IRE1α deficiency impairs autophagy in chondrocytes by upregulating calcium homeostasis endoplasmic reticulum protein / 南方医科大学学报

OBJECTIVE@#To explore the mechanism by which inositol-requiring enzyme-1α (IRE1α) regulates autophagy function of chondrocytes through calcium homeostasis endoplasmic reticulum protein (CHERP).@*METHODS@#Cultured human chondrocytes (C28/I2 cells) were treated with tunicamycin, 4μ8c, rapamycin, or both 4μ8c and rapamycin, and the expressions of endoplasmic reticulum (ER) stress- and autophagy-related proteins were detected with Western blotting. Primary chondrocytes from ERN1 knockout (ERN1 CKO) mice and wild-type mice were examined for ATG5 and ATG7 mRNA expressions, IRE1α and p-IRE1α protein expressions, and intracellular calcium ion content using qPCR, Western blotting and flow cytometry. The effect of bafilomycin A1 treatment on LC3 Ⅱ/LC3 Ⅰ ratio in the isolated chondrocytes was assessed with Western blotting. Changes in autophagic flux of the chondrocytes in response to rapamycin treatment were detected using autophagy dual fluorescent virus. The changes in autophagy level in C28/I2 cells overexpressing CHERP and IRE1α were detected using immunofluorescence assay.@*RESULTS@#Tunicamycin treatment significantly up-regulated ER stress-related proteins and LC3 Ⅱ/LC3 Ⅰ ratio and down-regulated the expression of p62 in C28/I2 cells (P < 0.05). Rapamycin obviously up-regulated LC3 Ⅱ/LC3 Ⅰ ratio (P < 0.001) in C28/I2 cells, but this effect was significantly attenuated by co-treatment with 4μ8c (P < 0.05). Compared with the cells from the wild-type mice, the primary chondrocytes from ERN1 knockout mice showed significantly down-regulated mRNA levels of ERN1 (P < 0.01), ATG5 (P < 0.001) and ATG7 (P < 0.001), lowered or even lost expressions of IRE1α and p-IRE1α proteins (PP < 0.01), and increased expression of CHERP (P < 0.05) and intracellular calcium ion content (P < 0.001). Bafilomycin A1 treatment obviously increased LC3 Ⅱ/ LC3 Ⅰ ratio in the chondrocytes from both wild-type and ERN1 knockout mice (P < 0.01 or 0.05), but the increment was more obvious in the wild-type chondrocytes (P < 0.05). Treatment with autophagy dual-fluorescence virus resulted in a significantly greater fluorescence intensity of LC3-GFP in rapamycin-treated ERN1 CKO chondrocytes than in wild-type chondrocytes (P < 0.05). In C28/I2 cells, overexpression of CHERP obviously decreased the fluorescence intensity of LC3, and overexpression of IRE1α enhanced the fluorescence intensity and partially rescued the fluorescence reduction of LC3 caused by CHERP.@*CONCLUSION@#IRE1α deficiency impairs autophagy in chondrocytes by upregulating CHERP and increasing intracellular calcium ion content.

Tunicamycin-induced Endoplasmic Reticulum Stress Upregulates the Expression of Pentraxin 3 in Human Retinal Pigment Epithelial Cells

PURPOSE: To investigate the production of long pentraxin 3 (PTX3) in response to tunicamycin-induced endoplasmic reticulum (ER) stress and its role in ER stress-associated cell death, PTX3 expression was evaluated in the human retinal pigment epithelial cell line, ARPE-19. METHODS: PTX3 production in ARPE-19 cells was analyzed in the absence or presence of tunicamycin treatment by enzyme-linked immunosorbent assay. PTX3 protein and mRNA levels were estimated using western blot analysis and real-time reverse transcription-polymerase chain reaction, respectively. Protein and mRNA levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and ARPE-19 cell viability were measured in the presence of tunicamycin-induced ER stress in control or PTX3 small hairpin RNA (shRNA)-transfected ARPE-19 cells. RESULTS: The protein and mRNA levels of PTX3 were found to be significantly increased by tunicamycin treatment. PTX3 production was significantly decreased in inositol-requiring enzyme 1α shRNA-transfected ARPE-19 cells compared to control shRNA-transfected cells. Furthermore, pretreatment with the NF-κB inhibitor abolished tunicamycin-induced PTX3 production. Decreased cell viability and prolonged protein and mRNA expression of CHOP were observed under tunicamycin-induced ER stress in PTX3 shRNA transfected ARPE-19 cells. CONCLUSIONS: These results suggest that PTX3 production increased in the presence of tunicamycin-induced ER stress. Therefore, PTX3 could be an important protector of ER stress-induced cell death in human retinal pigment epithelial cells. Inositol-requiring enzyme 1α and the NF-κB signaling pathway may serve as potential targets for regulation of PTX3 expression in the retina. Therefore, their role in PTX3 expression needs to be further investigated.

Mecanismos celulares e moleculares envolvidos com alterações na expressão de glicanos durante a progressão do câncer colorretal / Cellular and molecular mechanisms involved with altered glycans expression colorectal cancer progression

O câncer colorretal representa uma das maiores causas de morbidade e mortalidade relacionadas ao câncer. No Brasil, é o terceiro tipo de câncer mais frequente em homens e mulheres. Muitos estudos estão sendo desenvolvidos no sentido de esclarecer os diversos aspectos moleculares que regulam as alterações fenotípicas exibidas pelas células que constituem o câncer colorretal, no entanto, comparativamente, ainda são poucos os que são dedicados a investigar o papel de modificações co- e pós-traducionais neste processo. Entre os vários tipos destas modificações que ocorrem em proteínas, a glicosilação é a mais comum. Cogita-se que aproximadamente cinquenta por cento de todas as proteínas são glicosiladas. Durante a transformação maligna, mudanças no perfil de expressão de glicanos (carboidratos covalentemente ligados a proteínas ou lipídios) estão envolvidas em uma variedade de mecanismos celulares, tais como: perda da adesão célula-célula e célula matriz, migração, invasão e evasão da apoptose. Neste estudo, foi investigada a atividade antitumoral de inibidores da biossíntese de N-glicanos, swainsonina e tunicamicina, em células derivadas de câncer colorretal (Caco-2, HCT-116 e HT-29). Os resultados obtidos mostram que o tratamento das células HCT-116 com tunicamicina inibe mecanismos celulares relacionados ao fenótipo maligno, como formação de colônia dependente e independente de ancoragem, migração e invasão. Estes resultados sugerem que modulação da biossíntese de N-glicanos parece ser uma potencial ferramenta terapêutica para o tratamento do câncer colorretal. Em outra etapa do trabalho, foram avaliados também o impacto da estimulação com insulina e IGF-1 na expressão N-glicanos bissectados em células tumorais MDA-MB-435. Os resultados obtidos confirmaram também a existência de uma relação entre a estimulação dos receptores de insulina e IGF-1 e a regulação da expressão de N-glicanos bissectados em células tumorais MDA-MB-435, fornecendo assim informações ...

Colorectal cancer is a major cause of cancer-related morbidity and mortality. In Brazil, it is the third most common cancer. Many studies have been developed to clarify several molecular features which regulate phenotypic changes exhibited by cells that constitute colorectal cancer, however, comparatively, there are few studies dedicated to investigate co- and post-translational modifications of proteins in this process. Glycosylation is the most common post-translational modification of proteins. Approximately fifty percent of all proteins are glycosylated. During malignant transformation, changes in the expression profile of glycans (carbohydrates covalently bound to proteins or lipids) may be involved in a variety of events, including the loss of cell–cell and cell–matrix adhesion, migration, invasion, and evasion of apoptosis. In this study, we investigated the in vitro anticancer activity of the N-glycan biosynthesis inhibitors, swainsonine and tunicamycin, in cells derived from colorectal cancer (Caco-2, HCT-116 e HT-29). Our results show that treatment with tunicamycin inhibits cellular mechanisms related to the malignant phenotype, such as anchorage-dependent and anchorage-independent colony formation, migration and invasion, in HCT-116 colon cancer cells. Given these results, we suggest that the modulation of N-glycan biosynthesis appears to be a potential therapeutic tool for CRC treatment. Moreover, we confirmthe existence of an interplay between stimulation with insulin and IGF-1 and bisecting GlcNAc N-glycans expression in MDA-MB435 cancer cells, providing new insights into the role that Insulin/IGF-I signaling play during carcinoma progression

Tunicamycin enhances TRAIL-induced apoptosis by inhibition of cyclin D1 and the subsequent downregulation of survivin

TNF-related apoptosis-inducing ligand (TRAIL) has been proposed as a promising cancer therapy that preferentially induces apoptosis in cancer cells, but not most normal tissues. However, many cancers are resistant to TRAIL by mechanisms that are poorly understood. In this study, we showed that tunicamycin, a naturally occurring antibiotic, was a potent enhancer of TRAIL-induced apoptosis through downregulation of survivin. The tunicamycin-mediated sensitization to TRAIL was efficiently reduced by forced expression of survivin, suggesting that the sensitization was mediated at least in part through inhibition of survivin expression. Tunicamycin also repressed expression of cyclin D1, a cell cycle regulator commonly overexpressed in thyroid carcinoma. Furthermore, silencing cyclin D1 by RNA interference reduced survivin expression and sensitized thyroid cancer cells to TRAIL; in contrast, forced expression of cyclin D1 attenuated tunicamycin-potentiated TRAIL-induced apoptosis via over-riding downregulation of survivin. Collectively, our results demonstrated that tunicamycin promoted TRAIL-induced apoptosis, at least in part, by inhibiting the expression of cyclin D1 and subsequent survivin. Of note, tunicamycin did not sensitize the differentiated thyroid epithelial cells to TRAIL-induced apoptosis. Thus, combined treatment with tunicamycin and TRAIL may offer an attractive strategy for safely treating resistant thyroid cancers.

Tunicamycin enhances TRAIL-induced apoptosis by inhibition of cyclin D1 and the subsequent downregulation of survivin

TNF-related apoptosis-inducing ligand (TRAIL) has been proposed as a promising cancer therapy that preferentially induces apoptosis in cancer cells, but not most normal tissues. However, many cancers are resistant to TRAIL by mechanisms that are poorly understood. In this study, we showed that tunicamycin, a naturally occurring antibiotic, was a potent enhancer of TRAIL-induced apoptosis through downregulation of survivin. The tunicamycin-mediated sensitization to TRAIL was efficiently reduced by forced expression of survivin, suggesting that the sensitization was mediated at least in part through inhibition of survivin expression. Tunicamycin also repressed expression of cyclin D1, a cell cycle regulator commonly overexpressed in thyroid carcinoma. Furthermore, silencing cyclin D1 by RNA interference reduced survivin expression and sensitized thyroid cancer cells to TRAIL; in contrast, forced expression of cyclin D1 attenuated tunicamycin-potentiated TRAIL-induced apoptosis via over-riding downregulation of survivin. Collectively, our results demonstrated that tunicamycin promoted TRAIL-induced apoptosis, at least in part, by inhibiting the expression of cyclin D1 and subsequent survivin. Of note, tunicamycin did not sensitize the differentiated thyroid epithelial cells to TRAIL-induced apoptosis. Thus, combined treatment with tunicamycin and TRAIL may offer an attractive strategy for safely treating resistant thyroid cancers.

Characterization of the Recombinant Proteins of Porcine Circovirus Type2 Field Isolate Expressed in the Baculovirus System

Porcine circovirus (PCV) type2 was isolated using primary porcine kidney cells from lymph node of piglets with typical PMWS. The presence of the virus was identified by PCR using primers specific to PCV type2. The ORFs 1 and 2 were amplified by PCR using primers corresponding to the target genes of the PCV type 2. Cloned genes were inserted into the baculovirus expression vector and PCV recombinant proteins were expressed using baculovirus expression system. Recombinant protein expression was determined by indirect immunofluorescent assay (IFA) and immunoblotting using polyclonal antiserum to PCV. ORF1 gene expressed two proteins with approximately 17 kDa and 31 kDa proteins in the baculovirus system. Recombinant protein of the ORF2 was similar to that of the native virus except minor bands with different molecular weight were detected. Recombinant protein expressed in the baculovirus system showed at least two glycosylation sites based on the tunicamycin treatment. Recombinant protein of the ORF2 assembled virus-like particle in recombinant virus infected insect cells.

Evolution of cell-surface acid phosphatase of Burkholderia pseudomallei.

Acid phosphatase active fractions were obtained from cell-free extract, outermembrane fraction and culture filtrate of Burkholderia pseudomallei by column chromatography with sepharose 6B and DEAE cellulose. The comparison of the elution patterns of protein, sugar and enzymatic activity among these three components suggested that the enzyme is a glycoprotein evolving from premature proteins through glycosylation and that the enzyme is translocated during glycosylation from the cytoplasm to the outer membrane and finally excreted into the environment. When tunicamycin, a glycosylation inhibitor, was added to the culture, the peaks of sugar and enzymatic activity were lowered concomitantly leaving the protein peak unchanged in the elution pattern of the culture filtrate. The affinity of the bacterial surface to antienzyme sera was demonstrated by immuno-fluorescence microscopy.

Kinetics of translation and glycosylation of pituitary glycoproteins

The present study determines the basal kinectics of synthesis of translation (by[(14)C] leucine incorporation. [(14)C] leu-PROT] and of proximal (by [(3)H] mannose incorparation. [(3)H] man-PROT) and distal (by [3H] galactose incorporation, [(3)H] gal-PROT) glycosylation of total adenohypophyseal glycoproteins, by rat pituitary cells in primary culture. In order to obtain more information regarding the role of both septs of glycosylation on the secretory process, the effects of cycloheximide (CH; translation inhibitor) and tunicamycin (TM; glycosylation inhibitor) on the kinetics of synthesis and release of pituitary glycoproteins were also studied. Cells were incubated in medium containing [(14)C] leu plus [(3)H] man or [(14)C] leu plus [(3)H] gal, for various time-intervals (from 0.5 to 5h) in the abscence (control) or presence of different doses od CH (1.0;4.0 or 16.0 mug/ml) or TM (0.5;1.0 or 2.0 mug/ml). The kinetics of synthesis (slope=3488) and release (slope=622) of [(14)C] le-PROT were higler than those of the sugar percursos (slopes: [(3)H] man-PROT=1751 and 526; [(3)H] gal-PROT=1231 and 506). Leucine or mannose-labeled protein was barely detectable in the medium after 2h incubation, whereas galactose-labeled protein had already been released into the incubation medium by 30min. Cycloheximide induced translation blockage and, concomitantly, produced a marked inhibition of [(3)H] man incorporation. On the other hand, TM inhibited the kinetics of synthesis and release of [(3)H] man-PROT without affecting those of [(14)C] leu-PROT. The kinetics of synthesis and release of [(3)H] gal-PROT, although diminished, maintained linearity and increased in function of time, even in the presence of the antibiotics. Thus, the present results on glycoproteins from the pituitary gland are consistent with the previous conclusion for other mammalian glycoproteins that carbohydrate attachment occurs in several steps to molecules destined to be secreted. Addition of mannose (proximal glycosylation) is a co-translational event and that of galactose (distal glycosylation) is post-translational and can be designated as final stages in carbohydrate assembly, occurring close to the time of release. Furthermore, it has been demonstrated that the absence of the carbohydrate side chains of the pituitary glycoprotein does not prevent the intracellular transport of the protein and its export from the cell.

Effects of tunicamycin on the pH-activity pattern of acid phosphatase in Pseudomonas pseudomallei.

The liquid culture of Pseudomonas pseudomallei shows a complex feature in in the pH-activity pattern of acid phosphatase, not a single peak curve. There was an evident tendency that the higher activity shifted to the higher pH range with the growth of culture. The culture in the presence of tunicamycin (20 micrograms/ml) showed a decreased activity selectively in the higher pH range, while the activity in the lower pH was more heat-labile. The bacterial cells grown on agar plates containing tunicamycin were more heat-labile than the untreated control cells. The glucosidase-treatment reduced the enzymatic activity (of the phosphatase-active fractions from the living cells) with the shift of the optimum pH to lower pH. These observations together with some collateral findings suggest that the pH-activity pattern of acid phosphatase in P. pseudomallei is associated with the development of precursor enzyme proteins to mature glycoproteins.