A limited window of receptivity is a prerequisite of reproductive success. Indispensable receptivity genes include cyclooxygenase 2 (COX2), an enzyme accomplishing formation of prostaglandin E2 (PGE2). A powerful regulator of PGE2 formation is Annexin A7 (ANXA7). The present study thus explored whether ANXA7 impacts on implantation and fertility. Here we show that ANXA7 is expressed in endometrial tissue and increases upon decidual transformation of human endometrial stromal cells (HESCs) in a time-dependent manner. Silencing ANXA7 significantly decreased the expression of PRL and IGFBP1, canonical decidual marker genes, but enhances COX2 and PGE2 levels. Genetic knockout of AnxA7 in mice significantly increases the number of implantation sites and litter sizes. Further, analysis of human endometrial biopsies showed that ANXA7 transcript and protein levels are decreased during the midluteal window of implantation in women suffering from recurrent pregnancy loss (RPL) when compared to subfertile patients. Taken together, the data indicate that ANXA7 has a conserved role in regulating endometrial receptivity and implantation.

Annexin A7 is a Ca2+- and phospholipid-binding protein expressed as a 47 and 51 kDa isoform, which is thought to be involved in membrane fusion processes. Recently the 47 kDa isoform has been identified in erythrocytes where it was proposed to be a key component in the process of the Ca2+-dependent vesicle release, a process with which red blood cells might protect themselves against an attack by for example complement components.

Autophagy involves multiple membrane trafficking and fusion events. Annexin A7 (ANXA7) is postulated to play a role in membrane fusion during exocytosis, while the contribution of ANXA7 to autophagy is poorly understood. Our recent studies demonstrated that ABO could promote autophagy via elevation of ANXA7 and triggering ANXA7 subcellular redistribution. However, little is known about the molecular mechanisms how ANXA7 regulates autophagy. As molecular disruption of ANXA7 in mice results in several unwished phenotypes, small molecule modulators may be efficacious in defining the mechanisms of ANXA7 action. However, so far no compounds that selectively target ANXA7 have been identified. So, we hypothesize that ABO might be a potent modulator of ANXA7. We also have detected the colocalization of ANXA7 and microtubule-associated protein 1 light chain 3 (LC3), and ANXA7 was essential for LC3 accumulation in VEC autophagy. As a GTPase, whether ANXA7 affects the phosphorylation of LC3 or other proteins needs further investigation. In this study, we performed site-directed mutagenesis and found that ABO directly bound to Thr(286) of ANXA7 and inhibited its phosphorylation. By yeast two-hybrid screening, we found that ANXA7 could interact with grancalcin (GCA). ABO promoted the interaction and inhibited GCA phosphorylation, leading to the decrease of intracellular Ca(2+) concentration. At the same time, ABO inhibited the phosphorylation of LC3. Hence, by identifying ABO as an unprecedented modulator of ANXA7 as well as GCA and LC3 as interacting proteins of ANXA7, we demonstrated the possible mechanisms how ANXA7 regulates autophagy for the first time.

Lysosomes are crucial organelles essential for various cellular processes, and any damage to them can severely compromise cell viability. This study uncovers a previously unrecognized function of the calcium- and phospholipid-binding protein Annexin A7 in lysosome repair, which operates independently of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Our research reveals that Annexin A7 plays a role in repairing damaged lysosomes, different from its role in repairing the plasma membrane, where it facilitates repair through the recruitment of ESCRT-III components. Notably, our findings strongly suggest that Annexin A7, like the ESCRT machinery, is dispensable for membrane contact site formation within the newly discovered phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway. Instead, we speculate that Annexin A7 is recruited to damaged lysosomes and promotes repair through its membrane curvature and cross-linking capabilities. Our findings provide new insights into the diverse mechanisms underlying lysosomal membrane repair and highlight the multifunctional role of Annexin A7 in membrane repair.

Annexin A7 is a member of the annexin protein family, which is characterized by its ability to interact with phospholipids in the presence of Ca2+-ions and which is thought to function in Ca2+-homeostasis. Results from mutant mice showed altered Ca2+-wave propagation in astrocytes. As the appearance and distribution of Annexin A7 during brain development has not been investigated so far, we focused on the distribution of Annexin A7 protein during mouse embryogenesis in the developing central nervous system and in the adult mouse brain.

The plasma membrane of eukaryotic cells forms the essential barrier to the extracellular environment, and thus plasma membrane disruptions pose a fatal threat to cells. Here, using invasive breast cancer cells we show that the Ca2+ - and phospholipid-binding protein annexin A7 is part of the plasma membrane repair response by enabling assembly of the endosomal sorting complex required for transport (ESCRT) III. Following injury to the plasma membrane and Ca2+ flux into the cytoplasm, annexin A7 forms a complex with apoptosis linked gene-2 (ALG-2) to facilitate proper recruitment and binding of ALG-2 and ALG-2-interacting protein X (ALIX) to the damaged membrane. ALG-2 and ALIX assemble the ESCRT III complex, which helps excise and shed the damaged portion of the plasma membrane during wound healing. Our results reveal a novel function of annexin A7 - enabling plasma membrane repair by regulating ESCRT III-mediated shedding of injured plasma membrane.

Annexin A7 (Anxa7) is a cytoskeletal protein interacting with Ca(2+) signaling which in turn is a crucial factor for cardiac remodeling following cardiac injury. The present study explored whether Anxa7 participates in the regulation of cardiac stress signaling. To this end, mice lacking functional Anxa7 (anxa7(-/-)) and wild-type mice (anxa7(+/+)) were investigated following pressure overload by transverse aortic constriction (TAC). In addition, HL-1 cardiomyocytes were silenced with Anxa7 siRNA and treated with isoproterenol. Transcript levels were determined by quantitative RT-PCR, transcriptional activity by luciferase reporter assay and protein abundance by Western blotting and confocal microscopy. As a result, TAC treatment increased the mRNA and protein levels of Anxa7 in wild-type mice. Moreover, TAC increased heart weight to body weight ratio and the cardiac mRNA levels of αSka, Nppb, Col1a1, Col3a1 and Rcan1, effects more pronounced in anxa7(-/-) mice than in anxa7(+/+) mice. Silencing of Anxa7 in HL-1 cardiomyocytes significantly increased nuclear localization of Nfatc1. Furthermore, Anxa7 silencing increased NFAT-dependent transcriptional activity as well as αSka, Nppb, and Rcan1 mRNA levels both, under control conditions and following β-adrenergic stimulation by isoproterenol. These observations point to an important role of annexin A7 in the regulation of cardiac NFAT activity and hypertrophic response following cardiac stress conditions.

The incidence of traumatic brain injury (TBI) has been increasing annually. Annexin A7 is a calcium-dependent phospholipid binding protein. It can promote melting of the cell membrane. Recent studies have shown that it plays an important role in atherosclerosis, other cardiovascular diseases, and a variety of tumors. However, few studies of ANXA7 in TBI have been performed. We here observed how ANXA7 changes after TBI and discuss whether brain injury is associated with the use of ANXA7 antagonist intervention. Experimental Results: 1. After TBI, ANXA7 levels were higher than in the sham group, peaking 24 h after TBI. 2. The use of siA7 was found to reduce the expression of A7 in the injured brain tissue, and also brain edema, BBB damage, cell death, and apoptosis relative to the sham group. Conclusion: ANXA7 promotes the development of secondary brain injury (SBI) after TBI.

The ubiquitin-protein ligase E3C (UBE3C) belongs to the E3 ligase enzyme family and implicates in the ubiquitin-proteasome pathway, thus regulates physiological and cancer-related processes. Here, we investigated the expression and roles of UBE3C in glioma. We demonstrated that UBE3C was overexpressed in glioma tissues and cell lines. Inhibition of UBE3C expression in glioma cells significantly decreased cell migration and invasion in vitro. Mechanistically, we disclosed that UBE3C physically interacted with and ubiquitinated tumor suppressor gene annexin A7 (ANXA7), resulting in ubiquitination and degradation of ANXA7. Our results also revealed that increased UBE3C expression was accompanied by a reduction in ANXA7 protein expression in glioma tissues, but not ANXA7 mRNA. Importantly, the inhibition of ANXA7 expression in gliomas cells with UBE3C interference could rescue the cell invasion. Clinically, UBE3C overexpression significantly correlated with high-grade tumors (p < 0.05), poor overall survival, and early tumor recurrence. Thus, our data reveal that high UBE3C expression contributes to glioma progression by ubiquitination and degradation of ANXA7, and thus presents a novel and promising target for glioma therapy.

Annexin A7 (synexin, annexin VII), a member of the annexin family of proteins, causes aggregation of membranes in a Ca2+-dependent manner and has been suggested to promote membrane fusion during exocytosis of lung surfactant, catecholamines, and insulin. Although annexin A7 (A7) was one of the first annexin proteins described, limited studies of its physical characteristics or of structural domains affecting any of its proposed functions have been conducted. As postulated for other annexin proteins, the unique NH2-domain possibly determines the functional specificity of A7. Therefore, we evaluated the effects of segmental deletions in the NH2-terminus on several characteristics associated with the COOH-terminus of A7. The COOH-terminus contains the only tryptophan residue, and all potential trypsin sites, and the Ca2+ and phospholipid binding sites. Recombinant rat A7 and its deletion mutants were expressed using constructs based on the cDNA sequence obtained by screening a rat lung cDNA library. Ca2+ increased the tryptophan fluorescence of A7 and caused a small red shift in the emission maximum (lambdamax), which was further increased in presence of phospholipid vesicles (PLV). NH2-terminal deletions of 29, 51, and 109 residues affected the peak width of fluorescence and lambdamax, surface-exposure of tryptophan residue, and caused a smaller Ca2+-dependent red shift in lambdamax of membrane-bound protein in comparison to A7. Limited proteolysis with trypsin showed that Ca2+ increased the proteolysis of all proteins, but the deletions also affected the pattern of proteolysis. The presence of PLV protected against Ca2+-dependent increase in proteolysis of all proteins. The deletion of first 29 residues also caused decreased membrane binding, aggregation, and fusion, when compared with A7. Collectively, these results suggest that specific NH2-terminus domains can alter those properties of A7 that are normally associated with the COOH-terminus. We speculate that interactions between the NH2- and COOH-termini are required for membrane binding, and aggregation and fusion properties of annexin A7.

This study was to investigate whether annexin A7 (AnnexinA7, ANXA7) and its co-related protein tumor cell death domain silencer [suppressor of death domains (SODD)] regulates the migratory phenotype of liver cancer cells.

A role for annexin A7 (A7) is postulated in the obligatory fusion between lamellar bodies and the plasma membrane during surfactant secretion in alveolar type II cells. This study investigated if surfactant secretagogues increase cell surface A7, which could support A7 insertion into plasma membrane as annexin proteins reportedly lack membrane penetration ability. In vivo trafficking of A7 to cell surface was determined by immuno-staining after non-permeabilizing fixation of alveolar type II cells. Stimulation with various secretagogues increased protein kinase-dependent staining for A7 and ABCA3 in comparison to control cells. Biotin-labeling of surface proteins showed ~4% of total A7 in control cells, which increased ~3-4 folds in stimulated type II cells. Increased cell surface A7 was also observed by protein cross-linking studies showing ~70kDa A7-adduct in the membranes but not in the cytosol fraction of PMA- or A23187-stimulated cells. In vitro phosphorylation increased the Ca(2+)-dependent binding of recombinant A7 to lung plasma membranes; and subsequent cross-linking showed increased levels of ~70kDa A7-adduct. PMA-stimulation of type II cells increased A7 trafficking to lipid rafts suggesting that the latter are involved in A7 trafficking to the cell surface. However, in vitro membrane insertion of recombinant A7 and its tryptophan mutants as determined by fluorescence quenching with doxylPC suggested only shallow membrane insertion by A7. Together, our studies support in vivo association between surfactant secretion and cell surface A7 occurring by insertion into plasma membrane and by fusion of A7 containing lamellar bodies.

Lung surfactant secretion involves lamellar body docking and fusion with the plasma membrane in alveolar type II cells. Annexin A7 (A7) is postulated to play a role in membrane fusion during exocytosis. Our recent studies demonstrated increased co-localization of A7 with ABCA3 in lamellar bodies in type II cells stimulated with established secretagogues of lung surfactant. In this study, we investigated in vivo and in vitro interactions of A7 with the t-SNARE protein, SNAP23. Immuno-fluorescence studies showed time-dependent increases in co-localization of A7 with SNAP23 in PMA- and in A23187-stimulated cells. PMA and A23187 also caused a time-dependent increase in co-localization of ABCA3 with SNAP23. The relocation of A7 to SNAP23 domains was inhibited in the presence of PKC inhibitor, similar to that previously reported for co-localization of A7 with ABCA3. The interaction of A7 and SNAP23 was confirmed by affinity binding and by in vitro interaction of recombinant A7 and SNAP23 proteins. The in vitro binding of recombinant A7 (rA7) to GST-SNAP23 fusion protein was calcium-dependent. Phosphorylation of rA7 with PKC increased its in vitro binding to SNAP23 suggesting that a similar mechanism may operate during A7 relocation to t-SNARE domains. Thus, our studies demonstrate that annexin A7 may function in co-ordination with SNARE proteins and that protein kinase activation may be required for annexin A7 trafficking to the interacting membranes (lamellar bodies and plasma membrane) to facilitate membrane fusion during surfactant secretion.

Knockdown of Annexin A7 (ANXA7) or C-Jun N-terminal kinase (JNK) inhibits the proliferation, migration, invasion, and lymphatic adhesion of hepatocellular carcinoma (HCC) cells, suggesting that ANXA7 and JNK signaling pathways contribute to HCC growth and lymph node metastasis (LNM). While the intervening molecular pathways are largely unknown, emerging evidence suggests that long noncoding RNAs (lncRNAs) participate in ANXA7 and JNK signaling. To identify potential therapeutic targets for HCC, we screened for lncRNAs differentially expressed among Hca-P cells stably expressing shRNA-ANXA7, shRNA-JNK, or control-shRNA. RNA sequencing identified 216 lncRNAs differentially expressed between shRNA-ANXA7 and control-shRNA cells, of which 101 were downregulated and 115 upregulated, as well as 436 lncRNAs differentially expressed between shRNA-JNK and control-shRNA cells, of which 236 were downregulated and 200 upregulated. Fifty-six lncRNAs were differentially expressed under both ANXA7 and JNK knockdown. We selected 4 of these for verification based on putative involvement in cancer regulation according to GO and KEEG analyses of target genes. Knockdown of ANXA7 or JNK suppressed expression of NONMMUT012084.2, NONMMUT024756.2, and ENSMUST00000130486, and enhanced expression of ENSMUST00000197932. These lncRNAs are intriguing candidate targets for mechanistic analysis of HCC progression and therapeutic intervention.

Annexin A7 is a member of the Annexin A family, which participates in various biological processes. Accumulating evidence has demonstrated that Annexin A7 serves an important role in tumorigenesis and is dysregulated in multiple types of cancer. However, the role of Annexin A7 in the tumorigenesis of gastric cancer remains to be determined. The present study revealed that Annexin A7 expression is downregulated in late-stage gastric cancer and is negatively correlated with the differentiation grade and apoptosis. There was a significant difference in Annexin A7 mRNA and protein expression in gastric cancer samples with distinct differentiation grades, with the lowest expression being observed in the highly differentiated cases. A terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling assay demonstrated that the apoptosis indices of highly, moderately and poorly differentiated gastric cancers were 18.12±2.40, 9.73±1.73 and 4.13±0.83%, respectively, with statistical significance (P<0.05). Flow cytometric analysis demonstrated that the apoptosis rates of gastric cancer MKN74, SGC7901 and BGC823 cells were 10.07±1.21, 7.11±1.04 and 4.25±1.02%, respectively, with statistical significance (P<0.05). Spearman's rank correlation analysis revealed that the Annexin A7 mRNA and protein levels were negatively correlated with the differentiation grade of the gastric cancer tissues, while the apoptosis index was positively correlated with the differentiation grade of the gastric cancer tissues. Furthermore, the apoptosis index was negatively correlated with Annexin A7 mRNA and protein expression. Similar associations were observed among Annexin A7 expression, differentiation grades and apoptosis in gastric cancer cell lines. The results of the present study demonstrated that Annexin A7 expression is downregulated, while apoptosis is upregulated, with the progression of gastric adenocarcinoma. These observations suggested that Annexin A7 may inhibit apoptosis during tumorigenesis and that it is a potential biomarker for the diagnosis, prognosis and treatment of gastric adenocarcinoma.

To understand the mechanisms underlying the regulating dyslipidemia action of Chinese propolis and Brazilian green propolis, we investigated their effects on phosphatidylcholine-specific phospholipase C (PC-PLC) activity and annexin a7 (ANXA7) level which play crucial roles in the control of the progress of atherosclerosis. Furthermore, active oxygen species (ROS) levels, nuclear factor-KappaB p65 (NF- κ B p65), and mitochondrial membrane potential (MMP) were also investigated in oxidized-LDL- (ox-LDL-) stimulated human umbilical vein endothelial cells (HUVECs). Our data indicated that the treatment of both types of propolis 12.5  μ g/mL significantly increased cell viability and attenuated apoptosis rate, increased ANXA7 level, and decreased PC-PLC activity. Both types of propolis also inhibited ROS generation as well as the subsequent MMP collapse, and NF- κ B p65 activation induced by ox-LDL in HUVECs. Our results also indicated that Chinese propolis and Brazilian green propolis had similar biological activities and prevented ox-LDL induced cellular dysfunction in HUVECs.

Our previous studies identified the calcium-dependent phospholipid binding protein Annexin A7 (ANXA7) as a critical mediator of hepatocellular carcinoma (HCC) lymph node metastasis (LNM) in mice, possibly through c-Jun N-terminal kinase (JNK) signaling. Activating transcription factor-2 (ATF2) is a downstream target of JNK, so we examined if modulation of LNM capacity by ANXA7 and JNK is associated with changes in ATF2 activity and its sequence-related long non-coding (lnc)RNA NONMMUT114121.1.

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a key factor in apoptosis and autophagy of vascular endothelial cells (VECs), and involved in atherosclerosis in apolipoprotein E⁻/⁻ (apoE⁻/⁻) mice. But the endogenous regulators of PC-PLC are not known. We recently found a small chemical molecule (6-amino-2, 3-dihydro-3-hydroxymethyl-1, 4-benzoxazine, ABO) that could inhibit oxidized low-density lipoprotein (oxLDL)-induced apoptosis and promote autophagy in VECs, and further identified ABO as an inhibitor of annexin A7 (ANXA7) GTPase. Based on these findings, we hypothesize that ANXA7 is an endogenous regulator of PC-PLC, and targeting ANXA7 by ABO may inhibit atherosclerosis in apoE⁻/⁻ mice. In this study, we tested our hypothesis. The results showed that ABO suppressed oxLDL-induced increase of PC-PLC level and activity and promoted the co-localization of ANXA7 and PC-PLC in VECs. The experiments of ANXA7 knockdown and overexpression demonstrated that the action of ABO was ANXA7-dependent in cultured VECs. To investigate the relation of ANXA7 with PC-PLC in atherosclerosis, apoE⁻/⁻ mice fed with a western diet were treated with 50 or 100 mg/kg/day ABO. The results showed that ABO decreased PC-PLC levels in the mouse aortic endothelium and PC-PLC activity in serum, and enhanced the protein levels of ANXA7 in the mouse aortic endothelium. Furthermore, both dosages of ABO significantly enhanced autophagy and reduced apoptosis in the mouse aortic endothelium. As a result, ABO significantly reduced atherosclerotic plaque area and effectively preserved a stable plaques phenotype, including reduced lipid deposition and pro-inflammatory macrophages, increased anti-inflammatory macrophages, collagen content and smooth muscle cells, and less cell death in the plaques. In conclusion, ANXA7 was an endogenous regulator of PC-PLC, and targeting ANXA7 by ABO inhibited atherosclerosis in apoE⁻/⁻ mice.

Introduction: The regulation of intracellular functions in mammalian cells involves close coordination of cellular processes. During recent years it has become evident that the sorting, trafficking and distribution of transport vesicles and mRNA granules/complexes are closely coordinated to ensure effective simultaneous handling of all components required for a specific function, thereby minimizing the use of cellular energy. Identification of proteins acting at the crossroads of such coordinated transport events will ultimately provide mechanistic details of the processes. Annexins are multifunctional proteins involved in a variety of cellular processes associated with Ca2+-regulation and lipid binding, linked to the operation of both the endocytic and exocytic pathways. Furthermore, certain Annexins have been implicated in the regulation of mRNA transport and translation. Since Annexin A2 binds specific mRNAs via its core structure and is also present in mRNP complexes, we speculated whether direct association with RNA could be a common property of the mammalian Annexin family sharing a highly similar core structure. Methods and results: Therefore, we performed spot blot and UV-crosslinking experiments to assess the mRNA binding abilities of the different Annexins, using annexin A2 and c-myc 3'UTRs as well as c-myc 5'UTR as baits. We supplemented the data with immunoblot detection of selected Annexins in mRNP complexes derived from the neuroendocrine rat PC12 cells. Furthermore, biolayer interferometry was used to determine the KD of selected Annexin-RNA interactions, which indicated distinct affinities. Amongst these Annexins, Annexin A13 and the core structures of Annexin A7, Annexin A11 bind c-myc 3'UTR with KDs in the nanomolar range. Of the selected Annexins, only Annexin A2 binds the c-myc 5'UTR indicating some selectivity. Discussion: The oldest members of the mammalian Annexin family share the ability to associate with RNA, suggesting that RNA-binding is an ancient trait of this protein family. Thus, the combined RNA- and lipid-binding properties of the Annexins make them attractive candidates to participate in coordinated long-distance transport of membrane vesicles and mRNAs regulated by Ca2+. The present screening results can thus pave the way for studies of the multifunctional Annexins in a novel cellular context.

Gonadotropin-releasing hormone (GnRH) regulates gonadotropin secretion. We previously demonstrated that the expression of annexin A5 (ANXA5) is stimulated by GnRH in gonadotropes and has a significant role in gonadotropin secretion. It is therefore of interest to know whether other members of the ANXA family, which consists of twelve structurally related members, are also regulated by GnRH. Therefore, the expression of all annexins was examined in LβT2 gonadotrope cells. ANXA4, A5, A6, A7 and A11 were detected in LβT2 cells. The expression of ANXA5 and A1 mRNA was stimulated by a GnRH agonist. An increase in ANXA1 protein by this agonist was demonstrated by western blotting. Immunohistochemistry showed that ANXA1 was present in the nucleus and to a lesser extent in the cytoplasm of some rat pituitary cells. The GnRH agonist induced translocation of ANXA1 to the periphery of LβT2 cells. The presence of ANXA1 in gonadotropes and its increase upon GnRH agonist treatment were confirmed in a primary pituitary cell culture. ANXA1 expression was also demonstrated in the ovary, the testis, the thyroid gland and the pancreas in a different manner to that of ANXA5. These data suggest that ANXA1 is a novel GnRH target gene in gonadotropes. ANXA1 also may be a target of local GnRH in peripheral tissues and may have a different role than that of ANXA5.