Dynamic Nrf2 Expression In CutaneousWounds Regulates Skin Regeneration
Alvaro P. Villarreal-Ponce, PhD1, Joshua A. David, BS2, Melat Worku-Tiruneh, BS2, Salma A. Abdou, BA1, Darren L. Sultan1, Jennifer Kwong, BA1, Joseph Kuhn, MD1, Piul S. Rabbani, PhD1, Daniel J. Ceradini, MD1.
1NYU Langone Health, New York, NY, USA, 2Rutgers Robert Wood Johnson School of Medicine, New Jersey, NJ, USA.
Diabetic ulcers are chronic cutaneous complications that afflict more than 4.5 million people in the United States, partly result from impaired intracellular redox homeostasis that disturbs the wound environment. We previously discovered that Nrf2, a critical transcriptional regulator of antioxidant response signaling, is necessary but deficient in cutaneous wounds of diabetic mice. This study aims to uncover the molecular mechanisms that underlie Nrf2 function in the epidermis, which are critical in wound repair and are defective in this disease.
Leprdb/db type II diabetic mice were used to elucidate cellular dysfunction that results in delayed wound healing. To study the functional role of epidermal Nrf2, we crossed Keratin14 (K14)-CreER and Nrf2flox/flox mice to generate K14-CreER;Nrf2flox/flox double transgenic mice and administered tamoxifen to induce conditional deletion of Nrf2 in only K14+ keratinocytes (KO) prior to wounding. Ten mm diameter full-thickness stented excisional wounds were created on the dorsum of 6-8-week-old WT, Leprdb/db, and KO mice and monitored until closure and characterized histologically. Whole wounds were also collected at various time points during healing to examine keratinocyte proliferation and migration, by immunohistochemistry, expression analysis, and functional assays.
Spatio-temporal analysis of Nrf2 expression in full-thickness excisional wounds reveals dynamic nuclear translocation of Nrf2 in K14+ keratinocytes of the wound edge during early repair in WT mice but is largely absent in diabetic wounds (WT 96.97±3.58% vs. Diabetic 22.5±16.40%), correlating with reduced expression of antioxidant Nrf2 gene targets MnSOD, Nqo1, Gpx, and HO1. Importantly, we find epidermal Nrf2 has a significant role in cutaneous wound regeneration, as its deletion results in a severe wound healing delay (WT 14±0days vs. KO 33±2.45days) that is reminiscent of wound closure in diabetic mice (31±1.41days).
We find that epidermal deletion of Nrf2 affects both keratinocyte proliferation and migration at the wound edge, two critical regenerative events that are compromised in diabetic mice. Ki67 expression, indicating proliferation in K14+ keratinocytes, is reduced in KO keratinocytes at the wound edge (Control 66.36±7.63% vs. KO 25.78±4.01% p=0.0004).
High-throughput transcriptome analysis and ex vivo functional analyses reveal a prominent role of Nrf2 in regulating epidermal migration by governing onset of epithelial-to-mesenchymal transition (EMT). Diabetic keratinocytes fail to downregulate protein expression of epithelial adhesion markers, such as E-cadherin, and transcriptional upregulation of mesenchymal markers Vimentin (3.9-fold reduction compared to WT), as well as that of EMT regulators, Snai1 and Twist1 (5.6 and 8.9-fold reduction compared to WT, respectively), after wounding. Keratinocytes in Nrf2 KO wounds follow the same phenomenon and we identify Nrf2 as a novel direct regulator of Snai1 and Twist1, which enhances binding to proximal promoter motifs in response to wounding.
We find that Nrf2 activity in the epidermis is essential for proper progression of critical events occurring in a wound regenerative niche. Our data presents strong evidence for the central role of Nrf2 in controlling both proliferation and directional migration of epidermal keratinocytes
to facilitate adult skin epithelial regeneration and repair. Our findings provide the basis for continued investigations to establish the therapeutic value in targeting diabetes.
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