Annexins - insights from knockout mice.

Annexins are a highly conserved protein family that bind to phospholipids in a calcium (Ca2+) - dependent manner. Studies with purified annexins, as well as overexpression and knockdown approaches identified multiple functions predominantly linked to their dynamic and reversible membrane binding behavior. However, most annexins are found at multiple locations and interact with numerous proteins. Furthermore, similar membrane binding characteristics, overlapping localizations and shared interaction partners have complicated identification of their precise functions. To gain insight into annexin function in vivo, mouse models deficient of annexin A1 (AnxA1), A2, A4, A5, A6 and A7 have been generated. Interestingly, with the exception of one study, all mice strains lacking one or even two annexins are viable and develop normally. This suggested redundancy within annexins, but examining these knockout (KO) strains under stress conditions revealed striking phenotypes, identifying underlying mechanisms specific for individual annexins, often supporting Ca2+ homeostasis and membrane transport as central for annexin biology. Conversely, mice lacking AnxA1 or A2 show extracellular functions relevant in health and disease that appear independent of membrane trafficking or Ca2+ signaling. This review will summarize the mechanistic insights gained from studies utilizing mouse models lacking members of the annexin family.

Annexin-1-deficient mice exhibit spontaneous airway hyperresponsiveness and exacerbated allergen-specific antibody responses in a mouse model of asthma.

BACKGROUND: Glucocorticoids are the mainstream drugs used in the treatment and control of inflammatory diseases such as asthma. Annexin-1 (ANXA1) is an anti-inflammatory protein which has been described as an endogenous protein responsible for some anti-inflammatory glucocorticoid effects. Previous studies have identified its importance in other immune diseases such as rheumatoid arthritis and cystic fibrosis. ANXA1-deficient ((-/-)) mice are Th2 biased, and ANXA1 N-terminus peptide exhibits anti-inflammatory activity in a rat model of pulmonary inflammation. OBJECTIVE: ANXA1 protein is found in bronchoalveolar lavage fluid from asthmatics. However, the function of ANXA1 in the pathological development of allergy or asthma is unclear. Thus, in this study we intended to examine the effect of ANXA1 deficiency on allergen-specific antibody responses and airway responses to methacholine (Mch). METHODS: ANXA1(-/-) mice were sensitized with ovalbumin (OVA) and challenged with aerosolized OVA. Airway resistance, lung compliance and enhanced pause (PenH) were measured in naïve, sensitized and saline or allergen-challenged wild-type (WT) and ANXA1(-/-) mice. Total and allergen-specific antibodies were measured in the serum. RESULTS: We show that allergen-specific and total IgE, IgG2a and IgG2b levels were significantly higher in ANXA1(-/-) mice. Furthermore, naïve ANXA1(-/-) mice displayed higher airway hypersensitivity to inhaled Mch, and significant differences were also observed in allergen-sensitized and allergen-challenged ANXA1(-/-) mice compared with WT mice. CONCLUSIONS: In conclusion, ANXA1(-/-) mice possess multiple features characteristic to allergic asthma, such as airway hyperresponsiveness and enhanced antibody responses, suggesting that ANXA1 plays a critical regulatory role in the development of asthma. CLINICAL RELEVANCE: We postulate that ANXA1 is an important regulatory factor in the development of allergic disease and dysregulation of its expression can lead to pathological changes which may affect disease progression.

Annexin B9 binds to ß(H)-spectrin and is required for multivesicular body function in Drosophila.

The role of the cytoskeleton in protein trafficking is still being defined. Here, we describe a relationship between the small Ca(2+)-dependent membrane-binding protein Annexin B9 (AnxB9), apical ß(Heavy)-spectrin (ß(H)) and the multivesicular body (MVB) in Drosophila. AnxB9 binds to a subset of ß(H) spliceoforms, and loss of AnxB9 results in an increase in basolateral ß(H) and its appearance on cytoplasmic vesicles that overlap with the MVB markers Hrs, Vps16 and EPS15. Similar colocalizations are seen when ß(H)-positive endosomes are generated either by upregulation of ß(H) in pak mutants or through the expression of the dominant-negative version of ß(H). In common with other mutations disrupting the MVB, we also show that there is an accumulation of ubiquitylated proteins and elevated EGFR signaling in the absence of AnxB9 or ß(H). Loss of AnxB9 or ß(H) function also causes the redistribution of the DE-Cadherin (encoded by shotgun) to endosomal vesicles, suggesting a rationale for the previously documented destabilization of the zonula adherens in karst (which encodes ß(H)) mutants. Reduction of AnxB9 results in degradation of the apical-lateral boundary and the appearance of the basolateral proteins Coracle and Dlg on internal vesicles adjacent to ß(H). These results indicate that AnxB9 and ß(H) are intimately involved in endosomal trafficking to the MVB and play a role in maintaining high-fidelity segregation of the apical and lateral domains.

Annexin 11 is required for midbody formation and completion of the terminal phase of cytokinesis.

Annexins are Ca(2+)-binding, membrane-fusogenic proteins with diverse but poorly understood functions. Here, we show that during cell cycle progression annexin 11 translocates from the nucleus to the spindle poles in metaphase and to the spindle midzone in anaphase. Annexin 11 is recruited to the midbody in late telophase, where it forms part of the detergent-resistant matrix that also contains CHO1. To investigate the significance of these observations, we used RNA interference to deplete cells of annexin 11. A combination of confocal and video time-lapse microscopy revealed that cells lacking annexin 11 fail to establish a functional midbody. Instead, daughter cells remain connected by intercellular bridges that contain bundled microtubules and cytoplasmic organelles but exclude normal midbody components such as MKLP1 and Aurora B. Annexin 11-depleted cells failed to complete cytokinesis and died by apoptosis. These findings demonstrate an essential role for annexin 11 in the terminal phase of cytokinesis.

The lack of annexin A7 affects functions of primary astrocytes.

Annexin A7 is a Ca(2+)- and phospholipid-binding protein, which is thought to function in membrane organization and Ca(2+)-dependent signaling processes. It localizes to different cellular compartments and exists in a 47- and 51-kDa isoform with the large isoform being expressed in brain, skeletal, and heart muscle. In human temporal brain annexin A7 was found exclusively in astroglial cells. As astrocytes are thought to play key roles in several processes of the brain we focused on Ca(2+)-dependent signaling processes and astrocyte proliferation. Primary astrocytes from an anxA7(-/-) mouse exhibited an increased velocity of mechanically induced astrocytic Ca(2+) waves as compared to wild type. We also observed a remarkably increased proliferation rate in cultured mutant astrocytes. A search for annexin A7 binding partners with advanced biochemical methods confirmed sorcin as the major binding protein. However, in vivo GFP-tagged annexin A7 and sorcin appeared to redistribute mainly independently from each other in wild type and in mutant astrocytes. Our results favor an involvement of annexin A7 in Ca(2+)-dependent signaling or Ca(2+) homeostasis in astrocytes.

Annexin A5 is not essential for skeletal development.

Annexins are highly conserved proteins that are characterized by their ability to interact with phospholipids in a calcium-dependent manner. Although diverse functions have been ascribed to annexins based on in vitro analyses, their in vivo functions still remain unclear. The intensively studied annexin A5 has been identified by its effects on blood coagulation, and subsequently, its function as a calcium-specific ion channel was described. In vitro experiments and expression studies suggested a potential role of annexin A5 during calcification processes in vivo, especially in endochondral ossification. To gain insights into the relevance of annexin A5 in this process, we generated an annexin A5-deficient mouse mutant. Mice lacking annexin A5 are viable, are fertile, and reveal no significant alterations in the biochemical parameters characteristic for metabolic or functional defects. Neither the development of skeletal elements nor the in vitro calcification properties of isolated chondrocytes is significantly impaired by the absence of annexin A5. Therefore, annexin A5 is dispensable for the formation and maintenance of skeletal elements in the mouse and may possibly be pointing to a compensatory effect of other members from the annexin family due to their high functional and structural similarity.