Loss of GTPase activating protein neurofibromin stimulates paracrine cell communication via macropinocytosis
Authors: Bhupesh Singla
Pushpankur Ghoshal
Rebekah Tritz
Farlyn Hudson
Caleb A Padgett
Mary Cherian-Shaw
Huiping Lin
Hanfang Zhang
Gábor Csányi
Brian K Stansfield
Journal: Redox Biology (2019)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Macrophages and exosomes; Macropinocytosis; NADPH oxidase; Neurofibromin.
Affiliations: Vascular Biology Center, Augusta University, Medical College of Georgia, Augusta, GA, 30912, USA., Vascular Biology Center, Augusta University, Medical College of Georgia, Augusta, GA, 30912, USA; Department of Pediatrics and Neonatal-Perinatal Medicine, Augusta University, Medical College of Georgia, Augusta, GA, 30912, USA., Vascular Biology Center, Augusta University, Medical College of Georgia, Augusta, GA, 30912, USA; Department of Pharmacology and Toxicology, Augusta University, Medical College of Georgia, Augusta, GA, 30912, USA. Electronic address: .

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. 

Retracted: No

PubPeer comments:
 Thanks for your interest in our manuscript. We do not see your comments. Please feel free to communicate with us via email. gcsanyi@augusta.edu , 
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  communicate with us via email . The entire purpose of PubPeer is to shine light so the broad public can evaluate and critique publications. Communicating in private defeats that purpose. , 
 Thank you for your interest in our manuscript. All samples were loaded at the same time. Both the heavy chain (50 kD) and light chain (25 kD) bands are present in the image (see fig). The 75 kDa band represents heavy and light chains together. Regarding the red (beta actin) bands, total lysate samples show strong staining. This is due to the high sensitivity of secondary ab we use for Li-Cor (IRDye 680RD). The red bands in the pull-down fractions are possibly due to actin contamination, which is common in standard immunoprecipitation experiments. Please note that the pull-down fractions show Nox2 protein bands only and not non-specific bands (present in whole lysates). Regarding your comment on p47phox, Fig. 3A indicates that p47phox expression is similar between Cre- and Cre+NF1f f macrophages. ![file] , 
 Why no light chain and heavy chain bands are visible in pull down fractions? u201cRegarding the red (beta actin) bands, total lysate samples show strong staining. This is due to the high sensitivity of secondary ab we use for Li-Cor (IRDye 680RD)u201d. Ratios of nox2 and beta actin bands seem similar on both sides of the gel. Contamination? He she is talking about p47phox levels in pull down fractions not in whole lysates. , 
 u201cPlease note that the pull-down fractions show Nox2 protein bands only and not non-specific bands (present in whole lysates)u201d. This is because lower amount of proteins were loaded to mimic pull down fractions (see differences in the levels of control beta actin). If you load equal amount of proteins, there will be non-specific bands as well. , 
 #4 #4 u201cBoth the heavy chain (50 kD) and light chain (25 kD) bands are present in the image (see fig). The 75 kDa band represents heavy and light chains together.u201d Why are no light chain and heavy chain bands visible in the pull down fractions? u201cRegarding the red (beta actin) bands, total lysate samples show strong staining. This is due to the high sensitivity of secondary ab we use for Li-Cor (IRDye 680RD). The red bands in the pull-down fractions are possibly due to actin contamination, which is common in standard immunoprecipitation experiments. Please note that the pull-down fractions show Nox2 protein bands only and not non-specific bands (present in whole lysates).u201d Contamination?? The ratios of nox2 and actin seem similar on both side of membrane. Less intense actin bands are due to loading of lower amount of lysates in pull down fraction lanes. This is why there is no non specific bands in pull down lanes but in whole Lysate lanes. If you load equal amount of proteins, you will see non-specific bands as well. u201c Regarding your comment on p47phox, Fig. 3A indicates that p47phox expression is similar between Cre- and Cre+NF1f f macrophages.u201d He she was talking about p47phox levels in pull down fractions not in total lysates. Was similar levels of p47phox present in pull down fractions? ,