Impairment of the Hif-1α regulatory pathway in Foxn1-deficient (Foxn1^−/−) mice affects the skin wound healing process

Abstract

Hypoxia and hypoxia-regulated factors [hypoxia-inducible factor-1α (Hif-1α), factor inhibiting Hif-1α (Fih-1)] have essential roles in skin wound healing. Using Foxn1-/- mice that can heal skin injuries in a unique scar-free manner, we investigated the interaction between Foxn1 and hypoxia-regulated factors. Foxn1-/- mice displayed impairments in the regulation of Hif-1α and Fih-1 during the healing process. An analysis of wounded skin showed that the skin of Foxn1-/- mice healed in a scarless manner, displaying rapid re-epithelialization and an increase in Tgfβ-3 and collagen III expression. An in vitro analysis revealed that Foxn1 overexpression in keratinocytes isolated from the skin of Foxn1-/- mice led to reduced Hif-1α expression in normoxic but not hypoxic cultures and inhibited Fih-1 expression exclusively under hypoxic conditions. These data indicate that in the skin, Foxn1 affects hypoxia-regulated factors that control the wound healing process and suggest that under normoxic conditions, Foxn1 is a limiting factor for Hif-1α.

Hypoxia reveals a new function of Foxn1 in the keratinocyte antioxidant defense system

“…Based on the functional protein association network (STRING database; see Figure 3), which indicated that Foxn1 strongly activates the redox system, we showed that Txn and Txnrd together with detoxification proteins/detox proteins, for example, Nqo1 (quinone dehydrogenase NAD[P]H1), Glrx5 and Sxrn1, form an antioxidant defense coherent system. The results of in vivo experiments from our previous 45 and present study showed that cytosolic isoforms of the Txn system, Txn1, and Txnrd1 are increased in noninjured and injured skin of the Foxn1 +/+ but not Foxn1 −/− mice. However, overexpression of Foxn1 in keratinocytes as well as hypoxic culture conditions did not affect the mRNA or protein levels of Txn1 or Txnrd1 in vitro, suggesting wide‐ranging action of Foxn1 in the skin beyond keratinocytes.…”

“…25 Using an in vitro and in vivo approach, we confirmed the increase in Ep300 mRNA expression in the Ad-Foxn1-stimulated keratinocytes as well as in the wounded skin of the Foxn1 +/+ mice. These results strongly correlate with the high levels of Hif-1α expression in the skin of Foxn1 +/+ mice, 45 indicating the possible role of Foxn1 and hypoxia in Hif-1α transcription through Ep300 activation.…”

“…However, Txn proteins that are involved in protection against oxidative stress are considered the strongest oxidoreduction system 61 . The potential role of Foxn1 in Txn system regulation was already examined in our previous study, which showed the presence of an active form of Txn in Foxn1‐stimulated keratinocytes (in vitro study, 57 ) and during the skin wound healing process in Foxn1 +/+ mice (in vivo study, 45 ). Herein, a detailed proteomic analysis of Foxn1‐stimulated keratinocytes allowed us to construct protein interaction networks (see Figure 3).…”

“…The total skin samples examination of intact (Day 0) and post-injured tissues (Days 1-21) showed low levels of Txnrd1 mRNA expression in Foxn1 −/− mice post-injured days 3-21 (Figure 4G, Table S1), consistent with the low levels of Txn1 protein previously identified in Foxn1 −/− mice skin. 45 Txn2, the mitochondrial form of Txn, displayed no differences in mRNA levels related to adenoviral transduction or culture conditions for Foxn1 +/+ and Foxn1 −/− keratinocytes (Figure 4D). The analysis of Txn2 expression in the skin showed slightly higher mRNA levels in the Foxn1 −/− injured skin (Days 5, 7, and 21), although the data did not reach statistical significance (Figure 4H).…”

“…21 A subsequent study in which we applied in vivo and in vitro approaches identified the transcription factor Foxn1 as a potential regulator of Hif-1α stability during cutaneous wound healing. 45 In the present study, using mass spectrometry (LC-MS/ MS)-based proteomics analysis, followed by functional assays, we aimed to identify a possible mechanism by which Foxn1 and/or hypoxia regulate physiological changes in keratinocytes at the molecular and functional levels.…”