Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type I (NF1), a disease characterized by the formation of cutaneous neurofibromas infiltrated with a high density of degranulating mast cells. A hallmark of cell lines generated from NF1 patients or Nf1-deficient mice is their propensity to hyperproliferate. Neurofibromin, the protein encoded by NF1, negatively regulates p21(ras) activity by accelerating the conversion of Ras-GTP to Ras-GDP. However, identification of alterations in specific p21(ras) effector pathways that control proliferation in NF1-deficient cells is incomplete and critical for understanding disease pathogenesis. Recent studies have suggested that the proliferative effects of p21(ras) may depend on signaling outputs from the small Rho GTPases, Rac and Rho, but the physiologic importance of these interactions in an animal disease model has not been established. Using a genetic intercross between Nf1(+/)- and Rac2(-)(/)- mice, we now provide genetic evidence to support a biochemical model where hyperactivation of the extracellular signal-regulated kinase (ERK) via the hematopoietic-specific Rho GTPase, Rac2, directly contributes to the hyperproliferation of Nf1-deficient mast cells in vitro and in vivo. Further, we demonstrate that Rac2 functions as mediator of cross-talk between phosphoinositide 3-kinase (PI-3K) and the classical p21(ras)-Raf-Mek-ERK pathway to confer a distinct proliferative advantage to Nf1(+/)- mast cells. Thus, these studies identify Rac2 as a novel mediator of cross-talk between PI-3K and the p21(ras)-ERK pathway which functions to alter the cellular phenotype of a cell lineage involved in the pathologic complications of a common genetic disease.

Neurofibromatosis type 1 (NF1) is a common autosomal-dominant disorder characterized by cutaneous neurofibromas infiltrated with large numbers of mast cells, melanocyte hyperplasia, and a predisposition to develop malignant neoplasms. NF1 encodes a GTPase activating protein (GAP) for Ras. Consistent with Knudson's "two hit" model of tumor suppressor genes, leukemias and malignant solid tumors in NF1 patients frequently demonstrate somatic loss of the normal NF1 allele. However, the phenotypic and biochemical consequences of heterozygous inactivation of Nf1 are largely unknown. Recently neurofibromin, the protein encoded by NF1, was shown to negatively regulate Ras activity in Nf1-/- murine myeloid hematopoietic cells in vitro through the c-kit receptor tyrosine kinase (dominant white spotting, W). Since the W and Nf1 locus appear to function along a common developmental pathway, we generated mice with mutations at both loci to examine potential interactions in vivo. Here, we show that haploinsufficiency at Nf1 perturbs cell fates in mast cells in vivo, and partially rescues coat color and mast cell defects in W(41) mice. Haploinsufficiency at Nf1 also increased mast cell proliferation, survival, and colony formation in response to Steel factor, the ligand for c-kit. Furthermore, haploinsufficiency was associated with enhanced Ras-mitogen-activated protein kinase activity, a major downstream effector of Ras, via wild-type and mutant (W(41)) c-kit receptors. These observations identify a novel interaction between c-kit and neurofibromin in vivo, and offer experimental evidence that haploinsufficiency of Nf1 alters both cellular and biochemical phenotypes in two cell lineages that are affected in individuals with NF1. Collectively, these data support the emerging concept that heterozygous inactivation of tumor suppressor genes may have profound biological effects in multiple cell types.