Sos1 Regulates Macrophage Podosome Assembly and Macrophage Invasive Capacity

Abstract

Podosomes are protrusive structures implicated in macrophage extracellular matrix degradation and three-dimensional migration through cell barriers and the interstitium. Podosome formation and assembly are regulated by cytoskeleton remodeling requiring cytoplasmic tyrosine kinases of the Src and the Abl families. Considering that Abl has been reported to phosphorylate the guanine nucleotide exchange factor Sos1, eliciting its Rac-guanine nucleotide exchange factor activity, and Rac regulates podosome formation in myeloid cells and invadopodia formation in cancer cells, we addressed whether Sos1 is implicated in podosome formation and function in macrophages. We found that ectopically expressed Abl or the Src kinase Fgr phosphorylate Sos1, and the Src kinases Hck and Fgr are required for Abl and Sos1 phosphorylation and Abl/Sos1 interaction in macrophages. Sos1 localizes to podosomes in both murine and human macrophages, and its silencing by small interfering RNA results in disassembly of murine macrophage podosomes and a marked reduction of GTP loading on Rac. Matrix degradative capacity, three-dimensional migration through Matrigel, and transmigration through an endothelial cell monolayer of Sos1-silenced macrophages were inhibited. In addition, Sos1- or Abl-silenced macrophages, or macrophages treated with the selective Abl inhibitor imatinib mesylate had a reduced capability to migrate into breast tumor spheroids, the majority of cells remaining at the margin and the outer layers of the spheroid itself. Because of the established role of Src and Abl kinases to regulate also invadopodia formation in cancer cells, our findings suggest that targeting the Src/Abl/Sos1/Rac pathway may represent a double-edged sword to control both cancer-invasive capacities and cancer-related inflammation.

Arnica montana Stimulates Extracellular Matrix Gene Expression in a Macrophage Cell Line Differentiated to Wound-Healing Phenotype

“…in this system we used primary mouse bone marrow derived macrophages rather that THP-1 cells. This because in the previous experience of our department laboratory, and in our own test assays, the mouse cells formed more stable monolayers and showed more consistent motility [25]. The left panels of Fig 6 show some typical features of the scratch test assay.…”

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“…Finally, cells were cultured in DMEM supplemented with 10% FBS and 20% L929 cell-conditioned medium as previously described. 52,53 After 3 days of culture, macrophages were detached, counted and plated for subsequent experiments.…”

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“…where F 0 is the fraction of negative wells, µ the mean cell concentration expressed as number of initially seeded cells per well and ε is the fraction of clonogenic cells. T47D spheroids were obtained by the liquid overlay method as described previously [12,13]. Briefly, cells (500 cells/well in 150 µl of standard culture medium) were plated in 96 round-bottomed well plates previously coated with an agar gel layer (50 µl of a 0.7% w/v solution of agar in complete medium) to initially prevent cell attachment.…”