Recent studies have shown that the transcription factor Foxn1, which is expressed in keratinocytes, is involved in the skin wound healing process, yet how Foxn1 functions remains largely unknown. Our latest data indicate that Foxn1 drives skin healing via engagement in re-epithelization and the epithelial-mesenchymal transition (EMT) process. In the present study, 2D-DIGE proteomic profiling analysis of in vitro cultured keratinocytes transfected with adenoviral vector carrying Foxn1-GFP or GFP alone (control) revealed forty proteins with differential abundance between the compared groups. Among the proteins with Foxn1-dependent expression, several enable adaptation to hypoxia. Subsequent experiments revealed that hypoxic conditions (1% O2) stimulate endogenous and exogenous (transfected Ad-Foxn1) Foxn1 expression in cultured keratinocytes. A proteomics analysis also identified proteins that can act as a factors controlling the balance between cell proliferation, differentiation and apoptosis in response to Foxn1. We also showed that in C57BL/6 keratinocytes, the stimulation of Foxn1 by hypoxia is accompanied by increases in Mmp-9 expression. These data corroborate the detected co-localization of Foxn1 and Mmp-9 expression in vivo in post-wounding skin samples of Foxn1::Egfp transgenic mice. Together, our data indicate that Foxn1 orchestrates cellular changes in keratinocytes in both physiological (self-renewal) and pathological (skin wound healing) contexts.

The transcription factor Foxn1 is essential for skin development. Our previous studies performed on young C57BL/6J mice model showed that Foxn1 acts as regulator of the skin wound healing process. The present study extended our initial research regarding the expression and potential role of Foxn1 in the intact and wounded skin as a function of animal age and stage of the wound healing process. We analyzed Foxn1 and Mmp-9 expression in the intact and postinjured skin of young, adult, and old C57BL/6J and transgenic Foxn1::Egfp mice. The similar levels of epidermal Foxn1 mRNA expression were detected in young and adult C57BL/6J mice and higher levels in old animals. Postinjured skin tissues displayed a gradual decrease of Foxn1 mRNA expression at Days 1, 5, and 7 after injury. Foxn1-eGFP positive cells were abundant at wound margin and in re-epithelialized epidermis at postwounded Days 1, 5, and 7 and colocalized with E-cadherin and Mmp-9. Postwounded skin at Days 14-36 displayed Foxn1-eGFP cells in the epidermis and in the dermal part of the skin (papillary dermis). A subset of Foxn1-eGFP positive cells in the papillary dermis expressed the myofibroblast marker αSMA. Flow cytometric analysis of cells isolated from postwounded (Day 7) skin tissues showed a significant increase in the percentage of Foxn1-eGFP positive cells with phenotype of double positivity for E-cadherin/N-cadherin (epithelial/mesenchymal markers). Collectively, these data identify the transcription factor Foxn1 as a potential key epidermal regulator modifying both epidermal and dermal healing processes after cutaneous wounding.

FOXN1, a transcription factor expressed in the epidermis, regulates keratinocyte differentiation and participates in skin wound healing. In this study, we explored the impact of FOXN1 insufficiency on diet-stimulated weight gain and dermal white adipose tissue regulation in the intact and wounded skin of FOXN1 eGFP/þ (heterozygotes, FOXN1-insufficient) mice in the context of age and diet. The results showed that on a high-fat diet, FOXN1 eGFP/þ mice gained significantly less body weight than their FOXN1 þ/þ counterparts (FOXN1-sufficient mice). The intact and wounded skin of FOXN1 eGFP/þ mice displayed abrogated expression of the master regulators of adipogenesis, PPARg, FABP4, and leptin, which decreased with age in FOXN1 þ/þ mice. FOXN1 insufficiency also resulted in a decreased percentage of adipocyte-committed precursor cells (CD24 þ) in the skin. The proadipogenic pathway genes Bmp2, Igf2, and Mest showed a gradual decrease in expression that accompanied the gradual inactivation of FOXN1 in the skin of FOXN1 þ/þ , FOXN1 eGFP/þ , and FOXN1 eGFP/eGFP (lack of FOXN1) mice. Bone morphogenetic protein 2 and insulin-like growth factor 2 signals colocalized with FOXN1-eGFP in the epidermis and in hair follicles. These data demonstrated that FOXN1 initiates the cascade of adipogenic signaling that regulates skin homeostasis and wound healing and affects susceptibility to dietinduced obesity.