Ruolo dell'annessina A1 nel processo di differenziamento muscolare

2008 - 2009Annessina A1 (ANXA1, lipocortina 1) è il primo membro caratterizzato della superfamiglia delle annessine, proteine così chiamate per la loro proprietà di legare (to annex) le membrane cellulari in maniera Ca2+-dipendente. ANXA1 è coinvolta in una vasta gamma di processi molecolari e cellulari, come le vie di segnale anti-infiammatorie, le attività chinasiche nelle vie di trasduzione del segnale, il mantenimento dell‟integrità del citoscheletro e della matrice extracellulare, la crescita cellulare, l‟apoptosi e il differenziamento. In base a queste evidenze, abbiamo analizzato i profili di espressione e il ruolo dell‟annessina A1 in un modello cellulare in vitro, la linea di mioblasti murini C2C12 e in un modello in vivo, i topi transgenici Tg:Pax7nGFP. I risultati mostrano che l‟espressione di ANXA1 aumenta durante il differenziamento e che l‟inibizione dell‟espressione della proteina riduce significativamente il processo di differenziamento. ANXA1 è espressa in vivo in cellule satellite quiescenti e attivate, mentre la sua espressione aumenta solo in cellule satellite attivate. ANXA1, inoltre, si localizza principalmente nelle cellule muscolari che migrano nel lume delle fibre muscolari in rigenerazione in seguito a danno muscolare acuto. A 3 giorni dall‟induzione del differenziamento, ANXA1 trasloca alla membrana cellulare delle cellule C2C12 ed è secreta in ambienti extracellulari in C2C12 a 5 giorni di differenziamento. L‟ANXA1 endogena co-localizza con le fibre di actina nelle protrusioni della membrana plasmatica di cellule muscolari non differenziate ma non di quelle differenziate suggerendo un ruolo per la proteina nei processi di migrazione probabilmente mediante attivazione del recettore FPR. Un anticorpo neutralizzante ANXA1, inoltre, riduce l‟espressione della proteina muscolo-specifica MyHC, riduce la formazione dei miotubi e inibisce significativamente la migrazione dei mioblasti murini C2C12. In un sistema ex vivo di fibre muscolari isolate, ANXA1 sembra localizzarsi principalmente in prossimità delle membrane cellulari di cellule Pax7-positive mentre un anticorpo neutralizzante ANXA1 sembra inibire la migrazione di cellule satellite attivate. I dati ottenuti durante questo progetto di dottorato suggeriscono per la prima volta che ANXA1 ha un ruolo nel processo di differenziamento muscolare come dimostrato in altri modelli e tessuti. Regolando la migrazione delle cellule satellite, ANXA1 potrebbe contribuire alla rigenerazione del tessuto muscolo-scheletrico. [a cura dell'autore]VIII n.s

Role of intracellular and extracellular annexin A1 in migration and invasion of human pancreatic carcinoma cells

Background: Annexin A1 (ANXA1), a 37 kDa multifunctional protein, is over-expressed in tissues from patients of pancreatic carcinoma (PC) where the protein seems to be associated with malignant transformation and poor prognosis. Methods: The expression and localization of ANXA1 in MIA PaCa-2, PANC-1, BxPC-3 and CAPAN-2 cells were detected by Western Blotting and Immunofluorescence assay. Expression and activation of Formyl Peptide Receptors (FPRs) were shown through flow cytometry/PCR and FURA assay, respectively. To investigate the role of ANXA1 in PC cell migration and invasion, we performed in vitro wound-healing and matrigel invasion assays. Results: In all the analyzed PC cell lines, a huge expression and a variable localization of ANXA1 in sub-cellular compartments were observed. We confirmed the less aggressive phenotype of BxPC-3 and CAPAN-2 compared with PANC-1 and MIA PaCa-2 cells, through the evaluation of Epithelial-Mesenchymal Transition (EMT) markers. Then, we tested MIA PaCa-2 and PANC-1 cell migration and invasiveness rate which was inhibited by specific ANXA1 siRNAs. Both the cell lines expressed FPR-1 and -2. Ac2-26, an ANXA1 mimetic peptide, induced intracellular calcium release, consistent with FPR activation, and significantly increased cell migration/invasion rate. Interestingly, in MIA PaCa-2 cells we found a cleaved form of ANXA1 (33 kDa) that localizes at cellular membranes and is secreted outside the cells, as confirmed by MS analysis. The importance of the secreted form of ANXA1 in cellular motility was confirmed by the administration of ANXA1 blocking antibody that inhibited migration and invasion rate in MIA PaCa-2 but not in PANC-1 cells that lack the 33 kDa ANXA1 form and show a lower degree of invasiveness. Finally, the treatment of PANC-1 cells with MIA PaCa-2 supernatants significantly increased the migration rate of these cells. Conclusion: This study provides new insights on the role of ANXA1 protein in PC progression. Our findings suggest that ANXA1 protein could regulate metastasis by favouring cell migration/invasion intracellularly, as cytoskeleton remodelling factor, and extracellularly like FPR ligand

Common variants in Alzheimer’s disease and risk stratification by polygenic risk scores

Funder: Funder: Fundación bancaria ‘La Caixa’ Number: LCF/PR/PR16/51110003 Funder: Grifols SA Number: LCF/PR/PR16/51110003 Funder: European Union/EFPIA Innovative Medicines Initiative Joint Number: 115975 Funder: JPco-fuND FP-829-029 Number: 733051061Genetic discoveries of Alzheimer's disease are the drivers of our understanding, and together with polygenetic risk stratification can contribute towards planning of feasible and efficient preventive and curative clinical trials. We first perform a large genetic association study by merging all available case-control datasets and by-proxy study results (discovery n = 409,435 and validation size n = 58,190). Here, we add six variants associated with Alzheimer's disease risk (near APP, CHRNE, PRKD3/NDUFAF7, PLCG2 and two exonic variants in the SHARPIN gene). Assessment of the polygenic risk score and stratifying by APOE reveal a 4 to 5.5 years difference in median age at onset of Alzheimer's disease patients in APOE ɛ4 carriers. Because of this study, the underlying mechanisms of APP can be studied to refine the amyloid cascade and the polygenic risk score provides a tool to select individuals at high risk of Alzheimer's disease

Annexin A1: Novel Roles in Skeletal Muscle Biology

Annexin A1 (ANXA1, lipocortin-1) is the first characterized member of the annexin superfamily of proteins, so called since their main property is to bind (i.e., to annex) to cellular membranes in a Ca2þ -dependent manner. ANXA1 has been involved in a broad range of molecular and cellular processes, including anti-inflammatory signalling, kinase activities in signal transduction, maintenance of cytoskeleton and extracellular matrix integrity, tissue growth, apoptosis, and differentiation. Newinsights show that endogenousANXA1 positively modulates myoblast cell differentiation by promoting migration of satellite cells and, consequently, skeletal muscle differentiation. This suggests that ANXA1 may contribute to the regeneration of skeletal muscle tissue and may have therapeutic implications with respect to the development of ANXA1 mimetics

Effects of Prisma® Skin dermal regeneration device containing glycosaminoglycans on human keratinocytes and fibroblasts

Prisma® Skin is a new pharmaceutical device developed by Mediolanum Farmaceutici S.p.a. It includes alginates, hyaluronic acid and mainly mesoglycan. The latter is a natural glycosaminoglycan preparation containing chondroitin sulfate, dermatan sulfate, heparan sulfate and heparin and it is used in the treatment of vascular disease. Glycosaminoglycans may contribute to the re-epithelialization in the skin wound healing, as components of the extracellular matrix. Here we describe, for the first time, the effects of Prisma® Skin in in vitro cultures of adult epidermal keratinocytes and dermal fibroblasts. Once confirmed the lack of cytotoxicity by mesoglycan and Prisma® Skin, we have shown the increase of S and G2 phases of fibroblasts cell cycle distribution. We further report the strong induction of cell migration rate and invasion capability on both cell lines, two key processes of wound repair. In support of these results, we found significant cytoskeletal reorganization, following the treatments with mesoglycan and Prisma® Skin, as confirmed by the formation of F-actin stress fibers. Additionally, together with a significant reduction of E-cadherin, keratinocytes showed an increase of CD44 expression and the translocation of ezrin to the plasma membrane, suggesting the involvement of CD44/ERM (ezrin-radixin-moesin) pathway in the induction of the analyzed processes. Furthermore, as showed by immunofluorescence assay, fibroblasts treated with mesoglycan and Prisma® Skin exhibited the increase of Fibroblast Activated Protein α and a remarkable change in shape and orientation, two common features of reactive stromal fibroblasts. In all experiments Prisma® Skin was slightly more potent than mesoglycan. In conclusion, based on these findings we suggest that Prisma® Skin may be able to accelerate the healing process in venous skin ulcers, principally enhancing re-epithelialization and granulation processes

The pharmaceutical device prisma® skin promotes in vitro angiogenesis through endothelial to mesenchymal transition during skin wound healing

Glycosaminoglycans are polysaccharides of the extracellular matrix supporting skin wound closure. Mesoglycan is a mixture of glycosaminoglycans such as chondroitin-, dermatan-, heparan-sulfate and heparin and is the main component of Prisma® Skin, a pharmaceutical device developed by Mediolanum Farmaceutici S.p.a. Here, we show the in vitro effects of this device in the new vessels formation by endothelial cells, since angiogenesis represents a key moment in wound healing. We found a strong increase of migration and invasion rates of these cells treated with mesoglycan and Prisma® Skin which mediate the activation of the pathway triggered by CD44 receptor. Furthermore, endothelial cells form longer capillary-like structures with a great number of branches, in the presence of the same treatments. Thus, the device, thanks to the mesoglycan, leads the cells to the Endothelial-to-Mesenchymal Transition, suggesting the switch to a fibroblast-like phenotype, as shown by immunofluorescence assays. Finally, we found that mesoglycan and Prisma® Skin inhibit inflammatory reactions such as nitric oxide secretion and NF-κB nuclear translocation in endothelial cells and Tumor Necrosis Factor-α production by macrophages. In conclusion, based on our data, we suggest that Prisma® Skin may be able to accelerate angiogenesis in skin wound healing, and regulate inflammation avoiding chronic, thus pathological, responses

Annexin A1 induces skeletal muscle cell migration acting through formyl peptide receptors.

Annexin A1 (ANXA1, lipocortin-1) is a glucocorticoid-regulated 37-kDa protein, so called since its main property is to bind (i.e. to annex) to cellular membranes in a Ca(2+)-dependent manner. Although ANXA1 has predominantly been studied in the context of immune responses and cancer, the protein can affect a larger variety of biological phenomena, including cell proliferation and migration. Our previous results show that endogenous ANXA1 positively modulates myoblast cell differentiation by promoting migration of satellite cells and, consequently, skeletal muscle differentiation. In this work, we have evaluated the hypothesis that ANXA1 is able to exert effects on myoblast cell migration acting through formyl peptide receptors (FPRs) following changes in its subcellular localization as in other cell types and tissues. The analysis of the subcellular localization of ANXA1 in C2C12 myoblasts during myogenic differentiation showed an interesting increase of extracellular ANXA1 starting from the initial phases of skeletal muscle cell differentiation. The investigation of intracellular Ca(2+) perturbation following exogenous administration of the ANXA1 N-terminal derived peptide Ac2-26 established the engagement of the FPRs which expression in C2C12 cells was assessed by qualitative PCR. Wound healing assay experiments showed that Ac2-26 peptide is able to increase migration of C2C12 skeletal muscle cells and to induce cell surface translocation and secretion of ANXA1. Our results suggest a role for ANXA1 as a highly versatile component in the signaling chains triggered by the proper calcium perturbation that takes place during active migration and differentiation or membrane repair since the protein is strongly redistributed onto the plasma membranes after an rapid increase of intracellular levels of Ca(2+). These properties indicate that ANXA1 may be involved in a novel repair mechanism for skeletal muscle and may have therapeutic implications with respect to the development of ANXA1 mimetics