Annexin A2 and PSF proteins interact with p53 IRES and regulate translation of p53 mRNA

p53 mRNA has been shown to be translated into two isoforms, full-length p53 (FL-p53) and a truncated isoform Delta N-p53, which modulates the functions of FL-p53 and also has independent functions. Previously, we have shown that translation of p53 and Delta N-p53 can be initiated at Internal Ribosome Entry Sites (IRES). These two IRESs were shown to regulate the translation of p53 and Delta N-p53 in a distinct cell-cycle phase-dependent manner. Earlier observations from our laboratory also suggest that the structural integrity of the p53 RNA is critical for IRES function and is compromised by mutations that affect the structure as well as RNA protein interactions. In the current study, using RNA affinity approach we have identified Annexin A2 and PTB associated Splicing Factor (PSF/SFPQ) as novel ITAFs for p53 IRESs. We have showed that the purified Annexin A2 and PSF proteins specifically bind to p53 IRES elements. Interestingly, in the presence of calcium ions Annexin A2 showed increased binding with p53 IRES. Immunopulldown experiments suggest that these two proteins associate with p53 mRNA ex vivo as well. Partial knockdown of Annexin A2 and PSF showed decrease in p53 IRES activity and reduced levels of both the p53 isoforms. More importantly the interplay between Annexin A2, PSF and PTB proteins for binding to p53mRNA appears to play a crucial role in IRES function. Taken together, our observations suggest pivotal role of two new trans-acting factors in regulating the p53-IRES function, which in turn influences the synthesis of p53 isoforms.

Investigating the role of Annexin A2 in Epidermal Growth Factor (EGF) induced signalling in cancer

Dissertação de mestrado, Qualidade em Análises, Faculdade de Ciências e Tecnologia, Universidade do Algarve; Universitat de Barcelona; Gdansk University of Technology, Universidad de Cádiz, Universitas Bergensis; 2015

Reperfusion-induced myocardial dysfunction is prevented by endogenous annexin-A1 and its N-terminal-derived peptide Ac-ANX-A12-26

BACKGROUND AND PURPOSE : Annexin-A1 (ANX-A1) is an endogenous, glucocorticoid-regulated anti-inflammatory protein. The N-terminal-derived peptide Ac-ANX-A12–26 preserves cardiomyocyte viability, but the impact of ANX-A1-peptides on cardiac contractility is unknown. We now test the hypothesis that ANX-A1 preserves post-ischaemic recovery of left ventricular (LV) function.

EXPERIMENTAL APPROACH : Ac-ANX-A12–26 was administered on reperfusion, to adult rat cardiomyocytes as well as hearts isolated from rats, wild-type mice and mice deficient in endogenous ANX-A1 (ANX-A1–/–). Myocardial viability and recovery of LV function were determined.

KEY RESULTS: Ischaemia–reperfusion markedly impaired both cardiomyocyte viability and recovery of LV function by 60%. Treatment with exogenous Ac-ANX-A12–26 at the onset of reperfusion prevented cardiomyocyte injury and significantly improved recovery of LV function, in both intact rat and wild-type mouse hearts. Ac-ANX-A12–26 cardioprotection was abolished by either formyl peptide receptor (FPR)-nonselective or FPR1-selective antagonists, Boc2 and cyclosporin H, but was relatively insensitive to the FPR2-selective antagonist QuinC7. ANX-A1-induced cardioprotection was associated with increased phosphorylation of the cell survival kinase Akt. ANX-A1−/− exaggerated impairment of post-ischaemic recovery of LV function, in addition to selective LV FPR1 down-regulation.

CONCLUSIONS AND IMPLICATIONS : These data represent the first evidence that ANX-A1 affects myocardial function. Our findings suggest ANX-A1 is an endogenous regulator of post-ischaemic recovery of LV function. Furthermore, the ANX-A1-derived peptide Ac-ANX-A12–26 on reperfusion rescues LV function, probably via activation of FPR1. ANX-A1-based therapies may thus represent a novel clinical approach for the prevention and treatment of myocardial reperfusion injury.

Annexin 1 peptides protect against experimental myocardial ischemia-reperfusion: analysis of their mechanism of action

Myocardial reperfusion injury is associated with the infiltration of blood-borne polymorphonuclear leukocytes. We have previous described the protection afforded by annexin 1 (ANXA1) in an experimental model of rat myocardial ischemia-reperfusion (IR) injury. We examined the 1) amino acid region of ANXA1 that retained the protective effect in a model of rat heart IR; 2) changes in endogenous ANXA1 in relation to the IR induced damage and after pharmacological modulation; and 3) potential involvement of the formyl peptide receptor (FPR) in the protective action displayed by ANXA1 peptides. Administration of peptide Ac2-26 at 0, 30, and 60 min postreperfusion produced a significant protection against IR injury, and this was associated with reduced myeloperoxidase activity and IL-1 beta levels in the infarcted heart. Western blotting and electron microscopy analyses showed that IR heart had increased ANXA1 expression in the injured tissue, associated mainly with the infiltrated leukocytes. Finally, an antagonist to the FPR receptor selectively inhibited the protective action of peptide ANXA1 and its derived peptides against IR injury. Altogether, these data provide further insight into the protective effect of ANXA1 and its mimetics and a rationale for a clinical use for drugs developed from this line of research.

Time-dependent expression of annexin 1 in a model of chronic granulomatous inflammation

Objective and design: To determine the expression pattern and distribution of the glucocorticoid-inducible protein annexin 1 (ANXA1) in a murine model of chronic granulomatous inflammation.Materials or subjects: TO Mouse.Treatment: Chronic granulomatous inflammation was induced by injecting into dorsal sub-cutaneous air-pouches in mice, a mixture of croton oil and Freund's complete adjuvant (CO/FCA).Methods: Western and northern analysis, corticosterone assay, and immunohistochemistry. Statistical analysis was performed using ANOVA followed by Tukey's pair-wise comparisons or Dunnett's multiple comparisons.Results: ANXA1 protein levels changed significantly throughout the 4-week time course, with an initial peak at day 7 and a later elevation at 28 days. ANXA1 mRNA levels peaked at days 1 and 3, with a significant decline at day 7 followed by an upward trend to day 28. Plasma corticosterone measurements taken throughout the time course revealed an increase from 14 days onward, suggesting that corticosterone does not influence ANXA1 expression during the initial stages of the model. Immunogold staining revealed that ANXA1 expression in the inflamed tissue was mainly in extravasated neutrophils, with intact protein (37 kDa) being predominantly observed on the cell membrane.Conclusions: the pattern of ANXA1 expression indicates that infiltrated neutrophils are responsible for the majority of ANXA1 present both at early and later stages of this model of granulomatous inflammation.

Formyl-peptide receptor is not involved in the protection afforded by annexin 1 in murine acute myocardial infarct

Recent interest in the annexin 1 field has come from the notion that specific G-protein-coupled receptors, members of the formyl-peptide receptor (FPR) family, appear to mediate the anti-inflammatory actions of this endogenous mediator. Administration of the annexin 1 N-terminal derived peptide Ac2-26 to mice after 25 min ischemia significantly attenuated the extent of acute myocardial injury as assessed 60 min postreperfusion. Evident at the dose of 1 mg/kg (similar to9 nmol per animal), peptide Ac2-26 cardioprotection was intact in FPR null mice. Similarly, peptide Ac2-26 inhibition of specific markers of heart injury (specifically myeloperoxidase activity, CXC chemokine KC contents, and endogenous annexin 1 protein expression) was virtually identical in heart samples collected from wild-type and FPR null mice. Mouse myocardium expressed the mRNA for FPR and the structurally related lipoxin A(4) receptor, termed ALX; thus, comparable equimolar doses of two ALX agonists (W peptide and a stable lipoxin A4 analog) exerted cardioprotection in wild-type and FPR null mice to an equal extent. Curiously, marked (>95%) blood neutropenia produced by an anti-mouse neutrophil serum did not modify the extent of acute heart injury, whereas it prevented the protection afforded by peptide Ac2-26. Thus, this study sheds light on the receptor mechanism(s) mediating annexin 1-induced cardioprotection and shows a pivotal role for ALX and circulating neutrophil, whereas it excludes any functional involvement of mouse FPR. These mechanistic data can help in developing novel therapeutics for acute cardioprotection.

Spatial expression of two anti-inflammatory mediators, annexin 1 and galectin-1, in nasal polyposis

Background There is renewed interest in the role played by specific counter-regulatory mechanisms to control the inflammatory host response, poorly investigated in human pathology. Here, we monitored the expression of two anti-inflammatory mediators, annexin 1 and galectin-1, and assessed their potential link to glucocorticoids' (GCs) effective control of nasal polyposis (NP).Methods Total patterns of mRNA and protein expression were analysed by quantitative real-time PCR (qPCR) and Western blotting analyses, whereas ultrastructural immunocytochemistry was used for spatial localization and quantification of each mediator, focusing on mast cells, eosinophils and epithelial cells.Results Up-regulation of the annexin 1 gene, and down-regulation of galectin-1 gene, was detected in polypoid tissue compared with nasal mucosa. Patient treatment with betamethasone augmented galectin-1 protein expression in polyps. At the cellular level, control mast cells and eosinophils displayed higher annexin 1 expression, whereas marked galectin-1 immunolabelling was detected in the granule matrix of mast cells. Cells of glandular duct epithelium also displayed expression of both annexin 1 and galectin-1, augmented after treatment.Conclusion Mast cells and epithelial cells appeared to be pivotal cell types involved in the expression of both annexin 1 and galectin-1. It is possible that annexin 1 and galectin-1 could be functionally associated with a specific mechanism in NP and that GC exert at least part of their beneficial effects on the airway mucosa by up-regulating, in a specific cell target fashion, these anti-inflammatory agonists.

An immunocytochemical and in situ hybridization analysis of annexin 1 expression in rat mast cells: Modulation by inflammation and dexamethasone

The presence and localization of the anti-inflammatory protein annexin 1 (also known as lipocortin 1) in perivenular rat mast cells was investigated here. Using the rat mesenteric microvascular bed and a combination of morphologic techniques ranging from immunofluorescence to electron microscopy analyses, we detected the presence of annexin 1 in discrete intracellular sites, both in the nucleus and in the cytoplasm. In resting mast cells, most of the protein pool (approximately 80% of the cytosolic portion) was localized to cytoplasmic granules. In agreement with other cell types, treatment of rats with dexamethasone (0.2 mg/kg, ip) increased annexin 1 expression in mast cells, inducing a remarkable appearance of dusters of protein immunoreactivity. This effect was most likely the result of de novo protein synthesis as determined by an increase in mRNA seen by in situ hybridization. Triggering an ongoing experimental inflammatory response (0.3 mg of carrageenin, ip) increased annexin 1 mRNA and protein levels. In conclusion, we report for the first time the localization of annexin 1 in connective tissue mast cells, and its susceptibility not only to glucocorticoid hormone treatment, but also to an experimental acute inflammatory response.

Annexin 1 localisation in tissue eosinophils as detected by electron microscopy

BACKGROUND: Human and rodent leukocytes express high levels of the glucocorticoid-inducible protein annexin 1 ( ANXA1) ( previously referred to as lipocortin 1). Neutrophils and monocytes have abundant ANXA1 levels.Aim: We have investigated, for the first time, ANXA1 ultrastructural expression in rat eosinophils and compared it with that of extravasated neutrophils. The effect of inflammation ( carrageenin peritonitis) was also monitored.Methods: Electron microscopy was used to define the sub-cellular localisation of ANXA1 in rat eosinophils and neutrophils extravasated in the mesenteric tissue. A pair of antibodies raised against the ANXA1 N-terminus (i.e. able to recognise intact ANXA1, termed LCPS1) or the whole protein ( termed LCS3) was used to perform the ultrastructural analysis.Results: the majority of ANXA1 was localised in the eosinophil cytosol (similar to 60%) and nucleus (30-40%), whereas a small percentage was found on the plasma membrane (< 10%). Within the cytosol, the protein was equally distributed in the matrix and in the granules, including those containing the typical crystalloid. The two anti-ANXA1 antibodies gave similar results, with the exception that LCPS1 gave a lower degree of immunoreactivity in the plasma membrane. Inflammation (i.e. carrageenin injection) produced a modest increase in eosinophil-associated ANXA1 reactivity ( significant only in the cytoplasm compartment). Extravasated neutrophils, used for comparative purposes, displayed a much higher degree of immunoreactivity for the protein.Conclusion: We describe for the first time ANXA1 distribution in rat eosinophil by ultrastructural analysis, and report a different protein mobilisation from extravasated neutrophils, at least in this acute model of peritonitis.