Global crotonylome identifies EP300-regulated ANXA2 crotonylation in cumulus cells as a regulator of oocyte maturation.

Lysine crotonylation (Kcr), a newly discovered post-translational modification, played a crucial role in physiology and disease progression. However, the roles of crotonylation in oocyte meiotic resumption remain elusive. As abnormal cumulus cell development will cause oocyte maturation arrest and female infertility, we report that cumulus cells surrounding human meiotic arrested oocytes showed significantly lower crotonylation, which was associated with decreased EP300 expression and blocked cumulus cell expansion. In cultured human cumulus cells, exogenous crotonylation or EP300 activator promoted cell proliferation and reduced cell apoptosis, whereas EP300 knockdown induced the opposite effect. Transcriptome profiling analysis in human cumulus cells indicated that functions of crotonylation were associated with activation of epidermal growth factor receptor (EGFR) pathway. Importantly, we characterized the Kcr proteomics landscape in cumulus cells by LC-MS/MS analysis, and identified that annexin A2 (ANXA2) was crotonylated in cumulus cells in an EP300-dependent manner. Crotonylation of ANXA2 enhanced the ANXA2-EGFR binding, and then activated the EGFR pathway to affect cumulus cell proliferation and apoptosis. Using mouse oocytes IVM model and EP300 knockout mice, we further confirmed that crotonylation alteration in cumulus cells affected the oocyte maturation. Together, our results indicated that EP300-mediated crotonylation is important for cumulus cells functions and oocyte maturation.

Nectin2 influences cell apoptosis by regulating ANXA2 expression in neuroblastoma.

Neuroblastoma (NB) is a pediatric cancer of the peripheral sympathetic nervous system and represents the most frequent solid malignancy in infants. Nectin2 belongs to the immunoglobulin superfamily and has been shown to play a role in tumorigenesis. In the current study, we demonstrate that serum Nectin2 level is increased in NB patients compared with that in healthy controls and Nectin2 level is correlated with neuroblastoma international neuroblastoma staging system (INSS) classification. There is a positive correlation between Nectin2 level and shorter overall survival in NB patients. Knockdown of Nectin2 reduces the migration of SH-SY5Y and SK-N-BE2 cells and induces their apoptosis and cell cycle arrest. RNA-seq analysis demonstrates that Nectin2 knockdown affects the expressions of 258 genes, including 240 that are upregulated and 18 that are downregulated compared with negative controls. qRT-PCR and western blot analysis confirm that ANXA2 expression is decreased in Nectin2-knockdown SH-SY5Y cells, consistent with the RNA-seq results. ANXA2 overexpression rescues the percentage of apoptotic NB cells induced by Nectin2 knockdown and compensates for the impact of Nectin2 knockdown on cleaved caspase3 and bax expressions. In addition, western blot analysis results show that ANXA2 overexpression rescues the effect of Nectin2 knockdown on MMP2 and MMP9 expressions. The current data highlight the importance of Nectin2 in NB progression and the potential of Nectin2 as a novel candidate target for gene therapy.

The p-STAT3/ANXA2 axis promotes caspase-1-mediated hepatocyte pyroptosis in non-alcoholic steatohepatitis.

BACKGROUND: To explore the roles of Annexin A2 (ANXA2) on hepatocyte pyroptosis and hepatic fibrosis in nonalcoholic steatohepatitis (NASH) and underlying molecular mechanism. METHODS: Bioinformatics analyses were performed on transcriptome data of liver tissues from mice and patients with liver fibrosis for screening the hepatocyte pyroptosis-related differential genes. The in vivo NASH mouse model and in vitro NASH cellular model were established. The expression levels of Anxa2/ANXA2 were quantified. Then, the upstream transcription factor of Anxa2 was screened by ChIP-Seq and experimentally verified. The effects of the p-STAT3/ANXA2 axis on Caspase-1 mediated pyroptosis and fibrosis were explored by in vivo and in vitro experiments. RESULTS: Bioinformatics analyses suggested that the expression of Anxa2/ANXA2 was significantly up-regulated in liver tissues of both NASH mice and patients scoring with high pyroptotic activity. Experimental data showed that the ANXA2 expression was positively associated with the development of hepatocyte pyroptosis and fibrosis. As a transcription factor of ANXA2, p-STAT3 can bind to the promoter of Anxa2 and promote its transcription. The inhibition of p-STAT3 can significantly suppress hepatocyte pyroptosis and fibrosis, which was significantly reversed after the over-expression of Anxa2. Caspase-1 was verified as the player of the p-STAT3/ANXA2 axis to promote pyroptosis and fibrosis. By specifically inhibiting Caspase-1, the promotion effect of the p-STAT3/ANXA2 axis on pyroptosis and fibrosis can be significantly weakened. CONCLUSION: The p-STAT3 promoted Anxa2 expression at the transcription level, thus activating the Caspase-1 mediated hepatocyte pyroptosis and fibrosis in NASH.

Annexin A2 promotes development of retinal neovascularization through PI3K/ AKT signaling pathway.

PURPOSE: Retinal Neovascularization (RNV) is a pathological characteristic of ocular diseases. Annexin A2 (ANXA2) plays important roles in RNV while the mechanism remains unclear. The study aimed to explore relationship between ANXA2 and PI3K/AKT signaling pathway in RNV. METHODS: We used human retinal vascular endothelial cells (HRECs) and oxygen-induced retinopathy (OIR) mice model to show ANXA2 can promote the development of RNV through PI3K/AKT signaling pathway. We divided HRECs into six groups by infecting lentivirus containing appropriate plasmid and adding corresponding solution. Assays showing ability of HRECs were performed in vitro. Mice were randomly divided into three groups and treated accordingly. RESULTS: Expression of ANXA2 and activity of PI3K/AKT signaling pathway in HRECs were detected. RNV and expression of ANXA2 in mice retinas were detected. Results showed that ANXA2 expression is positively related with RNV-forming ability of HRECs in vitro and development of RNV in vivo while low activity of PI3K/AKT signaling pathway could attenuate the role of ANXA2. CONCLUSIONS: We can make ANXA2 and PI3K/ AKT signaling pathway as a promising target for the regulation of pathological neovascularization of the retina, which also provides a novel idea for effective prevention and treatment of diseases related to RNV in future.

Annexin A2 (ANXA2) interacts with nonstructural protein 1 and promotes the replication of highly pathogenic H5N1 avian influenza virus.

BACKGROUND: Non-structural protein 1 (NS1) is a multifunctional protein and a crucial regulatory factor in the replication and pathogenesis of avian influenza virus (AIV). Studies have shown that NS1 can interact with a variety of host proteins to modulate the viral life cycle. We previously generated a monoclonal antibody against NS1 protein; In the current research study, using this antibody, we immunoprecipitated host proteins that interact with NS1 to better understand the roles played by NS1 in communications between virus and host. RESULTS: Co-immunoprecipitation experiments identified annexin A2 (ANXA2) as a target molecule interacting with NS1. Results from confocal laser scanning microscopy indicated that NS1 co-localized with ANXA2 in the cell cytoplasm. Overexpression of ANXA2 significantly increased the titer of H5N1 subtype HPAIV, whereas siRNA-mediated knockdown of ANXA2 markedly inhibited the expression of viral proteins and reduced the progeny virus titer. CONCLUSIONS: Our results indicate that ANXA2 interacts with NS1 and ANXA2 expression increases HPAIV replication.

Annexin A2 Plus Low-Dose Tissue Plasminogen Activator Combination Attenuates Cerebrovascular Dysfunction After Focal Embolic Stroke of Rats.

Previous studies showed recombinant annexin A2 (rA2) in combination with low-dose tissue-type plasminogen activator (tPA) improved thrombolytic efficacy and long-term neurological outcomes after embolic focal ischemia in rats. The objective of this study was to investigate the effects and mechanisms of the combination in early BBB integrity and cerebrovascular patency in the rat focal embolic stroke model. Ischemic brain infarct volume and hemorrhagic transformation were quantified at 24 h after stroke. At an earlier time point, 16 h after stroke, BBB integrity was evaluated by IgG extravasation, and the involved mechanisms were assessed for tight junction ZO-1 and adhesion junction ve-cadherin protein expression, matrix metalloproteinase activation, extracellular matrix collagen IV and endothelial barrier antigen expression, and activation of microglia/macrophages and astrocytes. While at the same time point, cerebrovascular patency was assessed by intravascular fibrin and platelet depositions. At 24 h after stroke, the combination showed significant reduction in brain infarction and intracerebral hemorrhage. At 16 h after stroke onset, the combination therapy significantly reduced BBB disruption, and improved preservation of the junction proteins ZO-1 and ve-cadherin, decreased activation of matrix metalloproteinase, inhibited degradation of extracellular matrix collagen IV and endothelial barrier antigen, and reduced microglia/macrophage and astrocytes activations. Meanwhile, the combination also significantly improved cerebrovascular patency by reducing intravascular fibrin and platelet depositions in the peri-infarct brain tissues. These results suggest the beneficial effects of the rA2 plus low-dose tPA combination may be mediated in part by the amelioration of BBB disruption and improvement of cerebrovascular patency.

Domains I and IV of annexin A2 affect the formation and integrity of in vitro capillary-like networks.

Annexin A2 (AnxA2) is a widely expressed multifunctional protein found in different cellular compartments. In spite of lacking a hydrophobic signal peptide, AnxA2 is found at the cell surface of endothelial cells, indicative of a role in angiogenesis. Increased extracellular levels of AnxA2 in tumours correlate with neoangiogenesis, metastasis and poor prognosis. We hypothesised that extracellular AnxA2 may contribute to angiogenesis by affecting endothelial cell-cell interactions and motility. To address this question, we studied the effect of heterotetrameric and monomeric forms of AnxA2, as well as its two soluble domains on the formation and maintenance of capillary-like structures by using an in vitro co-culture system consisting of endothelial and smooth muscle cells. In particular, addition of purified domains I and IV of AnxA2 potently inhibited the vascular endothelial growth factor (VEGF)-dependent formation of the capillary-like networks in a dose-dependent manner. In addition, these AnxA2 domains disrupted endothelial cell-cell contacts in preformed capillary-like networks, resulting in the internalisation of vascular endothelial (VE)-cadherin and the formation of VE-cadherin-containing filopodia-like structures between the endothelial cells, suggesting increased cell motility. Addition of monoclonal AnxA2 antibodies, in particular against Tyr23 phosphorylated AnxA2, also strongly inhibited network formation in the co-culture system. These results suggest that extracellular AnxA2, most likely in its Tyr phosphorylated form, plays a pivotal role in angiogenesis. The exogenously added AnxA2 domains most likely mediate their effects by competing with endogenous AnxA2 for extracellular factors necessary for the initiation and maintenance of angiogenesis, such as those involved in the formation/integrity of cell-cell contacts.

Withaferin A binds covalently to the N-terminal domain of annexin A2.

Annexin A2 (AnxA2), a 38-kDa member of the Ca2+-binding annexin family, has been implicated in numerous cancer pathways. Withaferin A (WithfA), a natural plant compound, has been reported previously to bind covalently to Cys133 of the AnxA2 core domain leading to a reduction of the invasive capabilities of cancer cells by altering their cytoskeleton. We show here that AnxA2 has an inhibitory effect on actin polymerization, and a modification with WithfA significantly increases this inhibitory role of AnxA2. Using mass spectrometry and single-site mutants, we localized the WithfA-AnxA2 interaction to the N-terminal domain of AnxA2 where WithfA binds covalently to Cys9. Whereas binding to F-actin filaments has been mapped to the C terminus of AnxA2, our results suggest that the N-terminal domain modified by WithfA may also play a role in the AnxA2-actin interaction. The binding of WithfA may regulate the AnxA2-mediated actin dynamics in two distinct ways: (i) the increase of F-actin bundling activity by the Anx2/p11 heterotetramer and (ii) the decrease of actin polymerization as a result of the increased affinity of AnxA2 to the barbed end of actin microfilaments. We demonstrate the susceptibility of Cys9 of AnxA2 to chemical modifications and exclude Cys133 as a binding site for WithfA.

Extracellular release of annexin II from pancreatic cancer cells and resistance to anticancer drug-induced apoptosis by supplementation of recombinant annexin II.

OBJECTIVES: Extracellular microenvironment plays crucial roles in the development of cancers and chemoresistance. Pancreatic carcinoma is resistant to almost all chemotherapeutic agents. In this study, we identified annexin II in the medium from pancreatic cancer cells as a protein released into the extracellular environment. METHODS: Medium from 5-hour cultures of various cancer cells was collected. Proteins in the medium were detected by molecular mass analysis and immunoblotting. Anticancer drug sensitivity of cells preincubated with or without recombinant annexin II (rANX II) was measured using crystal violet assay and colony survival assay. Apoptosis-related molecules were analyzed by immunoblotting. RESULTS: Recombinant ANX II supplementation in the medium confers resistance to anticancer drugs, including cisplatin, 5-fluorouracil, and gemcitabine, in MiaPaCa-2 and AsPC-1 cells. In MiaPaCa-2 cells, rANX II supplementation resulted in suppression of caspase-3 activation associated with increased Bcl-2/Bax ratios. Suppression of cisplatin-induced cell death by rANX II supplementation was canceled by inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase signal pathways. CONCLUSIONS: The current study is the first report to demonstrate that supplementation of rANX II in the medium increased resistance to anticancer drugs in pancreatic cancer cells. Recombinant ANX II exerts cell death-suppressive function by antagonizing cisplatin-induced apoptosis.

Methionine-induced hyperhomocysteinemia reverts fibrinolytic pathway activation in a murine model of acute promyelocytic leukemia.

Increased fibrinolysis is an important component of acute promyelocytic leukemia (APL) bleeding diathesis. APL blasts overexpress annexin II (ANXII), a receptor for tissue plasminogen activator (tPA), and plasminogen, thereby increasing plasmin generation. Previous studies suggested that ANXII plays a pivotal role in APL coagulopathy. ANXII binding to tPA can be inhibited by homocysteine and hyperhomocysteinemia can be induced by L-methionine supplementation. In the present study, we used an APL mouse model to study ANXII function and the effects of hyperhomocysteinemia in vivo. Leukemic cells expressed higher ANXII and tPA plasma levels (11.95 ng/mL in leukemic vs 10.74 ng/mL in wild-type; P = .004). In leukemic mice, administration of L-methionine significantly increased homocysteine levels (49.0 µmol/mL and < 6.0 µmol/mL in the treated and nontreated groups, respectively) and reduced tPA levels to baseline concentrations. The latter were also decreased after infusion of the LCKLSL peptide, a competitor for the ANXII tPA-binding site (11.07 ng/mL; P = .001). We also expressed and purified the p36 component of ANXII in Pichia methanolica. The infusion of p36 in wild-type mice increased tPA and thrombin-antithrombin levels, and the latter was reversed by L-methionine administration. The results of the present study demonstrate the relevance of ANXII in vivo and suggest that methionine-induced hyperhomocysteinemia may reverse hyperfibrinolysis in APL.

Annexin II interactions with the annexin II receptor enhance multiple myeloma cell adhesion and growth in the bone marrow microenvironment.

Multiple myeloma (MM) is an incurable B-cell malignancy in which the marrow microenvironment plays a critical role in our inability to cure MM. Marrow stromal cells in the microenvironment support homing, lodging, and growth of MM cells through activation of multiple signaling pathways in both MM and stromal cells. Recently, we identified annexin II (AXII) as a previously unknown factor produced by stromal cells and osteoclasts (OCL) that is involved in OCL formation, HSC and prostate cancer (PCa) homing to the BM as well as mobilization of HSC and PCa cells. AXII expressed on stromal cells supports PCa cell lodgment via the AXII receptor (AXIIR) on PCa cells, but the role of AXII and AXIIR in MM is unknown. In this study, we show that MM cells express AXIIR, that stromal/osteoblast-derived AXII facilitates adhesion of MM cells to stromal cells via AXIIR, and OCL-derived AXII enhances MM cell growth. Finally, we demonstrate that AXII activates the ERK1/2 and AKT pathways in MM cells to enhance MM cell growth. These results demonstrate that AXII and AXIIR play important roles in MM and that targeting the AXII/AXIIR axis may be a novel therapeutic approach for MM.

Extracellular recombinant annexin II confers UVC-radiation resistance and increases the Bcl-xL to Bax protein ratios in human UVC-radiation-sensitive cells.

In this study, we found that refractoriness to ultraviolet (UVC) light-induced cell death was increased in UVC-radiation-sensitive cells derived from Cockayne syndrome patients when the cells were precultured in medium supplemented with recombinant annexin II (rANX II). In CS3BES cells, an immortal cell line derived from Cockayne syndrome patients, the rANX II supplementation-induced UVC-radiation resistance was suppressed by treatment with an anti-annexin II antibody and EGTA. The amount of biotinylated annexin II on the cell surface increased in the rANX II-supplemented cells but did not increase in the cells that were cotreated with rANX II and EGTA. The capacity to remove UVC-radiation-damaged DNA, (6-4) photoproducts and cyclobutane pyrimidine dimers, was the same in cells that were precultured with rANX II and in control cells that did not receive rANX II supplementation. The rANX II supplementation-induced UVC-radiation resistance was also observed in nucleotide excision repair-deficient cells and xeroderma pigmentosum group A-downregulated cells. The Bcl-xL to Bax protein ratios, an index of survival activity in cells exposed to lethal stresses, were increased in the cells that had been precultured in rANX II for 24 h prior to UVC irradiation. Treatment with a phosphatidylinositol 3-kinase inhibitor suppressed the increased UVC-radiation resistance and Bcl-xL to Bax ratios in the cells with rANX II supplementation. Furthermore, downregulation of Bcl-xL by siRNA transfection also suppressed the UVC-radiation resistance that was induced by rANX II supplementation. These results suggest that the increase in the Bcl-xL to Bax ratios may be associated with enhanced resistance to UVC-radiation-induced cell death.

Visualization of clot lysis in a rat embolic stroke model: application to comparative lytic efficacy.

BACKGROUND AND PURPOSE: The purpose of this study was to develop a novel MRI method for imaging clot lysis in a rat embolic stroke model and to compare tissue plasminogen activator (tPA)-based clot lysis with and without recombinant Annexin-2 (rA2). METHODS: In experiment 1 we used in vitro optimization of clot visualization using multiple MRI contrast agents in concentrations ranging from 5 to 50 µL in 250 µL blood. In experiment 2, we used in vivo characterization of the time course of clot lysis using the clot developed in the previous experiment. Diffusion, perfusion, angiography, and T1-weighted MRI for clot imaging were conducted before and during treatment with vehicle (n=6), tPA (n=8), or rA2 plus tPA (n=8) at multiple time points. Brains were removed for ex vivo clot localization. RESULTS: Clots created with 25 µL Magnevist were the most stable and provided the highest contrast-to-noise ratio. In the vehicle group, clot length as assessed by T1-weighted imaging correlated with histology (r=0.93). Clot length and cerebral blood flow-derived ischemic lesion volume were significantly smaller than vehicle at 15 minutes after treatment initiation in the rA2 plus tPA group, whereas in the tPA group no significant reduction from vehicle was observed until 30 minutes after treatment initiation. The rA2 plus tPA group had a significantly shorter clot length than the tPA group at 60 and 90 minutes after treatment initiation and significantly smaller cerebral blood flow deficit than the tPA group at 90 minutes after treatment initiation. CONCLUSIONS: We introduce a novel MRI-based clot imaging method for in vivo monitoring of clot lysis. Lytic efficacy of tPA was enhanced by rA2.

Plasmin-clipped beta(2)-glycoprotein-I inhibits endothelial cell growth by down-regulating cyclin A, B and D1 and up-regulating p21 and p27.

beta(2)-Glycoprotein-I (beta(2)gpI), an abundant plasma glycoprotein, functions as a regulator of thrombosis. Previously, we demonstrated that plasmin-clipped beta(2)gpI (cbeta(2)gpI) exerts an anti-angiogenic effect on human umbilical vein endothelial cells (HUVEC). The present study was focused on the molecular background responsible for this phenomenon. cbeta(2)gpI strongly reduced HUVEC growth and proliferation as evidenced by the MTT and BrdU assay and delayed cell cycle progression arresting HUVEC in the S-and G2/M-phase. Western blot analysis indicated that cbeta(2)gpI inhibited cyclin A, B and D1, and enhanced p21 and p27 expression. Activity of p38 was down-regulated independently from the cbeta(2)gpI incubation time. Phosphorylation of ERK1/2 was not changed early (30 and 60 min) but became enhanced later (90 min, 4h). JNK activity was reduced rapidly after cbeta(2)gpI treatment but compared to controls, increased thereafter. Annexin II blockade prevented growth inhibition and cell cycle delay evoked by cbeta(2)gpI. We assume that cbeta(2)gpI's effects on HUVEC growth is mediated via cyclin A, B and D1 suppression, up-regulation of p21 and p27 and coupled to modifications of the mitogen-activated protein (MAP) kinase signalling pathway. cbeta(2)gpI may represent a potential endogenous angiogenesis-targeted compound, opening the possibility of a novel tool to treat cancer.

Annexin A2 combined with low-dose tPA improves thrombolytic therapy in a rat model of focal embolic stroke.

Recent studies showed that soluble annexin A2 dramatically increases tissue plasminogen activator (tPA)-mediated plasmin generation in vitro, and reduces thrombus formation in vivo. Here, we hypothesize that combining annexin A2 with tPA can significantly enhance thrombolysis efficacy, so that lower doses of tPA can be applied in ischemic stroke to avoid neurotoxic and hemorrhagic complications. In vitro activity assays confirmed tPA-specific amplification of plasmin generation by recombinant annexin A2. In a rat focal embolic stroke model, combination therapy with tPA and recombinant annexin A2 protein at 2 h post-ischemia decreased the effective dose required for tPA by four-fold and reduced brain infarction. Combining annexin A2 with tPA also lengthened the time window for thrombolysis. Compared with tPA (10 mg/kg) alone, the combination of annexin A2 (5 mg/kg) plus low-dose tPA (2.5 mg/kg) significantly enhanced fibrinolysis, attenuated mortality, brain infarction, and hemorrhagic transformation, even when administered at 4 h post-ischemia. Combination with recombinant annexin A2, the effective thrombolytic dose of tPA can be decreased. As a result, brain hemorrhage and infarction are reduced, and the time window for stroke reperfusion prolonged. Our present findings provide a promising new approach for enhancing tPA-based thrombolytic stroke therapy.

Homocysteine inhibits neoangiogenesis in mice through blockade of annexin A2-dependent fibrinolysis.

When plasma levels of homocysteine (HC), a thiol amino acid formed upon methionine demethylation, exceed 12 muM, individuals are at increased risk of developing large vessel atherothrombosis and small vessel dysfunction. The annexin A2 complex (termed "A2") is the cell surface coreceptor for plasminogen and TPA and accelerates the catalytic activation of plasmin, the major fibrinolytic agent in mammals. We previously showed that HC prevents A2-mediated, TPA-dependent activation of plasminogen in vitro by disulfide derivatization of the "tail" domain of A2. We also demonstrated that fibrinolysis and angiogenesis are severely impaired in A2-deficient mice. We now report here that, although hyperhomocysteinemic mice had a normal coagulation profile and normal platelet function, fibrin accumulated in their tissues due to reduced perivascular fibrinolytic activity and angiogenesis was impaired. A2 isolated from hyperhomocysteinemic mice failed to fully support TPA-dependent plasmin activation. However, infusion of hyperhomocysteinemic mice with fresh recombinant A2, which localized to neoangiogenic endothelial cells, resulted in normalization of angiogenesis and disappearance of peri- and intravascular fibrin. We therefore conclude that hyperhomocysteinemia impairs postnatal angiogenesis by derivatizing A2, preventing perivascular fibrinolysis, and inhibiting directed endothelial cell migration. These findings provide a mechanistic explanation for microvascular dysfunction and macrovascular occlusion in individuals with hyperhomocysteinemia.

Annexin A2 is a soluble mediator of macrophage activation.

On the surface of the macrophage, annexin A2 tetramer (A2t) serves as a docking protein or recognition element for bacterial and viral pathogens. Plasma levels of free A2t have been reported to increase following infection, although the mechanistic significance of this observation is unclear. Although annexin A2 had generally been thought to play an anti-inflammatory role, soluble A2t stimulates MAP kinase activity in bone marrow stromal cells downstream of a recently cloned receptor. This raises the question of whether A2t activates human macrophages via MAP kinases and whether it might be capable of acting as an inflammatory mediator. To this end, human monocyte-derived macrophages were treated with soluble A2t and MAP kinase phosphorylation, p65 NF-kappaB activation, and inflammatory mRNA and protein levels were measured. It was found that A2t caused rapid phosphorylation of several MAP kinases, as well as translocation of p65 NF-kappaB to the nucleus. A2t stimulated the production of TNF-alpha, IL-1beta, and IL-6, as well as several members of the chemokine family within 24 h, which are capable of recruitment and/or activation of a broad range of leukocyte classes. Furthermore, A2t-activated macrophages demonstrated enhanced phagocytic ability for the ingestion of GFP-expressing Escherichia coli. These data are the first to suggest the participation of an annexin in microbial clearance, as well as the establishment of inflammation and the immune response, including the recruitment and activation of immune cells to the site of infection.

Recombinant annexin-2 inhibits the progress of diabetic nephropathy in a diabetic mouse model via recovery of hypercoagulability.

Diabetic nephropathy, a major complication of diabetes mellitus that leads to mortality, has been shown to involve a dysregulation of the coagulation system. Annexin-2, a co-receptor for plasminogen and tissue plasminogen activator on endothelial cells, is one of the molecules required to maintain the antithrombogenic properties of endothelial cells. Previously, we showed that recombinant annexin-2 protein (rAN II) modulated impaired fibrinolytic activity in the carotid arteries of rats. In the present study, to investigate its protective effects against diabetic nephropathy, rAN II was administered to KK-Ay mice, a murine model of type 2 diabetes, for eight weeks, and albuminuria, kidney size, and histological glomerular lesions were investigated. The mean weight of kidneys from KK-Ay mice treated with rAN II was significantly less than that of those treated with PBS (control) (p < 0.02). Furthermore, the level of albuminuria observed in rAN II-treated KK-Ay mice was significantly less than that of the control group (rAN II, 0.90+/-0.12 microg/day; PBS, 1.55+/-0.31 microg/day; p < 0.01); also, the area of diffuse glomerular lesions was significantly smaller (rAN II, 41.51+/-4.54%; PBS, 81.81+/-8.10%; p < 0.01). Bleeding time, prothrombin time (PT), and active partial thromboplastin time (APTT) did not significantly differ between the two groups. Our results suggest that rAN II may inhibit the progression of diabetic nephropathy in KK-Ay mice without influencing the coagulation system, indicating that annexin-2 may be considered as a possible new therapeutic tool for patients with diabetic nephropathy.

Annexin 2-mediated enhancement of cytomegalovirus infection opposes inhibition by annexin 1 or annexin 5.

Biochemical studies have suggested that annexin 2 (A2) may participate in cytomegalovirus (CMV) infection. In the current work, effects of A2 monomer (p36) and heterotetramer (A2t; p36(2)p11(2)) were investigated. Demonstrating a role for endogenous A2, the four stages of infection that were followed were each inhibited by anti-p36 or anti-p11 at 37 degrees C. Immuno-inhibition was attenuated when the virus and cells were pre-incubated at 4 degrees C to coordinate virus entry initiated afterwards at 37 degrees C, reconciling controversy in the literature. As an explanation, CMV-induced phosphorylation of p36 was prevented by the 4 degrees C treatment. Supporting these immuno-inhibition data, purified A2t or p11 increased CMV infectious-progeny generation and CMV gene expression. A specific role for A2t was indicated by purified p36 having no effect. Unlike other steps, primary plaque formation was not enhanced by purified A2t or p11, possibly because of undetectable phosphorylation. As annexins 1 (A1) and 5 (A5) interact with A2, their effect on CMV was also tested. Both purified proteins inhibited CMV infection. In each experiment, the concentration of A1 required for half-maximal inhibition was five- to 10-fold lower than that of A5. Addition of A2 opposed A1- or A5-mediated inhibition of CMV, as did certain A2-specific antibodies that had no effect in the absence of added A1 or A5. Transfection of the p36-deficient cell line HepG2 increased CMV infection and was required for inhibition by the other annexins. These data suggest that CMV exploits A2t at physiological temperature to oppose the protection of cells conferred by A1 or A5.

Annexin II stimulates RANKL expression through MAPK.

UNLABELLED: We report that AX-II, in addition to inducing GM-CSF expression, also increases membrane-bound RANKL synthesis by marrow stromal cells and does so through a previously unreported MAPK-dependent pathway. Thus, both GM-CSF and RANKL are required for AX-II stimulation of OCL formation. INTRODUCTION: Annexin II (AX-II) is an autocrine/paracrine factor secreted by osteoclasts (OCLs) that stimulates human OCL formation and bone resorption in vitro by inducing bone marrow stromal cells and activated CD4+ T cells to produce granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF in turn increases OCL precursor proliferation and further enhances OCL formation. However, the induction of GM-CSF by AX-II cannot fully explain its effects on OCL formation. In this study, we tested the capacity of AX-II to induce the expression of RANKL and the corresponding signaling pathways AX-II employs in human marrow stromal cells to induce RANKL. We also showed that both GM-CSF and RANKL are required for OCL formation induced by AX-II. MATERIALS AND METHODS: Real-time RT-PCR and Western blot analysis were used to detect RANKL and osteoprotegerin (OPG) mRNA and protein expression in unfractionated human bone marrow mononuclear cells stimulated with AX-II. Soluble RANKL in the conditioned medium was analyzed by ELISA. Activation of the MAPK pathway by AX-II was tested by Western blot. The effects of OPG and anti-GM-CSF on AX-II-induced OCL formation were also examined. RESULTS AND CONCLUSION: In addition to upregulating GM-CSF mRNA, AX-II increased RANKL mRNA expression dose-dependently in unfractionated human bone marrow mononuclear cells and modestly increased soluble RANKL in unfractionated human bone marrow mononuclear cell conditioned medium. However, AX-II markedly increased membrane-bound RANKL on human bone marrow stromal cells. Treatment of marrow stromal cells with AX-II activated MAP-kinase (ERKs) and PD 98059 abolished the effect but did not block the increase in GM-CSF. Interestingly, OPG, a natural decoy receptor for RANKL, or anti-GM-CSF partially inhibited OCL formation by AX-II in human bone marrow cells, and the combination of OPG and anti-GM-CSF completely blocked AX-II-induced OCL formation. These data show that AX-II stimulates both the proliferation and differentiation of OCL precursors through production of GM-CSF and RANKL respectively.