Journal: The Journal of Neuroscience

Article Title: Astrocytic GAP43 Induced by the TLR4/NF-κB/STAT3 Axis Attenuates Astrogliosis-Mediated Microglial Activation and Neurotoxicity

doi: 10.1523/JNEUROSCI.3457-15.2016

Figure Lengend Snippet: LPS-induced astrocytic GAP43 expression was mediated by both NF-κB and STAT3. A, Quantitative RT-PCR analysis showed Gap43 mRNA expression at different time points in LPS (50 ng/ml)-treated primary rat HB astrocytes. B, The increase of Gap43 mRNA expression after LPS treatment (50 ng/ml, 16 h) was inhibited by the anti-inflammatory glucocorticoid MP (1 μm), NF-κB inhibitor PTN (10 μm), and JAK inhibitor AG490 (10 μm), respectively). n = 3. Note that the control group (CTL) was treated with 0.1% DMSO, the vehicle for MP, PTN, and AG490. C, Schematic representation of rat Gap43 (rGAP43) promoter fragment. The predicted NF-κB and STATx binding sites locate in promoter region at a nearby TATA box. The binding activities of NF-κB and STAT3 in this promoter region were examined by ChIP assay with the indicated primers. A luciferase reporter DNA construct of rat Gap43 promoter fragment (rGap43 promoter-Luc) was prepared for promoter activity determination. D, ChIP assay of NF-κB binding on rGap43 promoter by using p65/RelA antibody to precipitate the protein–DNA complex in HB astrocytes treated with 60 min 0.1% DMSO (CTL) or LPS (50 ng/ml) with or without 90 min pretreatment with PTN (10 μm). E, ChIP assay of STAT3 and p65/RelA binding on rGap43 promoter in HB astrocytes treated with LPS at 0, 30, and 60 min. The PCR primers (−633 to −414 bp; C) were used for the DNA fragment amplification by semi-quantitative PCR. The band intensity of the gel images in D and E was quantified and normalized to the input control to obtain the enrichment of PCR products ratio to the CTL or the 0 min values (bottom panel in D and E). n = 3. F, Luciferase reporter assay of LPS (50 ng/ml)-treated HB astrocytes transfected with rGap43 promoter-Luc. The increased luciferase activity after 24 h of LPS treatment was completely blocked by the AG490 (10 μm) pretreatment. n = 6. G, The effects of siRNA-mediated STAT3 knockdown (siSTAT3) on LPS-induced GAP43 expression were determined by Western blotting. n = 3. H, ELISA of IL-6 release at different time point of LPS (50 ng/ml)-treated cultured rat astrocytes. The concentrations of IL-6 released in the culture medium were reported in picograms per milliliter. n = 3. I, The effects of siRNA-mediated IL-6 knockdown (si-IL6) on IL6 and Gap43 mRNA expression at 16 h after the LPS treatment were determined by qRT-PCR. n = 3.

Article Snippet: For the NF-κB pathway inhibitor peptide (NBD), TAT-NBD (ygrkkrrqrr–TALD W S W LQTE; Nijboer et al., 2008 ) and its mutant peptide TAT-NBD mut (ygrkkrrqrr–TALD A S A LQTE) were synthesized by Kelowna International Scientific.

Techniques: Expressing, Quantitative RT-PCR, Control, Binding Assay, Luciferase, Construct, Activity Assay, Amplification, Real-time Polymerase Chain Reaction, Reporter Assay, Transfection, Knockdown, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: The Journal of Neuroscience

Article Title: Astrocytic GAP43 Induced by the TLR4/NF-κB/STAT3 Axis Attenuates Astrogliosis-Mediated Microglial Activation and Neurotoxicity

doi: 10.1523/JNEUROSCI.3457-15.2016

Figure Lengend Snippet: NF-κB mediated LPS-induced GAP43 expression in the astrocytic processes. A, Western blotting of GAP43 in primary HB astrocytes treated with vehicle of TAT-peptide (CTL; 0.1% DMSO) or LPS (50 ng/ml) for 24 h with the pretreatment of 20 μm TAT-NF-κB inhibitory peptide (TAT-NBD) or its mutant control, TAT-NBDmut, for 2 h. Lamin A was used as the internal control. The bar graphs showed the quantification of band intensity normalized by the respective internal control. n = 3 in each group. B, Immunofluorescent double staining of GAP43 and GFAP in LPS-treated HB astrocytes with TAT-NBDmut or TAT-NBD pretreatments under the same condition as in A. C, D, Imaging analysis and quantification of the average number of astrocytic processes per cell (C) and the percentage of GAP43 signals in the GFAP-positive astrocytic process [GAP43 intensity/(GAP43+GFAP) intensity × 100%] (D) obtained from B. The number of microscopic images counted in each group was as indicated. E, F, Cell viability (WST-1 assay; E) and damage (LDH release assay; F) of the LPS-treated astrocytes with or without TAT-NBDmut or TAT-NBD pretreatments. n = 3.

Article Snippet: For the NF-κB pathway inhibitor peptide (NBD), TAT-NBD (ygrkkrrqrr–TALD W S W LQTE; Nijboer et al., 2008 ) and its mutant peptide TAT-NBD mut (ygrkkrrqrr–TALD A S A LQTE) were synthesized by Kelowna International Scientific.

Techniques: Expressing, Western Blot, Mutagenesis, Control, Double Staining, Imaging, WST-1 Assay, Lactate Dehydrogenase Assay

Journal: The Journal of Neuroscience

Article Title: Astrocytic GAP43 Induced by the TLR4/NF-κB/STAT3 Axis Attenuates Astrogliosis-Mediated Microglial Activation and Neurotoxicity

doi: 10.1523/JNEUROSCI.3457-15.2016

Figure Lengend Snippet: A proposed model of astrocytic GAP43 functions in mediating astrocytic plasticity; and regulating microglial activation, axonal plasticity, and neuronal survival. In astrocytes, inflammatory stimuli that activate TLR4 trigger both NF-κB and STAT3 signaling pathways and lead to their binding onto the Gap43 gene promoter. NF-κB also mediates the expression and release of IL-6 to activate IL-6 receptor (IL-6R), which then activates a second-phase JAK/STAT3 signaling to give rise to the upregulation of astrocytic GAP43 expression. Astrocytic GAP43, especially its S41-phosphorylated form that promotes actin polymerization, functions to mediate morphological plasticity, including process arborization and elongation in stellate, not flat-shaped, astrocytes during astrogliosis. Furthermore, astrocytic GAP43 may attenuate the release of proinflammatory cytokines (IL-6 and TNF-α), thereby dampening the activation and proinflammatory response in microglia. Astrocytic GAP43 can also facilitate axonal plasticity by attenuating the inhibitory effect of reactivated astrocytes on axon growth and neuronal GAP43 phosphorylation possibly involved the regulation of astrocyte-derived inhibitory factors (In.F.). Finally, an actin-dependent MKL1 activation that contributes to the GAP43-mediated transcriptional activation of EAAT2 was revealed, which increases the glutamate uptake activity and may lead to reduction of excessive glutamate to attenuate the astrogliosis-induced neurotoxicity.

Article Snippet: For the NF-κB pathway inhibitor peptide (NBD), TAT-NBD (ygrkkrrqrr–TALD W S W LQTE; Nijboer et al., 2008 ) and its mutant peptide TAT-NBD mut (ygrkkrrqrr–TALD A S A LQTE) were synthesized by Kelowna International Scientific.

Techniques: Activation Assay, Protein-Protein interactions, Binding Assay, Expressing, Phospho-proteomics, Derivative Assay, Activity Assay

Journal: The Journal of investigative dermatology

Article Title: MiR-146a negatively regulates TLR2-induced inflammatory responses in keratinocytes.

doi: 10.1038/jid.2014.89

Figure Lengend Snippet: Figure 5. MiR-146a downregulates IRAK1 and TRAF6 in keratinocytes. Keratinocytes were transfected with miR-146a precursor (pre-miR-146a), scrambled control precursor (pre-miR-Ctrl), miR-146a inhibitor (anti-miR-146a), or scrambled control inhibitor (anti-miR-Ctrl) for 72hours, and the expression of IRAK1 mRNA and protein was determined by (a) qRT–PCR and (b) western blotting, respectively. Densitometry quantification is normalized to actin. (c) Keratinocytes were co-transfected with miR-146a inhibitor (anti-miR-146a) or scrambled control inhibitor (anti-miR-Ctrl) and with IRAK1-specific siRNA (siRNA IRAK1) or scrambled control siRNA (siRNA Ctrl) for 48hours, and were subsequently treated with zymosan for 3 hours. *Po0.05; ***Po0.001. (d) Schematic summary of the regulation and function of miR-146a in keratinocytes. IRAK1, interleukin-1 receptor-associated kinase 1; mRNA, messenger RNA; qRT–PCR, quantitative real-time reverse-transcriptase–PCR; siRNA, small interfering RNA; TRAF6, TNF receptor associated factor 6.

Article Snippet: As IRAK1 and TRAF6 are important parts of the signaling cascade between TLRs and the NF-kB pathway (Takeda and Akira, 2004), we hypothesized that the observed effect of miR-146a could be mediated by repression of IRAK1 and TRAF6 in keratinocytes.

Techniques: Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Reverse Transcription, Small Interfering RNA