Bone Abstracts

Interleukin-34 (IL-34) is a newly discovered cytokine which regulates, like macrophage-colony stimulating factor (M-CSF), the differentiation/activation of the myeloid lineage. IL-34 and M-CSF are homodimers known to bind to the M-CSF receptor (M-CSFR) in a competitive manner. In this system, IL-34 can substitute for the M-CSF to induce osteoclastic differentiation. The aim of the present work was to study the functional relationships of these cytokines on cells expressing the M-CSFR.

Intracellular pathways activated by either M-CSF or IL-34 were analyzed by western Blot. Cellular proliferation/survival (CD14⁺ cells, erythroblastic cell line TF-1 overexpressing M-CSFR) was assessed by manual counting or by measuring the cell metabolic activity (alamar blue). Protein interactions were studied by surface plasmon resonance (Biacore), in situ ‘Proximity Ligation Assay’ technology (Olink) and bioinformatic modeling. The cellular localization of the M-CSFR was followed by immunocytochemistry and confocal microscopy.

Simultaneous addition of M-CSF and IL-34 induced higher phosphorylation of the tyrosine residues of the M-CSFR, showing an additive rather than a competitive effect of both cytokines. Similar results were observed on cellular proliferation. Biacore experiments revealed the possible binding of M-CSF on immobilized IL-34, with a weak affinity around 100 nM. Molecular docking studies predicted the formation of a heteromeric M-CSF/IL-34 cytokine which was confirmed by proximity ligation assay. Moreover, the co-expression of the M-CSFR and its ligands differentially regulated the M-CSFR trafficking into the cell.

This is the first report demonstrating the direct interaction between IL-34 and M-CSF and their ability to form a new heteromeric cytokine. This heteromeric structure is biologically active and may modify specifically the tridimensional conformation of the M-CSFR chains. Furthermore, cellular expression of M-CSFR ligands may regulate the receptor glycosylation, as M-CSF expression stops the glycosylation process in the Golgi apparatus, leading to a decrease of the M-CSFR membrane expression.