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Review
. 2016 Nov;164:379-385.
doi: 10.1016/j.jsbmb.2015.08.011. Epub 2015 Aug 14.

Vitamin D and Calcium Regulation of Epidermal Wound Healing

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Free PMC article
Review

Vitamin D and Calcium Regulation of Epidermal Wound Healing

Yuko Oda et al. J Steroid Biochem Mol Biol. .
Free PMC article

Abstract

Wound healing is essential for survival. This is a multistep process involving a number of different cell types. In the skin wounding triggers an acute inflammatory response, with the innate immune system contributing both to protection against invasive organisms and to triggering the invasion of inflammatory cells into the wounded area. These cells release a variety of cytokines and growth factors that stimulate the proliferation and migration of dermal and epidermal cells to close the wound. In particular, wounding activates stem cells in the interfollicular epidermis (IFE) and hair follicles (HF) to proliferate and send their progeny to re-epithelialize the wound. β-catenin and calcium signaling are important for this activation process. Mice lacking the VDR when placed on a low calcium diet have delayed wound healing. This is associated with reduced β-catenin transcriptional activity and proliferation in the cells at the leading edge of wound closure. These data suggest that vitamin D and calcium signaling are necessary components of the epidermal response to wounding, likely by regulating stem cell activation through increased β-catenin transcriptional activity.

Keywords: Calcium; Epidermis; Keratinocytes; Stem cells; Vitamin D receptor; Wound repair.

Figures

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1. The impact of keratinocyte specific deletion of VDR on wound healing
A. 3 month old epiVDRKO mice and their control littermates (CON) underwent 6mm full thickness skin biopsies. B. The wound area was calculated by measurement of wound size each day, and shown as percent of 0 time control. The bars enclose mean +/− SD, *p<0.05 (n=7–8). C. Photographs were taken of the wounds daily through 9 days. Representative photographs from KO and control mice are shown. D. 3 mm wounds excised at day 3 were examined histologically to evaluate re-epithelization. The representative H&E stained sections across the anterior/posterior diameter of the wounds are shown. The yellow dotted line outlines the epidermal tongue. The red lines show the edges of the epidermal tongues crossing the wound, and the red double headed arrow shows the distance to be traveled to close the wound. Percentage re-epithelialization was defined as the distance traveled by both epithelial tongues divided by the distance needed to travel to fully re-epithelialize the wound. Bar=400µm.
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2. Expression of VDR in the HF and IFE of control mice
Immunohistochemistry (IHC) was performed on the skin of 3mo control mice. VDR was detected in the nuclei of cells in the basal, suprabasal, and infundibular regions of the HF and basal layer of the IFE.
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3. The decreased expression of signaling pathways promoting proliferation and HF differentiation in mice null for VDR in their keratinocytes
The mRNA levels in the skin of epiVDRKO mice and their controls were measured by QPCR. The PCR results are normalized to the expression of L19 and expressed as a ratio of KO to control (WT) littermates.
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4. β-catenin signaling is down regulated in epiVDRKO skin wounds that are delayed in their closure rate compared to the control wounds
RNA was extracted from skin wounds and in epiVDRKO and littermate control mice 3 days post wounding. The RNA was evaluated by Illumina microarray (Mouse Ref 8 v 2.0), and fold changes were calculated. Pathway analysis by Ingenuity IPA software demonstrated that the β-catenin signaling pathway is decreased in epiVDRKO wounds as indicated by the genes in green. The TCF/β-catenin transcriptional complex is shown by the white double circle in the center. The β-catenin target genes are aligned under the complex. They are categorized by their function such as cell cycle or stem cell fate. The arrows denote the regulation through β-catenin signaling found in the IPA database. The crosses symbolize DNA. The potential location of VDR to regulate β-catenin signaling is added. Green represents down regulation, red represents up regulation, and the intensity of the hue indicates the degree of change.
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5. The injury induced expression of β-catenin target genes is blunted in mice lacking VDR
To confirm the microarray results skin biopsies were taken from 3mo epiVDRKO and control littermates (CON). RNA was extracted from wounded skin and adjacent control skin. The expression of β-catenin target genes Axin2 and Ccnd1 (cyclin D1) was measured by QPCR. The results are expressed as percentage over the expression in the non-wounded skin (CON mice) (mean + SD), and statistically significant decreases in wounded skin in VDR KO compared to control wounded skin are shown by asterisks. *p<0.05 (n=3). The unwounded and wounded skin are shown by open bars and grey or black bars, respectively. Lower magnification (upper panels) Bar=100µm; higher magnification upper panels and lower panels Bar=25µm
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6. epiVDRKO reduces the proliferative and axin 2 responses to wounding
Three days after wounding sections of the wound were made and evaluated for PCNA and axin 2 expression by immunohistochemistry as markers of proliferation and β-catenin signaling, respectively. At the edge of the wound the epidermis was hyperplastic in both genotypes. PCNA and axin 2 expression was limited to the basal layer of the epidermis. However, PCNA and axin 2 expression was markedly reduced in the epiVDRKO mice compared to their littermate controls. *p<0.05 (n=3).

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