Fibronectin (FN) is a glycoprotein of approximately 220 kDa, whose mRNA has three alternative splicing sites (termed EDA, EDB and IIICS) that allow 20 different isoforms of FN mRNA. Circulating plasma FN lacks both EDA and EDB segments and is a soluble form secreted by the hepatocytes, while cellular FN contains variable proportions of EDA and EDB segments and is organized as fibrils in the tissue matrix. FN containing EDA segment (EDA FN) presents unique biochemical properties as compared to the isoform lacking this domain, specifically: 1) pro-fibrotic, as the EDA-containing isoform becomes predominantly expressed during wound healing to sustain myofibroblast differentiation; 2) pro-inflammatory, as inclusion of EDA domain activates Toll Like Receptor 4 (TLR4), involved in the defense of the innate immune response following recognition of pathogens associated molecular patterns resulting in NF-κβ-dependent cytokine release; 3) pro-thrombotic, as EDA FN was shown to promote agonist-induced platelet aggregation and thrombus formation in vivo through TLR4 dependent mechanisms. EDA FN is instrumental in fibrogenesis but, to date, its expression and function in bone marrow (BM) fibrosis have not been explored. We identified the up-regulation, at both molecular and protein levels, of EDA FN in an experimental mouse model of BM fibrosis treated with supra-pharmacological doses of the thrombopoietin (TPO) mimetic Romiplostim (TPOhigh). Higher expression levels of EDA FN were detected in endothelial and stromal cells after inducing experimental fibrosis in wild type (WT) mice as compared to untreated controls. To unravel the role of EDA segment on the progression of BM fibrosis in vivo, we exploited two unique transgenic mouse models that present an aberrant splicing of the EDA exon. Mice with constitutive inclusion of EDA exon (EIIIA+/+), in tissues, were more prone to BM fibrosis development as compared to knockout mice (EIIIA-/-). Upon fibrosis induction with TPO mimetic treatment, EIIIA+/+ mice presented extensive reticulin deposition, thrombocytosis, splenomegaly and increased extra-medullary hematopoiesis.
Applying numerous in vitro methods, we demonstrated that EDA FN as opposed to plasma FN lacking the EDA domain, induces megakaryocyte (Mk) proliferation in a TPO-independent fashion through engagement of TLR4 and activation of STAT5 and ERK 1/2 signalling pathways. Additionally, in vitro activation of EDA FN/TLR4 axis on Mks resulted in lipopolysaccharide-like responses, such as NF-kb activation, the release of pro-fibrotic Interleukin-6 (IL-6), and no anti-fibrotic TNF-a. Pharmacological inhibition of TLR4 in TPOhigh and EIIIA+/+/TPOhigh mice or TLR4 deletion in TPOhigh mice abrogated Mk hyperplasia, BM fibrosis, IL-6 release, extramedullary hematopoiesis and splenomegaly. Finally, developing a novel ELISA assay, we analysed samples from patients affected by primary myelofibrosis (PMF). PMF is one of the Philadelphia-negative Myeloproliferative Neoplasms (MPNs), a group of heterogeneous clonal disorders affecting the hematopoietic stem cell. MPNs are characterised by neoplastic proliferation of the myeloid lineage leading to an abnormal increase of platelet count in essential thrombocythemia (ET), red blood cells in polycythemia vera (PV), or Mks with BM fibrosis in primary myelofibrosis (PMF). To date, three major driver mutations have been identified affecting, respectively, the TPO receptor gene (MPL), the intracellular Janus Kinase 2 gene (JAK2) and the latest discovered mutations affecting the endoplasmic reticulum (ER) molecular chaperone calreticulin gene (CALR). All of these mutations induce a permanent activation of the JAK/STAT signalling pathways. We found that the EDA FN is increased in plasma and BM biopsies of PMF patients, as compared to healthy controls and ET patients, correlating with the fibrotic phase. In conclusion, we identified EDA FN/TLR4 as a new pathological axis that sustains Mk expansion and inflammation during BM fibrosis progression. EDA FN and TLR4 targeting has very promising potential for new therapeutic approaches in PMF patients with high phase BM fibrosis.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.