The role of the integrated stress response in keratinocyte migration

Background and Hypothesis: Cutaneous wound healing involves: hemostatic, inflammatory, proliferative, and tissue remodeling phases. Re-epithelialization can be modeled in vitro using human keratinocytes and artificial wounds. Previous work showed undifferentiated keratinocytes closing wounds in vitro using individual cell migration (ICM), whilst differentiated keratinocytes utilize collective cell migration (KCCM). Therefore, we hypothesize that ICM in vitro is equivalent to keratinocyte migration during squamous cell carcinoma metastasis in vivo and KCCM is a model for wound re-epithelialization. Furthermore, we hypothesize that the integrated stress response (ISR) is important in ICM and KCCM. The ISR is activated by environmental stresses that protein kinases (GCN2 and PERK) can detect and phosphorylate translation factor, eIF2a. Our goal is to define how the ISR, specifically GCN2 and PERK, influence keratinocyte migration. Methods: We will evaluate in vitro wound healing and kinetic variation in protein expression and cytoskeleton remodeling. We will utilize four keratinocyte cell lines, control human keratinocyte NTERTs, and CRISPR-derived gene knockouts of GCN2, PERK, and ISR effector gene ATF4. Quantitative analysis of wound healing is accomplished using an IncuCyte ZOOM instrument. Protein expression is measured via immunoblots following high density wounding. Cytoskeletal analyses was done by immunofluorescence. Results: Preliminary results show PERK-KO and GCN2-KO cells have reduced expression of F-actin. Immunoblots showed actin-binding protein, phospho-cofilin, at lower levels in PERK-KO and GCN2-KO cells than in NTERT cells. Wound healing assays showed differentiated keratinocytes healing faster than undifferentiated in all cells, except GCN2-KO. GCN2-KO cells healed significantly slower than other differentiated cells and undifferentiated GCN2-KO cells. Wound healing assays showed undifferentiated PERK-KO cells healing slower than other undifferentiated cell lines. Conclusion/Potential Impact: The results indicate PERK and GCN2 could be key components in ICM and CCM respectfully. GCN2 and PERK could thus be potential therapeutic targets to provide cost-effective therapeutics to enhance/inhibit keratinocyte migration.

Spatiotemporal dynamics of PIEZO1 localization controls keratinocyte migration during wound healing

Keratinocytes, the predominant cell type of the epidermis, migrate to reinstate the epithelial barrier during wound healing. Mechanical cues are known to regulate keratinocyte re-epithelialization and wound healing however, the underlying molecular transducers and biophysical mechanisms remain elusive. Here, we show through molecular, cellular and organismal studies that the mechanically-activated ion channel PIEZO1 regulates keratinocyte migration and wound healing. Epidermal-specific Piezo1 knockout mice exhibited faster wound closure while gain-of-function mice displayed slower wound closure compared to littermate controls. By imaging the spatiotemporal localization dynamics of endogenous PIEZO1 channels we find that channel enrichment at some regions of the wound edge induces a localized cellular retraction that slows keratinocyte collective migration. In migrating single keratinocytes, PIEZO1 is enriched at the rear of the cell, where maximal retraction occurs, and we find that chemical activation of PIEZO1 enhances retraction during single as well as collective migration. Our findings uncover novel molecular mechanisms underlying single and collective keratinocyte migration that may suggest a potential pharmacological target for wound treatment. More broadly, we show that nanoscale spatiotemporal dynamics of Piezo1 channels can control tissue-scale events, a finding with implications beyond wound healing to processes as diverse as development, homeostasis, disease and repair.

Promoting effect of acylated homoserine lactone on the healing of tissue damage in model rats with incontinence-associated dermatitis

Objective: One of the most common complications in patients with incontinence is incontinence-associated dermatitis (IAD). This study was conducted to determine the pathophysiology of the healing process of IAD and to develop an effective therapeutic approach according to its pathophysiology. Method: IAD was reproduced on a dorsal rat skin by applying agarose gel containing water and enzymes, and inoculating it with bacteria. Examination of the IAD healing process suggested that the promotion of keratinocyte migration and improvement of basement membrane enhance keratinocyte layer elongations, which contribute to IAD healing. A therapeutic approach using N-(3-oxotetradecanoyl)-L-homoserine lactone, which is one of the acylated homoserine lactones (AHLs) and can promote keratinocyte migration in vitro, was applied on the IAD area in rats. Results: AHL treatment after IAD development resulted in an earlier tipping point for recovery than the vehicle treatment. Histological and immunohistological analyses revealed that the tissue surface was already covered by the epidermis, indicating the results of elongation of the keratinocyte layer from hair follicles. The characteristics of the alignment of basal keratinocytes, the existence of stratum corneum, and the membrane-like distribution of the components of basement membrane were similar to those of a normal epidermis. Conclusion: These results suggested that AHL application possibly contributed to earlier IAD healing before progressing to a severe state. Although elongation of the keratinocyte layer was observed in both the AHL and vehicle groups, the possibility that AHL application promotes IAD healing was suggested. The new concept of the enhancement of keratinocyte migration as a therapeutic approach for IAD would change the skin care strategy for IAD in the healthcare setting.

Glucocorticoid-mediated induction of caveolin-1 disrupts cytoskeletal organization, inhibits cell migration and re-epithelialization of non-healing wounds

Although impaired keratinocyte migration is a recognized hallmark of chronic wounds, the molecular mechanisms underpinning impaired cell movement are poorly understood. Here, we demonstrate that both diabetic foot ulcers (DFUs) and venous leg ulcers (VLUs) exhibit global deregulation of cytoskeletal organization in genomic comparison to normal skin and acute wounds. Interestingly, we found that DFUs and VLUs exhibited downregulation of ArhGAP35, which serves both as an inactivator of RhoA and as a glucocorticoid repressor. Since chronic wounds exhibit elevated levels of cortisol and caveolin-1 (Cav1), we posited that observed elevation of Cav1 expression may contribute to impaired actin-cytoskeletal signaling, manifesting in aberrant keratinocyte migration. We showed that Cav1 indeed antagonizes ArhGAP35, resulting in increased activation of RhoA and diminished activation of Cdc42, which can be rescued by Cav1 disruption. Furthermore, we demonstrate that both inducible keratinocyte specific Cav1 knockout mice, and MβCD treated diabetic mice, exhibit accelerated wound closure. Taken together, our findings provide a previously unreported mechanism by which Cav1-mediated cytoskeletal organization prevents wound closure in patients with chronic wounds.

Kajian Etnosains Pada Potensi Penggunaan Saliva untuk Penyembuhan Luka Ringan di Lampung

Traditional medicine is the local wisdom of the Indonesian people. The people of the Jaya Sakti area of Central Lampung use saliva as a medicine to heal minor wounds. This study aims to determine the potential use of saliva in healing minor wounds. This study uses the method of observing the community. The results showed that based on the percentage of data from field observations related to the use of saliva for minor injuries in communities around Jayasakti village, Anak Tuha District, Central Lampung Regency, it is known that most people have used saliva to treat minor wounds, while other communities have never used it. or seeing other people use saliva as a remedy for minor wounds. Based on the literature review, the content contained in saliva, especially histatin, plays a major role in improving wound healing by increasing the revitalization phase, especially through increasing keratinocyte migration and proliferation. Besides, people use saliva as a medicine to heal minor wounds because they follow the habits of people who have been passed down from generation to generation and already know the content of saliva that can heal minor wounds, namely histatin and alkaline acids. In conclusion, saliva is effective to use as a wound-healing drug because of the compounds contained in saliva and people's experiences when using it.

Intracellular Ca2+-Mediated AE2 Is Involved in the Vectorial Movement of HaCaT Keratinocyte

Keratinocyte migration is initiated toward the wound skin barrier as a crucial process in wound healing. However, the migratory machinery used by keratinocytes is relatively unknown. Histamine signaling, including an increase in the Ca2+ signal, mediated the enhanced protein expression and chloride/bicarbonate exchange activity of anion exchanger AE2 in keratinocytes. In this study, we applied an agarose spot assay to induce a vectorial motion. The vectorial stimulation of the histamine-containing agarose spot enhanced the HaCaT keratinocyte migration, compared to non-directional stimulation. AE2 is associated with the vectorial movement of HaCaT keratinocytes. Enhanced expression of AE2 was mainly associated with an increase in Ca2+ and was abolished by the treatment with the Ca2+ chelating agent BAPTA-AM. These findings revealed that the directionality of Ca2+-exerted stimulation can play a prominent role in facilitating migration through the involvement of AE2 as a migratory machinery in HaCaT keratinocytes.

Author Correction: The Japanese herbal medicine Hangeshashinto enhances oral keratinocyte migration to facilitate healing of chemotherapy-induced oral ulcerative mucositis

An amendment to this paper has been published and can be accessed via a link at the top of the paper.