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

Pushpankur Ghoshal, Bhupesh Singla, Huiping Lin, Mary Cherian-Shaw, Rebekah Tritz, Caleb A. Padgett, Farlyn Hudson, Hanfang Zhang, Brian Kevin Stansfield, Gabor Csanyi

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

Original languageEnglish (US)
Article number101224
JournalRedox Biology
DOIs
StatePublished - Jan 1 2019

Fingerprint

Neurofibromin 1
Paracrine Communication
GTPase-Activating Proteins
Macrophages
Cell Communication
Communication
Exosomes
Neurofibromatosis 1
Myeloid Cells
Phenotype
Phosphorylation
Flow cytometry
NADPH Oxidase
Endothelial cells
Peritoneal Cavity
Tumor Suppressor Genes
Angiotensin II
Electron Scanning Microscopy
Genetic Recombination
Blood Vessels

Keywords

  • Macrophages and exosomes
  • Macropinocytosis
  • NADPH oxidase
  • Neurofibromin

ASJC Scopus subject areas

  • Biochemistry
  • Organic Chemistry

Cite this

Loss of GTPase activating protein neurofibromin stimulates paracrine cell communication via macropinocytosis. / Ghoshal, Pushpankur; Singla, Bhupesh; Lin, Huiping; Cherian-Shaw, Mary; Tritz, Rebekah; Padgett, Caleb A.; Hudson, Farlyn; Zhang, Hanfang; Stansfield, Brian Kevin; Csanyi, Gabor.

In: Redox Biology, 01.01.2019.

Research output: Contribution to journalArticle

Ghoshal, Pushpankur ; Singla, Bhupesh ; Lin, Huiping ; Cherian-Shaw, Mary ; Tritz, Rebekah ; Padgett, Caleb A. ; Hudson, Farlyn ; Zhang, Hanfang ; Stansfield, Brian Kevin ; Csanyi, Gabor. / Loss of GTPase activating protein neurofibromin stimulates paracrine cell communication via macropinocytosis. In: Redox Biology. 2019.
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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.",
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author = "Pushpankur Ghoshal and Bhupesh Singla and Huiping Lin and Mary Cherian-Shaw and Rebekah Tritz and Padgett, {Caleb A.} and Farlyn Hudson and Hanfang Zhang and Stansfield, {Brian Kevin} and Gabor Csanyi",
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AU - Ghoshal, Pushpankur

AU - Singla, Bhupesh

AU - Lin, Huiping

AU - Cherian-Shaw, Mary

AU - Tritz, Rebekah

AU - Padgett, Caleb A.

AU - Hudson, Farlyn

AU - Zhang, Hanfang

AU - Stansfield, Brian Kevin

AU - Csanyi, Gabor

PY - 2019/1/1

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AB - 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.

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KW - Neurofibromin

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