Phase Separation and Fibrillization of Human Annexin A7 Are Mediated by Its Proline-Rich Domain.

Human annexin A7, a calcium- and phospholipid-binding protein, governs calcium homeostasis, plasma membrane repair, apoptosis, and tumor progression. A7 contains an N-terminal proline-rich domain (PRD; 180 residues, ∼24% prolines) that determines its functional specificity. Using microscopy and dye-binding assays, we show that recombinant A7 and its isolated PRD spontaneously phase separate into spherical condensates, which subsequently transform into ß-sheet-rich fibrils. We demonstrate that fibrillization of A7-PRD proceeds via primary nucleation and fibril-catalyzed secondary nucleation processes, as determined by chemical kinetics, providing a mechanistic basis for its amyloid assembly. This study confirms and highlights a subclass of eukaryotic PRDs prone to forming aggregates with important physiological and pathological implications.

ANXA7 Regulates Platelet Lipid Metabolism and Ca2+ Release in Arterial Thrombosis.

[Figure: see text].

ZBM-H-induced activation of GRP78 ATPase promotes apoptosis via annexin A7 in A549 lung cancer cells.

Hypochlorous acid (HOCl) is an essential signal for the regulation of cancer cell fate, including autophagy and apoptosis. HOCl regulated autophagy by affecting the oxidation modification of glucose-regulated protein 78 (GRP78) and the activity of GRP78 ATPase. The mechanism of GRP78 ATPase in cell apoptosis has however not yet been clarified. Here we reported that ZBM-H, as a probe of HOCl, was able to directly bind to GRP78 in the presence or absence of ATP. Following ZBM-H treatment, the interaction between GRP78 and annexin A7 (ANXA7) was promoted, and this was accompanied by increased phosphorylation of integrin ß4 (ITGB4). In addition, ZBM-H enhanced the phosphorylation of ANXA7. ABO, an inhibitor of ANXA7, inhibited ZBM-H-induced ITGB4 phosphorylation and apoptosis, while ANXA7 activator SEC had opposite effect. Collectively, these data provide new evidence for the mechanism by which ZBM-H-induced activation of GRP78 ATPase regulates apoptosis of A549 lung cancer cells.

Expression Characteristics of SODD and ALG-2 as Possible Biomarkers for Evaluating Lymphatic Metastasis Potential of Hepatocarcinoma in a Mouse Model.

Annexin A7 has been confirmed in our previous research to be an important factor in lymph node metastasis (LNM) of hepatocellular carcinoma (HCC). SODD and ALG-2 are the binding proteins of Annexin A7 and can work in protein complexes. The present study was carried out with the constructed cell lines in mouse model of metastasis for further elaboration of possible mechanisms and identification of associated genes in the LNM of HCC. This experiment used inbred Chinese 615 mice, as well as Hca-F and Hca-P cells. Quantification of the relative messenger RNA (mRNA) expression of SODD and ALG-2 was realized by using qRT-PCR. Quantification of the protein expressions of SODD and ALG-2 was achieved by using western blot. Experimental mice (n=160) (6-8weeks old, 18-22g, SCXK [LIAO] 2008-0002) were randomly classified into four groups equally, which were separately inoculated with Hca-F, Hca-P, FAnxa7-upregulated, and PAnxa7-upregulated cells. Serum levels of SODD and ALG-2 were measured by ELISA. Immunohistochemical analysis of SODD and ALG-2 was further conducted. Tumor LNM-related factors of SODD and ALG-2 showed the same tendency in their expression correspondingly with the up-regulated expression of Annexin A7. Our experiment further explored the roles of SODD and ALG-2 based on Annexin A7 up-regulation vectors construction and the establishment of corresponding controls in vivo. Furthermore, the mouse model of primary tumors was constructed by injecting Hca-F, FAnxa7-upregulated and Hca-P, PAnxa7-upregulated cells into the mouse footpad. Mice were sacrificed at the designated time points for detecting SODD and ALG-2 expression in tumor tissue and serum samples. Collectively, our work indicates SODD in tumors and in serum and ALG-2 in serum are valuable in evaluating LNM in mice with HCC.

ON-bipolar cell gene expression during retinal degeneration: Implications for optogenetic visual restoration.

PURPOSE: Retinal bipolar cells survive even in the later stages of inherited retinal degenerations (IRDs) and so are attractive targets for optogenetic approaches to vision restoration. However, it is not known to what extent the remodelling that these cells undergo during degeneration affects their function. Specifically, it is unclear if they are free from metabolic stress, receptive to adeno-associated viral vectors, suitable for opsin-based optogenetic tools and able to propagate signals by releasing neurotransmitter. METHODS: Fluorescence activated cell sorting (FACS) was performed to isolate labelled bipolar cells from dissociated retinae of litter-mates with or without the IRD mutation Pde6brd1/rd1 selectively expressing an enhanced yellow fluorescent protein (EYFP) as a marker in ON-bipolar cells. Subsequent mRNA extraction allowed Illumina® microarray comparison of gene expression in bipolar cells from degenerate to those of wild type retinae. Changes in four candidate genes were further investigated at the protein level using retinal immunohistochemistry over the course of degeneration. RESULTS: A total of sixty differentially expressed transcripts reached statistical significance: these did not include any genes directly associated with native primary bipolar cell signalling, nor changes consistent with metabolic stress. Four significantly altered genes (Srm2, Slf2, Anxa7 & Cntn1), implicated in synaptic remodelling, neurotransmitter release and viral vector entry had immunohistochemical staining colocalising with ON-bipolar cell markers and varying over the course of degeneration. CONCLUSION: Our findings suggest relatively few gene expression changes in the context of degeneration: that despite remodelling, bipolar cells are likely to remain viable targets for optogenetic vision restoration. In addition, several genes where changes were seen could provide a basis for investigations to enhance the efficacy of optogenetic therapies.

Promoting TTC4 and HSP70 interaction and translocation of annexin A7 to lysosome inhibits apoptosis in vascular endothelial cells.

We previously demonstrated that Tetraticopeptide 4 (TTC4) inhibited apoptosis in vascular endothelial cells (VEC) deprived of serum and fibroblast growth factor 2 (FGF-2). In this study, we aimed to resolve the mechanism of TTC4 inhibiting VEC apoptosis. TTC4, predicted as a HSP70 co-chaperone protein, may regulate the fate of cells by affecting the activity of HSP70, however, there is no experimental evidence showing the interaction of TTC4 and HSP70. Using Co-immunoprecipitation (Co-IP), we demonstrated that TTC4 interacted with HSP70. If HSP70 was knockdown, TTC4 no longer suppressed apoptosis. Furthermore, we found ABO, an inhibitor of annexin A7 (ANXA7) GTPase, could promote the interaction of TTC4 and HSP70 and the translocation of ANXA7 to lysosome. At the same time, ABO inhibited the interaction of HSP70 and ANXA7. Moreover, Akt, as a downstream effector of HSP70 was upregulated, and ANXA7 translocating to lysosome protected the stability of lysosomal membrane. Here, we discovered a special mechanism by which TTC4 inhibited apoptosis via HSP70 in VECs. On the one hand, increasing TTC4 and HSP70 interaction upregulated Akt that inhibited apoptosis. On the other hand, decreasing HSP70 and ANXA7 interaction promoted the translocation of ANXA7 to lysosome, which inhibited apoptosis through protecting the lysosomal membrane stability.

Quantitative Mitochondrial Proteomics Reveals ANXA7 as a Crucial Factor in Mitophagy.

Mitochondria are involved in many crucial cellular processes. Maintaining healthy mitochondria is essential for cellular homeostasis. Parkin-dependent mitophagy plays an important role in selectively eliminating damaged mitochondria in mammalian cells. However, mechanisms of Parkin-dependent mitophagy remain elusive. In this research, we performed data-independent acquisition-based quantitative mitochondrial proteomics to study the proteomic alterations of carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced Parkin-mediated mitophagy. We identified 222 differentially expressed proteins, with 76 upregulations and 146 downregulations, which were potentially involved in mitophagy. We then demonstrated that annexin A7 (ANXA7), a calcium-dependent phospholipid-binding protein, can translocate to impaired mitochondria upon CCCP treatment, where it played a pivotal part in the process of Parkin-dependent mitophagy via interacting with BASP1. As a mitochondrial uncoupling agent, CCCP indirectly regulated ANXA7 and BASP1 to induce Parkin-dependent mitophagy.

Effects of annexin A7 inhibitor-ABO on the expression and distribution of long noncoding RNA-CERNA1 in vascular endothelial cells apoptosis.

More and more studies reported that diverse biological roles of long noncoding RNAs were usually dependent on their subcellular location. In our previous study, long noncoding RNA CERNA1 was identified both located in cytoplasm and nucleus of vascular endothelial cells (VECs). And CERNA1 in cytoplasm, which functioned as competitive endogenous RNA (ceRNA), alleviated the apoptosis of VECs. However, the function of CERNA1 in nucleus was still unclear. In this study, we found that nuclear CERNA1 positively regulated BCL2L10, which accelerated the serum and FGF-2 starvation-induced apoptosis of VECs, by enhancing the histone modification level of H3K9ac and H3K4me3 in BCL2L10 promoter region. Furthermore, due to the paradoxical function, we investigated the variation of CERNA1 subcellular location in VECs. The results showed that, as the change of apoptosis status, CERNA1 altered the cellular distribution in VECs. And the annexin A7 inhibitor, ABO (6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine), not only increased the expression of CERNA1 by TIA-1, but also specifically improved its cytoplasm distribution proportion so as to inhibit the apoptosis of VECs. This evidence suggested that the subcellular location of CERNA1 played an important role in the VECs apoptosis and ABO might be a potential chemical molecule for therapy of VECs apoptosis related cardiovascular diseases.

Annexin A7 is correlated with better clinical outcomes of patients with breast cancer.

The clinical prognosis of breast cancer (BC) patients remains poor. Studies on BC microarrays GSE37751, GSE7390, and GSE21653 were reanalyzed to characterize the expressions of annexin A7 (ANXA7) in BC patients and the corresponding normal breast tissues and the correlation between ANXA7 expression and clinical characteristics and survivals of BC patients. Gene set enrichment analysis (GSEA) was applied to investigate the exact mechanisms as for the expression of ANXA7 and the proliferation of BC cells. The level of ANXA7 expression was significantly decreased in BC patients than that in normal controls (P < .0001). BC patients in the ANXA7 high-expression group were associated with better clinical features such as tumor size; histopathological grading; estrogen receptors; and clinical risk groups according to St Gallen criteria, Nottingham prognostic index criteria, and Veridex signature compared with those in the ANXA7 low-expression group. Higher expression of ANXA7 predicted better prognosis of BC patients. The result of GSEA indicated that ANXA7 might inhibit the proliferation of BC cells through biological processes involved in androgen response, heme metabolism, and oxidative phosphorylation. The messenger RNA and protein levels of ANXA7 were decreased in BC tissues compared with those in normal breast tissues. Our results proved that ANXA7 was downregulated in BC cells and that a higher expression of ANXA7 was associated with better prognosis of BC patients.

Critical role for Annexin A7 in secondary brain injury mediated by its phosphorylation after experimental intracerebral hemorrhage in rats.

Glutamate excitotoxicity has been implicated in intracerebral hemorrhage (ICH)-induced secondary brain injury (SBI). Synaptosome associated protein 23 (SNAP23) and SNAP25 are respectively participate in presynaptic glutamate release and postsynaptic glutamate receptor (NMDA receptor) trafficking, both of which are essential for glutamate-mediated excitatory toxicity. SNAP23 and SNAP25 exhibit high homology and SNAP23 has been shown to interact with Annexin A7 (ANXA7). This study was to examine the role of ANXA7 in ICH-induced neuronal damage. A collagenase ICH model was performed in adult male Sprague Dawley rats. First, a possible relationship between ANXA7 and ICH pathology was confirmed by an increase in the protein and mRNA level of ANXA7 in the brain tissue around hematoma of ICH rats and the rescue effects of ANXA7 knockdown in vivo on neuronal death, blood-brain barrier damage, brain edema, neurobehavioral deficient, and inflammatory response. In addition, the rescue effect of ANXA7 knockdown on neurobehavioral deficient was also verified in rat autologous blood injection ICH model. Second, we found that ICH significantly increased the phosphorylation ratio of ANXA7 at the threonine residues mainly in neurons. Finally, based on site-specific mutagenesis, we identified that ANXA7 phosphorylation at threonine 286 is required for its interaction with SNAP25 at presynaptic axon terminal and SNAP23 at postsynaptic axon terminal. Collectively, our findings suggest that ANXA7 contributed to SBI at least partially through regulating glutamate toxicity after ICH. Selective inhibition of ANXA7 phosphorylation may be a novel approach to ameliorate ICH-induced SBI.

High co-expression of the SDF1/CXCR4 axis in hepatocarcinoma cells is regulated by AnnexinA7 in vitro and in vivo.

BACKGROUND: SDF1/CXCR4 and AnnexinA7 play important roles in many physiological and pathological conditions, but the molecular association between them in cancer cells has not been studied thus far. METHODS: The expression changes of SDF1/CXCR4 were detected by gene transcriptome sequencing, qRT-PCR, Western blotting, cytoimmunofluorescence and immunohistochemistry in mouse hepatocarcinoma F/P cells, AnnexinA7 downregulated expression F (FA7DOWN) cells, AnnexinA7 overexpression P (PA7UP) cells, AnnexinA7 unrelated sequence F (FSHUS) cells, empty vector P (PNCEV) cells and normal liver cells in vitro and in vivo. RESULTS: SDF1 and CXCR4 were co-expressed in hepatocarcinoma cells. SDF1 was localized mainly in the cytoplasm of cells, while CXCR4 was mainly localized in the cell membrane. Both in vitro and in vivo, expression levels of SDF1/CXCR4 in F and P cells were higher than in normal liver cells, and expression levels of SDF1/CXCR4 in F cells with high lymphatic metastatic potential were higher than those in P cells with low lymphatic metastatic potential. Expression of SDF1 was higher than that of CXCR4 in P cells and normal liver cells, while expression of CXCR4 was higher than that of SDF1 in F cells. Expression levels of SDF1/CXCR4 were completely consistent with AnnexinA7 regulation. After the AnnexinA7 gene was downregulated or upregulated, expression levels of SDF1/CXCR4 in FA7DOWN/PA7UP cells were lower or higher than those in FSHUS/PNCEV cells. Furthermore, CXCR4 was more sensitively modulated by AnnexinA7 regulation than SDF1. CONCLUSIONS: High co-expression of SDF1/CXCR4 is a molecular characteristic of hepatocarcinoma cells, especially those with high lymphatic metastatic potential. AnnexinA7 positively regulates expression levels of SDF1/CXCR4, in particular CXCR4, and AnnexinA7 is a functional regulator of SDF1/CXCR4.

Effect of annexin A7 suppression on the apoptosis of gastric cancer cells.

Understanding the molecular mechanism of gastric cancer cell apoptosis is pivotal for the development of precise therapies targeting this disease. In the present study, we examined the effects of annexin A7 inhibition on the apoptosis of gastric cancer cells and the growth of tumour xenografts in vivo. Expression of annexin A7 in BGC823 cells was suppressed by small interference RNA, and cells apoptosis was assessed by flow cytometry. The mechanism by which annexin A7 mediates apoptosis in BGC823 cells was explored by determining the expression of key apoptosis regulators. In addition, by suppressing annexin A7 in BGC823 cells with small hairpin RNA, we studied the effects of annexin A7 inhibition on in vivo tumour growth. Our results showed that inhibiting annexin A7 expression induced more than fivefold increase in BGC823 cell apoptosis in vitro. This was in concord with a significant decrease of Bcl-2 expression and increases of Bax, Caspase-3, and Caspase-9. The activities of caspase-3 and caspase-9 were increased by 2.95 ± 0.18 and 3.70 ± 0.33 times, respectively, upon the annexin A7 downregulation in BGC823 cells. Importantly, suppressing annexin A7 showed the same apoptotic mechanism in vivo and significantly inhibited the growth of BGC823 xenografts in mice. These data suggest that annexin A7 likely protects gastric cells from apoptosis and targeting it may represent a valuable strategy in future therapeutic development.

In vivo and in vitro effect of hepatocarcinoma lymph node metastasis by upregulation of Annexin A7 and relevant mechanisms.

We unveiled the association of Annexin A7 with vascular endothelial growth factor-C (VEGF-C) and the effect of upregulation of Annexin A7 in Hca-F and Hca-P cells on inhibiting hepatocarcinoma (HCC) lymph node metastasis (LNM) in vitro and in vivo. A total of 200 inbred 615 mice were randomly divided into four equal groups inoculated with Hca-F, Hca-P, FAnxa7-upregulated, and PAnxa7-upregulated cells, respectively. The primary tumor, popliteal, inguinal, and iliac lymph nodes were prepared for immunohistochemical (IHC) staining, real-time quantitative polymerase chain reaction (qRT-PCR) analysis, Western blot, and hematoxylin-eosin (H&E) staining. There was over 50 % increase both in the number of FAnxa7-upregulated and PAnxa7-upregulated cells migrated through the filter compared to their controls (FAnxa7-control, Hca-F and PAnxa7-control, Hca-P). However, no significant differences were noted in invasion ability between them (all P > 0.05). Tumor lymph vessels were significantly reduced in FAnxa7-upregulated and PAnxa7-upregulated tumors when compared with Hca-F and Hca-P tumors (all P < 0.05). Blood vessel density did not differ significantly between FAnxa7-upregulated and PAnxa7-upregulated tumors and Hca-F and Hca-P tumors. Enzyme-linked immunosorbent assay (ELISA) for VEGF-C showed that upregulating Annexin A7 decreased VEGF-C secretion in FAnxa7-upregulated and PAnxa7-upregulated cells (P < 0.05). The IHC staining result showed that the level of serum Annexin A7 was found to be statistically higher in all experimental groups than that in the control group (P < 0.05). The present results indicated that alterations in serum Annexin A7 expression may be of prognostic relevance in HCC lymphatic metastasis.

Impact of Annexin A 7 Deficiency on FGF23 Plasma Concentrations.

BACKGROUND/AIMS: The release of fibroblast growth factor FGF23, a powerful regulator of 1,25(OH)2D3 formation and mineral metabolism, is stimulated by store-operated Ca2+ entry (SOCE), which is accomplished by the pore forming Ca2+ release activated channel protein Orai1. Regulators of Orai1 and thus FGF23 release include serum & glucocorticoid inducible kinase SGK1, a kinase up-regulated by glucocorticosteroids. Some effects of glucocorticoids require the presence of annexin A7, such as suppression of prostaglandin E2 in gastric glands. The present study thus explored whether annexin A7 impacts on FGF23 plasma levels. METHODS: Comparisons were made between gene targeted mice lacking functional annexin A7 (Anx7-/-) and their wild type littermates (Anx7+/+). Serum C-terminal-FGF23, intact FGF23, 1,25(OH)2D3 and PTH concentrations were measured by ELISA or EIA. The serum and urinary phosphate concentrations were measured by colorimetry, the serum Ca2+ concentration and the urinary Ca2+ concentration by flame photometry. RESULTS: Serum C-terminal FGF23 levels and corticosterone levels were significantly higher and serum 1,25(OH)2 D3 and PTH levels were significantly lower in Anx7-/- than in Anx7+/+ mice. Water intake was slightly but significantly higher in Anx7-/- mice than in Anx7+/+ mice. No significant difference was observed between Anx7-/- and Anx7+/+ mice in urinary fluid excretion, plasma Ca2+ concentration, plasma phosphate concentration and urinary Ca2+ output. The urinary phosphate output was significantly lower in Anx7-/- mice than in Anx7+/+ mice. CONCLUSION: Annexin A7 deficiency upregulates FGF23 plasma levels, an effect paralleled by increased corticosterone plasma levels, as well as decreased 1,25(OH)2 D3 and PTH plasma levels.

[Expression of annexin A7 and its clinical significance in gastric carcinoma].

OBJECTIVE: To investigate the expression of annexin A7 (ANXA7) in the differentiation and lymphatic metastasis of gastric cancer (GC), and to investigate the relationship between ANXA7 and biological characteristics of GC. METHODS: The clinicopathological data of 124 patients with gastric cancer who underwent surgical treatment in our hospital were retrospectively reviewed and analyzed. Immunohistochemical staining and Western blot were performed to analyze the expression of ANXA 7 in primary GC tissues. Logistic regression analysis was conducted to evaluate the association between ANXA7 expression level and differentiation of the GC. RESULTS: A total of 124 GC patients were enrolled in this study, and the expression rate of ANXA7 was 65.3% in the GC. The survival rate of ANXA7-positive patients was significantly lower than that in the patients with negative expression (P<0.001). The results of Cox regression analysis showed that the positive expression of ANXA7, submucosal confinement and pathological stage of GC were associated with poor clinical outcomes. The ratio of pixel density value of primary GC tissues with lymph node metastasis was significantly higher than those in the tissues without lymph node metastasis (0.51±0.07 vs. 0.39±0.06, P<0.001). ROC analysis showed a high area under the curve for the ratio of pixel density value of annexin A7 in the primary GC tissues. At a cut-off level of >0.419, the ratio of pixel density value of ANXA7 exhibited a sensitivity of 91.2% and a specificity of 72.7% for detecting lymph node metastasis of GC. CONCLUSION: High annexin A7 expression is associated with poor differentiation of gastric cancer, and it may become a predictor for lymphatic metastasis of GC.

Influence of annexin A7 on insulin sensitivity of cellular glucose uptake.

Insulin sensitivity is decreased by prostaglandin E2 (PGE2), a major product of cyclooxygenase (COX). As shown in erythrocytes, PGE2 formation is inhibited by annexin A7. The present study defined the role of annexin A7 in glucose metabolism. Gene-targeted mice lacking annexin A7 (annexin7 (-/-)) were compared to wild-type mice (annexin7 (+/+)). The serum 6-Keto-prostaglandin-F1α (6-Keto-PGF1α) concentration was measured by ELISA and hepatic COX activity determined by an enzyme assay. Expression of COX-1, COX-2, prostaglandin E synthase, GLUT-4, and insulin receptor was determined by Western blotting. Glucose and insulin serum concentrations were analyzed following an intraperitoneal glucose load and glucose serum levels after intraperitoneal injection of insulin. Experiments were done without and with pretreatment of the mice with COX-inhibitor aspirin. The serum 6-Keto-PGF1α level and hepatic COX activity were significantly higher in annexin7 (-/-) than in annexin7 (+/+) mice. Hepatic COX-1 expression was higher in annexin7 (-/-) mice. Glucose tolerance was decreased in annexin7 (-/-) mice. Intraperitoneal insulin injection decreased the serum glucose level in both genotypes, an effect significantly less pronounced in annexin7 (-/-) mice. Glucose-induced insulin secretion was higher in annexin7 (-/-) mice. GLUT-4 expression in skeletal muscle from annexin7 (-/-) mice was reduced. Aspirin pretreatment lowered the increase in insulin concentration following glucose injection in both genotypes and virtually abrogated the differences in serum insulin between the genotypes. Aspirin pretreatment improved glucose tolerance in annexin7 (-/-) mice. In conclusion, annexin A7 influences insulin sensitivity of cellular glucose uptake and thus glucose tolerance. These effects depend on COX activity.

Identification of a small molecule targeting annexin A7.

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.

Annexin A7 trafficking to alveolar type II cell surface: possible roles for protein insertion into membranes and lamellar body secretion.

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.

Guanine nucleotide-binding protein subunit beta-2-like 1, a new Annexin A7 interacting protein.

We report for the first time that Guanine nucleotide-binding protein subunit beta-2-like 1 (RACK1) formed a complex with Annexin A7. Hca-F and Hca-P are a pair of syngeneic mouse hepatocarcinoma cell lines established and maintained in our laboratory. Our previous study showed that both Annexin A7 and RACK1 were expressed higher in Hca-F (lymph node metastasis >70%) than Hca-P (lymph node metastasis <30%). Suppression of Annexin A7 expression in Hca-F cells induced decreased migration and invasion ability. In this study, knockdown of RACK1 by RNA interference (RNAi) had the same impact on metastasis potential of Hca-F cells as Annexin A7 down-regulation. Furthermore, by co-immunoprecipitation and double immunofluorescence confocal imaging, we found that RACK1 was in complex with Annexin A7 in control cells, but not in the RACK1-down-regulated cells, indicating the abolishment of RACK1-Annexin A7 interaction in Hca-F cells by RACK1 RNAi. Taken together, these results suggest that RACK1-Annexin A7 interaction may be one of the means by which RACK1 and Annexin A7 influence the metastasis potential of mouse hepatocarcinoma cells in vitro.

Annexin A7 deficiency potentiates cardiac NFAT activity promoting hypertrophic signaling.

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 ß-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.