Layilin enhances the invasive ability of malignant glioma cells via SNAI1 signaling.

BACKGROUND: Malignant gliomas are characterized by high invasive ability. In this study, we investigated roles of layilin, a C-type lectin-homologous protein, in the invasive ability of malignant glioma cells. METHODS: Expression of layilin was investigated by western blotting in the malignant glioma cell lines of U251-MG, A172, and T98G and in astrocytes. The effects of layilin-knockdown on the expression and protein levels of snail family transcriptional repressor 1 (SNAI1), a transcriptional factor involved in the acquisition and enhancement of invasive ability in malignant gliomas, and on the expression of its target genes, matrix metalloproteinase 2 (MMP2), MMP9, and collagen type I alpha 1 chain (COL1A1), were investigated by qPCR and/or western blotting. Furthermore, the effects of layilin-knockdown on the expression and protein levels of metastasis associated 1 family member 3 (MTA3), a transcriptional repressor of SNAI1, were also investigated by qPCR and western blotting. Finally, the effects of layilin-knockdown on the invasive ability of the cells were investigated by a wound healing assay. RESULTS: All the tested malignant glioma cells highly expressed layilin, compared to astrocytes, one of representative glial cell types. Layilin-knockdown reduced SNAI1 both at the mRNA and protein levels in A172 cells, and consequently mRNA levels of MMP2, MMP9, and COL1A1 were also reduced. Furthermore, layilin-knockdown increased nuclear protein levels of MTA3 in A172 cells. Notably, layilin-knockdown suppressed the invasive ability of the cells. CONCLUSION: Layilin up-regulates the expression of SNAI1 via down-regulation of MTA3. This process enhances the invasive ability of malignant glioma cells.

Effects of salazosulfapyridine on the profile of cell surface proteins, revealed by biotinylation of cell surface proteins and 2-dimentional electrophoresis.

OBJECTIVE: We investigated effects of salazosulfapyridine (SASP) on the protein profile of cell surface (CS)-proteins of SW982, a human synovial sarcoma cell line, using biotinylation of CS-proteins and 2-dimensional fluorescence difference gel electrophoresis (2D-DIGE). METHODS: SW982 cells were treated with SASP and its metabolites, sulfapyridine (SP) and 5-aminosalicylic acid (5ASA). Then the cells were treated with a membrane-impermeable biotinylating reagent. Biotinylated CS-proteins were isolated using NeutrAvidin-bound beads. CS-proteins affected by the drugs were detected by 2D-DIGE and subjected to mass spectrometry. RESULTS: By the 2D-DIGE analysis, in total 576 spots were detected, 29 out of which showed more than ±1.5-fold different intensity in the SASP-, SP-, and 5ASA-treated cells, compared to non-treated cells (p < 0.05). Interestingly, 7 out of the 29 spots changed their intensity only by SASP and 17 spots changed their intensity only by SP. We identified 9 protein from 15 out of the 29 spots, most of which were evidenced to exist on the cell surface by flow cytometry. CONCLUSION: We found novel effects of SASP and its metabolites on SW982 cells by the combination of biotinylation of cell surface proteins and 2D-DIGE analysis. These data would help understanding of anti-rheumatic actions of SASP. Furthermore, the combination would be a useful method for the analysis of CS-proteins in various conditions.

Serum peptides as putative modulators of inflammation in psoriasis.

BACKGROUND: Psoriasis is a refractory inflammatory disease, however, its pathophysiology is still not fully understood. OBJECTIVE: We tried to identify novel serum peptides associated with the pathophysiology of psoriasis. METHODS: Serum peptides from 24 patients with psoriasis vulgaris (PV), 10 patients with psoriatic arthritis (PsA), 14 patients with atopic dermatitis (AD), and 23 healthy control (HC) subjects were analyzed by mass spectrometry. The effects of some peptides on the secretion of humoral factors from dermal cells were investigated by cytokine arrays and ELISAs. RESULTS: A total of 93 peptides were detected. 24, 20, 23, and 2 peptides showed at least 1.2-fold difference in ion intensity between the psoriasis (PV+PsA) and HC groups, between the PV+PsA and AD groups, between the PV and PsA groups, and between patients with severe-to-moderate PV (n=6) and those with mild PV (n=18), respectively (p<0.05). 13 out of 27 peptides that showed at least 1.5-fold ion intensity difference in the abovementioned 4 comparisons were identified. The parent proteins of the identified peptides included a coagulation factor, proteins involved in the maintenance of skin, and a protein relating to cytoskeleton. We focused on 2 peptides that were increased in the PV+PsA group: a fibrinogen α chain-derived peptide (1462m/z), the unmodified form of which was fibrinopeptide A-des-alanine (FPAdA), and a filaggrin (FLG)-derived peptide (1977m/z), a modified form of FLG2099-2118 (Q2099pE, Q2115E; FLG-pEE). FPAdA stimulation increased the secretion of GROα from dermal microvascular endothelial cells (dMVECs) and decreased the secretion of lipocalin-2 from keratinocytes in comparison to FPAdA-resequenced peptide stimulation (GROα, 280.9±7.3pg/mL vs. 229.6±5.0pg/mL, p<0.001; lipocalin-2, 273±13pg/mL vs. 350±10pg/mL, p<0.01). Interestingly, FLG-pEE stimulation decreased the secretion of GROα, IL-8, and MCP-1 from dMVECs in comparison to FLG-derived control peptide stimulation (GROα, 844.3±47.5pg/mL vs. 1038.5±96.9pg/mL, p<0.05; IL-8, 2240.1±172.6pg/mL vs. 3221.8±523.7pg/mL, p<0.05; MCP-1, 4057.8±157.2pg/mL vs. 4619.1±213.4pg/mL, p<0.05). CONCLUSIONS: The results suggested that some serum peptides are involved in the pathophysiology of psoriasis, regulating the secretion of inflammatory chemokines and an antimicrobial protein. The modulation of serum peptides may be a potential therapeutic strategy for psoriasis.

Hypoxia-induced production of peptidylarginine deiminases and citrullinated proteins in malignant glioma cells.

BACKGROUND: Recently, it has been reported that hypoxia highly enhances expression of peptidylarginine deiminase (PAD) 4 and production of citrullinated proteins in some tumor cells. However, little is known about malignant gliomas on this issue. Therefore, we here investigated whether expression of PADs was induced by hypoxia and whether PADs citrullinated intracellular proteins if induced using U-251 MG cells of a human malignant glioma cell line. METHODS: Expression of PADs in U-251 MG cells, cultured under hypoxia or normoxia for 24 h, was investigated by quantitative polymerase chain reaction (qPCR). Citrullination of proteins in the cells and the cell lysates incubated for 48 h with or without Ca2+ was detected by western blotting. Citrullinated proteins were identified by mass spectrometry. RESULTS: The mRNA levels of PAD1, 2, 3, and 4 were up-regulated by hypoxia in a hypoxia-inducible factor-1-dependent manner in U-251 MG cells. In spite of the increased expression, intracellular proteins were not citrullinated. However, the induced PADs citrullinated U-251 MG cell-derived proteins when the cells were lysed. Multiple proteins citrullinated by hypoxia-induced PADs were identified. In addition, the extracellular domain of vascular endothelial growth factor receptor 2 was citrullinated by human PAD2 in vitro. CONCLUSION: Our data may contribute to understanding of pathophysiology of malignant gliomas from the aspects of protein citrullination.

Effects of methotrexate and salazosulfapyridine on protein profiles of exosomes derived from a human synovial sarcoma cell line of SW982.

PURPOSE: To elucidate effects of salazosulfapyridine (SASP) and methotrexate (MTX), major anti-rheumatic drugs, on exosomes derived from SW982 of a human synovial sarcoma cell line. EXPERIMENTAL DESIGN: SW982 was treated with SASP and/or MTX under interleukin-1ß (IL-1ß)-treated or nontreated conditions. Exosomes were isolated from the culture media, and exosomal proteome was analyzed by 2D-DIGE. Protein spots whose intensity was significantly altered by the above treatments were identified by MS. RESULTS: Two hundred ninety-four protein spots were detected in the exosome preparations by 2D-DIGE. Compared to the nontreated cells, SASP-, MTX-, and (SASP + MTX)-treated cells displayed 8, 10, and 21 exosomal protein spots with more than ±2.0-fold intensity differences (p < 0.05), respectively. Similarly, the IL-1ß-treated cells displayed 58 exosomal protein spots with more than ±1.5-fold intensity differences (p < 0.05). In about half of the 58 spots, the IL-1ß-induced intensity changes were suppressed by simultaneous addition of SASP and/or MTX. Most of the identified proteins were immunity- or anti-oxidation-related proteins. CONCLUSIONS AND CLINICAL RELEVANCE: The SASP and/or MTX treatments altered the protein profiles of exosomes and suppressed the effects of IL-1ß on the exosomal proteome. Exosomes may play roles in the actions of these anti-rheumatic drugs.

Roles of layilin in TNF-α-induced epithelial-mesenchymal transformation of renal tubular epithelial cells.

BACKGROUND: Tumor necrosis factor (TNF)-α is suggested to induce epithelial-mesenchymal transformation (EMT) of renal tubular epithelial cells that possibly exacerbates renal interstitial fibrosis in glomerulonephritis (GN). We here investigated whether layilin (LAYN), a c-type lectin-homologous protein, was involved in the EMT process. METHODS: Expression of LAYN was investigated in kidneys of mice administered with TNF-α and in a clear cell renal carcinoma cell line of KMRC-1 stimulated with TNF-α by quantitative polymerase chain reaction (qPCR) and/or western blotting. Expression of LAYN was assessed immunohistochemically in renal biopsy samples of patients with various types of GN. Changes of EMT markers and cell morphology by TNF-α and transforming growth factor (TGF)-ß in LAYN-knocked down KMRC-1 cells were investigated by qPCR and immunocytochemistry. RESULTS: Administration of TNF-α increased expression of LAYN in renal tubular epithelia in mice. TNF-α but not TGF-ß increased expression of LAYN in KMRC-1 cells. Renal biopsy samples from the patients with GN showed high expression of LAYN in tubular epithelial cells. TNF-α induced up-regulation of vimentin, down-regulation of E-cadherin, and fibroblast-like morphological change in KMRC-1 cells, indicating occurrence of EMT. These changes were not observed in the LAYN-knocked down cells. In contrast, similarly occurred TGF-ß-induced EMT was not affected by the LAYN knockdown. CONCLUSION: Our data indicate that LAYN is involved in the TNF-α-induced EMT of renal tubular epithelial cells. LAYN may play roles in the generation of renal interstitial fibrosis in GN via TNF-α-induced EMT.

Serum peptides as candidate biomarkers for dementia with Lewy bodies.

OBJECTIVE: For diagnosis of dementia with Lewy bodies (DLB), we tried to find blood biomarkers for the disease. METHODS: Serum peptides were comprehensively detected by mass spectrometry. Peptides of interest were identified by tandem mass spectrometry. RESULTS: One hundred forty-six peptides were detected in a training set consisting of 30 DLB patients, 30 patients with Alzheimer's disease (AD), and 28 healthy control (HC) subjects. Multivariate analysis for discriminating the DLB group from the non-DLB (AD and HC) group using ion intensity of four peptides (2898, 4052, 4090, and 5002 m/z) showed sensitivity of 93.3% and specificity of 87.9% (DLB/nonDLB-4P model). In a testing set consisting of 20 DLB patients, 30 AD patients, and 14 HC subjects, this model showed sensitivity of 90.0% and specificity of 88.6%. DLB/nonDLB-4P model detected 86.7% and 90.0% of the AD patients as non-DLB in the training and testing sets, respectively, and discriminated all the 15 patients with amnestic mild cognitive impairment as non-DLB. Notably, a combination of two peptides (1737 and 5002 m/z) showed sensitivity of 95.0% and specificity of 93.3% for discriminating the DLB group from the AD group (DLB/nonDLB-2P model) in the testing set. The peptides used in these models included fragments from complement 4b, Wnt-2b, and lipopolysaccharide-binding protein, which were reported to be involved in the pathology of DLB or Parkinson's disease and hippocampal neurogenesis. CONCLUSIONS: Serum peptide profiles would provide useful DLB biomarker candidates, which may be implicated in the pathophysiology of the disease.

Effects of tofacitinib on nucleic acid metabolism in human articular chondrocytes.

OBJECTIVE: In our previous screening of chondrocyte protein profiles, the amount of adenosine monophosphate deaminase (AMPD) 2 was found to be decreased by tofacitinib. Extending the study, here we confirmed the decrease of AMPD2 by tofacitinib and further investigated effects of tofacitinib on purine nucleotide metabolism. METHODS: Human articular chondrocytes and a chondrosarcoma cell line: OUMS-27 were stimulated with tofacitinib. Then the levels of AMPD2 and its related enzymes were investigated by Western blot. The levels of AMP and adenosine were assessed by mass spectrometry. RESULTS: We confirmed the significant decrease of AMPD2 by tofacitinib in chondrocytes (p = 0.025). The levels of adenosine kinase and 5'-nucleotidase were decreased in chondrocytes, although they did not meet statistical significance (p = 0.067 and p = 0.074, respectively). The results from OUMS-27 were similar to those from the chondrocytes. The cellular adenosine levels were significantly decreased by tofacitinib in OUMS-27 (p = 0.014). The cellular AMP levels were increased, although they did not meet statistical significance in OUMS-27 (p = 0.066). CONCLUSION: Our data indicate that tofacitinib increases the cellular levels of adenosine, which is known to have anti-inflammatory activity, through the downregulation of AMPD2. This would be a novel functional aspect of tofacitinib.

Secretion of inflammatory factors from chondrocytes by layilin signaling.

Layilin (LAYN) is thought to be involved in reorganization of cytoskeleton structures, interacting with merlin, radixin, and talin. Also, LAYN is known to be one of the receptors for hyaluronic acid (HA). In rheumatoid arthritis (RA), inflammatory cytokines like tumor necrosis factor α (TNF-α) have been known to play pathological roles. HA with low molecular weight is speculated to exacerbate inflammation in RA. In this context, differences of quantity and functions of HA receptors would affect the severity of inflammation in RA. Chondrocytes, which play critical roles in maintaining articular cartilage and are affected in RA, express at least kinds of HA receptors like CD44 and LAYN. However, roles and regulation of LAYN in articular chondrocytes have been poorly understood. To clarify regulation of LAYN in chondrocytes, we here investigated whether TNF-α affected expression levels of LAYN in human articular chondrocytes. Next, to clarify LAYN-specific roles in chondrocytes, we investigated whether binding of antibodies to the extracellular domain of LAYN affected secretion of inflammatory cytokines using a chondrosarcoma cell line. As a result, we found that TNF-α up-regulated expression levels of LAYN in the chondrocytes. Further, the LAYN signaling was found to enhance secretion of inflammatory factors, IL-8 and complement5 (C5)/C5a, from the cells. Our results indicate that LAYN would be involved in the enhancement of inflammation and degradation of cartilage in joint diseases such as RA and OA.

Proteomic analysis of whole glomeruli in patients with IgA nephropathy using microsieving.

BACKGROUND: To promote understanding of immunoglobulin A nephropathy (IgAN) pathophysiology, we tried to elucidate glomerular protein profiles in IgAN, using microsieving that we established recently to isolate glomeruli from renal biopsy samples and proteomic approaches. METHODS: Glomeruli were isolated from renal biopsy samples of patients with IgAN (n = 5) and with minimal change nephrotic syndrome (MCNS; n = 5) using microsieving. Proteins extracted from the isolated glomeruli were separated by 2-dimensional differential gel electrophoresis (2D-DIGE). Proteins with different amounts between the two groups were identified by mass spectrometry. One of the identified proteins, α-actinin-4 (ACTN4), was further analyzed by Western blotting, RT-polymerase chain reaction (PCR), and immunohistochemistry. RESULTS: By 2D-DIGE, 72 out of the detected 1,170 protein spots showed significantly different intensity between the two groups (p < 0.05). Thirty-four out of the 72 protein spots showed more than 1.5-fold or less than 1/1.5-fold intensity, out of which 16 protein spots were successfully identified. No microbial protein was identified. ACTN4 molecules with a low molecular weight of approximately 77 kDa were found to increase in the IgAN group. Lack of an N-terminal part of ACTN4 was demonstrated by Western blotting. No defect of mRNA for ACTN4 was evidenced by RT-PCR. Predominant existence of ACTN4 in capillary walls of glomeruli of IgAN patients was demonstrated by immunohistochemistry in glomerular sections of patients with IgAN. CONCLUSION: Use of microsieving enabled us to biochemically analyze glomerular proteins in renal biopsy samples from patients with glomerular diseases. With this method, we demonstrated skewed glomerular protein profiles in IgAN.

Protein profiles of peripheral blood mononuclear cells as a candidate biomarker for Behçet's disease.

OBJECTIVES: To investigate the pathophysiology of Behçet's disease (BD) and find biomarkers for the disease, we analysed protein profiles of peripheral blood mononuclear cells (PBMCs). METHODS: Proteins, extracted from PBMCs, were comprehensively analysed in 16 patients with BD, 16 patients with rheumatoid arthritis (RA), 12 patients with Crohn's disease (CD), and 16 healthy control subjects (HC) by 2-dimensional differential gel electrophoResis (2D-DIGE). Differently expressed proteins were identified by mass spectrometry. RESULTS: 563 protein spots were detected. We completely discriminated between the BD and HC groups, between the BD and RA groups, and between the BD and CD groups by multivariate analysis of intensity of 23, 35, and 1 spots, respectively. The spots contributing to the differences included proteins related to cytoskeleton, transcription/translation, T cell activation, bone turnover, regulating apoptosis, and microbial infection. Intensity of 3 spots (tyrosine-protein phosphatase non-receptor type 4, threonine synthase-like 2, and ß-actin) provided area under the receiver operating characteristic curves (AUROC) of 0.889 for discrimination between the BD group and the non-BD groups. Informatively, intensity of the above 1 spot completely discriminated the CD group from the other groups (AUROC 1.000). This spot, identified as ß-actin, had different pI from the above ß-actin-spot probably due to different post-translational modification. CONCLUSIONS: PBMC protein profiles, especially the profile of the 3 spots, would be candidate biomarkers for BD. The latter ß-actin subtype would be useful for discriminating inflammatory bowel diseases from BD and other diseases. The identified proteins may play important roles in the pathophysiology of BD.

Comparative proteomic analysis of neutrophils from patients with microscopic polyangiitis and granulomatosis with polyangiitis.

Both microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) belong to ANCA-associated vasculitis (AAV), in which neutrophils play a key role in their pathology. In this study, in order to discriminate between MPA and GPA, protein profiles of peripheral blood polymorphonuclear cells (PMNs) of 11 MPA patients and 9 GPA patients and 10 healthy controls (HC) were analyzed by 2D-DIGE. In all the 864 spots detected, intensity of 55 spots was significantly different (p<0.05) among the three groups by ANOVA. 31 out of the 55 spots were identified by mass spectrometry. Orthogonal partial-least-squares-discriminate analysis revealed that the abundance profile of the protein spots discriminated the AAV group from the HC group, and the MPA group from the GPA group completely. 13 protein spots were considered as biomarker candidates to distinguish between MPA and GPA. In those, spots whose intensity was higher in MPA than in GPA included actin with various pI values, while a considerable part of spots whose intensity was higher in GPA were proteins related with the activity of neutrophils. Among the candidate proteins, ROC analysis showed that a combination of neutrophil gelatinase-associated lipocalin and a-kinase anchor protein 7 isoforms beta had a high diagnostic potential. BIOLOGICAL SIGNIFICANCE: In this study, protein profiles of polymorphonuclear cells (PMNs) of microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) patients and healthy controls (HC) were investigated by 2D-DIGE, and MS analysis. As a result, we found that the protein profiles of PMNs were useful for distinguishing between patients (MPA and GPA) and HC, and between patients with MPA and patients with GPA. Especially, we found that the 13 protein spots that consisted of 10 proteins considerably contributed to the discrimination between MPA and GPA. This is the first to demonstrate that protein profiles of PMNs are different among MPA, GPA and healthy control. The 10 proteins we identified in this study would be new biomarkers for the diagnosis of the diseases, and may be reflect the pathology difference between MPA and GPA.

AC13, a C-terminal fragment of apolipoprotein A-I, is a candidate biomarker for microscopic polyangiitis.

OBJECTIVE: Microscopic polyangiitis (MPA) is necrotizing vasculitis of unknown etiology. We analyzed the serum peptide profile of MPA to find a biomarker for this disease. METHODS: Serum peptides from 33 patients with MPA, 7 with granulomatosis with polyangiitis (Wegener's), 7 with Churg-Strauss syndrome, 6 with giant cell arteritis, and 25 with systemic lupus erythematosus (SLE) were comprehensively analyzed by mass spectrometry. Peptide function on human microvascular endothelial cells (HMVECs) was examined by enzyme-linked immunosorbent assay and real-time polymerase chain reaction. RESULTS: A total of 102 serum peptides were detected from the 78 patients. One of the peptides, peptide 1,523, showed significantly higher ion intensity in MPA (mean ± SD 46.8 ± 39.3 arbitrary units [AU]) than in the other systemic vasculitides (14.1 ± 12.2 AU) (P < 0.05) or in SLE (17.0 ± 12.1 AU) (P < 0.05). In MPA, peptide 1,523 showed significantly higher ion intensity before treatment than 1 week (P < 0.05) and 6 weeks (P < 0.05) after the initiation of treatment. Peptide 1,523 was identified as 13 C-terminal amino acid residues of apolipoprotein A-I (Apo A-I) and was designated "AC13." Validation of AC13 ion intensity using another MPA cohort (n = 14) similarly showed significantly higher ion intensity (90.1 ± 167.9 AU) compared to 14 patients with rheumatoid arthritis (8.6 ± 5.4 AU) (P < 0.01) and 14 healthy subjects (11.8 ± 6.1 AU) (P < 0.01). Serum concentrations of Apo A-I and high-density lipoprotein cholesterol were down-regulated in MPA before treatment and returned to their normal ranges 6 weeks after the initiation of treatment (both P < 0.01). Stimulation of HMVECs with AC13 significantly up-regulated secretion of interleukin-6 (IL-6) (P < 0.05) and IL-8 (P < 0.01). CONCLUSION: AC13, a candidate biomarker for MPA, may be useful for monitoring disease activity and may exacerbate vascular inflammation through up-regulation of proinflammatory cytokines.

Arthritogenicity of annexin VII revealed by phosphoproteomics of rheumatoid synoviocytes.

OBJECTIVE: To identify novel proteins involved in the pathogenesis of rheumatoid arthritis (RA) and to characterise the identified proteins based on pathogenic and therapeutic aspects. METHODS: The authors applied differential phosphoproteomic analysis to articular synoviocytes between RA and osteoarthritis (OA) to identify proteins differently phosphorylated between RA and OA. Focusing on annexin VII (Anx7), one of the highly phosphorylated proteins in RA, the authors prepared Anx7-transgenic C57BL/6 (Anx7-Tg-B6) mice to evaluate their susceptibility to collagen-induced arthritis (CIA). In addition, the authors examined the effect of anti-Anx7 antibodies (Abs) on CIA and serum levels of cytokines in wild-type DBA/1J mice, which are known to be susceptible to CIA, and in Anx7-Tg-B6 mice. In vitro, the authors examined the effect of the Anx7 knockdown by small interfering RNA on the secretion of cytokines in rheumatoid synoviocytes and the human synovial sarcoma cell line SW982. RESULTS: The Anx7 transgene altered the CIA-resistant B6 mice to CIA-susceptible ones. The Abs treatment suppressed CIA even in the wild-type DBA/1J mice. The serum levels of cytokines including interleukin 6 (IL-6) and TNFα were not altered by the Abs treatment in vivo. On the other hand, the knockdown of Anx7 by small interfering RNA caused downregulation of IL-8 secretion in vitro. CONCLUSIONS: These results indicate that Anx7 participates in the pathogenesis of RA partly through the secretion of IL-8. The study data have demonstrated the pathogenic roles and therapeutic significance of Anx7 in RA for the first time.

Protein profiles of peripheral blood mononuclear cells are useful for differential diagnosis of ulcerative colitis and Crohn's disease.

BACKGROUND: Effective biomarkers for discrimination between ulcerative colitis (UC) and Crohn's disease (CD) have not been established yet. In this study, we analyzed protein profiles of peripheral blood mononuclear cells (PBMCs) of the patients to find such a biomarker. METHODS: Peripheral blood mononuclear cell proteins from 17 UC patients, 13 CD patients, and 17 healthy controls were separated by two-dimensional gel electrophoresis. The intensities of individual protein spots were subjected to discriminant analysis of UC and CD using the SIMCA-P+program. RESULTS: We found that 547 protein spots were commonly detected among the UC, CD, and healthy groups. Orthogonal partial least squares-discriminant analysis using 276 protein spots clearly discriminated the UC patients from the CD patients (R (2) 0.994; Q (2) 0.462). A similar analysis using a further selected 58 protein spots showed higher performance for discrimination of the diseases (R (2) 0.948; Q (2) 0.566). Eleven out of the 58 protein spots were successfully identified; these were functionally related to inflammation, oxidation/reduction, the cytoskeleton, endocytotic trafficking, and transcription. In addition, the PBMC protein profiles were useful for the prediction of disease activity in the UC and the CD patients, and they were also useful for predicting disease severity and responses to treatments in the UC patients. CONCLUSIONS: PBMC protein profiles are useful for the discrimination of UC from CD. The profiles could be a potent biomarker for the differential diagnosis of these diseases. Further investigation of the proteins which contributed to the discrimination could promote elucidation of the pathophysiology of UC and CD.

Comprehensive analysis of short peptides in sera from patients with IgA nephropathy.

We analyzed serum short peptides comprehensively to know whether they were useful to characterize IgA nephropathy (IgAN). Serum samples from 26 patients with untreated IgAN and 25 healthy donors were tested. Short peptides with molecular weights of approximately 7 kDa, purified from the serum samples by magnetic-beads-based weak cation exchange, were detected by mass spectrometry. Then the peptide peaks detected were subjected to the multivariate data analysis by SIMCA-P+ containing principal component analysis (PCA) and orthogonal partial-least-squares-discriminate analysis (OPLS-DA). A total of 92 peptide peaks were detected in the tested serum samples. The OPLS-DA analysis revealed that the profile of all the peptide peak intensities discriminated the IgAN group and the healthy group completely with a high R2 value (0.919) and a high Q2 value (0.861). Further, the profile of only five peptide peaks was found to discriminate the two groups. By tandem mass spectrometry and database searching, three of the five peptides which increased in the IgAN group were identified as fragments of fibrinogen alpha chain, and the two peptides which increased in the healthy group were identified as fragments of complement C3f and kininogen-1 light chain. Taken together, the profile of the serum short peptides would be useful to discriminate IgAN and healthy conditions. Further, the five peptides may be candidate serum markers for IgAN and may be related to pathogenesis of IgA.

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.

Growth factor-induced phosphorylation of sterol regulatory element-binding proteins inhibits sumoylation, thereby stimulating the expression of their target genes, low density lipoprotein uptake, and lipid synthesis.

The destiny and activity of sterol regulatory element-binding proteins (SREBPs) in the nucleus are regulated by modification with ubiquitin, small ubiquitin-like modifier (SUMO), or phosphorus. ERK-dependent phosphorylation causes an increase in their transcriptional activity, whereas SUMO modification halts it. We hypothesized a causal linkage between phosphorylation and sumoylation because their sites are very closely located in SREBP-1 and -2 molecules. When Ser(455), a phosphorylation site in SREBP-2, was substituted with Ala, this SREBP-2 mutant was more efficiently modified by SUMO-1. On the other hand, substitution of Asp inhibited SUMO conjugation, mimicking phosphoserine. When cells were cultured with insulin-like growth factor-1, sumoylation of SREBP-2 was decreased with an increase in its phosphorylation, but SREBP-2(S455A) was continuously sumoylated. An ERK cascade inhibitor, U0126, inversely augmented SUMO modification of SREBP-2. Insulin-like growth factor-1 treatment stimulated the expression of SREBP target genes such as the low density lipoprotein (LDL) receptor, squalene synthase, and hydroxymethylglutaryl-CoA synthase genes. These results indicate that growth factor-induced phosphorylation of SREBP-2 inhibits sumoylation, thereby facilitating SREBP transcriptional activity. Glutathione S-transferase pulldown assays revealed that wild-type SREBP-2, but not a mutant lacking Lys(464), interacts with HDAC3 preferentially among the histone deacetylase family members. HDAC3 small interfering RNA induced gene expression of the LDL receptor and thereby augmented fluorescently labeled LDL uptake in HepG2 cells. In summary, growth factors inhibit sumoylation of SREBPs through their phosphorylation, thus avoiding the recruitment of an HDAC3 corepressor complex and stimulating the lipid uptake and synthesis required for cell growth.

Interaction between sterol regulatory element-binding proteins and liver receptor homolog-1 reciprocally suppresses their transcriptional activities.

In previous studies it was demonstrated that sterol regulatory element-binding proteins (SREBPs) are able to interact with one of the nuclear receptors, hepatocyte nuclear receptor (HNF)-4, and that this interaction regulates transcriptional activities of these proteins (Misawa, K., Horiba, T., Arimura, N., Hirano, Y., Inoue, J., Emoto, N., Shimano, H., Shimizu, M., and Sato, R. (2003) J. Biol. Chem. 278, 36176-36182; Yamamoto, T., Shimano, H., Nakagawa, Y., Ide, T., Yahagi, N., Matsuzaka, T., Nakakuki, M., Takahashi, A., Suzuki, H., Sone, H., Toyoshima, H., Sato, R., and Yamada, N. (2004) J. Biol. Chem. 279, 12027-12035). In an attempt to identify other nuclear receptor family members affecting the SREBP transcriptional activities, we found that the liver receptor homolog (LRH)-1 suppresses them. Several types of luciferase assays revealed that coexpression of these two proteins (LRH-1 and SREBP-1a, -1c, or -2) results in reciprocal inhibition of the transcriptional activity of each protein. It was confirmed that suppression in endogenous LRH-1 by small interference RNA stimulates the mRNA levels of certain SREBP target genes and that elevation in active SREBPs in the nucleus in response to cholesterol depletion suppresses the LRH-1 activity. In vitro/in vivo glutathione S-transferase pulldown experiments demonstrated that the basic helix-loop-helix-leucine zipper domain in SREBP-2 binds to the ligand-binding domain in LRH-1. Furthermore, we found that SREBP-2 interferes with the recruitment of a coactivator of LRH-1, the peroxisome proliferator-activated receptor gamma coactivator-1alpha, thereby leading to the inhibition of the LRH-1 transcriptional activity. These results clearly indicate that the interaction between SREBPs and LRH-1 exerts a suppressive influence on their target gene expression responsible for cholesterol and bile acid metabolism.