Lysyl oxidases directly control cell surface abundance of platelet-derived growth factor receptors and signaling in osteoblasts

Abstract Introduction Platelet-derived Growth Factor Receptors α and β (PDGFRs) are fundamental regulators of cell development and differentiation in the bone marrow, supporting hematopoiesis, angiogenesis and bone formation. The osteoblast niche is a significant regulator of myeloid differentiation, and signaling through osteoblast PDGFRs contributes to bone marrow fibrosis by driving stromal proliferation as well as the deposition and cross-linking of extracellular matrix proteins. Consequently, PDGFRs are strongly involved in the progression of myeloproliferative neoplasms (MPN). Lysyl oxidases (LOX, LOXL1-4) are a family of enzymes that facilitate collagen and elastin cross-linking through oxidation of the lysine side chains in these matrix proteins. We previously reported that LOX is upregulated in MPN and can oxidize PDGFRs to enhance signaling and cellular proliferation. While this provides an interesting route for pharmacological intervention in the treatment of hematological malignancies, the various contributory cellular mechanisms/pathways have not been fully explored. Objective The aim of this study was to interrogate the effects of amsulostat (previously called SNT-5505 or PXS-5505), a small molecule lysyl oxidase inhibitor currently in a Phase 2 clinical trial for the treatment of MPN (NCT04676529) on oxidation of lysyl residues within mitogenic growth factor receptors and the regulation of PDGF downstream signalling events. Methods The extracellular moieties of several recombinant growth factor receptors [including PDGF, Vascular Endothelial Growth Factor Receptor (VEGFR) and Epidermal Growth Factor Receptor (EGFR)] were incubated with recombinant lysyl oxidase like 2 (LOXL2) in the presence and absence of amsulostat. Receptor oxidation was measured by fluorescent aldehyde scavenger probes or mass spectrometry. To explore the impact of lysyl oxidases on osteoblast signaling, the human osteosarcoma cell line MG-63, which displays osteoblast-like characteristics, was chosen as it expresses PDGFR α and β. As MG-63 cells secrete low amounts of lysyl oxidases, they were cultured in the presence of amsulostat for 5 days. The media was then replaced with the addition of recombinant LOXL2 in the presence or absence of amsulostat for 24 hours. PDGFR was then stimulated with its ligand PDGFab and cell surface expression was determined using biotinylated membrane-impermeable reagents (sulfo-NHS-SS-biotin). The extracts as well as downstream signaling were measured by a modified version of Western blotting (Jess, BioTechne). All measurements were normalized against unphosphorylated or total proteins as appropriate. Results Amsulostat prevented the formation of aldehydes at the extracellular moiety of three growth factors, including PDGFR, VEGFR and EGFR, suggestive of a common structural feature. Despite the heterogeneous structure of the growth factor receptors, the oxidation of lysine residues occurs in areas related to ligand binding and receptor arrangement. As anticipated from previous reports, PDGFab stimulation of MG-63 cells caused a reduction in the cellular surface expression of PDGFRs. Here, we found that this was more pronounced when lysyl oxidase activity was blocked by amsulostat, importantly suggesting that active LOX preserves this receptor access to its ligand. Accordingly, intracellular signalling was analysed after PDGFR stimulation with PDGFab. The presence of LOX activity enhanced MEK phosphorylation when compared to conditions in which the activity was blocked by amsulostat. Interestingly, classical upstream (cRAF) and downstream (ERK) kinase pathways were not significantly reduced by amsulostat, pointing to a novel mechanism of PDGFR signal modulation by lysyl oxidases. ConclusionThis study provides new direct evidence that lysyl oxidase activity leads to aldehyde formation at lysine residues on the extracellular domains of growth factor receptors, including PDGFR. Such oxidations enhance overall cell surface expression after PDGF stimulation, leading to a prolonged response and augmented signaling. Importantly, these effects are blocked by amsulostat, suggesting a powerful mode of action beyond the extracellular inhibition of cross-linking