Loss of GTPase activating protein neurofibromin stimulates paracrine cell communication via macropinocytosis

Abstract

Neurofibromin, the protein product of the neurofibromatosis type 1 (NF1) tumor suppressor gene, is a negative regulator of Ras signaling. Patients with mutations in NF1 have a strong predisposition for cardiovascular disease, which contributes to their early mortality. Nf1 heterozygous (Nf1+/−) bone marrow to wild type chimeras and mice with heterozygous recombination of Nf1 in myeloid cells recapitulate many of the vascular phenotypes observed in Nf1+/− mutants. Although these results suggest that macrophages play a central role in NF1 vasculopathy, the underlying mechanisms are currently unknown. In the present study, we employed macrophages isolated from either Nf1+/− or Lysm Cre+/Nf1f/f mice to test the hypothesis that loss of Nf1 stimulates macropinocytosis in macrophages. Scanning electron microscopy and flow cytometry analysis of FITC-dextran internalization demonstrated that loss of Nf1 in macrophages stimulates macropinocytosis. We next utilized various cellular and molecular approaches, pharmacological inhibitors and genetically modified mice to identify the signaling mechanisms mediating macropinocytosis in Nf1-deficient macrophages. Our results indicate that loss of Nf1 stimulates PKCδ-mediated p47phox phosphorylation via RAS activation, leading to increased NADPH oxidase 2 activity, reactive oxygen species generation, membrane ruffling and macropinocytosis. Interestingly, we also found that Nf1-deficient macrophages internalize exosomes derived from angiotensin II-treated endothelial cells via macropinocytosis in vitro and in the peritoneal cavity in vivo. As a result of exosome internalization, Nf1-deficient macrophages polarized toward an inflammatory M1 phenotype and secreted increased levels of proinflammatory cytokines compared to controls. In conclusion, the findings of the present study demonstrate that loss of Nf1 stimulates paracrine endothelial to myeloid cell communication via macropinocytosis, leading to proinflammatory changes in recipient macrophages.

The endocytic pathways of Dictyostelium discoideum

“…Disruption of Dictyostelium NF1 causes hyperactivation of Ras, increasing the size of the cup-shaped protrusions that generate macropinosomes and therefore the volume of fluid engulfed (Bloomfield et al, 2015). Importantly, this regulatory role in macropinocytosis and therefore inflammatory signalling was also recently confirmed in mouse macrophages (Ghoshal et al, 2019). Mutations in NF1 also cause the genetic condition Neurofibratomatosis type 1 in humans, resulting in tumours in the nervous system (Xu et al, 1990).…”

Scavenger receptor MARCO contributes to cellular internalization of exosomes by dynamin-dependent endocytosis and macropinocytosis

“…Macropinocytosis, which is accompanied by membrane ruffling, is an actin-driven process that permits uptake of larger materials and extracellular non-specific fluids and proteins. It also has been reported that endothelial cell-derived exosomes 35 or oligodendrocyte-derived exosomes 36 are internalized by macrophages or microglia via membrane ruffling and macropinocytosis, respectively. Our study indicated that exosomes and 20-nm nanoparticles were co-localized with lamellipodia, cellular domains that exhibit membrane ruffling (Supplemental Fig.…”

Functional and morphological cardiovascular alterations associated with neurofibromatosis 1

“…Among the most interesting observations, some are worth mentioning as multiple research groups have independently documented them. Partial or complete loss of the NF1 gene results in increased PKCδ-mediated p47phox phosphorylation through the activation of p21(Ras), resulting in increased NADPH oxidase 2 activity, alterations in the redox balance and pro-inflammatory effects 32 – 34 . Acceleration of Ras activity also has an impact on multiple kinases of its downstream signaling pathways, including Erk and Akt, both of which are involved in the modulation of the phenotype of cells making up the vascular wall in NF1 heterozygous (Nf1 +/− ) animal model 35 , 36 .…”