Nuclear protein-1 is the common link for pathways activated by aging and obesity in chondrocytes: A potential therapeutic target for osteoarthritis.

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.

Co-Immunoprecipitation-Blotting: Analysis of Protein-Protein Interactions.

Immunoprecipitation of protein complexes, also known as co-immunoprecipitation (Co-IP), is a powerful technique to analyze protein-protein interactions. Commercial availability of Dynabeads Protein A magnetic beads provides a fast, convenient, and efficient method for protein interaction studies by Co-IP followed by immunoblotting (Co-IP-blotting). Recently, the Co-IP-blotting technique helped us to investigate complicated protein interactions/networks involving nuclear protein 1 (Nupr1), a recently discovered regulator of apoptosis in human cartilage cells.

Spaceflight and hind limb unloading induces an arthritic phenotype in knee articular cartilage and menisci of rodents.

Reduced knee weight-bearing from prescription or sedentary lifestyles are associated with cartilage degradation; effects on the meniscus are unclear. Rodents exposed to spaceflight or hind limb unloading (HLU) represent unique opportunities to evaluate this question. This study evaluated arthritic changes in the medial knee compartment that bears the highest loads across the knee after actual and simulated spaceflight, and recovery with subsequent full weight-bearing.

Dietary saturated fatty acid palmitate promotes cartilage lesions and activates the unfolded protein response pathway in mouse knee joints.

Increased intake of dietary saturated fatty acids has been linked to obesity and the development of Osteoarthritis (OA). However, the mechanism by which these fats promote cartilage degradation and the development of OA is not clearly understood. Here, we report the effects of consumption of common dietary saturated and unsaturated fatty acids, palmitate and oleate, respectively, on body weight, metabolic factors, and knee articular cartilage in a mouse model of diet-induced obesity.

Age-Related Decline in Expression of Molecular Chaperones Induces Endoplasmic Reticulum Stress and Chondrocyte Apoptosis in Articular Cartilage.

Aging is a major risk factor for the development of osteoarthritis (OA). One hallmark of aging is loss of proteostasis resulting in increased cellular stress and cell death. However, its effect on the development of OA is not clear.

Role of the Hypoxia-Inducible Factor Pathway in Normal and Osteoarthritic Meniscus and in Mice after Destabilization of the Medial Meniscus.

Objective: Meniscus injury and the hypoxia-inducible factor (HIF) pathway are independently linked to osteoarthritis pathogenesis, but the role of the meniscus HIF pathway remains unclear. We sought to identify and evaluate HIF pathway response in normal and osteoarthritic meniscus and to examine the effects of Epas1 (HIF-2α) insufficiency in mice on early osteoarthritis development.

Methods: Normal and osteoarthritic human meniscus specimens were obtained and used for immunohistochemical evaluation and cell culture studies for the HIF pathway.

High-fat diet induces endoplasmic reticulum stress to promote chondrocyte apoptosis in mouse knee joints.

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.

Nupr1 regulates palmitate-induced apoptosis in human articular chondrocytes.

Obesity, a major risk factor for the development of osteoarthritis (OA), is associated with increased circulating levels of free fatty acids (FFA). However, the role of these FFAs in OA pathophysiology is not clearly understood. In the present study, we found that palmitate treatment of human primary articular chondrocytes increased the expression of ER stress markers [activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP)] and apoptosis markers [cytochrome and cleaved caspase-3 (CC3)].

HO oxidation of cysteine residues in c-Jun N-terminal kinase 2 (JNK2) contributes to redox regulation in human articular chondrocytes.

Reactive oxygen species (ROS), in particular HO, regulate intracellular signaling through reversible oxidation of reactive protein thiols present in a number of kinases and phosphatases. HO has been shown to regulate mitogen-activated protein kinase (MAPK) signaling depending on the cellular context. We report here that in human articular chondrocytes, the MAPK family member c-Jun N-terminal kinase 2 (JNK2) is activated by fibronectin fragments and low physiological levels of HO and inhibited by oxidation due to elevated levels of HO The kinase activity of affinity-purified, phosphorylated JNK2 from cultured chondrocytes was reversibly inhibited by 5-20 μm HO Using dimedone-based chemical probes that react specifically with sulfenylated cysteines (RSOH), we identified Cys-222 in JNK2, a residue not conserved in JNK1 or JNK3, as a redox-reactive site.

Saturated fatty acid palmitate negatively regulates autophagy by promoting ATG5 protein degradation in meniscus cells.

Obesity and associated metabolic factors are major risk factors for the development of osteoarthritis. Previously, we have shown that the free fatty acid palmitate induces endoplasmic reticulum (ER) stress and induces apoptosis in meniscus cells. However, the molecular mechanisms involved in these effects are not clearly understood.

Cysteine-Mediated Redox Regulation of Cell Signaling in Chondrocytes Stimulated With Fibronectin Fragments.

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.

Therapeutic exercise attenuates neutrophilic lung injury and skeletal muscle wasting.

Early mobilization of critically ill patients with the acute respiratory distress syndrome (ARDS) has emerged as a therapeutic strategy that improves patient outcomes, such as the duration of mechanical ventilation and muscle strength. Despite the apparent efficacy of early mobility programs, their use in clinical practice is limited outside of specialized centers and clinical trials. To evaluate the mechanisms underlying mobility therapy, we exercised acute lung injury (ALI) mice for 2 days after the instillation of lipopolysaccharides into their lungs.

Brief report: stress-inducible nuclear protein 1 regulates matrix metalloproteinase 13 expression in human articular chondrocytes.

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.

Extracellular nicotinamide phosphoribosyltransferase (NAMPT/visfatin) inhibits insulin-like growth factor-1 signaling and proteoglycan synthesis in human articular chondrocytes.

Introduction: Obesity is one of the major risk factors for the development of osteoarthritis (OA). Although the mechanical factors appear to be critical, recent studies have suggested a role for adipokines in cartilage degradation. Chondrocytes from osteoarthritic cartilage respond poorly to insulin-like growth factor-1 (IGF-1) and the molecular mechanism(s) involved is not clearly understood.

S100 proteins in cartilage: role in arthritis.

S100 proteins are low molecular weight calcium binding proteins expressed in vertebrates. The family constitutes 21 known members that are expressed in several tissues and cell types and play a major role in various cellular functions. Uniquely, members of the S100 family have both intracellular and extracellular functions.

Sumoylation and nuclear translocation of S100A4 regulate IL-1beta-mediated production of matrix metalloproteinase-13.

S100A4, a member of the S100 family of proteins, plays an important role in matrix remodeling by up-regulating the expression of matrix metalloproteinases (MMPs). We have previously shown that S100A4 is overexpressed in diseased cartilage and that extracellular S100A4 stimulates MMP-13 production, a major type II collagen-degrading enzyme, via activation of receptor for advanced glycation end product signaling. In the present study, using human articular chondrocytes, we show that intracellular S100A4 translocated into the nucleus upon interleukin-1β (IL-1β) stimulation and translocation required post-translational modification of S100A4 by the sumo-1 protein.

Interleukin-7 stimulates secretion of S100A4 by activating the JAK/STAT signaling pathway in human articular chondrocytes.

Objective: S100A4 has been shown to be increased in osteoarthritic (OA) cartilage and to stimulate chondrocytes to produce matrix metalloproteinase 13 (MMP-13) through activation of the receptor for advanced glycation end products (RAGE). The aim of this study was to examine the mechanism of S100A4 secretion by chondrocytes.

Methods: Human articular chondrocytes isolated from ankle cartilage were stimulated with 10 ng/ml of interleukin-1beta (IL-1beta), IL-6, IL-7, or IL-8.

Increased expression of the Akt/PKB inhibitor TRB3 in osteoarthritic chondrocytes inhibits insulin-like growth factor 1-mediated cell survival and proteoglycan synthesis.

Objective: The chondrocyte response to insulin-like growth factor 1 (IGF-1) is reduced with aging and in osteoarthritis (OA). IGF-1 signals through the phosphatidylinositol 3-kinase/Akt pathway. TRB3, a tribbles homolog, has been shown to inhibit IGF-1-mediated activation of Akt in HEK 293 cells.

Interactions of cubilin with megalin and the product of the amnionless gene (AMN): effect on its stability.

Cubilin, a 456 kDa multipurpose receptor lacking in both transmembrane and cytoplasmic domains is expressed in the apical BBMs (brush border membranes) of polarized epithelia. Cubilin interacts with two transmembrane proteins, AMN, a 45-50 kDa protein product of the amnionless gene, and megalin, a 600 kDa giant endocytic receptor. In vitro, three fragments of cubilin, the 113-residue N-terminus and CUB domains 12-17 and 22-27, demonstrated Ca2+-dependent binding to megalin.

Plasma membrane delivery, endocytosis and turnover of transcobalamin receptor in polarized human intestinal epithelial cells.

Cells that are metabolically active and in a high degree of differentiation and proliferation require cobalamin (Cbl: vitamin B(12)) 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 (T(1/2)) of 8 h following its delivery to the basolateral plasma membrane (BLM).

Increase in production of matrix metalloproteinase 13 by human articular chondrocytes due to stimulation with S100A4: Role of the receptor for advanced glycation end products.

Objective: S100 proteins have been implicated in various inflammatory conditions, including arthritis. The aims of this study were to determine whether chondrocytes produce S100A4 and whether S100A4 can stimulate the production of matrix metalloproteinase 13 (MMP-13) by articular chondrocytes via receptor for advanced glycation end products (RAGE)-mediated signaling.

Methods: The expression of chondrocyte S100A4 was analyzed by immunohistochemistry using normal and osteoarthritic (OA) cartilage and by immunoblotting of chondrocyte cell lysates.

Articular chondrocytes express the receptor for advanced glycation end products: Potential role in osteoarthritis.

Objective: The receptor for advanced glycation end products (RAGE) binds multiple ligands, including S100 proteins, high mobility group box chromosomal protein 1 (HMGB-1), and AGEs, all of which are present in articular cartilage. Stimulation of RAGE signaling can lead to MAP kinase activation and increased NF-kappaB activity. The objective of the present study was to determine if chondrocytes express functional RAGE.

Cobalamin (vitamin B12) binding, phylogeny, and synteny of human transcobalamin.

Selected residues in a highly conserved 15-residue region, 174SVDTAAMAGLAFTC L188 of human transcobalamin (TC), a cobalamin (Cbl: vitamin B12) binding protein, were subjected to site-directed mutagenesis. The mutant constructs were expressed in TC-deficient fibroblasts or in vitro to assess the effect of these mutations on Cbl binding. Phylogenetic analyses and protein parsimony indicated that TC evolved earlier than other mammalian Cbl-binding proteins, intrinsic factor and haptocorrins, and divergence occurred between mouse/rat and human dispersing TC gene to different chromosomes.

Rat transcobalamin: cloning and regulation of mRNA expression.

Transcobalamin (TC) has been cloned and used for studying its gene expression in the rat. TC mRNA is distributed widely in adult rat tissues, but at different levels (kidney > liver > lung > yolk sac > intestine > heart > brain > spleen > muscle). TC mRNA levels were 4-fold higher in the jejunum and ileum compared to its levels in the duodenum.

Cobalamin transport proteins and their cell-surface receptors.

The primary function of cobalamin (Cbl; vitamin B12) is the formation of red blood cells and the maintenance of a healthy nervous system. Before cells can utilise dietary Cbl, the vitamin must undergo cellular transport using two distinct receptor-mediated events. First, dietary Cbl bound to gastric intrinsic factor (IF) is taken up from the apical pole of ileal epithelial cells via a 460 kDa receptor, cubilin, and is transported across the cell bound to another Cbl-binding protein, transcobalamin II (TC II).