The neurofibromin GAP-related domain rescues endothelial but not neural crest development in Nf1–/– mice
Fraz A. Ismat, … , Min Min Lu, Jonathan A. Epstein
Fraz A. Ismat, … , Min Min Lu, Jonathan A. Epstein
Published September 1, 2006
Citation Information: J Clin Invest. 2006;116(9):2378-2384. https://doi.org/10.1172/JCI28341.
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Research Article Cardiology

The neurofibromin GAP-related domain rescues endothelial but not neural crest development in Nf1–/– mice

Abstract

Neurofibromatosis type I (NF1; also known as von Recklinghausen’s disease) is a common autosomal-dominant condition primarily affecting neural crest–derived tissues. The disease gene, NF1, encodes neurofibromin, a protein of over 2,800 amino acids that contains a 216–amino acid domain with Ras–GTPase-activating protein (Ras-GAP) activity. Potential therapies for NF1 currently in development and being tested in clinical trials are designed to modify NF1 Ras-GAP activity or target downstream effectors of Ras signaling. Mice lacking the murine homolog (Nf1) have mid-gestation lethal cardiovascular defects due to a requirement for neurofibromin in embryonic endothelium. We sought to determine whether the GAP activity of neurofibromin is sufficient to rescue complete loss of function or whether other as yet unidentified functions of neurofibromin might also exist. Using cre-inducible ubiquitous and tissue-specific expression, we demonstrate that the isolated GAP-related domain (GRD) rescued cardiovascular development in Nf1–/– embryos, but overgrowth of neural crest–derived tissues persisted, leading to perinatal lethality. These results suggest that neurofibromin may possess activities outside of the GRD that modulate neural crest homeostasis and that therapeutic approaches solely aimed at targeting Ras activity may not be sufficient to treat tumors of neural crest origin in NF1.

Authors

Fraz A. Ismat, Junwang Xu, Min Min Lu, Jonathan A. Epstein

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Figure 2

HA-GRD expression downregulates Ras activity and normalizes downstream effectors.

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HA-GRD expression downregulates Ras activity and normalizes downstream e...
(A) HA-GRD expression downregulates activated Ras in cells. Cos7 cells were transfected with expression vectors for constitutively active (CA Ras, rasV12), dominant-negative (DN Ras, rasN17), and wild-type Ras (WT Ras). Cells with wild-type Ras were also transfected with an expression construct for HA-GRD (WT Ras + NF1 GRD). Protein samples were blotted with anti-Ras antibody before and after pulldown with the Ras-binding domain of raf-1. All bands in A were from a single scan of the same blot. (B) HA-GRD expression rescues abnormally activated Ras in Nf1–/– embryos. Ras pulldowns of protein lysates from E12.5 littermates: wild type; Nf1–/–; and Nf1–/–, HA-GRDk/+, CMV-cre+. The intensities of the bands were normalized to total Ras in the graph. (C) Embryonic HA-GRD expression rescues down-stream Ras effector abnormalities. Western blots of E12.5 embryos of wild-type; Nf1–/–; and Nf1–/–, HA-GRDk/+, CMV-cre+ genotypes. There was a reduction of phosphorylated mTOR (p-mTOR), phosphorylated S6K, and phosphorylated S6RP in Nf1–/– relative to wild type. CMV-cre–mediated expression of NF1 HA-GRD in Nf1–/– rescued this reduction in activated mTOR, s6K, and s6RP. eIF4E served as a loading control.