Specific down-regulation of annexin II expression in human cells interferes with cell proliferation

The protein-tyrosine kinase substrate annexin II is a growth regulated gene whose expression is increased in several human cancers. While the precise function of this protein is not understood, annexin II is proposed to be involved in multiple physiological activities, including DNA synthesis and cell proliferation. Targeted disruption of the annexin II gene affects calcium signaling, tyrosine phosphorylation and apoptosis, indicating the important physiological role of this protein. We used a transient co-transfection assay to regulate annexin II expression in human HeLa, 293 and 293T cells, and measured the effects of annexin II down regulation on DNA synthesis and proliferation. Transfection of cells with an antisense annexin II vector results in inhibition of cell division and proliferation, with concomitant reduction in annexin II message and protein levels. Cellular DNA synthesis is significantly reduced in antisense transfected cells. Replication extracts made from antisense transfected cells have significantly reduced efficiency to support SV40 in vitro DNA replication, while the extracts made from sense transfected cells are fully capable of replication. Our results indicate an important role of annexin II in cellular DNA synthesis and cell proliferation.     

Annexin I and dexamethasone effects on phospholipase and cyclooxygenase activity in human synoviocytes

Annexin I is a glucocorticoid-induced mediator with anti-inflammatory activity in animal models of arthritis. We studied the effects of a bioactive annexin I peptide, ac 2-26, dexamethasone (DEX), and interleukin-1beta (IL-1beta) on phospholipase A2 (PLA2) and cyclooxygenase (COX) activities and prostaglandin E2 (PGE2) release in cultured human fibroblast-like synoviocytes (FLS). Annexin I binding sites on human osteoarthritic (OA) FLS were detected by ligand binding flow cytometry. PLA2 activity was measured using 3H-arachidonic acid release, PGE2 release and COX activity by ELISA, and COX2 content by flow cytometry. Annexin I binding sites were present on human OA FLS. Annexin I peptide ac 2-26 exerted a significant concentration-dependent inhibition of FLS constitutive PLA2 activity, which was reversed by IL-1beta. In contrast, DEX inhibited IL-1beta-induced PLA2 activity but not constitutive activity. DEX but not annexin I peptide inhibited IL-1beta-induced PGE2 release. COX activity and COX2 expression were significantly increased by IL-1beta. Annexin I peptide demonstrated no inhibition of constitutive or IL-1beta-induced COX activity. DEX exerted a concentration-dependent inhibition of IL-1beta-induced but not constitutive COX activity. Uncoupling of inhibition of PLA2 and COX by annexin I and DEX support the hypothesis that COX is rate-limiting for PGE2 synthesis in FLS. The effect of annexin I but not DEX on constitutive PLA2 activity suggests a glucocorticoid-independent role for annexin I in autoregulation of arachidonic acid production. The lack of effect of annexin I on cytokine-induced PGE2 production suggests PGE2-independent mechanisms for the anti-inflammatory effects of annexin I in vivo.     

Annexins in cardiac tissue: cellular localization and effect on phospholipase activity

Stimulation of cardiac phospholipid metabolism has diverse biological effects, ranging from subtle changes in cellular function to severe cellular damage. Accordingly, knowledge of the factors governing the activity of cardiac phospholi pases is of great biological importance. A possible role of annexins, intracellular proteins that bind to membranes in a calcium dependent manner, as modulators of phospholipase activity has been proposed. In this study we investigated the cell type specific distribution of Annexin V and VIII in the heart. Recombinant Annexin V was used to examine the effect of this type of Annexin on cardiac phospholipase activity. Western blot analysis shows that annexin V is abundantly present in the heart. Using isolated myocytes and cultured cardiac endothelial and fibroblast-like cells, it is demonstrated that the localization of Annexin V is confined to non-myocytes. No positive bands matching the Mw of recombinant Annexin VIII are found in any of the cell types examined.In vitro studies demonstrate that recombinant Annexin V potently inhibits the activity of cardiac membrane-bound phospholipases, acting on their natural sur rounding substrate, in a calcium dependent manner. Interestingly, annexin V also inhibits triacylglycerol hydrolysis. In conclusion, the expression of annexins is cell-type specific and suggests a cell-type specific function of these proteins in the heart. The absence of Annexin V in cardiac myocytes dismisses involvement of this annexin in cardiomyocyte phospholipid metabolism. The presence of Annexin V in cardiac endothelial and fibroblasts suggests a regulating role in the phospholipid homeostasis of non-myocyte cell types in the heart. (Mol Cell Biochem116: 95–101, 1992)     

Phospholipase C-gamma1 is required for calcium-induced keratinocyte differentiation.

Phospholipase C-gamma1 is the most abundant member of the phospholipase C family in keratinocytes and is induced by calcium. Phospholipase C-gamma1, therefore, may be involved in the signal transduction system leading to calcium regulation of keratinocyte differentiation. To test this hypothesis, expression of phospholipase C-gamma1 in human keratinocytes was blocked by transfecting cells with the antisense human phospholipase C-gamma1 cDNA construct. These cells demonstrated a dramatic reduction in phospholipase C-gamma1 protein level compared with the empty vector-transfected cells and a marked reduction in the mRNA and protein levels of the differentiation markers involucrin and transglutaminase following administration of calcium. Similarly, cotransfection of antisense phospholipase C-gamma1 constructs with a luciferase reporter vector containing involucrin or transglutaminase promoters led to a substantial reduction in calcium-stimulated involucrin and transglutaminase promoter activities. Similar results were seen following treatment with a specific phospholipase C inhibitor U73122. To determine whether phospholipase C-gamma1 regulated differentiation by controlling intracellular calcium, we examined the ability of antisense phospholipase C-gamma1 to block the calcium-induced rise in intracellular calcium and found that it could. These findings indicate that phospholipase C-gamma1 is a critical component of the signaling pathway mediating calcium regulation of keratinocyte differentiation via its mobilization of intracellular calcium.     

Changes in annexin (lipocortin) content in human amnion and chorion at parturition.

Arachidonic acid is mobilized from fetal membrane phospholipids at parturition leading to increased production of oxytocic prostaglandins which may initiate or maintain myometrial contractions. Phospholipid mobilization requires activation of phospholipase A2 or C, both of which require calcium for activity. The annexins (lipocortins) are a superfamily of proteins which bind to calcium and phospholipids and thereby may alter phospholipase activity through two mechanisms: modulation of intracellular free Ca2+ concentrations or regulation of the accessibility of phospholipids to hydrolyzing enzymes. Using Western immunoblotting with monospecific polyclonal antibodies, annexins I-VI were identified in human amnion and chorion/decidua at term in tissues obtained from patients in labor or not in labor. Each annexin was present in two distinct pools: a pool which only associated with the membrane in the presence of calcium (calcium-dependent pool) and a calcium-independent pool that remained membrane bound in the presence of calcium chelators. Annexin I was present as two species, resolving at 36 kDa and 68 kDa. The total concentration of annexin I in both amnion and chorion/decidua was significantly decreased with labor, while the total concentration of annexin V in chorion significantly increased with labor. The size of individual pools of annexins also changed with labor: the calcium-dependent pool of annexins I and II in both amnion and chorion significantly decreased; the calcium-dependent pool of annexin V increased in chorion; and calcium-independent pools of annexin I in amnion and annexins I, II, and V in chorion significantly decreased with labor. The decrease in total annexin I concentration with labor in amnion reflects a substantial decrease (80-90%) in the pool tightly bound to the membrane in a calcium-independent manner. This striking change distinguishes annexin I as a potential candidate inhibitor which is specifically downregulated at parturition, potentially leading to increased access of phospholipases to substrate phospholipids and increased prostaglandin production at labor.     

The surface receptor is involved in annexin I-stimulated insulin secretion in MIN6N8a cells

This study investigated the effect of extracellular annexin I on regulating insulin secretion in MIN6N8a (an insulin secreting cell line) cells. The properties of annexin I receptor in MIN6N8a cells were also determined. Annexin I stimulated insulin release in MIN6N8a cells, regardless of the presence or absence of extracellular Ca(2+). Confocal microscopy revealed that annexin I bound to the surface of MIN6N8a cells. In addition, FACs analysis showed that annexin I bound to the surface of MIN6N8a cells in a dose-dependent manner. However, the annexin I-stimulated insulin secretion and the annexin I binding were abolished in MIN6N8a cells treated with proteases. Annexin I receptors were regenerated time-dependently. Furthermore, annexin I-stimulated insulin secretion was inhibited by cycloheximide but not by actinomycin D. These results showed that annexin I binds to the surface receptor in order to regulate the stimulation of insulin release in MIN6N8a cells.     

Dexamethasone alters arachidonate release from human epithelial cells by induction of p11 protein synthesis and inhibition of phospholipase A2 activity.

The effect of the glucocorticosteroid, dexamethasone, on arachidonic acid (AA) release and on protein levels of p11 and cytosolic phospholipase A2 (cPLA2) was studied in two epithelial cell lines, HeLa cells and BEAS-2B cells. Dexamethasone treatment of HeLa cells and BEAS-2B cells increased cellular p11 protein and mRNA levels in a time- and dose-dependent manner. It had little effect on levels of cPLA2 protein. In order to determine if increased p11 protein expression resulted in increased interaction between p11 and cPLA2, anti-cPLA2 antibodies were used to immunoprecipitate p11.cPLA2 complexes and Western blots of the immunoprecipitate were used to detect p11. In cells treated with dexamethasone, more p11 was detected in the anti-cPLA2 immunoprecipitate compared with control cells. Dexamethasone treatment of HeLa cells prelabeled with [3H]AA decreased the release of [3H]AA under basal conditions and after stimulation with the calcium ionophore A23187 (10(-6) M). In order to determine if altering the p11 protein levels in HeLa cells independent of glucocorticosteroid treatment could also produce an effect on [3H]AA release, cells were stably transfected with plasmids expressing either p11 antisense mRNA or p11 mRNA. Cloned HeLa cells expressing p11 antisense mRNA exhibited less cellular p11 protein compared with control cells and greater [3H]AA release compared with cells transfected with a control vector. Cloned HeLa cells stably transfected with a p11 expression vector exhibited increased p11 cellular protein and diminished [3H]AA release under basal conditions and in response to A23187. Therefore, dexamethasone alteration of epithelial cell AA release may be due in part to induction of p11 protein expression.     

DISTRIBUTION OF ANNEXIN I DURING NON-PATHOGEN OR PATHOGEN PHAGOCYTOSIS BY CONFOCAL IMAGING AND IMMUNOGOLD ELECTRON MICROSCOPY

Annexin I is an abundant protein in U937 cells differentiated towards a macrophagic phenotype. These cells become able to killEscherichia coli, however, the intracellular pathogenBrucella suis, known to interfere with phagosome maturation, multiply in these differentiated cells. We have analysed by confocal and electron microscopy the cellular localization of annexin I during phagocytosis of yeast, non-pathogenicE. coliand the intracellular pathogenB. suis. Using immunocytochemical detections annexin I was found mainly as patches in the cytoplasm of uninfected cells. Upon phagocytosis of yeast orE. coliorganisms, annexin I rapidly translocated and concentrated around phagosomes. On the other hand, annexin I was never detected around liveB. suis-containing phagosomes. However, when dead brucellae were used, annexin I did translocate to the periphagosomal region. Our results suggest that annexin I could play a role in the molecular mechanism of phagosome maturation, which is impaired by some intracellular pathogens.     

Annexin I is a stress protein induced by heat, oxidative stress and a sulfhydryl-reactive agent.

Annexin I (also called lipocortin 1) is a 37-kDa member of the annexin family of proteins. It has been proposed to be involved in the regulation of cell growth and differentiation, apoptosis, and inflammation. Previously, we have reported that annexin I displays a chaperone-like function (Kim, G.Y., Lee, H.B., Lee, S.O., Rhee, H.J. & Na, D.S. (1997) Biochem. Mol. Biol. Int. 43, 521-528). To determine the possibility that annexin I is a stress protein, we examined whether expression of annexin I and annexin I mRNA increases in response to stresses in A549 and HeLa cells. Treatments of cells with heat, hydrogen peroxide or sodium arsenite resulted in (a) an increase in annexin I and annexin I mRNA and (b) translocation of annexin I from the cytoplasm to the nucleus and perinuclear region. The annexin I gene promoter region, cloned upstream of a reporter gene, was inducible in response to heat, hydrogen peroxide, and sodium arsenite. These results indicate that annexin I serves as a stress protein and annexins may constitute a new class of stress proteins.     

Bradykinin enhances reactive oxygen species generation, mitochondrial injury, and cell death induced by ATP depletion--a role of the phospholipase C-Ca(2+) pathway

This study aimed to study the effect of bradykinin on reactive oxygen species (ROS) generation, mitochondrial injury, and cell death induced by ATP depletion in cell culture. Renal tubular cells were subjected to ATP depletion. Cell death was evaluated with LDH release, sub-G0/G1 fraction, Hoechst staining, and annexin V binding assay. ROS generation, mitochondrial membrane potential (DeltaPsi(m)), and intramitochondrial calcium were evaluated with flow cytometry. Translocation of cytochrome c and activation of apoptotic protein were analyzed with cell fractionating and Western blotting. Intracellular calcium was measured with a spectrofluorometer. Bradykinin enhanced cellular LDH release, apoptosis, generation of superoxide, and hydrogen peroxide induced by ATP depletion. Bradykinin also enhanced the loss of DeltaPsi(m), translocation of cytochrome c into cytosol, and activation of apoptotic protein. The intracellular/mitochondrial calcium was higher in bradykinin-treated cells. All these effects were reversed by coadministration with bradykinin B2 receptor (B2R) antagonist. Besides, blocking the phospholipase C (PLC) could reverse the synergistic effect of bradykinin with ATP depletion on ROS generation, mitochondrial damage, accumulation of intracellular/mitochondrial calcium, and apoptosis. Activation of B2R aggravates ROS generation, mitochondrial damage, and cell death induced by ATP depletion. These effects may act through the PLC-Ca(2+) signaling pathway.     

An annexin 2 phosphorylation switch mediates p11-dependent translocation of annexin 2 to the cell surface

Annexin 2 is a profibrinolytic co-receptor for plasminogen and tissue plasminogen activator that stimulates activation of the major fibrinolysin, plasmin, at cell surfaces. In human subjects, overexpression of annexin 2 in acute promyelocytic leukemia leads to a bleeding diathesis reflective of excessive cell surface annexin 2-dependent generation of plasmin (Menell, J. S., Cesarman, G. M., Jacovina, A. T., McLaughlin, M. A., Lev, E. A., and Hajjar, K. A. (1999) N. Engl. J. Med. 340, 994-1004). In addition, mice completely deficient in annexin 2 display fibrin accumulation within blood vessels and impaired clearance of injury-induced thrombi (Ling Q., Jacovina, A.T., Deora, A.B., Febbraio, M., Simantov, R., Silverstein, R. L., Hempstead, B. L., Mark, W., and Hajjar, K. A. (2004) J. Clin. Investig. 113, 38-48). Here, we show that endothelial cell annexin 2, a protein that lacks a typical signal peptide, translocates from the cytoplasm to the extracytoplasmic plasma membrane in response to brief temperature stress both in vitro and in vivo in the absence of cell death or cell lysis. This regulated response is independent of new protein or mRNA synthesis and does not require the classical endoplasmic reticulum-Golgi pathway. Temperature stress-induced annexin 2 translocation is dependent on both expression of protein p11 (S100A10) and tyrosine phosphorylation of annexin 2 because annexin 2 release is completely eliminated on depletion of p11, inactivation of tyrosine kinase, or mutation of tyrosine 23. Translocation of annexin 2 to the cell surface dramatically increases tissue plasminogen activator-dependent plasminogen activation potential and may represent a novel stress-induced protein secretion pathway.     

Differential expression of annexins I and II in normal and malignant human mammary epithelial cells

Abstract. Collagen-binding proteins ( CBPs ) of rat mammary tumors are identical to Ca²⁺-binding annexins [49]. We have now isolated a protein of 38 kDa from the human mammary tumor cell line ALAB by collagen type I affinity chromatography as well as by extraction of calcium-binding proteins. The 38-kDa band of both preparations was identified as annexin II (calpactin I) by its reaction with an annexin II-specific monoclonal antibody in Western blot analysis. Annexin I (lipocortin I) was not detectable in these cells. Two other human cell lines, the SV40-transformed cell line SV3 and cell line HBL-100, both established from normal mammary glands, were also positive for annexin II and negative for annexin I.
In vivo expression of annexins was investigated by immunohistological staining of normal and malignant human mammary tissue. The annexin II-specific mAb reacted with normal and tumor parenchyme whereas the annexin I-specific mAb reacted with acini and ductal myoepithelium of the normal mammary gland but showed no reaction with tumor tissue. Immunolocalization studies also showed annexin II expression in both normal and tumor stroma while only tumor stromal cells were found to be reactive with the antibody against annexin I. The differential expression of annexins in normal and malignant human mammary tissue suggests special functions of these proteins in the mammary gland.     

The association of annexin I with early endosomes is regulated by Ca2+ and requires an intact N-terminal domain.

Annexin I is a member of a multigene family of Ca2+/phospholipid-binding proteins and a major substrate for the epidermal growth factor (EGF) receptor kinase, which has been implicated in membrane-related events along the endocytotic pathway, in particular in the sorting of internalized EGF receptors occurring in the multivesicular body. We analyzed in detail the intracellular distribution of this annexin by cell fractionation and immunoelectron microscopy. These studies used polyclonal as well as a set of species-specific monoclonal antibodies, whose epitopes were mapped to the lateral surface of the molecule next to a region thought to be involved in vesicle aggregation. Unexpectedly, the majority of annexin I was identified on early and not on multivesicular endosomes in a form that required micromolar levels of Ca2+ for the association. The specific cofractionation with early endosomes was also observed in transfected baby hamster kidney cells when the intracellular fate of ectopically expressed porcine annexin I was analyzed by using the species-specific monoclonal antibodies in Western blots of subcellular fractions. Interestingly, a truncation of the N-terminal 26, but not the N-terminal 13 residues of annexin I altered its intracellular distribution, shifting it from fractions containing early to those containing late and multivesicular endosomes. These findings underscore the regulatory importance of the N-terminal domain and provide evidence for an involvement of annexin I in early endocytotic processes.     

Annexin I and involucrin are cross-linked by particulate transglutaminase into the cornified cell envelope of squamous cell carcinoma Y1.

The squamous cell carcinoma line, SqCC/Y1, like natural squamous epithelia, forms a cornified cell envelope during differentiation which can be directly correlated with an increase in particulate transglutaminase activity. When transglutaminase is activated in these cells by calcium ionophore X-537A, annexin I and involucrin become incorporated into the cornified cell envelope and cannot be extracted with solutions containing sodium dodecyl sulfate (SDS) and beta-mercaptoethanol. This effect is specific for annexin I; thus, the amounts of annexins II and IV that were extractable from cells by SDS and beta-mercaptoethanol did not change following treatment with ionophore X-537A. Annexin I could be cross-linked in vitro to itself and to other endogenous proteins by transglutaminase extracted from the particulate fraction of SqCC/Y1 cells. Immunofluorescence studies showed that cross-linked annexin I and involucrin form an envelope-like structure in SqCC/Y1 cells during differentiation that cannot be extracted by EGTA and Triton X-100. The amount of staining of this envelope structure corresponded directly to the particulate transglutaminase activity of these cells. Annexin I monoclonal and polyclonal antibodies were shown to bind to purified cornified cell envelopes from SqCC/Y1. These studies suggest that particulate transglutaminase regulates a function of annexin I during the differentiation of SqCC/Y1 cells by covalently cross-linking this protein into the cornified cell envelope.     

Pseudomonas aeruginosa autoinducer modulates host cell responses through calcium signalling

The opportunistic pathogen Pseudomonas aeruginosa utilizes a cell density-dependent signalling phenomenon known as quorum sensing (QS) to regulate several virulence factors needed for infection. Acylated homoserine lactones, or autoinducers, are the primary signal molecules that mediate QS in P. aeruginosa. The autoinducer N-3O-dodecanoyl-homoserine lactone (3O-C12) exerts effects on mammalian cells, including upregulation of pro-inflammatory mediators and induction of apoptosis. However, the mechanism(s) by which 3O-C12 affects mammalian cell responses is unknown. Here we report that 3O-C12 induces apoptosis and modulates the expression of immune mediators in murine fibroblasts and human vascular endothelial cells (HUVEC). The effects of 3O-C12 were accompanied by increases in cytosolic calcium levels that were mobilized from intracellular stores in the endoplasmic reticulum (ER). Calcium release was blocked by an inhibitor of phospholipase C, suggesting that release occurred through inositol triphosphate (IP3) receptors in the ER. Apoptosis, but not immunodulatory gene activation, was blocked when 3O-C12-exposed cells were co-incubated with inhibitors of calcium signalling. This study indicates that 3O-C12 can activate at least two independent signal transduction pathways in mammalian cells, one that involves increases in intracellular calcium levels and leads to apoptosis, and a second pathway that results in modulation of the inflammatory response.     

Increased apoptosis in U937 cells over-expressing lipocortin 1 (annexin I)

The potential involvement of endogenous lipocortin 1 in the process of cellular apoptosis, particularly in cells of the myelo-monocytic lineage, has been investigated. U937 cells were transfected either with an antisense or a sense DNA for lipocortin 1 and the stable clones 36.4AS clone (20-40% lower lipocortin 1 levels) and 15S (30% higher lipocortin 1 levels) were obtained. Cell apoptosis was induced by incubation with tumor necrosis factor-alpha: optimal responses were observed within a 24 h incubation period at a 5 ng/ml concentration. Apoptosis was assessed both morphologically, by annexin V binding and cell cycle analysis with propidium iodide. Whilst no consistent difference was seen between wild type cells and clone 36.4AS, a higher incidence of apoptosis (ranging from +30% to + 60%) was observed in the 15S clone. Release of arachidonic acid from loaded cells was promoted by 24 h incubation with the cytokine, and a higher degree of release was measured in the 15S clone. These data indicate that endogenous intracellular lipocortin 1 is involved in the promotion of apoptosis in cells of the myelo-monocytic derivation.     

Cloning and characterization of the annexin II receptor on human marrow stromal cells

Annexin II is a heterotetramer, consisting of two 11-kDa (p11) and two 36-kDa (p36) subunits, that is produced by osteoclasts and stimulates osteoclast formation. However, its receptor is unknown. We showed that annexin II binds to normal primary human marrow stromal cells and the Paget's marrow-derived PSV10 stromal cell line to induce osteoclast formation. 125I-Labeled annexin II binding assays with PSV10 cells demonstrated that there was a single class of annexin II receptors with a Kd of 5.79 nm and Bmax of 2.13 x 10(5) receptors/cell. Annexin III or annexin V did not bind this receptor. Using 125I-labeled annexin II binding to screen NIH3T3 transfected with a human marrow cDNA expression library, we identified a putative annexin II receptor clone, which encoded a novel 26-kDa type I membrane receptor protein when expressed in HEK 293 cells. HEK 293 cells transformed with the cloned annexin II receptor cDNA showed a similar binding affinity to annexin II as that observed in PSV10 cells. Chemical cross-linking experiments with biotinylated annexin II and intact PSV10 cells identified a 55-kDa band on Western blot analysis that reacted with both an anti-p11 antibody and streptavidin but not anti-p36 antibody. A rabbit polyclonal antibody raised against the putative recombinant annexin II receptor also recognized the same 26-kDa protein band detected in PSV10 cells. Importantly, the annexin II receptor antibody dose-dependently blocked the stimulatory effects of annexin II on human osteoclast formation, demonstrating that the receptor mediates the effects of annexin II on osteoclast formation.     

Screening,purification, and identification of annexin B1 mutants with high phosphatidylserine-binding activity and reduced immunogenicity

Annexin B1 has many potential biomedical applications based on its high affinity for negatively charged phospholipid (phosphatidylserine, PS) in the presence of physiological concentrations of calcium. Low immunogenicity is prerequisite for the in vivo application of a nonhuman protein as a novel-imaging agent. In the present study, three sequence-deleted mutants with different numbers of functional domains were designed and expressed according to the predicted three-dimensional structure of annexin B1. The mutants of annexin B1, as well as the wild-type annexin B1, were expressed as Glutathione-S-transferase (GST)-fusion proteins. Two mutants with their purity above 80% could be obtained after one-step primary purification procedure on basis of the PS-binding activity. The immunogenicity of the two mutants was evaluated in mice by detecting the titers of elicited antigen-specific IgG. A member of three mutants of annexin B1, M12, which involved N-terminal amino-acid sequence and double functional domain I and II of annexin B1, was finally selected to detect apoptosis that is due to its lowest immunogenicity among the candidate mutants. Flourescein isothiocyanate-labeled M12 could bind the outer membranes of apoptotic cells and discriminate apoptotic cells in the early stage from necrotic cells when used with propidium iodide. ^99mTc-labeled M12 could recognize the apoptotic hepatocytes induced by anti-Fas antibody treatment. Our data in vitro and in vivo demonstrated that M12 could be applied as a promising agent for the detection of apoptosis.     

Dichotomy of Ca2+ signals triggered by different phospholipid pathways in antigen stimulation of human mast cells

Mast cell activation triggers Ca(2+) signals and the release of enzyme-containing granules, events that play a major role in allergic/hypersensitivity reactions. However, the precise molecular mechanisms that regulate antigen-triggered degranulation and Ca(2+) fluxes in human mast cells are still poorly understood. Here we show, for the first time, that a receptor can trigger Ca(2+) via two separate molecular mechanisms. Using an antisense approach, we show that IgE-antigen stimulation of human bone marrow-derived mast cells triggers a sphingosine kinase (SPHK) 1-mediated fast and transient Ca(2+) release from intracellular stores. However, phospholipase C (PLC) gamma1 triggers a second (slower) wave of calcium release from intracellular stores, and it is this PLCgamma1-generated signal that is responsible for Ca(2+) entry. Surprisingly, FcepsilonRI (a high affinity receptor for IgE)-triggered mast cell degranulation depends on the first, sphingosine kinase-mediated Ca(2+) signal. These two pathways act independently because antisense knock down of either enzyme does not interfere with the activity of the other enzyme. Of interest, similar to PLCgamma1, SPHK1 translocates rapidly to the membrane after FcepsilonRI cross-linking. Here we also show that SPHK1 activity depends on phospholipase D1 and that FcepsilonRI-triggered mast cell degranulation depends primarily on the activation of both phospholipase D1 and SPHK1.     

Translocation of annexin B1 in response to the stimulation of PMA and ionomycin in cervical cancer cells

Annexin B1 is a novel member of the annexin superfamily which was isolated from a Cysticercus cellulosae cDNA library. To investigate the physiological roles of annexin B1, we firstly performed immunohistochemical analysis on frozen Cysticercus cellulosae sections and found that annexin B1 was present not only in the tegument of the bladder wall, but also in the host-derived inflammatory layer; In addition, ELISA analysis revealed that annexin B1 could be detected in the cystic fluid of Cysticercus cellulosae and the sera of pigs with cysticercosis. These findings indicated that annexin B1 might be a secretary protein. We further constructed a pEGFP-annexin B1 plasmid and transfected it into SiHa cells. We found that GFP-annexin B1 was stimulated to translocate to the plasma membrane by phorbol 12-myristate 13-acetate (PMA). By contrast, it was induced to distribute at the plasma and nuclear membranes by treatment with calcium ionophore ionomycin. PMA increased annexin B1 membrane binding, which might facilitate exocytosis. Moreover, translocation of the protein to the plasma and nuclear membranes after stimulated by ionomycin, was predicted to be related to an additional function.