Foxn1 Transcription Factor Regulates Wound Healing of Skin through Promoting Epithelial-Mesenchymal Transition

Abstract

Transcription factors are key molecules that finely tune gene expression in response to injury. We focused on the role of a transcription factor, Foxn1, whose expression is limited to the skin and thymus epithelium. Our previous studies showed that Foxn1 inactivity in nude mice creates a pro-regenerative environment during skin wound healing. To explore the mechanistic role of Foxn1 in the skin wound healing process, we analyzed post-injured skin tissues from Foxn1::Egfp transgenic and C57BL/6 mice with Western Blotting, qRT-PCR, immunofluorescence and flow cytometric assays. Foxn1 expression in non-injured skin localized to the epidermis and hair follicles. Post-injured skin tissues showed an intense Foxn1-eGFP signal at the wound margin and in leading epithelial tongue, where it co-localized with keratin 16, a marker of activated keratinocytes. This data support the concept that suprabasal keratinocytes, expressing Foxn1, are key cells in the process of re-epithelialization. The occurrence of an epithelial-mesenchymal transition (EMT) was confirmed by high levels of Snail1 and Mmp-9 expression as well as through co-localization of vimentin/E-cadherin-positive cells in dermis tissue at four days post-wounding. Involvement of Foxn1 in the EMT process was verified by co-localization of Foxn1-eGFP cells with Snail1 in histological sections. Flow cytometric analysis showed the increase of double positive E-cadherin/N-cadherin cells within Foxn1-eGFP population of post-wounded skin cells isolates, which corroborated histological and gene expression analyses. Together, our findings indicate that Foxn1 acts as regulator of the skin wound healing process through engagement in re-epithelization and possible involvement in scar formation due to Foxn1 activity during the EMT process.

FOXN1 Transcription Factor in Epithelial Growth and Wound Healing

“…In the last, remodeling phase, all the signals that were rapidly evoked are ceased, and disorganized collagen fibers form scar tissue (6). FOXN1 was reported by our team as a damage response signal during the first two phases of wound healing, and we consider FOXN1 responsible for scar formation (40). In our studies, the levels of FOXN1 expression have clearly been raised in all keratinocytes except basal ones at the wound margin and the postinjury epithelium.…”

“…Similarly to FOXN1, Notch is a prodifferentiation factor (48), and recently it has been reported to act as an injury response signal in the inflammatory and proliferation phases of wound healing in suprabasal keratinocytes (49). This process mirrors the postwounding expression of Foxn1 (40). This observation supports their common function in the BMP/Notch signaling pathway suggested in HFs (45).…”

“…In addition to its role in the regulation of keratinocyte differentiation and inflammation, FOXN1 can be involved in skin carcinogenesis and wound healing through yet another process, EMT. Our recent data implicate FOXN1 in the EMT process, resulting in the formation of scar tissue (40). The expression of Foxn1 during mouse wound healing was found to colocalize with various EMT markers, such as Snail1 and MMP-9.…”

Epithelial-mesenchymal transition in tissue repair and fibrosis

“…Foxn1, a potent mammalian wound healing factor, also appears to be involved in EMT-driven re-epithelialization during repair, as evidenced by studies in Foxn1 transgenic mice. In these mice, the induction of EMT post-wounding was demonstrated though the upregulation of EMT transcriptional regulator Snail1, increased MMP9 expression, presence of vimentin+/E-cadherin+ cells, and migratory keratinocytes at the wound edge expressed Foxn1 which co-localized with Snail (Gawronska-Kozak, et al, 2016). Finally, zebrafish keratocytes in explant culture, which serve as a well-studied model of epithelial wound healing, display evidence of EMT (McDonald, et al, 2013).…”

Neotenic phenomenon in gene expression in the skin of Foxn1- deficient (nude) mice - a projection for regenerative skin wound healing

“…Moreover, our recent data revealed that Foxn1 can act as a regulator of the skin wound healing process. The experiments performed on Foxn1::Egfp transgenic mice showed that Foxn1-bearing cells participate in the wound healing process through engagement in re-epithelialization and possible involvement in scar formation due to Foxn1 activity during the epithelial-to-mesenchymal transition (EMT) [12]. In the present study using next-generation high-throughput DNA sequencing techniques, we aimed to test the hypothesis that Foxn1 expression in the skin is an essential condition for establishing the adult skin phenotype and that a lack of Foxn1 maintains skin in a neoteny stage (immature stage of development).…”