Acceleration of inflammatory responses by ER stress.

<p>(A and B) RT-PCR analysis of (A) <i>Bip</i> and (B) <i>Chop</i> in LS174T human colon carcinoma cells treated with ER stress inducer tunicamycin (Tm) and TUDCA for 24 h (<i>n</i> = 4). The expression levels of both <i>Bip</i> and <i>Chop</i> were downregulated by TUDCA. (C–E) RT-PCR analysis of (C) <i>Tnfα</i>, (D) <i>IL-1</i>, and (E) <i>IL-6</i> in LS174T cells treated with Tm and TUDCA (<i>n</i> = 4). Note that the expression levels of these inflammatory cytokines were upregulated in LS174T cells treated with Tm, and downregulated by treatment with TUDCA. (F) Luciferase assay using LS174T cells transfected with the p-Luc reporter plasmid containing the NF-κB binding sequence. Relative activities were increased by treatment of LS174T cells with Tm, and decreased by treatment with TUDCA (<i>n</i> = 4). Values represent the means ± s.d. *<i>P</i><0.05; **P<0.01; ***<i>P</i><0.001.</p

Increased Susceptibility to Dextran Sulfate Sodium-Induced Colitis in the Endoplasmic Reticulum Stress Transducer OASIS Deficient Mice

<div><p>OASIS is a basic leucine zipper (bZIP) transmembrane transcription factor that is activated in response to endoplasmic reticulum (ER) stress. Previously, we showed that OASIS regulates final maturation of goblet cells in the large intestine. In the present study, to elucidate the roles of OASIS under pathophysiological conditions, we examined the stress response and inflammatory responses in <i>Oasis</i> deficient (<i>Oasis</i>−/−) mice exposed to dextran sulfate sodium (DSS) to induce colitis. A significant loss of body weight and an increase of mortality were observed in <i>Oasis</i>−/− mice with DSS-induced colitis compared with those in WT mice. The mucosa of the large intestine in <i>Oasis</i>−/− mice exhibited severe damage involving inflammatory cell infiltration. The expression levels of ER stress and apoptosis markers in intestinal epithelial cells were upregulated in <i>Oasis</i>−/− mice. These abnormalities were improved by treatment with tauroursodeoxycholic acid, a chemical chaperone that facilitates protein folding. Taken together, our findings demonstrate that OASIS plays important roles in protection of the large intestinal mucosa in DSS-induced colitis through attenuation of ER stress and inflammation.</p></div

ER stress is accelerated in <i>Oasis</i>−/− mice with DSS-induced colitis.

<p>(A) <i>In situ</i> hybridization of ER stress markers <i>Bip</i> (upper panels) and <i>Chop</i> (lower panels) in the large intestinal mucosa of WT and <i>Oasis</i>−/− mice exposed to 3.5% DSS for 5 days. In WT mice, both ER stress markers were mainly observed in the basal crypt. In contrast, these markers were expressed in both the basal and apical crypts of <i>Oasis</i>−/− mice. (B) The number of <i>Bip</i>- and <i>Chop</i>-positive cells per crypt in (A) (<i>n</i> = 5). The number of cells positive for each ER stress marker was increased by about 1.5-fold in the <i>Oasis</i>−/− large intestinal mucosa compared with that in WT mice. (C) RT-PCR analysis of ER stress markers <i>Bip</i> and <i>Chop</i> in the large intestine of WT and <i>Oasis</i>−/− mice exposed to 3.5% DSS for 5 days (n = 5). (D) Western blotting of cleaved caspase-12 and -3 in the large intestinal mucosa of WT and <i>Oasis</i>−/− mice exposed to 3.5% DSS for 5 days. (E) TUNEL staining of the large intestinal mucosa in WT and <i>Oasis</i>−/− mice exposed to 3.5% DSS for 5 days. (F) The number of TUNEL-positive cells per 10 crypts in (E) (n = 4). The number of TUNEL-positive cells was increased in the apical portion of the mucosa in <i>Oasis</i>−/− mice. Values represent the means ± s.d. *<i>P</i><0.05.</p

Decreased number of mature goblet cells in the large intestine of adult <i>Oasis</i>−/− mice.

<p>(A) Peptide features of mouse OASIS and ATF6. (B) RT-PCR analysis of <i>Oasis</i> mRNA in various tissues from 8-week-old mice. <i>Oasis</i> mRNA was highly expressed in the large intestine. (C) Western blotting of OASIS in the large intestine from 8-week-old mice. Both full-length OASIS (full OASIS) and the N-terminal OASIS (N-OASIS) were detected in the large intestine. (D) HE (upper panels) and PAS (lower panels) staining of the large intestines from 8-week-old WT and <i>Oasis</i>−/− mice. In the <i>Oasis</i>−/− large intestine, the number of mature goblet cells containing large mucosal granules was markedly decreased compared with that in WT mice. (E and F) Quantification of (E) PAS-positive cells and (F) areas of PAS-positive cells in (D) (<i>n</i> = 9). Values represent the means ± s.d. ***<i>P</i><0.001.</p

TUDCA alleviates DSS-induced colitis.

<p>All the <i>Oasis</i>−/− mice received 3.5% DSS and some were given TUDCA (+TUDCA) and others were given the same volume of PBS (vehicle) daily by oral administration for 5 days. (A) HE (upper panels) and PAS (lower panels) staining of the large intestinal mucosa of <i>Oasis</i>−/− mice exposed to 3.5% DSS and TUDCA or the vehicle. (B) Higher magnification of HE staining in (A). Arrowheads show inflammatory cells. (C) Histological scores of control and TUDCA-treated <i>Oasis</i>−/− mice that received 3.5% DSS (<i>n</i> = 4). The pathological findings were markedly improved in <i>Oasis</i>−/− mice treated with TUDCA. (D) RT-PCR analysis of <i>Bip</i> and <i>Chop</i> in the large intestinal mucosa of <i>Oasis</i>−/− mice. The expression levels of these ER stress markers in the large intestinal mucosa of <i>Oasis</i>−/− mice were decreased by treatment with TUDCA. (E) Quantification of the expression levels of <i>Bip</i> and <i>Chop</i> in (D) (<i>n</i> = 4). (F) RT-PCR analysis of inflammatory cytokines in the large intestinal mucosa of <i>Oasis</i>−/− mice exposed to 3.5% DSS and TUDCA or the vehicle for 5 days (<i>n</i> = 4). Note that the expression levels of inflammatory cytokines were decreased in <i>Oasis</i>−/− mice that received TUDCA. (G) Western blotting of cleaved caspase-12 and -3 in <i>Oasis</i>−/− large intestinal mucosa exposed to 3.5% DSS and TUDCA or the vehicle for 5 days. (H) TUNEL staining of the large intestinal mucosa in <i>Oasis</i>−/− mice that received 3.5% DSS and TUDCA or the vehicle. (I) The number of TUNEL-positive cells in (H) (n = 4). The number of TUNEL-positive cells was decreased in <i>Oasis</i>−/− mice treated with TUDCA. Values represent the means ± s.d. *<i>P</i><0.05; **<i>P</i><0.01.</p

Sec16A, a key protein in COPII vesicle formation, regulates the stability and localization of the novel ubiquitin ligase RNF183

<div><p>We identified 37 ubiquitin ligases containing RING-finger and transmembrane domains. Of these, we found that RNF183 is abundantly expressed in the kidney. RNF183 predominantly localizes to the endoplasmic reticulum (ER), Golgi, and lysosome. We identified Sec16A, which is involved in coat protein complex II vesicle formation, as an RNF183-interacting protein. RNF183 colocalized with Sec16A and interacted through the central conserved domain (CCD) of Sec16A. Although Sec16A is not a substrate for RNF183, RNF183 was more rapidly degraded by the ER-associated degradation (ERAD) in the absence of Sec16A. Sec16A also stabilized the interacting ubiquitin ligase RNF152, which localizes to the lysosome and has structural similarity with RNF183. These results suggest that Sec16A appears to regulate the protein stability and localization of lysosomal ubiquitin ligases.</p></div

Transcriptional Regulation of VEGFA by the Endoplasmic Reticulum Stress Transducer OASIS in ARPE-19 Cells

<div><h3>Background</h3><p>Vascular endothelial growth factor-A (VEGFA) is the main mediator of angiogenesis. Angiogenesis plays important roles not only in many physiological processes, but also in the pathophysiology of many diseases. VEGFA is one of the therapeutic targets of treatment for ocular diseases with neovascularization. Therefore, elucidation of the regulatory mechanisms for VEGFA expression is important for the development of pharmaceutical drugs. Recent studies have demonstrated that the unfolded protein response is involved in the transcriptional regulation of VEGFA. However, the precise regulation of VEGFA in the human retina is not fully understood.</p> <h3>Principal Findings</h3><p>When human retinal pigment epithelial cells, ARPE-19, were exposed to endoplasmic reticulum stressors, VEGFA mRNA was significantly upregulated. The unfolded protein response-related transcription factors XBP1, ATF4, ATF6, and OASIS were expressed in ARPE-19 cells. To determine which transcription factors preferentially contribute to the induction of VEGFA expression after endoplasmic reticulum stress, we carried out reporter assays using an approximately 6-kbp 5′-upstream region of the human VEGFA gene. Among these transcription factors, OASIS acted most effectively on the VEGFA promoter in ARPE-19 cells. Based on data obtained for certain deleted and mutated reporter constructs, we determined that OASIS promoted VEGFA expression by acting on a cyclic AMP-responsive element-like site located at around –500 bp relative to the VEGFA transcription start site. Furthermore, we confirmed that OASIS directly bound to the promoter region containing this site by chromatin immunoprecipitation assays.</p> <h3>Conclusions and Significance</h3><p>We have demonstrated a novel regulatory mechanism for VEGFA transcription by OASIS in human retinal pigment epithelial cells. Chemical compounds that regulate the binding of OASIS to the promoter region of the VEGFA gene may have potential as therapeutic agents for ocular diseases with neovascularization.</p> </div

Effects of Sec16 on RNF183 subcellular localization.

<p>(A) Effect of Sec16A downregulation on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, 5th panels) or Sec16A (2nd, 4th, 6th panels) siRNAs. At 48 h after transfection, cells were subjected to immunofluorescence staining with anti-V5 (<i>green</i>) and anti-calnexin, GM130, or LAMP1 (<i>red</i>) antibodies, and DAPI (<i>blue</i>). (B) Effect of proteasome inhibition on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC (<i>top</i> panels) or Sec16A (<i>middle</i> and <i>bottom</i> panels) siRNAs. At 36 h after transfection, cells were incubated with (<i>bottom</i> panels) or without (<i>top</i> and <i>middle</i> panels) 10 μM MG132 for 12 h.</p

Proteomics analysis of the RNF183 interacting protein.

<p>Proteomics analysis of the RNF183 interacting protein.</p

Interaction and colocalization of RNF183 and Sec16A.

<p>(A) Interaction of RNF183 and Sec16A. Cell lysates from HEK293 cells stably expressing V5-tagged RNF183 were immunoprecipitated using anti-V5 antibody, and the immune complexes were analyzed by Western blotting with anti-Sec16A (<i>upper</i> panel) or anti-V5 (<i>lower</i> panel) antibodies. (B) Colocalization of RNF183 with Sec16A. HeLa cells stably transfected with RNF183-V5 were subjected to immunofluorescence staining with anti-Sec16A (<i>green</i>) and DAPI (<i>blue</i>). (C) Effect of RNF183 knockdown on Sec16A protein. RNF183 expression in HEK293 cells stably transfected with RNF183-V5 was suppressed using siRNA. Endogenous Sec16A protein levels were detected using Western blotting with anti-Sec16A antibody. NC, negative control. (D) Schematic diagram of the predicted domains of Sec16A. (E) Interaction domain of Sec16A with RNF183. Lysates from HEK293 cells stably expressing RNF183 transiently transfected with GFP-tagged full-length Sec16A (Full) or its deletion mutant constructs were subjected to immunoprecipitation with anti-V5 antibody, followed by immunoblotting with anti-GFP antibody.</p