Layilin promotes mitochondrial fission by cyclin-dependent kinase 1 and dynamin-related protein 1 activation in HEK293T cells.

OBJECT: Functions of layilin, a type 1 transmembrane protein with a C-type lectin motif, remain to be clarified. We here investigated precise intracellular localization of layilin and the location-related functions. METHODS: We used HEK293T cells to assess the co-localization of layilin with different individual organelle markers by double immunostaining. We then investigated mitochondrial morphology in layilin-knockdown (KD) conditions, also with immunostaining. Next, we measured amounts of proteins involved in regulation of mitochondrial dynamics, DRP1, pS616-DRP1, mitofusin1, mitofusin2, CDK1, pY15-CDK1, and cyclin B1, in layilin-KD cells versus control cells by Western blot. Furthermore, by using layilin-knockout (KO) cells, amounts of CDK1 and pY15-CDK1 as well as mitochondrial morphology were investigated. RESULT: We found that layilin localized to mitochondria rather than the other organelles. Small round-shape mitochondria were observed in control cells, whereas elongated and highly connected mitochondria were observed in layilin-KD cells. Amounts of active DRP1 (pS616-DRP1) and total DRP1 were significantly smaller in layilin-KD cells than in controls. Amounts of inactive CDK1 (pY15-CDK1) were significantly larger in layilin-KD cells than in controls. No other tested molecules were significantly altered in layilin-KD cells. Amounts of inactive CDK1 were significantly larger in layilin-KO cells than in wild type (WT) cells. Small round-shape mitochondria were observed in WT cells, whereas elongated and highly connected mitochondria were observed in layilin-KO cells. CONCLUSION: We here demonstrated that layilin played a role in the maintenance of fragmented mitochondria in mitochondrial dynamics and that this function needed CDK1 and DRP1 activation. Our data unveiled a novel function for layilin, regulation of mitochondrial dynamics.

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.

Aberrant Glycosylation of Lumican in Aortic Valve Stenosis Revealed by Proteomic Analysis.

To identify proteins related to the pathophysiology of aortic valve stenosis (AS), we investigated the protein profiles of AS aortic valves. Specifically, proteins were extracted from a thickened and calcified area (AS-C) and an apparently non-thickened and non-calcified area (AS-N) in an identical aortic valve leaflet in each of 6 AS patients. The proteins were then separated by 2-dimensional gel electrophoresis (2DE). Protein spots detected by 2DE were compared between the AS-C and AS-N samples. Protein spots of interest were subjected to protein identification by mass spectrometry.In total, 670 protein spots were detected by 2DE, 28 of which showed more than 1.5-fold different intensity (P < 0.05) between the AS-C and AS-N samples. Proteins were identified in 17 out of the 28 spots. Fibrinogen and lumican were identified in 9 and 3 spots, respectively. Intensity of these 12 spots was lower in the AS-C samples than in the AS-N samples. In the 1D-Western blot analysis, 4 lumican bands (80 kDa, 75 kDa, 65 kDa, and 53 kDa) were detected, of which 2 bands with 80 kDa and 75 kDa showed lower intensity in the AS-C samples than in the AS-N samples. When de-glycosylated protein samples were used in the 1D-Western blot, only a single lumican band with ~40 kDa was detected, indicating that lumican was variously glycosylated and that highly glycosylated lumican molecules were decreased in AS-C.Collectively, insufficient glycosylation of lumican in the thickened and calcified areas of AS aortic valves may be involved in the pathophysiology of AS.

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.

Roles of serum fibrinogen α chain-derived peptides in Alzheimer's disease.

OBJECTIVE: To find a blood biomarker and disease-related peptides in Alzheimer's disease (AD), we comprehensively detected serum peptides. METHODS: Ion intensity of serum peptides from 62 AD patients and 82 control subjects was measured by mass spectrometry. RESULTS: A total of 157 peptides were detected from 30 AD patients and 30 healthy control (HC) subjects. Sixty out of the 157 peptide profiles discriminated between the AD and HC groups. Sixteen out of the 60 peptides were identified, 10 out of which were fragments of a fibrinogen α chain (FIBA). Among the 10 peptides, four and six peptides were derived from fibrinopeptide A (FPA, Aα1-16) and the C-terminal region of the αC-domain (αCDC, Aα557-610), respectively. The profile of 10 FIBA-derived peptides combined with age discriminated between the two groups with an area under the receiver operating characteristic curve (AUROC) of 0.940. Validation of this model using a testing set of 32 AD patients and 19 HC subjects showed an AUROC of 0.717, sensitivity of 65.6%, and specificity of 73.7% by a cutoff value of 0.56420. Another value, 0.04029, showed sensitivity of 96.9%, suggesting that subjects with values less than 0.04029 rarely possess AD. FPA and αCDC showed increased ion intensity in the AD group compared with the HC group (p < 0.05). CONCLUSIONS: The profile of 10 FIBA-derived peptides combined with age would be a candidate biomarker for AD, which facilitates screening of the disease. The significant release of FPA and αCDC may be involved in the aberrant coagulation that leads to vascular damage in AD.

Effects of sulfasalazine and tofacitinib on the protein profile of articular chondrocytes.

OBJECTIVE: Sulfasalazine (SSZ) and tofacitinib are effective for treating rheumatoid arthritis, however, their effects on chondrocytes have not been fully understood. We here tried to elucidate their effects on chondrocyte proteins. METHODS: We treated chondrocytes from five osteoarthritis patients with IL-1ß, IL-1ß+ SSZ, IL-1ß+ tofacitinib, SSZ alone, and tofacitinib alone. Then, we compared protein profiles of the chondrocytes using two-dimensional differential gel electrophoresis. Further, we identified altered proteins by mass spectrometry. RESULTS: Out of 892 detected protein spots, the IL-1ß stimulation changed intensity of 43 spots more than 1.3-fold or less than 1/1.3-fold significantly. SSZ suppressed the IL-1ß-induced intensity alteration in 16 (37%) out of the 43 protein spots. Tofacitinib suppressed the IL-1ß-induced alteration in 4 (9.3%) out of the 43 spots. The production of AMP deaminase 2 and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 were increased by IL-1ß and the increase was suppressed by SSZ and by tofacitinib. SSZ alone altered intensity of 273 (31%) out of the 852 spots significantly, whereas tofacitinib alone altered intensity of only 24 (2.7%) out of them. CONCLUSION: SSZ and, to lesser extent, tofacitinib suppress the effects of IL-1ß on the protein profiles of chondrocytes. Our data would promote understanding of effects of the drugs on chondrocytes.

Role of layilin in regulating mitochondria-mediated apoptosis: a study on B cell lymphoma (BCL)-2 family proteins.

BACKGROUND: Malignant gliomas exhibit rapid tumor progression and resistance to treatment, leading to high lethality. One of the causes is the reduced progression of apoptosis in glioma cells. Layilin is a type 1 transmembrane protein with a C-type lectin motif in its extracellular domain. We previously reported that layilin is mainly localized to mitochondria or their close proximity and that layilin is essential for maintaining of the fragmented type of mitochondria. This study investigates the effects of layilin on mitochondria-mediated apoptosis, focusing on B cell lymphoma (BCL)-2 family proteins in a glioma cell line of A172 cells. RESULTS: We compared the levels of pro-apoptotic BCL-2 family proteins of BAD, BAK, BAX, and BIM and anti-apoptotic BCL-2 family proteins of BCL-2 and BCL-XL between layilin- knockdown (KD) cells and control cells using western blot. The protein levels of BAD were significantly smaller in layilin-KD cells than in control cells, while those of BCL-2 were significantly larger. We then compared the mitochondrial membrane potential (ΔΨm) under p-trifluoromethoxyphenyl hydrazone (FCCP)-treated conditions using MT-1 staining. In layilin-KD cells, ΔΨm was significantly larger and FCCP-induced ΔΨm reduction was significantly lower than in control cells. Furthermore, we examined the levels of cell membrane-bound Annexin V and DNA-bound propidium idodide (PI) in layilin-KD cells with/without staurosporine (STS) treatment. Layilin-KD significantly decreased levels of cell membrane-bound Annexin V with/without STS treatment. On the other hand, PI levels were not changed by layilin-KD. We also investigated the amounts of the active caspase (CASP)-3, CASP-6, CASP-7, and poly (ADP-ribose) polymerase-1 (PARP1, cleaved form), as well as DNA fragmentation in layilin-KD cells under apoptotic conditions induced by STS, using western blot and the DNA ladder method, respectively. Under STS-treated conditions, the amounts of active CASP-3, CASP-7, and poly (ADP-ribose) PARP1 were significantly smaller in layilin-KD cells than in control cells. Accordingly, DNA fragmentation was significantly suppressed in layilin-KD cells compared to control cells under STS-treated conditions. CONCLUSION: This study demonstrates that layilin contributes to ΔΨm reduction to promote apoptosis by up-regulating BAD and down-regulating BCL-2 in glioma cells. Our data elucidates a new function of layilin: regulation of mitochondria-mediated apoptosis.

Serum peptides, represented by complement 3f des-arginine, are useful for prediction of the response to pegylated interferon-α plus ribavirin in patients with chronic hepatitis C.

AIM: Biomarkers predicting sustained virological response (SVR) to pegylated interferon-α plus ribavirin (PEG IFN-α/RBV) were investigated. METHODS: Peptides in pretreatment sera from 107 patients with hepatitis C virus (HCV) genotype 1 were comprehensively analyzed by mass spectrometry. Ion intensity of the peptides was used to generate discriminant models between the responders who achieved SVR (R) and the non-responders (NR) to PEG IFN-α/RBV. RESULTS: In total, 107 peptides were detected in a training set (n = 23). A discriminant model using a peptide, complement 3f des-arginine (C3f-dR), showed sensitivity of 35% and specificity of 94% for SVR prediction in a testing set (n = 68). In all the R and NR (n = 96), an area under the receiver-operator curve (AUROC) of 0.64 in the C3f-dR model was increased to 0.78 by addition of platelet (PLT) counts (C3f-dR/PLT model). Another model using the 107 peptides (AUROC, 0.77) also showed higher AUROC (0.79) by addition of hemoglobin (Hb), body mass index (BMI) and age (107P/Hb/BMI/Age model). The sensitivity and specificity of the C3f-dR/PLT model were 59% and 88%, and those of the 107P/Hb/BMI/Age model were 70% and 92%, respectively. The C3f-dR/PLT model showed high AUROC (0.82), similar to that of interleukin-28B rs8099917 genotype analysis (0.86) in the 45 tested patients. Prediction by the combination of the C3f-dR/PLT model, the 107P/Hb/BMI/Age model and the rs8099917 genotype analysis was accurate in 44 out of the 45 patients (AUROC, 0.95). CONCLUSION: Serum peptides, especially C3f-dR, would be useful predictors for SVR to PEG IFN-α/RBV. The complements may be involved in the HCV elimination.

Altered posttranslational modification on U1 small nuclear ribonucleoprotein 68k in systemic autoimmune diseases detected by 2D Western blot.

Anti-ribonucleoprotein (anti-RNP) antibodies are one of the representative autoantibodies detectable in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Generally, posttranslational modifications (PTMs) on autoantigens are proposed to be involved in the production of autoantibodies. In this study, we tried to detect the alteration in PTMs on a U1 small nuclear RNP 68k subunit (U1-68k), a major antigen of anti-RNP antibodies. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with MCTD, SLE, and rheumatoid arthritis (RA), and from healthy donors. U1-68ks in the PBMCs were detected by 2D Western blot (WB), where extracted nuclear proteins were separated by 2DE, followed by the detection of U1-68k using WB. More than 20 PTM isoforms were detected with different molecular weights of 65.0 , 66.5, and 68.0kDa, and different pIs between 6.0 and 8.5. Importantly, the relative intensity of the spot with 66.5 kDa and pI 7.5 was significantly increased in the MCTD and SLE groups compared to the RA and healthy groups. Further, this U1-68k isoform, in particular, in its RS domain, was found to have significantly decreased phosphorylation compared to the other isoforms. The PTM alternation may be one of the steps to generate the anti-RNP antibodies.

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.

Identification of novel substrates of a disintegrin and metalloprotease 17 by specific labeling of surface proteins.

A disintegrin and metalloprotease 17 (ADAM17) catalyzes the cleavage and release of the ectodomains of its substrates at the cell surface in a process termed ectodomain shedding. However, not all ADAM17 substrates have been identified. Here, we used cell surface protein-specific labeling and proteomic approaches to detect and identify ADAM17 substrates. HeLa cell surface proteins were labeled with a fluorescent dye and cultured with or without TAPI-2, an ADAM17 inhibitor. Labeled proteins released into the culture medium were detected by 2-dimensional gel electrophoresis (2DE). Protein spots showing decreased intensity in response to TAPI-2 were selected as substrates of ADAM17 or their binding proteins, and identified by mass spectrometry. ADAM17 knockdown was preformed to examine the behavior of identified proteins. Of 347 proteins detected by 2DE, 49 showed lower intensity in TAPI-2 (+) than in TAPI-2 (-) samples (p < 0.05), and were considered as candidate substrates of ADAM17. Mass spectrometric analysis of 14 protein spots showing >50% decreased intensity identified clusterin as a novel ADAM17 substrate, in addition to known substrates such as desmoglein-2. Western blot analysis showed that ADAM17 knockdown decreased the levels of clusterin fragments cleaved and released from the cell surface. The results identified clusterin as a novel ADAM17 substrate. The method used to identify clusterin could be used to identify the substrates of other sheddases involved in ectodomain shedding.

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.

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.

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.