Human Parathyroid Hormone Analog (3-34/29-34) promotes wound re-epithelialization through inducing keratinocyte migration and epithelial-mesenchymal transition via PTHR1-PI3K/AKT activation

Abstract

Background: Re-epithelialization is important in the process of wound healing. Various methods have been identified to expedite the process, but their clinical application remains limited. While parathyroid hormone (PTH) has shown promising results in wound healing due to its role in promoting collagen deposition and cell migration, application is limited by its potentially inhibitive effects when being continuously and locally administrated. Herein, we developed a novel PTH analog, Human parathyroid hormone (hPTH) (3-34/29-34) (henceforth MY-1), by partially replacing and repeating the amino acid sequences of hPTH (1-34), and evaluated its effect on skin wound re-epithelialization.

Methods: CCK-8, colony formation unit assay, and Ki67 immunofluorescent staining were performed to evaluate the effect of MY-1 on HaCaT cell proliferation. Then, wound scratch assay, Transwell assay and lamellipodia staining were carried out to evaluate the effect of MY-1 on cell migration. Moreover, the epithelial-mesenchymal transition (EMT) markers were measured using qPCR and western blot analysis. For in-vivo drug delivery, gelatin methacryloyl (GelMA) hydrogel was employed to load the MY-1, with the physicochemical characteristics evaluated prior to its application in wound models. Then, MY-1's role in wound healing was determined via acute skin wound models. Finally, the mechanism that MY-1 activated was also detected on HaCaT cells and in-vivo wound models.

Results: In-vitro, MY-1 accelerated the migration and EMT of HaCaT cells, while having little effect on cell proliferation. GelMA and MY-1-incorporated GelMA hydrogels showed similar physicochemical characteristics and were used in the in-vivo studies, where the results revealed that MY-1 led to a stronger re-epithelialization by inducing basal keratinocyte migration and EMT. Further studies on in-vivo wound models and in-vitro HaCaT cells revealed that MY-1 regulated cell migration and EMT through activating PI3K/AKT signaling. The parathyroid hormone type 1 receptor (PTHR1), the main receptor of PTH, was found to be the upstream of PI3K/AKT signaling, through interfering PTHR1 expression with a small interference RNA following detection of the PI3K/AKT activation.

Conclusion: Collectively, our study demonstrated that MY-1 accelerates skin wound re-epithelialization by inducing keratinocyte migration and EMT via PTHR1-PI3K/AKT axis activation. Video Abstract.

Keywords: Cell migration; Epithelial-mesenchymal transition; PI3K/AKT; Re-epithelialization; Wound healing; hPTH (3–34/29–34).