Published as doi: 10.1096/fj.06-7464rev.
(The FASEB Journal. 2007;21:968-975.)
© 2007 FASEB
Annexin 1: the new face of an old molecule
Lina H. K. Lim* and
Shazib Pervaiz*,
,1
* Department of Physiology, Yong Loo Lin School of Medicine, and
NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore
1Correspondence: Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 2 Medical Dr., Bldg MD9, Singapore 117597. E-mail: phssp{at}nus.edu.sg
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ABSTRACT
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The annexin superfamily consists of 13 calcium or calcium and phospholipid binding proteins with a significant degree of biological and structural homology (40–60%). First described in the late 1970s and subsequently referred to as macrocortin, renocortin, lipomodulin, lipocortin-1, and more recently Annexin 1, this 37 kDa calcium and phospholipid binding protein is a strong inhibitor of glucocorticoid-induced eicosanoid synthesis and PLA2. Recent interest in the biological activity of this intriguing molecule has unraveled important functional attributes of Annexin 1 in a variety of inflammatory pathways, on cell proliferation machinery, in the regulation of cell death signaling, in phagocytic clearance of apoptosing cells, and most importantly in the process of carcinogenesis. Here we attempt to present a short review on these diverse biological activities of an interesting and important molecule, which could be a potential target for novel therapeutic intervention in a host of disease states.— Lim, L. H. K., Pervaiz, S. Annexin 1: the new face of an old molecule.
Key Words: inflammation • glucocorticoids • apoptosis • cancer
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INTRODUCTION
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ANNEXIN-1 (ANXA1), THE first characterized member of the annexin superfamily, was described in the late 1970s, originally known as macrocortin (1)
, renocortin (2)
, and lipomodulin (3)
, before it was named lipocortin-1 and, subsequently, ANXA1. This 37 kDa protein was found to have calcium and phospholipids binding properties and was actively involved in the inhibition of eicosanoid synthesis and PLA2, induced by glucocorticoids (GC). The gene encoding this protein is located on chromosome 19q24.
The annexin superfamily consists of 13 calcium or calcium and phospholipid binding proteins with high biological and structural homology (40–60%) (4)
. In theory, their main biochemical property is the binding or "annexing" to phospholipid membranes in a calcium-dependent manner. The inherent defining feature of the family is a core domain with four conserved 70 amino acid motifs, where the calcium- or phospholipid-binding consensus sequence lies. The only exception is LC6, with 8 repeats of 70 amino acid motif instead of four. The N terminus is unique for each member of the superfamily with varying amino acid sequence and length, and is thought to be responsible for the biological activity and specific function of the annexins. In fact, the N terminus of ANXA1 (49 residues) is the regulatory region and contains the sites for phosphorylation and proteolysis (4)
.
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ANTIINFLAMMATORY ACTIVITY OF ANXA1
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ANXA1 is undetectable in plasma but found in many cells and tissues (lung, bone marrow, intestine) at concentrations <50 ng/ml, with the highest levels in the seminal fluid (150 µg/ml) (5)
. It makes up for 2–4% of the total cytosolic protein and is found in gelatinase granules in neutrophils. ANXA1 is secreted on cellular adhesion to the endothelium when these gelatinase granules are released, whereas extravasated neutrophils have consistently lower levels of ANXA1 (6)
. On release, ANXA1 is thought to bind to its receptor, which brings about cell detachment, and inhibits transmigration of leukocytes thereby blocking the inflammatory response (7
, 8)
. The f-Met-Leu-Phe (FMLP) receptors FPR (formyl peptide receptor) and FPRL1 (FMLP receptor like) have recently been shown to bind the N terminus of ANXA1 (9)
and may regulate the effects of exogenous ANXA1. In this regard, the N-terminal peptide, Ac 2–26 (constructed with an acetyl blocked N terminus for stability and delay of proteolytic degradation), protected against myocardial ischemia-reperfusion injury (10)
, and inhibited cell adhesion and migration in FPR-dependent manner; the peptide had no effect in FPR-deficient animals (11)
. Human recombinant ANXA1 exhibited antiinflammatory characteristics, mirroring GCs when tested in a carragenin-induced edema model of inflammation in the rat paw (12)
. Similarly, the N terminus peptide also exhibited antiinflammatory effects, as assessed in the mouse air-pouch and rat paw edema models of inflammation (13
, 14)
. Furthermore, systemic treatment of mice with the GC dexamethasone (DEX) inhibited polymorphonuclear cell (PMN) influx in response to interleukin-1 (IL1), but passive immunization with specific antibodies against ANXA1 abrogated the inhibitory effect of DEX (15
, 16)
. However, when the effects of ANXA1 and GCs were examined on eosinophils in vivo, DEX inhibited eosinophil influx to allergen challenge independent of ANXA1 action (16
, 17)
.
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ANXA1 INTERACTION WITH PLA2
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The originally identified activity of ANXA1 as an inhibitor of phospholipase A2 (PLA2) was initially proposed to be responsible for its antiinflammatory actions (18)
. This activity is nonspecifically regarded now, as most or all the other annexins display similar PLA2 inhibition (4)
. The inhibition of PLA2 activity, including the production of arachadonic acid, was thought to be a result of ANXA1 binding to the substrate rather than directly to the enzyme, leading to the depletion of substrate sites and a subsequent reduction of PLA2 activity (19)
. However, this idea was reconsidered and it is now apparent that a secretory form (sPLA2) as well as a cytosolic form of PLA2 (cPLA2) exist. PLA2 cleaves the sn-2-acyl bond of phospholipids producing equimolar amounts of lysophospholipids and free fatty acids (20)
. cPLA2 is calcium-sensitive and exhibits partiality for the arachidonyl-containing phospholipids. cPLA2 is predominantly involved in the production of inflammatory lipid mediators, and arachadonic acid and PLA2 activation is a rate-limiting step in the process of lipid mediator synthesis (21)
. ANXA1 has now been shown to inhibit PLA2 activity directly, rather than by substrate depletion. Studies by Kim et al. (22)
showed direct interaction between ANXA1 and PLA2, and this was further confirmed where prevention of cPLA2 phosphorylation and activation was shown with ANXA1 (23)
. However, this interaction between PLA2 and ANXA1 is not clear-cut and is observed in a cell-specific manner. A more recent study demonstrated coimmunoprecipitation of and functional link between phosphorylated ANXA1 with cPLA2 in a hepatocyte cell line (24)
.
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MODULATION AND REGULATION OF ANXA1 BY GCs
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Although the endogenous function(s) of ANXA1 is/are still unclear, many postulations have been formulated, mostly focusing on its antiinflammatory and antimigratory properties. Recent studies on ANXA1 knockout mice revealed that cycloxoygenase-2 (COX-2) mRNA and protein levels were constitutively increased and that GCs were ineffective in these mice in two models of acute inflammation, suggesting that ANXA1 is in fact an endogenous protein mediating the antiinflammatory actions of GCs (25)
. After purification, ANXA1 was found to mimic some of the antiinflammatory effects of GCs (18)
. The protein itself is GC-inducible, as several studies have shown increased synthesis of ANXA1 in response to GCs in different cell types (26
27
28)
. ANXA1 levels were found to be increased 2–3 fold with a peak at 2 h after incubation with the steroid and this was blocked by the addition of the GC receptor antagonist RU486 (29)
. GC treatment results in fast exocytosis of ANXA1 from the cytoplasm (28)
and rapid de novo synthesis of ANXA1 in both the periphery and central tissues (30)
. GCs can also induce ANXA1 serine phosphorylation and translocation to the membrane through PKC, PI3K, and MAP kinase dependent pathways (31)
.
ANXA1 synthesis could also be modulated by the hypothalamic-pituitary axis (32)
, and endogenous GCs control the basal ANXA1 expression in several organs and cells as demonstrated by the reduced ANXA1 levels in leukocytes after adrenalectomy in rats (33)
. However, ANXA1 itself modulates the GC-induced secretion of adrenocorticotrophic hormone (ACTH) from the anterior pituitary gland in vitro (34)
and in vivo (35)
, suggesting an intricate interplay between ANXA1 and the corticosteroid hormones.
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ANXA1 AS A MEDIATOR OF GC FUNCTION
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In rheumatoid arthritis, an impaired induction of ANXA1 by GCs in monocytes (36)
as well as a lower capacity of ANXA1 binding to monocytes and neutrophils is observed (37)
. ANXA1 is present in arthritic synovium and mediates the effects of exogenous GCs on neutrophil migration in carrageenan-induced acute arthritis. Injection of anti-ANXA1 antibodies directly exacerbates the condition and mediates GC-induced inhibition of neutrophil activation (38)
, whereas adrenalectomized animals have reduced levels of ANXA1 in leukocytes, as well as severe exacerbation of arthritis (33)
. Similarly, in ischemic conditions, treatment of rats with ANXA1 or the N-terminal peptide Ac 2–26 protected against ischemia-reperfusion injury and shock by inhibiting neutrophil migration and accumulation (39)
. ANXA1 has also been shown to be involved in the antipyretic actions of GCs (40)
and capable of blocking the hyperalgesic effect mediated via COX-2 (41)
. Evidence also indicates increased levels of ANXA1 in lung secretions (42)
and in broncho-alveolar lavage fluid following prednisolone administration (43)
. However, ANXA1 expression is not altered on clinical treatment with steroids but is increased in smokers (44)
.
In summary, ANXA1 has antiinflammatory and antimigratory effects on neutrophils and monocytes in several models in vitro (45)
and in vivo (46
47
48)
and could have functions in cellular homeostasis, as immuno-neutralization of ANXA1 in mice intensified the acute inflammatory response (29)
. In addition, the fact that its expression can be induced by GCs in several cell types and that ANXA1 knockout mice do not respond to GCs indicates its role as an endogenous homeostatic antiinflammatory protein recruited to aid in the resolution of inflammation (6)
.
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ANXA1 AS A REGULATOR OF CELL PROLIFERATION, DIFFERENTIATION, AND APOPTOSIS
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Earlier in vitro work suggested that ANXA1 had inhibitory roles in cell growth and proliferation in A549 lung cancer cells (49)
, mostly correlating with GC effects. ANXA1 is also involved in hepatocyte proliferation and regeneration where ANXA1 expression is up-regulated in proliferative hepatocytes (24)
. ANXA1 has antiproliferative activity in macrophages due to the constitutive activation of the MAPK/ERK pathway, which was linked to its phosphorylation by epidermal growth factor (EGF) (50)
. An interesting function of ANXA1 on proliferation is its role in acting as a substrate for the EGF receptor tyrosine kinase (51)
, thereby inhibiting EGF-mediated proliferation (52)
. The EGF receptor family of tyrosine kinases plays important roles in cell differentiation and proliferation and in cancer development. ANXA1 is thought to have a src-homology 2 (SH2) domain and can bind to the Grb-2 adaptor protein, which is upstream of the MAPK pathway (50)
. ANXA1 is thought to exert its antiproliferative activity via ERK-mediated disruption of the actin cytoskeleton and inhibition of cyclin D1, but not by induction of p21cip1/waf1 (53)
. ANXA1 is known to be the phosphorylation target for various signal transducing kinases such as those associated with the platelet-derived growth factor (PDGF) receptor, hepatocyte growth factor receptor (54)
, and protein kinase C (55)
as well as TRPM7 channel kinase (56)
, thus contributing to its importance in proliferation. The phosphorylation of ANXA1 in hepatocytes was linked to the proliferating signal of hepatocyte growth factor where ANXA1 acts as a signal amplifier to enhance proliferation, chemotaxis and remodeling (54)
. A recent paper by Hsiang et al. studied the biological role of ANXA1 in prostate cancer cell lines, which express low levels of ANXA1 (57)
. Re-expression of ANXA1 reduced cell viability and colony formation, and inhibited the proliferative effect of EGF. These data suggest tumor suppressor function of ANXA1 to inhibit proliferation.
Aside from its role as a mediator of GC action, ANXA1 has been implicated in the regulation of apoptosis in a number of studies. Some groups have shown ANXA1 to be proapoptotic, whereas other evidence links ANXA1 to resistance of cells to apoptosis. The reason behind this discrepancy is unknown but could be dependent on cell type or cellular differentiation status. The possible importance of ANXA1 in cell survival was first demonstrated in postlactating mammary ducts undergoing apoptosis where ANXA1 expression was increased (58)
. Exogenous ANXA1 promoted cellular apoptosis induced by hydrogen peroxide, while it protected cells from necrotic death. Similarly, an anti-ANXA1 antibody inhibited hydrogen peroxide-induced apoptosis, resulting in enhanced necrosis (59)
. These observations were seemingly dependent on the effect of ANXA1 on PLA2 regulation and not membrane lipid peroxidation, as similar results were obtained with PLA2 inhibitors. Over-expression of ANXA1 in monocytic cells (with sense DNA to the N terminus) enhanced apoptosis induced by TNF-
and etoposide (60)
. Interestingly, the transfection with full length ANXA1 in these cells induced spontaneous apoptosis with 
70% of cells undergoing cell death by day 5. This was not related to changes in basal Bcl-2 or Bax expression or changes in these apoptosis-related proteins after stimulation with TNF-. However, changes in expression of ANXA1 affected caspase 3 activation (61)
and calcium release (62)
. Exogenous ANXA1 induced neutrophil apoptosis thorough intracellular calcium release and dephosphorylation of BAD, allowing BAD to associate with the mitochondria (62)
. Furthermore, ANXA1 translocates to the nucleus during apoptosis, which is inhibited by the over-expression of the antiapoptotic protein BCL2 (63)
. The precise mechanism and functional relevance of the nuclear localization of ANXA1 is not understood. Interestingly, recent findings also implicate ANXA1 in TRAIL-induced apoptosis; silencing ANXA1 with siRNA inhibited TRAIL-mediated signaling in prostate cancer cells (64)
. In contradistinction, Wu et al. demonstrated that ANXA1, as a mediator of GC action, rendered monocytic cells resistant to TNF--induced apoptosis, and that ANXA1 levels were constitutively higher in TNF- resistant cells than cells sensitive to TNF- (65)
. This was linked to a possible mechanism for evading immune surveillance. Similarly, transfection of drug-sensitive MCF-7 cells with ANXA1 rendered these cells resistant to adriamycin and etoposide. These findings are similar to another study where DEX treatment, through ANXA1-dependent mechanisms, led to resistance to doxorubicin and etoposide in prostate cancer cells (66)
.
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ANNEXIN-1 AND THE "EAT ME" SIGNAL
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ANXA1 has been implicated in the apoptotic cell "eat me" signal and ensuing phagocytosis, and evidence is accumulating to support a role in the resolution phase of inflammation. Specific "eat me" signals on apoptotic cells serve as markers for phagocytes to recognize and ingest them as a means for clearing dead debris. Exposure of phosphatidylserine (PS) on the outer leaflet of plasma membrane is one of the characteristic hallmarks of apoptotic cells. PS-dependent ingestion of apoptotic cells inhibits the release of proinflammatory cytokines, such as TNF-, but IL-1ß stimulates the release of antiinflammatory cytokines such as TGFß and IL-10. A recent paper by Arur et al. elegantly demonstrated that ANXA1 might serve as an endogenous PS ligand, mediating engulfment of apoptotic cells (67)
. ANXA1 is recruited to the PS-rich region of apoptotic cell surface in a caspase-mediated mechanism and involves the release of intracellular calcium. Supporting these findings, siRNA-mediated silencing of ANXA1 gene expression resulted in defective engulfment of apoptotic cells (67)
.
Another member of the annexin superfamily, ANX-A5 is a well-known commercial ligand for PS and is commonly used to identify apoptotic cells. Interestingly, ANXA1 is also thought to bind to PS in a bivalent manner (i.e., ANXA1 can act as a bridging protein between 2 PS molecules) via several possible mechanisms. The N terminus of ANXA1 is exposed on the membrane-binding of the core protein and can either (i) bind to a second membrane (68
, 69)
; (ii) bind to each other via their N terminus domains, thus pulling 2 membranes together (70)
; or (iii) bind to other proteins such as S100 to produce complexes that can cross-link two membranes (71
72
73)
. Monoclonal antibodies to ANXA1 inhibited phagocytosis of apoptotic lymphocytes mediated by PS (74)
, while normal Fc-mediated phagocytosis was unaffected, implying that ANXA1 was involved only in apoptotic cells or PS-mediated phagocytosis. Interestingly, the PS receptor is only found on the surface of the phagocytic cells, while the PS molecule is expressed on both the apoptotic as well as phagocytic cells. Thus, it may be reasonable to deduce that ANXA1 is a receptor on phagocytes utilized for the recognition of exposed PS on cells undergoing apoptosis; ANXA1 is expressed, albeit at low levels on the surface of phagocytes. Thus, ANXA1 exposure on the cell surface may be functionally relevant in the tethering of the apoptotic cell to the phagocytic cell. The N-terminal peptide of ANXA1 stimulates phagocytosis of apoptotic neutrophils by macrophages, and similarly, stimulation of endogenous ANXA1 expression by GCs increases the phagocytic ability of macrophages, which is inhibited by blocking antibodies against ANXA1 (64)
. This indicates that ANXA1 could, indeed, be involved in the phagocytosis of apoptotic cells.
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SIGNIFICANCE OF ANXA1 IN CANCER
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ANXA1 may have important regulatory roles in tumor development and progression. Evidence for this lies in the clear observations that its expression and the expression of another annexin (ANXA2) are reduced in certain cancers, while it is increased in other cancer types. Table 1
illustrates the studies done on clinical cancer tissues and cells. Xia et al. demonstrated an important role of ANXA1 in esophageal cancers, where decreases in ANXA1 mRNA and protein were reported (75)
. Mutational studies on esophageal cancers showed that allelic loss of ANX-AI occurs frequently, whereas somatic mutations are rare. The loss of ANXA1 expression in prostate cancer is particularly obvious and correlates with the early onset of tumorigenesis (76)
. In head and neck cancers, the expression of ANXA1 has been associated with advanced stage of the disease, metastasis, and differentiation status and could be an effective differentiation marker for the detection of epithelial dysplasia and histopathological grading of head and neck squamous cell carcinomas (77)
. In breast cancer, overexpression of ANXA1 was reported in tumor ducts when compared to normal ducts (78)
. However, a recent report on tissue microarray (which contains 1158 informative breast tissue cores of different histologies, including normal, hyperplasia, in situ and invasive tumors, and lymph node metastases) demonstrated a significant reduction in ANXA1 expression in ductal carcinoma in situ and invasive ductal carcinoma, compared to normal or hyperplastic tissues (79)
. The conflicting reports on the expression of ANXA1 in breast cancers may be correlated with estrogen receptor status, although a clear correlation has not been reported.
Many clinical cases (see Table 1
) on ANXA1 expression changes report a loss of ANXA1, apart from those that may be hormonally regulated (e.g., breast and pituitary tumors). This may link to the known functions of ANXA1 in proliferation and apoptosis, and the fact that cancer cells down-regulate antiproliferative and/or proapoptotic protein(s) may lead them to a more tumorigenic phenotype. Similarly, loss of ANXA1 may lead to cancer cell resistance to apoptosis induced by chemotherapeutic agents. Thus, it may be possible to suggest the importance of ANXA1 as a negative biomarker in cancer development and progression.
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CONCLUSIONS
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ANXA1, an endogenous antiinflammatory protein, has roles in many diverse cellular functions, such as membrane aggregation, inflammation, phagocytosis, proliferation, and apoptosis (Fig. 1
). ANXA1 also possesses phosphorylation sites for important proliferative signaling molecules e.g., EGF receptor tyrosine kinase and PKC. This suggests that ANXA1 may have a role in certain signaling pathways important in cancer. However, the exact mechanisms through which ANXA1 exerts some or all of its effects are still not clearly understood, and may reveal important functions of ANXA1 in tumorigenesis and cancer development.
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Figure 1. The diverse biological actions of ANXA1. A) ANXA1 was found to inhibit the activity of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase 2 (COX-2), thus exhibiting antiinflammatory, antipyretic, and antihyperalgesic activity. B) Exogenous ANXA1 acts on its receptor, recently identified as the formyl peptide receptor (FPR) and the formyl peptide receptor like-1 (FPRL1) to inhibit cell adhesion and migration, and induce detachment of adherent cells. When cells (particularly neutrophils) adhere onto the endothelium, ANXA1 is released together with the gelatinase granules and can act like exogenous ANXA1. C) ANXA1 expression can be up-regulated with GC treatment through the glucocorticoid receptor (GR), which contributes to its antiinflammatory activity. GCs can also induce rapid ANXA1 phosphorylation and membrane translocation. D) ANXA1 is recruited to the cell surface, where it binds to PS and mediates the engulfment of apoptotic cells. E) ANXA1 can be phosphorylated by a number of kinases, including EGF-R tyrosine kinase, protein kinase C (PKC), platelet-derived growth factor receptor tyrosine kinase (PDGFR-TK), and hepatocyte growth factor receptor tyrosine kinase (HGFR-TK) to mediate proliferation. F) Overexpression of ANXA1 induces apoptosis. ANXA1 can mediate apoptosis by inducing the dephosphorylation of BAD, allowing BAD to translocate to the mitochondria. During apoptosis, ANXA1 itself translocates to the nucleus, which can be inhibited by BCL2.
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ACKNOWLEDGMENTS
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S. P. is supported by research grants from the National Medical Research Council, Biomedical Research Council, and the Academic Research Fund (ARF), NUS, Singapore and L. L. is a recipient of funding support from the National Medical Research Council and the ARF.
Received for publication October 31, 2006.
Accepted for publication November 28, 2006.
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