Cytokine modulation of liver annexin 1 expression during experimental endotoxemia.

Annexin 1 (ANXA1) is a calcium-binding protein endowed with anti-inflammatory properties. Using an extra-hepatic system, we showed that interleukin (IL)-6 regulates ANXA1 expression at the transcriptional level. The purpose of this study was to determine whether ANXA1 synthesis was modulated by IL-6 during experimental inflammation. We have compared liver ANXA1 expression during systemic and localized inflammatory reaction, using lipopolysaccharide (LPS) and turpentine. LPS treatment strongly induced ANXA1 expression in the liver of wild-type (WT) animals (+600%) whereas a modest increase (+60%) was measured in IL-6 knockout (KO) animals. Turpentine treatment did not affect the expression of ANXA1 in either animal type. LPS enhanced serum corticosteroid levels equally in WT and IL-6 KO mice, whereas higher tumor necrosis factor (TNF)-alpha and IL-1beta levels were released in IL-6 KO animals. Injection of mouse recombinant IL-6 to IL-6 KO animals before LPS or TNF-alpha challenge, replenished ANXA1 liver synthesis to that of WT animals. Exogenous ANXA1 but not ANXA5, administered to IL-6 KO mice before LPS challenge inhibited TNF-alpha release. We propose that ANXA1 acts as a novel acute phase protein, which is controlled in the liver by TNF-alpha and IL-6, and which may contribute to the resolution of systemic endotoxemia through a negative feedback on TNF-alpha release.

Mitochondrial membrane potential and apoptosis peripheral blood monocytes in severe human sepsis.

UNLABELLED: Reduced mitochondrial membrane potential (Delta(Psi)m), which is considered as an initial and irreversible step towards apoptosis, as well as cell death regulating proteins, such as Fas, Hsp70, or Bcl-2, may play an important role in sepsis. We studied the relationship between sepsis severity and peripheral blood monocyte Delta(Psi)m, cell death (necrosis and apoptosis), soluble Fas ligand, Hsp70, and Bcl-2 expression over time in 18 patients with sepsis, and compared these data with those of a group of 17 healthy control subjects. All measurements were performed within 3 d of the onset of severe sepsis (T1), then 7 to 10 d later (T2), and finally at hospital discharge (T3). Delta(Psi)m was expressed as the percent monocytes with altered Delta(Psi)m (%Delta(Psi)m). Patients with sepsis had greater %Delta(Psi)m at T1 and T2 but not at T3 (14.6 +/- 2.6% and 15.9 +/- 2%, respectively, versus control 6.6 +/- 0.2%, p < 0.01). Septic patients exhibited greater cell death in their monocytes and had greater Hsp70 expression only at T1. Bcl-2 levels were similar in septic and control subjects. Comparing survivors with non-survivors of sepsis, nonsurvivors had a greater %Delta(Psi)m at T1 (26.4 +/- 5.3% versus 10.1 +/- 2.7%, p < 0.01) and a significant decrease in Bcl-2 expression, whereas no difference was found in Hsp70 levels. These results indicate that mitochondrial dysfunction and subsequent cell death occur in severe sepsis and suggest that %Delta(Psi)m is a marker of severity in human sepsis. KEYWORDS: mitochondria; apoptosis; sepsis; heat-shock protein 70; proto-oncogene protein c-Bcl-2

Annexin A5 D226K structure and dynamics: identification of a molecular switch for the large-scale conformational change of domain III.

The domain III of annexin 5 undergoes a Ca(2+)- and a pH-dependent conformational transition of large amplitude. Modeling of the transition pathway by computer simulations suggested that the interactions between D226 and T229 in the IIID-IIIE loop on the one hand and the H-bond interactions between W187 and T224 on the other hand, are important in this process [Sopkova et al. (2000) Biochemistry 39, 14065-14074]. In agreement with the modeling, we demonstrate in this work that the D226K mutation behaves as a molecular switch of the pH- and Ca(2+)-mediated conformational transition. In contrast, the hydrogen bonds between W187 and T224 seem marginal.

CREB is involved in mouse annexin A1 regulation by cAMP and glucocorticoids.

A cAMP and some glucocorticoid response elements have been underlined in the promoter of mouse annexin A1. To analyse the function of these DNA sequences, the role of cAMP and glucocorticoids, as well as the transcription factors involved in their activation, were investigated. A construct containing 1381 bp of the DNA 5'-flanking annexin A1 gene fused to LacZ was used. The level of activation of the reporter gene was analysed by transient transfection of the JEG3 cell line. Activation of beta-galactosidase expression was observed with both dibutyryl cAMP and dexamethasone when compared with cells treated with serum only. Simultaneous addition of dexamethasone and dibutyryl cAMP did not result in a synergistic effect but rather in a competitive one. Gel-shift assays with a probe including the cAMP response element-like element of the annexin A1 promoter revealed a main specific DNA-protein complex when cells were stimulated with dibutyryl cAMP and/or dexamethasone. In all cases CREB protein was identified by supershift analysis. We therefore conclude that this cAMP response element sequence plays a prominent role in the transactivation of the annexin A1 promoter by dibutyryl cAMP and that it is involved in the response to glucocorticoids.

Annexin 1 binds to U937 monocytic cells and inhibits their adhesion to microvascular endothelium: involvement of the alpha 4 beta 1 integrin.

Annexin 1 (ANX1), a calcium-binding protein, participates in the regulation of early inflammatory responses. Whereas some of its effects depend on intracellular interactions, a growing number of observations indicate that ANX1 may also act via autocrine/paracrine functions following externalization to the outer side of the plasma membrane. We studied the effects of ANX1 on leukocyte adhesion to endothelial cells using as a model system the monocytic cell line U937 and human bone marrow microvascular endothelial cells. Exogenous rANX1, as well as endogenous ANX1 externalized by U937 differentiated in vitro, inhibited monocyte firm adhesion to vascular endothelium. Both binding of ANX1 to U937 cells and ANX1-mediated inhibition of cell adhesion involved the short N-terminal domain of the ANX1 molecule. Under experimental conditions in which ANX1 inhibited U937 adhesion to human bone marrow microvascular endothelial cells, this protein specifically colocalized with the alpha 4 integrin, and a direct interaction between ANX1 and the alpha 4 integrin could be documented by immunoprecipitation experiments. Moreover, ANX1 competed with the endothelial integrin counterreceptor, VCAM-1, for binding to alpha 4 integrin. These results indicate that ANX1 plays an important physiological role in modulating monocyte firm adhesion to the endothelium.

The annexin A3-membrane interaction is modulated by an N-terminal tryptophan.

The crystal structure of annexin A3 (human annexin III) solved recently revealed a well-ordered folding of its N-terminus with the side chain of tryptophan 5 interacting with residues at the extremity of the central pore. Since the pore of annexins has been suggested as the ion pathway involved in membrane permeabilization by these proteins, we investigated the effect of the N-terminal tryptophan on the channel activity of annexin A3 by a comparative study of the wild-type and the W5A mutant in structural and functional aspects. Calcium influx and patch-clamp recordings revealed that the mutant exhibited an enhanced membrane permeabilization activity as compared to the wild-type protein. Analysis of the phospholipid binding behavior of wild-type and mutant protein was carried out by cosedimentation with lipids and inhibition of PLA(2) activity. Both methods reveal a much stronger binding of the mutant to phospholipids. The structure is very similar for the wild-type and the mutant protein. The exchange of the tryptophan for an alanine results in a disordered N-terminal segment. Urea-induced denaturation of the wild-type and mutant monitored by intrinsic fluorescence indicates a separate unfolding of the N-terminal region which occurs at lower urea concentrations than unfolding of the protein core. We therefore conclude that the N-terminal domain of annexin A3, and especially tryptophan 5, is involved in the modulation of membrane binding and permeabilization by annexin A3.

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.

System A neutral amino acid transporter regulation by interleukin-1beta in human osteoarthritic synovial cells: evidence for involvement of prostaglandin E(2) as a second messenger.

We studied the long-terms effects of interleukin-1beta (IL-1beta; 3 to 6 h) on alpha-(methylamino) isobutyric acid (MeAIB), a nonmetabolizable amino acid transported by system A. We found that IL-1beta induced a large decrease in MeAIB uptake by human osteoarthritic synovial cells and a concomitant increase in prostaglandin E(2) (PGE(2)) synthesis. Therefore, we investigated whether PGE(2) acts as a mediator for the long-term action of IL-1beta. We found that exogenous PGE(2) inhibited MeAIB uptake, and that AH6809, a PGE(2) receptor antagonist, inhibited IL-1beta-mediated MeAIB uptake. To identify the enzymes involved in the IL-1beta-mediated synthesis of PGE(2) that inhibits MeAIB uptake, we studied the expression of secreted (s) and cytosolic (c) phospholipase A(2) (PLA(2)). Because both were expressed, we selected a broad spectrum of inhibitors to determine which of the two PLA(2)s was involved. We used AACOCF3, a cPLA(2) inhibitor, and dithiothreitol (DTT) and bromophenacyl bromide (BPB), which are sPLA(2) inhibitors. Our results suggest that the PLA(2) involved in the IL-1beta-mediated synthesis of PGE(2) was sPLA(2). We also showed the expression of cyclooxygenase (COX)-2 and its partial involvement using a potent selective COX-2 inhibitor, L-745337. These findings provide insight into the mechanisms underlying the IL-1beta-mediated regulation of transport system A. The Il-1beta-induced inhibition of MeAIB uptake in human osteoarthritic synovial cells thus seems to be essentially mediated by PGE(2) production via the activation of sPLA(2) and the partial activation of COX-2.

Expression and localization of the annexins II, V, and VI in myocardium from patients with end-stage heart failure.

Annexins II, V, and VI belong to a family of Ca(2+)-dependent phospholipid-binding proteins that have been involved mainly in signal transduction, differentiation, membrane trafficking events, or binding to the extracellular matrix, or that might be effective as Ca(2+)-channels. They are abundant in the mammalian myocardium and might play a role in ventricular remodeling and altered calcium handling during heart failure. To test this hypothesis, we compared the expression and distribution of these annexins in nonfailing (n = 9) and failing human hearts with idiopathic dilated cardiomyopathy (n = 11). Northern blot and slot blot analysis were used to determine the annexin mRNA levels and Western blots were used to quantify the amounts of annexin proteins. Distribution of annexins was studied by immunohistofluorescence labeling and compared with that of a sarcolemmal marker (Na+/K(+)-ATPase) and of a myofibrillar protein (alpha-actinin). We showed that nonfailing hearts contained a higher amount of annexin VI than of annexin V or II (13.5 +/- 1.8, 3.7 +/- 0.2, and 2.5 +/- 0.5 microg/mg protein, respectively). In failing hearts, there was a parallel increase in both mRNA and protein levels of annexin II (146% and 132%, p < 0.05, respectively) and annexin V (152%, p < 0.01, 147%, p < 0.005, respectively); the protein level of annexin VI was also increased (117%, p < 0.05), whereas the increase of its mRNA level was statistically insignificant. We observed a predominant localization of annexin II in interstitium, and of annexins V and VI in cardiomyocytes at the level of the sarcolemma, T-tubules, and intercalated disks in nonfailing hearts, whereas in failing hearts enlarged interstitium contained all three annexins. Furthermore, annexin V staining at the level of cardiomyocytes almost disappeared. In conclusion, we showed that heart failure is accompanied by marked overexpression of annexins II and V, as well as translocation of annexin V from cardiomyocytes to interstitial tissue. The data suggest that annexins may contribute to ventricular remodeling and annexin V to impaired Ca2+ handling in failing heart.

Structural basis of the Ca(2+)-dependent association between S100C (S100A11) and its target, the N-terminal part of annexin I.

BACKGROUND: S100C (S100A11) is a member of the S100 calcium-binding protein family, the function of which is not yet entirely clear, but may include cytoskeleton assembly and dynamics. S100 proteins consist of two EF-hand calcium-binding motifs, connected by a flexible loop. Like several other members of the family, S100C forms a homodimer. A number of S100 proteins form complexes with annexins, another family of calcium-binding proteins that also bind to phospholipids. Structural studies have been undertaken to understand the basis of these interactions. RESULTS: We have solved the crystal structure of a complex of calcium-loaded S100C with a synthetic peptide that corresponds to the first 14 residues of the annexin I N terminus at 2.3 A resolution. We find a stoichiometry of one peptide per S100C monomer, the entire complex structure consisting of two peptides per S100C dimer. Each peptide, however, interacts with both monomers of the S100C dimer. The two S100C molecules of the dimer are linked by a disulphide bridge. The structure is surprisingly close to that of the p11-annexin II N-terminal peptide complex solved previously. We have performed competition experiments to try to understand the specificity of the S100-annexin interaction. CONCLUSIONS: By solving the structure of a second annexin N terminus-S100 protein complex, we confirmed a novel mode of interaction of S100 proteins with their target peptides; there is a one-to-one stoichiometry, where the dimeric structure of the S100 protein is, nevertheless, essential for complex formation. Our structure can provide a model for a Ca(2+)-regulated annexin I-S100C heterotetramer, possibly involved in crosslinking membrane surfaces or organising membranes during certain fusion events.

Annexin 1 expression and phosphorylation are upregulated during liver regeneration and transformation in antithrombin III SV40 T large antigen transgenic mice.

We have used a transgenic animal model, which constitutively develops hepatocarcinoma (Antithrombin III SV40 T large Antigen: ASV), to study the involvement of Annexin 1 (ANX1) in liver regeneration and malignant transformation. Primary hepatocytes isolated from normal mice did not express ANX1. In contrast, ANX1 was strongly expressed in hepatocytes of transgenic mice during constitutive development of hepatocarcinoma. In ASV transgenic mice, an elevated ANX1 level preceded the appearance of the tumor, indicating that it could be a good marker in the diagnosis of cancer. One-third hepatectomy in normal mice resulted in stimulation of ANX1 synthesis and phosphorylation. This upregulation correlated with increased synthesis of EGF and consequently with increased phosphorylation of the EGF receptor (EGF-R). Stable transfection of a hepatocyte cell line derived from ASV transgenic mice (mhAT2) with antisense complementary DNA for ANX1 reduced the proliferation rate as well as cytosolic phospholipase A(2) (cPLA(2)) activity. Thus, ANX1 expression and phosphorylation could be a factor implicated in liver regeneration and tumorigenesis, either through modulation of cPLA(2) activity or EGF-R function.

Annexin V counteracts apoptosis while inducing Ca(2+) influx in human lymphocytic T cells.

We have previously shown that when annexin V is present during the execution of a cell death program, apoptosis is delayed. This is reflected by the inhibition of DNA cleavage and of the release of apoptotic membrane particles, and by reduction of the proteolytic processing of caspase-3. Here, we have studied the mechanism(s) through which annexin V counteracts apoptosis in the human CEM T cell line. The degree of apoptosis inhibition was associated with an increase of intracellular Ca(2+) concentration ([Ca(2+)](i)). Reduction of the extracellular Ca(2+) concentration by EGTA abolished the anti-apoptotic effect, suggesting that annexin V favors Ca(2+) influx and that Ca(2+) acts as an inhibitor rather than an activator of apoptosis in CEM T cells. The effects on apoptosis and [Ca(2+)](i) of several modified annexins with different electrophysiological properties indicate that the N-terminal domain of annexin V is necessary for the Ca(2+)-dependent anti-apoptotic action of annexin V. These results suggest that annexin V regulates membrane Ca(2+) permeability and is protective against apoptosis by increasing [Ca(2+)](i) in CEM T cells.

Annexin V and phospholipid metabolism.

Annexins, protein kinases C and cytosolic phospholipase A2 belong to three families of ubiquitous cytoplasmic proteins involved in signal transduction. All annexins share the property of binding to phospholipids in the presence of calcium. Most annexins are substrates for protein kinases C except annexin V, the most ubiquitous and abundant annexin. Protein kinases C (PKC) belong to three distinct groups of kinases, conventional PKCs (cPKCs) that depend on calcium, diacylglycerol and negatively charged phospholipids for their activity, novel PKCs (nPKCs) and atypical PKCs (aPKCs), that do not require calcium for their activity, although they both require negatively charged phospholipids. Cytosolic phospholipase A2 (cPLA2) depends on calcium for its catalytic activity as well as on serine phosphorylation by MAP kinases. We report that annexin V modulates the activity of cPKCs as well as of cPLA2 by interfering with their ability to bind to negatively charged phospholipids and calcium. We propose that annexin V could interfere with the calcium and phospholipid signalling pathway.

Localization and quantitation of cardiac annexins II, V, and VI in hypertensive guinea pigs.

Annexins are characterized by Ca2+-dependent binding to phospholipids. Annexin II mainly participates in cell-cell adhesion and signal transduction, whereas annexins V and VI also seem to regulate intracellular calcium cycling. Their abundance and localization were determined in left ventricle (LV) and right ventricle (RV) from hypertensive guinea pigs, during the transition from compensatory hypertrophy to heart failure. Immunoblot analysis of annexins II, V, and VI revealed an increased accumulation (2.6-, 1.45-, and 2.3-fold, respectively) in LV from hypertensive guinea pigs and no modification in RV. Immunofluorescent labeling of annexins II, V, and VI; of Na+-K+-ATPase; and of sarcomeric alpha-actinin showed that in control LV and RV, 1) annexin II is present in nonmuscle cells; 2) annexins V and VI are mainly observed in the sarcolemma and intercalated disks of myocytes; 3) annexins II, V, and VI strongly label endothelial cells and adventitia of coronary arteries; and 4) annexin VI is present in the media. At the onset of heart failure, the most striking changes are the increased protein accumulation in LV and the very strong labeling of annexins II, V, and VI in interstitial tissue, suggesting a role in fibrosis development and cardiac remodeling.

Anti-inflammatory mechanism of alminoprofen: action on the phospholipid metabolism pathway.

Alminoprofen is a nonsteroidal anti-inflammatory drug (NSAID) of the phenylpropionic acid class. It has anti-inflammatory properties different from the classical NSAID. Using both in vitro systems of cells in culture and in vivo models of inflammation, we report here that alminoprofen possesses both antiphospholipase A2 (PLA2) activity and anti-cycloxygenase (COX) activity. The PLA2 targeted by alminoprofen is likely the secretory phospholipase A2 (sPLA2) while the COX targeted is the COX-2.

The crystal structure of a complex of p11 with the annexin II N-terminal peptide.

The aggregation and membrane fusion properties of annexin II are modulated by the association with a regulatory light chain called p11.p11 is a member of the S100 EF-hand protein family, which is unique in having lost its calcium-binding properties. We report the first structure of a complex between p11 and its cognate peptide, the N-terminus of annexin II, as well as that of p11 alone. The basic unit for p11 is a tight, non-covalent dimer. In the complex, each annexin II peptide forms hydrophobic interactions with both p11 monomers, thus providing a structural basis for high affinity interactions between an S100 protein and its target sequence. Finally, p11 forms a disulfide-linked tetramer in both types of crystals thus suggesting a model for an oxidized form of other S100 proteins that have been found in the extracellular milieu.

[Glucocorticoids and acute phase proteins]. / Glucocorticoïdes et protéines de la phase aiguë.

Glucocorticoids as well as acute phase proteins participate in non-specific host defence as well as in restoring host integrity after injury. Plasma levels of both compounds augment during the inflammatory reaction. However, glucocorticoids also have physiological effects that share similar molecular mechanisms with the family of steroids. During the inflammatory reaction, and for participating in host defense, glucocorticoids, together with augmented cytokines, use new signalling pathways. In doing so, they participate in the positive or negative control of inflammatory mediator synthesis. For example, they induce the synthesis of acute phase proteins in synergy with interleukin 6, interleukin 1 and TNF alpha.

Promoting detachment of neutrophils adherent to murine postcapillary venules to control inflammation: effect of lipocortin 1.

In this study we investigated, using intravital microscopy, how neutrophil extravasation across mouse mesenteric postcapillary venules is inhibited by the glucocorticoid-regulated protein lipocortin (LC; also termed annexin) 1. Intraperitoneal injection of 1 mg of zymosan into mice induced neutrophil rolling on the activated mesenteric endothelium followed by adhesion (maximal at 2 hr: 5-6 cells per 100-micrometers of vessel length) and emigration (maximal at 4 hr: 8-10 cells per high-powered field). Treatment of mice with human recombinant LC1 (2 mg/kg s.c.) or its mimetic peptide Ac2-26 (13 mg/kg s.c.) did not modify cell rolling but markedly reduced (>/=50%) the degree of neutrophil adhesion and emigration (P < 0.05). Intravenous treatment with peptide Ac2-26 (13 mg/kg) or recombinant human LC1 (0.7-2 mg/kg) promoted detachment of neutrophils adherent to the endothelium 2 hr after zymosan administration, with adherent cells detaching within 4.12 +/- 0.75 min and 2.36 +/- 0.31 min, respectively (n = 20-25 cells). Recruitment of newly adherent cells to the endothelium was unaffected. The structurally related protein LC5 was inactive in this assay, whereas a chimeric molecule constructed from the N terminus of LC1 (49 aa) attached to the core region of LC5 produced cell detachment with kinetics similar to LC1. Removal of adherent neutrophils from activated postcapillary endothelium is a novel pharmacological action, and it is at this site where LC1 and its mimetics operate to down-regulate this aspect of the host inflammatory response.