(B) Phosphorylated and total c-Jun was detected by western blotting in whole cell extracts. binding analysis and luciferase reporter assay. Rabbit Polyclonal to TNF14 Protein interaction was examined by coimmunoprecipitation. == Results == Inhibition of GSK-3 by selective GSK-3 inhibitors or by RNA interference attenuated LPS-induced TNF- production in cultured microglia. Exploration of the mechanisms by which GSK-3 positively regulates inflammatory response showed that LPS-induced IB- degradation, NF-Bp65 nuclear translocation, and p65 DNA binding activity were not affected by inhibition of GSK-3 activity. However, GSK-3 inactivation inhibited transactivation activity of p65 by deacetylating p65 at lysine 310. Furthermore, we also demonstrated a functional interaction between mixed lineage kinase 3 (MLK3) and GSK-3 during LPS-induced TNF- production in microglia. The phosphorylated levels of HLCL-61 MLK3, MKK4, and JNK were increased upon LPS treatment. Decreasing GSK-3 activity blocked MLK3 signaling cascades through disruption of MLK3 dimerization-induced autophosphorylation, ultimately leading to a decrease in TNF- secretion. == Conclusion == These results suggest that inactivation of GSK-3 might represent a potential strategy to downregulate microglia-mediated inflammatory processes. == Background == Inflammatory processes, involving a host of cytokines, have been shown to be associated with ongoing neuronal degeneration in several neurodegenerative diseases. Activation of HLCL-61 glial cells such as microglia and astrocytes is a characteristic finding in brain inflammation. Microglia, as the immunocompetent resident cells of the brain, possess properties particularly suitable for mediating cellular inflammatory responses. The secretion of pro-inflammatory and neurotoxic mediators from activated microglia is believed to contribute to progressive damage in neurodegenerative disorders [1-3]. Therefore, deciphering the mechanisms that govern inflammation caused by microglial activation and its effects on brain are vital for understanding the pathogenesis of these diseases. Glycogen synthase kinase 3 (GSK-3) is a multifunctional serine/threonine kinase found in all eukaryotes. There are two highly homologous mammalian isoforms of GSK-3, GSK-3 and GSK-3. GSK-3 is a key regulator of numerous signaling pathways, and is involved in a wide range of cellular processes ranging from glycogen metabolism to the regulation of cell survival and neuronal polarity [4,5]. Furthermore, the function of GSK-3 in signaling mechanisms that activate nuclear factor B (NF-B), as well as the resulting effects on NF-B-mediated gene expression, indicate that GSK-3 acts as a regulator of inflammation [6-8]. Although an effect of GSK-3 in modulation of inflammation has been identified, the potential role and mechanism for this effect are still controversial. Inhibition of GSK-3 by pharmacological inhibitors or by overexpression of a dominant negative mutant of GSK-3 enhances tumor necrosis factor- (TNF-) expression in lipopolysaccharide- (LPS-)stimulated cardiomyocytes [9]. Another report has implicated GSK-3 in inhibition of TNF– and interleukin- (IL-)1-induced inflammatory gene expression [10]. Conversely, the identification of GSK-3 as a major regulator of peripheral inflammatory responses has shown that GSK-3 promotes the stimulus-induced HLCL-61 production of several cytokines and the subsequent development of disease symptoms in animal models of inflammatory conditions [11]. Recently, GSK-3 inactivation has been shown to downregulate the inflammatory response induced by microglial activation [12,13]. However, the molecular mechanisms of downstream signal transduction leading to this anti-inflammatory effect of GSK-3 inhibition in microglia are not yet clearly understood. TNF- is a pro-inflammatory cytokine that is upregulated in the brain in response to various insults or injury. Activated microglia around an injured area have been shown to be the major source of this cytokine. Within the brain, inflammatory processes might be modulated by TNF- through further activation of microglia and astrocytes [14,15]. TNF- is known to induce generation of reactive oxygen intermediates associated with necrotic cell death, and it also induces changes in mitochondrial ultrastructure and function [16,17]. In addition, TNF- also directly induces neuronal death by binding to TNF receptor 1 to trigger intracellular death-related signaling pathways [18]. Increased TNF- production is seen in several neurodegenerative diseases and may contribute to secondary damage that further worsens a disease state [19-22]. For example, in Parkinson’s disease (PD), significant increases in the expression of TNF- and its receptors have been reported in the caudate and putamen of postmortem brain samples from patients with PD [20]. HLCL-61 Several studies have demonstrated that blocking soluble TNF signaling attenuates loss of dopaminergic neurons in cellular and.
