Effects of Retinoic Acid on Wingless (WNT) Signaling in the Hair Follicles of Mice

Many cancers in the skin arise from aberrant hair follicle stem cells. WNT (wingless related mouse mammary tumor virus) signaling is important in regulating these stem cells, as well as in initiating the growth phase of the hair cycle (anagen). Follicular localization sites (including hair follicle stem cells) of several WNT signaling molecules are similar to those of synthesis enzymes of retinoic acid (RA), a vitamin A metabolite. As vitamin A and metabolites (retinoids/RA) are essential for growth, vision, reproduction, cell proliferation, differentiation, and apoptosis, exogenous retinoid treatments are currently used for various human skin diseases. However, the specific effects of endogenous retinoids (particularly RA) on hair follicles remain uncertain. To gain a better understanding of how RA interacts with WNT signaling, we performed 3 preliminary studies. First, we analyzed the localization of ß-catenin in hair follicles of mice, as nuclear ß-catenin marks sites of WNT signaling. Mice were wax-stripped to synchronize the hair cycle and tissues were collected. Immunohistochemistry (IHC) determined that nuclear ß-catenin peaked 6 days after wax stripping, when hair was in mid-anagen. Second, we determined a drug and dose that topically inhibited RA synthesis, and whether this inhibition affected WNT signaling. Additional mice were wax-stripped, and 6 days later, 4 doses of citral or disulfiram were applied to this area. IHC determined that the highest dose of each drug best inhibited RAR-ß, a RA target gene, and citral provided stronger inhibition overall. However, citral did not completely inhibit RA synthesis, indicating that further studies should be conducted using higher doses in order to determine the optimal amount to accomplish this. IHC analysis on the dorsal skin tissue from these mice also found that when RA is inhibited, WNT signaling is active. Third, we determined the easiest and most cost effective manner to measure WNT signaling. Two additional mice were wax stripped, and 6 days later, dorsal skin was collected. IHC determined that the ß-galactosidase antibody provided an easier way to determine active WNT signaling when compared to nuclear ß-catenin localization. A large-scale experiment, analyzing the effects of topically applied RA inhibitors on WNT signaling, can be performed once the optimal dose is determined. Interactions between RA and WNT signaling may impact the development of treatments for hair diseases and skin cancer.This work was supported by the National Institutes of Health (NIH) grant # AR052009No embarg