Neurofibromin-deficient Schwann cells secrete a potent migratory stimulus for Nf1+/– mast cells
Yang Feng-Chun, David A. Ingram, Shi Chen, Cynthia M. Hingtgen, Nancy Ratner, Kelly R. Monk, Travis Clegg, Hilary White, Laura Mead, Mary Jo Wenning, David A. Williams, Reuben Kapur, Simon J. Atkinson, D. Wade Clapp
Yang Feng-Chun, David A. Ingram, Shi Chen, Cynthia M. Hingtgen, Nancy Ratner, Kelly R. Monk, Travis Clegg, Hilary White, Laura Mead, Mary Jo Wenning, David A. Williams, Reuben Kapur, Simon J. Atkinson, D. Wade Clapp
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Article Oncology

Neurofibromin-deficient Schwann cells secrete a potent migratory stimulus for Nf1+/– mast cells

Abstract

The NF1 tumor suppressor gene encodes a GTPase-activating protein called neurofibromin that negatively regulates Ras signaling. Mutations in NF1 cause neurofibromatosis type 1 (NF1). The development of neurofibromas, which are complex tumors composed of multiple cell types, is a hallmark of NF1. Somatic inactivation of murine Nf1 in Schwann cells is necessary, but not sufficient, to initiate neurofibroma formation. Neurofibromas occur with high penetrance in mice in which Nf1 is ablated in Schwann cells in the context of a heterozygous mutant (Nf1+/–) microenvironment. Mast cells infiltrate neurofibromas, where they secrete proteins that can remodel the ECM and initiate angiogenesis. Thus, identification of mechanisms responsible for mast cell migration to tumor microenvironments is important for understanding tumorigenesis and for designing potential therapies. Here, we show that homozygous Nf1 mutant (Nf1–/–) Schwann cells secrete Kit ligand (KitL), which stimulates mast cell migration, and that Nf1+/– mast cells are hypermotile in response to KitL. Furthermore, we link hyperactivation of the Ras-class IA-PI3K-Rac2 pathway to increased Nf1+/– mast cell migration. Thus, these studies identify a novel interaction between Nf1–/– Schwann cells and Nf1+/– mast cells that is likely to be important in neurofibroma formation.

Authors

Yang Feng-Chun, David A. Ingram, Shi Chen, Cynthia M. Hingtgen, Nancy Ratner, Kelly R. Monk, Travis Clegg, Hilary White, Laura Mead, Mary Jo Wenning, David A. Williams, Reuben Kapur, Simon J. Atkinson, D. Wade Clapp

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Figure 4

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Effect of heterozygosity of Nf1 on mast cell haptotaxis and F-actin cont...
Effect of heterozygosity of Nf1 on mast cell haptotaxis and F-actin content in response to rmKitL. (a) Haptotaxis of WT and Nf1+/– mast cells in response to 10 ng/ml rmKitL. Results represent the mean ± SEM of five independent experiments. *P < 0.05 for Nf1+/– versus WT by the Student’s paired t test. (b) Sequential exposures of Nf1+/– and WT mast cells taken at 2.5-minute intervals during exposure to a 10 ng/ml KitL gradient (from left to right) for 1 hour. Mast cells were allowed to adhere to FN-coated glass coverslips for 15 minutes before being loaded into the chemotactic gradient of a Zigmond chamber. Nf1+/– mast cells demonstrated greatly enhanced movement to the rmKitL gradient compared with WT cells. (c) Percentage of Nf1+/– or WT cells moving at various speeds during videomicroscopy. The average speed of directed movement was calculated from data collected from the second 30-minute interval of videomicroscopy from WT and Nf1+/– samples (n >100 cells analyzed per genotype). Histogram depicts the results from one representative experiment. Two other experiments showed similar results. (d) F-actin content in BMMCs. Mast cells were stimulated with 10 ng/ml rmKitL and fixed, at the time points indicated, by addition of formaldehyde. WT and Nf1+/– cells were examined in triplicate. Results are expressed as mean channel fluorescence (MCF) and represent the mean of four independent experiments per genotype. *P < 0.001 for all comparisons between genotypes using a Student’s paired t test.