Abstract
Macrophage multinucleation (MM) is essential for various biological processes such as osteoclast-mediated bone resorption and multinucleated giant cell-associated inflammatory reactions. Here we study the molecular pathways underlying multinucleation in the rat through an integrative approach combining MS-based quantitative phosphoproteomics (LC-MS/MS) and transcriptome (high-throughput RNA-sequencing) to identify new regulators of MM. We show that a strong metabolic shift toward HIF1-mediated glycolysis occurs at transcriptomic level during MM, together with modifications in phosphorylation of over 50 proteins including several ARF GTPase activators and polyphosphate inositol phosphatases. We use shortest-path analysis to link differential phosphorylation with the transcriptomic reprogramming of macrophages and identify LRRFIP1, SMARCA4, and DNMT1 as novel regulators of MM. We experimentally validate these predictions by showing that knock-down of these latter reduce macrophage multinucleation. These results provide a new framework for the combined analysis of transcriptional and post-translational changes during macrophage multinucleation, prioritizing essential genes, and revealing the sequential events leading to the multinucleation of macrophages.
Highlights
Macrophage multinucleation (MM) is essential for various biological processes such as osteoclast-mediated bone resorption and multinucleated giant cell-associated inflammatory reactions
At day 5 the multinucleation process is well underway with formation of multinucleated giant cells (MGCs) and further culture of these cells lead to almost complete fusion of all cells in the plate at day 7 (Fig. 1A)
In order to capture the dynamics of molecular events underlying of MGC formation, we have quantified the phosphopeptides by LC-MS/MS and mRNA by RNA-seq at day 3 and day 5 in fusing macrophages. (Fig. 1A and 1B)
Summary
Maxime; Ko, Jeong-hun; Srivastava, Prashant K.; Kerloc'h, Audrey; Montoya, Alex; Mauro, Claudio; Faull, Peter; Cutillas, Pedro R.; Petretto, Enrico; Behmoaras, Jacques.
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