Activity of Nrf2 in the human hair follicle: understanding Dkk-1 and mast cells regulation by Nrf2

UoM administered thesis: Phd

  • Authors:
  • Laura Jadkauskaite

Abstract

The human hair follicle (HF) is a mini-organ that can be affected by excessive levels of reactive oxygen species (ROS) resulting in hair graying and hair loss. The transcription factor nuclear factor (erythroid-derived-2) like 2 (Nrf2) is the “master regulator” of redox homeostasis and is involved in counteracting oxidative stress by upregulating downstream targets, yet Nrf2 activity in human HFs is unknown. We sought to investigate whether Nrf2 is expressed and active within human HFs, using microarray, immunofluorescence and qRT-PCR techniques. Activation of Nrf2 using sulforaphane (SFN) increased the transcription of Nrf2 downstream targets. In addition, Nrf2 knockdown in human HFs confirmed that Nrf2 directly regulates the transcription of downstream targets. Thus, increasing the intrafollicular Nrf2 activity by SFN also reduced HF damage induced by H2O2. Therefore, previously unknown role of Nrf2 in human HFs has been discovered. This encouraged the hypothesis that Nrf2 activity may also regulate other centrally important signalling pathways in HF biology. Luminex analysis and qRT-PCR revealed that Nrf2 activation with SFN in human HFs significantly downregulated, the secretion and transcription of Wnt canonical pathway inhibitor, Dkk-1. In addition, using bioinformatics and ChIP it was found that Dkk-1 promoter contains ARE sequence that Nrf2 is able to bind and that downregulation of Dkk-1 is Nrf2 dependent. This demonstrated that Nrf2 regulates the canonical Wnt/β-catenin pathway by controlling Dkk-1 transcription, translation and secretion. Finally, we also sought to investigate whether Nrf2 can control perifollicular mast cell (MC) activity using immunohistochemistry, flow cytometry and qRT-PCR. We found that perifollicular MCs express Nrf2 and its downstream target HO-1 upon stimulation with SFN and substance P (SP). In addition, Nrf2 activation via SFN prior stimulation with SP suppressed pro-inflammatory mediators transcription and degranulation in human MCs. Here we have demonstrated that human HFs express Nrf2 that is involved in not only counteracting oxidative stress but also controlling Wnt canonical pathway via Dkk-1 and regulating the activity of perifollicular human MCs.

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Original languageEnglish
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Award date1 Aug 2018