EGFR family members are essential for proper peripheral nervous system development. A role for EGFR itself in peripheral nervous system development in vivo, however, has not been reported. We investigated whether EGFR is required for cutaneous innervation using Egfr null and skin-targeted Egfr mutant mice. Neuronal markers; including PGP9.5, GAP-43, acetylated tubulin, and neurofilaments; revealed that Egfr null dorsal skin was hyperinnervated with a disorganized pattern of innervation. In addition, receptor subtypes such as lanceolate endings were disorganized and immature. To determine whether the hyperinnervation phenotype resulted from a target-derived effect of loss of EGFR, mice lacking EGFR expression in the cutaneous epithelium were examined. These mice retained other aspects of the cutaneous Egfr null phenotype but exhibited normal innervation. The sensory deficits in Egfr null dorsal skin were not associated with any abnormality in the morphology or density of dorsal root ganglion (DRG) neurons or Schwann cells. However, explant and dissociated cell cultures of DRG revealed more extensive branching in Egfr null cultures. These data demonstrate that EGFR is required for proper cutaneous innervation during development and suggest that it limits axonal outgrowth and branching in a DRG-autonomous manner.

The epidermal growth factor receptor (EGFR) is necessary for normal involution of hair follicles after the growth phase of anagen, although the mechanisms through which it acts are not well understood. In this report, we used transcriptional profiling of microdissected hair follicles from mice with skin-targeted deletion of Egfr to investigate how EGFR activation triggers catagen. Immunofluorescence for phospho-EGFR in mouse skin revealed increased activation of EGFR in follicular keratinocytes at catagen onset. Consistent with other models of EGFR deficiency, mice with skin-targeted deletion of Egfr (Krt14-Cre(+) /Egfr(fl/fl) ) exhibited a delayed and asynchronous catagen entry. Transcriptional profiling at the time of normal catagen onset at post-natal day (P) 17 revealed increased expression of the mitotic regulator Rcc2 in hair follicles lacking EGFR. Rcc2 protein was strongly immunopositive in the nuclei of control follicular keratinocytes at P16 then rapidly decreased until it was undetectable between P18 and 21. In contrast, Rcc2 expression continued in Egfr mutant follicles throughout this period. Proliferation, measured by bromodeoxyuridine incorporation, was also significantly increased in Egfr mutant follicular keratinocytes compared to controls at P18-21. Similarly, Rcc2-regulated mitotic regulator Stathmin 1 was strikingly reduced in control but not Egfr mutant follicles between P17 and P19. Deletion of Stmn1, in turn, accelerated catagen entry associated with premature cessation of proliferation in the hair follicles. These data reveal EGFR suppression of mitotic regulators including Rcc2 and Stathmin 1 as a mechanism for catagen induction in mouse skin.

Treatment of cancer patients with chemotherapeutics like cyclophosphamide often causes alopecia as a result of premature and aberrant catagen. Because the epidermal growth factor receptor (EGFR) signals anagen hair follicles to enter catagen, we hypothesized that EGFR signaling may be involved in cyclophosphamide-induced alopecia. To test this hypothesis, skin-targeted Egfr mutant mice were generated by crossing floxed Egfr and Keratin 14 promoter-driven Cre recombinase mice. Cyclophosphamide treatment of control mice resulted in alopecia while Egfr mutant skin was resistant to cyclophosphamide-induced alopecia. Egfr mutant skin entered catagen normally, as indicated by dermal papilla condensation and decreased follicular proliferation, but did not progress to telogen as did Egfr wild type follicles. Egfr mutant follicles responded with less proliferation, apoptosis, and fewer p53-positive cells after cyclophosphamide. Treatment of control mice with the EGFR inhibitors erlotinib or gefitinib similarly suppressed alopecia and catagen progression by cyclophosphamide. Secondary analysis of clinical trials utilizing EGFR-targeted therapies and alopecia-inducing chemotherapy also revealed evidence for involvement of EGFR in chemotherapy-induced alopecia. Taken together, our results demonstrated the involvement of EGFR signaling in chemotherapy-induced alopecia, which will help in the design of novel therapeutic regimens to minimize chemotherapy-induced alopecia.

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Treatment of cancer patients with epidermal growth factor receptor (EGFR) inhibitors causes perifollicular inflammation in the skin. Mouse models with EGFR deficiency predicted this folliculitis and revealed that abrogation of EGFR disrupts hair follicle cycling. We hypothesized that the defect in progression through catagen resulting from EGFR deficiency causes cutaneous inflammation. EGFR regulation of the hair cycle and inflammation was investigated using skin-targeted deletion of Egfr (Krt14-Cre+/Egfrfl/fl mice). Hair cycle stage was assessed using histological criteria, analysis of follicular apoptosis and the position of the dermal papillae. Dorsal hair follicles of control mice synchronously entered catagen at 17 d and progressed to telogen by 21 d. In contrast, Egfr mutant follicles asynchronously entered catagen, with some follicles beginning catagen at 18 d or later, and others remaining in anagen through 24 d. Mast cells were examined because follicular mast cell precursors can facilitate the transition into catagen after their maturation and degranulation. Failed catagen progression preceded an inflammatory response in the mutant skin, which was characterized by a 40% or more increase in mast cell numbers beginning at 21 d. Mast cells in mutant skin were more likely to be degranulated as well. Neutrophils and macrophages increased in mutant skin by 28 d, subsequent to the increase in mast cell numbers. Transcriptional profiling was performed at 17 d, the time of normal catagen onset, using follicular RNA obtained from laser capture microdissection to identify EGFR-regulated mediators of the inflammatory response. In silico analyses of these data identified increased expression of 12 immune function genes in mutant hair follicles including the mast cell proteases Chymase and Tryptase. Additionally, the class I major histocompatibility (MHC) molecules H2-Q9, H2-Q7 and H2-D1 and class II MHC molecules H2-Eb1, H2-Dmb1 and H2-Dmb1/2 were over-expressed in mutant hair follicles consistent with a role for adaptive immunity in cutaneous inflammation following asynchronous catagen. Thus, an aberrant catagen transition and upregulation of pro-inflammatory genes preceded folliculitis in Egfr mutant skin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1219. doi:1538-7445.AM2012-1219