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Abstract
15-deoxy-delta12,14 prostaglandin J2 (15d-PGJ2) may hold promise in treatment of the pathologies associated with human immunodeficiency virus (HIV) infection of the central nervous system. However, its precise role and neuroprotective mechanism in the hippocampus remain poorly understood. In the present study, rat hippocampal slices were stimulated with HIV-1 Tat protein to investigate the protective role of 15d-PGJ2 on the hippocampal cytotoxicity. Full-length HIV-1 Tat protein (Tat1-86), but neither its Tat32-62 nor Tat30-86 fragment, significantly induced cytotoxicity in the hippocampus, the brain region most commonly damaged in HIV-associated dementia. This Tat-induced cytotoxicity was associated with inactivation of MEK/extracellular signal-regulated kinase (ERK) signaling pathway. In contrast, Tat1-86 did not alter Wnt signaling pathway necessary for cell survival. Pretreatment of slices with 15d-PGJ2 markedly reduced Tat-driven cytotxicity. Interestingly, this reduction was accompanied by suppression of ERK inactivation in response to Tat. Moreover, the inhibition of the MEK/ERK pathway with SL327 enhanced the Tat-induced cytotoxicity, confirming the ERK-dependent mechanism of Tat-driven cytotoxicity. Collectively, these data demonstrate that the protective action of 15d-PGJ2 against the hippocampal cytotoxicity upon Tat stimulation is exerted through suppression of Tat-mediated ERK1/2 inactivation.
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Figure 1.
Influences of Tat on cell toxicity of hippocampal slices. Hippocampal slices were exposed to 1 μM of Tat1-86, Tat fragments, or heat inactivated Tat1-86 (Tat1-86hi) as indicated for 3 days. MTT reduction activity in Tat-treated slices were assayed as described in materials and methods. Values are expressed as the means ± SEM of triplicate experiments. *, p < 0.05 versus vehicle-treated samples.
Figure 2.
Tat1-86 decreased the activation of ERK1/2 signaling pathway. The slices were treated for 3 days with Tat1-86 (1 μM) or vehicle only. (A) Immunoblots showing the Tat-induced inactivation of the ERK1/2 signaling pathway in hippocampal slices. Total lysates were analyzed via immunoblotting with phospho-antibody against the activated form of ERK1/2 or anti-total ERK1/2 evidencing decreased ERK1/2 phosphorylation. The blots were stripped and developed with anti-β-actin for equal protein loading. The data represent three independent experiments. (B) A normalized densitometric quantification of ERK1/2 activity against-β-actin for equal protein loading. The results of triplicate experiments are expressed as the means ± SEM. **, p < 0.01 versus vehicle-treated samples.
Figure 3.
Effects of Tat1-86 on the Wnt signaling pathway in the hippocampus. (A) Western blotting was used to measure the proteolysis of β-catenin and GSK-3β using total lysates of hippocampal slices treated for 3 days with Tat1-86 (1 μM) or vehicle only as described for Fig. 2. Representative western blots showed that β-catenin level was not changed after Tat treatment for 3 days. Similarly, levels of the active form of GSK-3β (phosphorylated GSK-3βtyr216) and non-phosphorylated GSK-3β were not altered. (B and C) Quantitative analysis of A, showing the levels of β-catenin (B) and GSK-3β (C) normalized to β-actin. Numbers represent the means ± SEM (n = 3). Representative gels from three experiments are shown.
Figure 4.
15d-PGJ2 attenuated Tat-dependent cytotoxicity in the hippocampus. Hippocampal slices were pretreated with 15d-PGJ2 (5 μM) and/or SL327 (10 μM) for 1 h and then stimulated with Tat1-86 (1 μM) for 3 days. MTT reduction activity in Tat-treated slices was measured as described in Fig. 1. Values are expressed as the means ± SEM (n=5). *, p < 0.05; **, p < 0.01 versus vehicle-treated samples; Z, p < 0.05 versus Tat-treated samples.
Figure 5.
15d-PGJ2 exerted its protective action against Tat-induced cytotoxicity through ERK1/2 activation. Hippocampal slices were pretreated with 15d-PGJ2 (5 μM) and/or SL327 (10 μM) for 1 h and then stimulated with Tat1-86 (1 μM) for 3 days. Total lysates were analyzed via immunoblotting as described in Fig. 2. (A) Western blots showed that 15d-PGJ2 suppressed Tat-induced inactivation of the ERK1/2 pathway in hippocampal slices. (B) A normalized densitometric quantification of ERK1/2 activity against β-actin for equal protein loading. The results of experiments are expressed as the means ± SEM (n= 5). **, p < 0.01 versus vehicle-treated samples; Z, p < 0.05 versus Tat-treated samples.
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