Evidence for investigating GSK-3 inhibitors as potential therapeutics for severe COVID-19.

A key component of severe COVID-19 is a "cytokine storm" i.e., the excessive expression of unneeded cytokines. Previous studies suggest that SARS-CoV-2 proteins can induce macrophages to secrete pro-inflammatory cytokines; a process that may involve Toll-like receptors (TLRs). Glycogen synthase kinase-3 (GSK-3) has been implicated in TLR signal transduction and a selective GSK-3 inhibitor, termed COB-187, dramatically attenuates cytokine expression induced by the TLR ligand lipopolysaccharide (LPS). In the present study, we provide evidence that the SARS-CoV-2 spike protein (S) and the S2 subunit (S2) induce production of CXCL10 (a chemokine elevated in severe COVID-19) by a human macrophage cell line. Further, we report that two clinically relevant GSK-3 inhibitors and COB-187 attenuate S and S2 protein-induced CXCL10 production. Combined, our observations provide impetus for investigating GSK-3 inhibitors as potential therapeutics for severe COVID-19.

A novel GSK-3 inhibitor binds to GSK-3ß via a reversible, time and Cys-199-dependent mechanism.

Glycogen synthase kinase-3 (GSK-3) has been implicated in numerous pathologies making GSK-3 an attractive therapeutic target. Our group has identified a compound termed COB-187 that is a potent and selective inhibitor of GSK-3. In this study, we probed the mechanism by which COB-187 inhibits GSK-3ß. Progress curves, generated via real-time monitoring of kinase activity, indicated that COB-187 inhibition of GSK-3ß is time-dependent and subsequent jump dilution assays revealed that COB-187 binding to GSK-3ß is reversible. Further, a plot of the kinetic constant (kobs) versus COB-187 concentration suggested that, within the range of concentrations studied, COB-187 binds to GSK-3ß via an induced-fit mechanism. There is a critical cysteine residue at the entry to the active site of GSK-3ß (Cys-199). We generated a mutant version of GSK-3ß wherein Cys-199 was substituted with an alanine. This mutation caused a dramatic decrease in the activity of COB-187; specifically, an IC50 in the nM range for wild type versus >100 µM for the mutant. A screen of COB-187 against 34 kinases that contain a conserved cysteine in their active site revealed that COB-187 is highly selective for GSK-3 indicating that COB-187's inhibition of GSK-3ß via Cys-199 is specific. Combined, these findings suggest that COB-187 inhibits GSK-3ß via a specific, reversible, time and Cys-199-dependent mechanism.

Toll-Like Receptors Signaling in the Tumor Microenvironment.

The involvement of inflammation in cancer progression is well-established. The immune system can play both tumor-promoting and -suppressive roles, and efforts to harness the immune system to help fight tumor growth are at the forefront of research. Of particular importance is the inflammatory profile at the site of the tumor, with respect to both the leukocyte population numbers, the phenotype of these cells, as well as the contribution of the tumor cells themselves. In this regard, the pro-inflammatory effects of pattern recognition receptor expression and activation in the tumor microenvironment have emerged as a relevant issue both for therapy and to understand tumor development.Pattern recognition receptors (PRRs) were originally recognized as components of immune cells, particularly innate immune cells, as detectors of pathogens. PRR signaling in immune cells activates them, inducing robust antimicrobial responses. In particular, toll-like receptors (TLRs) constitute a family of membrane-bound PRRs which can recognize pathogen-associated molecular patterns (PAMPs) carried by bacteria, virus, and fungi. In addition, PRRs can recognize products generated by stressed cells or damaged tissues, namely damage-associated molecular patterns or DAMPS. Taking into account the role of the immune system in fighting tumors together with the presence of immune cells in the microenvironment of different types of tumors, strategies to activate immune cells via PRR ligands have been envisioned as an anticancer therapeutic approach.In the last decades, it has been determined that PRRs are present and functional on nonimmune cells and that their activation in these cells contributes to the inflammation in the tumor microenvironment. Both tumor-promoting and antitumor effects have been observed when tumor cell PRRs are activated. This argues against nonspecific activation of PRR ligands in the tumor microenvironment as a therapeutic approach. Therefore, the use of PRR ligands for anticancer therapy might benefit from strategies that specifically deliver these ligands to immune cells, thus avoiding tumor cells in some settings. This review focuses on these aspects of TLR signaling in the tumor microenvironment.

Identification of a novel selective and potent inhibitor of glycogen synthase kinase-3.

Glycogen synthase kinase-3 (GSK-3) is a multitasking protein kinase that regulates numerous critical cellular functions. Not surprisingly, elevated GSK-3 activity has been implicated in a host of diseases including pathological inflammation, diabetes, cancer, arthritis, asthma, bipolar disorder, and Alzheimer's. Therefore, reagents that inhibit GSK-3 activity provide a means to investigate the role of GSK-3 in cellular physiology and pathophysiology and could become valuable therapeutics. Finding a potent inhibitor of GSK-3 that can selectively target this kinase, among over 500 protein kinases in the human genome, is a significant challenge. Thus there remains a critical need for the identification of selective inhibitors of GSK-3. In this work, we introduce a novel small organic compound, namely COB-187, which exhibits potent and highly selective inhibition of GSK-3. Specifically, this study 1) utilized a molecular screen of 414 kinase assays, representing 404 unique kinases, to reveal that COB-187 is a highly potent and selective inhibitor of GSK-3; 2) utilized a cellular assay to reveal that COB-187 decreases the phosphorylation of canonical GSK-3 substrates indicating that COB-187 inhibits cellular GSK-3 activity; and 3) reveals that a close isomer of COB-187 is also a selective and potent inhibitor of GSK-3. Taken together, these results demonstrate that we have discovered a region of chemical design space that contains novel GSK-3 inhibitors. These inhibitors will help to elucidate the intricate function of GSK-3 and can serve as a starting point for the development of potential therapeutics for diseases that involve aberrant GSK-3 activity.

Wnt5a, TLR2 and TLR4 are elevated in advanced human atherosclerotic lesions.

OBJECTIVE AND DESIGN: Atherosclerosis (ATH) is a chronic inflammatory disease that involves cascades of signaling events mediated by various effector proteins. Here we sought to determine if the expression of Wnt5a, a secreted glycoprotein, is altered in discrete regions of the arterial plaque. METHODS: Atherosclerotic plaque tissues from 14 human subjects undergoing elective carotid endarterectomy were used in this study. Immunohistochemistry and laser capture microdissection combined with quantitative real-time PCR were used to determine the expression of Wnt5a and Toll-like receptors (TLRs) in different sections of the arterial lesions. Atherosclerotic serum samples (n = 30) and serum from healthy subjects (n = 16) were quantified for Wnt5a using an enzyme-linked immunosorbent assay (ELISA). RESULTS: The data analysis revealed that Wnt5a transcripts and protein were elevated in advanced arterial lesions relative to less advanced arterial lesions; that Wnt5a expression correlated with the presence of TLR4 and TLR2 transcripts; and that the average amount of Wnt5a protein present in atherosclerotic patient serum was significantly higher compared to healthy controls. CONCLUSIONS: This study is the first to provide evidence that the expression of Wnt5a increases as the disease progresses to a more advanced stage, and that this expression is coincident with that of TLR2 and TLR4. In addition, we found that the average Wnt5a levels in the serum of atherosclerotic patients are elevated relative to healthy controls, which is consistent with the hypothesis that Wnt5a plays a role in ATH.

Inhibition of LPS-induced TLR4 signaling products in murine macrophages by phenylmethimazole: an assay methodology for screening potential phenylmethimazole analogs.

Preclinical Research Phenylmethimazole (C10) is an inhibitor of Toll-like receptor (TLR3 and TLR4) expression and signaling. In this study, we carried out a detailed investigation of the effect of C10 on TLR4 and its molecular signaling products in RAW 264.7 macrophages using quantitative real-time polymerase chain reaction (PCR), ELISA and cell toxicity assays, a set of in vitro assays that may be used to screen future C10 analogs. C10 exhibited an inhibitory effect on TLR4 MyD88-dependent and MyD88-independent pathways. Within the TLR4 pathway, C10 inhibited the expression of cytokines, cytokine receptors, kinases, adapter molecules and transcription factors, suggesting a pathway-wide inhibitory effect. We also found that C10 dose-dependently inhibited the expression of TLR4 signaling products, specifically IL-6, inducible nitric oxide (NO) synthase and IFNß. Additionally, pre-treatment of RAW 264.7 cells with C10 resulted in protection from lipopolysaccharide (LPS) insults, suggesting C10 may be bound to the target thus exhibiting activity during/following LPS stimulation. Also, dimethyl sulfoxide, the solvent for C10 exhibited inhibitory effect on TLR4 signaling products independent from the effects of C10. Combined, this study enhances understanding of the actions of C10 on the TLR4 signaling pathway providing a path for the development of new C10 analogs for inhibiting TLR expression and signaling [corrected].

Capturing the Effects of Single Atom Substitutions on the Inhibition Efficiency of Glycogen Synthase Kinase-3ß Inhibitors via Markov State Modeling and Experiments.

Small modifications in the chemical structure of ligands are known to dramatically change their ability to inhibit the activity of a protein. Unraveling the mechanisms that govern these dramatic changes requires scrutinizing the dynamics of protein-ligand binding and unbinding at the atomic level. As an exemplary case, we have studied Glycogen Synthase Kinase-3ß (GSK-3ß), a multifunctional kinase that has been implicated in a host of pathological processes. As such, there is a keen interest in identifying ligands that inhibit GSK-3ß activity. One family of compounds that are highly selective and potent inhibitors of GSK-3ß is exemplified by a molecule termed COB-187. COB-187 consists of a five-member heterocyclic ring with a thione at C2, a pyridine substituted methyl at N3, and a hydroxyl and phenyl at C4. We have studied the inhibition of GSK-3ß by COB-187-related ligands that differ in a single heavy atom from each other (either in the location of nitrogen in their pyridine ring, or with the pyridine ring replaced by a phenyl ring), or in the length of the alkyl group joining the pyridine and the N3. The inhibition experiments show a large range of half-maximal inhibitory concentration (IC50) values from 10 nM to 10 µM, implying that these ligands exhibit vastly different propensities to inhibit GSK-3ß. To explain these differences, we perform Markov State Modeling (MSM) using fully atomistic simulations. Our MSM results are in excellent agreement with the experiments in that they accurately capture differences in the binding propensities of the ligands. The simulations show that the binding propensities are related to the ligands' ability to attain a compact conformation where their two aromatic rings are spatially close. We rationalize this result by sampling numerous binding and unbinding events via funnel metadynamics simulations, which show that indeed while approaching the bound state, the ligands prefer to be in their compact conformation. We find that the presence of nitrogen in the aromatic ring increases the probability of attaining the compact conformation. Protein-ligand binding is understood to be dictated by the energetics of interactions and entropic factors, like the release of bound water from the binding pockets. This work shows that changes in the conformational distribution of ligands due to atom-level modifications in the structure play an important role in protein-ligand binding.

Mechanistic insights into SARS-CoV-2 spike protein induction of the chemokine CXCL10.

During a SARS-CoV-2 infection, macrophages recognize viral components resulting in cytokine production. While this response fuels virus elimination, overexpression of cytokines can lead to severe COVID-19. Previous studies suggest that the spike protein (S) of SARS-CoV-2 can elicit cytokine production via the transcription factor NF-κB and the toll-like receptors (TLRs). In this study, we found that: (i) S and the S2 subunit induce CXCL10, a chemokine implicated in severe COVID-19, gene expression by human macrophage cells (THP-1); (ii) a glycogen synthase kinase-3 inhibitor attenuates this induction; (iii) S and S2 do not activate NF-κB but do activate the transcription factor IRF; (iv) S and S2 do not require TLR2 to elicit CXCL10 production or activate IRF; and (v) S and S2 elicit CXCL10 production by peripheral blood mononuclear cells (PBMCs). We also discovered that the cellular response, or lack thereof, to S and S2 is a function of the recombinant S and S2 used. While such a finding raises the possibility of confounding LPS contamination, we offer evidence that potential contaminating LPS does not underly induced increases in CXCL10. Combined, these results provide insights into the complex immune response to SARS-CoV-2 and suggest possible therapeutic targets for severe COVID-19.

Phenylmethimazole suppresses dsRNA-induced cytotoxicity and inflammatory cytokines in murine pancreatic beta cells and blocks viral acceleration of type 1 diabetes in NOD mice.

Accumulating evidence supports a role for viruses in the pathogenesis of type 1 diabetes mellitus (T1DM). Activation of dsRNA-sensing pathways by viral dsRNA induces the production of inflammatory cytokines and chemokines that trigger beta cell apoptosis, insulitis, and autoimmune-mediated beta cell destruction. This study was designed to evaluate and describe potential protective effects of phenylmethimazole (C10), a small molecule which blocks dsRNA-mediated signaling, on preventing dsRNA activation of beta cell apoptosis and the inflammatory pathways important in the pathogenesis of T1DM. We first investigated the biological effects of C10, on dsRNA-treated pancreatic beta cells in culture. Cell viability assays, quantitative real-time PCR, and ELISAs were utilized to evaluate the effects of C10 on dsRNA-induced beta cell cytotoxicity and cytokine/chemokine production in murine pancreatic beta cells in culture. We found that C10 significantly impairs dsRNA-induced beta cell cytotoxicity and up-regulation of cytokines and chemokines involved in the pathogenesis of T1DM, which prompted us to evaluate C10 effects on viral acceleration of T1DM in NOD mice. C10 significantly inhibited viral acceleration of T1DM in NOD mice. These findings demonstrate that C10 (1) possesses novel beta cell protective activity which may have potential clinical relevance in T1DM and (2) may be a useful tool in achieving a better understanding of the role that dsRNA-mediated responses play in the pathogenesis of T1DM.

Anti-Inflammatory and Therapeutic Effects of a Novel Small-Molecule Inhibitor of Inflammation in a Male C57BL/6J Mouse Model of Obesity-Induced NAFLD/MAFLD.

Purpose: Non-alcoholic fatty liver disease (NAFLD), recently renamed metabolic (dysfunction) associated fatty liver disease (MAFLD), is the most common chronic liver disease in the United States. Presently, there is an intense and ongoing effort to identify and develop novel therapeutics for this disease. In this study, we explored the anti-inflammatory activity of a new compound, termed IOI-214, and its therapeutic potential to ameliorate NAFLD/MAFLD in male C57BL/6J mice fed a high fat (HF) diet. Methods: Murine macrophages and hepatocytes in culture were treated with lipopolysaccharide (LPS) ± IOI-214 or DMSO (vehicle), and RT-qPCR analyses of inflammatory cytokine gene expression were used to assess IOI-214's anti-inflammatory properties in vitro. Male C57BL/6J mice were also placed on a HF diet and treated once daily with IOI-214 or DMSO for 16 weeks. Tissues were collected and analyzed to determine the effects of IOI-214 on HF diet-induced NAFL D/MAFLD. Measurements such as weight, blood glucose, serum cholesterol, liver/serum triglyceride, insulin, and glucose tolerance tests, ELISAs, metabolomics, Western blots, histology, gut microbiome, and serum LPS binding protein analyses were conducted. Results: IOI-214 inhibited LPS-induced inflammation in macrophages and hepatocytes in culture and abrogated HF diet-induced mesenteric fat accumulation, hepatic inflammation and steatosis/hepatocellular ballooning, as well as fasting hyperglycemia without affecting insulin resistance or fasting insulin, cholesterol or TG levels despite overall obesity in vivo in male C57BL/6J mice. IOI-214 also decreased systemic inflammation in vivo and improved gut microbiota dysbiosis and leaky gut. Conclusion: Combined, these data indicate that IOI-214 works at multiple levels in parallel to inhibit the inflammation that drives HF diet-induced NAFLD/MAFLD, suggesting that it may have therapeutic potential for NAFLD/MAFLD.

Phenylmethimazole inhibits production of proinflammatory mediators and is protective in an experimental model of endotoxic shock*.

BACKGROUND: One form of sepsis, or endotoxic shock, is a hyperactivated systemic response caused by excessive expression of proinflammatory mediators, which results from Gram-negative bacterial lipopolysaccharide-stimulated Toll-like receptor-4 signaling. This lipopolysaccharide signaling is known to consist of a MyD88-dependent nuclear factor-κB-mediated pathway that results in production of proinflammatory mediators (tumor necrosis factor-α, interleukin-6, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, inducible nitric oxide synthase, cyclooxygenase-2) and a MyD88-independent interferon regulatory factor-mediated pathway that regulates production of Type 1 interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1). In prior studies, phenylmethimazole markedly decreased virally induced Toll-like receptor-3 expression and signaling and significantly suppressed murine colitis in an experimental model wherein lipopolysaccharide is known to play an important role. OBJECTIVE: In this study, we probed the hypothesis that phenylmethimazole inhibits lipopolysaccharide-mediated Toll-like receptor-4 signaling and is efficacious in attenuating inflammatory changes and improving survival in an in vivo murine model of endotoxic shock. DESIGN: Experimental animal model. SETTING: University laboratory. SUBJECTS: Male C57BL/6J mice weighing 18-22 g. INTERVENTIONS: Phenylmethimazole (1 mg/kg) was administered intraperitoneally to mice before a lethal lipopolysaccharide challenge (25 mg/kg). RAW264.7 mouse macrophage cells were pretreated with phenylmethimazole followed by lipopolysaccharide stimulation. MEASUREMENTS AND MAIN RESULTS: : Macroscopic observations revealed that phenylmethimazole was significantly protective in controlling clinical manifestations of endotoxic shock and death under conditions wherein flunixin of meglumine and prednisolone were marginally effective. A combination of enzyme-linked immunosorbent assay, Northern blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blot analyses showed that phenylmethimazole attenuated lipopolysaccharide-induced increases in production of proinflammatory cytokines (tumor necrosis factor-α, interleukin-6, interferon-γ), endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), inducible nitric oxide synthase and cyclooxygenase-2, interferon regulatory factor-1, interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1), and signal transducer and activator of transcription-1 phosphorylation in multiple tissues in mice. Consistent with these observations, electrophoretic mobility shift assay demonstrated that phenylmethimazole inhibited in vitro lipopolysaccharide-induced nuclear factor-κB and interferon regulatory factor-1 activation in RAW 264.7 mouse macrophages. CONCLUSIONS: Collectively, these results provide direct evidence that phenylmethimazole diminishes lipopolysaccharide-induced MyD88-dependent as well as MyD88-independent signaling pathways and is protective in an experimental model of endotoxic shock.

Phenylmethimazole blocks dsRNA-induced IRF3 nuclear translocation and homodimerization.

Previous studies revealed that phenylmethimazole (C10) inhibits IRF3 signaling, preventing dsRNA-induction of type 1 interferon gene expression, production, and downstream signaling. In the present study, we investigated the molecular basis for C10 inhibition of dsRNA-stimulated IRF3 signaling. IRF-3 Trans-AM assays were used to measure C10 effects on dsRNA induction of IRF3 DNA binding. Green fluorescent protein-labeled IRF3 was used to measure C10 effects on dsRNA-induced IRF3 nuclear translocation. Native PAGE, SDS PAGE, and western blotting were used to identify effects of C10 on IRF3 homodimer formation and phosphorylation, respectively. There was a significant impairment of dsRNA-induced IRF3 DNA binding activity in human embryonic kidney and pancreatic cancer cells with C10 treatment. C10 also blocked dsRNA-induced IRF3 nuclear translocation and homodimer formation without blocking serine 396 phosphorylation of IRF3. Together, these results indicate that C10 interferes with IRF3 signaling by blocking dsRNA-induced IRF3 homodimer formation, a prerequisite for nuclear translocation and DNA binding activities.

Toll-Like Receptor 3 Is Critical to the Pancreatic Islet Milieu That Is Required for Coxsackievirus B4-Induced Type 1 Diabetes in Female Nonobese Diabetic Mice.

OBJECTIVE: Genetic and environmental influences play a role as triggers of type 1 diabetes mellitus (T1DM). Female nonobese diabetic (NOD) mice are useful for studying T1DM as they spontaneously develop T1DM, which can be accelerated by some viruses. Toll-like receptor 3 (TLR3) is believed to play a critical role in viral-induced T1DM and ß-cell destruction, because female Tlr3 knockout (Tlr3-/-) NOD mice are protected from Coxsackievirus B4 (CVB4)-induced acceleration of T1DM. However, the exact role(s) TLR3 plays in the pathogenesis of CVB4-induced T1DM remain unknown. METHODS: This longitudinal study used immunostaining, laser capture microdissection, and reverse transcription real-time polymerase chain reaction of islets from female uninfected and CVB4-infected Tlr3+/+ and Tlr3-/- NOD mice. RESULTS: Islets isolated from female Tlr3+/+ NOD mice 4 to 8 weeks of age had higher amounts of insulitis, Cxcl10, Il1b, Tnfa, and Tgfb1 expression compared with Tlr3-/- NOD mice. After CVB4 infection, Tlr3+/+ NOD mice had higher amounts of insulitis and T-cell infiltration at 3 days after infection compared with Tlr3-/- CVB4-infected NOD mice. CONCLUSIONS: Toll-like receptor 3 is necessary for establishment of a pancreatic islet inflammatory microenvironment by increasing insulitis and cytokine expression that facilitates CVB4-induced T1DM in female NOD mice.

Consequences of Both Coxsackievirus B4 and Type 1 Diabetes on Female Non-Obese Diabetic Mouse Kidneys.

Despite the 2019 Executive Order on Advancing American Kidney Health Initiative, kidney disease has moved up in rank from the 9th to the 8th leading cause of death in the United States. A recent push in the field of nephrology has been to identify molecular markers and/or molecular profiles involved in kidney disease process or injury that can help identify the cause of injury and predict patient outcomes. While these studies have had moderate success, they have not yet considered that many of the health conditions that cause kidney disease (diabetes, hypertension, etc.) can also be caused by environmental factors (such as viruses), which in and of themselves can cause kidney disease. Thus, the goal of this study was to identify molecular and phenotypic profiles that can differentiate kidney injury caused by diabetes (a health condition resulting in kidney disease) and coxsackievirus B4 (CVB4) exposure (which can cause diabetes and/or kidney disease), both alone and together. Non-obese diabetic (NOD) mice were used for this study due to their susceptibility to both type 1 diabetes (T1D)- and CVB4-mediated kidney injury, in order to glean a better understanding of how hyperglycemia and viral exposure, when occurring on their own and in combination, may alter the kidneys' molecular and phenotypic profiles. While no changes in kidney function were observed, molecular biomarkers of kidney injury were significantly up- and downregulated based on T1D and CVB4 exposure, both alone and together, but not in a predictable pattern. By combining individual biomarkers with function and phenotypic measurements (i.e., urinary albumin creatinine ratio, serum creatinine, kidney weight, and body weight), we were able to perform an unbiased separation of injury group based on the type of injury. This study provides evidence that unique kidney injury profiles within a kidney disease health condition are identifiable, and will help us to identify the causes of kidney injury in the future.

Modulation of LPS-induced inflammatory cytokine production by a novel glycogen synthase kinase-3 inhibitor.

Sepsis is a serious condition that can lead to long-term organ damage and death. At the molecular level, the hallmark of sepsis is the elevated expression of a multitude of potent cytokines, i.e. a cytokine storm. For sepsis involving gram-negative bacteria, macrophages recognize lipopolysaccharide (LPS) shed from the bacteria, activating Toll-like-receptor 4 (TLR4), and triggering a cytokine storm. Glycogen synthase kinase-3 (GSK-3) is a highly active kinase that has been implicated in LPS-induced cytokine production. Thus, compounds that inhibit GSK-3 could be potential therapeutics for sepsis. Our group has recently described a novel and highly selective inhibitor of GSK-3 termed COB-187. In the present study, using THP-1 macrophages, we evaluated the ability of COB-187 to attenuate LPS-induced cytokine production. We found that COB-187 significantly reduced, at the protein and mRNA levels, cytokines induced by LPS (e.g. IL-6, TNF-α, IL-1ß, CXCL10, and IFN-ß). Further, the data suggest that the inhibition could be due, at least in part, to COB-187 reducing NF-κB (p65/p50) DNA binding activity as well as reducing IRF-3 phosphorylation at Serine 396. Thus, COB-187 appears to be a potent inhibitor of the cytokine storm induced by LPS.

Coxsackievirus B4 Exposure Results in Variable Pattern Recognition Response in the Kidneys of Female Non-Obese Diabetic Mice Before Establishment of Diabetes.

End-stage renal disease (ESRD) is described by four primary diagnoses, diabetes, hypertension, glomerulonephritis, and cystic kidney disease, all of which have viruses implicated as causative agents. Enteroviruses, such as coxsackievirus (CV), are a common genus of viruses that have been implicated in both diabetes and cystic kidney disease; however, little is known about how CVs cause kidney injury and ESRD or predispose individuals with a genetic susceptibility to type 1 diabetes (T1D) to kidney injury. This study evaluated kidney injury resulting from coxsackievirus B4 (CVB4) inoculation of non-obese diabetic (NOD) mice to glean a better understanding of how viral exposure may predispose individuals with a genetic susceptibility to T1D to kidney injury. The objectives were to assess acute and chronic kidney damage in CVB4-inoculated NOD mice without diabetes. Results indicated the presence of CVB4 RNA in the kidney for at least 14 days post-CVB4 inoculation and a coordinated pattern recognition receptor response, but the absence of an immune response or cytotoxicity. CVB4-inoculated NOD mice also had a higher propensity to develop an increase in mesangial area 17 weeks post-CVB4 inoculation. These studies identified initial gene expression changes in the kidney resulting from CVB4 exposure that may predispose to ESRD. Thus, this study provides an initial characterization of kidney injury resulting from CVB4 inoculation of mice that are genetically susceptible to developing T1D that may one day provide better therapeutic options and predictive measures for patients who are at risk for developing kidney disease from T1D.

Evidence that knock down of GSK-3ß in Chronic Myelogenous Leukemia cells augments IFN-γ-induced apoptosis.

The role of interferon-gamma (IFN-γ) in Chronic Myelogenous/Myeloid Leukemia (CML) and in the treatment of CML remains unclear; specifically, the effect of IFN-γ on apoptosis. There is reported interplay between IFN-γ and glycogen synthase kinase-3 (GSK-3), a kinase which has been implicated in both cell death and, conversely, cell survival. Thus, we utilized the CML-derived HAP1 cell line and a mutant HAP1 GSK-3ß knocked-down cell line (GSK-3ß 31bp) to investigate whether GSK-3 modulates IFN-γ's action on CML cells. Significantly less GSK-3ß 31bp cells, relative to HAP1 cells, were present after 48 h treatment with IFN-γ. IFN-γ treatment significantly decreased GSK-3ß 31bp substrate adhesiveness (relative to HAP1 cells); an observation often correlated with cell death. Fluorescence microscopy revealed that IFN-γ induces a modest level of apoptosis in the HAP1 cells and that IFN-γ induced apoptosis is significantly enhanced in GSK-3ß 31bp cells. Utilizing a complementary GSK-3ß knocked-down cell line (8bp) we found, via flow cytometric analysis, that IFN-γ induced apoptosis is significantly enhanced in GSK-3ß 8bp cells relative to HAP1 cells. Combined, our findings suggest that IFN-γ induces apoptosis of CML cells and that loss of GSK-3ß significantly augments IFN-γ-induced apoptosis.

Phenylmethimazole abrogates diet-induced inflammation, glucose intolerance and NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of both metabolic and inflammatory diseases and has become the leading chronic liver disease worldwide. High-fat (HF) diets promote an increased uptake and storage of free fatty acids (FFAs) and triglycerides (TGs) in hepatocytes, which initiates steatosis and induces lipotoxicity, inflammation and insulin resistance. Activation and signaling of Toll-like receptor 4 (TLR4) by FFAs induces inflammation evident in NAFLD and insulin resistance. Currently, there are no effective treatments to specifically target inflammation associated with this disease. We have established the efficacy of phenylmethimazole (C10) to prevent lipopolysaccharide and palmitate-induced TLR4 signaling. Because TLR4 is a key mediator in pro-inflammatory responses, it is a potential therapeutic target for NAFLD. Here, we show that treatment with C10 inhibits HF diet-induced inflammation in both liver and mesenteric adipose tissue measured by a decrease in mRNA levels of pro-inflammatory cytokines. Additionally, C10 treatment improves glucose tolerance and hepatic steatosis despite the development of obesity due to HF diet feeding. Administration of C10 after 16 weeks of HF diet feeding reversed glucose intolerance, hepatic inflammation, and improved hepatic steatosis. Thus, our findings establish C10 as a potential therapeutic for the treatment of NAFLD.

High basal levels of functional toll-like receptor 3 (TLR3) and noncanonical Wnt5a are expressed in papillary thyroid cancer and are coordinately decreased by phenylmethimazole together with cell proliferation and migration.

High basal levels of TLR3 and Wnt5a RNA are present in papillary thyroid carcinoma (PTC) cell lines consistent with their overexpression and colocalization in PTC cells in vivo. This is not the case in thyrocytes from normal tissue and in follicular carcinoma (FC) or anaplastic carcinoma (AC) cells or tissues. The basally expressed TLR3 are functional in PTC cells as evidenced by the ability of double-strand RNA (polyinosine-polycytidylic acid) to significantly increase the activity of transfected NF-kappaB and IFN-beta luciferase reporter genes and the levels of two end products of TLR3 signaling, IFN-beta and CXCL10. Phenylmethimazole (C10), a drug that decreases TLR3 expression and signaling in FRTL-5 thyrocytes, decreases TLR3 levels and signaling in PTC cells in a concentration-dependent manner. C10 also decreased Wnt5a RNA levels coordinate with decreases in TLR3. E-cadherin RNA levels, whose suppression may be associated with high Wnt5a, increased with C10 treatment. C10 simultaneously decreased PTC proliferation and cell migration but had no effect on the growth and migration of FC, AC, or FRTL-5 cells. C10 decreases high basal phosphorylation of Tyr705 and Ser727 on Stat3 in PTC cells and inhibits IL-6-induced Stat3 phosphorylation. IL-6-induced Stat3 phosphorylation is important both in up-regulating Wnt5a levels and in cell growth. In sum, high Wnt5a levels in PTC cells may be related to high TLR3 levels and signaling; and the ability of phenylmethimazole (C10) to decrease growth and migration of PTC cells may be related to its suppressive effect on TLR3 and Wnt5a signaling, particularly Stat3 activation.

Overexpression of Wnt-1 in thyrocytes enhances cellular growth but suppresses transcription of the thyroperoxidase gene via different signaling mechanisms.

Wnt binding to cell surface receptors can activate a 'canonical' pathway that increases cellular beta-catenin or a 'noncanonical' Ca(++) pathway which can increase protein kinase C (PKC) activity. Although components of both Wnt/beta-catenin-signaling pathways exist in thyrocytes, their biological role is largely unknown. In evaluating the biological role of Wnt signaling in differentiated FRTL-5 thyroid cells, we showed that TSH increased canonical Wnt-1 but, surprisingly, decreased the active form of beta-catenin. Transient overexpression of Wnt-1 or beta-catenin in FRTL-5 cells increased active beta-catenin (ABC), decreased thyroperoxidase (TPO) mRNA, and suppressed TPO-promoter activity. The target of beta-catenin suppressive action was a consensus T cell factor/lymphoid enhancing factor (TCF/LEF)-binding site 5'-A/T A/T CAAAG-3', -137 to -129 bp on the rat TPO promoter. beta-Catenin overexpression significantly increased complex formation between beta-catenin/TCF-1 and an oligonucleotide containing the TCF/LEF sequence, suggesting that the beta-catenin/TCF-1 complex acts as a transcriptional repressor of the TPO gene. Stable over-expression of Wnt-1 in FRTL-5 cells significantly increased the growth rate without increasing beta-catenin levels. Increased growth was blunted by a PKC inhibitor, staurosporin. Wnt-1 overexpression increased serine phosphorylation, without affecting tyrosine phosphorylation, of signal transducers and activators of transcription 3 (STAT3) protein. In addition, these final results suggest that TSH-induced increase in Wnt-1 levels in thyrocytes contributes to enhanced cellular growth via a PKC pathway that increases STAT3 serine phosphorylation and activation, whereas TSH-induced decrease in activation of beta-catenin simultaneously relieves transcriptional suppression of TPO. We hypothesize that Wnt signaling contributes to the ability of TSH to simultaneously increase cell growth and functional, thyroid-specific, gene expression.