The Role of Neurofibromin as a GTPase Activating Protein for K-ras in Mast Cells.

Neurofibromatosis type 1 (NF1), encoded by the NF1 tumor suppressor gene, is one of the most common autosomal dominant genetic disorders. Neurofibromin, the protein product of NF1, functions as a GTPase activating protein (GAP) for p21 ^Ras (Ras); however, its specific GAP activity towards the different Ras isoforms is not fully understood. We have previously established that Nf1 haploinsufficiency alters mast cell fates both in vitro and in vivo, and modulates the recruitment of mast cells to Nf1-/- Schwann cells, the tumor initiating cell type in neurofibromas (Yang, JCI 2003). We have also found high expression of both K-ras and N-ras, but undetectable levels of H-ras, in mast cells. We examined the role of Nf1 as a GAP for K-ras utilizing fetal liver-derived mast cells. Consistent with previous studies, haploinsufficency of Nf1 results in a two-fold increase in proliferation of mast cells associated with an increase in ERK activity as compared to WT controls. However, genetic inactivation of K-ras rescues the proliferative gain in function of Nf1 +/− mast cells, associated with lower biochemical activation of ERK as compared to WT levels. Additionally, utilizing Boyden chamber assays, as well as videomicroscopy, Nf1 +/− mast cells exhibits increased migration to kit-L, while genetic inactivation of K-ras abrogates this gain in function. Further, degranulation in response to crosslinking the high affinity Fc receptor on the surface of mast cells is markedly reduced in Nf1+/−; K-ras-/- cells as compared to Nf1 +/− mast cells. A key gain in function observed in Nf1+/− and Nf1 -/- hematopoietic cells is increased survival associated with an increase in PI3-K activity. While H-ras has been shown to differentially activate the PI3-K pathway compared to K-ras in other cell systems, we have found that genetic inactivation of K-ras reduces Akt activity, a sensitive measure of PI3-K activity, in Nf1+/− mast cells. This decrease in activity is associated with a two- to three-fold reduction in survival as compared to Nf1 +/− mast cells and is comparable to WT mast cells. Additionally, an intermediate in vitro phenotype is observed in cells that are heterozygous at both the Nf1 and K-ras loci (Nf1 +/−; K-ras +/−). To evaluate the interaction between Nf1 and K-ras in vivo, two model systems were utilized. Since K-ras-/- embryos die in utero, adult K-ras heterozygotes were utilized to examine the in vivo mast cell response to kit-L using microsmotic pumps placed subcutaneously in the dorsal side of the animals. Consistent with previous studies, Nf1 +/− mice have increased mast cell numbers in response to kit-L as compared to WT controls. However, genetic disruption of a single allele of K-ras restores mast cell numbers to WT levels. As a second model system, we are currently evaluating the role of genetic disruption of both alleles of K-ras in mast cells in vivo by adoptively transferring hematopoietic cells of the respective genotypes into lethally irradiated mice that are genetically deficient in the c-kit receptor and lack mast cells (W/Wv mice). Collectively, these findings suggest that Nf1 acts as a GAP for K-ras in mast cells and that K-ras is integral to a range of mast cell functions. Further, these data suggest that K-ras may be a useful molecular target for the treatment of neurofibromas as well as other disorders that involve mast cells in disease pathogenesis.