Phosphoproteome analysis of synoviocytes from patients with rheumatoid arthritis.

AIM: To explore disease-associated molecules in rheumatoid arthritis (RA), we comprehensively analyzed phosphoproteins purified from RA synoviocytes. METHOD: Synoviocytes were obtained from three patients with RA and three patients with osteoarthritis (OA). Profiles of phosphoproteins purified from the synoviocytes were compared by two-dimensional differential gel electrophoresis (2D-DIGE) between the RA and OA groups. Protein spots with significantly different phosphorylation levels were identified by mass spectrometry. Recombinant protein of annexin A4 (ANXA4), one of the identified phosphoproteins, was transfected into synoviocytes from an OA patient to mimic RA synoviocytes and humoral factor secretion was compared between rANXA4-transfected and non-transfected synoviocytes under a tumor necrosis factor-α (TNFα)-stimulated condition. RESULTS: In 2D-DIGE, 318 phosphoprotein spots were detected, of which 94 spots showed significantly different intensities between the two groups (P < 0.05). Among the 94 spots, 22 spots showed two-fold or higher intensity and one spot showed less than 1/2-fold intensity in the RA group compared to the OA group. From the 22 spots, 11 phosphoproteins were identified, which included kinases, carrier and chaperone proteins, cytoskeletal proteins, proteases and calcium-binding proteins. One of the identified calcium-binding proteins was ANXA4, an exocytosis-regulating protein. The transfected rANXA4 was found to be phosphorylated intracellularly, and secretion of chemokine (C-X-C motif) ligand 1 and interleukin-8 induced by TNFα stimulation was significantly suppressed by the transfection (P < 0.01). CONCLUSION: The phosphoprotein profile of RA synoviocytes was different from that of OA synoviocytes. This difference would reflect the different pathophysiologies of the diseases. ANXA4 may be one of therapeutic targets in RA.

Increased expression of S100 calcium binding protein A8 in GM-CSF-stimulated neutrophils leads to the increased expressions of IL-8 and IL-16.

OBJECTIVES: In our previous proteomic surveillance, we found that at least 11 proteins in neutrophils were increased more than 2.5-fold by the stimulation of GM-CSF. In this paper, focusing on one of the 11 proteins, S100 calcium binding protein A8 (S100A8), we tried to elucidate the effect of S100A8 and the cooperative effect of S100A8 and GM-CSF on production and secretion of cytokines of neutrophils. METHODS: S100A8 in neutrophil was detected by western blotting, and concentrations of S100A8 in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were measured by ELISA. Cytokine levels in the culture medium of neutrophils incubated with and without S100A8 were measured by an antibody array. IL-8 and IL-16 levels in the culture medium of neutrophils stimulated with S100A8, GM-CSF, and the combination of S100A8 and GM-CSF were measured by ELISA. The mRNA levels of IL-8 and IL-16 in the stimulated neutrophils were analysed by real-time PCR. RESULTS: The western blotting analysis confirmed that S100A8 is up-regulated in neutrophil by the stimulation of GM-CSF. Furthermore, the ELISA analysis confirmed that S100A8 was significantly elevated in SF of patients with RA compared to SF of patients with OA. S100A8 induced mRNA expression and secretion of IL-8 and IL-16. S100A8 further enhanced production of IL-8 by GM-CSF but not that of IL-16. CONCLUSIONS: These data suggest that S100A8 may be involved in the exacerbation of RA, and that S100A8 may be a therapeutic target of RA.

Quality control and monitoring for the isolation process of mesenchymal stem cells and their differentiation into osteoblasts.

We developed a method of quality control and monitoring for the isolation of mesenchymal stem cells (MSCs) from bone marrow and their differentiation into osteoblasts. After dividing the cell culture process into five groups based on cell types such as MSCs and osteoblasts, we used microarray analysis to select genes with expression profiles characteristic of each group and quantitative polymerase chain reaction for confirming the expression profiles of these genes. Comparing multiple gene expression profiles per cell from quantitative polymerase chain reaction permitted us to distinguish (1) different groups of cell culture including MSCs and osteoblasts; (2) MSCs that had differentiated cells other than osteoblasts such as chondroblasts, adipocytes, or skin-derived fibroblasts; and (3) desirable MSCs from undesirable MSCs occurring under different culture conditions. These findings suggest that it is possible to standardize MSCs and osteoblasts on the basis of multiple gene expression profiles and to check the quality of these cells. We believe that our methods can be applied to cells cultured for transplants.

Implication of granulocyte-macrophage colony-stimulating factor induced neutrophil gelatinase-associated lipocalin in pathogenesis of rheumatoid arthritis revealed by proteome analysis.

INTRODUCTION: In rheumatoid arthritis (RA), synovial fluid (SF)contains a large number of neutrophils that contribute to the inflammation and destruction of the joints. The SF also contains granulocyte-macrophage colony-stimulating factor (GM-CSF),which sustains viability of neutrophils and activates their functions. Using proteomic surveillance, we here tried to elucidate the effects of GM-CSF on neutrophils. METHODS: Neutrophils stimulated by GM-CSF were divided in to four subcellular fractions: cytosol, membrane/organelle, nuclei,and cytoskeleton. Then, proteins were extracted from each fraction and digested by trypsin. The produced peptides were detected using matrix-assisted laser desorption ionisation-time of-flight mass spectrometry (MALDI-TOF MS). RESULTS: We detected 33 peptide peaks whose expression was upregulated by more than 2.5-fold in GM-CSF stimulated neutrophils and identified 11 proteins out of the 33 peptides using MALDI-TOF/TOF MS analysis and protein database searches. One of the identified proteins was neutrophil gelatinase-associated lipocalin (NGAL). We confirmed that the level of NGAL in SF was significantly higher in patients with RA than in those with osteoarthritis. We next addressed possible roles of the increased NGAL in RA. We analysed proteome alteration of synoviocytes from patients with RA by treatment with NGAL in vitro. We found that, out of the detected protein spots (approximately 3,600 protein spots), the intensity of 21 protein spots increased by more than 1.5-fold and the intensity of 10 protein spots decreased by less than 1 to 1.5-fold as a result of the NGAL treatment. Among the 21 increased protein spots, we identified 9 proteins including transitional endoplasmic reticulum ATPase (TERA), cathepsin D, and transglutaminase 2 (TG2), which increased to 4.8-fold, 1.5-fold and 1.6-fold, respectively. Two-dimensional electrophoresis followed by western blot analysis confirmed the upregulation of TERA by the NGAL treatment and, moreover, the western blot analysis showed that the NGAL treatment changed the protein spots caused by post-translational modification of TERA.Furthermore, NGAL cancelled out the proliferative effects of fibroblast growth factor (FGF)-2 and epidermal growth factor(EGF) on chondrocytes from a patient with RA and proliferative effect of FGF-2 on chondrosarcoma cells.Conclusions Our results indicate that GM-CSF contributes to the pathogenesis of RA through upregulation of NGAL in neutrophils, followed by induction of TERA, cathepsin D and TG2 in synoviocytes. NGAL and the upregulated enzymes may therefore play an important role in RA.

Crosslinking of the CD69 molecule enhances S100A9 production in activated neutrophils.

Expression of CD69 on neutrophils and generation of anti-CD69 autoantibodies in patients with rheumatoid arthritis (RA) have been reported. Thus natural ligands for CD69 not yet identified and/or the anti-CD69 autoantibodies possibly affect neutrophils by evoking CD69 signaling, which may further affect joint-composing cells in RA. However, the effect of the CD69 signaling in neutrophils remains largely unclear. To elucidate the issue, we tried to identify proteins affected by the crosslinking of CD69 on neutrophils using a proteomic approach. Specifically, CD69 on granulocyte-macrophage colony stimulating factor (GM-CSF)-activated neutrophils was crosslinked by anti-CD69 monoclonal antibodies, and then intracellular proteins were detected using 2-dimensional electrophoresis and further identified by mass spectrometry and subsequent protein database searching. As a result, we successfully identified multiple proteins that increased their production by the CD69 signaling. Among the proteins, we focused on one of the up-regulated proteins, S100A9 calcium binding protein (S100A9), and investigated proteome changes brought by a recombinant S100A9 in a human synovial sarcoma cell line (SW982), a human chondrosarcoma cell line (OUMS-27), and a human T leukemia cell line (Jurkat). This revealed that the recombinant S100A9 altered proteomes of SW982 and OUMS-27, and to a lesser extent, that of the Jurkat cells. Further, S100A9 induced IL-1beta production from neutrophils and the SW982 cells. These data suggest that unidentified natural ligands for CD69 and/or the anti-CD69 autoantibodies possibly affect joint-composing cell types through the increased production of S100A9 in neutrophils, providing a new insight into functions of CD69 on neutrophils in RA.

Ribozymes targeting serine/threonine kinase Akt1 sensitize cells to anticancer drugs.

The serine/threonine kinase Akt is a key component of the cellular signaling pathway for survival and drug-resistance in cancer cells. In the present study we confirmed this view by expressing an antagonist of Akt, a dominant negative form of Akt, in HCT116 colon carcinoma cells and observing apoptosis induction in cells in which expression of the mutant protein had been induced. Three isoforms of Akt have been found: Akt1/PKBalpha, Akt2/PKBbeta and Akt3/PKBgamma. However, the function of individual isoforms with respect to tumorigenicity and drug-resistance of cancer cells is largely unknown. We designed ribozymes targeting the Akt1 protein in mammalian cells. Our data indicate that Akt1 ribozymes downregulate Akt1 expression to less than half that of control cells. Downregulation of Akt1 expression appears to sensitize HEK293 and HeLa cells to typical chemotherapeutic agents. However, Akt1 ribozymes had little effect on the proliferative activity of the cells. Thus, Akt as a whole and even just the Akt1 isozyme is an excellent target for chemotherapy. We further suggest a synergistic effect for combination therapy targeting Akt and other vital molecules such as tubulins, topoisomerases and protein kinases.