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Reactive astrogliosis is a prominent neuropathologic feature of manganism, a neurodegenerative disorder caused by excessive accumulation of manganese (Mn) in the basal ganglia. Activation of astrocytes has been linked to neuronal injury in manganism resulting from overproduction of inflammatory mediators, including tumor necrosis factor-alpha (TNFalpha), interferon-gamma (IFNgamma), interleukin-1beta (IL-1beta), and nitric oxide (NO), but the signaling mechanisms by which Mn regulates these factors remain poorly understood. We previously reported that Mn enhances production of NO in activated astrocytes that promotes apoptosis in cocultured neuronal cells by a mechanism involving the transcription factor nuclear factor-kappaB (NF-kappaB) (Liu et al., 2005). Because NF-kappaB-dependent expression of inducible nitric oxide synthase (NOS2) can be antagonized by the nuclear orphan receptor peroxisome proliferator-activated receptor-gamma (PPARgamma), we postulated that a novel agonist of this receptor, 1,1-bis(3'-indolyl)-1-(p-trifluoromethylphenyl)methane (cDIM1), would suppress expression of NOS2 in astrocytes and protect cocultured neuronal cells from apoptosis. Submicromolar concentrations of cDIM1 potently suppressed production of NO and expression of NOS2 in cultured astrocytes exposed to Mn and IFNgamma/TNFalpha and prevented apoptosis in cocultures of differentiated PC12 cells, but this neuroprotective effect was lost in the absence of astrocytes. By using fluorescence reporter and chromatin immunoprecipitation (ChIP) assays, we found that cDIM1 prevented activation of NF-kappaB in astrocytes by a mechanism involving stabilization of the nuclear corepressor 2 (NCoR2) on the proximal NF-kappaB binding site of the NOS2 promoter. These data suggest that PPARgamma may be an effective target for limiting inflammatory activation of astrocytes during neurologic injury., ((c) 2007 Wiley-Liss, Inc.)

Neuronal injury in manganism is accompanied by activation of astroglia within the basal ganglia that is thought to increase production of inflammatory mediators such as nitric oxide (NO). The present studies postulated that astroglial-derived NO mediates neuronal apoptosis induced by manganese (Mn) and pro-inflammatory cytokines. Pheochromocytoma (PC12) cells differentiated with nerve growth factor (NGF) were co-cultured with primary astrocytes and exposed to Mn and tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma). Mn enhanced cytokine-induced expression of inducible nitric oxide synthase (NOS2, EC 1.14.13.39) and production of NO in astrocytes that correlated with apoptosis in co-cultured neurons, as determined by caspase activity, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), and nuclear morphology. Apoptosis in PC12 neurons required the presence of astrocytes and was blocked by overexpression of a phosphorylation-deficient mutant of IkappaBalpha (S32/36A) in astrocytes that prevented induction of NOS2. Pharmacologic inhibition of NOS2 with (+/-)-2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) significantly reduced neuronal apoptosis, and the addition of low concentrations of the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), to neurons cultured without astrocytes was sufficient to recover the apoptotic phenotype following exposure to Mn and TNF-alpha/IFN-gamma. It is concluded that Mn- and cytokine-dependent apoptosis in PC12 neurons requires astroglial-derived NO and NF-kappaB-dependent expression of NOS2.

Objective To study the effect of down-regulation of long non-coding RNA (lncRNA) HOXA cluster antisense RNA 2 (HOXA-AS2)/miR-20a pathway on apoptosis and invasion of skin squamous cell carcinoma cells in vitro. Methods qRT-PCR was used to detect the expression of HOXA-AS2 and miR-20a in 50 clinical specimens of skin squamous cell carcinoma and paired adjacent tissues. Skin squamous carcinoma cell line A431 was transfected with HOXA-AS2 siRNA, and the changes in cell proliferation, clonogenic ability, apoptosis, migration and invasion were evaluated by using MTT assay, clone formation experiment, flow cytometry, and Transwell assay respectively. Western blotting was performed to detect the expression of MMP-2, MMP-9, and cleaved caspase-3 proteins in the cells. Bioinformatics software predicted that HOXA-AS2 and miR-20a had complementary binding sites, and a luciferase reporter system was employed to verify their targeting relationship. A431 cells were co-transfected with HOXA-AS2 siRNA and miR-20a inhibitor, and the changes in cell proliferation, clone formation, apoptosis, migration and invasion were assessed. Results The expression level of HOXA-AS2 was increased in skin squamous cell carcinoma tissues. Transfection of A431 cells with HOXA-AS2 siRNA significantly suppressed the cell proliferation, clone formation, invasion and migration, promoted cell apoptosis, decreased the expression of MMP-2 and MMP-9 proteins, and increased the expression of cleaved caspase-3 protein. Down-regulation of HOXA-AS2 significantly up-regulated miR-20a expression. The expression level of miR-20a in skin squamous cell carcinoma tissues was negatively correlated with that of HOXA-AS2. In A431 cells, miR-20a inhibitor obviously reversed the effects of HOXA-AS2 siRNA on proliferation, clone formation, invasion, migration and apoptosis of the cells. Conclusion Down-regulation of HOXA-AS2/miR-20a pathway can promote the expression of miR-20a, suppress the proliferation, clone formation, invasion and migration, and induce apoptosis of skin squamous cell carcinoma cells in vitro.