Regulation of Lysyl Oxidase, a Key Regulator of Bone Marrow Fibrosis, in Myelofibrosis

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that arises from clonal proliferation of hematopoietic stem cells, which leads to expansion of immature megakaryocytes (MKs), progressive bone marrow (BM) fibrosis and cytopenias. Our previous work showed that lysyl oxidase (LOX), a matrix cross-linking enzyme highly expressed by MKs in PMF, contributes to disease progression. GATA1 is a transcription factor that is important for MK development and has been found to be downregulated in MKs of patients with PMF and in mouse models of PMF. We observed an inverse correlation between GATA-1 and LOX levels in MKs of PMF mice. GATA1 can act as an activator as well as a repressor, and our chromatin precipitation study showed that it binds to the Lox gene. To determine whether GATA-1 suppresses LOX expression in MKs, BM cells induced to differentiate to MKs were transfected with a retrovirus containing GATA-1 with GFP reporter or empty virus with GFP. MKs were isolated using a BSA gradient and RNA was extracted for LOX mRNA expression via qRT-PCR. GATA-1 transduced MKs suppressed LOX mRNA levels. Further, to assess the effect of GATA1 on Lox gene promoter activity, we transfected a murine pluripotent mesenchymal cell line (C3H10T1/2 cells) with the murine Lox gene promoter linked to a reporter gene encoding luciferase. Co-transfection with a vector expressing mouse GATA-1 down-regulated basal LOX promoter activity compared to empty vector. Given that PMF MKs express high levels of TGF-β, and TGF-β has been shown to stimulate LOX expression, we next examined the ability of GATA-1 to suppress TGF-β-mediated LOX expression. Indeed, transfection of GATA-1 suppressed LOX expression even in the presence of TGF-β compared to empty vector. Taken together, we identified a key role for GATA-1 in LOX regulation, which has significant relevance to fibrosis in PMF.