Signal Transducer And Activator Of Transcription 3 Knockout Research Articles

An Integrated Computational and Experimental Binding Study Identifies the DNA Binding Domain as the Putative Binding Site of Novel Pyrimidinetrione Signal Transducer and Activator of Transcription 3 (STAT3) Inhibitors

Objective: Signal transducer and activator of transcription 3 (STAT3) is as a potential drug target for cancer and inflammation. Strategies to block STAT3 dimerization have dominated STAT3 inhibitor discovery, while inhibition of direct STAT3-DNA binding has not been much explored. This study was to identify novel STAT3 inhibitors that may be developed into probes or therapeutics, and to investigate their putative binding site. Methods: A library of in-house compounds was screened against STAT3-DNA binding using an electrophoretic mobility shift assay (EMSA). Inhibition of DNA and STAT3:STAT3 or STAT3:STAT1 interaction and antiproliferative activity in STAT3 expressing or STAT3 knockout cells were tested. Pharmacophore modeling and 3D-QSAR; and docking with molecular mechanics generalized born surface area (MM-GBSA) refinement were also undertaken at the STAT3 SH2 and STAT3 DNA binding domains. Surface plasmon resonance (SPR) analysis was done to determine STAT3 domain interactions. Organic synthesis was also undertaken. Results: Pyrimidinetriones derivatives were identified as novel STAT3 inhibitors, with activity in low micromolar concentrations. Application of 3D-QSAR and docking analysis with MM-GBSA refinement suggested that the compounds bind at the STAT3-DNA binding interface, and not at the SH2 domain where most current STAT3 inhibitors are thought to bind. The results were confirmed with SPR analysis, pointing to the DNA binding domain (DBD) as the putative binding site of this novel class of STAT3 inhibitors. The analysis guided the synthesis of active novel compounds. Conclusion: Pyrimidinetrione were identified as new STAT3 inhibitors that putatively bind at the STAT3 DBD. This study is an excellent example of the use of QSAR and structure-based design to aid the identification of putative ligand binding sites on proteins. The compounds will provide new tools for studying STAT3 biology; and also serve as potential leads for the development of new therapeutics against STAT3 to fight or study diseases such as cancer and inflammation.

Cardiac STAT3 Deficiency Impairs Contractility and Metabolic Homeostasis in Hypertension.

Signal transducer and activator of transcription 3 (STAT3) protects the heart from acute ischemic stress. However, the importance of STAT3 to the heart in chronic stress, such as hypertension, is not known. To study this, we used cardiomyocyte-targeted STAT3 knockout (KO) mice and Angiotensin II (ANG II) infusion by osmotic minipumps. After 4 weeks, ANG II induced similar cardiac hypertrophy in wild type (WT) and cardiac Cre-expressing control (CTRL) mice with no impairment of cardiac function. In contrast, STAT3 KO mice exhibited reduced contractile function but similar hypertrophy to CTRL mice. Ejection fraction and fractional shortening decreased by 22.5 and 27.3%, respectively. Since STAT3 has direct protective effects on mitochondrial function, we examined rates of glucose and oleate oxidation by isolated perfused hearts using a Langendorff system. Hearts of ANG II-treated STAT3 KO and CTRL mice had similar rates of oleate oxidation as saline-infused WT mice. Rates of glucose oxidation were similar between hearts of WT plus saline and CTRL plus ANG II mice; however, glucose oxidation was increased by 66% in hearts of ANG II-treated STAT3 KO mice. The ratio of maximal ATP yield from glucose to fatty acid oxidation was 21.1 ± 3.1 in hearts of ANG II-treated STAT3 KO mice vs. 12.6 ± 2.2 in hearts of ANG II-treated CTRL mice. Lactate production was also elevated in hearts of ANG II-treated STAT3 KO mice by 162% compared to ANG II-treated CTRL mice. Our findings indicate that STAT3 is important for maintaining contractile function and metabolic homeostasis in the hypertensive heart, and STAT3 deficiency promotes a switch toward glucose utilization.

Leptin Signaling Is Not Required for Anorexigenic Estradiol Effects in Female Mice.

Estradiol and leptin are critical hormones in the regulation of body weight. The aim of this study was to determine whether this cross talk between leptin receptor (LepRb) and estrogen receptor-α (ERα) signaling is critical for estradiol's anorexigenic effects. Leprb-Cre mice were crossed with Cre-dependent Tau-green fluorescent protein (GFP) reporter, Stat3-flox or Erα-flox mice to generate female mice with GFP expression, signal transducer and activator of transcription 3 (STAT3) knockout (KO), or ERα KO, specifically in LepRb-expressing cells. The proportion of Leprb-GFP cells colocalizing ERα was high (∼80%) in the preoptic area but low (∼10%) in the mediobasal hypothalamus, suggesting that intracellular cross talk between these receptors is minimal for metabolic regulation. To test whether estradiol enhanced arcuate leptin sensitivity, ovarectomized mice received varying levels of estradiol replacement. Increasing estrogenic states did not increase the degree of leptin-induced STAT3 phosphorylation. LepRb-specific STAT3 KO mice and controls were ovarectomized and given either chronic estradiol or vehicle treatment to test whether STAT3 is required for estrogen-induced body weight suppression. Both groups of estradiol-treated mice showed an equivalent reduction in body weight and fat content compared with vehicle controls. Finally, mice lacking ERα specifically in LepRb-expressing neurons also showed no increase in body weight or impairments in metabolic function compared with controls, indicating that estradiol acts independently of leptin-responsive cells to regulate body weight. However, fecundity was impaired in in Leprb-ERα KO females. Contrary to the current dogma, we report that estradiol has minimal direct actions on LepRb cells in the mediodasal hypothalamus and that its anorexigenic effects can occur entirely independently of LepRb-STAT3 signaling in female mice.

Knockout of STAT3 in skeletal muscle does not prevent high-fat diet-induced insulin resistance.

ObjectiveIncreased signal transducer and activator of transcription 3 (STAT3) signaling has been implicated in the development of skeletal muscle insulin resistance, though its contribution, in vivo, remains to be fully defined. Therefore, the aim of this study was to determine whether knockout of skeletal muscle STAT3 would prevent high-fat diet (HFD)-induced insulin resistance.MethodsWe used Cre-LoxP methodology to generate mice with muscle-specific knockout (KO) of STAT3 (mKO). Beginning at 10 weeks of age, mKO mice and their wildtype/floxed (WT) littermates either continued consuming a low fat, control diet (CON; 10% of calories from fat) or were switched to a HFD (60% of calories from fat) for 20 days. We measured body composition, energy expenditure, oral glucose tolerance and in vivo insulin action using hyperinsulinemic-euglycemic clamps. We also measured insulin sensitivity in isolated soleus and extensor digitorum longus muscles using the 2-deoxy-glucose (2DOG) uptake technique.ResultsSTAT3 protein expression was reduced ∼75–100% in muscle from mKO vs. WT mice. Fat mass and body fat percentage did not differ between WT and mKO mice on CON and were increased equally by HFD. There were also no genotype differences in energy expenditure or whole-body fat oxidation. As determined, in vivo (hyperinsulinemic-euglycemic clamps) and ex vivo (2DOG uptake), skeletal muscle insulin sensitivity did not differ between CON-fed mice, and was impaired similarly by HFD.ConclusionsThese results demonstrate that STAT3 activation does not underlie the development of HFD-induced skeletal muscle insulin resistance.

Cardiac preconditioning with sphingosine-1-phosphate requires activation of signal transducer and activator of transcription-3.

SummaryAimsSphingosine-1-phosphate (S1P) is a cardioprotective agent. Signal transducer and activator of transcription 3 (STAT-3) is a key mediator of many cardioprotective agents. We aimed to explore whether STAT-3 is a key mediator in S1P-induced preconditioning.MethodsLangendorff-perfused hearts from Wistar rats and wild-type or cardiomyocyte-specific STAT-3 knockout mice were pre-treated with S1P (10 nmol/l), with or without the STAT-3 pathway inhibitor AG490, before an ischaemia–reperfusion insult. Triphenyltetrazolium chloride and Evans blue staining were used for the determination of infarct size. Western blot analysis was carried out on the S1P pre-treated hearts for detection of cytosolic, nuclear and mitochondrial phosphorylated and total STAT-3 proteins.ResultsPre-treatment with S1P decreased the infarct size in isolated rat (5 ± 3% vs control 26 ± 8%, p < 0.01) and wild-type mouse hearts (13 ± 1% vs control 33 ± 3%, p < 0.05). This protective effect was abolished in the rat hearts pre-treated with AG490 (30 ± 10%, p = ns vs control) and in the hearts from STAT-3 knockout mice (35 ± 4% vs control 30 ± 3%, p = ns). Levels of phosphorylated STAT-3 were significantly increased in both the nuclear (p < 0.05 vs control) and mitochondrial (p < 0.05 vs control) fractions in the S1P pre-treated hearts, but remained unchanged in the cytosolic fraction (p = ns vs control).ConclusionThese novel results demonstrate that pharmacological preconditioning with S1P in the isolated heart is mediated by activation of mitochondrial and nuclear STAT-3, therefore suggesting that S1P may be a novel therapeutic target to modulate mitochondrial and nuclear function in cardiovascular disease in order to protect the heart against ischaemia–reperfusion.

OP0240 Signal Transducer and Activator of Transcription 3 Regulates Fibroblast Activation as A Downstream Mediator of Transforming Growth Factor-Beta

BackgroundSystemic sclerosis (SSc) is an autoimmune disease characterized by uncontrolled activation of fibroblasts with increased deposition of extracellular matrix. Persistent activation of TGFβ signalling is thought to play a key-role...

Neither Signal Transducer and Activator of Transcription 3 (STAT3) or STAT5 Signaling Pathways Are Required for Leptin's Effects on Fertility in Mice

The hormone leptin is critical for the regulation of energy balance and fertility. The long-form leptin receptor (LepR) regulates multiple intracellular signaling cascades, including the classic Janus kinase-signal transducer and activator of transcription (STAT) pathways. Previous studies have shown that deletion of STAT3 or the closely related STAT5 from the brain results in an obese phenotype, but their roles in fertility regulation are not clear. This study tested whether STAT3 and STAT5 pathways of leptin signaling are required for fertility, and whether absence of one pathway might be compensated for by the other in a redundant manner. A Cre-loxP approach was used to generate 3 models of male and female transgenic mice with LepR-specific deletion of STAT3, STAT5, or both STAT3 and STAT5. Body weight, puberty onset, estrous cyclicity, and fertility were measured in all knockout (KO) mice and their control littermates. Knocking out STAT3 or both STAT3 and 5 from LepR expressing cells, but not STAT5 alone, led to significant increase in body weight. All STAT3 and STAT5 single KO mice exhibited normal puberty onset and subsequent fertility compared to their control littermates. Surprisingly, all STAT3 and STAT5 double KO mice also exhibited normal puberty onset, estrous cyclicity, and fertility, although they had severely disrupted body weight regulation. These results suggest that, although STAT3 signaling is crucial for body weight regulation, neither STAT3 nor STAT5 is required for the regulation of fertility by leptin. It remains to be determined what other signaling molecules mediate this effect of leptin, and whether they interact in a redundant manner.

Pancreatic STAT3 Protects Mice against Caerulein-Induced Pancreatitis via PAP1 Induction

The signal transducer and activator of transcription 3 (STAT3) is a transcription factor that controls expressions of several genes involved in cell survival, proliferation and differentiation, and tissue inflammation. However, the significance of pancreatic STAT3 in acute pancreatitis remains unclear. We generated conditional STAT3 knockout (stat3(Δ/Δ)) mice by crossing stat3(flox/flox) mice with Pdx1-promoter Cre transgenic mice. Caerulein administration activated pancreatic STAT3 and induced acute pancreatitis as early as 3 hours in wild-type mice, and full recovery from the induced pancreatic injury was observed within 7 days. The levels of serum amylase and lipase and histologic scores of pancreatic necrosis and inflammatory cell infiltration were significantly higher at 3 hours in stat3(Δ/Δ) mice than in stat3(flox/flox) mice. Pancreatic recovery after pancreatitis was significantly delayed in stat3(Δ/Δ) mice compared with stat3(flox/flox) mice. Although stat3(flox/flox) mice had marked production in the pancreas of pancreatitis-associated protein 1 (PAP1), a serum acute phase protein, this induction was completely abrogated in stat3(Δ/Δ) mice. Enforced production of PAP1 by a hydrodynamic procedure in the liver significantly suppressed pancreatic necrosis and inflammation and also promoted pancreatic regeneration and recovery in stat3(Δ/Δ) mice to levels similar to those observed in stat3(flox/flox) mice. In conclusion, pancreatic STAT3 is indispensable for PAP1 production, and this STAT3/PAP1 pathway plays a protective role in caerulein-induced pancreatitis.

STAT3 is critical to promote inflammatory cytokines and RANKL expression in inflammatory arthritis

Rheumatoid arthritis (RA) causes sever joint damage and significant disability of daily living. The symptoms of RA patients are mainly from chronic inflammation and continuous joint destruction, however, the mechanisms underlying how inflammation and joint destruction in RA develop and are sustained chronically remain largely unclear. In this study, we show that signal transducer and activator of transcription 3 (STAT3) plays a critical role in both chronic inflammation and joint destruction in RA. We found that inflammatory cytokines, such as IL-1β, TNFα and IL-6, activated STAT3 either directly or indirectly and induced expression of inflammatory cytokines, further activating STAT3. STAT3 activation also induced expression of receptor activator of nuclear factor kappa B ligand (RANKL), an essential cytokine for osteoclast differentiation. STAT3 knockout or pharmacological inhibition resulted in significant reduction of the expression of both inflammatory cytokines and RANKL in vitro. STAT3 inhibition was also effective in treating an RA model, collagen induced arthritis (CIA), in vivo through significant reduction in expression of inflammatory cytokines and RANKL, inhibiting both inflammation and joint destruction. Thus our data provide new insight into pathogenesis of RA and provide evidence that inflammatory cytokines induce a cytokine amplification loop via STAT3 that promotes sustained inflammation and joint destruction.

Intracoronary Mesenchymal Stem Cells Promote Postischemic Myocardial Functional Recovery, Decrease Inflammation, and Reduce Apoptosis via a Signal Transducer and Activator of Transcription 3 Mechanism

Signal transducer and activator of transcription 3 (STAT3) regulates myocardial apoptosis, cellular proliferation, and the immune response after ischemia/reperfusion (I/R). STAT3 is also necessary for the production of vascular endothelial growth factor (VEGF) by mesenchymal stem cells (MSCs), which are known to reduce myocardial injury after I/R. However, it remains unknown whether STAT3 is an important mediator of MSC-based cardioprotection. We hypothesized that knockout of stem cell STAT3 would reduce MSC-derived myocardial functional recovery and increase myocardial inflammatory and apoptotic signaling. With a Langendorff apparatus, male rat hearts were subjected to 15 minutes of equilibration and 25 minutes of ischemia, followed by 40 minutes of reperfusion. Immediately before ischemia, hearts received intracoronary infusions of vehicle, wild-type MSCs (WT MSCs) or STAT3 knockout MSCs (STAT3KO MSCs). Heart function was measured continuously. Myocardial homogenates were analyzed for production of interleukin (IL)-1, IL-6, and tumor necrosis factor-α (TNF-α). Additionally, MSC production of hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) were measured in vitro. Hearts treated with WT MSCs exhibited the greatest functional recovery, and those treated with STAT3KO MSCs had equivalent recovery to vehicle. The highest proinflammatory cytokine levels were seen in vehicle-treated hearts, and the lowest in the WT MSC group. STAT3KO MSCs produced less IGF-1, but more HGF than WT MSCs. Finally, hearts treated with STAT3KO MSCs or vehicle had significantly higher caspase-3 levels than those treated with WT MSCs. Intracoronary infusions of MSCs improve postischemic left ventricular function and reduce proapoptotic and proinflammatory signaling via a STAT3-dependent mechanism.

Involvement of STAT3-regulated hepatic soluble factors in attenuation of stellate cell activity and liver fibrogenesis in mice

Glycoprotein 130 (gp130)/signal transducer and activator of transcription 3 (STAT3) signaling in hepatocytes controls a variety of physiological and pathological processes including liver regeneration, apoptosis resistance and metabolism. Recent research has shed light on the importance of acute phase proteins (APPs) regulated by hepatic gp130/STAT3 in host defense through suppression of innate immune responses during systemic inflammation. To examine whether these STAT3-regulated soluble factors directly affect liver fibrogenic responses during liver injury, hepatocyte-specific STAT3 knockout (L-STAT3 KO) mice and control littermates were subjected to bile duct ligation (BDL) and examined 10 days later. In contrast to controls, L-STAT3 KO mice failed to produce APPs, such as serum amyloid A and haptoglobin, after BDL. Whereas L-STAT3 KO mice displayed similar levels of cholestasis, inflammatory cell infiltration and regeneration in the liver, they developed exacerbated liver injury and fibrosis with significant increases in expression of alpha-smooth muscle actin and type I collagen genes. In vitro experiments revealed that attenuated expression of APPs in primary hepatocytes isolated from L-STAT3 KO mice with IL-6 exposure, compared to wild-type hepatocytes. The cultured supernatant from IL-6-treated wild-type hepatocytes inhibited expression of alpha-smooth muscle actin and type I collagen genes in activated hepatic stellate cells (HSCs), whereas this did not occur with the supernatant from IL-6-treated knockout hepatocytes or with control medium. In conclusion, the absence of STAT3 in hepatocytes leads to exacerbation of liver fibrosis during cholestasis. Soluble factors released from hepatocytes, dependent on STAT3, collectively play a protective role in liver fibrogenesis through an inhibitory effect on activated HSCs.

Ethanolamine is a novel STAT-3 dependent cardioprotective agent

Ethanolamine is a biogenic amine found naturally in the body as part of membrane lipids and as a metabolite of the cardioprotective substances, sphingosine-1-phosphate (S1P) and anandamide. In the brain, ethanolamine, formed from the breakdown of anandamide protects against ischaemic apoptosis. However, the effects of ethanolamine in the heart are unknown. Signal transducer and activator of transcription 3 (STAT-3) is a critical prosurvival factor in ischaemia/reperfusion (I/R) injury. Therefore, we investigated whether ethanolamine protects the heart via activation of STAT-3. Isolated hearts from wildtype or cardiomyocyte specific STAT-3 knockout (K/O) mice were pre-treated with ethanolamine (Etn) (0.3 mmol/L) before I/R insult. In vivo rat hearts were subjected to 30 min ischaemia/2 h reperfusion in the presence or absence of 5 mg/kg S1P and/or the FAAH inhibitor, URB597. Infarct size was measured at the end of each protocol by triphenyltetrazolium chloride staining. Pre-treatment with ethanolamine decreased infarct size in isolated mouse or rat hearts subjected to I/R but this infarct sparing effect was lost in cardiomyocyte specific STAT-3 deficient mice. Pre-treatment with ethanolamine increased nuclear phosphorylated STAT-3 [control 0.75 ± 0.08 vs. Etn 1.50 ± 0.09 arbitrary units; P < 0.05]. Our findings suggest a novel cardioprotective role for ethanolamine against I/R injury via activation of STAT-3.

Anti‐Inflammatory and Anti‐Apoptotic Roles of Endothelial Cell STAT3 in Alcoholic Liver Injury

It is generally believed that the hepatoprotective effect of interleukin-6 (IL-6) is mediated via activation of signal transducer and activator of transcription 3 (STAT3) in hepatocytes. IL-6-deficient mice are more susceptible to alcohol-induced hepatocyte apoptosis and steatosis and elevation of serum alanine transaminase (ALT); however, whereas hepatocyte-specific STAT3 knockout mice are more susceptible to alcohol-induced hepatic steatosis, they have similar hepatocyte apoptosis and serum ALT after alcohol feeding compared with wild-type mice. This suggests that the hepatoprotective effect of IL-6 in alcoholic liver injury may be mediated via activation of STAT3-independent signals in hepatocytes, activation of STAT3 in nonparenchymal cells, or both. We have previously shown that IL-6 also activates STAT3 in sinusoidal endothelial cells (SECs). Thus, the purpose of this study was to investigate whether STAT3 in endothelial cells also plays a protective role in alcoholic liver injury. Wild-type and endothelial cell-specific STAT3 knockout (STAT3(E-/-)) mice were pair-fed and fed ethanol containing diet for 4 weeks. Liver injury and inflammation were determined. Feeding mice with ethanol-containing diet for 4 weeks induced greater hepatic injury (elevation of serum ALT) and liver weight in STAT3(E-/-) mice than wild-type control groups. In addition, ethanol-fed STAT3(E-/-) mice displayed greater hepatic inflammation and substantially elevated serum and hepatic levels of IL-6 and TNF-alpha compared with wild-type mice. Furthermore, ethanol-fed STAT3(E-/-) mice displayed a greater abundance of apoptotic SECs and higher levels of serum hyaluronic acid than wild-type controls. These data suggest that endothelial cell STAT3 plays important dual functions of attenuating hepatic inflammation and SEC death during alcoholic liver injury.

Dissociation Between Liver Inflammation and Hepatocellular Damage Induced by Carbon Tetrachloride in Myeloid Cell–Specific Signal Transducer and Activator of Transcription 3 Gene Knockout Mice

Liver injury is associated with inflammation, which is generally believed to accelerate the progression of liver diseases; however, clinical data show that inflammation does not always correlate with hepatocelluar damage in some patients. Investigating the cellular mechanisms underlying these events using an experimental animal model, we show that inflammation may attenuate liver necrosis induced by carbon tetrachloride (CCl(4)) in myeloid-specific signal transducer and activator of transcription 3 (STAT3) knockout mice. As an important anti-inflammatory signal, conditional deletion of STAT3 in myeloid cells results in markedly enhanced liver inflammation after CCl(4) injection. However, these effects are also accompanied by reduced liver necrosis, correlating with elevated serum interleukin-6 (IL-6) and hepatic STAT3 activation. An additional deletion of STAT3 in hepatocytes in myeloid-specific STAT3 knockout mice restored hepatic necrosis but decreased liver inflammation. Inflammation-mediated STAT3 activation attenuates hepatocellular injury induced by CCl(4) in myeloid-specific STAT3 knockout mice, suggesting that inflammation associated with a predominance of hepatoprotective cytokines that activate hepatic STAT3 may reduce rather than accelerate hepatocellular damage in patients with chronic liver diseases.

Dual activation of STAT-3 and Akt is required during the trigger phase of ischaemic preconditioning

During preconditioning by tumour necrosis factor-alpha (TNFalpha), activation of the signal transducer and activator of transcription-3 (STAT-3) but not Akt, is essential, whereas ischaemic cardiac preconditioning (IPC) requires both STAT-3 and Akt at the time of reperfusion. However, it is not known whether the same signalling pattern occurs during the preconditioning stimulus (trigger phase) and whether links exist between STAT-3 and Akt. Hence, our hypothesis is that concomitant activation or co-interaction between these two key signals is required during the trigger phase for IPC. Conversely, we proposed that there would be no such interaction when preconditioning was induced by TNFalpha (TNF-PC). Cardiomyocytes, isolated from adult wild-type (WT) and cardiac-specific STAT-3 knockout (KO) mice, were exposed to simulated ischaemia (SI) reperfusion. Cells were preconditioned either by 30 min SI or by 30 min TNFalpha (0.5 ng/mL) in the presence or absence of AG490 (100 nM) or wortmannin (100 nM) to inhibit STAT-3 or Akt, respectively. Cell viability was evaluated by trypan blue, and phosphorylation levels of STAT-3 and Akt were measured by Western blot analysis. Similar experiments were conducted in isolated rat hearts subjected to an ischaemia-reperfusion insult. Both preconditioning stimuli failed to protect KO cardiomyocytes, and addition of AG490 abolished preconditioning in WT cardiomyocytes or isolated hearts. Wortmannin abolished the protection afforded by IPC, but did not affect TNF-PC in both models. Western blot analysis demonstrated that added wortmannin during IPC stimulus decreased STAT-3 phosphorylation while, conversely, AG490 reduced Akt phosphorylation. STAT-3 activation could be achieved independent of Akt during TNF-PC. In contrast, during an IPC stimulus, both prosurvival signalling molecule cascades acted in concert so that inhibiting activation of STAT-3 also inhibited that of Akt and vice versa.

Cell Type–Dependent Pro- and Anti-Inflammatory Role of Signal Transducer and Activator of Transcription 3 in Alcoholic Liver Injury

Signal transducer and activator of transcription 3 (STAT3) is known to be activated in human alcoholic liver disease, but its role in the pathogenesis of alcoholic liver injury remains obscure. The role of STAT3 in alcoholic liver injury was investigated in hepatocyte-specific STAT3 knockout (H-STAT3KO) mice and macrophage/neutrophil-specific STAT3 KO (M/N-STAT3KO) mice. Alcoholic liver injury was achieved by feeding mice a liquid diet containing 5% ethanol for up to 8 weeks. Compared with wild-type mice, feeding H-STAT3KO mice with an ethanol-containing diet induced greater hepatic steatosis, hypertriglyceridemia, and hepatic expression of lipogenic genes (sterol regulatory element-binding protein, fatty acid synthase, acetyl-CoA carboxylase-1, and stearoyl-CoA desaturase 1), but less inflammation and lower expression of hepatic proinflammatory cytokines. In contrast, ethanol-fed M/N-STAT3KO mice showed more hepatic inflammation, worse injury, and increased hepatic expression of proinflammatory cytokines compared with wild-type mice. Kupffer cells isolated from ethanol-fed H-STAT3KO mice produced similar amounts of reactive oxygen species and tumor necrosis factor alpha, whereas Kupffer cells from M/N-STAT3KO mice produced more reactive oxygen species and tumor necrosis factor alpha compared with wild-type controls. These findings suggest that STAT3 regulates hepatic inflammation in a cell type-dependent manner during alcoholic liver injury: STAT3 in hepatocytes promotes whereas STAT3 in macrophages/Kupffer cells suppresses inflammation. In addition, activation of hepatocellular STAT3 ameliorates alcoholic fatty liver via inhibition of sterol regulatory element-binding protein 1c expression.

Identification of de novo STAT3 target gene in liver regeneration

The process of liver regeneration is regulated by complex mechanisms. Although signal transducer and activator of transcription-3 (STAT3), a transcription factor which targets mainly mitotic genes, definitely plays an important role in liver regeneration, the exact roles of STAT3 are not completely understood. In this report, we tried to search for a new target of STAT3 involved in liver regeneration in mice. We generated liver-specific STAT3 knockout (L-S3KO) mice and a STAT3 knockout cell line of mouse origin. Using chromatin immunoprecipitation, we screened 12 genes to which STAT3 binds after partial hepatectomy (PH). Of these genes, we analyzed the S3-IE3 clone that is located on chromosome-3 and possesses STAT3 binding sites in it. We showed that STAT3 binds to a specific site on S3-IE3, and that interleukin-6 (IL-6) stimulates its transcriptional activity. The mRNA and protein levels of the net gene, which is located downstream of S3-IE3, were negatively regulated in the control cells, but not in the STAT3 knockout cells after IL-6 stimulation. Similarly in in vivo mouse PH, the mRNA and protein levels of net were also negatively regulated after PH, but not in L-S3KO mice. The net gene is located downstream of a newly-recognized STAT3 binding site (S3-IE3) and negatively regulated after IL-6 stimulation and PH, although its role is still unclear.

Role of STAT3 in liver regeneration: survival, DNA synthesis, inflammatory reaction and liver mass recovery

The hepatoprotective effect of interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) has been well documented. However, reports on the role of IL-6/STAT3 in liver regeneration are conflicting probably due to the fact that the model of Stat3 knockout mice were complicated with obesity and fatty liver, which may cause some secondary effects on liver regeneration. To study the direct role of STAT3 and to circumvent the problems of obesity and fatty liver in liver regeneration, we generated conditional STAT3 knockout in the liver (L-Stat3−/−) using a transthyretin-driven Cre-lox method. The L-Stat3−/− mice were born with the expected Mendelian frequency and showed no obesity or other obvious phenotype. After partial hepatectomy, mortality in the L-Stat3−/− mice was significantly higher than the littermate Stat3f/+ controls in the early time points (<24 h). Hepatocyte DNA synthesis in the survived L-Stat3−/− mice slightly decreased as compared with Stat3f/+ mice at 40 h after partial hepatectomy, whereas similar hepatocyte DNA synthesis was found at other time points and liver mass could be completely recovered in the L-Stat3−/− mice. In another model of liver regeneration induced by subcutaneous injection of carbon tetrachloride (CCl4), hepatocyte DNA synthesis in the CCl4-treated L-Stat3−/− mice also decreased as compared with Stat3f/+ mice at 40 h after injection but not at other time points. In addition, infiltration of neutrophils and monocyte increased in the liver of CCl4-treated L-Stat3−/− mice compared to wild-type mice. In conclusion, STAT3 is required for survival in the acute stage after 70% hepatectomy and plays a role in inflammatory reaction after hepatocyte necrosis. However, the hepatocytic STAT3 may have limited role in liver mass recovery although DNA synthesis may be impaired.

Signal transducer and activator of transcription 3 is required for myocardial capillary growth, control of interstitial matrix deposition, and heart protection from ischemic injury.

The transcription factor signal transducer and activator of transcription 3 (STAT3) participates in a wide variety of physiological processes and directs seemingly contradictory responses such as proliferation and apoptosis. To elucidate its role in the heart, we generated mice harboring a cardiomyocyte-restricted knockout of STAT3 using Cre/loxP-mediated recombination. STAT3-deficient mice developed reduced myocardial capillary density and increased interstitial fibrosis within the first 4 postnatal months, followed by dilated cardiomyopathy with impaired cardiac function and premature death. Conditioned medium from STAT3-deficient cardiomyocytes inhibited endothelial cell proliferation and increased fibroblast proliferation, suggesting the presence of paracrine factors attenuating angiogenesis and promoting fibrosis in vitro. STAT3-deficient mice showed enhanced susceptibility to myocardial ischemia/reperfusion injury and infarction with increased cardiac apoptosis, increased infarct sizes, and reduced cardiac function and survival. Our study establishes a novel role for STAT3 in controlling paracrine circuits in the heart essential for postnatal capillary vasculature maintenance, interstitial matrix deposition balance, and protection from ischemic injury and heart failure.

Cardiomyocyte-restricted knockout of STAT3 results in higher sensitivity to inflammation, cardiac fibrosis, and heart failure with advanced age.

Cytokines and inflammation have been implicated in the pathogenesis of heart failure. For example, IL-6 family cytokines and the gp130 receptor play important roles in cardiac myocyte survival and hypertrophy. Signal transducer and activator of transcription 3 (STAT3) is a major signaling protein that is activated through gp130. We have created mice with a cardiomyocyte-restricted deletion of STAT3. As measured by serial echocardiograms, mice with cardiac specific deletion of STAT3 are significantly more susceptible to cardiac injury after doxorubicin treatment than age-matched controls. Intriguingly, STAT3 appears to have a critical role in protection of inflammation-induced heart damage. STAT3-deficient mice treated with lipopolysaccharide demonstrated significantly more apoptosis than their WT counterparts. At the cellular level, cardiomyocytes with STAT3 deleted secrete significantly more tumor necrosis factor in response to lipopolysaccharide than those with WT STAT3. Furthermore, histologic examination of the cardiomyocyte-restricted STAT3-deficient mice reveals a dramatic increase in cardiac fibrosis in aged mice. Although no overt signs of heart failure are present in young STAT3-deficient mice, they spontaneously develop heart dysfunction with advancing age. These results indicate the crucial functions of STAT3 in cardiomyocyte resistance to inflammation and other acute injury and in pathogenesis of age-related heart failure.