Inhibition of Androgen Receptor Signaling Promotes Prostate Cancer Cell Migration via Upregulation of Annexin A1 Expression.

BACKGROUND: Recent studies indicate that androgen deprivation therapy (ADT), the main therapeutic approach for metastatic prostate cancer (PCa), accelerates PCa invasion and metastasis. Annexin A1 (ANXA1) is a Ca2+-regulated phospholipid-binding protein that can promote PCa migration and invasion. AIM OF THE STUDY: The aim of this study is to determine whether ANXA1 is regulated by ADT and participates in PCa progression after ADT, and to explore the possible mechanism of ANXA1-mediated PCa migration. METHODS: Expression of ANXA1 and androgen receptor (AR) in PCa cell lines and tissues was detected, and the association between these two proteins were analyzed. Expression of ANXA1 was evaluated after AR knockdown or AR inhibition in PCa cell lines. Cell migration of PCa cell liness after ANXA1 knockdown or overexpression was determined by in vitro migration assay. Transcriptome analysis was used to explore the possible mechanism of ANXA1-mediated PCa migration. RESULTS: ANXA1 expression in PCa cell lines and tissues was reversely associated with AR. In vitro studies revealed an increase in ANXA1 expression after AR knockdown or treatment with AR antagonist. Moreover, functional assays indicated that ANXA1 knockdown in PCa cells significantly inhibited cell migration, while ANXA1 overexpression in PCa cells significantly accelerated cell migration. Transcriptome analysis showed that ANXA1 regulated multiple genes involved in cell junction organization, such as CADM1, LIMCH1 and PPM1F. CONCLUSIONS: Our results indicate that ADT might accelerate PCa metastasis via ANXA1 expression and PCa cell migration.

Expression of the Annexin A1 and its correlation with matrix metalloproteinases and the receptor for formylated peptide-2 in diffuse astrocytic tumors.

Astrocytomas represent the majority of cerebral gliomas. Studies show that the anti-inflammatory protein Annexin-A1 (ANXA1) is associated with the tumor invasion process and that its actions can be mediated by the receptor for formylated peptides (FPR). Therefore, we evaluated the expression of ANXA1, the receptor FPR2 and matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) in brain astrocytomas. Detection of proteins was performed in sections of diffuse astrocytomas (grade II), anaplastic astrocytomas (grade III) and glioblastomas (GBM, grade IV) and quantifications were made by densitometry. Our analyses showed increased expression of ANXA1 in astrocytomas of all grades, but especially in GBM. The expression of FPR2 is similar to that found for ANXA1, being higher in GBM. Immunostaining for MMPs is also stronger as the degree of malignancy increases, especially with respect to MMP-9. The positive correlation between ANXA1/FPR2 and ANXA1/MMP-9 was observed in all tumors studied. The data indicate the possible action of ANXA1 and FPR2 on the development and progression of astrocytomas, related to increased expression of MMP-9. Thereby, ANXA1 and FPR2 are involved in the biology and malignancy of diffuse astrocytic tumors.

Mast cell heterogeneity and anti-inflammatory annexin A1 expression in leprosy skin lesions.

Mast cells (MCs) have important immunoregulatory roles in skin inflammation. Annexin A1 (ANXA1) is an endogenous anti-inflammatory protein that can be expressed by mast cells, neutrophils, eosinophils, monocytes, epithelial and T cells. This study investigated MCs heterogeneity and ANXA1 expression in human dermatoses with special emphasis in leprosy. Sixty one skin biopsies from 2 groups were investigated: 40 newly diagnosed untreated leprosy patients (18 reaction-free, 11 type 1 reaction/T1R, 11 type 2 reaction/T2R); 21 patients with other dermatoses. Tryptase/try+ and chymase/chy + phenotypic markers and toluidine blue stained intact/degranulated MC counts/mm2 were evaluated. Try+/chy+ MCs and ANXA1 were identified by streptavidin-biotin-peroxidase immunostaining and density was reported. In leprosy, degranulated MCs outnumbered intact ones regardless of the leprosy form (from tuberculoid/TT to lepromatous/LL), leprosy reactions (reactional/reaction-free) and type of reaction (T1R/T2R). Compared to other dermatoses, leprosy skin lesions showed lower numbers of degranulated and intact MCs. Try+ MCs outnumbered chy+ in leprosy lesions (reaction-free/reactional, particularly in T2R), but not in other dermatoses. Compared to other dermatoses, ANXA1 expression, which is also expressed in mast cells, was higher in the epidermis of leprosy skin lesions, independently of reactional episode. In leprosy, higher MC degranulation and differential expression of try+/chy+ subsets independent of leprosy type and reaction suggest that the Mycobacterium leprae infection itself dictates the inflammatory MCs activation in skin lesions. Higher expression of ANXA1 in leprosy suggests its potential anti-inflammatory role to maintain homeostasis preventing tissue and nerve damage.

Unravelling Pathophysiology of Crystalline Nephropathy in Ceftriaxone-Associated Acute Kidney Injury: A Cellular Proteomic Approach.

BACKGROUND: Previous studies showed that ceftriaxone can cause acute kidney injury (AKI) in the pediatric population. This study proposed a cellular model of crystalline nephropathy in ceftriaxone-associated AKI and explored the related pathophysiology by using a proteomic approach. METHODS: Ceftriaxone was crystallized with calcium in artificial urine. Madin-Darby Canine Kidney (MDCK) cells, a model of distal renal tubular cell, were cultured in the absence (untreated control) or presence of ceftriaxone crystals for 48-h (n = 5 each). MDCK cells were harvested and subsequently analyzed by proteomic analysis. Protein bioinformatics (i.e., STRING and Reactome) was used to predict functional alterations, and subsequently validated by Western blotting and cellular studies. p < 0.05 was considered statistically significant. RESULTS: Phase-contrast microscopy showed increased intracellular vesiculation and cell enlargement as a result of ceftriaxone crystal exposure. Proteome analysis revealed a total of 20 altered proteins (14 increased, 5 decreased and 1 absent) in ceftriaxone crystal-treated MDCK cells as compared to untreated cells (p < 0.05). Protein bioinformatics and validation studies supported heat stress response mediated by heat shock protein 70 (Hsp70) and downregulation of annexin A1 as the proposed pathophysiology of crystalline nephropathy in ceftriaxone-associated AKI, in which impaired proliferation and wound healing of crystal-induced distal tubular cells were outcomes. CONCLUSIONS: This study, for the first time, used the in vitro model of crystalline nephropathy to investigate the underlying pathophysiology of ceftriaxone-associated AKI, which should be investigated in vivo for potential clinical benefits in the future.

Upregulation of annexin A1 expression by butyrate in human melanoma cells induces invasion by inhibiting E-cadherin expression.

Epithelial to mesenchymal transition (EMT) is a critical step in the metastasis of epithelial cancer cells. Butyrate, which is produced from dietary fiber by colonic bacterial fermentation, has been reported to influence EMT. However, some studies have reported that butyrate promotes EMT, while others have reported an inhibitory effect. To clarify these controversial results, it is necessary to elucidate the mechanism by which butyrate can influence EMT. In this study, we examined the potential role of annexin A1 (ANXA1), which was previously reported to promote EMT in breast cancer cells, as a mediator of EMT regulation by butyrate. We found that ANXA1 mRNA and protein were expressed in highly invasive melanoma cell lines (A2058 and A375), but not in SK-MEL-5 cells, which are less invasive. We also showed that butyrate induced ANXA1 mRNA and protein expression and promoted EMT-related cell invasion in SK-MEL-5 cells. Downregulation of ANXA1 expression using specific small interfering RNAs in butyrate-treated SK-MEL-5 cells resulted in increased expression of the epithelial marker E-cadherin and decreased cell invasion. Moreover, overexpressing ANXA1 decreased the expression of the E-cadherin. Collectively, these results indicate that butyrate induces the expression of ANXA1 in human melanoma cells, which then promotes invasion through activating the EMT signaling pathway.

Divergent Annexin A1 expression in periphery and gut is associated with systemic immune activation and impaired gut immune response during SIV infection.

HIV-1 disease progression is paradoxically characterized by systemic chronic immune activation and gut mucosal immune dysfunction, which is not fully defined. Annexin A1 (ANXA1), an inflammation modulator, is a potential link between systemic inflammation and gut immune dysfunction during the simian immunodeficiency virus (SIV) infection. Gene expression of ANXA1 and cytokines were assessed in therapy-naïve rhesus macaques during early and chronic stages of SIV infection and compared with SIV-negative controls. ANXA1 expression was suppressed in the gut but systemically increased during early infection. Conversely, ANXA1 expression increased in both compartments during chronic infection. ANXA1 expression in peripheral blood was positively correlated with HLA-DR+CD4+ and CD8+ T-cell frequencies, and negatively associated with the expression of pro-inflammatory cytokines and CCR5. In contrast, the gut mucosa presented an anergic cytokine profile in relation to ANXA1 expression. In vitro stimulations with ANXA1 peptide resulted in decreased inflammatory response in PBMC but increased activation of gut lymphocytes. Our findings suggest that ANXA1 signaling is dysfunctional in SIV infection, and may contribute to chronic inflammation in periphery and with immune dysfunction in the gut mucosa. Thus, ANXA1 signaling may be a novel therapeutic target for the resolution of immune dysfunction in HIV infection.

The ANXA1 released from intestinal epithelial cells alleviate DSS-induced colitis by improving NKG2A expression of Natural Killer cells.

Inflammatory bowel disease (IBD) arises when intestinal immune homeostasis is broken, the maintenance of such homeostasis is principally controlled by cross talk between commensal bacteria, mucosal immune cells and intestinal epithelial cells (IECs). IECs can prevent the contact between luminal bacteria with immune cells through the formation of a physical barrier and the expression of antimicrobial peptides to maintain intestinal immune homeostasis. During Colitis the IECs can express increased ANXA1, which is important for regeneration of intestinal mucosa and function as a potent anti-inflammatory protein. Natural Killer (NK) cells can also suppress the progression of colitis. It is uncertain about the effect of the cross-talk between injured IECs and recruited NK cells during colitis. In this study, the expression of ANXA1 in IECS from DSS treated mice was increased, and more NK cells were recruited to intestinal mucosa. In addition, the expression of NKG2A was upregulated when co-cultured with NK cells. The results further proved that overexpression of NKG2A in NK cells was important for inhibiting the recruitment and activity of neutrophils to alleviate DSS-induced colitis. Here, we provide a new anti-inflammation mechanism about ANXA1 secreted from injured IECs, where ANXA1 can stimulate the expression of NKG2A in NK cells that affect the recruitment and activity of neutrophils necessary for pathology of colitis.

The Role and Effects of ANXA1 in Temporal Lobe Epilepsy: A Protection Mechanism?

BACKGROUND The endogenous protein annexin A1 (ANXA1) is an anti-inflammatory mediator in the brain that is thought to contribute to the progression of many neurological conditions. However, its exact role in temporal lobe epilepsy (TLE) remains unclear. We hypothesized that ANXA1 exerts negative actions on TLE by alleviating inflammatory damage in neurons. To identify the potential mechanism of TLE by assessing ANXA1 expression in TLE rats. MATERIAL AND METHODS TLE was induced in rats (n=70) via an intraperitoneal injection of lithium chloride (LiCl) and pilocarpine (PILO). The control group (n=10) received an injection of the equivalent amount of saline. ANXA1 expression was detected via immunohistochemistry and Western blotting. RESULTS Successful establishment of the TLE model in rats resulted in epileptic seizures. ANXA1 was immunohistochemically detected as brownish yellow particles in the dentate gyrus and the CA1 region of the door zone; this expression was predominantly localized to the cytoplasm of glia rather than neurons. ANXA1 expression was stronger in TLE rats compared with the control group. ANXA1 expression in TLE was also assessed via Western blotting, and compared between groups at various time points. ANXA1 expression was significantly increased in the acute (the first 24 h) and chronic (after 1 month) phases (P<0.001) but significantly decreased during the recovery phase (72 h, 1 week, and 2 weeks) (P<0.001). These findings suggest that ANXA1 expression is correlated with TLE activity. CONCLUSIONS Our data suggest that ANXA1 plays an important role in TLE by alleviating inflammatory damage and protecting neurons.

An update on Anti-inflammatory Compounds: A Review.

Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process. Also, it has been reported to be associated with the onset of various cancers. An effective anti-inflammatory drug should be able to inhibit the development of inflammation without interfering in normal homeostasis. Current approaches to overcome the inflammation include the use of immune selective anti-inflammatory derivatives, selective glucocorticoid receptor agonist, resolvins and protectins and TNF inhibitors. A number of herbal drugs have been identified in the past that can target inflammatory cytokines. This review mainly focuses on the newer molecules to combat the inflammation and also emphasise on various studies carried out in the past. Thus, the high prevalence of inflammation obliges the development of new drugs; therefore, a safe and efficient drug molecule to confer protection against inflammation is urgently needed.

Upregulated Annexin A1 promotes cellular invasion in triple-negative breast cancer.

Annexin A1 (ANXA1) is a calcium-dependent phospholipid-linked protein, involved in anti-inflammatory effects, regulation of cellular differentiation, proliferation and apoptosis. While many studies have investigated the ANXA1 expression in various tumor types, the role of ANXA1 is not fully understood. Therefore, in the present study, we evaluated the ANXA1 expression in 211 breast cancer patients and compared the levels with clinicopathological factors. ANXA1 was positively expressed in 31 (14.7%) of the 211 cases in our cohort, and these positive cases were associated with triple-negative breast cancer (TNBC) (P=0.007) and venous invasion (P=0.028). The in vitro cell experiment found that the MDA-MB-231 cell line, which is a TNBC cell line, highly expressed ANXA1. Using this cell line, the functional role of ANXA1 in breast cancer was revealed and the knockdown of ANXA1 by specific siRNA demonstrated a significant reduction in cellular invasion. Further experiments indicated that ANXA1 was induced by hypoxia with hypoxia-inducible factor-1α induction. These results suggested that ANXA1, which enhanced breast cancer invasion and metastasis under hypoxia, were significantly associated with the worst patient outcome. This is particularly noted in TNBC, the group of breast cancer with the worst outcome for which new therapeutic implications are required.

Down-regulation of Ras-related protein Rab 5C-dependent endocytosis and glycolysis in cisplatin-resistant ovarian cancer cell lines.

Drug resistance poses a major challenge to ovarian cancer treatment. Understanding mechanisms of drug resistance is important for finding new therapeutic targets. In the present work, a cisplatin-resistant ovarian cancer cell line A2780-DR was established with a resistance index of 6.64. The cellular accumulation of cisplatin was significantly reduced in A2780-DR cells as compared with A2780 cells consistent with the general character of drug resistance. Quantitative proteomic analysis identified 340 differentially expressed proteins between A2780 and A2780-DR cells, which involve in diverse cellular processes, including metabolic process, cellular component biogenesis, cellular processes, and stress responses. Expression levels of Ras-related proteins Rab 5C and Rab 11B in A2780-DR cells were lower than those in A2780 cells as confirmed by real-time quantitative PCR and Western blotting. The short hairpin (sh)RNA-mediated knockdown of Rab 5C in A2780 cells resulted in markedly increased resistance to cisplatin whereas overexpression of Rab 5C in A2780-DR cells increases sensitivity to cisplatin, demonstrating that Rab 5C-dependent endocytosis plays an important role in cisplatin resistance. Our results also showed that expressions of glycolytic enzymes pyruvate kinase, glucose-6-phosphate isomerase, fructose-bisphosphate aldolase, lactate dehydrogenase, and phosphoglycerate kinase 1 were down-regulated in drug resistant cells, indicating drug resistance in ovarian cancer is directly associated with a decrease in glycolysis. Furthermore, it was found that glutathione reductase were up-regulated in A2780-DR, whereas vimentin, HSP90, and Annexin A1 and A2 were down-regulated. Taken together, our results suggest that drug resistance in ovarian cancer cell line A2780 is caused by multifactorial traits, including the down-regulation of Rab 5C-dependent endocytosis of cisplatin, glycolytic enzymes, and vimentin, and up-regulation of antioxidant proteins, suggesting Rab 5C is a potential target for treatment of drug-resistant ovarian cancer. This constitutes a further step toward a comprehensive understanding of drug resistance in ovarian cancer.

Regulatory mechanisms of annexin-induced chemotherapy resistance in cisplatin resistant lung adenocarcinoma.

Adenocarcinoma of lung has high incidence and a poor prognosis, woith chemotherapy as the main therapeutic tool, most commonly with cisplatin. However, chemotherapy resistance develops in the majority of patients during clinic treatment. Mechanisms of resistance are complex and still unclear. Although annexin play important roles in various tumor resistance mechanisms, their actions in cisplatin-resistant lung adenocarcinoma remain unclear. Preliminary studies by our group found that in cisplatin-resistant lung cancer A549 cells and lung adenocarcinoma tissues, both mRNA and protein expression of annexins A1, A2 and A3 is increased. Using a library of annexin A1, A2 and A3 targeting combined molecules already established by ourselves we found that specific targeting decreased cisplatin-resistance. Taken together, the underlined effects of annexins A1, A2 and A3 on drug resistance and suggest molecular mechanisms in cisplatin-resistant A549 cells both in vivo and in vitro. Furthermore, the study points to improved research on occurrence and development of lung adenocarcinoma, with provision of effective targets and programmes for lung adenocarcinoma therapy in the clinic.

Analysis of lymphocytes in patients with Plasmodium vivax malaria and its relation to the annexin-A1 and IL-10.

BACKGROUND: Malaria is the most prevalent parasitic disease in the world. In Brazil, the largest number of malaria cases (98%) is within the Legal Amazon region, where Plasmodium vivax is responsible for over 80% of diagnosed cases. The aim of this study was to investigate the annexin-A1 expression in CD4+, CD8+ T cells, regulatory T cells (Treg) and cytokine IL-10 quantification in plasma from patients with malaria caused by P. vivax. METHODS: The quantification of the cytokine IL-10 of patients infected with P. vivax and healthy controls were evaluated by enzyme-linked immunosorbent assay (ELISA). The determination of the expression of annexin-A1 in lymphocytes from patients and healthy controls was determined by immunofluorescence staining. All results were correlated with the parasitaemia and the number of previous episodes of malaria. RESULTS: The cytokine IL-10 plasma levels showed a significant increase in both patients with low (650.4 ± 59.3 pg/mL) and high (2870 ± 185.3 pg/mL) parasitaemia compared to the control (326.1 ± 40.1 pg/mL). In addition, there was an increase of this cytokine in an episode dependent manner (individuals with no previous episodes of malaria--primoinfected: 363.9 ± 31.1 pg/mL; individuals with prior exposure: 659.9 ± 49.4 pg/mL). The quantification of annexin-A1 expression indicated a decrease in CD4+ and CD8+ T cells and an increase in Treg in comparison with the control group. When annexin-A1 expression was compared according to the number of previous episodes of malaria, patients who have been exposed more than once to the parasite was found to have higher levels of CD4+ T cells (96.0 ± 2.5 A.U) compared to primoinfected (50.3 ± 1.7). However, this endogenous protein had higher levels in CD8+ (108.5 ± 3.1) and Treg (87.5 ± 2.5) from patients primoinfected. CONCLUSION: This study demonstrates that in the patients infected with P. vivax the release of immunoregulatory molecules can be influenced by the parasitaemia level and the number of previous episodes of malaria. annexin-A1 is expressed differently in lymphocyte sub-populations and may have a role in cell proliferation. Furthermore, annexin-A1 may be contributing to IL-10 release in plasma of patients with vivax malaria.

Neonatal hyperoxic exposure persistently alters lung secretoglobins and annexin A1.

Altered functions of the lung epithelial surface likely contribute to the respiratory morbidities in infants with bronchopulmonary dysplasia (BPD). Infants with BPD exhibit decreased expressions of secretoglobins (SCGBs), including Clara cell secretory protein (CCSP). Expression of lung SCGB and annexin A1 (ANXA1) is persistently altered in CCSP knockout mice suggesting that CCSP indirectly influences innate immune responses. The present studies tested the hypothesis that neonatal hyperoxic exposure induces deficits in CCSP expression that are associated with persistent alterations in lung SCGB and ANXA1 expression. Newborn C3H/HeN mice were exposed to room air (RA) or 85% O2 from birth and were sacrificed at 14 d or returned to RA for 14 d. Neonatal hyperoxia followed by RA recovery was associated with decreased lung CCSP and SCGB3A1 protein but not mRNA expression. Hyperoxia-induced alterations in the charge characteristics of ANXA1 were unchanged by RA recovery and were associated with elevated lung macrophage numbers. These findings support a model in which hyperoxia-induced alterations in Clara cell function influence lung innate immune function through effects on immunomodulatory proteins. Studies to determine the mechanism(s) by which CCSP alterations affect SCGBs, ANXA1, and innate immune responses in BPD are warranted.

Annexin-A1 regulates TLR-mediated IFN-ß production through an interaction with TANK-binding kinase 1.

TLRs play a pivotal role in the recognition of bacteria and viruses. Members of the family recognize specific pathogen sequences to trigger both MyD88 and TRIF-dependent pathways to stimulate a plethora of cells. Aberrant activation of these pathways is known to play a critical role in the development of autoimmunity and cancer. However, how these pathways are entirely regulated is not fully understood. In these studies, we have identified Annexin-A1 (ANXA1) as a novel regulator of TLR-induced IFN-ß and CXCL10 production. We demonstrate that in the absence of ANXA1, mice produce significantly less IFN-ß and CXCL10, and macrophages and plasmacytoid dendritic cells have a deficiency in activation following polyinosinic:polycytidylic acid administration in vivo. Furthermore, a deficiency in activation is observed in macrophages after LPS and polyinosinic:polycytidylic acid in vitro. In keeping with these findings, overexpression of ANXA1 resulted in enhanced IFN-ß and IFN-stimulated responsive element promoter activity, whereas silencing of ANXA1 impaired TLR3- and TLR4-induced IFN-ß and IFN-stimulated responsive element activation. In addition, we show that the C terminus of ANXA1 directly associates with TANK-binding kinase 1 to regulate IFN regulatory factor 3 translocation and phosphorylation. Our findings demonstrate that ANXA1 plays an important role in TLR activation, leading to an augmentation in the type 1 IFN antiviral cytokine response.

Low Annexin A1 expression predicts benefit from induction chemotherapy in oral cancer patients with moderate or poor pathologic differentiation grade.

BACKGROUND: The benefit of induction chemotherapy in locally advanced oral squamous cell carcinoma (OSCC) remains to be clearly defined. Induction chemotherapy is likely to be effective for biologically distinct subgroups of patients and biomarker development might lead to identification of the patients whose tumors are to respond to a particular treatment. Annexin A1 may serve as a biomarker for responsiveness to induction chemotherapy. The aim of this study was to investigate Annexin A1 expression in pre-treatment biopsies from a cohort of OSCC patients treated with surgery and post-operative radiotherapy or docetaxel, cisplatin and 5-fluorouracil (TPF) induction chemotherapy followed by surgery and post-operative radiotherapy. Furthermore we sought to assess the utility of Annexin A1 as a prognostic or predictive biomarker. METHODS: Immunohistochemical staining for Annexin A1 was performed in pre-treatment biopsies from 232 of 256 clinical stage III/IVA OSCC patients. Annexin A1 index was estimated as the proportion of tumor cells (low and high, <50% and ≥50% of stained cells, respectively) to Annexin A1 cellular membrane and cytoplasm staining. RESULTS: There was a significant correlation between Annexin A1 expression and pathologic differentiation grade (P=0.015) in OSCC patients. The proportion of patients with low Annexin A1 expression was significantly higher amongst those with moderate/poorly differentiated tumor (78/167) compared to those with well differentiated tumor (18/65). Multivariate Cox model analysis showed clinical stage (P=0.001) and Annexin A1 expression (P=0.038) as independent prognostic risk factors. Furthermore, a low Annexin A1 expression level was predictive of longer disease-free survival (P=0.036, HR=0.620) and locoregional recurrence-free survival (P=0.031, HR=0.607) compared to high Annexin A1 expression. Patients with moderate/poorly differentiated tumor and low Annexin A1 expression benefited from TPF induction chemotherapy as measured by distant metastasis-free survival (P=0.048, HR=0.373) as well as overall survival (P=0.078, HR=0.410). CONCLUSIONS: Annexin A1 can be used as a prognostic biomarker for OSCC. Patients with moderate/poorly differentiated OSCC and low Annexin A1 expression can benefit from the addition of TPF induction chemotherapy to surgery and post-operative radiotherapy. Annexin A1 expression can potentially be used as a predictive biomarker to select OSCC patients with moderate/poorly differentiated tumor who may benefit from TPF induction chemotherapy.

Macrophage migration inhibitory factor counter-regulates dexamethasone-induced annexin 1 expression and influences the release of eicosanoids in murine macrophages.

Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine and glucocorticoid (GC) counter-regulator, has emerged as an important modulator of inflammatory responses. However, the molecular mechanisms of MIF counter-regulation of GC still remain incomplete. In the present study, we investigated whether MIF mediated the counter-regulation of the anti-inflammatory effect of GC by affecting annexin 1 in RAW 264.7 macrophages. We found that stimulation of RAW 264.7 macrophages with lipopolysaccharide (LPS) resulted in down-regulation of annexin 1, while GC dexamethasone (Dex) or Dex plus LPS led to significant up-regulation of annexin 1 expression. RNA interference-mediated knockdown of intracellular MIF increased annexin 1 expression with or without incubation of Dex, whereas Dex-induced annexin 1 expression was counter-regulated by the exogenous application of recombinant MIF. Moreover, recombinant MIF counter-regulated, in a dose-dependent manner, inhibition of cytosolic phospholipase A2α (cPLA2α) activation and prostaglandin E2 (PGE2 ) and leukotriene B4 (LTB4 ) release by Dex in RAW 264.7 macrophages stimulated with LPS. Endogenous depletion of MIF enhanced the effects of Dex, reflected by further decease of cPLA2α expression and lower PGE2 and LTB4 release in RAW 264.7 macrophages. Based on these data, we suggest that MIF counter-regulates Dex-induced annexin 1 expression, further influencing the activation of cPLA2α and the release of eicosanoids. These findings will add new insights into the mechanisms of MIF counter-regulation of GC.

Annexin A1 is a biomarker of T-tubular repair in skeletal muscle of nonmyopathic patients undergoing statin therapy.

Skeletal muscle complaints are a common consequence of cholesterol-lowering therapy. Transverse tubular (T-tubular) vacuolations occur in patients having statin-associated myopathy and, to a lesser extent, in statin-treated patients without myopathy. We have investigated quantitative changes in T-tubular morphology and looked for early indicators of T-tubular membrane repair in skeletal muscle biopsy samples from patients receiving cholesterol-lowering therapy who do not have myopathic side effects. Gene expression and protein levels of incipient membrane repair proteins were monitored in patients who tolerated statin treatment without myopathy and in statin-naive subjects. In addition, morphometry of the T-tubular system was performed. Only the gene expression for annexin A1 was up-regulated, whereas the expression of other repair genes remained unchanged. However, annexin A1 and dysferlin protein levels were significantly increased. In statin-treated patients, the volume fraction of the T-tubular system was significantly increased, but the volume fraction of the sarcoplasmic reticulum remained unchanged. A complex surface structure in combination with high mechanical loads makes skeletal muscle plasma membranes susceptible to injury. Ca(2+)-dependent membrane repair proteins such as dysferlin and annexin A1 are deployed at T-tubular sites. The up-regulation of annexin A1 gene expression and protein points to this protein as a biomarker for T-tubular repair.

Annexin A1 mediates the anti-adhesive effects of the dexamethasone-treated promyelocytic leukemic cells.

Annexin A1 (AnxA1) is an important anti-inflammatory mediator during granulocytic differentiation in all trans-retinoic acid (ATRA) treated acute promyelocytic leukemic (APL) cells. Dexamethasone has been used successfully to prevent complications in ATRA-treated APL patients, although its mechanism of action is still not clear. In the present study, we have examined the effect of dexamethasone on the modulation of AnxA1 in ATRA-APL NB4 (ATRA-NB4) cells, ATRA-NB4 cells-derived microparticles (MPs) and its role during cell-cell interaction between ATRA-NB4 cells and endothelial cells. Our results have shown that dexamethasone can inhibit the percentage of ATRA-NB4 cells expressing surface AnxA1 and its receptor FPR2/ALX in a time-dependent manner based on flow cytometric analysis. However, dexamethasone treatment of ATRA-NB4 cells has no significant effect on the level of AnxA1 mRNA, the total cellular level of AnxA1 protein or the release of AnxA1 from these cells, as determined by RT-PCR, Western blotting, and ELISA, respectively. Further studies demonstrate that dexamethasone is able to significantly inhibit the adhesion of ATRA-NB4 cells to endothelial cells, and this anti-adhesive effect can be inhibited if the cells were pre-treated with a neutralizing antibody specific for AnxA1. Finally, dexamethasone also enhances the release of AnxA1-containing MPs from ATRA-NB4 cells which can in turn prevent the adhesion of the ATRA-NB4 cells to endothelial cells. We conclude that biologically active AnxA1 originating from dexamethasone-treated ATRA-APL cells and their MPs plays an anti-adhesive effect and this contributes to inhibit the adhesion of ATRA-APL cell to endothelial cells.