The skin, as the outermost barrier of the body, is exposed to threats from the external environment including injuries, oxidative stress and UV irradiation. Therefore, efficient systems of repair and antioxidant protection of damaged skin that are achieved by an extraordinary mechanism involving the interaction of cells, cytokines and growth factors are vital for survival. In the present study, we focused on keratinocytes, the transcription factor Foxn1 and oxygen availability, the components that collectively sustain skin homeostasis and regulate the response to injury.
To investigate a possible mechanism by which Foxn1 under hypoxic or normoxic conditions regulates physiological changes in keratinocytes at the molecular and functional levels, we used detailed mass spectrometry analysis (LC-MS/MS) followed by in vitro and in vivo experiments. Primary cultures of mouse keratinocytes transduced with Ad-Foxn1 or Ad-GFP (control) were cocultured with mouse dermal fibroblasts (DFs) under hypoxia (1% O2) or normoxia (21% O2) for 24 h. Afterwards, keratinocytes were used to detailed analyzes: proteomics (LC-MS/ MS mass spectrometry), flow cytometry, qRT-PCR and colorimetric tests. To confirm the proteomics results at the systemic level, uninjured and injured skin collected from the Foxn1-/- and Foxn1+/+ mice using qRT-PCR, Western blot and immunohistochemistry were analyzed.
We demonstrated that Foxn1 in keratinocytes regulates elements of the electron transport chain and participates in thioredoxin system (Txn2, Txnrd3, Srxn1) induction, particularly in a hypoxic environment. Remarkably, the activation of Txnrd3, the reductase which expression has been considered to be limited to the genitals, was detected upon Foxn1 stimulation in keratinocytes and in the skin of Foxn1+/+ mice. We also showed that Foxn1 strongly downregulated the Ccn2 protein expression, participating in epidermal reconstruction after injury. An in vitro assay revealed that Foxn1 controls keratinocyte migration, stimulating it under normoxia and suppressing it under hypoxia. Angiogenesis assay showed that Foxn1 action reaches beyond epidermis limiting under hypoxic conditions HUVECells angiogenic properties by downregulating Vegfa expression.
This study showed a new mechanism in which Foxn1, along with hypoxia, participates in the activation of antioxidant defence and controls the functional properties of keratinocytes. Future studies aiming to restore Foxn1 and its regulatory pathway in the skin of older individuals may provide new solutions for antioxidative skin protection and improvement of skin wound healing.