Abstract B59: Role of Nf1 signaling in regulating pigmentation in the mouse

Abstract

Introduction: Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominantly inherited disorders affecting 1 in 3500 individuals. The NF1 gene encodes a protein called neurofibromin that has a Ras-GAP activity and acts as a tumor suppressor by negatively regulating Ras. Loss of neurofibromin has a pleiotropic effect affecting a number of cell types. We are interested in using the elegance and sensitivity of the pigmentary system to understand the mechanisms by which Nf1 regulates cell survival and proliferation in vivo. We have found that an ENU-induced point mutation (Nf1Dsk9) or a targeted knockout of one Nf1 allele specifically and uniformly darken the mouse skin dermis. The Endothelin receptor B (Ednrb) is essential for melanocyte (pigment producing cell) survival in the dermis between E10–E12.5. Since Nf1Dsk9 causes uniform hyper pigmentation of the dermis, we wanted to know whether Nf1Dsk9 depends on Ednrb signaling to cause dermal hyper pigmentation. In an attempt to understand the effect of homozygous loss of Nf1 on melanocytes, we knocked Nf1 out specifically in melanocytes. Methods: The Nf1Dsk9/+ mice were crossed to Ednrbs-l/s-l mice that have a complete deletion of the Ednrb gene. Pigmentation of the dermal tail skin and coat color spotting of 2–3 week old mice obtained from the above cross was quantified using ImageJ. A melanocyte-specific knockout of Nf1 was made in vivo in the mouse by crossing existing Mitf-Cre line to the floxed-Nf1 line. Pigmentation of the dermal tail skin was quantified using ImageJ. Results: A significant (p< 0.05, T-Test) hyper pigmentation of the dermis was observed in the Mitf-Cre/+; Nf1flox/flox homozygotes (now called Nf1mcko/mcko) with no change in epidermal pigmentation. In contrast to the Nf1Dsk9/+(germline heterozygote), the Nf1mcko/+ (melanocyte-specific knockout heterozygote) does not show dermal hyper pigmentation. Comparison of the tail dermis of Nf1Dsk9/+;Ednrbs-l/s-l mice with +/+;Ednrbs-l/s-l mice showed that Nf1Dsk9 was unable to give a dark dermis in the absence of Ednrb. However, the Nf1Dsk9/+;Ednrbs-l/s-l mice showed a significant (p< 0.05, T-Test) increase in the percentage of coat that was pigmented. Discussion: A significant darkening of the dermis in the Nf1mcko/mcko compared to wild type or Nf1mcko/+ littermates suggests a cell autonomous mechanism of dermal hyper pigmentation in the homozygote caused by the loss of Nf1 in melanocytes. The Nf1mcko/+ heterozygote does not show dermal hyper pigmentation, unlike the Nf1Dsk9/+ heterozygote. This suggests that the effect of loss of Nf1 in melanocytes acts in a non-cell autonomous mechanism in the heterozygote. The lack of dermal hyper pigmentation but an increased percentage of coat color spotting in the Nf1Dsk9/+;Ednrbs-l/s-l mice suggests that Nf1 regulates pigmentation by two independent mechanisms, one that depends on Ednrb and one that does not. Citation Information: Cancer Res 2009;69(23 Suppl):B59.