n-(4-methoxybenzyl)-n--(5-nitro-1-3-thiazol-2-yl)urea and 6-bromoindirubin-3--oxime

Either glycogen synthase kinase (GSK)-3β or nuclear factor (NF)-κB regulates interferon (IFN)-γ-induced nitric oxide (NO) biosynthesis; however, the inter-regulation between GSK-3β and NF-κB is unknown. We have previously shown that IFN-γ-activated GSK-3β negatively regulates Src homology-2 domain-containing phosphatase (SHP) 2 to facilitate Janus kinase (Jak) 2-signal transducer and activator of transcription (STAT) 1 activation. Because Jaks-IFN-inducible dsRNA-activated serine-threonine protein kinase (PKR) axis signaling is essential for IFN-γ-activation of NF-κB, in this study we investigate the potential mechanism for GSK-3β-facilitated NF-κB activation in IFN-γ-stimulated RAW264.7 murine macrophages. Pharmacological inhibitors of GSK-3β or NF-κB signaling, such as the inhibitor of κB (IκB) kinase β (IKKβ) and IκBα, inhibited IFN-γ-induced inducible NO synthase (iNOS) and thus NO biosynthesis. Inhibiting GSK-3β decreased IFN-γ-induced NF-κB phosphorylation (Ser536) and activation. The upstream regulators for GSK-3β activation, including okadaic acid-sensitive protein phosphatase and proline-rich tyrosine kinase 2, were also important for IFN-γ-induced IκBα phosphorylation (Ser32) and degradation. Under IFN-γ stimulation, Jak2-PKR axis signaling induced IκBα inactivation as well as iNOS/NO biosynthesis. It is notable that inhibiting GSK-3β caused SHP2-mediated dephosphorylation of PKR (Thr446), IKKβ (Ser180), and NF-κB (Ser536). Taken together, we provide the first evidence to demonstrate that GSK-3β indirectly facilitates IFN-γ-induced NF-κB activation by inhibiting SHP2, in turn activating the PKR-IKKβ-IκBα axis signaling pathway.

Topics: Animals; Blotting, Western; Cell Line; Electrophoretic Mobility Shift Assay; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Indoles; Interferon-gamma; Mice; NF-kappa B; Nitric Oxide; Oximes; RNA Interference; Signal Transduction; Thiazoles; Urea

Glycogen synthase kinase-3beta (GSK3beta) role in human immunodeficiency virus(HIV)-associated neurodegeneration has been evidenced by previous investigations. In this study, we investigated the specificity of two GSK3beta-specific inhibitors, AR-A014418 (A) and B6B30 (B) to prevent direct neurotoxicity in primary human neurons exposed to HIV (BaL). Neurons were exposed to HIV (500 pg/ml) for 12-h and 6-day periods in the presence and absence of A (1 microM, 100 nM, 10 nM) and B (50 nM, 5 nM, 500 pM) to investigate acute and ongoing mechanisms of HIV neurotoxicity. Using an lactate dehydrogenase (LDH) assay to assess cytotoxicity, we observed a significant neurotoxic effect of HIV from control values (P < .01) that was not restored via coexposures of all concentrations of A and B. Additionally, no change in LDH levels were observed after 6 days. However, activity of the acute proapoptotic markers caspases 3 and 7 using a luminescence assay were measured and found to be increased by exposure to HIV (BaL) compared to controls (P = .022). This effect was ameliorated via coexposure to all concentrations of A and 50 nM B after 12 h (P < .01) and to all concentrations of A and B after 6 days (P < .01). Overall, the results from this study provide further evidence for the ability of GSK3beta inhibition to be neuroprotective against HIV-associated neurotoxicity by reducing HIV associated procaspase induction. These data support a role for GSK3beta as a potential therapeutic target and may have important clinical implications for treatment of HIV-associated neurocognitive disorder.

Topics: AIDS Dementia Complex; Caspase 3; Caspase 7; Cells, Cultured; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Indoles; L-Lactate Dehydrogenase; Macrophages; Necrosis; Nerve Degeneration; Neurons; Oximes; Thiazoles; Urea