"<div>Introduction. The dermal white adipose tissue (dWAT) is the population of intradermal adipocytes within dermal part of the skin which actively participates in physiological and pathological processes i.e. hair regeneration, thermoregulation, immune response in skin infections and wound healing. Despite the growing interest in this population of adipocytes and identification of their impact on skin physiology, dWAT regulatory pathways have not been fully recognized. It has been shown that activation of epidermal Wnt/β-catenin pathway correlate with dWAT thickness and stimulate adipogenic differentiation by induction of pro-adipogenic ligands: BMP2 (Bone Morphogenetic Protein 2) and IGF2 (Insulin-like Growth Factor 2). Our previous study revealed that epidermally expressed transcription factor Foxn1 regulates homeostasis of epidermis and affects the phenotype and functional characteristics of dermal fibroblasts (DFs). In the present study we investigated the role of transcription factor Foxn1 on intradermal adipocytes differentiation and lipid metabolism in intact and post wounded skin.
Methodology. Experiments were performed on young (8-11 weeks old) Foxn1+/+ (Balb /c; with active Foxn1 factor) and Foxn1-/- (with inactive Foxn1 factor) mice. For skin wound healing model, four (4 mm diameter) full-thickness excisional wounds were made on the back of mice. The skin samples were collected from intact skin (day 0) and during the process of healing. At post- injured days 1, 3, 5, 7 and 14, mice were sacrificed and skin samples from the back of the mice were collected using biopsy punches with a diameter of 8 mm. Tissue samples were frozen and stored in liquid nitrogen until RNA and protein isolation or fixed for immunohistochemical and immunofluorescence analyses.
Results. Histological analysis of intact skin showed an increase in the adipocyte number and the percentage of dWAT area in Foxn1+/+ mice compared to Foxn1-/- mice. Immunofluorescence staining pattern of LipidTOX fluorescence dye displayed lipid accumulation exclusively in the lower layer of the dermis, particularly in the skin of Foxn1+/+ mice. The expression profile of genes related to the process of adipogenesis and lipid metabolism demonstrated increased levels of lipolysis markers in Foxn1-deficient mice. Injury increased levels of adipogenic and lipid metabolism genes exclusively in mice with active Foxn1. Interestingly, Foxn1-deficient mice were characterized by lower in comparison to Foxn1+/+ mice expression of adipogenesis regulators (Pparγ, Fabp4 and Mest) during the entire healing process. In contrast, the expression levels of lipogenic and lipolytic genes were elevated at later stage of wound healing (day 14) in Foxn1-/- mice in comparison to Foxn1+/+ animals. Western Blot and immunofluorescence analyses of two elements of adipogenic stimulatory pathway revealed higher BMP2 and IGF2 protein content in the skin of Foxn1+/+ mice. Additionally, Foxn1+/+ animals demonstrated peak of Bmp2 expression at 14 day post injury which corresponded with increased Foxn1 mRNA levels during wound healing process.
Conclusions. The results indicate that: (i) Foxn1 modulates dWAT morphology and lipid profile; (ii) stimulated by wounding Foxn1 affects intradermal adipocytes activation during early phase of wound healing; (iii) Foxn1 participates in transcriptional regulation of lipogenesis and lipolysis; (iiii) Foxn1 contributes in stimulation of pro-adipogenic pathways: BMP2 and IGF2.</div>"