YAP-dependent proliferation by a small molecule targeting annexin A2.

The transcriptional coactivator Yes-associated protein 1 (YAP) orchestrates a proproliferative transcriptional program that controls the fate of somatic stem cells and the regenerative responses of certain tissues. As such, agents that activate YAP may hold therapeutic potential in disease states exacerbated by insufficient proliferative repair. Here we report the discovery of a small molecule, termed PY-60, which robustly activates YAP transcriptional activity in vitro and promotes YAP-dependent expansion of epidermal keratinocytes in mouse following topical drug administration. Chemical proteomics revealed the relevant target of PY-60 to be annexin A2 (ANXA2), a protein that directly associates with YAP at the cell membrane in response to increased cell density. PY-60 treatment liberates ANXA2 from the membrane, ultimately promoting a phosphatase-bound, nonphosphorylated and transcriptionally active form of YAP. This work reveals ANXA2 as a previously undescribed, druggable component of the Hippo pathway and suggests a mechanistic rationale to promote regenerative repair in disease.

(20S) Ginsenoside Rh2 Inhibits STAT3/VEGF Signaling by Targeting Annexin A2.

Signal transducers and activators of transcription 3 (STAT3) acts as a transcriptional signal transducer, converting cytokine stimulation into specific gene expression. In tumor cells, aberrant activation of the tyrosine kinase pathway leads to excessive and continuous activation of STAT3, which provides further signals for tumor cell growth and surrounding angiogenesis. In this process, the tumor-associated protein Annexin A2 interacts with STAT3 and promotes Tyr705 phosphorylation and STAT3 transcriptional activation. In this study, we found that (20S) ginsenoside Rh2 (G-Rh2), a natural compound inhibitor of Annexin A2, inhibited STAT3 activity in HepG2 cells. (20S) G-Rh2 interfered with the interaction between Annexin A2 and STAT3, and inhibited Tyr705 phosphorylation and subsequent transcriptional activity. The inhibitory activity of STAT3 leaded to the negative regulation of the four VEGFs, which significantly reduced the enhanced growth and migration ability of HUVECs in co-culture system. In addition, (20S)G-Rh2 failed to inhibit STAT3 activity in cells overexpressing (20S)G-Rh2 binding-deficient Annexin A2-K301A mutant, further proving Annexin A2-mediated inhibition of STAT3 by (20S)G-Rh2. These results indicate that (20S)G-Rh2 is a potent inhibitor of STAT3, predicting the potential activity of (20S)G-Rh2 in targeted therapy applications.

Preeclampsia: a defect in decidualization is associated with deficiency of Annexin A2.

BACKGROUND: Decidualization defects in the endometrium have been demonstrated at the time of delivery in women with severe preeclampsia and to linger for years, which suggests a maternal contribution to the pathogenesis of this condition. Global transcriptional profiling reveals alterations in gene expression, which includes down-regulation of Annexin A2 in severe preeclampsia patients with decidualization resistance. OBJECTIVE: We investigated the functional role of Annexin A2 deficiency during endometrial decidualization and its potential contribution to shallow trophoblast invasion during implantation and subsequent placentation using in vitro and in vivo modeling. STUDY DESIGN: Annexin A2 gene and protein levels were assessed during in vitro decidualization of human endometrial stromal cells isolated from biopsy specimens that were collected from women with previous severe preeclampsia (n=5) or normal obstetric outcomes (n=5). Next, Annexin A2 was inhibited with small interference RNA in control human endometrial stromal cells that were isolated from endometrial biopsy specimens (n=15) as an in vitro model to analyze decidualization defects at the morphologic level and the secretion of prolactin and insulin-like growth binding protein-1. Annexin A2-inhibited cells were used to evaluate motility and promotion of embryo invasion. Decidualization and placentation defects of Annexin A2 deficiency were confirmed with the use of an Annexin A2-null mouse model. RESULTS: Annexin A2 gene and protein levels were down-regulated during in vitro decidualization of human endometrial stromal cells from women with previous severe preeclampsia compared with control individuals. To assess its role in the endometrial stroma, we inhibited Annexin A2 expression and detected decidualization failure as evidenced by impaired morphologic transformation, which was associated with altered actin polymerization and low prolactin and insulin-like growth binding protein-1 secretions. Functionally, in vitro models demonstrated that Annexin A2 inhibition failed to support embryo invasion. This finding was corroborated by reduced trophoblast spreading through human endometrial stromal cells, lack of motility of these cells, and reduced trophoblast invasion in the presence of conditioned media from Annexin A2-inhibited cells. Extending our discovery to an animal model, we detected that Annexin A2-null mice have a functional deficiency in decidualization and placentation that impairs fetal growth as a feature that is associated with severe preeclampsia. CONCLUSION: Together, in vitro and in vivo results suggest that endometrial defects in Annexin A2 expression impair decidualization of endometrial stromal cells as well as the uterine microenvironment that promotes embryo implantation and placentation. Our findings highlight the maternal contribution to the pathogenesis of severe preeclampsia and suggest that evaluation of Annexin A2 may provide a novel strategy to assess a woman's risk of experiencing this disease and perhaps discover therapeutic interventions to improve decidualization.

Preclinical Activity of Embryonic Annexin A2-Specific Chimeric Antigen Receptor T Cells Against Ovarian Cancer.

Chimeric antigen receptors (CARs) have found clinical success in B cell malignancies, but a dearth of potential targets limits their wider clinical application, especially in solid tumours. Here, we describe the development of an anti-annexin A2 CAR, CAR(2448), derived from an antibody found to have activity against epithelial ovarian cancer cell lines. The spacer length of CAR(2448) was optimised based on in vitro cytotoxic activity against ovarian cancer (OC) cell lines via a real-time cytotoxicity assay. The longer spacer CAR(2448)L T cells exhibit significant effector activity, inducing inflammatory cytokine release and cytotoxicity against OC cell lines. Furthermore, CAR(2448)L-BBz T cells induced enhanced survival in an in vivo OC xenograft model and reduced tumour volume by 76.6%. Our preclinical studies of CAR(2448) suggest its potential for the unmet need of novel strategies for the treatment of ovarian cancer.

Anxa2 gene silencing attenuates obesity-induced insulin resistance by suppressing the NF-κB signaling pathway.

Insulin resistance (IR) continues to pose a major threat to public health due to its role in the pathogenesis of metabolic syndrome and its ever-increasing prevalence on a global scale. The aim of the current study was to investigate the efficacy of Anxa2 in obesity-induced IR through the mediation of the NF-κB signaling pathway. Microarray analysis was performed to screen differentially expressed genes associated with obesity. To verify whether Anxa2 was differentially expressed in IR triggered by obesity, IR mouse models were established in connection with a high-fat diet (HFD). In the mouse IR model, the role of differentially expressed Anxa2 in glycometabolism and IR was subsequently detected. To investigate the effect of Anxa2 on IR and its correlation with inflammation, a palmitic acid (PA)-induced IR cell model was established, with the relationship between Anxa2 and the NF-κB signaling pathway investigated accordingly. Anxa2 was determined to be highly expressed in IR. Silencing Anxa2 was shown to inhibit IR triggered by obesity. When Anxa2 was knocked down, elevated expression of phosphorylated insulin receptor substrate 1 (IRS1), IRS1 and peroxisome proliferator-activated receptor coactivator-1a, and glucose tolerance and insulin sensitivity along with 2-deoxy-d-glucose uptake was detected, whereas decreased expression of suppressor of cytokine signaling 3, IL-6, IL-1ß, TNF-α, and p50 was observed. Taken together, the current study ultimately demonstrated that Anxa2 may be a novel drug strategy for IR disruption, indicating that Anxa2 gene silencing is capable of alleviating PA or HFD-induced IR and inflammation through its negative regulatory role in the process of p50 nuclear translocation of the NF-κB signaling pathway.

Cell-surface translocation of annexin A2 contributes to bleomycin-induced pulmonary fibrosis by mediating inflammatory response in mice.

Bleomycin, a widely used anti-cancer drug, may give rise to pulmonary fibrosis, a serious side effect which is associated with significant morbidity and mortality. Despite the intensive efforts, the precise pathogenic mechanisms of pulmonary fibrosis still remain to be clarified. Our previous study showed that bleomycin bound directly to annexin A2 (ANXA2, or p36), leading to development of pulmonary fibrosis by impeding transcription factor EB (TFEB)-induced autophagic flux. Here, we demonstrated that ANXA2 also played a critical role in bleomycin-induced inflammation, which represents another major cause of bleomycin-induced pulmonary fibrosis. We found that bleomycin could induce the cell surface translocation of ANXA2 in lung epithelial cells through exosomal secretion, associated with enhanced interaction between ANXA2 and p11. Knockdown of ANXA2 or blocking membrane ANXA2 mitigated bleomycin-induced activation of nuclear factor (NF)-κB pathway and production of pro-inflammatory cytokine IL-6 in lung epithelial cells. ANXA2-deficient (ANXA2-/-) mice treated with bleomycin exhibit reduced pulmonary fibrosis along with decreased cytokine production compared with bleomycin-challenged wild-type mice. Further, the surface ANXA2 inhibitor TM601 could ameliorate fibrotic and inflammatory response in bleomycin-treated mice. Taken together, our results indicated that, in addition to disturbing autophagic flux, ANXA2 can contribute to bleomycin-induced pulmonary fibrosis by mediating inflammatory response.

Annexin A2 is critical for blood-testis barrier integrity and spermatid disengagement in the mammalian testis.

Throughout spermatogenesis, two important processes occur at late stage VIII of the seminiferous epithelial cycle in the rat testis: preleptotene spermatocytes commence entry into the adluminal compartment and step 19 spermatids release from the seminiferous epithelium. Presently, it is not clear how these processes, which involve extensive restructuring of unique Sertoli-Sertoli and Sertoli-germ cell junctions, are mediated. We aimed to determine whether annexin A2 (ANXA2), a Ca2+-dependent and phospholipid-binding protein, participates in cell junction dynamics. To address this, in vitro and in vivo RNA interference studies were performed on prepubertal Sertoli cells and adult rat testes. The endpoints of Anxa2 knockdown were determined by immunoblotting, morphological analyses, fluorescent immunostaining, and barrier integrity assays. In the testis, ANXA2 localized to the Sertoli cell stalk, with specific staining at the blood-testis barrier and the concave (ventral) surface of elongated spermatids. ANXA2 also bound actin when testis lysates were used for immunoprecipitation. Anxa2 knockdown was found to disrupt the Sertoli cell/blood-testis barrier in vitro and in vivo. The disruption in barrier function was substantiated by changes in the localization of claudin-11, zona occludens-1, N-cadherin, and ß-catenin. Furthermore, Anxa2 knockdown resulted in spermiation defects caused by a dysfunction of tubulobulbar complexes, testis-specific actin-rich ultrastructures that internalize remnant cell junction components prior to spermiation. Additionally, there were changes in the localization of several tubulobulbar complex component proteins, including actin-related protein 3, cortactin, and dynamin I/II. Our results indicate that ANXA2 is critical for the integrity of the blood-testis barrier and the timely release of spermatids.

Effects of MicroRNA-206 on Osteosarcoma Cell Proliferation, Apoptosis, Migration and Invasion by Targeting ANXA2 Through the AKT Signaling Pathway.

BACKGROUND/AIMS: This study aimed to investigate the mechanism by which microRNA-206 (miR-206) affects the proliferation, apoptosis, migration and invasion of osteosarcoma (OS) cells by targeting ANXA2 via the AKT signaling pathway. METHODS: A total of 132 OS tissues and 120 osteochondroma tissues were examined in this study. The targeting relationship between miR-206 and ANXA2 was verified with a dual-luciferase reporter assay. The miR-206 expression and ANXA2, AKT, PARP, FASN, Survivin, Bax, Mcl-1 and Bcl-1 mRNA and protein expression in the above two groups were examined by qRT-PCR and western blotting. The cultured OS cells were divided into 6 groups: a blank group, negative control (NC) group, miR-206 mimic group, miR-206 inhibitor group, si-ANXA2 group and miR-206 inhibitor + si-ANXA2 group. Cell cycle and apoptosis were assessed by flow cytometry, cell migration was examined with a wound-healing assay, and cell invasion was assessed with a Transwell assay. Pearson correlation analysis was used to determine the correlation between ANXA2 mRNA expression and miR-206 expression in OS. RESULTS: OS tissues exhibited increased mRNA and protein expression of ANXA2, AKT, PARP, FASN, Survivin, Mcl-1 and Bcl-2; decreased miR-206 expression; and decreased Bax mRNA and protein expression. ANXA2 mRNA expression was strongly negatively correlated with miR-206 expression in OS. ANXA2 was found to be a miR-206 target gene. In the miR-206 mimic group and the si-ANXA2 group, the mRNA and protein expression of ANXA2, AKT, PARP, FASN, Survivin, Mcl-1 and Bcl-1 decreased markedly, cell proliferation was inhibited, apoptosis was promoted, higher cell growth in G1 phase and decreased growth in S phase was detected, and decreased cell migration and invasion were observed compared with those in the blank group. CONCLUSION: The current results demonstrate that miR-206 overexpression inhibits OS cell proliferation, migration and invasion and promotes apoptosis through targeting ANXA2 by blocking the AKT signaling pathway.

Annexin A2 is required for the early steps of cytokinesis.

Cytokinesis requires the formation of an actomyosin contractile ring between the two sets of sister chromatids. Annexin A2 is a calcium- and phospholipid-binding protein implicated in cortical actin remodeling. We report that annexin A2 accumulates at the equatorial cortex at the onset of cytokinesis and depletion of annexin A2 results in cytokinetic failure, due to a defective cleavage furrow assembly. In the absence of annexin A2, the small GTPase RhoA-which regulates cortical cytoskeletal rearrangement-fails to form a compact ring at the equatorial plane. Furthermore, annexin A2 is required for cortical localization of the RhoGEF Ect2 and to maintain the association between the equatorial cortex and the central spindle. Our results demonstrate that annexin A2 is necessary in the early phase of cytokinesis. We propose that annexin A2 participates in central spindle-equatorial plasma membrane communication.

Annexin A2 antibodies but not inhibitors of the annexin A2 heterotetramer impair productive HIV-1 infection of macrophages in vitro.

During sexual transmission of human immunodeficiency virus (HIV), macrophages are initial targets for HIV infection. Secretory leukocyte protease inhibitor (SLPI) has been shown to protect against HIV infection of macrophages through interactions with annexin A2 (A2), which is found on the macrophage cell surface as a heterotetramer (A2t) consisting of A2 and S100A10. Therefore, we investigated potential protein-protein interactions between A2 and HIV-1 gp120 through a series of co-immunoprecipitation assays and a single molecule pulldown (SiMPull) technique. Additionally, inhibitors of A2t (A2ti) that target the interaction between A2 and S100A10 were tested for their ability to impair productive HIV-1 infection of macrophages. Our data suggest that interactions between HIV-1 gp120 and A2 exist, though this interaction may be indirect. Furthermore, an anti-A2 antibody impaired HIV-1 particle production in macrophages in vitro, whereas A2ti did not indicating that annexin A2 may promote HIV-1 infection of macrophages in its monomeric rather than tetrameric form.

Receptor role of the annexin A2 in the mesothelial endocytosis of crocidolite fibers.

Asbestos-induced mesothelioma is a worldwide problem. Parietal mesothelial cells internalize asbestos fibers that traverse the entire lung parenchyma, an action that is linked to mesothelial carcinogenesis. Thus far, vitronectin purified from serum reportedly enhances the internalization of crocidolite by mesothelial cells via integrin αvß5. To reveal another mechanism by which mesothelial cells endocytose (phagocytose) asbestos, we first evaluated the effects of serum on asbestos uptake, which proved to be nonessential. Thereafter, we undertook a study to identify proteins on the surface of mesothelial cells (MeT5A) that act as receptors for asbestos uptake based on the assumption that receptors bind to asbestos with physical affinity. To this end, we incubated the membrane fraction of MeT5A cells with crocidolite or chrysotile and evaluated the adsorbed proteins using sodium dodecyl sulfate polyacrylamide gel analysis. Next, we extensively identified the proteins using an in-solution or in-gel digestion coupled with mass spectrometry. Among the identified proteins, annexin A2 (ANXA2) and transferrin receptor protein 1 (TFRC) were distinguished because of their high score and presence at the cell surface. Crocidolite uptake by MeT5A cells was significantly decreased by shRNA (short hairpin RNA)-induced knockdown of ANXA2 and direct blockade of cell surface ANXA2 using anti-ANXA2 antibody. In addition, abundant ANXA2 protein was present on the cell membrane of mesothelial cells, particularly facing the somatic cavity. These findings demonstrate that ANXA2 has a role in the mesothelial phagocytosis of crocidolite and may serve as its receptor.

Small molecule inhibitors of the annexin A2 heterotetramer prevent human papillomavirus type 16 infection.

OBJECTIVES: High-risk human papillomavirus (HPV) infection leads to the development of several human cancers that cause significant morbidity and mortality worldwide. HPV type 16 (HPV16) is the most common of the cancer-causing genotypes and gains entry to the basal cells of the epithelium through a non-canonical endocytic pathway that involves the annexin A2/S100A10 heterotetramer (A2t). A2t is composed of two annexin A2 monomers bound to an S100A10 dimer and this interaction is a potential target to block HPV16 infection. Here, recently identified small molecule inhibitors of A2t (A2ti) were investigated for their ability to prevent HPV16 infection in vitro. METHODS: A2ti were added to HeLa cells in increasing concentrations prior to the addition of HPV16. Cytotoxicity was evaluated via trypan blue exclusion. HPV16 pseudovirion infection and fluorescently labelled HPV16 capsid internalization was measured with flow cytometry. RESULTS: A2ti blocked HPV16 infection by 100% without substantial cellular toxicity or reduction in cell growth. Furthermore, A2ti blocked HPV16 entry into epithelial cells by 65%, indicating that the observed inhibition of HPV16 infection is in part due to a block in entry and that non-infectious entry may occur in the absence of A2t binding. CONCLUSIONS: These results demonstrate that targeting A2t may be an effective strategy to prevent HPV16 infection.

Antiphospholipid syndrome: an update.

BACKGROUND: Antiphospholipid syndrome (APS) or 'Hughes syndrome' is a prothrombotic disease characterized by thrombosis and pregnancy morbidity in the presence of antiphospholipid antibodies (aPL). More than three decades have passed, and experts are still uncovering new pieces of this disease complex pathogenesis and management. MATERIALS AND METHODS: We searched in literature using MEDLINE and PubMed databases focusing on the latest development on disease pathogenesis, risk assessment of thrombosis and treatment of APS. RESULTS: The phosphatidylinositol 3-kinase (PI3K)-AKT-mTORC pathway was most recently identified to have a crucial role in activating inflammation among endothelial vessel wall causing vascular lesions in APS. Additionally, new variables are being implemented to assess the risk of thrombosis in patients with APS. Global APS Score (GAPSS) utilizes cardiovascular risk factors and new autoimmune antibodies as part of the score assessment and is the most valid so far. It can be a promising tool in the future for prediction of thrombosis. Anticoagulation remains the cornerstone in APS; however, many new potential therapeutic agents are developing and are currently under investigation. CONCLUSIONS: The most recent advances in pathogenesis, risk stratification and treatment provide a platform for high yield studies with the ultimate goal of providing the optimal management to patients with APS.

Tissue-type plasminogen activator deficiency delays bone repair: roles of osteoblastic proliferation and vascular endothelial growth factor.

Further development in research of bone regeneration is necessary to meet the clinical demand for bone reconstruction. Recently, we reported that plasminogen is crucial for bone repair through enhancement of vessel formation. However, the details of the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) in the bone repair process still remain unknown. Herein, we examined the effects of plasminogen activators on bone repair after a femoral bone defect using tPA-deficient (tPA(-/-)) and uPA-deficient (uPA(-/-)) mice. Bone repair of the femur was delayed in tPA(-/-) mice, unlike that in wild-type (tPA(+/+)) mice. Conversely, the bone repair was comparable between wild-type (uPA(+/+)) and uPA(-/-) mice. The number of proliferative osteoblasts was decreased at the site of bone damage in tPA(-/-) mice. Moreover, the proliferation of primary calvarial osteoblasts was reduced in tPA(-/-) mice. Recombinant tPA facilitated the proliferation of mouse osteoblastic MC3T3-E1 cells. The proliferation enhanced by tPA was antagonized by the inhibition of endogenous annexin 2 by siRNA and by the inhibition of extracellular signal-regulated kinase (ERK)1/2 phosphorylation in MC3T3-E1 cells. Vessel formation as well as the levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were decreased at the damaged site in tPA(-/-) mice. Our results provide novel evidence that tPA is crucial for bone repair through the facilitation of osteoblast proliferation related to annexin 2 and ERK1/2 as well as enhancement of vessel formation related to VEGF and HIF-1α at the site of bone damage.

Design, synthesis and SAR exploration of tri-substituted 1,2,4-triazoles as inhibitors of the annexin A2-S100A10 protein interaction.

Recent target validation studies have shown that inhibition of the protein interaction between annexin A2 and the S100A10 protein may have potential therapeutic benefits in cancer. Virtual screening identified certain 3,4,5-trisubstituted 4H-1,2,4-triazoles as moderately potent inhibitors of this interaction. A series of analogues were synthesized based on the 1,2,4-triazole scaffold and were evaluated for inhibition of the annexin A2-S100A10 protein interaction in competitive binding assays. 2-[(5-{[(4,6-Dimethylpyrimidin-2-yl)sulfanyl]methyl}-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)sulfanyl]-N-[4-(propan-2-yl)phenyl]acetamide (36) showed improved potency and was shown to disrupt the native complex between annexin A2 and S100A10.

'Shedding' light on mechanisms of hyperphosphatemic vascular dysfunction.

Cardiovascular disease is prevalent in chronic kidney disease (CKD) populations. Vascular dysfunction characterized by endothelial-cell damage, inflammation, and impaired angiogenesis is described in CKD, and hyperphosphatemia has been associated with vascular disease in CKD and general populations. We discuss the findings of elegant studies by Di Marco et al., linking hyperphosphatemia to downregulation of annexin II, an important protein implicated in fibrinolysis, cell adhesion, signal transduction, and angiogenesis.

High phosphate directly affects endothelial function by downregulating annexin II.

Hyperphosphatemia is associated with increased cardiovascular risk in patients with renal disease and in healthy individuals. Here we tested whether high phosphate has a role in the pathophysiology of cardiovascular events by interfering with endothelial function, thereby impairing microvascular function and angiogenesis. Protein expression analysis found downregulation of annexin II in human coronary artery endothelial cells, an effect associated with exacerbated shedding of annexin II-positive microparticles by the cells exposed to high phosphate media. EAhy926 endothelial cells exposed to sera from hyperphosphatemic patients also display decreased annexin II, suggesting a negative correlation between serum phosphate and annexin II expression. By using endothelial cell-based assays in vitro and the chicken chorioallantoic membrane assay in vivo, we found that angiogenesis, vessel wall morphology, endothelial cell migration, capillary tube formation, and endothelial survival were impaired in a hyperphosphatemic milieu. Blockade of membrane-bound extracellular annexin II with a specific antibody mimicked the effects of high phosphate. In addition, high phosphate stiffened endothelial cells in vitro and in rats in vivo. Thus, our results link phosphate and adverse clinical outcomes involving the endothelium in both healthy individuals and patients with renal disease.

A competitive hexapeptide inhibitor of annexin A2 prevents hypoxia-induced angiogenic events.

Extracellular proteolysis is an indispensable requirement for the formation of new blood vessels during neovascularization and is implicated in the generation of several angiogenic regulatory molecules. Anti-proteolytic agents have become attractive therapeutic strategies in diseases associated with excessive neovascularization. Annexin A2 (AnxA2) is an endothelial cell-surface receptor for the generation of active proteolytic factors, such as plasmin. Here, we show that AnxA2 is abundantly expressed in the neovascular tufts in a murine model of neovascularization. Exposure to hypoxic conditions results in elevation of AnxA2 and tissue plasminogen activator (tPA) in human retinal microvascular endothelial cells (RMVECs). We show that the hexapeptide competitive inhibitor LCKLSL, which targets the N-terminal tPA-binding site of AnxA2, binds efficiently to cell-surface AnxA2 compared with binding of the control peptide LGKLSL. Treatment with the competitive peptide inhibits the generation of plasmin and suppresses the VEGF-induced activity of tPA under hypoxic conditions. Application of the competitive peptide in two in vivo models of angiogenesis demonstrated suppression of the angiogenic responses, which was also associated with significant changes in the vascular sprouting. These results suggest that AnxA2-mediated plasmin generation is an important event in angiogenesis and is inhibited by a specific competitive peptide that inhibits the binding of tPA to AnxA2.

Annexin A2 is critical for embryo adhesiveness to the human endometrium by RhoA activation through F-actin regulation.

Annexin A2 (ANXA2) is present in vivo in the mid- and late-secretory endometria and is mainly localized in the luminal epithelium. Our aim was to evaluate its function in regulating the human implantation process. With an in vitro adhesion model, constructed to evaluate how the mouse embryo and JEG-3 spheroids attach to human endometrial epithelial cells, we demonstrated that ANXA2 inhibition significantly diminishes embryo adhesiveness. ANXA2 is also implicated in endometrial epithelial cell migration and trophoblast outgrowth. ANXA2 was seen to be linked to the RhoA/ROCK pathway and to regulate cell adhesion. We noted that ANXA2 inhibition significantly reduces active RhoA, although RhoA inactivation does not alter the ANXA2 levels. RhoA inactivation and ROCK inhibition also moderate embryo adhesiveness to endometrial epithelial cells. We corroborated that the induction of constitutively active RhoA partially reverses the effects of ANXA2 inhibition on endometrial adhesiveness. These molecules colocalize on the plasma membrane of endometrial epithelial cells, and a large proportion of ANXA2 and RhoA are colocalized in the F-actin networks. The functional effects of ANXA2 inhibition and RhoA/ROCK inactivation are associated with significant alterations in F-actin organization and its depolymerization. ANXA2 may act upstream of the RhoA/ROCK pathway by regulating F-actin remodeling and is a key factor in human endometrial adhesiveness.

The involvement of annexin II in resistance to UVB-induced cell death and in the increased nucleotide excision repair capacity of UV-damaged DNA in human cells.

Annexin II, an HSP27-interacting protein, is involved in the protection of human cells against UVC. UVB is concerned with deleterious actions on human health. In this study, we attempted to confirm the anti-UVB effect of annexin II, and to elucidate the mechanisms underlying annexin II-involving UV resistance. The RSa cells were more sensitive to UVB lethality than the AP(r)-1 cells. Overproduction of annexin II in RSa cells resulted in increased resistance to UVB lethality, while annexin II siRNA-transfected AP(r)-1 cells were sensitized to UVB lethality. The excision capacity of the two major types (CPD and 6-4PP) of UVC- and UVB-damaged DNA in AP(r)-1 cells was greater than in RSa cells. The excision capacity of the RSa cells improved following upregulation of annexin II, while the capacity of the AP(r)-1 cells decreased after annexin II downregulation. Our results suggest that annexin II is involved in the UV resistance of human cells, via functioning in nucleotide excision repair.