Endoplasmic reticulum stress in human chronic wound healing: Rescue by 4-phenylbutyrate

Abstract

During wound healing, cells have a high rate of protein synthesis and many proteins need to be folded post-translationally to function, which occurs in the endoplasmic reticulum (ER). In addition to proliferation, several cellular stress conditions, such as hypoxia, in the wound micro-environment lead to the accumulation of unfolded or misfolded proteins in the ER, causing ER stress. Eukaryotic cells have a signalling system to manage ER stress called the unfolded protein response (UPR). Mild UPR activation has a beneficial homeostatic effect; however, excessive UPR induces cell death. Herein, we examined venous leg ulcer biopsies versus normal acute incisional wounds in age-matched elderly subjects and found a large increase in ER stress markers. To study the underlying mechanism, we established several cell cultures from amputated legs from the elderly that showed inherent ER stress. While both keratinocytes and fibroblasts migration was impaired by ER stress, migration of elderly leg skin keratinocytes was markedly improved after treatment with the chemical chaperone and clinically established drug 4-phenylbutyrate (4-PBA) and demonstrated a reduction in ER stress markers. In a full-thickness human skin wound healing model, 4-PBA improved the reepithelialisation rate, which suggests it as a promising drug repurposing candidate for wound healing.

Keywords: ER stress; chronic leg ulcers; drug repurposing; skin; wound healing.

FIGURE 1

Endoplasmic reticulum (ER) stress is increased in chronic venous ulcers and acute normal wounds. A, Biopsy technique of human lower leg skin. Example of healthy volunteer 60+ years old, in which two 4 mm punch biopsies were taken thus creating two wounds, followed by one of the wounds being excised with a 6 mm punch biopsy knife on day 1, and this was repeated 7 days post incision. This generates skin samples of acute normal wounds at different time points. B, Biopsy technique of chronic venous leg ulcer. Example of 60+ years patient in which the biopsy was taken at the wound edge as indicated by the orange circle. C, Spliced XBP‐1 expression analysed by qPCR in acute leg wounds 1/7 days post incision and chronic venous leg ulcers (n = 7 and n = 11, respectively). Data are normalised to 18S and presented as a ratio between spliced XBP‐1 to unspliced XBP‐1. D, Western blot analysis of the key ER stress markers CHOP, ERO1α, XBP‐1s in acute leg wounds after 1/7 days and in chronic venous leg ulcers. Densitometric quantifications corrected to β‐actin shown below the western blots, normalised to day 1 samples (1.0). E, Gene expression analysis by nanoString nCounter of multiple genes primarily related to unfolded protein response and ER stress as well as genes related to apoptosis (since ER stress can induce apoptosis) and collagen (control for biopsy quality, upregulated in wounds). mRNA expression values were normalised to two stable housekeeping genes in each sample, that is, each samples' gene expression was normalised to its housekeeping genes. Relative gene expression shown. Note that CW versus D1 appears twice (but in different gene order) because of limitations of the analysis software. All biopsies from subjects ≥60 years. N = 7 for D1 and D7, n = 11 for CW. *P < .05, **P < .01, ***P < .001. CW, chronic wound (chronic venous leg ulcer)

FIGURE 2

Endoplasmic reticulum (ER) stress impairs cell migration in fibroblasts and keratinocytes while chemical chaperones improve cellular migration only in elderly skin keratinocytes. Scratch wound assay of fibroblasts and keratinocytes derived from both young and aged skin and treated with different pharmacological ER stressors and chemical chaperones. A, Young trunk skin fibroblasts ± Tm and 4‐PBA for 48 to 72 hours. B, Old leg skin fibroblasts ± Tm and 4‐PBA for 48 to 72 hours. C, Young trunk skin keratinocytes ± Tm, 4‐PBA and TUDCA for 48 hours. D, Elderly skin keratinocytes ± Tm and 4‐PBA for 72 hours. E, Representative images of elderly skin keratinocytes ± 62.5 μM 4‐PBA for 72 hours (yellow scale bar 300 μm). (F) Elderly skin keratinocytes ± 4‐PBA and TUDCA for 12 hours pre‐scratching followed by ± Tm, 4‐PBA and TUDCA for 48 hours post‐scratching. N = 3 with mean ± SE. *P < .05, **P < .01, ***P < .001, ****P < .0001 compared with ctrl unless indicated otherwise by brackets, by two way‐analysis of variance (ANOVA). 4‐PBA, 4‐phenylbutyrate; Ctrl, control; Tm, tunicamycin

FIGURE 3

Endoplasmic reticulum (ER) stress primarily impairs keratinocyte migration and not proliferation. Young trunk skin keratinocytes. A, ± MMC 1 hour pre‐scratching. B, ± MMC 1 hour pre‐scratching followed by ± Tm 48 hours post‐scratching. C, ± MMC 1 hour pre‐scratching followed by ± Tg 48 hours post‐scratching. D, Representative images (yellow scale bar 300 μm). N = 3 with mean ± SE. *P < .05, **P < .01, ***P < .001, ****P < .0001 compared with ctrl, unless indicated otherwise by brackets, by two way‐analysis of variance (ANOVA). Ctrl, control; MMC, mitomycin C; Tm, tunicamycin; Tg, thapsigargin

FIGURE 4

The endoplasmic reticulum (ER) is dilated in keratinocytes adjacent to the wound edge. A, Cartoon illustrating transmission electron microscopy imaging of wound edge zone and intact epidermal zone, as well as how punch biopsies were taken (50% intact epidermis, 50% wound bed). B‐E, Transmission electron microscopy images. The rough ER is identified by ribosomes (black punctae). Note the ER dilation in the basal layer keratinocytes adjacent to the wound edge zone. ER, endoplasmic reticulum; N, nucleus; K, keratin. Yellow arrows indicate ER. Black arrows and yellow ovals indicate cells imaged. Star indicates migrating epidermal tongue

FIGURE 5

Endoplasmic reticulum (ER) stress is increased in elderly leg skin keratinocytes and 4‐PBA alleviates it. CHOP, GRP78, spliced XBP‐1, unspliced XBP‐1, PERK, ERO1α, ATF6β, ERN1, eIF2α and ATF‐4 in primary keratinocytes derived from both young trunk skin and elderly leg skin. mRNA expression levels measured by qRT‐PCR and corrected to β‐actin following 24 and 48 hours treatments with and without 62.5 μM 4‐PBA, 200 μM TUDCA, ± 0.12 μg/mL Tm. For each time point n = 4 with mean ± SE. *P < .05, **P < .01, ***P < .001 by unpaired two‐tailed Student's t test. Ctrl‐control, Tm‐tunicamycin, s/unXBP‐1‐spliced/unspliced XBP‐1

FIGURE 6

4‐PBA improves wound reepithelialisation and model of endoplasmic reticulum (ER) stress in wound healing. A, Representative images of haematoxylin and eosin staining of human ex vivo full thickness wounds treated with topical or transdermal (media) delivered 4‐PBA or vehicle. Black arrows point to representative newly formed epidermal tongues 1 day after incision. ×4 (500 μm) and ×20 objectives used (100 μm). B, Reepithelialisation quantified for day 1, 5 and 7 compared with the initial wound size as shown in day 0. For each time point n ≥ 5 with mean ± SE. *P < .05; **P < .01; ***P < .001 by unpaired two‐tailed Student's t test. C, Model of ER stress in wound healing. During normal and chronic incision, there is an increase in ER stress followed by activation of the UPR. To restore protein homeostasis, the three classical UPR signalling arms [ATF6, IRE1α/β (ERN1, ERN2‐the latter one is selectively expressed in epithelial cells ¹⁵ ), PERK] are all activated to different levels (green boxes). Transcription factors such as spliced XBP1 (XBP1s), ATF6b, and ATF4 ¹⁶ migrate to the nucleus to transactivate genes of the ERAD (HERPUD1, EDEM2, ERLEC1), chaperones (BIP) and HSPs (HSPA6, A2, 1 L DNAJ gene family), as well as, genes responsible for oxidative protein folding in the ER such as ERO1α, ERP44, and PDI. The latter genes upregulation contributes to the cells' ability to cope with ER stress. However, when the stress is prolonged the oxidative activity of ERO1α and other oxireductases burden cells with potentially toxic reactive oxygen species, ¹⁰ which may contribute to healing impairment. Chronic UPR activation may also lead to apoptotic death through CHOP (red box), a master regulator of ER stress induced apoptosis that inhibits pro‐survival BCL2 and GADD34, which exacerbates cell death signalling. IRE1α can also contribute to cell death by activating the JNK pathway through a direct interaction with TRAF2 and ASK1 (red boxes) followed by induction of the pro‐apoptotic factor BAX (which can also bind to IRE1 α and release Ca²⁺ from the ER). 4‐PBA, a small molecular chemical chaperone that hampers protein misfolding and aggregation, as well as, promotes intracellular trafficking and secretion, ¹⁵ reduces ER stress and may thus improve healing of chronic wounds