In long-term bone marrow cultures, rAnxA1 did not alter the percentage of LSK cells but enhanced the Gr-1+Mac-1+ population; treatment with a PLC (U73122), but not with a PKC (“type”:”entrez-nucleotide”,”attrs”:”text”:”GF109203″,”term_id”:”295317075″,”term_text”:”GF109203″GF109203), inhibitor reduced rAnxA1-induced phosphorylation of ERK1/2 and Elk1. the percentage of cells in the S/G2/M cell cycle phases and reducing Notch-1 expression. Simultaneous treatment with WRW4, a selective FPR2 antagonist, reversed the in vitro effects elicited by rAnxA1. Treatment of LSK cells with rAnxA1 led to phosphorylation of PCL2, PKC, RAS, MEK, and ERK1/2 with increased expression of NFAT2. In long-term bone marrow cultures, rAnxA1 did not alter the percentage of LSK cells but enhanced the Gr-1+Mac-1+ population; treatment with a PLC (U73122), but not with a PKC (“type”:”entrez-nucleotide”,”attrs”:”text”:”GF109203″,”term_id”:”295317075″,”term_text”:”GF109203″GF109203), inhibitor reduced rAnxA1-induced phosphorylation of ERK1/2 and Elk1. Therefore, we identify here rAnxA1 as an inducer of HSC/progenitor cell differentiation, favouring differentiation of the myeloid/granulocytic lineage, via Ca2+/MAPK signalling transduction pathways. Subject terms: Calcium signalling, Recombinant protein therapy Introduction Annexin A1 (AnxA1) is a member of the annexin superfamily, composed of 13 proteins. The protein binds to acidic phospholipids on the cell surface of activated cells and can also activate specific the G-protein-coupled receptors (GPCRs) termed formyl peptide receptors (FPR). AnxA1 displays high affinity for this target partners in the presence of Ca2+ (ref. 1). Secreted AnxA1 is a mediator of the anti-inflammatory activity of glucocorticoids, especially through regulation of neutrophil influx to the site of inflammation and stimulating Epirubicin resolution of the inflammatory process2. AnxA1 mediates a broad range of molecular and cellular processes, including intracellular vesicle trafficking3, tissue growth4,5, maintenance of the cytoskeleton and extracellular matrix integrity, kinase activities in signal transduction and differentiation6. The effects of AnxA1 on blood cells seem to be specifically due to activation of FPR2 (refs. 7,8). By direct binding of AnxA1 on FPR2, heterotrimeric G proteins rapidly dissociate into and subunits. The subunit initiates a series of signal transduction pathways, such as the one mediated by phospholipase C (PLC)9, which could result in positive modulation of the mitogen-activated protein kinase (MAPK) pathway, particularly extracellular signal-related kinases 1 and 2 (ERK1/2)10. Another way to positively modulate MAPK signalling is through activation of Ca2+-sensing proteins, such as protein kinase C (PKC) and calmodulin11,12. Some of these mechanisms underlie the multiple biological properties of AnxA1 especially in the context of immune and hematopoietic settings. Herein, AnxA1 augments the activation of T cells with the involvement of the ERK and protein kinase B (AKT) pathways, favours their differentiation into Th1 cells13; affects the differentiation of semi-mature dendritic cells14; mediates the clearance of apoptotic neutrophils in the bone marrow15; inhibits neutrophil tissue accumulation by reducing leucocyte infiltration, activates neutrophil apoptosis16 and modulates secretion of stromal-derived factor- (SDF-1) by bone marrow stromal cells17. In the bone marrow, haematopoiesis is initiated by a rare multipotent population called hematopoietic stem cells (HSCs), which, at each cell division, must decide whether Epirubicin to self-renew, differentiate, migrate, or Epirubicin die. HSCs can differentiate into common myeloid progenitors (CMPs), which then produce granulocytes and monocytes/macrophages. Endogenous chemical mediators control these processes by binding to specific cell-surface receptors in a stage-specific and lineage-specific manner, resulting in the activation of intracellular signal transduction pathways that are important for proliferation, survival, and differentiation18,19. Definition of the mechanisms that regulate haematopoiesis is essential for successful mobilization of cells under stress conditions to defend the host20 and to rescue components of the blood during cancer chemotherapy or during haematological and immunosuppressive diseases21,22. Herein, we examined the role of exogenously administered Epirubicin AnxA1 in differentiation of murine HSCs/progenitor cells in the bone marrow, and our results identify this protein as an effective determinant to differentiate the myeloid/granulocytic lineage, through the engagement of the Ca2+/MAPK signalling transduction pathway. Results In vivo rAnxA1 treatment accelerates myeloid/granulocytic Cast differentiation AnxA1 is an endogenous modulator of neutrophil trafficking between compartments15,17. To determine its effects on bone marrow cell maturation, mice were treated intraperitoneally with rAnxA1 once daily over 4 days, followed by bone marrow and.