Regulation of S100A10 Gene Expression.

S100A10, a member of the S100 family of Ca2+-binding proteins, is a widely distributed protein involved in many cellular and extracellular processes. The best recognized role of S100A10 is the regulation, via interaction with annexin A2, of plasminogen conversion to plasmin. Plasmin, together with other proteases, induces degradation of the extracellular matrix (ECM), which is an important step in tumor progression. Additionally, S100A10 interacts with 5-hydroxytryptamine 1B (5-HT1B) receptor, which influences neurotransmitter binding and, through that, depressive symptoms. Taking this into account, it is evident that S100A10 expression in the cell should be under strict control. In this work, we summarize available literature data concerning the physiological stimuli and transcription factors that influence S100A10 expression. We also present our original results showing for the first time regulation of S100A10 expression by grainyhead-like 2 transcription factor (GRHL2). By applying in silico analysis, we have found two highly conserved GRHL2 binding sites in the 1st intron of the gene encoding S100A10 protein. Using chromatin immunoprecipitation (ChIP) and luciferase assays, we have shown that GRHL2 directly binds to these sites and that this DNA region can affect transcription of S100A10.

Expression of p11 and Heteromeric TASK Channels in Rat Carotid Body Glomus Cells and Nerve Growth Factor-differentiated PC12 Cells.

TWIK-related acid-sensitive K+ (TASK) homomeric channels, TASK1 and TASK3, are present in PC12 cells. The channels do not heteromerize due plausibly to a lack of p11 protein. Single-channel recording reveals that most of the rat carotid body (CB) glomus cells express heteromeric TASK1-TASK3 channels, but the presence of p11 in glomus cells has not yet been verified. TASK1, but not TASK3, binds to p11, which has a retention signal for the endoplasmic reticulum. We hypothesized that p11 could facilitate heteromeric TASK1-TASK3 formation in glomus cells. We investigated this hypothesis in isolated immunocytochemically identified rat CB glomus cells. The findings were that glomus cells expressed p11-like immunoreactive (IR) material, and TASK1- and TASK3-like IR material mainly coincided in the cytoplasm. The proximity ligation assay showed that TASK1 and TASK3 heteromerized. In separate experiments, supporting evidence for the major role of p11 for channel heteromerization was provided in PC12 cells stimulated by nerve growth factor. p11 production took place there via multiple signaling pathways comprising mitogen-activated protein kinase and phospholipase C, and heteromeric TASK1-TASK3 channels were formed. We conclude that p11 is expressed and TASK1 and TASK3 heteromerize in rat CB glomus cells.

Two tales of Annexin A2 knock-down: One of compensatory effects by antisense RNA and another of a highly active hairpin ribozyme.

Besides altering its own expression during cell transformation, Annexin A2 is upregulated during the progression of many cancer types and also plays key roles during viral infection and multiplication. Consequently, there has been great interest in Annexin A2 as a potential drug target. The successful design of efficient in vivo delivery systems constitutes an obstacle in full exploitation of antisense and RNA-cleaving technologies for the knock-down of specific targets. Efficiency is dependent on the method of delivery and accessibility of the target. Here, hairpin ribozymes and an antisense RNA against rat annexin A2 mRNA were tested for their efficiencies in a T7-driven coupled transcription/translation system. The most efficient ribozyme and antisense RNA were subsequently inserted into a retroviral vector under the control of a tRNA promoter, in a cassette inserted between retroviral Long Terminal Repeats for stable insertion into host DNA. The Phoenix package system based on defective retroviruses was used for virus-mediated gene transfer into PC12 cells. Cells infected with the ribozyme-containing particles died shortly after infection. However, the same ribozyme showed a very high catalytic effect in vitro in cell lysates, explained by its loose hinge helix 2 region. This principle can be transferred to other ribozymes, such as those designed to cleave the guide RNA in the CRISPR/Cas9 technology, as well as to target specific viral RNAs. Interestingly, efficient down-regulation of the expression of Annexin A2 by the antisense RNA resulted in up-regulation of Annexin A7 as a compensatory effect after several cell passages. Indeed, compensatory effects have previously been observed during gene knock-out, but not during knock-down of protein expression. This highlights the problems in interpreting the phenotypic effects of knocking down the expression of a protein. In addition, these data are highly relevant when considering the effects of the CRISPR/Cas9 approach.

High expression of Anxa2 and Stat3 promote progression of hepatocellular carcinoma and predict poor prognosis.

AIM: To elucidate whether the interaction between Anxa2 and Stat3 could promote the progression of hepatocellular carcinoma (HCC) and that high co-expression of Anxa2 and Stat3 could predict poor prognosis in HCC patients. METHODS: We investigated Anxa2 and Stat3 expression using Western blot analysis in 4 HCC and adjacent nontumor tissues and using immunohistochemistry in 100 patients' paraffin sections. Then we assessed the expression of Stat3, Anxa2 and co-expression of Stat3 and Anxa2 with relevant clinical pathological parameters and their prognostic value in HCC patients. The recurrence and overall survival rates were estimated using the Kaplan-Meier method and compared with the log-rank test. The prognostic analysis was carried out with univariate and multivariate Cox regressions models. RESULTS: The incidence of high Stat3 expression in HCC tissues (35%) was significantly higher than that in non-HCC tissues (8%) (P < 0.001). The same result was observed in Anxa2 (P < 0.001). Also, the overexpression of Stat3 or Anxa2 showed a significant relationship with the recurrence of the 100 HCC patients (P = 0.012; P = 0.003). Additionally, tumor size >3 cm in diameter, multiple tumor number, and the presence of microvascular tumor thrombus were also significantly associated with recurrence in 100 patients. Then, all enrolled patients were divided into four groups according to IHC score of Stat3 and Anxa2, and the results indicated a significant difference in recurrence time between the subgroups (P < 0.001). What's more, co-highexpression of Stat3 and Anxa2 was related to the presence of microvascular tumor thrombus (P = 0.003) and poor tumor differentiation (P < 0.001), but not relevant with other clinical features (All P > 0.05). CONCLUSION: The expression of Stat3, Anxa2, or co-high-expression of the two proteins was associated with HCC recurrence and survival.

iTRAQ-based proteomic analysis of DMH-induced colorectal cancer in mice reveals the expressions of ß-catenin, decorin, septin-7, and S100A10 expression in 53 cases of human hereditary polyposis colorectal cancer.

PURPOSE: The aim of this study is to explore the roles of ß-catenin, decorin, septin-7, and S100A10 expression in colorectal cancer development. METHODS: Twenty-five BALB/c mice were divided into five groups; four groups were administrated N,N-dimethylhydrazine for 0, 10, 15, and 20 weeks, and one group was administrated normal saline for 20 weeks. The colons were collected for histopathological analysis. Protein samples prepared from the frozen colon tissues of mice treated with N,N-dimethylhydrazine for the different time points were evaluated using the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique coupled with the 2D liquid chromatography-tandem mass spectrometry analysis. Based on the proteomic analysis results, immunohistochemical staining of ß-catenin, decorin, septin-7, and S100A10 was performed in paraffin-embedded mice colorectal tissue, and 53 cases of human hereditary polyposis colorectal cancer samples. RESULTS: Colorectal cancer was observed in mice treated with N,N-dimethylhydrazine for 20 weeks, and adenomas were observed in mice subjected to the 10-, and 15-week treatments. Seventy-two differentially expressed proteins were involved in the development of cancer as per the iTRAQ and spectrometry analysis. In normal epithelium, adenoma, and cancer from human hereditary polyposis colorectal cancer, S100A10 expression (c2 = 100.989, P = 0.000) was highest in cancer, whereas decorin (c2 = 12.852, P = 0.002) and septin-7 (c2 = 66.519, P = 0.002) expressions were highest in the normal epithelium, which was confirmed via immunohistochemical staining. CONCLUSIONS: The subcellular localization of ß-catenin and decorin, septin-7, and S100A10 expressions are associated with the development of colorectal cancer in mice after N,N-dimethylhydrazine treatment and in human hereditary polyposis colorectal cancers.

ANXA2 expression in African American triple-negative breast cancer patients.

PURPOSE: Our aim was to determine the role of Annexin A2 (AnxA2), which we have previously found to contribute to the aggressiveness of TNBC, with AA TNBC patients and clinical outcome. METHODS: We analyzed TCGA breast cancer database (n = 1098) to observe AnxA2 expression within breast cancer subtypes and is correlation with overall survival. Further, we examined breast tissue specimens (n = 119) through chromogenic in situ hybridization (CISH) and specimen were scored independently by two pathologists in a blinded study. RESULTS: In our TCGA analysis, high expression of AnxA2 was correlated with poor survival in patients with TNBC. AnxA2 gene expression was not correlated with poor survival in other breast cancer subtypes. AnxA2 average CISH intensity score (CISH score = 0, null expression to 3, high expression) for TNBC was significantly higher in comparison to estrogen receptor and/or progesterone receptor positive, human epidermal growth factor positive, and non-malignant tissues. Furthermore, AnxA2 average score was significantly higher in AA TNBC patients (CISH average score = 2.45 ± 0.3266) in comparison to Caucasian TNBC patients (CISH average score = 1.1 ± 0.4069). CONCLUSION: AnxA2 is overexpressed in TNBC, implicating AnxA2 as a contributor to the aggressive biology of TNBC in AA women.

ANXA2 promotes esophageal cancer progression by activating MYC-HIF1A-VEGF axis.

BACKGROUND: ANXA2 (Annexin A2) is a pleiotropic calcium-dependent phospholipid binding protein that is abnormally expressed in various cancers. We previously found that ANXA2 is upregulated in esophageal squamous cell carcinoma (ESCC). This study was designed to investigate the functional significance of ANXA2 dysregulation and underlying mechanism in ESCC. METHODS: Proliferation, migration, invasion and metastasis assay were performed to examine the functional roles of ANXA2 in ESCC cells in vitro and in vivo. Real-time RT-PCR, immunoblotting, ChIP, reporter assay, confocal-immunofluorescence staining, co-immunoprecipitation and ubiquitination assay were used to explore the molecular mechanism underlying the actions of deregulated ANXA2 in ESCC cells. RESULTS: Overexpression of ANXA2 promoted ESCC cells migration and invasion in vitro and metastasis in vivo through activation of the MYC-HIF1A-VEGF cascade. Notably, ANXA2 phosphorylation at Tyr23 by SRC led to its translocation into the nucleus and enhanced the metastatic potential of ESCC cells. Phosphorylated ANXA2 (Tyr23) interacted with MYC and inhibited ubiquitin-dependent proteasomal degradation of MYC protein. Accumulated MYC directly potentiated HIF1A transcription and then activated VEGF expression. Correlation between these molecules were also found in ESCC tissues. Moreover, dasatinib in combination with bevacizumab or ANXA2-siRNA produced potent inhibitory effects on the growth of ESCC xenograft tumors in vivo. CONCLUSIONS: This study provides evidence that highly expressed p-ANXA2 (Tyr23) contributes to ESCC progression by promoting migration, invasion and metastasis, and suggests that targeting the SRC-ANXA2-MYC-HIF1A-MYC axis may be an efficient strategy for ESCC treatment.

Hydrogen peroxide is a neuronal alarmin that triggers specific RNAs, local translation of Annexin A2, and cytoskeletal remodeling in Schwann cells.

Schwann cells are key players in neuro-regeneration: They sense "alarm" signals released by degenerating nerve terminals and differentiate toward a proregenerative phenotype, with phagocytosis of nerve debris and nerve guidance. At the murine neuromuscular junction, hydrogen peroxide (H2O2) is a key signal of Schwann cells' activation in response to a variety of nerve injuries. Here we report that Schwann cells exposed to low doses of H2O2 rewire the expression of several RNAs at both transcriptional and translational levels. Among the genes positively regulated at both levels, we identified an enriched cluster involved in cytoskeleton remodeling and cell migration, with the Annexin (Anxa) proteins being the most represented family. We show that both Annexin A2 (Anxa2) transcript and protein accumulate at the tips of long pseudopods that Schwann cells extend upon H2O2 exposure. Interestingly, Schwann cells reply to this signal and to nerve injury by locally translating Anxa2 in pseudopods, and undergo an extensive cytoskeleton remodeling. Our results show that, similarly to neurons, Schwann cells take advantage of local protein synthesis to change shape and move toward damaged axonal terminals to facilitate axonal regeneration.

Differential expression of Annexin 2, SPINK1, and Hsp60 predict progression of prostate cancer through bifurcated WHO Gleason score categories in African American men.

BACKGROUND: Although studies have observed several markers correlate with progression of prostate cancer (PCa), no specific markers have been identified that accurately predict the progression of this disease, even in African American (AA) men who are generally at higher risk than other ethnic groups. The primary goal of this study was to explore whether three markers could predict the progression of PCa. METHOD: We investigated protein expression of Annexin 2 (ANX2), serine peptidase inhibitor, kazal type 1(SPINK1)/tumor-associated trypsin inhibitor (TATI), and heat shock protein 60 (Hsp60) in 79 archival human prostate trans-rectal ultrasound (TRUS) biopsy tissues according to a modified World Health Organization (WHO) classification: normal (WHO1a), Gleason Score (GS6 (WHO1b), GS7 subgroups (WHO2 = 3 + 4, WHO3 = 4 + 3), GS8 (WHO4), and GS9-10 (WHO5). AA men aged 41-90 diagnosed from 1990 to 2013 at Howard University were included. Automated staining assessed expression of each biomarker. Spearman correlation assessed the direction and relationship between biomarkers, WHO and modified WHO GS, age, and 5-year survival. A two-tailed t-test and ANOVA evaluated biomarkers expression in relationship to WHO normal and other GS levels, and between WHO GS levels. A logistic and linear regression analysis examined the relationship between biomarker score and WHO GS categories. Kaplan-Meier curves graphed survival. RESULTS: ANX2 expression decreased monotonically with the progression of PCa while expression of SPINK1/TATI and Hsp60 increased but had a more WHO GS-specific effect; SPINK1/TATI differed between normal and GS 2-6 and HSP60 differed between GS 7 and GS 2-6. WHO GS was found to be significantly and negatively associated with ANX2, and positively with SPINK1/TATI and Hsp60 expression. High SPINK1/TATI expression together with the low ANX2 expression at higher GS exhibited a bi-directional relationship that is associated with PCa progression and survival. CONCLUSION: Importantly, the data reveal that ANX2, and SPINK1/TAT1 highly associate with WHO GS and with the transition from one stage of PrCa to the next in AA men. Future research is needed in biracial and larger population studies to confirm this dynamic relationship between ANX2 and SPINK1 as independent predictors of PCa progression in all men.

Annexin A2 and cancer: A systematic review.

Annexin A2 is a 36-kDa protein interfering with multiple cellular processes especially in cancer progression. The present review aimed to show the relations between Annexin A2 and cancer. A systematic search for studies investigating cancer and Annexin A2 expression was conducted using PubMed. Acute lymphoblastic leukaemia, acute promyelocytic leukaemia, clear cell renal cell carcinoma, breast, cervical, colorectal, endometrial, gastric cancer, glioblastoma, hepatocellular carcinoma, lung, multiple myeloma, oesophageal squamous cell carcinoma, ovarian cancer, pancreatic duct adenocarcinoma, prostate cancer and urothelial carcinoma were evaluated. Annexin A2 expression correlates with resistance to treatment, binding to the bone marrow, histological grade and type, TNM-stage and shortened overall survival. The regulation of Annexin A2 is of interest due to its potential as target for a more individualized cancer management.

Characterization of the adrenocorticotrophic hormone - induced mouse model of resistance to antidepressant drug treatment.

Approximately 30-60% of patients treated with existing antidepressants fail to achieve remission of depressive symptoms leading to Treatment Resistant Depression (TRD). There is an urgent need to develop novel medications, which is highly limited by the non-availability of relevant animal models with good predictive validity. ACTH administration has been shown to result in the resistance to acute and chronic effects of imipramine. However, the pharmacology of the model and the mechanisms contributing to the resistance are not completely understood. Furthermore, it is not known whether the ACTH administered animals show signs of depression-like behavior. Accordingly, we characterized the behavioral profile and sensitivity to antidepressants in BALB/c mice treated with ACTH and to evaluate some of the mechanisms responsible for the behavioral effects. Daily treatment with ACTH for 14, 21 or 28days failed to produce a depression-like phenotype in the sucrose preference test, voluntary wheel running or FST. In contrast, the acute antidepressant response in the FST was no longer observed in ACTH mice treated with fluoxetine, imipramine, duloxetine or bupropion. Interestingly, the combination of fluoxetine and a low dose of olanzapine, or the combination of fluoxetine and bupropion was efficacious in ACTH treated mice. Further, the sensitivity to a GluN2B receptor antagonist, radiprodil was retained in the ACTH model. To understand the mechanism responsible for the diminished response in these mice, we evaluated p11 (S100A10) mRNA expression and 5-HT2A protein expression. p11 expression was decreased and 5-HT2A protein content increased in ACTH treated mice. In summary, this model may have utility for the identification of novel treatments for TRD.

Circular RNA profiling reveals that circular RNAs from ANXA2 can be used as new biomarkers for multiple sclerosis.

Multiple sclerosis is an autoimmune disease, with higher prevalence in women, in whom the immune system is dysregulated. This dysregulation has been shown to correlate with changes in transcriptome expression as well as in gene-expression regulators, such as non-coding RNAs (e.g. microRNAs). Indeed, some of these have been suggested as biomarkers for multiple sclerosis even though few biomarkers have reached the clinical practice. Recently, a novel family of non-coding RNAs, circular RNAs, has emerged as a new player in the complex network of gene-expression regulation. MicroRNA regulation function through a 'sponge system' and a RNA splicing regulation function have been proposed for the circular RNAs. This regulating role together with their high stability in biofluids makes them seemingly good candidates as biomarkers. Given the dysregulation of both protein-coding and non-coding transcriptome that have been reported in multiple sclerosis patients, we hypothesised that circular RNA expression may also be altered. Therefore, we carried out expression profiling of 13.617 circular RNAs in peripheral blood leucocytes from multiple sclerosis patients and healthy controls finding 406 differentially expressed (P-value < 0.05, Fold change > 1.5) and demonstrate after validation that, circ_0005402 and circ_0035560 are underexpressed in multiple sclerosis patients and could be used as biomarkers of the disease.

Cell- and region-specific expression of depression-related protein p11 (S100a10) in the brain.

P11 (S100a10), a member of the S100 family of proteins, has widespread distribution in the vertebrate body, including in the brain, where it has a key role in membrane trafficking, vesicle secretion, and endocytosis. Recently, our laboratory has shown that a constitutive knockout of p11 (p11-KO) in mice results in a depressive-like phenotype. Furthermore, p11 has been implicated in major depressive disorder (MDD) and in the actions of antidepressants. Since depression affects multiple brain regions, and the role of p11 has only been determined in a few of these areas, a detailed analysis of p11 expression in the brain is warranted. Here we demonstrate that, although widespread in the brain, p11 expression is restricted to distinct regions, and specific neuronal and nonneuronal cell types. Furthermore, we provide comprehensive mapping of p11 expression using in situ hybridization, immunocytochemistry, and whole-tissue volume imaging. Overall, expression spans multiple brain regions, structures, and cell types, suggesting a complex role of p11 in depression. J. Comp. Neurol. 525:955-975, 2017. © 2016 Wiley Periodicals, Inc.

Annexin A2, up-regulated by IL-6, promotes the ossification of ligament fibroblasts from ankylosing spondylitis patients.

BACKGROUND: Annexin A2, a calcium-dependent phospholipid binding protein, is involved in osteogenesis. The objective of the present study was to explore the expression of Annexin A2 in spinal ligament tissues (LT) of ankylosing spondylitis (AS) patients and determine its pathological functions. METHODS: mRNA and protein expression of Annexin A2 was detected by real-time PCR and Western blotting, respectively. Interleukin-6 (IL-6) concentration in serum was assessed by enzyme linked immunosorbent assay. Alkaline phosphatase (ALP) activity was measured with ALP activity kit on a microplate reader. RESULTS: mRNA and protein expression of Annexin A2 in LT, and IL-6 concentration in serum were significantly increased in AS patients. Moreover, exogenous IL-6 treatment significantly up-regulated Annexin A2 expression and ALP activity. Silencing of Annexin A2 expression significantly ameliorated IL-6-induced ossification of fibroblasts from AS patients, as indicated by ALP activity, expression of proteins associated with osteogenic differentiation, including bone morphogenetic protein-2, osteocalcin and osterix, and the ratio of osteoprotegerin to receptor activator of NF-κB ligand. Further MEK inhibitor experiments suggested that Annexin A2 may exert its function through extracellular signal-related kinase pathway. CONCLUSIONS: Annexin A2, up-regulated by IL-6, may promote ligament ossification of AS patients.

AHNAK enables mammary carcinoma cells to produce extracellular vesicles that increase neighboring fibroblast cell motility.

Extracellular vesicles play important roles in tumor development. Many components of these structures, including microvesicles and exosomes, have been defined. However, mechanisms by which extracellular vesicles affect tumor progression are not fully understood. Here, we investigated vesicular communication between mammary carcinoma cells and neighboring nontransformed mammary fibroblasts. Nonbiased proteomic analysis found that over 1% of the entire proteome is represented in these vesicles, with the neuroblast differentiation associated protein AHNAK and annexin A2 being the most abundant. In particular, AHNAK was found to be the most prominent component of these vesicles based on peptide number, and appeared necessary for their formation. In addition, we report here that carcinoma cells produce vesicles that promote the migration of recipient fibroblasts. These data suggest that AHNAK enables mammary carcinoma cells to produce and release extracellular vesicles that cause disruption of the stroma by surrounding fibroblasts. This paradigm reveals fundamental mechanisms by which vesicular communication between carcinoma cells and stromal cells can promote cancer progression in the tumor microenvironment.

Expression and clinical role of chemoresponse-associated genes in ovarian serous carcinoma.

OBJECTIVE: To validate our earlier observation that 11 chemoresistance-associated mRNAs are molecular markers of poor overall survival in ovarian serous carcinoma. METHODS: Ovarian serous carcinomas (n=112) and solid metastases (n=63; total=175) were analyzed for mRNA expression of APC, BAG3, EGFR, S100A10, ITGAE, MAPK3, TAP1, BNIP3, MMP9, FASLG and GPX3 using quantitative real-time PCR. mRNA expression was studied for association with clinicopathologic parameters and survival. Tumor heterogeneity was assessed in 20 cases with >1 specimen per patient. APC, BAG3, S100A10 and ERK1 protein expression by immunohistochemistry was analyzed in 58 specimens (38 primary carcinomas, 20 metastases). RESULTS: BAG3 (p=0.013), TAP1 (p=0.014), BNIP3 (p<0.001) and MMP9 (p=0.036) were overexpressed in primary tumors, whereas S100A10 (p=0.027) and FASLG (p=0.006) were overexpressed in metastases. Analysis of patient-matched primary carcinomas and metastases showed overexpression of APC (p=0.022), MAPK3 (p=0.002) and BNIP3 (p=0.004) in the former. In primary carcinomas, higher APC (p=0.003) and MAPK3 (p=0.005) levels were related to less favorable chemoresponse. Higher S100A10 (p=0.029) and MAPK3 (p=0.041) levels were related to primary chemoresistance. Higher BAG3 (p=0.026) and APC (p=0.046) levels in primary carcinomas were significantly related to poor overall survival in univariate, though not in multivariate survival analysis. S100A10 protein expression was related to poor chemoresponse (p=0.002) and shorter overall (p=0.005) and progression-free (p<0.001) survival, the latter finding retained in multivariate analysis (p=0.035). CONCLUSIONS: Our data provide evidence of heterogeneity in ovarian serous carcinoma and identify APC, MAPK3, BAG3 and S100A10 as potential biomarkers of poor chemotherapy response and/or poor outcome in this cancer.

Comprehensive protein profiling of synovial fluid in osteoarthritis following protein equalization.

OBJECTIVE: The aim of the study was to characterise the protein complement of synovial fluid (SF) in health and osteoarthritis (OA) using liquid chromatography mass spectrometry (LC-MS/MS) following peptide-based depletion of high abundance proteins. DESIGN: SF was used from nine normal and nine OA Thoroughbred horses. Samples were analysed with LC-MS/MS using a NanoAcquity™ LC coupled to an LTQ Orbitrap Velos. In order to enrich the lower-abundance protein fractions protein equalisation was first undertaken using ProteoMiner™. Progenesis-QI™ LC-MS software was used for label-free quantification. In addition immunohistochemistry, western blotting and mRNA expression analysis was undertaken on selected joint tissues. RESULTS: The number of protein identifications was increased by 33% in the ProteoMiner™ treated SF compared to undepleted SF. A total of 764 proteins (462 with≥2 significant peptides) were identified in SF. A subset of 10 proteins were identified which were differentially expressed in OA SF. S100-A10, a calcium binding protein was upregulated in OA and validated with western blotting and immunohistochemistry. Several new OA specific peptide fragments (neopeptides) were identified. CONCLUSION: The protein equalisation method compressed the dynamic range of the synovial proteins identifying the most comprehensive SF proteome to date. A number of proteins were identified for the first time in SF which may be involved in the pathogenesis of OA. We identified a distinct set of proteins and neopeptides that may act as potential biomarkers to distinguish between normal and OA joints.

Annexin II-dependent actin remodelling evoked by hydrogen peroxide requires the metalloproteinase/sphingolipid pathway.

Actin remodeling is a dynamic process associated with cell shape modification occurring during cell cycle and proliferation. Oxidative stress plays a role in actin reorganization via various systems including p38MAPK. Beside, the mitogenic response evoked by hydrogen peroxide (H2O2) in fibroblasts and smooth muscle cells (SMC) involves the metalloproteinase (MMPs)/sphingomyelinase 2 (nSMase2) signaling pathway. The aim of this work was to investigate whether this system plays a role in actin remodeling induced by H2O2. Low H2O2 dose (5µM) rapidly triggered a signaling cascade leading to nSMase2 activation, src and annexin 2 (AnxA2) phosphorylation, and actin remodeling, in fibroblasts and SMC. These events were blocked by pharmacological inhibitors of MMPs (Ro28-2653) and p38MAPK (SB203580), and were lacking in MMP2(-/-) and in nSMase2-mutant (fro) fibroblasts. Likewise, H2O2 was unable to induce actin remodeling in fro and MMP2(-/-) fibroblasts or in cells pretreated with p38MAPK, or MMP inhibitors. Finally we show that nSMase2 activation by H2O2, depends on MMP2 and p38MAPK, and is required for the src-dependent phosphorylation of AnxA2, and actin remodeling. Taken together, these findings indicate for the first time that AnxA2 phosphorylation and actin remodeling evoked by oxidative stress depend on the sphingolipid pathway, via MMP2 and p38MAPK.

Identification of novel viral receptors with cell line expressing viral receptor-binding protein.

The viral cell receptors and infection can be blocked by the expression of the viral receptor-binding protein. Thus, the viral cell receptor is an attractive target for anti-viral strategies, and the identification of viral cell receptor is critical for better understanding and controlling viral disease. As a model system for viral entry and anti-retroviral approaches, avian sarcoma/leukosis virus (ASLV, including the A-J ten subgroups) has been studied intensively and many milestone discoveries have been achieved based on work with ASLV. Here, we used a DF1 cell line expressed viral receptor-binding protein to efficiently identify chicken Annexin A2 (chANXA2) as a novel receptor for retrovirus ALV-J (avian leukosis virus subgroup J). Our data demonstrate that antibodies or siRNA to chANXA2 significantly inhibited ALV-J infection and replication, and over-expression of chANXA2 permitted the entry of ALV-J into its non-permissible cells. Our findings have not only identified chANXA2 as a novel biomarker for anti-ALV-J, but also demonstrated that cell lines with the expression of viral receptor-binding protein could be as efficient tools for isolating functional receptors to identify novel anti-viral targets.