Methods: Datamining was performed on NCBI GEO datasets GSE23006, GSE28914, GSE153596 and our prior dataset deposited as PRJNA1077073. Immunoblots and immunohistochemistry for SerpinB3 were performed on full-thickness wounds tissues from Balb/c mice at 6 hours, and 2-, 4-, and 7-days post-wounding and from HaCaT monolayer scratch wounds. Balb/c wounds were treated with recombinant Serpinb3a and collagen morphometry was quantified post-healing by Picrosirius red staining and polarization microscopy. HaCaT monolayer scratches were treated with EGF or recombinant Serpinb3a and wound closure was quantified. HaCaT colonies were treated with Serpinb3a and proliferation was measured. HaCaTs overexpressing FLAG-tagged SerpinB3 were co-immunostained for E-cadherin or phospho-STAT3 and morphology quantified by confocal microscopy. Cell circularity from overexpressing and exogenously treated cultures was evaluated by FIJI/ImageJ. Wound tissues were collected form C57BL6/J healthy and db/db diabetic wounds at 4-, 7-, and 10-days post-wounding and RNAseq transcriptomics was performed.
Results: Data mining indicates a temporal upregulation of Serpinb3a in mouse skin and SerpinB3/B4 in human skin after wounding, which we confirmed in mice at the protein level by immunoblotting. Interestingly, treatment mouse wounds with recombinant SerpinB3 resulted in a significantly more native-like collagen architecture after healing. A significantly increased, robust expression of Serpinb3a in migrating wound-edge keratinocytes was observed by immunohistochemistry, indicating a distinct role for SerpinB3 epithelialization. A scratch-induced epidermal expression of SerpinB3 after wounding by immunoblotting of HaCaT keratinocyte scratches in vitro was also observed. Exogenous Serpinb3a significantly accelerating HaCaT wound migration, equivalent to EGF, and reduced cellular proliferation, and overexpression of SerpinB3 in HaCaT cells induced an EMT-like response with reduced E-cadherin and increased nuclear phospho-STAT3. Both modalities of exposure also significantly reduced cell circularity and more mesenchymal phenotype. Transcriptomic studies found that diabetic wounds and acute wounds have a similar Serpinb3a expression during the early stages of healing, but that by day 10 there is a distinct dysregulation of expression control in diabetic wounds with significantly increased Serpinb3a levels in diabetic wounds, while acute wounds have resolved their Serpinb3a expression.
Conclusions/Discussion: We identify the first non-cancer, physiological role for SerpinB3 signaling in the epidermis upon wounding, and that SerpinB3-dependent signaling results in an EMT-like phenotype in migrating keratinocytes associated with epithelialization and wound closure. Ongoing studies indicates that regulation of SerpinB3 expression is dysfunctional in diabetic wounds by a mechanism under active investigation.
Acknowledgement of Support: The authors gratefully acknowledge funding from the Arizona Biomedical Research Centre Grant # RFGA2022-010-03 and the 2022 Wound Healing Foundation 3M Fellowship to JRY and NIH grant R01AR074627 to KR.