaired intestinal barrier.

Topics: Adult; Biopsy; Caco-2 Cells; Case-Control Studies; Crohn Disease; Cyclic S-Oxides; Down-Regulation; Female; Humans; Intestinal Mucosa; Leptin; Male; MARVEL Domain Containing 2 Protein; Middle Aged; Pyridines; Receptors, Lipoprotein; STAT3 Transcription Factor; Transcription Factors; Tyrphostins; Young Adult

This experiment was designed to explore the protection of sphingosine1-phosphate (S1P) postconditioning on rat myocardial cells injured by hypoxia/reoxygenation acting via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signal pathway. The data showed that S1P could significantly increase cell viability, lower the rate of apoptosis, decrease the content of lactate dehydrogenase (LDH) and caspase3 activity in the culture medium, increase the activity of total superoxide dismutase (T-SOD) and manganese superoxide dismutase (Mn-SOD), reduce the loss of mitochondrial membrane potential and the fluorescence intensity of intracellular calcium, as well as increase the phosphorylation of JAK2 and STAT3 in comparison with the H/R group. When the JAK inhibitor AG490 or the STAT inhibitor stattic were added, the effects of S1P were inhibited. Our date shows that S1P protects H9c2 cells from hypoxia/reoxygenation injury and that the protection by S1P was inhibited by AG490 and stattic. Therefore S1P protects H9c2 cells against hypoxia/reoxygenation injury via the JAK-STAT pathway.

Topics: Animals; Apoptosis; Calcium; Caspase 3; Cell Hypoxia; Cell Line; Cyclic S-Oxides; Gene Expression Regulation, Enzymologic; Intracellular Space; Ischemic Postconditioning; Janus Kinases; Lysophospholipids; Membrane Potential, Mitochondrial; Myocardial Reperfusion Injury; Myocytes, Cardiac; Oxygen; Phosphoproteins; Rats; Signal Transduction; Sphingosine; STAT Transcription Factors; Superoxide Dismutase; Tyrphostins

Deoxynivalenol (DON) and T-2 toxin commonly affect cells of the immune system and cause inflammation and apoptosis. Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is highly associated with inflammatory process and apoptosis and is worth investigating its role when cells were exposed to trichothecenes. The results showed that DON and T-2 upregulated the messenger RNA (mRNA) expressions of interleukin (IL)-6, IL-1β, tumor necrosis factor-α, JAK1-2, STAT1-3, and suppressors of cytokine signaling members and activated the tyrosine phosphorylation of STAT1 and STAT3 with a dose-dependent manner in RAW264.7 cells. AG490 and Stattic, the specific inhibitors of JAK/STAT pathway, blocked the STAT1 and STAT3 tyrosine phosphorylation and decreased the gene expressions of proinflammatory cytokines induced by trichothecenes. Interestingly, the time when the mRNA levels of STAT1 and STAT3 were significantly upregulated was at 12 h, which was much later than the time when mitogen-activated protein kinase was activated, indicating that STATs might be the downstream targets of the trichothecenes. With the intervention of AG490 and Stattic, DON and T-2 toxin induced apoptosis in a strengthened way, with the loss of mitochondrial membrane potential and the decrease ratios of the B-cell leukemia/lymphoma 2 (Bcl-2)/bcl-2-associated X (Bax) and B-cell lymphoma-extra large (Bcl-xL)/Bax. After exposing to DON and T-2 toxin, cells exhibited G2/M and G0/G1 phase arrest, respectively. The increased mRNA expressions of STAT target genes p21 and cyclin D1 for DON and the increases in p21 mRNA and the decreases in cyclin D1 for T-2 toxin were observed. These results demonstrated for the first time that the activation of JAK/STAT might be a critical mediator to induce the inflammatory response and apoptosis in macrophage in response to trichothecenes.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Cell Line; Cell Survival; Cyclic S-Oxides; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cytokines; Dose-Response Relationship, Drug; Gene Expression Regulation; Inflammation Mediators; Janus Kinases; Macrophages; Membrane Potential, Mitochondrial; Mice; Phosphorylation; Protein Kinase Inhibitors; RNA, Messenger; Signal Transduction; STAT Transcription Factors; STAT1 Transcription Factor; STAT3 Transcription Factor; Suppressor of Cytokine Signaling Proteins; T-2 Toxin; Time Factors; Trichothecenes; Tyrphostins

Hepatic ischemia/reperfusion (I/R) injury is a serious complication of liver surgery and transplantation. Regulation of this injury response occurs at the cellular and molecular levels. Previous studies have shown that interleukin-6 (IL-6) is a negative regulator of the acute inflammatory injury occurring as a result of hepatic I/R. The signal transducer and activator of transcription-3 (STAT3) is a key target of receptor signaling for IL-6. Both IL-6 and STAT3 have been implicated in the protective effects of ischemic preconditioning of the liver. However, there have been no studies that have directly addressed the potential role of STAT3 in regulating acute inflammatory liver injury induced by I/R. In the current study, we investigated whether blockade of STAT3 phosphorylation altered the injury response to hepatic I/R injury.. Male Balb/c mice were subjected to 90 min of partial hepatic ischemia followed by reperfusion with or without treatment with specific inhibitors of STAT3 activation, AG490 (selective JAK2 inhibitor), or STATTIC (direct inhibitor of STAT3 phosphorylation). Mice were sacrificed at 8 and 24 h after reperfusion.. STAT3 activation was induced by I/R. This activation was partially inhibited by administration of AG490 and almost completely abrogated by treatment with STATTIC. Despite the blockade of STAT3, neither AG490 nor STATTIC had any effect on acute liver injury induced by I/R. Treatment with STATTIC did reduce hepatic neutrophil accumulation.. The data suggest that STAT3 is not a central regulator of acute liver injury induced by I/R.

Topics: Acute Disease; Animals; Cyclic S-Oxides; Enzyme Inhibitors; Hepatitis; Janus Kinase 2; Male; Mice; Mice, Inbred BALB C; Neutrophils; Phosphorylation; Reperfusion Injury; STAT3 Transcription Factor; Tyrphostins

Timely restoration of coronary blood flow is the only way to salvage myocardium from infarction, but reperfusion per se brings on additional injury. Such reperfusion injury and the resulting size of myocardial infarction is attenuated by ischemic postconditioning, ie, the repeated brief interruption of coronary blood flow during early reperfusion. The signal transduction of ischemic postconditioning is under intense investigation, but no signaling step has yet been identified as causal for such protection in larger mammals in situ.. We have now in an in situ pig model of regional myocardial ischemia/reperfusion addressed the role of mitochondrial signal transducer and activator of transcription 3 (STAT3).. We demonstrated reduction of infarct size by ischemic postconditioning (26 ± 3% of area at risk versus 38 ± 2% in controls with immediate full reperfusion) along with more markedly increased tyrosine(705) phosphorylation of STAT3 in myocardial biopsies (at 10 minutes reperfusion: 9.2 ± 3.0-fold from baseline versus 6.6 ± 2.9-fold in controls with immediate full reperfusion). Increased tyrosine(705) phosphorylation of STAT3 and better preservation of complex 1 respiration and calcium retention capacity were also present in isolated mitochondria from postconditioned myocardium in vitro. Prior janus kinase/STAT inhibition with AG490 in vivo abrogated the infarct size reduction and the better preservation of mitochondrial function, and the STAT3 inhibitor Stattic in vitro also abrogated better preservation of mitochondrial function.. Our data support a causal role for mitochondrial STAT3 activation to mediate cardioprotection through better mitochondrial function.

Topics: Animals; Calcium; Cyclic S-Oxides; Electron Transport Complex IV; Ischemic Postconditioning; Janus Kinases; Mitochondria, Heart; Models, Animal; Oxygen Consumption; Phosphorylation; Protein Processing, Post-Translational; Signal Transduction; STAT Transcription Factors; STAT3 Transcription Factor; Sus scrofa; Swine; Tyrphostins

Both interleukin 1 beta (IL-1beta) and interleukin-6 (IL-6) are pro-inflammatory cytokines that play a major role in inflammatory diseases as well as cancer. In this work we investigated the signaling pathway involving lipopolysaccharide (LPS)-mediated IL-1beta and IL-6 production in murine macrophage cell lines and primary macrophages. We show that in response to LPS, the JAK/STAT pathway is activated, leading to tyrosine phosphorylation at residue 705 on STAT3 and at residue 701 on STAT1, respectively. A newly developed STAT3 specific inhibitor (stattic) blocked LPS-mediated STAT3 tyrosine phosphorylation and led to inhibition of LPS-mediated IL-1beta and IL-6 production but not TNF-alpha production. Knockdown of STAT3 expression via small interfering RNA (siRNA) decreased the level of STAT3 expression in Raw 264.7 cells and decreased STAT3 tyrosine phosphorylation in response to LPS treatment. Quantitative real time PCR and Western analysis of cells treated with inhibitor or STAT3 siRNA after LPS treatment showed a significant reduction of IL-1beta and IL-6 mRNA and protein compared to cells treated with LPS alone. Moreover stattic abrogated IL-1beta formation in response to extracellular bacteria Staphylococcus aureus and Escherichia coli in murine peritoneal macrophages. This inhibition did not affect caspase-1 activation. These results highlight the complex role of STAT3 in cytokine production and the key role of STAT3 tyrosine phosphorylation in IL-1beta and IL-6 production in response to inflammation.

Topics: Animals; Bacteria; Bacterial Physiological Phenomena; Cells, Cultured; Cyclic S-Oxides; Enzyme Inhibitors; Female; Inflammation; Interleukin-1beta; Interleukin-6; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Microbial Viability; Phosphorylation; Protein-Tyrosine Kinases; STAT3 Transcription Factor; Tumor Necrosis Factor-alpha; Tyrosine; Tyrphostins

STAT3 (signal transducer and activator of transcription 3) is an important transcription factor involved in many biological events, including apoptosis, tumorigenesis, angiogenesis and epithelial-to-mesenchymal transition. However, no direct evidence for a role of STAT3 in 3T3-L1 adipocyte differentiation has been reported.. In the present study, we found that rapid activation of STAT3, lasting for more than 48 h, was elicited upon induction of adipogenesis. Both the STAT3-selective inhibitor stattic and the JAK2 (Janus kinase 2)/STAT3-selective inhibitors AG490 and Gö6976 inhibited STAT3 activation, leading to the suppression of adipocyte differentiation. Adipocyte differentiation was also suppressed by STAT3 siRNA (small interfering RNA) or dominant-negative STAT3. Interestingly, the PPARgamma (peroxisome-proliferator-activated receptor gamma) agonist TAZ (troglitazone) abolished the STAT3-inhibitor- and RNAi (RNA interference)-mediated suppression of adipogenesis. However, TAZ treatment had no effect on the stattic- and AG490-mediated down-regulation of STAT3 activation, suggesting that STAT3 regulates adipocyte differentiation through signalling that occurs upstream of PPARgamma.. These data indicate that STAT3 functions as a critical factor for adipogenesis via a mechanism involving the PPARgamma activation pathway.

Topics: 3T3 Cells; Adipocytes; Animals; Carbazoles; Cell Differentiation; Cells, Cultured; Chromans; Cyclic S-Oxides; Enzyme Inhibitors; Gene Expression Regulation; Mice; PPAR gamma; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; STAT3 Transcription Factor; Thiazolidinediones; Troglitazone; Tyrphostins

Postconditioning (POC), a novel strategy of cardioprotection against ischemia-reperfusion injury, is clinically attractive because of its therapeutic application at the predictable onset of reperfusion. POC activates several intracellular kinase signaling pathways, including phosphatidylinositol 3-kinase (PI3K)-Akt (RISK). The regulation of POC-induced survival kinase signaling, however, has not been fully characterized. JAK-STAT activation is integral to cardiac ischemic tolerance and may provide upstream regulation of RISK. We hypothesized that POC requires the activation of both JAK-STAT and RISK signaling. Langendorff-perfused mouse hearts were subjected to 30 min of global ischemia and 40 min of reperfusion, with or without POC immediately after ischemia. A separate group of POC hearts was treated with AG 490, a JAK2 inhibitor, Stattic, a specific STAT3 inhibitor, or LY-294002, a PI3K inhibitor, at the onset of reperfusion. Cardiomyocyte-specific STAT3 knockout (KO) hearts were also subjected to non-POC or POC protocols. Myocardial performance (+dP/dt(max), mmHg/s) was assessed throughout each perfusion protocol. Phosphorylated (p-) STAT3 and Akt expression was analyzed by Western immunoblotting. POC enhanced myocardial functional recovery and increased expression of p-STAT3 and p-Akt. JAK-STAT inhibition abrogated POC-induced functional protection. STAT3 inhibition decreased expression of both p-STAT3 and p-Akt. PI3K inhibition also attenuated POC-induced cardioprotection and reduced p-Akt expression but had no effect on STAT3 phosphorylation. Interestingly, STAT3 KO hearts undergoing POC exhibited improved ischemic tolerance compared with KO non-POC hearts. POC induces myocardial functional protection by activating the RISK pathway. JAK-STAT signaling, however, is insufficient for effective POC without PI3K-Akt activation.

Topics: Animals; Chromones; Cyclic S-Oxides; Janus Kinases; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morpholines; Myocardial Contraction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Recovery of Function; Signal Transduction; STAT3 Transcription Factor; Time Factors; Tyrphostins; Ventricular Function, Left