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.

Essential roles of S100A10 in Toll-like receptor signaling and immunity to infection.

Toll-like receptors (TLRs) are key pattern recognition receptors that mediate innate immune responses to infection. However, uncontrolled TLR activation can lead to severe inflammatory disorders such as septic shock. The molecular mechanisms through which TLR responses are regulated are not fully understood. Here, we demonstrate an essential function of S100A10 in TLR signaling. S100A10 was constitutively expressed in macrophages, but was significantly downregulated upon TLR activation. S100A10-deficient macrophages were hyperresponsive to TLR stimulation, and S100A10-deficient mice were more sensitive to endotoxin-induced lethal shock and Escherichia coli-induced abdominal sepsis. Mechanistically, S100A10 regulated macrophage inflammatory responses by interfering with the appropriate recruitment and activation of the receptor-proximal signaling components and eventually inhibited TLR-triggered downstream signaling. These findings expand our understanding of TLR signaling and establish S100A10 as an essential negative regulator of TLR function and a potential therapeutic target for treating inflammatory diseases.

Annexin A2 Deficiency Exacerbates Neuroinflammation and Long-Term Neurological Deficits after Traumatic Brain Injury in Mice.

Our laboratory and others previously showed that Annexin A2 knockout (A2KO) mice had impaired blood-brain barrier (BBB) development and elevated pro-inflammatory response in macrophages, implying that Annexin A2 (AnxA2) might be one of the key endogenous factors for maintaining homeostasis of the neurovascular unit in the brain. Traumatic brain injury (TBI) is an important cause of disability and mortality worldwide, and neurovascular inflammation plays an important role in the TBI pathophysiology. In the present study, we aimed to test the hypothesis that A2KO promotes pro-inflammatory response in the brain and worsens neurobehavioral outcomes after TBI. TBI was conducted by a controlled cortical impact (CCI) device in mice. Our experimental results showed AnxA2 expression was significantly up-regulated in response to TBI at day three post-TBI. We also found more production of pro-inflammatory cytokines in the A2KO mouse brain, while there was a significant increase of inflammatory adhesion molecules mRNA expression in isolated cerebral micro-vessels of A2KO mice compared with wild-type (WT) mice. Consistently, the A2KO mice brains had a significant increase in leukocyte brain infiltration at two days after TBI. Importantly, A2KO mice had significantly worse sensorimotor and cognitive function deficits up to 28 days after TBI and significantly larger brain tissue loss. Therefore, these results suggested that AnxA2 deficiency results in exacerbated early neurovascular pro-inflammation, which leads to a worse long-term neurologic outcome after TBI.

Axon Guidance Molecules Promote Perineural Invasion and Metastasis of Orthotopic Pancreatic Tumors in Mice.

BACKGROUND & AIMS: Little is known about mechanisms of perineural invasion (PNI) by pancreatic ductal adenocarcinomas (PDAs) or other tumors. Annexin A2 (ANXA2) regulates secretion of SEMA3D, an axon guidance molecule, which binds and activates the receptor PLXND1 to promote PDA invasion and metastasis. We investigated whether axon guidance molecules promote PNI and metastasis by PDA cells in mice. METHODS: We performed studies in a dorsal root ganglion (DRG) invasion system, wild-type C57BL/6 mice (controls), mice with peripheral sensory neuron-specific disruption of PlxnD1 (PLAC mice), LSL-KRASG12D/+;LSL-TP53R172H/+;PDX-1-CRE+/+ (KPC) mice, and KPC mice crossed with ANXA2-knockout mice (KPCA mice). PDA cells were isolated from KPC mice and DRG cells were isolated from control mice. Levels of SEMA3D or ANXA2 were knocked down in PDA cells with small hairpin and interfering RNAs and cells were analyzed by immunoblots in migration assays, with DRGs and with or without antibodies against PLXND1. PDA cells were injected into the pancreas of control and PLAC mice, growth of tumors was assessed, and tumor samples were analyzed by histology. DRG cells were incubated with SEMA3D and analyzed by live imaging. We measured levels of SEMA3D and PLXND1 in PDA specimens from patients with PNI and calculated distances between tumor cells and nerves. RESULTS: DRG cells increase the migration of PDC cells in invasion assays; knockdown of SEMA3D in PDA cells or antibody blockade of PLXND1 on DRG cells reduced this invasive activity. In mice, orthotopic tumors grown from PDA cells with knockdown of SEMA3D, and in PLAC mice, orthotopic tumors grown from PDA cells, had reduced innervation and formed fewer metastases than orthotopic tumors grown from PDA cells in control mice. Increased levels of SEMA3D and PLXND1 in human PDA specimens associated with PNI. CONCLUSIONS: DRG cells increase the migratory and invasive activities of pancreatic cancer cells, via secretion of SEMA3D by pancreatic cells and activation of PLXND1 on DRGs. Knockdown of SEMA3D and loss of neural PLXND1 reduces innervation of orthotopic PDAs and metastasis in mice. Increased levels of SEMA3D and PLXND1 in human PDA specimens associated with PNI. Strategies to disrupt the axon guidance pathway mediated by SEMA3D and PLXND1 might be developed to slow progression of PDA.

A new role for host annexin A2 in establishing bacterial adhesion to vascular endothelial cells: lines of evidence from atomic force microscopy and an in vivo study.

Understanding bacterial adhesion is challenging and critical to our understanding of the initial stages of the pathogenesis of endovascular bacterial infections. The vascular endothelial cell (EC) is the main target of Rickettsia, an obligately intracellular bacterium that causes serious systemic disease in humans and animals. But the mechanism(s) underlying bacterial adherence to ECs under shear stress from flowing blood prior to activation are unknown for any bacteria. Although host surface annexin a2 (ANXA2) has been identified to participate in efficient bacterial invasion of epithelial cells, direct evidence is lacking in the field of bacterial infections of ECs. In the present study, we employ a novel, anatomically based, in vivo quantitative bacterial-adhesion-to-vascular-EC system, combined with atomic force microscopy (AFM), to examine the role of endothelial luminal surface ANXA2 during rickettsial adherence to ECs. We also examined whether ANXA2 antibody affected binding of Staphylococcus aureus to ECs. We found that deletion of ANXA2 impeded rickettsial attachment to the ECs in vitro and blocked rickettsial adherence to the blood vessel luminal surface in vivo. The AFM studies established that EC surface ANXA2 acts as an adherence receptor for rickettsiae, and that rickettsial adhesin OmpB is the associated bacterial ligand. Furthermore, pretreatment of ECs with anti-ANXA2 antibody reduced EC surface-associated S. aureus. We conclude that the endothelial surface ANXA2 plays an important role in initiating pathogen-host interactions, ultimately leading to bacterial anchoring on the vascular luminal surface.

Anxa2- and Ctsd-knockout CHO cell lines to diminish the risk of contamination with host cell proteins.

Chinese hamster ovary (CHO) cells have been used as host cells in the production of a range of recombinant therapeutic proteins, including monoclonal antibodies and Fc-fusion proteins. Host cell proteins (HCP) represent impurities that must be removed from therapeutic formulations because of their potential risks for immunogenicity. While the majority of HCP impurities are effectively removed in typical downstream purification processes, clearance of a small population of HCP remains challenging. In this study, we knocked out the Anxa2 and Ctsd genes to assess the feasibility of knockout approaches for diminishing the risk of contamination with HCP. Using the CRISPR/Cas9 system, Anxa2-, and Ctsd-knockout CHO cell lines were successfully established, and we confirmed the complete elimination of the corresponding HCP in cell lysates. Importantly, all knockout cell lines showed similar growth and viability to those of the wild-type control during 8 days of cultivation. Thus, knockout of unrequired genes can reduce contamination with HCP in the production of recombinant therapeutic proteins.

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.

Lack of p11 expression facilitates acidity-sensing function of TASK1 channels in mouse adrenal medullary cells.

External acidity induces catecholamine secretion by inhibiting TASK1-like channels in rat adrenal medullary (AM) cells. TASK channels can function as a heteromer or homomer in the TASK subfamily. In this study, we elucidate the molecular identity of TASK1-like channels in mouse AM cells using gene knockout. Genetic deletion of TASK1, but not TASK3, abolished the depolarizing inward current and catecholamine secretion in response to acidity, whereas it did not affect the resting current level. Immunocytochemistry revealed that AM cells exhibited predominantly TASK1-like and little TASK3-like immunoreactivity. A proximity ligation assay showed that TASK1/3 heteromeric channels were not formed in AM cells or PC12 cells. However, the exogenous expression of p11 in PC12 cells resulted in the heteromeric formation of TASK isoforms, which were mainly located in the cytoplasm, and p11 was not expressed in rat adrenal medullae or PC12 cells. In AM cells, genetic deletion of TASK1 resulted in enhancement of the immunoreactivity of the TALK2 channel, but not TASK3. The results indicate that TASK1 homomeric channels function as acidity sensors in AM cells, and that function is facilitated by the lack of p11 expression.-Inoue, M., Matsuoka, H., Lesage, F., Harada, K. Lack of p11 expression facilitates acidity-sensing function of TASK1 channels in mouse adrenal medullary cells.

Identification of natural compounds targeting Annexin A2 with an anti-cancer effect.

Annexin A2, a multifunctional tumor associated protein, promotes nuclear factor-kappa B (NF-κB) activation by interacting with NF-κB p50 subunit and facilitating its nuclear translocation. Here we demonstrated that two ginsenosides Rg5 (G-Rg5) and Rk1 (G-Rk1), with similar structure, directly bound to Annexin A2 by molecular docking and cellular thermal shift assay. Both Rg5 and Rk1 inhibited the interaction between Annexin A2 and NF-κB p50 subunit, their translocation to nuclear and NF-κB activation. Inhibition of NF-κB by these two ginsenosides decreased the expression of inhibitor of apoptosis proteins (IAPs), leading to caspase activation and apoptosis. Over expression of K302A Annexin A2, a mutant version of Annexin A2, which fails to interact with G-Rg5 and G-Rk1, effectively reduced the NF-κB inhibitory effect and apoptosis induced by G-Rg5 and G-Rk1. In addition, the knockdown of Annexin A2 largely enhanced NF-κB activation and apoptosis induced by the two molecules, indicating that the effects of G-Rg5 and G-Rk1 on NF-κB were mainly mediated by Annexin A2. Taken together, this study for the first time demonstrated that G-Rg5 and G-Rk1 inhibit tumor cell growth by targeting Annexin A2 and NF-κB pathway, and G-Rg5 and G-Rk1 might be promising natural compounds for targeted cancer therapy.

Annexin A2 facilitates endocytic trafficking of antisense oligonucleotides.

Chemically modified antisense oligonucleotides (ASOs) designed to mediate site-specific cleavage of RNA by RNase H1 are used as research tools and as therapeutics. ASOs modified with phosphorothioate (PS) linkages enter cells via endocytotic pathways. The mechanisms by which PS-ASOs are released from membrane-enclosed endocytotic organelles to reach target RNAs remain largely unknown. We recently found that annexin A2 (ANXA2) co-localizes with PS-ASOs in late endosomes (LEs) and enhances ASO activity. Here, we show that co-localization of ANXA2 with PS-ASO is not dependent on their direct interactions or mediated by ANXA2 partner protein S100A10. Instead, ANXA2 accompanies the transport of PS-ASOs to LEs, as ANXA2/PS-ASO co-localization was observed inside LEs. Although ANXA2 appears not to affect levels of PS-ASO internalization, ANXA2 reduction caused significant accumulation of ASOs in early endosomes (EEs) and reduced localization in LEs and decreased PS-ASO activity. Importantly, the kinetics of PS-ASO activity upon free uptake show that target mRNA reduction occurs at least 4 hrs after PS-ASOs exit from EEs and is coincident with release from LEs. Taken together, our results indicate that ANXA2 facilitates PS-ASO trafficking from early to late endosomes where it may also contribute to PS-ASO release.

[Depression and addiction comorbidity: towards a common molecular target?]. / Comorbidité entre la dépression et l'addiction - Vers une cible moléculaire commune ?

The comorbidity of depression and cocaine addiction suggests shared mechanisms and anatomical pathways. Specifically, the limbic structures, such as the nucleus accumbens (NAc), play a crucial role in both disorders. P11 (S100A10) is a promising target for manipulating depression and addiction in mice. We summarized the recent genetic and viral strategies used to determine how the titration of p11 levels within the NAc affects hedonic behavior and cocaine reward learning in mice. In particular, p11 in the ChAT+ cells or DRD1+ MSN of the NAc, controls depressive-like behavior or cocaine reward, respectively. Treatments to counter maladaptation of p11 levels in the NAc could provide novel therapeutic opportunities for depression and cocaine addiction in humans.

Plasminogen-stimulated inflammatory cytokine production by airway smooth muscle cells is regulated by annexin A2.

Plasminogen has a role in airway inflammation. Airway smooth muscle (ASM) cells cleave plasminogen into plasmin, a protease with proinflammatory activity. In this study, the effect of plasminogen on cytokine production by human ASM cells was investigated in vitro. Levels of IL-6 and IL-8 in the medium of ASM cells were increased by incubation with plasminogen (5-50 µg/ml) for 24 hours (P < 0.05; n = 6-9), corresponding to changes in the levels of cytokine mRNA at 4 hours. The effects of plasminogen were attenuated by α2-antiplasmin (1 µg/ml), a plasmin inhibitor (P < 0.05; n = 6-12). Exogenous plasmin (5-15 mU/ml) also stimulated cytokine production (P < 0.05; n = 6-8) in a manner sensitive to serine-protease inhibition by aprotinin (10 KIU/ml). Plasminogen-stimulated cytokine production was increased in cells pretreated with basic fibroblast growth factor (300 pM) in a manner associated with increases in urokinase plasminogen activator expression and plasmin formation. The knockdown of annexin A2, a component of the putative plasminogen receptor comprised of annexin A2 and S100A10, attenuated plasminogen conversion into plasmin and plasmin-stimulated cytokine production by ASM cells. Moreover, a role for annexin A2 in airway inflammation was demonstrated in annexin A2-/- mice in which antigen-induced increases in inflammatory cell number and IL-6 levels in the bronchoalveolar lavage fluid were reduced (P < 0.01; n = 10-14). In conclusion, plasminogen stimulates ASM cytokine production in a manner regulated by annexin A2. Our study shows for the first time that targeting annexin A2-mediated signaling may provide a novel therapeutic approach to the treatment of airway inflammation in diseases such as chronic asthma.

Bidirectional regulation of emotional memory by 5-HT1B receptors involves hippocampal p11.

Cognitive impairments are common in depression and involve dysfunctional serotonin neurotransmission. The 5-HT1B receptor (5-HT(1B)R) regulates serotonin transmission, via presynaptic receptors, but can also affect transmitter release at heterosynaptic sites. This study aimed at investigating the roles of the 5-HT(1B)R, and its adapter protein p11, in emotional memory and object recognition memory processes by the use of p11 knockout (p11KO) mice, a genetic model for aspects of depression-related states. 5-HT(1B)R agonist treatment induced an impairing effect on emotional memory in wild type (WT) mice. In comparison, p11KO mice displayed reduced long-term emotional memory performance. Unexpectedly, 5-HT(1B)R agonist stimulation enhanced memory in p11KO mice, and this atypical switch was reversed after hippocampal adeno-associated virus mediated gene transfer of p11. Notably, 5-HT(1B)R stimulation increased glutamatergic neurotransmission in the hippocampus in p11KO mice, but not in WT mice, as measured by both pre- and postsynaptic criteria. Magnetic resonance spectroscopy demonstrated global hippocampal reductions of inhibitory GABA, which may contribute to the memory enhancement and potentiation of pre- and post-synaptic measures of glutamate transmission by a 5-HT(1B)R agonist in p11KO mice. It is concluded that the level of hippocampal p11 determines the directionality of 5-HT(1B)R action on emotional memory processing and modulates hippocampal functionality. These results emphasize the importance of using relevant disease models when evaluating the role of serotonin neurotransmission in cognitive deficits related to psychiatric disorders.

Annexin A2 modulates radiation-sensitive transcriptional programming and cell fate.

We previously established annexin A2 as a radioresponsive protein associated with anchorage independent growth in murine epidermal cells. In this study, we demonstrate annexin A2 nuclear translocation in human skin organotypic culture and murine epidermal cells after exposure to X radiation (10-200 cGy), supporting a conserved nuclear function for annexin A2. Whole genome expression profiling in the presence and absence of annexin A2 [shRNA] identified fundamentally altered transcriptional programming that changes the radioresponsive transcriptome. Bioinformatics predicted that silencing AnxA2 may enhance cell death responses to stress in association with reduced activation of pro-survival signals such as nuclear factor kappa B. This prediction was validated by demonstrating a significant increase in sensitivity toward tumor necrosis factor alpha-induced cell death in annexin A2 silenced cells, relative to vector controls, associated with reduced nuclear translocation of RelA (p65) following tumor necrosis factor alpha treatment. These observations implicate an annexin A2 niche in cell fate regulation such that AnxA2 protects cells from radiation-induced apoptosis to maintain cellular homeostasis at low-dose radiation.

Annexin A2 is a natural extrahepatic inhibitor of the PCSK9-induced LDL receptor degradation.

Proprotein convertase subtilisin/kexin-9 (PCSK9) enhances the degradation of hepatic low-density lipoprotein receptor (LDLR). Deletion of PCSK9, and loss-of-function mutants in humans result in lower levels of circulating LDL-cholesterol and a strong protection against coronary heart disease. Accordingly, the quest for PCSK9 inhibitors has major clinical implications. We have previously identified annexin A2 (AnxA2) as an endogenous binding partner and functional inhibitor of PCSK9. Herein, we studied the relevance of AnxA2 in PCSK9 inhibition and lipid metabolism in vivo. Plasma analyses of AnxA2(-/-) mice revealed: i) a ∼1.4-fold increase in LDL-cholesterol without significant changes in VLDLs or HDLs, and ii) a ∼2-fold increase in circulating PCSK9 levels. Western blotting and immunohistochemistry of AnxA2(-/-) tissues revealed that the LDLR was decreased by ∼50% in extrahepatic tissues, such as adrenals and colon. We also show that AnxA2-derived synthetic peptides block the PCSK9≡LDLR interaction in vitro, and adenoviral overexpression of AnxA2 in mouse liver increases LDLR protein levels in vivo. These results suggest that AnxA2 acts as an endogenous regulator of LDLR degradation, mostly in extrahepatic tissues. Finally, we identified an AnxA2 coding polymorphism, V98L, that correlates with lower circulating levels of PCSK9 thereby extending our results on the physiological role of AnxA2 in humans.

Plasminogen receptor S100A10 is essential for the migration of tumor-promoting macrophages into tumor sites.

Macrophages are critical drivers of tumor growth, invasion, and metastasis. Movement of macrophages into tumors requires the activity of cell surface proteases such as plasmin. In this study, we offer genetic evidence that plasminogen receptor S100A10 is essential for recruitment of macrophages to the tumor site. Growth of murine Lewis lung carcinomas or T241 fibrosarcomas was dramatically reduced in S100A10-deficient mice compared with wild-type mice. The tumor growth deficit corresponded with a decrease in macrophage density that could be rescued by intraperitoneal injection of wild-type but not S100A10-deficient macrophages. Notably, macrophages of either genotype could rescue tumor growth if they were injected into the tumor itself, establishing that S100A10 was required specifically for the migratory capability needed for tumor homing. Conversely, selective depletion of macrophages from wild-type mice phenocopied the tumor growth deficit seen in S100A10-deficient mice. Together, our findings show that S100A10 is essential and sufficient for macrophage migration to tumor sites, and they define a novel rate-limiting step in tumor progression.

Annexin-2 is a regulator of stromal cell-derived factor-1/CXCL12 function in the hematopoietic stem cell endosteal niche.

OBJECTIVE: Previously, we reported that annexin-2 (anxa2) plays an important role in hematopoietic stem cell (HSC) localization to the endosteal/osteoblastic marrow niche. This study explored the role that annexin-2 plays in presenting stromal cell-derived factor-1 (or CXCL12) to HSCs. MATERIALS AND METHODS: Competitive long-term bone marrow transplant assays were used to determine if HSC engraftment is altered in annexin-2-deficient animals. Colony-forming cell assays, CXCL12 enzyme-linked immunosorbent assay, and real-time reverse transcription polymerase chain reaction analyses were used to determine stem or progenitor cell mobilization by granulocyte colony-stimulating factor. Immunohistochemistry, immunoprecipitation, binding assays, and chemotactic assays were employed to determine if annexin-2 is associated with CXCL12. Degradation assays were also used to determine if annexin-2 and CXCL12 protect each other from proteolytic degradation. RESULTS: Anxa2(-/-) animals had fewer HSCs in their marrow, and the HSCs in anxa2(-/-) animals express less CXCR4 and CXCR7, suggesting a cell intrinsic defect. Transplantation studies of wild-type marrow into anxa2(-/-) animals demonstrated a cell-extrinsic defect in the anxa2(-/-) animals. CXCL12 binds directly to annexin-2, and this interaction facilitates presentation of CXCL12 to HSCs. Yet the binding of CXCL12 to annexin-2 did not protect CXCL12 from proteolytic cleavage after stem or progenitor cell mobilization by granulocyte colony-stimulating factor. CONCLUSIONS: These results suggest that annexin-2 serves as an anchor for CXCL12 to help in the localization of HSCs to the niche.

Annexin 2 is not required for human immunodeficiency virus type 1 particle production but plays a cell type-dependent role in regulating infectivity.

During assembly and budding of retroviruses, host cell proteins are incorporated into viral particles. Identification of virion-associated proteins may help pinpoint key cellular components required for virus production and function. The cellular protein annexin 2 (Anx2) is incorporated into HIV-1 particles, and knockdown of Anx2 has been reported to cause defects in Gag processing and infectivity of HIV-1 particles in macrophages. Here, we tested whether Anx2 was required for HIV-1 production in other cell types capable of producing HIV-1 virions. Endogenous Anx2 levels were knocked down by approximately 98% using lentivirus encoding short hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) targeting Anx2. Under these conditions, there was no reduction in HIV-1 virus-like particle (VLP) production in either COS-1, 293T, or Jurkat T cells or primary human monocyte-derived macrophages (MDMs). Murine embryonic fibroblasts derived from Anx2(-/-) mice produced the same levels of VLPs as matched cells from wild-type mice. The calcium-mediated spike in VLP production still occurred in Anx2-depleted COS-1 cells, and there was no apparent alteration in the intracellular Gag localization. Overexpression of Anx2 in trans had no effect on Gag processing or VLP production. Neither Anx2 depletion nor Anx2 overexpression altered the infectivity of HIV-1 particles produced by COS-1 or 293T cells. However, supernatants containing virus from Anx2 siRNA-treated primary human MDMs exhibited decreased infectivity. These data indicate that Anx2 is not required for HIV-1 assembly or Gag processing but rather plays a cell type-dependent role in regulating production of infectious HIV-1 by macrophages.

Signal transducer and activator of transcription 6 (STAT6) is a novel interactor of annexin A2 in prostate cancer cells.

Annexin A2 (AnxA2) is a multifunctional Ca(2+)-dependent phospholipid-binding protein, and its overexpression is implicated in malignant transformation of several cancers. In prostate cancer, however, the expression of AnxA2 is lost in prostate intraepithelial neoplasia and reappears in the high-grade tumors, suggesting a complex regulation of AnxA2 in the prostate microenvironment. Since a majority of the biological functions of AnxA2 are mediated by its interaction with other proteins, we performed a yeast two-hybrid assay to search for novel interactors of AnxA2. Our studies revealed that signal transducer and activator of transcription 6 (STAT6), a member of the STAT family of transcription factors, is a binding partner of AnxA2. We confirmed AnxA2-STAT6 interaction by in vitro co-immunoprecipitation and fluorescence resonance energy transfer (FRET) studies and demonstrated that AnxA2 interacts with phosphorylated STAT6. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed that AnxA2 is associated with the STAT6 DNA-binding complex, and luciferase reporter assays demonstrated that AnxA2 upregulates the activity of STAT6. Upon interleukin-4 treatment, AnxA2 stabilizes the cytosolic levels of phosphorylated STAT6 and promotes its nuclear entry. These findings suggest that AnxA2-STAT6 interactions could have potential implications in prostate cancer progression. This report is the first to demonstrate the interaction of AnxA2 with STAT6 and suggests a possible mechanism by which AnxA2 contributes to the metastatic processes of prostate cancer.

Homocysteine-mediated thrombosis and angiostasis in vascular pathobiology.

The mechanisms by which homocysteine contributes to atherothrombosis are complex and their in vivo relevance uncertain. In this issue of the JCI, Jacovina and colleagues report a unique in vivo mechanism by which homocysteine may contribute to vascular disease (see the related article beginning on page 3384). This group had previously reported that homocysteine impairs endothelial cell surface plasminogen activation by posttranslationally modifying annexin A2, the coreceptor for plasminogen and tissue plasminogen activator. They now show that an annexin A2-deficient mouse rendered hyperhomocysteinemic by dietary means has impaired fibrinolysis, perivascular fibrin persistence, and attenuated angiogenesis (angiostasis). Potential mechanisms by which homocysteine-dependent changes in endothelial phenotype link thrombosis to angiostasis are reviewed and their relationship to homocysteine-dependent vascular disease considered.