Do annexins participate in lipid messenger mediated intracellular signaling? A question revisited

Abstract

Annexins are physiologically important proteins that play a role in calcium buffering but also influence membrane structure, participate in Ca²⁺-dependent membrane repair events and in remodelling of the cytoskeleton. Thirty years ago several peptides isolated from lung perfusates, peritoneal leukocytes, neutrophiles and renal cells were proven inhibitory to the activity of phospholipase A₂. Those peptides were found to derive from structurally related proteins: annexins AnxA1 and AnxA2. These findings raised the question whether annexins may participate in regulation of the production of lipid second messengers and, therefore, modulate numerous lipid mediated signaling pathways in the cell. Recent advances in the field of annexins made also with the use of knock-out animal models revealed that these proteins are indeed important constituents of specific signaling pathways. In this review we provide evidence supporting the hypothesis that annexins, as membrane-binding proteins and organizers of the membrane lateral heterogeneity, may participate in lipid mediated signaling pathways by affecting the distribution and activity of lipid metabolizing enzymes (most of the reports point to phospholipase A₂) and of protein kinases regulating activity of these enzymes. Moreover, some experimental data suggest that annexins may directly interact with lipid metabolizing enzymes and, in a calcium-dependent or independent manner, with some of their substrates and products. On the basis of these observations, many investigators suggest that annexins are capable of linking Ca²⁺, redox and lipid signaling to coordinate vital cellular responses to the environmental stimuli.

Keywords::

• Annexins
• lipid metabolism
• phospholipases
• lipid mediated signaling
• membrane heterogeneity

Introduction

The annexins belong to a multigenic family of calcium-dependent, pH-sensitive, membrane-binding proteins which are present in all eukaryotic organisms examined to date, including plants (Konopka-Postupolska et al. 2011, Laohavisit and Davies 2011), yeasts (Morgan et al. 2004), lower eukaryotes such as parasites (Hofmann et al. 2010) and bacteria (Morgan et al. 2006). In mammals there are currently 12 identified genes encoding structurally related annexins (Gerke and Moss 2002). Within the annexin molecule unique calcium-binding sites embedded in the highly conserved annexin repeat motifs, building their conserved C-terminal core, have been identified (Gerke and Moss 2002). In addition to the C-terminal core annexins contain a variable N-terminal domain containing binding sites for various protein ligands, as well as sites for functionally relevant posttranslational modifications, such as proteolysis and phosphorylation (Gerke and Moss 2002).

It is a strong belief of many annexin investigators that the reason for annexin multiplicity in various organisms may lie in the large number and diversity of their functions. First of all, as proteins that bind membranes in a calcium-dependent and -independent manner, they integrate calcium signaling with membrane-related cellular events including membrane repair mechanisms (Draeger et al. 2011). Furthermore, they participate in vesicular traffic (Futter and White 2007) and various signaling pathways that lead to cell differentiation, migration, and proliferation (Mussunoor and Murray 2008, Grewal and Enrich 2009, Monastyrskaya et al. 2009b, Grewal et al. 2010). Annexins also contribute to the maintenance of cellular calcium homeostasis (Babiychuk and Draeger 2000, Gerke et al. 2005, Monastyrskaya et al. 2009a).

Some members of the annexin family are ubiquitously expressed and function as intracellular Ca²⁺ concentration ([Ca²⁺]_c) sensors endowed with individual Ca²⁺-sensitivity (Monastyrskaya et al. 2009a, 2009b). The Ca²⁺ sensitivity of annexins is modulated by their interaction with other proteins (Gerke et al. 2005), especially those of the S100 family of Ca²⁺-binding proteins (Rescher et al. 2004, Gokhale et al. 2005, Hayes et al. 2009, Illien et al. 2010, Cmoch et al. 2011, Rezvanpour et al. 2011), and by proteolytic cleavage (Chung et al. 2004, Khau et al. 2011, Blume et al. 2012). Perhaps due to their variable calcium sensitivity, most cells contain multiple annexins (Monastyrskaya et al. 2007, Potez et al. 2011).

A growing number of evidence suggests that annexins exert their biological functions through influencing membrane dynamics, promoting membrane segregation and membrane fusion (Babiychuk and Draeger 2000, Gerke et al. 2005). Furthermore, annexins were found to participate in plasma membrane repair mechanisms by reacting to Ca²⁺ influx and rise in intracellular calcium concentration upon mechanical stress, and to the presence of toxins, pathogens, etc (Gerke et al. 2005, Draeger et al. 2011). In addition, annexins perform unique functions such as remodeling of cytoskeleton (Hayes et al. 2004a, 2006), and regulation of membrane ion conductance (Riquelme et al. 2004, Muimo 2009, Monastyrskaya et al. 2009a). Annexins have also been implicated in a range of pathologies, such as the progression of cancer, diabetes, the autoimmune disorder, anti-phospholipid syndrome and others frequently related to deregulated vesicular traffic (Hayes et al. 2007, Fatimathas and Moss 2010).

Originally, some annexins, such as annexin A7 (AnxA7), were reported to support vesicle aggregation (Chander et al. 2006). Later, the plasma membrane attachment, vesicle association and involvement in vesicular traffic have been shown for several other annexins, including AnxA1, AnxA2, AnxA5 and AnxA6. These proteins were described as participating in intracellular vesicle movement, formation of multivesicular bodies and endosomal functioning, including endocytic membrane traffic and the proper positioning of recycling endosomes (Futter et al. 1993, Gerke and Moss 2002, Mayran et al. 2003, Zobiack et al. 2003, Hayes et al. 2004b, White et al. 2006, Futter and White 2007, Lim and Pervaiz 2007, Morel et al. 2009). AnxA6, like AnxA1, has been associated with the EGF receptor signaling (Grewal and Enrich 2009, Vilá de Muga et al. 2009, Grewal et al. 2010).

For many years after their discovery, annexins were thought to bind to membrane anionic phospholipids, especially to phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂ or PIP₂) in a calcium-dependent manner (Gerke and Moss 2002, Hayes et al. 2009). Changes in intracellular calcium ion concentration were found to regulate the interaction of annexins with membranes. On the basis of these observations it was suggested that multiple annexins of different Ca²⁺ sensitivity present in a single cell, may interact with distinct plasma and/or internal membrane regions promoting processes important for cell function such as membrane segregation, membrane fusion and membrane repair, in response to membrane injury (Draeger et al. 2011). It is also worth mentioning calcium-independent annexin-membrane interactions, modulated by cholesterol, as these could be of particular importance in cellular signaling involving receptors localized in cholesterol and sphingomyelin-enriched membrane microdomains (Cornely et al. 2011).

A well described feature of annexins, i.e., their ability to interact with anionic phospholipids is commonly employed in the laboratory practice to recognize apoptotic cells characterized by the exposure of PS molecules on the external leaflet of the plasma membrane (Schutters and Reutelingsperger 2010). Biological relevance of identification and clearance of apoptotic cells could be related to the fact that this prevents the release of harmful cell contents thereby suppressing inflammation and autoimmune reactions (Rosenbaum et al. 2011). It is postulated that annexins may modulate cell removal by phagocytosis by acting as bridging molecules to PS, a characteristic phagocytosis signal of dying cells. Indeed, it has been shown that AnxA3, AnxA4, AnxA5 and AnxA13 can bind to PS in apoptotic cells, while the evolutionary younger AnxA8 cannot (Rosenbaum et al. 2011).

It must be emphasized that the repertoire of lipid molecules interacting with annexins is not limited to PS. It includes a relatively large number of phospholipids, such as phosphatidic acid (PA), phosphatidylinositol (PI), PtdIns(4,5)P₂ (PIP₂), fatty acids (arachidonic acid), ceramides and lipid-derived metabolites (Russo-Marie 1999, Monastyrskaya et al. 2009b, Cornely et al. 2011, Domon et al. 2012). Almost all of these molecules as second messengers are important constituents of lipid mediated signal transduction pathways that regulate vital cellular processes (Schug et al. 2012).

Lipid mediated transduction pathways can be defined as signaling events involving a lipid messenger that binds a protein target, which in turn exerts a specific effect on a given signaling pathway. Lipid messengers are not stored but rather are formed ‘on demand' at their site of action. They cannot circulate freely in solution but, rather, exist bound to special carrier proteins or remain within the membrane. In this respect, annexins which possess the ability to interact with amphipathic ligands and with membranes, seem to be well suited to play an intermediary role.

Since the discovery of annexins as molecules interfering with phospholipase A₂ activity, a source of important lipid messenger molecules, more than 30 years ago (Flower and Blackwell 1979), the current literature has provided little updated information on this topic. Therefore, in this review we present available evidence concerning the participation of annexins in regulation of lipid signaling via combination of various mechanisms including: (1) regulation of membrane lateral heterogeneity thus providing appropriate environment for enzymes and proteins involved in signal transduction; (2) interference with the activity of phospholipase A₂ and perhaps other phospholipases or lipid kinases by limiting their access to membranous substrates or directly interacting with these proteins; and (3) interaction with lipid mediators of signal transduction pathways.

The role of membrane lateral heterogeneity in lipid mediated signaling

One of the most intriguing feature of annexins, recently gaining attention of many laboratories worldwide, is their role in assembly, organization and stabilization of cholesterol and sphingomyelin enriched microdomains, frequently called rafts, which provide platforms for specific signaling pathways, as well as a proper environment for membrane enzymes and transport proteins, including ion channels and ion pumps (Cornely et al. 2011, Reverter et al. 2011, Domon et al. 2012). In addition, annexins were described as proteins whose intracellular distribution is affected by the distribution of cholesterol (Grewal et al. 2000, de Diego et al. 2002, te Vruchte et al. 2004, Grewal et al. 2010, Enrich et al. 2011); these proteins were found to co-localize with cholesterol at the plasma membrane and to follow intracellular cholesterol trafficking. Moreover, some evidence has been presented suggesting participation of annexins as cholesterol interacting proteins in the mechanism of formation of cholesterol-rich domains not only in the plasma membrane but also in membranes of the intracellular organelles (Ayala-Sanmartin et al. 2001, Lambert et al. 2004, Cubells et al. 2007, Domon et al. 2010, Vats et al. 2010, Cornely et al. 2011, Domon et al. 2012).

The opinion existing in the literature in the late '90s of the twentieth century about membrane binding properties of annexins and the consequences of this binding on membrane structure and properties, was based mostly on the results of in vitro experiments showing calcium-dependent binding of annexins to membranes and their ability to self-assemble on the membrane surface (Skrahina et al. 2008). These results led to the creation of the model depicted in Figure 1. The model considers a calcium signal, i.e., the rise in [Ca²⁺]_c concentration upon cell stimulation as a trigger of annexin interaction with membranes. At low resting [Ca²⁺]_c annexins exist in the soluble state. Upon cell stimulation annexins bind to the membrane surface and self-associate. The authors of the model also considered the lateral reorganization of lipids in the cytosolic leaflet of the membrane into lipid-protein microdomains as an additional trigger for annexin-membrane interactions (Seaton and Dedman 1998). A consequence of this model is that the presence of organized arrays of surface-bound annexins alters membrane properties and the properties of other membrane-bound proteins and enzymes (Kaetzel and Dedman 1995, Seaton and Dedman 1998, Russo-Marie 1999, Kaetzel and Dedman 2004). Additionally, one may consider the role of such surface-bound annexin aggregates in the lateral organization of the membrane, right beneath the arrays of the protein.

Figure 1. Schematic visualization of annexins as regulators of formation of lipid second messengers interfering with the activity of cytosolic phospholipase A₂ (PLA₂) and other phospholipases, as well as mediators of the activity of various protein and lipid kinases (including proteins kinase C isoforms) playing regulatory roles in different lipid mediated signaling pathways. The scheme illustrates the idea formulated by Dedman and his co-workers (Kaetzel and Dedman 1995, Seaton and Dedman 1998) suggesting that upon a rise in intracellular [Ca²⁺]_c annexin monomers (depicted in green), bind initially to certain lipid constituents of the inner leaflet of the plasma membrane, preferably to lipid microdomains enriched either in anionic phospholipids (such as phosphatidylserine, phosphatidylinositol or phosphatidylinositol 4,5-bisphosphate) or to cholesterol and sphingomyelin or ceramides (depicted in the figure in various colors). Then, annexins bound to the membranes interact with each other to form larger arrays on the membrane surface and, therefore, prevent the interaction of phospholipases, other lipid hydrolyzing enzymes, as well as protein and lipid kinases, with their substrates at the membrane. Other explanations are in the text. Redrawn from Kaetzel and Dedman (1995), modified. This Figure is reproduced in color in the online version of Molecular Membrane Biology.

In fact, membrane lipid and protein lateral distribution and their assembly into membrane microdomains of variable life-time, are tightly regulated processes (Lemmon 2008, Levental et al. 2010, Surma et al. 2011). A growing number of evidence suggests that the optimal function of many membrane proteins depends on their residence at the lipid rafts. Examples of these proteins include ion pumps (Baron et al. 2010) and ion channels such as human ether-à-go-go-related gene (HERG) (Ganapathi et al. 2010), and calcium-activated chloride channels (Sones et al. 2010), protein complexes involved in calcium entry (Orai1, TRPCs and STIM1) (Galan et al. 2010), membrane transporters (Mrp1 or ABCC1) (Hummel et al. 2011), membrane receptors (Lasley 2011), enzymes (NADPH oxidase subunits) (Zhang and Li 2010), as well as caveolin 1 (Lin et al. 2010), cadherins (Gentil-dit-Maurin et al. 2010), supervillin (Fang et al. 2010) and other proteins associated with various signaling pathways. As a consequence of such localization many vital signaling pathways are regulated at membrane microdomains where the signal complexes are assembled, organized and fine-tuned (Staubach and Hanisch 2011). Examples of such microdomain-dependent processes include signaling triggered by a member of the tumor necrosis factor (TNF) receptor family, CD40, expressed on a variety of immune and non-immune cells, and its ligand, CD154, playing a role in humoral and cell-mediated immunity (Nadiri et al. 2011). Activation of this signaling pathway causes changes in the compartmentalization of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway normally residing within plasma membrane microdomains. This, in turn, may lead to development of insulin resistance (Gao et al. 2011) and involvement of PI3K-associated cholesterol-enriched microdomains in CD204-mediated low density lipoprotein (LDL) uptake in human macrophages, that may be critical in inflammatory processes (Kiyanagi et al. 2011).

Despite the results of in vitro experiments (using liposomes, monolayer technique, solid supported membranes) showing interaction of some annexins (AnxA2, AnxA5, AnxA6 and AnxA13) with the lipid constituents of membrane microdomains, controversy still exists whether this process occurs in vivo and what its functional outcome might be (te Vruchte et al 2004, Gokhale et al. 2005, Grewal and Enrich 2009). The nature of the signal triggering annexin re-localization from the cytoplasm to the membranes, in particular microdomain-enriched regions, is not well understood either. Differential membrane binding ability of annexins is suggested to be the result of their different calcium and pH sensitivity, lipid and partner protein specificity, and posttranslational modifications (for review, see Domon et al. 2012).

Many members of the annexin family of proteins have been localized at membrane microdomains resembling rafts (Domon et al. 2012). This was observed for AnxA2 and its heterotetramer with the S100A10 protein (AnxA2₂S100A10₂); the latter is most probably implicated in microdomain-supported exocytosis of neurotransmitters and promotes the lateral association of glycosphingolipid- and cholesterol-enriched lipid microdomains into larger assemblies (Umbrecht-Jenck et al. 2010). The association of AnxA2 with lipid rafts was found to be influenced not only by intracellular [Ca²⁺] but also by N-terminal phosphorylation at Tyr23 residue of annexin. In addition it has been observed that the binding of AnxA2 to the lipid rafts is followed by the transport of protein along the endocytic pathway (Valapala and Vishwanatha 2011).

In neuroendocrine cells, AnxA2 and its heterotetramer were found to be implicated in the assembly of monosialotetrahexosylganglioside (GM1)-containing lipid microdomains as well as PIP₂-enriched microdomains that are required for calcium-regulated exocytosis (Chasserot-Golaz et al. 2005). Furthermore, upon stimulation of adrenergic chromaffin cells the translocation of cytosolic AnxA2 to the plasma membrane, where it co-localizes with a member of the SNARE family of proteins, SNAP-25, and S100A10 (a partner protein of VAMP2), was observed, suggesting close relation between AnxA2-mediated lipid microdomains and SNAREs during exocytosis (Umbrecht-Jenck et al. 2010). In addition, AnxA2 and HIV-1 Gag interact at the PIP₂-enriched membrane microdomains, sites of Gag-mediated viral assembly; this suggests that AnxA2 supports viral assembly, and functions in the late stages of HIV-1 replication (Harrist et al. 2009). AnxA13b, much like AnxA2, has been assigned to raft localization and functions in raft-dependent apical traffic in MDCK cells, as first described by Simons and co-workers (Lafont et al. 1998, Lecat et al. 2000) and further confirmed by other investigators (Astanina et al. 2010). AnxA8, another member of the family, was shown to bind, in a Ca²⁺-dependent manner, to F-actin-associated PIP₂-enriched membrane microdomains in HeLa cells. The appearance of PIP₂-enriched membrane microdomains seems to be stimulated upon infection with Escherichia coli (Goebeler et al. 2006). Similar observation was earlier made for AnxA2 (Zobiack et al. 2002).

The existing data on intracellular distribution and function of AnxA6 suggest that this protein, as a protein involved in the lateral organization of the plasma membrane, interacting with cholesterol and modulating the remodeling of actin cytoskeleton, is also involved in the vesicular transport along endocytotic and exocytotic pathways by interacting with the target membrane SNAP receptors (t-SNAREs) (Umbrecht-Jenck et al. 2010, Reverter et al. 2011) and working as a scaffold protein for several signaling proteins (Cornely et al. 2011). The best characterized to date is the Ca²⁺-dependent membrane targeting of p120^GAP to down-regulate Ras activity (Enrich et al. 2011) and the stimulatory effect of AnxA6 on protein kinase C (PKC) important in the regulation of HRas and epidermal growth factor receptor (EGFR) signal transduction pathways (Grewal et al. 2010). One of the most important and intriguing observations is that AnxA6, by sequestering cholesterol in the late endosomes, as for example in skin fibroblasts of patients suffering from the Niemann-Pick type C disease (Grewal et al. 2000, 2010, de Diego et al. 2002, te Vruchte et al. 2004, Sztolsztener et al. 2010, Enrich et al. 2011), may contribute to lowering the levels of cholesterol in the Golgi and the plasma membrane. This leads to reduced cytoplasmic phospholipase A₂ (cPLA₂) activity, retention of caveolin in the Golgi apparatus and a reduced number of caveolae at the cell surface (Cubells et al. 2008).

The latter observation raises the question whether lipid microdomains play a role in regulation of signaling pathways in which phospholipases are engaged. It has been shown for example that phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) selectively accumulates in the plasma membrane of HER2-overexpressing cells, where it co-localizes and associates with the EGFR family member, HER2, in raft domains. This may be of particular importance for targeting the molecular mechanisms controlling HER2 overexpression in the membrane of breast cancer cells by altering the rates of its endocytosis and lysosomal degradation (Paris et al. 2010). Furthermore, PI-specific PLC (PI-PLC) isoform δ1, which is localized in caveolae/rafts where it hydrolyzes PIP₂ and initiates calcium mobilization and PKC activation, was found to be involved in noradrenaline-induced PIP₂ hydrolysis and sustained contraction of vascular tissue (Clarke et al. 2008).

Taking together observations described in this paragraph it is worth to note the participation of annexins in the lateral organization of membranes, especially their ability to participate in the assembly and organization of lipid microdomains that constitute specific signaling platforms, may affect the function of lipid microdomain-associated proteins, including phospholipases. Bearing in mind that various phospholipases exert their activities also through lipid microdomains, in the next paragraph we address the question how annexins, either in a membrane-bound raft-associated or a soluble cytosolic form, may affect the functioning of phospholipases in certain signaling pathways in the cell.

Annexins as regulators of lipid-hydrolyzing enzyme activities

Cytosolic phospholipases A₂

Phospholipases A₂ release fatty acids (frequently arachidonic acid) from the second carbon of glycerol because they specifically recognize the sn-2 acyl bond of phospholipids. The superfamily includes several unrelated protein subfamilies with common enzymatic activity. The two most notable subfamilies are the secreted PLA₂ (they require Ca²⁺ for their activity) and the cytosolic PLA₂ (Dennis et al. 2011). The latter family is also Ca²⁺-dependent, but its members have a different structure (a C2 domain and a large catalytic domain) and are significantly larger than the secreted PLA₂. Cytosolic PLA₂ are involved in cell signaling processes, such as inflammatory responses. Arachidonic acid is both a signaling molecule and the precursor for other signalling molecules: eicosanoids (leukotrienes and prostaglandins). Other subfamilies include Ca²⁺-independent PLA₂ (i PLA₂) and lipoprotein-associated PLA₂ (lp-PLA₂), also known as platelet activating factor acetylhydrolases (PAF-AH) (Freedman et al. 2004, Saavedra et al. 2006, Leslie et al. 2010, Dennis et al. 2011).

Among many factors regulating their activity, the most important are phosphorylation (Hirabayashi et al. 2004), changes in [Ca²⁺], and interactions with various proteins, including vimentin (Hirabayashi et al. 2004), NADPH oxidase (Hirabayashi et al. 2004), PLA₂-activating protein (PLAA) (Zhang et al. 2008) and annexins (Buckland and Wilton 2003). As for annexins, ever since the publication of Flower and Blackwell more than 30 years ago, describing for the first time a factor present in lung perfusate that was induced as a result of treatment with anti-inflammatory steroids and was an inhibitor of PLA₂ activity (further identified as AnxA1) (Flower and Blackwell 1979, Blackwell et al. 1980, de Caterina et al. 1993, Hayashi et al. 1993, Croxtall et al. 1996a, 1996b, 1998), the dilemma over the mechanism by which annexins exert their inhibitory effect on PLA₂ activity still persists. Various hypotheses were formulated so far including reduced availability of membrane substrates for PLA₂ due to the presence of annexins (substrate depletion concept), an indirect mechanism in which signaling pathways leading to modulation of phospholipase activity are affected and, last but not least, a direct interaction of annexins with lipid-hydrolyzing enzymes (Buckland and Wilton 2000a, 2000b, 2003).

The first hypothesis considering substrate depletion or interfacial competition as a mechanism of PLA₂ activity inhibition by annexins was formulated with respect to the ability of annexins to bind in a calcium-dependent manner to membranous anionic phospholipids, thus preventing PLA₂ binding to the membrane (Kaetzel and Dedman 1995, 2004, Seaton and Dedman 1998). Furthermore, it has been postulated that membrane-bound annexins self-associate to form trimers that further assemble into sheets that cover the membrane surface and alter crucial properties of the membrane such as fluidity and permeability to metabolites and solutes. According to this concept, this submembranous annexin shield alters the functions of membrane integral proteins such as ion pumps and channels, and protects the membrane surface from phospholipid binding proteins such as cytosolic (or secreted) phospholipases and PKC isoforms (Kaetzel and Dedman 2004). This mechanism was analyzed in detail by Buckland and Wilton, and a positive conclusion has been reached concerning inhibition of the secreted PLA₂ activity by annexins that could be best explained by competition of the two proteins for the same membrane surface (Buckland and Wilton 1998a, 2003). The same investigators asked this question in relation to cytosolic PLA₂ activity (Buckland and Wilton 1998b, 2003). They stated that the regulation of cytosolic PLA₂ was more complex and could involve both direct protein-protein interactions and indirect effects involving various signaling pathways.

The concept of interfacial competition was further extended into a hypothesis that annexins could interfere with the calcium and phospholipid signaling pathways by affecting the activity of various calcium-dependent and independent, anionic phospholipid-stimulated isoforms of PKC, and cytosolic PLA₂ (Russo-Marie 1999).

Buckland and Wilton in their excellent review (Buckland and Wilton 2003) analyzed the glucocorticoid effects in relation to annexins and cPLA₂ interactions. These authors discussed the potential roles of these interactions taking into account that glucocorticoids control up to 1% of the genome and thus exert profound physiological effects. These effects include regulation of the production of mediators of inflammation and related metabolic disturbances. The authors also provided numerous evidence showing that annexins could control PLA₂ activity via protein-protein or protein-lipid interactions or through signalling cascades (Buckland and Wilton 2003). In this context, it is worth to note that annexins, especially AnxA1, may target the cytosolic PLA₂ both by direct enzyme inhibition and by suppression of cytokine-induced activation of phospholipase. AnxA1 inhibits the expression and activity of other inflammatory enzymes such as the inducible nitric oxide synthase (iNOS) in macrophages and inducible cyclooxygenase (COX-2) in activated microglia (Parente and Solito 2004). This, together with the role of AnxA1 in cell migration (Meliton et al. 2008) and its neuroprotective effects (Liu et al. 2005), point to the complexity of cellular events affected by the interplay between annexins and cPLA₂ (Chen et al. 2010).

Below we would like to focus on the results of experiments showing a direct interaction of annexins with PLA₂ and its possible physiological consequences.

It has been proposed that cPLA₂ activity is inhibited by AnxA1 by a direct interaction rather than by substrate depletion (Kim et al. 1994). By systematic approach it has been shown by the same investigators that inhibition of cPLA₂ by a specific interaction is not a general function of all annexins. It was characteristic for AnxA1, N-terminally truncated AnxA1, heterotetramer AnxA2₂S100A10₂ and to a lesser extent for AnxA5, but not AnxA2 and AnxA3 (Kim et al. 2001a). The interaction site within the AnxA1 molecule was then identified using AnxA1 deletion mutants. It was observed that the deletion mutant AnxA1-(1–274), exhibited no cPLA₂ inhibitory activity, whereas the deletion mutant AnxA1-(275–346), containing the C terminus, retained full activity. In addition, the AnxA1-(1–274) mutant did not interact with cPLA₂ (Kim et al. 2001b).

The mechanism of direct interaction of the AnxA2₂S100A10₂ heterotetramer with PLA₂ in terms of regulatory capacity is described for group IVA cPLA₂α. Group IVA cPLA₂α translocates to intracellular membranes, including the Golgi apparatus, in response to a rise in [Ca²⁺]_c (Leslie et al. 2010). Group IVA cPLA₂α has been reported to be phosphorylated at multiple serine residues, of which phosphorylation of the Ser727 residue was found to modulate protein-protein interactions. It has been shown that the AnxA2₂S100A10₂ heterotetramer but not S100A10 or AnxA2 alone, directly binds cPLA₂α via Ser727. This interaction prevents binding of the phospholipase to the membrane and inhibits its activity. On the other hand phosphorylation of the Ser727 residue of PLA₂ disrupts the interaction and activates cPLA₂α (Figure 2) (Tian et al. 2008).

Figure 2. The activity of ubiquitous mammalian group IVA cPLA₂ (cPLA₂α) is tightly regulated by various factors, such as phosphorylation at multiple serine residues, [Ca²⁺], lipid mediators and various proteins, including annexin A2/S100A10 heterotetramer (AnxA2₂S100A10₂). A cPLA₂α calcium-dependent binding to the plasma membrane is assured by the presence of an NH₂-terminal C2 domain (C2) within the enzyme molecule. According to the mechanism proposed by Cho and his co-workers (Tian et al. 2008) upon rise in intracellular [Ca²⁺] AnxA2₂S100A10₂ heterotetramer interacts with cPLA₂α (the binding occurs via the hydroxyl group of Ser727 (S727) of cPLA₂α that forms hydrogen bonds with S100A10 in the heterotetramer) and thus prevents its binding to the membrane and inhibits the enzyme. Phosphorylation of the Ser727 residue affects the cPLA₂α-AnxA2₂S100A10₂ interaction, thereby allowing phospholipase activation and subsequent lipid hydrolysis through binding to the membrane. Phosphorylation of another residue of cPLA₂α, Ser505 at C2 domain, does not appear to influence the cPLA₂α-AnxA2₂S100A10₂ interaction. On the Figure the lipid microdomain (pink) enriched in phosphatidylinositol 4,5-bisphosphate, a well known partner molecule for AnxA2, is depicted as a potential membrane target for the heterotetramer. Such domain could be stabilized by cholesterol molecules (Gokhale et al. 2005). Other explanations are in the text. Adapted from Tian et al. (2008), with some modifications. This Figure is reproduced in color in the online version of Molecular Membrane Biology.

The results of subsequent in vitro experiments using Calu-3 cells and the plasmon surface resonance technique revealed interaction of S100A10 with CFTR (NBD1 domain), AnxA1 and cPLA₂α. This was accompanied in Calu-3 cells by a partial redistribution of all four proteins into detergent resistant membranes induced by tumor necrosis factor-α (TNF-α), increased IL-8 synthesis, activation of cPLA₂α and overproduction of eicosanoids. This suggests that the putative cPLA₂α/AnxA1/S100A10/CFTR complex may participate in the regulation of the levels of inflammation mediators (Borot et al. 2009).

Using Madin-Darby canine kidney (MDCK) type II cells it has been observed that the stoichiometry of S100A10-cPLA₂ interaction may be controlled by cell confluence, therefore the phospholipase activation can be controlled by the establishment of cell-cell contacts (Bailleux et al. 2004). In addition, it has been found that the confluence-dependent interaction of cPLA₂α and AnxA1 at the Golgi membranes in endothelial cells may act as a molecular switch controlling cPLA₂α activity and endothelial cell prostaglandin generation (Herbert et al. 2007).

In Chinese hamster ovary (CHO) cells overexpressing AnxA6, sequestration of cholesterol in late endosomes led to reduced amounts of cholesterol and cPLA₂ in the Golgi membranes, and to inhibition of cPLA₂ activity. This correlated with an impairment of caveolin export suggesting that AnxA6 interfered with caveolin transport through inhibition of cPLA₂ activity; (Cubells et al. 2008). It is suggested that the AnxA6/cPLA₂ interaction may regulate vital physiological processes including proliferation, differentiation, inflammation and cell migration although a direct interaction between AnxA6 and cPLA₂ in the cell has not been demonstrated (Grewal et al. 2010).

The importance of potential cPLA₂/annexin interactions in pathological processes is supported by observations suggesting that in prostate cancer cPLA₂ function is affected by aberrant upregulation of the secreted enzymes and concomitant downregulation of their endogenous inhibitors: AnxA1 and AnxA2 (Dong et al. 2006).

Moreover, it was reported that in normal human keratinocytes (NHK), Ca²⁺-dependent binding of S100A11 to its partner annexin, AnxA1, facilitated the binding of the latter to cPLA₂, resulting in inhibition of cPLA₂ activity, which is essential for the growth of NHK cells. Upon NHK exposure to the epidermal growth factor, AnxA1 was cleaved at the Trp12 residue by cathepsin D, and this was accompanied by growth inhibition (Sakaguchi et al. 2007).

Other phospholipases

In contrast to the cytoplasmic and secretory PLA₂ isoforms, the number of reports related to the possible effects of annexins on phospholipase C (PLC), phospholipase D (PLD) and sphingomyelinase (SMase) activities is very limited. The reports mostly refer to the molecular events that are related to the potential role of annexins in the formation of membrane microdomains that provide a proper environment for the lipid hydrolysing enzymes. No data supporting a direct interaction of annexins with these enzymes is available so far. Therefore, further studies are required to answer the question whether annexins may modulate the activity of other lipid-hydrolysing enzymes. A brief analysis of the properties of these enzymes, i.e., their intracellular localization, substrates and interacting partners, makes the presumption about annexins as important factors for lipid-hydrolyzing enzymes very likely.

Phospholipases C belong to a class of enzymes that cleave phospholipids into diacylglycerol (DAG) and phosphoethanolamine or phosphocholine. When PIP₂ is subjected to digestion, DAG and inositol 1,4,5-trisphosphate (IP₃) are produced. In this respect, PLC is a key enzyme regulating intracellular calcium homeostasis and modulating phosphoinositide balance. Thirteen isoforms of mammalian PLC are classified into six isotypes (β, γ, δ, ε, ζ, η) (Fukami et al. 2010). In the next paragraph the ability of annexins to interact with phosphatidylinositol and phosphoinositides will be analyzed in conjunction with the PLC activity and cellular functions. Here, we refer to a spectacular example of biological activity of AnxA7 in the alveolar type II cells responsible for secretion of lung surfactants. It has been shown that the membrane fusion activity of AnxA7 involved in the fusion of lamellar body membranes with the plasma membrane during the secretory process is greatly modulated when DAG and PIP₂, the product and the substrate of PLC, respectively, are present (Chander et al. 2007).

Phospholipases D (PLD) are enzymes which are located in the plasma membrane and catalyze the hydrolysis of glycerophospholipids and PA (McDermott et al. 2004). Phosphatidylcholine-specific PLD isoforms cleave PC to form PA and a soluble choline head group which is released into the cytoplasm. Two mammalian isoforms of phospholipase D: PLD1 and PLD2, were identified. Mammalian PLD directly interacts with several kinases including PKC isoforms and control signaling pathways mediated by these kinases. PLD isoforms are activated by ADP-ribosylation factor (Arf) (Selvy et al. 2011). In the course of studies using neutrophiles several PA binding proteins were identified including vesicle amine transport protein-1 (VAT-1), AnxA3, Rac2, Cdc42 and RhoG, suggesting the role of PLD-derived PA in cell physiology, also in processes that are annexin-dependent (Faugaret et al. 2011).

SMase, especially the acid SMase (also known as sphingomyelin phosphodiesterase-1), plays an important role in normal membrane turnover through the hydrolysis of sphingomyelin (SM), and is one of the key enzymes responsible for the production of ceramide (Schiffman 2010, Schuchman 2010). Some results of biochemical studies and studies using SMase knockout mice suggest that acid SMase may be involved in the pathogenesis of several common diseases through its ability to reorganize membrane microdomains (Claus et al. 2009, Jin and Zhou 2009, Schuchman 2010). It has been reported that the Niemann-Pick disease type A or B, a lysosomal storage disorder characterized at the cellular level by accumulation of SM within the late endosome/lysosome compartment, may arise because of the dysfunction and deficit of acid SMase as a result of a mutation in the enzyme encoding gene, SMPD-1 (Jenkins et al. 2009, Tamasawa et al. 2012). Although highly speculative at this stage it is possible that the effect of SMase deficit could be related to the proposed role of annexins in membrane microdomain organization and their involvement in several pathologies related to deregulated intracellular vesicular transport and storage of metabolites (Monastyrskaya et al. 2009b, Grewal et al. 2010, Enrich et al. 2011, Domon et al. 2012).

Lipid-derived second messengers as ligands for annexins

Phosphatidylinositol-derived second messengers

In the course of in vitro studies numerous evidence have been presented that annexins bind to anionic phospholipids (for review see Babiychuk and Draeger 2000, Gerke and Moss 2002, Gerke et al. 2005, Draeger et al. 2011), including phosphatidylinositol and an important second messenger, phosphatidylinositol 3,4-bisphosphate (PI(4,5)P₂) (Rescher et al. 2004, Hayes et al. 2004a, 2004b, 2009). Data using mutated proteins revealed that individual annexin repeat domains may have characteristic affinities for different lipids, including PI(4,5)P₂ (Sohma et al. 2001). In the cell, the PI(4,5)P₂ messenger system consists of the G-protein coupled receptors and two effectors, PI-PLC and phosphoinositide 3-kinase (PI3K). PI-PLC produces two different second messengers, inositol trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ diffuses freely into the cytoplasm and is recognized by the inositol trisphosphate receptor (IP₃R) family in the endoplasmic reticulum membrane. Thus IP₃ participates in the overall intracellular calcium homoeostasis. DAG remains bound to the membrane where it recruits and activates both conventional and non conventional members of the PKC family of protein kinases (Nishizuka 1995). PI3K, as an effector enzyme in the system, phosphorylates PIP₂ to phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P₃) (Kanaho and Suzuki 2002). PI3K belongs to a family of phosphoinositide kinases that catalyze the phosphorylation of PI and its phosphorylated forms to produce phosphoinositides. These phosphoinositides have specific physiological functions, such as reorganization of actin cytoskeleton, vesicular transport, cell proliferation and survival (Kanaho and Suzuki 2002).

Among different annexins, AnxA2 was identified as a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂)-interacting protein, targeted together with its ligand, the S100A10 protein, to specific PI(4,5)P₂-enriched membrane microdomains where also F-actin was found to accumulate. Membrane-associated AnxA2 may play a role in membrane-associated actin assembly and bacterial adhesion during bacteria invasion. In vitro the AnxA2₂S100A10₂ heterotetramer binds to PI(4,5)P₂ with an affinity comparable to that of the PH domain of PLCδ1 (Rescher et al. 2004). Moreover, AnxA2 was stimulating macropinocytic rocketing in a calcium-dependent manner in fibroblasts from patients with Lowe Syndrome. This suggests that AnxA2 plays a role of an actin nucleator on PI(4,5)P₂-enriched membranes (Hayes et al. 2009). Recombinant AnxA2 binds to PI(4,5)P₂, but not to other poly- and monophosphoinositides, with affinity comparable to that of other PI(4,5)P₂-binding proteins (Hayes et al. 2004a, 2004b). It has been observed that in pro-B cell acute lymphoblastic leukemia cells AnxA2 expression was regulated by the Ras/PI3K pathway (Matsunaga et al. 2004).

In epithelium PI(4,5)P₂-AnxA2 interactions were found to be important in the generation of an apical region of the plasma membrane, and thereby in integrating cellular polarity with epithelial architecture (Martin-Belmonte and Mostov 2007). Furthermore, AnxA2 is recruiting other proteins such as GTPase Cdc42 and aPKC into this region of the plasma membrane where PTEN (phosphatase and tensin homolog deleted on chromosome ten), a phosphatase of a tumor suppressor function which plays a role in dephosphorylation of PI(3,4,5)P3 to PI(4,5)P₂, is also residing (Choi et al. 2002, Mahimainathan and Choudhury 2004). PTEN was found to be present in the apical region of the plasma membrane. Its malfunction prevented development of the apical domain of the plasma membrane (Martin-Belmonte et al. 2007).

Whether annexins may participate in regulation of kinases which phosphorylate various forms of PI remains to be elucidated. However, the data obtained so far on the skeletal muscle triad system responsible for propagation of the calcium signal point to the fact that AnxA6, despite its capacity to bind to negatively charged phospholipids including PI and PI(4,5)P₂, does not affect the kinase activities responsible for their generation (Barrientos and Hidalgo 2002). On the other hand, 1α,25-dihydroxyvitamin D3, via binding to its membrane receptor, AnxA2, induces activation of the PI3K/Ras/MEK/extracellular signal regulated kinase 1/2 and c-Jun N-terminal kinase 1 signal transduction pathway resulting in increased expression of c-Fos, Fra1, and c-Jun, and subsequently, increased activator protein 1 DNA binding activity and gene transcription (Johansen et al. 2003).

Other lipid-derived second messengers

Information about interaction of annexins with other lipid-derived second messengers such as lysophosphatidic acid (LPA), platelet activating factor (PAF), prostaglandins, leukotrienes, sphingosine-1-phosphate (S1P), ceramide and ceramide-1-phosphate, glucosylceramide, as well as retinol derivatives, endocannabinoids, and steroid hormones is very limited. Interaction of annexins with membrane regions enriched in precursors of the above mentioned compounds, i.e., specific lipids including sphingomyelin, phosphatidic acid, phospholipids acylated in the sn-2 position with polyunsaturated fatty acids, other anionic phospholipids, etc. (Zschörnig et al. 2007, Monastyrskaya et al. 2009b, Draeger et al. 2011, Domon et al. 2011, 2012), makes this presumption very likely. Moreover, steroid hormones are synthesized from isoprenoids and structurally resemble cholesterol, a molecule identified as a ligand for annexins (Domon et al. 2010, 2012).

Annexins may participate in the metabolism of arachidonic acid derivatives, such as leukotrienes, which are produced by 5-lipoxygenase. For example, it has been shown that AnxA7 possesses a tumor suppressor function demonstrated by cancer susceptibility of ANX7 +/- mice and by the loss of ANXA7 expression in human cancers (Torosyan et al. 2009). It was observed that AnxA7, by regulating 5-lipoxygenase transcription that is potentially relevant to the arachidonic acid-mediated cell growth control in tumor suppression, is associated with the arachidonic acid cascade (Torosyan et al. 2006).

In addition, it has been shown that vitamin A (all-trans retinol) and all-trans retinoid acid interact in vitro with recombinant human AnxA6, and stimulated the calcium-dependent binding of AnxA6 to liposomes, accompanied by oligomerization of AnxA6. This may support the hypothesis of a direct implication of AnxA6 in vitamin A-dependent tissue mineralization (Balcerzak et al. 2006).

Conclusions and future perspectives

In this review the possible participation of annexins in lipid metabolism, with a special focus on the metabolism of secondary lipid messengers, has been revisited. Our renewed interest in this subject is due to the growing number of observations suggesting that annexins, as membrane-binding proteins, participate in the regulation of membrane lateral organization, assembly of enzyme and signaling complexes, vesicular traffic, membrane repair mechanisms, and, therefore, may play a role in lipid-mediated signal transduction. As a consequence of these functions, deregulation of annexin expression or mistargeting of these proteins in the cell accompany or may directly lead to development of serious pathologies, including certain types of cancer or lipid storage diseases, such as the Niemann-Pick type C disease. Identification of other diseases in which malfunction of annexins is the pathological factor is only a question of time.

The focus of the review is on lipid-hydrolyzing enzymes, especially PLA₂ isoforms, that regulate the amount of important lipid messengers, or of their precursors, such as fatty acids, especially arachidonic acid. Of equal importance are also the secondary products of the reaction catalyzed by PLA₂, i.e., lysophospholipds that are gaining attention of many investigators as potent biologically active lipid mediators that exert a wide range of cellular effects through specific G protein-coupled receptors. These receptors are expressed in a large number of tissues and cell types, allowing for a wide variety of cellular responses including cell adhesion, cell motility, reorganization of cytoskeleton, proliferation, angiogenesis and cell survival (Rivera and Chun 2008). Their activation initiates different intracellular pathways that induce profound changes in [Ca²⁺], intracellular pH and production of lipid second messengers (Monastyrskaya et al. 2009b). In this respect, annexins, as Ca²⁺-sensitive proteins binding to a large variety of phospholipids, cholesterol and lipid-derived molecules, and playing important roles in organization and function of lipid microdomains, are key elements of the cellular machinery responsible for signal transduction during stress response. In summary, on the basis of available data, a still complex picture is emerging as to the relationship between annexins and PLA₂. It is strongly believed that detailed studies of cell lines (Solito et al. 2001, Croxtall et al. 2003) and knockout animals (especially ANXA1 -/- and ANXA2 -/-) (Bensalem et al. 2005, Damazo et al. 2007, Ayoub et al. 2008, Flood and Hajjar, 2011, He et al. 2011) should clarify the role of the annexin/phospholipase interaction in regulation of the amount as well as spatial and temporal distribution of lipid-derived second messengers in the cell.

Acknowledgements

The annexin project in the authors' laboratories is supported in part by a grant NN401642740 from the National Science Center to JBP, and by the Nencki Institute of Experimental Biology statutory grant to SP. The authors would like to apologize for not being able to cite all original articles related to the topic, due to space limitations.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.