Recombinant annexin A6 promotes membrane repair and protects against muscle injury.

Membrane repair is essential to cell survival. In skeletal muscle, injury often associates with plasma membrane disruption. Additionally, muscular dystrophy is linked to mutations in genes that produce fragile membranes or reduce membrane repair. Methods to enhance repair and reduce susceptibility to injury could benefit muscle in both acute and chronic injury settings. Annexins are a family of membrane-associated Ca2+-binding proteins implicated in repair, and annexin A6 was previously identified as a genetic modifier of muscle injury and disease. Annexin A6 forms the repair cap over the site of membrane disruption. To elucidate how annexins facilitate repair, we visualized annexin cap formation during injury. We found that annexin cap size positively correlated with increasing Ca2+ concentrations. We also found that annexin overexpression promoted external blebs enriched in Ca2+ and correlated with a reduction of intracellular Ca2+ at the injury site. Annexin A6 overexpression reduced membrane injury, consistent with enhanced repair. Treatment with recombinant annexin A6 protected against acute muscle injury in vitro and in vivo. Moreover, administration of recombinant annexin A6 in a model of muscular dystrophy reduced serum creatinine kinase, a biomarker of disease. These data identify annexins as mediators of membrane-associated Ca2+ release during membrane repair and annexin A6 as a therapeutic target to enhance membrane repair capacity.

Identification and characterization of the acidic pH binding sites for growth regulatory ligands of low density lipoprotein receptor-related protein-1.

The type V TGF-beta receptor (TbetaR-V) plays an important role in growth inhibition by IGFBP-3 and TGF-beta in responsive cells. Unexpectedly, TbetaR-V was recently found to be identical to the LRP-1/alpha(2)M receptor; this has disclosed previously unreported growth regulatory functions of LRP-1. Here we demonstrate that, in addition to expressing LRP-1, all cells examined exhibit low affinity but high density acidic pH binding sites for LRP-1 growth regulatory ligands (TGF-beta(1), IGFBP-3, and alpha(2)M(*)). These sites, like LRP-1, are sensitive to receptor-associated protein and calcium depletion but, unlike LRP-1, are also sensitive to chondroitin sulfate and heparin and capable of directly binding ligands, which do not bind to LRP-1. Annexin VI has been identified as a major membrane-associated protein capable of directly binding alpha(2)M(*) at acidic pH. This is evidenced by: 1) structural and Western blot analyses of the protein purified from bovine liver plasma membranes by alpha(2)M(*) affinity column chromatography at acidic pH, and 2) dot blot analysis of the interaction of annexin VI and (125)I-alpha(2)M(*). Cell surface annexin VI is involved in (125)I-TGF-beta(1) and (125)I-alpha(2)M(*) binding to the acidic pH binding sites and (125)I-alpha(2)M(*) binding to LRP-1 at neutral pH as demonstrated by the sensitivity of cells to pretreatment with anti-annexin VI IgG. Cell surface annexin VI is also capable of mediating internalization and degradation of cell surface-bound (125)I-TGF-beta(1) and (125)I-alpha(2)M(*) at pH 6 and of forming ternary complexes with (125)I-alpha(2)M(*) and LRP-1 at neutral pH as demonstrated by co-immunoprecipitation. Trifluoperazine and fluphenazine, which inhibit ligand binding to the acidic pH binding sites, block degradation after internalization of cell surface-bound (125)I-TGF-beta(1) or (125)I-alpha(2)M(*). These results suggest that cell surface annexin VI may function as an acidic pH binding site or receptor and may also function as a co-receptor with LRP-1 at neutral pH.

Evidence for the Involvement of annexin 6 in the trafficking between the endocytic compartment and lysosomes.

Annexins are a family of calcium-dependent phospholipid-binding proteins, which have been implicated in a variety of biological processes including membrane trafficking. The annexin 6/lgp120 prelysosomal compartment of NRK cells was loaded with low-density lipoprotein (LDL) and then its transport from this endocytic compartment and its degradation in lysosomes were studied. NRK cells were microinjected with the mutated annexin 6 (anx6(1-175)), to assess the possible involvement of annexin 6 in the transport of LDL from the prelysosomal compartment. The results indicated that microinjection of mutated annexin 6, in NRK cells, showed the accumulation of LDL in larger endocytic structures, denoting retention of LDL in the prelysosomal compartment. To confirm the involvement of annexin 6 in the trafficking and the degradation of LDL we used CHO cells transfected with mutated annexin 6(1-175). Thus, in agreement with NRK cells the results obtained in CHO cells demonstrated a significant inhibition of LDL degradation in CHO cells expressing the mutated form of annexin 6 compared to controls overexpressing wild-type annexin 6. Therefore, we conclude that annexin 6 is involved in the trafficking events leading to LDL degradation.

Annexin VI stimulates endocytosis and is involved in the trafficking of low density lipoprotein to the prelysosomal compartment.

Annexins are calcium-binding proteins with a wide distribution in most polarized and nonpolarized cells that participate in a variety of membrane-membrane interactions. At the cell surface, annexin VI is thought to remodel the spectrin cytoskeleton to facilitate budding of coated pits. However, annexin VI is also found in late endocytic compartments in a number of cell types, indicating an additional important role at later stages of the endocytic pathway. Therefore overexpression of annexin VI in Chinese hamster ovary cells was used to investigate its possible role in endocytosis and intracellular trafficking of low density lipoprotein (LDL) and transferrin. While overexpression of annexin VI alone did not alter endocytosis and degradation of LDL, coexpression of annexin VI and LDL receptor resulted in an increase in LDL uptake with a concomitant increase of its degradation. Whereas annexin VI showed a wide intracellular distribution in resting Chinese hamster ovary cells, it was mainly found in the endocytic compartment and remained associated with LDL-containing vesicles even at later stages of the endocytic pathway. Thus, data presented in this study suggest that after stimulating endocytosis at the cell surface, annexin VI remains bound to endocytic vesicles to regulate entry of ligands into the prelysosomal compartment.

Inhibition of EGF-dependent calcium influx by annexin VI is splice form-specific.

Annexin VI is a widely expressed calcium- and phospholipid-binding protein that lacks a clear physiological role. We now report that A431 cells expressing annexin VI are defective in their ability to sustain elevated levels of cytosolic Ca(2+) following stimulation with EGF. Other aspects of EGF receptor signaling, such as protein tyrosine phosphorylation and induction of c-fos are normal in these cells. However, EGF-mediated membrane hyperpolarization is attenuated and Ca(2+) entry abolished in cells expressing annexin VI. This effect of annexin VI was only observed for the larger of the two annexin VI splice forms, the smaller splice variant had no discernable effect on either cellular phenotype or growth rate. Inhibition of Ca(2+) influx was specific for the EGF-induced pathway; capacitative Ca(2+) influx initiated by emptying of intracellular stores was unaffected. These results provide the first evidence that the two splice forms of annexin VI have different functions.

Annexin VI binds S100A1 and S100B and blocks the ability of S100A1 and S100B to inhibit desmin and GFAP assemblies into intermediate filaments.

Annexin VI, a member of a family of Ca(2+)-dependent phospholipid- and membrane-binding proteins, interacts with the Ca(2+)-regulated EF-hand proteins, S100A1 and S100B, and blocks the ability of these two proteins to inhibit the assembly of desmin and glial fibrillary acidic protein (GFAP) into intermediate filaments in a Ca(2+)- and dose-dependent manner. S100A1 and S100B each possess one annexin VI binding site, characterized by an affinity for annexin VI in the submicromolar range. Binding of annexin VI to either S100 protein occurs at a site that appears to differ in some parts from that recognizing desmin and GFAP. As S100A1 and S100B exist in solution as homodimers in which the two monomers are related by a 2-fold symmetry axis, each of the above S100 homodimers likely crosslinks two annexin VI molecules, a situation that appears typical of all the annexin-S100 protein complexes described thus far. However, whereas in the cases of other annexin-S100 complexes the C-terminal extension of the S100 molecule appears indispensable for annexin binding, the annexin VI binding site cannot be restricted to the S100A1 and S100B C-terminal extension. We speculate that the annexin VI site on S100A1/B may only partially overlap to the desmin/GFAP site. In contrast, no effects of annexin V on the ability of S100A1 or S100B to affect the desmin and GFAP assemblies could be documented, although binding of annexin V to S100A1 and S100B could be detected at relatively high Ca2+ concentrations. The present data suggest that annexin VI might regulate S100A1 and S100B activities and vice versa.

Modulation of human type II secretory phospholipase A2 by sphingomyelin and annexin VI.

Conjectural results have been reported on the capacity of inflammatory secreted phospholipase A2 (sPLA2) to hydrolyse mammalian membrane phospholipids. Development of an assay based on the release of non-esterified fatty acids by the enzyme acting on the organized phospholipid mixture constituting the membrane matrix has led to the identification of two prominent effectors, sphingomyelin (SPH) and annexin. Recombinant human type II sPLA2 hydrolyses red-cell membrane phospholipids with a marked preference for the inner leaflet. This preference is apparently related to the high content of SPH in the outer leaflet, which inhibits sPLA2. This inhibition by SPH is specific for sPLA2. Cholesterol counteracts the inhibition of sPLA2 by SPH, suggesting that the SPH-to-cholesterol ratio accounts in vivo for the variable susceptibility of cell membranes to sPLA2. Different effects were observed of the presence of the non-hydrolysable D-alpha-dipalmitoyl phosphatidylcholine (D-DPPC), which renders the membranes rigid but does not inhibit sPLA2. Annexin VI was shown, along with other annexins, to inhibit sPLA2 activity by sequestering the phospholipid substrate. The present study has provided the first evidence that annexin VI, in concentrations that inhibit hydrolysis of purified phospholipid substrates, stimulated the hydrolysis of membrane phospholipids by sPLA2. The activation requires the presence of membrane proteins. The effect is specific for type II sPLA2 and is not reproducible with type I PLA2. The activation by annexin VI of sPLA2 acting on red cell membranes results in the preferential release of polyunsaturated fatty acids. It suggests that type II sPLA2, in conjunction with annexin VI, might be involved in the final step of endocytosis and/or exocytosis providing the free polyunsaturated fatty acids acting synergistically to cause membrane fusion.

Annexin VI is secreted in human bile.

A calcium-binding protein of 68 kDa was isolated from human bile that precipitates upon addition of 5 mM CaCl2. This protein was recognized by an immunoaffinity purified anti-annexin VI antibody and it had a similar aminoacid composition as annexin VI. Phospholipase A2 activity was inhibited in vitro in a dose-dependent manner as reported for annexin VI. It is the first time that an annexin secretion is demonstrated in bile.

Chromaffin granules release calcium on contact with annexin VI: implications for exocytosis.

Chromaffin granules represent a substantial and exchangeable intracellular calcium pool which is thought to be regulated by a sodium/calcium exchange protein and also by a putative inositol trisphosphate-activated calcium channel. A family of calcium-binding proteins, called the annexins, has been shown to bind to chromaffin granules. We have therefore investigated the possible involvement of these proteins in the regulation of chromaffin granule sequestered calcium. Annexin VI (A-VI) produced a concentration-dependent release of 45Ca2+ from chromaffin granules; half-maximal release occurred at 1 microM A-VI, with near-maximum release being at 20 microM A-VI. The A-VI-induced release of 45Ca2+ was rapid, being essentially complete by our first time point of 7 s, and corresponded to 40% of the total sequestered 45Ca2+. A-VI-induced release occurred at extravesicular Ca2+ concentrations ranging from a pCa2+ of 4.12 to 6.86 and also appeared specific to this protein since neither annexin I nor annexin II (tetramer) could evoke any 45Ca2+ release. Given the predominant localization of A-VI to the apical plasmalemma, these results suggest that this protein could participate in the secretory event by mediating the localized release of Ca2+ at sites of contact between the chromaffin granule and plasma membrane.

[Effect of coagulation inhibitor proteins (Calphobindins) on tissue factor expression of endothelial cells].

We established a method for measuring procoagulant action on human umbilical vein endothelial cells (HUVEC). HUVEC (2.5 x 10(4)/well) were stimulated with 1 microgram/ml endotoxin (lipopolysaccharide: LPS) for 6 hours at 37 degrees C in 5% CO2. After washing, the HUVEC were incubated with assay buffer containing Proplex ST 1 unit (factor VII)/ml, S2222 0.6 mg/ml and CaCl2 6.6 mM, for 30 minutes at 37 degrees C. The procoagulant activity was determined by measuring the supernatant at OD405. Calphobindin I, II and III (CPB I, CPB II and CPB III) are the calcium dependent phospholipid binding proteins that exhibit anticoagulant activity in vitro. In this study, we investigated the effects of CPB I, CPB II and CPB III on procoagulant activity (PCA) expressed on HUVEC. The results are as follows 1) CPBI inhibits the procoagulant activity on HUVEC in a dose-dependent manner (IC 50% less than 0.4 microM). The same doses (0.4 microM) of CPBII and CPBIII decreased the procoagulant activity to 28.1% (CPBII), and to 84.6% (CPB III). CPB anticoagulant activities were, CPBII greater than CPBI greater than CPBIII, in that order. 2) When 0.05% H2O2 was added to the cell culture medium wells, concentrations of CPBI in supernatants increased in a time-dependent manner, and they reached to the maximum after 8 hours. CPBI in supernatants after 24 hours were not detected without H2O2, but concentrations of 4.88 ng/ml/10(4) cells with 0.01% H2O2, and 9.60 ng/ml/10(4) cells with 0.05% H2O2 were detected.

Effects of calphobindin II (annexin VI) on procoagulant and anticoagulant activities of cultured endothelial cells.

Effects of human placental calphobindin II (CPB-II) on the protein C activation and prothrombin activation on the cell surface of cultured calf pulmonary arterial endothelial cells have been investigated. CPB-II inhibited thrombin generation by factor Xa bound to the surface of the cultured endothelial cells in a dose-dependent manner. The amount (IC50) of CPB-II causing the inhibition at 50% was estimated to be approximately 10 nM. CPB-II was found to be ineffective, however, in the protein C activation by thrombin-thrombomodulin (TM) complex on the cell surface. Assay using purified TM revealed that CPB-II was able to exhibit the inhibitory potency for the protein C activation exclusively in the reconstituted system with negatively charged phospholipids. These results suggest that the neutral phospholipids participate in the protein C activation through the thrombin-TM system on the endothelial cell surface. The ability of CPB-II to inhibit procoagulant activity without affecting anticoagulant activity on the cultured endothelial cells is probably related to its potential physiological function, while it is able to exert various degrees of influence upon these activities in blood coagulation by interacting with negatively charged phospholipids in vitro.