The Schizophrenia-Related Protein Dysbindin-1A Is Degraded and Facilitates NF-Kappa B Activity in the Nucleus

<div><p>Dystrobrevin-binding protein 1 (<i>DTNBP1</i>), a gene encoding dysbindin-1, has been identified as a susceptibility gene for schizophrenia. Functioning with partners in synapses or the cytoplasm, this gene regulates neurite outgrowth and neurotransmitter release. Loss of dysbindin-1 affects schizophrenia pathology. Dysbindin-1 is also found in the nucleus, however, the characteristics of dysbindin in the nucleus are not fully understood. Here, we found that dysbindin-1A is degraded in the nucleus via the ubiquitin-proteasome system and that amino acids 2-41 at the N-terminus are required for this process. By interacting with p65, dysbindin-1A promotes the transcriptional activity of NF-kappa B in the nucleus and positively regulates MMP-9 expression. Taken together, the data obtained in this study demonstrate that dysbindin-1A protein levels are highly regulated in the nucleus and that dysbindin-1A regulates transcription factor NF-kappa B activity to promote the expression of MMP-9 and TNF-α.</p></div

Dysbindin-1A up-regulates the MMP-9 expression.

<p>(A) NF-kappa B target gene products were examined in dysbindin-1A knockdown cells. Dysbindin-1A was silenced in N2a cells using si-dys2#, and mRNA levels of NF-kappa B downstream genes were examined using real-time PCR. The values shown represent the means ± S.E. of three independent groups. *, <i>P</i><0.05; one-way ANOVA. (B) p65 overexpression restores gene expression resulting from dysbindin-1A deficiency. EGFP or EGFP-p65 was co-transfected with si-control or si-dys 2# into N2a cells. Forty-eight hours later, MMP-9 mRNA levels were detected using real-time PCR. The values shown represent the means ± S.E. (error bars) of three independent groups. **, <i>P</i><0.01; EGFP/si-control vs. EGFP/si-dys; one-way ANOVA. ##, <i>p</i><0.01; EGFP/si-dys vs. EGFP-p65/si-dys; one-way ANOVA. (C) NF-kappa B targeting gene products were examined in dysbindin-1A overexpressed cells. EGFP, dysbindin-1A-EGFP, dysbindin-1A-NLS-EGFP were transfected into N2a cells, the NF-kappa B downstream genes were examined using real-time PCR. *, <i>P</i><0.05, **, <i>P</i><0.01; EGFP vs. dysbindin-1A-NLS-EGFP; one-way ANOVA.</p

EGFP-NLS-dysbindin-1A del 2–41 is stable in nucleus.

<p>(A) The degradation of EGFP-dysbindin-1A del 2–41 or EGFP-NLS-dysbindin-1A del 2–41. (B) The quantitative analysis of (A). The values shown represent the means ± S.E. of three independent experiments. (C) The C-terminus of dysbindin-1A was not ubiquitinated in nucleus. The deletion mutants of dysbindin were transfected into cells. The ubiquitination of deletion mutants were examined.</p

The N-terminal 2–41 amino acids of dysbindin-1A are important for its nuclear degradation.

<p>(A) The N-terminal sequence of dysbindin-1A (amino acids 1–42). The sequence was scored using Ubpred and BDM-PUB, and potential ubiquitination sites are colored red. (B) The potential dysbindin-1A ubiquitination site, lysine 21, was mutated to arginine. The ubiquitination of K21R and WT forms of dysbindin-1A was examined. (C) Dysbindin-1A-EGFP, EGFP-dysbindin (residues 1–189) or EGFP-dysbindin-1A residue 2–41 deletion mutant were co-transfected with HA-Ub into HEK293 cells, respectively. After culturing for 24 hours, 10 μM MG132 was added, and the cells were then cultured for an additional 12 hours. After lysis, the proteins were immunoprecipitated using an anti-GFP antibody and immunoblotted with an HA antibody. (D) The EGFP-dysbindin-1A residue 2–41 deletion mutant was more stable in the nucleus. HEK293 cells expressing dysbindin-1A-EGFP or EGFP-dysbindin-1A residue 2–41 deletion mutant were treated with leptomycin B (20 ng/ml) for 1 hour. The cells were then treated with CHX for the indicated time. Finally, cell extracts were subjected to immunoblot analysis. (E) The band intensity of the dysbindin-1A-EGFP and EGFP-dysbindin-1A residue 2–41 deletion mutant is shown, relative to GAPDH. The values shown represent the means ± S.E. of three independent experiments. *, <i>P</i><0.05; one-way ANOVA. (F) The localization of EGFP-dysbindin-1A del 2–41 under treatment of Ethanol or LMB. The nuclei were stained with DAPI; The bar represents 10 μm.</p

Dysbindin-1A is degraded in the nucleus.

<p>(A) HEK293 cells that had been transfected with dysbindin-1A-EGFP were pre-treated with leptomycin B 20 ng/ml or equal volumes of ethanol for 1 hour, respectively. The cells were then treated with CHX for the indicated time, and cell extracts were subjected to immunoblot analysis. (B) The band intensity of dysbindin-1A-EGFP relative to GAPDH is shown. The values shown represent the means ± S.E. of three independent experiments. *, <i>P</i><0.05; **, <i>P</i><0.01; one-way ANOVA. (C) Subcellular localization of dysbindin-1A-EGFP and its variants that harbor a nuclear localization signal and/or a nuclear export signal mutant. HEK293 cells were transfected with the indicated plasmids, and the nuclei were stained with DAPI; The bar represents 10 μm. (D) The half-life of dysbindin-1A-NLS or the NES mutant was shorter than that of wild type dysbindin-1A. Dysbindin-1A-EGFP, dysbindin-1A-NLS-EGFP and dysbindin-1A-NES mutant-NLS-EGFP were transfected into HEK293 cells for 24 hours; the cells were then treated with CHX (100 μg/ml) for the indicated time. (E) The data from three independent experiments of (D) were quantified. The values shown represent means ± S.E. (F) The HEK293 cells were pre-treated with leptomycin B 20 ng/ml or equal volumes of ethanol for 1 hour, respectively. The cells were then treated with CHX for the indicated time, and cell extracts were subjected to immunoblot analysis. (G) The quantified analysis from three independent experiments of (F). The values shown represent the means ± S.E. *, <i>P</i><0.05; one-way ANOVA.</p

Dysbindin-1A is degraded via the ubiquitin-proteasome pathway in the nucleus.

<p>(A) Nuclear dysbindin-1A was degraded via the ubiquitin-proteasome pathway. Twenty-four hours after transfection with dysbindin-1A-EGFP, dysbindin-1A-NLS-EGFP and dysbindin-1A-NES mutant-NLS-EGFP, cells were treated with DMSO or MG132 (10 μM) for 12 hours, respectively. (B and C) Dysbindin-1A-NLS-EGFP and dysbindin-1A-NES mutant-NLS-EGFP were transfected into HEK293 cells for 24 hours. The cells were then treated with MG132 (10 μM) for 12 hours. Cell lysates were immunoprecipitated with GFP antibody and immunoblotted with ubiquitin antibody.</p

Dysbindin-1A interacts with Rel A (p65) and promotes NF-kappa B activity.

<p>(A) A dual luciferase reporter assay shows that dysbindin-1A promotes NF-kappa B transcriptional activity. (B) Dysbindin-1A knockdown does not influence the protein levels of p65 in N2a cells. N2a cells were transfected with si-dys1# or si-dys2#. The resulting cell lysates were subjected to immunoblot analysis using antibodies to dysbindin-1A or p65. (C) p65 interacts with Flag-dysbindin-1A. In HEK293 cells that were co-transfected with Flag-dysbindin-1A and EGFP-p65, EGFP or EGFP-p65 was immunoprecipitated using an anti-GFP antibody. The immunoprecipitants were subjected to immunoblot analysis with the indicated antibodies. (D) Dysbindin-1A interacts with p65 in a GST-pulldown assay. (E) Endogenous dysbindin-1A interacts with endogenous p65 in SH-SY5Y cells. (F) EGFP-p65 interacts with Flag-dysbindin-1A in nucleus. The cytoplasm fraction or nuclear fraction of HEK293 cells transfected with EGFP/EGFP-p65 and Flag-dysbindin-1A were immunoprecipitated using an anti-GFP antibody. The immunoprecipitants were subjected to immunoblot analysis with the indicated antibodies. <i>IB</i>, immunoblot; <i>IP</i>, immunoprecipitation.</p

Biomaterial-engineered intra-articular drug delivery systems for osteoarthritis therapy

Osteoarthritis (OA) is a progressive and degenerative disease, which is no longer confined to the elderly. So far, current treatments are limited to symptom relief, and no valid OA disease-modifying drugs are available. Additionally, OA relative joint is challenging for drug delivery, since the drugs experience rapid clearance in joint, showing a poor bioavailability. Existing therapeutic drugs, like non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids, are not conducive for long-term use due to adverse effects. Though supplementations, including chondroitin sulfate and glucosamine, have shown beneficial effects on joint tissues in OA, their therapeutic use is still debatable. New emerging agents, like Kartogenin (KGN) and Interleukin-1 receptor antagonist (IL-1 ra), without a proper formulation, still will not work. Therefore, it is urgent to establish a suitable and efficient drug delivery system for OA therapy. In this review, we pay attention to various types of drug delivery systems and potential therapeutic drugs that may escalate OA treatments.</p

Additional file 1 of Targeting of G-protein coupled receptor 40 alleviates airway hyperresponsiveness through RhoA/ROCK1 signaling pathway in obese asthmatic mice

Additional file 1: Fig. S1. Effects of GPR40 inhibition on airway inflammation in asthmatic mice. (A) The number of total inflammatory cells in BALFs were calculated, and a minimum of 200 cells were employed to classify eosinophils (A), Neutrophils (B), macrophages (C) macrophages (D) and lymphocytes (E) after the last OVA challenge. BALFs were harvested to measure IL-4 (F), IL-13 (G) and IL-8 (H) release by ELISA. The data are expressed as the mean ± S.E.M (n=6). *P<0.05, **P<0.01 and ***P<0.001 compared with the control group, #P<0.05, ##P<0.01 and ###P<0.001 compared with the OVA group. Fig. S2. Mouse lung tissues were collected for the extraction of protein. The expression of GPR40 was measured by western blot. The data are expressed as the mean ± S.E.M (n=4). **P<0.01 compared with the control group, ##P<0.01 compared with the OVA model group