Angptl4 is upregulated by microenvironmental factors during the wound healing process and promotes epidermal stem cell proliferation via PRL8a6

Abstract

Angiopoietin-like 4 (ANGPTL4) expression is increased in wound tissue and contributes to wound healing. However, the underlying mechanisms are not fully understood. Here, we demonstrate that ANGPTL4 expression is significantly increased in epidermal stem cells (EpSCs) in the periwound epidermis during wound healing in mice. Increased Angptl4 expression is positively correlated with increased expressions of tumor growth factor-α, interleukin-1β, epidermal growth factor, nerve growth factor, fibroblast growth factor 7, and transforming growth factor-β1. Each of these molecules induces Angptl4 expression in mouse EpSCs. RNA sequencing of EpSCs derived from wild-type and Angptl4 knockout (Angptl4–/–) mice reveals altered expressions of genes involved in the cell cycle and cell proliferation in Angptl4–/– EpSCs, including a decrease in cyclin E2/A2/B1 and cyclin-dependent kinase 1 (Cdk1) expression; an increase in Cdk inhibitor 2a (Cdkn2a) and Cdkn2b expression; and a decrease in the prolactin (PRL) family members Prl2a1, Prl8a1, Prl8a9, and Prl8a6. Mechanistic studies reveal that ANGPTL4 stimulates EpSC proliferation via PRL8a6-mediated upregulation of cyclins A2/E2/B1 and Cdk1, downregulation of Cdkn2a, and acceleration of cell cycle progression from the G1 to the S and G2 phases. In vivo studies demonstrate that Prl8a6 mRNA is upregulated by ANGPTL4 in mouse periwound tissue during skin wound healing. Knockdown of Angptl4 or Prl8a6 in periwound skin tissue impairs EpSC proliferation and delays wound re-epithelialization. In conclusion, our study demonstrates that, after skin injury, elevated levels of proinflammatory cytokines and growth factors in periwound tissue stimulate Angptl4 expression in EpSCs and that ANGPTL4 promotes EpSC proliferation by increasing Prl8a6 expression, thereby accelerating wound re-epithelialization.

1. Introduction

Epidermal stem cells (EpSCs) are essential for the homeostasis of the skin and for wound repair. In the basal layer of the epidermis, EpSCs maintain skin homeostasis by self-renewing and differentiating into keratinocytes [1]. After skin injury, EpSCs located in the periwound tissue contribute to epidermal regeneration by proliferating, migrating to the wound site, and differentiating into keratinocytes [2]. A deeper comprehension of the regulatory processes and mechanisms governing the proliferation of EpSCs by endogenous factors could provide valuable strategies to enhance skin wound healing.

Angiopoietin-like 4 (ANGPTL4) belongs to the angiopoietin-like protein family. It is a secreted protein expressed in multiple tissues and cells, including the skin, adipose tissue, liver, skeletal muscle, heart, macrophages, and vascular endothelial cells [3]. ANGPTL4 plays a critical role in regulating lipid metabolism [4] and is involved in angiogenesis [5] and a variety of diseases, including diabetes, cardiovascular disease, pulmonary fibrosis, psoriasis and cancer [6–10]. In addition, ANGPTL4 has been reported to facilitate wound healing by regulating monocyte differentiation, promoting angiogenesis, and influencing keratinocyte migration and differentiation [11–14]. ANGPTL4 is upregulated in wound tissue within the first few days after skin injury [14,15]. We previously reported that ANGPTL4 expression is increased in cells located in the basal layer of the periwound epidermis during the early stage of wound healing in mice and that deletion of the Angptl4 gene in mice results in a reduction in EpSCs in the periwound epidermis and a delay in wound re-epithelialization [16]. In addition, overexpression of Angptl4 in EpSCs stimulates their migration and proliferation [16]. These results suggest that after skin injury, ANGPTL4 expression may increase in EpSCs located in the periwound epidermis, which in turn stimulates the migration and proliferation of EpSCs in an autocrine/paracrine manner. However, the regulatory factors governing the expression of ANGPTL4 in the wound healing process and the molecular mechanisms involved in ANGPTL4-induced proliferation of EpSCs remain to be elucidated.

The process of wound healing is divided into four distinct but overlapping phases: hemostasis, inflammation, proliferation, and remodeling. During the proliferative phase, re-epithelialization is critical for restoring the skin barrier. EpSCs in the periwound epidermis are activated to proliferate and migrate into the wound bed, where they differentiate into keratinocytes. Understanding the molecular cues that drive this process is essential for developing novel therapeutic strategies to accelerate wound closure, particularly in chronic wounds where healing is impaired.