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Objective Nuclear protein 1 (Nupr1) is a stress-inducible protein that is involved in gene transcription. The present study was undertaken to determine whether chondrocytes express Nupr1 and whether Nupr1 regulates matrix metalloproteinase 13 (MMP-13) expression. Methods Paraffin-embedded cartilage sections from normal human and osteoarthritic (OA) cartilage were immunostained using anti-Nupr1 antibody. To measure Nupr1 expression, total RNA was isolated from joint tissue obtained 8 weeks after surgery from young (12-week-old) and older (12-month-old) mice that underwent destabilization of the medial meniscus (DMM) to induce OA. Human chondrocytes were stimulated with 1-10 ng/ml interleukin-1β (IL-1β), 25 μ M tert-butyl-hydroperoxide (tBHP), or 2 μ M thapsigargin, and Nupr1 expression was analyzed by quantitative polymerase chain reaction. In addition, chondrocytes were transfected with small interfering RNA to knock down Nupr1 expression and then stimulated overnight with IL-1β. After incubation, the conditioned medium was collected and MMP levels measured. Results Increased Nupr1 immunostaining was noted in human OA cartilage compared to normal cartilage. Expression was also increased in joint tissue from 12-month-old mice that underwent DMM surgery compared to sham-operated controls. Stimulation of chondrocytes with IL-1β induced a 2-fold increase in Nupr1 messenger RNA (mRNA) within 1 hour, with the increase peaking to 4-fold at 6 hours. Treatment of chondrocytes with tBHP to induce oxidative stress increased Nupr1 mRNA expression by >2-fold; treatment with thapsigargin to induce endoplasmic reticulum stress did not produce a similar effect. Knockdown of Nupr1 inhibited IL-1β-mediated induction of MMP-13. Conclusion Nupr1 is expressed in cartilage, and its levels are increased in OA. Nupr1 expression is required for IL-1β-mediated expression of MMP-13. These findings provide evidence of a novel pathway for regulation of IL-1β-mediated production of MMPs in chondrocytes. [ABSTRACT FROM AUTHOR]

Pathways leading to osteoarthritis (OA) are diverse depending on the risk factors involved; thus, developing OA therapeutics has been challenging. Here we report that nuclear protein‐1 (Nupr1), a stress‐inducible protein/transcription factor, is activated by pathways associated with obesity and aging in chondrocytes. Treatment of human chondrocytes with free fatty acids (palmitate and oleate; a model for high‐fat diet/obesity) induced PERK signaling and increased expression of caspase‐3, TRB3, and Nupr1. On the other hand, treatment of chondrocytes with menadione (oxidative stress inducer) induced oxidation of IRE1, activated antioxidant response (higher Nrf2 expression), and increased expression of Nupr1 and matrix metalloproteinases. Experimental OA was induced by destabilization of the medial meniscus (DMM) in the knee joints of Nupr1+/+ and Nupr1−/− mice. Loss of Nupr1 expression reduced the severity of cartilage lesions in this model. Together, our findings suggest that Nupr1 is a common factor activated by signaling pathways activated by obesity (ER stress) and age (oxidative stress) and a potential drug target for OA resulting from various risk factors. [ABSTRACT FROM AUTHOR]

Cells that are metabolically active and in a high degree of differentiation and proliferation require cobalamin (Cbl: vitamin B12) and they obtain it from the circulation bound to transcobalamin (TC) via the transcobalamin receptor (TC-R). This study has investigated the plasma membrane dynamics of TC-R expression in polarized human intestinal epithelial Caco-2 cells using techniques of pulse-chase labelling, domain-specific biotinylation and cell fractionation. Endogenously synthesized TC-R turned over with a half-life ( T1/2) of 8 h following its delivery to the basolateral plasma membrane (BLM). The T1/2 of BLM delivery was 15 min and TC-R delivered to the BLM was endocytosed and subsequently degraded by leupeptin-sensitive proteases. However, about 15% of TC-R endocytosed from the BLM was transcytosed ( T1/2, 45 min) to the apical membranes (BBM) where it underwent endocytosis and was degraded. TC-R delivery to both BLM and BBM was inhibited by Brefeldin A and tunicamycin, but not by wortmannin or leupeptin. Colchicine inhibited TC-R delivery to BBM, but not BLM. At steady state, apical TC-R was associated with megalin and both these proteins were enriched in an intracellular compartment which also contained Rab5 and transferrin receptor. These results indicate that following rapid delivery to both plasma membrane domains of Caco-2 cells, TC-R undergoes constitutive endocytosis and degradation by leupeptin-sensitive proteases. TC-R expressed in apical BBM complexes with megalin during its transcytosis from the BLM. [ABSTRACT FROM AUTHOR]

Transcobalamin (TC) has been cloned and used for studying its gene expression in the rat. IC mRNA is distributed widely in adult rat tissues, but at different levels (kidney > liver > lung > yolk sac > intestine > heart > brain > spleen > muscle). IC mRNA levels were 4-fold higher in the jejunum and ileum compared to its levels in the duodenum. During postnatal development, TC mRNA levels in the ileum declined 4-fold from day 4 to day 12, but increased by 5-fold between days 12 and 24. In contrast, IC mRNA levels increased by 2.5-fold in the kidney from day 4 to day 12 and then declined by 2-fold by day 24. Adrenalectomy of adult rats resulted in a 4-fold decline in ileal levels of TC mRNA and a 50% decline in the ileal mucosal formation of the TC-[57Co] cobalamin (Cbl) complex following oral administration of [57Co]Cbl complexed to gastric intrinsic factor (IF). Cortisone treatment reversed these changes noted in the ileum. In contrast to ileum, kidney TC mRNA levels were not altered significantly in adrenalectomized rats before and after cortisone treatment. Taken together, this study has provided evidence for the regulation of IC gene expression in the rat kidney and intestine during their postnatal development, and cortisone selectively regulates deal but not kidney IC mRNA levels. [ABSTRACT FROM AUTHOR]

Mice fed a high‐fat diet (HFD) become obese and develop osteoarthritis (OA)‐like lesions, including chondrocyte apoptosis, in the knee joints. However, the mechanism by which HFD/obesity induces chondrocyte apoptosis is not clearly understood. In the present study, male mice were fed a low‐fat diet (LFD, 10% kcal), HFD (45% kcal), or a HFD administered with 0.5 g/kg bodyweight of 4‐phenyl butyric acid (PBA, a small chaperone known to ease endoplasmic reticulum [ER] stress), via the drinking water. At the end of the 18‐week study, stifle (knee) joints from all animals were collected, fixed, paraffin embedded, and sectioned. Immunostaining of joints from the HFD group showed increased expression of ER stress and apoptotic markers and increased expression of nuclear protein 1 and tribbles related protein‐3 compared to the LFD group. Mice on HFD also showed higher percentage of chondrocyte death, lower chondrocyte numbers per cartilage area, and thickening of subchondral bone. Administration of PBA alleviated all of the HFD‐induced symptoms. Our study demonstrated that HFD induces ER stress to promote chondrocyte death and subchondral bone thickening, which could be relieved by alleviating ER stress via PBA administration, suggesting that ER stress could play an important role in obesity‐linked OA and could be targeted for OA therapeutics. [ABSTRACT FROM AUTHOR]

Objective Oxidative posttranslational modifications of intracellular proteins can potentially regulate signaling pathways relevant to cartilage destruction in arthritis. In this study, oxidation of cysteine residues to form sulfenic acid (S-sulfenylation) was examined in osteoarthritic (OA) chondrocytes and investigated in normal chondrocytes as a mechanism by which fragments of fibronectin (FN-f) stimulate chondrocyte catabolic signaling. Methods Chondrocytes isolated from OA and normal human articular cartilage were analyzed using analogs of dimedone that specifically and irreversibly react with protein S-sulfenylated cysteines. Global S-sulfenylation was measured in cell lysates with and without FN-f stimulation by immunoblotting and in fixed cells by confocal microscopy. S-sulfenylation in specific proteins was identified by mass spectroscopy and confirmed by immunoblotting. Src activity was measured in live cells using a fluorescence resonance energy transfer biosensor. Results Proteins in chondrocytes isolated from OA cartilage were found to have elevated basal levels of S-sulfenylation relative to those of chondrocytes from normal cartilage. Treatment of normal chondrocytes with FN-f induced increased levels of S-sulfenylation in multiple proteins, including the tyrosine kinase Src. FN-f treatment also increased the levels of Src activity. Pretreatment with dimedone to alter S-sulfenylation function or with Src kinase inhibitors inhibited FN-f-induced production of matrix metalloproteinase 13. Conclusion These results demonstrate for the first time the presence of oxidative posttranslational modification of proteins in human articular chondrocytes by S-sulfenylation. Due to the ability to regulate the activity of a number of cell signaling pathways, including catabolic mediators induced by fibronectin fragments, S-sulfenylation may contribute to cartilage destruction in OA and warrants further investigation. [ABSTRACT FROM AUTHOR]

The article discussess the study which determines the increased expression of the Akt/PKB inhibitor TRB3 in osteoarthritic chondrocytes inhibits insulin-like growth factor 1-mediated cell survival and proteoglycan synthesis. The study indicates the role of TRB3 in the increased cell death and reduced response to insulin-like growth factor observed in osteoarthritis cartilage. It shows that TRB3 messenger RNA detects normal human chondrocytes from normal cartilage induced endoplasmic reticulum.