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.

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 stages: hemostasis, inflammation, proliferation, and tissue maturation/remodeling. Some proinflammatory cytokines and growth factors produced by cells at the wound site have been shown to contribute to wound re-epithelialization by regulating keratinocyte migration and/or proliferation, such as tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), IL-6, chemokines, fibroblast growth factor (FGF) family members, epidermal growth factor (EGF), nerve growth factor (NGF), macrophage colony-stimulating factor (GM-CSF), and transforming growth factor-β (TGF-β) [ 17– 19]. Among these factors, TNFα and IL-1β have been demonstrated to stimulate Angptl4 expression in adipocytes [ 20, 21], and EGF and TGF-β1 have been reported to upregulate ANGPTL4 expression in cancer cells [ 22– 24]. It is of interest to investigate whether TNFα, IL-1β, TGF-β1 and the other growth factors mentioned above influence Angptl4 expression in EpSCs during wound healing.

Prolactin (PRL) is synthesized and secreted primarily by lactotrophic cells in the anterior pituitary. It is well known as a regulator of the development of the mammary gland and lactation. The human PRL is encoded by a single PRL gene. However, the mouse Prl family consists of more than 30 paralogous genes that are closely related to PRL [ 25, 26]. In addition to the anterior pituitary gland, many extra-pituitary tissues and organs express PRL or mouse Prl family members, which perform various physiological functions in an autocrine and paracrine manner [ 27– 29]. PRL/Prl and its receptor are expressed in the epidermis, sebaceous gland, hair follicle, sweat gland and dermal fibroblasts [ 30– 34]. The widespread expression of the receptor for PRL/Prl in the epidermis and appendages suggests that locally produced PRL/Prl may regulate the function of various cells in the skin. PRL has been reported to regulate hair growth [30]. However, the reported effect of PRL on keratinocytes is controversial. Girolomoni et al. [35] reported that 0.01–100 ng/mL PRL stimulated keratinocyte proliferation in vitro. Langan et al. [36] reported that high concentration of PRL (400 ng/mL) delayed re-epithelialization in experimentally wounded human skin organ cultures. A comprehensive evaluation of PRL or Prl family expression, regulation, and function in skin wound healing is lacking.

In the present study, we investigated the expressions of proinflammatory cytokines (Tnfα and Il-1β) and growth factors (Ngf, Egf, Fgf7, and Tgf-β1) in periwound tissues, their relationship with Angptl4 expression in the skin wound healing process in mice, and their effect on Angptl4 proliferation in EpSCs. We further explored the mechanisms underlying the proproliferative effect of ANGPTL4 on EpSC via RNA sequencing of EpSCs isolated from Angptl4-knockout and wild-type mice. Our results demonstrated that the aforementioned proinflammatory cytokines and growth factors were elevated in periwound tissue and that these molecules upregulated Angptl4 expression in EpSCs in vitro. ANGPTL4 facilitates EpSC proliferation by upregulating Prl8a6 expression, thereby promoting wound re-epithelialization and healing.

Materials and Methods

Animals and skin wound healing

Angptl4 ^–/– mice on the C57BL/6 background were provided by Professor Weijuan Gong at Yangzhou University Medical College (Yangzhou, China). C57BL/6 mice were crossed with Angptl4 ^–/– mice to produce Angptl4 ^+/– mice. Angptl4 ^–/– mice and their wild-type (WT) littermates were generated by intercrossing Angptl4 ^+/– mice. For the wounding experiments, male mice aged between 6 and 8 weeks were utilized. All procedures involving animals were conducted in compliance with the guidelines of the Animal Care and Use Committee of Suzhou Ruihua Orthopedic Hospital (No. RHGK2022047).

A skin wound was induced in the dorsal region of the mouse following established protocols [16]. Briefly, the mice were anesthetized via intraperitoneal injection with a 25% pentobarbital sodium solution at a dosage of 35 mg/kg body weight. The dorsal fur was shaved, and the area was sterilized with povidone-iodine. A full-thickness wound was then excised from the back of each animal via an 8 mm diameter biopsy punch. Animals were anesthetized, and uninjured skin (day 0) and periwound skin tissues were harvested at various time points after wounding to determine the expressions of Angptl4, Tnfα, Il-1β, Egf, Ngf, Fgf7 , Tgf-1β, and Prl8a6. Alternatively, periwound skin tissues were fixed in 10% formalin and subsequently embedded in paraffin for further detection of ANGPTL4 and EpSC marker β1 integrin expression via immunohistochemical staining.

To determine the contributions of ANGPTL4 and PRL8a6 to skin wound healing, the mice were divided into control siRNA, Angptl4 siRNA, and Prl8a6 siRNA groups and treated with the corresponding siRNAs by intradermal injection into the periwound tissue. The Entranster ^TM- in vivo transfection reagent (Engreen Biosystem Co., Ltd., Beijing, China) was utilized for the delivery of siRNA. A transfection mixture containing 5 μg/μL siRNA was prepared in accordance with the manufacturer’s guidelines. Forty microliters of the transfection mixture was administered intradermally at four distinct sites within the periwound tissue following wounding. The images of wound area were captured every two days until complete healing was observed. The dimensions of the wound area were determined via ImageJ software (NIH Image, Bethesda, USA). In addition, on day 4 post-wounding, the mice were euthanized under anesthesia, and the periwound skin tissues were collected for the detection of Angptl4 and Prl8a6 expression or fixed in 10% formalin for subsequent histological and immunohistochemical analyses. Control siRNA, Angptl4 siRNA and Prl8a6 siRNA were procured from General Biol (Anhui, China). The sequences of the siRNAs are detailed in Supplementary Table S1.

Histology and immunohistochemistry

Skin tissues embedded in paraffin were sectioned to a thickness of 4 μm via a rotary microtome. To observe the regeneration of the epidermis, the tissue sections were subjected to hematoxylin and eosin (H&E) staining (Leica Biosystems, Wetzlar, Germany). Immunohistochemical staining was performed according to previously described methods [37]. To assess the expression of ANGPTL4 in EpSCs and the proliferation of these cells within the regenerated epidermis, serial sections were subjected to staining with primary antibodies targeting ANGPTL4, β1 integrin, or proliferating cell nuclear antigen (PCNA) (Abcam, Cambridge, UK), followed by the application of a horseradish peroxidase-conjugated anti-rabbit IgG antibody (MXB, Fuzhou, China). The sections were subsequently stained with hematoxylin, observed, and photographed under a microscope. ImageJ software (NIH Image) was used to conduct a quantitative analysis of the regenerated epidermis area in the H&E-stained images and the positive staining signals in the immunohistochemical staining images.

Isolation, culture and identification of mouse EpSCs

Newborn C57BL/6 mice were euthanized, and the skin tissues were harvested to isolate EpSCs following established protocols [ 16, 37]. In brief, the epidermis was separated from the dermis by incubating the skin tissue with a 0.25% dispase II solution (Sigma‒Aldrich, St. Louis, USA) at 4°C for 12--14 h. The epidermis was then minced and treated with 0.05% trypsin at 37°C for 15 min. The resulting mixture was filtered through a 70-μm strainer and centrifuged. The resulting cell pellet was suspended in Keratinocyte Growth Medium-2 Bullet Kit (KGM2 medium; Lonza, Basel, Switzerland), and the cells were seeded onto collagen IV-coated Petri dishes. Non-adherent cells were removed following a 10-min incubation at 37°C. The remaining adherent cells, identified as EpSCs, were then cultured in KGM2 medium supplemented with 10 μM Y-27632 (STEMCELL Technologies China Co., Ltd. Shanghai, China).

Reverse transcription and quantitative real-time polymerase chain reaction (RT-qPCR)

RT-qPCR was conducted to evaluate the expression levels of Angptl4 , Tnfα, Il-1β, Egf, Ngf, Fgf7, Tgf-1β, and Prl8a6 in periwound tissues, as well as Angptl4, Prl family members, Ccne2, Ccna2, Ccnb1, Cdk1 , Cdkn2a, and Cdkn2b in EpSCs. In brief, total RNA was extracted from periwound tissues or EpSCs via TRIzol reagent (Invitrogen, Carlsbad, USA). The reverse transcription of mRNA was carried out with the PrimeScript™ RT Reagent Kit (TaKaRa, Dalian, China). The ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) was used for qPCR on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, USA). The 2 ^–ΔΔCT method was used to quantify the relative expression levels of the target genes. Gapdh was utilized as an internal control for measuring mRNA levels. The PCR primers used were obtained from General Biol (Chuzhou, China), and their sequences are provided in Supplementary Table S2.

Western blot analysis

Proteins were extracted from EpSCs with RIPA lysis buffer supplemented with a protease and phosphatase inhibitor cocktail (Beyotime Biotechnology, Shanghai, China). The protein concentration was measured via a BCA protein quantification kit (Vazyme Biotech, Nanjing, China). Western blotting was performed according to routine protocols. Briefly, proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membranes were incubated in 5% nonfat milk (Sangon Biotech, Shanghai, China) at room temperature for 2 h, followed by incubation with anti-ANGPTL4 (Abcam) or anti-GAPDH (Proteintech, Chicago, USA) antibodies at 4°C for 16 h. The membranes were washed and then incubated with an HRP-conjugated goat anti-rabbit IgG (H+L) antibody (ImmunoWay Biotechnology, Plano, USA) for 1 h at room temperature. ANGPTL4 and GAPDH proteins were detected via a SuperPico ECL chemiluminescence kit (Vazyme Biotech), and quantification was conducted via ImageJ software (NIH Image).

RNA sequencing and data analysis

TRIzol reagent (Invitrogen) was used for total RNA extraction from Angptl4 ^-/- and WT EpSCs. RNA sequencing was performed on the BGISEQ-500 platform at BGI Genomics Co. Ltd. (Shenzhen, China), which provided a single-end read length of 50 bp and generated 23.92×10 ⁶ raw reads per sample. The average number of clean reads per sample was 23.82×10 ⁶. The ClusterProfiler package was utilized for GO pathway enrichment analysis of differentially expressed genes [38] .

Cell transfection

The plasmid expressing mouse Angptl4 was constructed as previously described [16]. The Prl8a6 expression plasmid was purchased from General Biol Co., Ltd. (Chuzhou, China). EpSCs were transfected with either control siRNA or various specific siRNAs, or with a control vector or plasmids encoding Angptl4 or Prl8a6 in combination with control siRNA or siPrl8a6, as illustrated in Figure 6 and Figure 7. Transfection was performed via Lipofectamine 3000 (Invitrogen). After 24 h of transfection, the cells were subcultured into 96-well plates and cultured for various durations to assess cell proliferation via the MTT assay. After 48 h of transfection, the cells were harvested for flow cytometric analysis of the cell cycle distribution and for the evaluation of gene expression related to cell cycle transition and regulation through RT-qPCR. The siRNAs targeting Prl8a1, Prl8a6, or Prl8a9 were obtained from GenePharma (Suzhou, China), and the sequences of the siRNAs are provided in Supplementary Table S1.

Figure 6 .

ANGPTL4 stimulates epidermal stem cell proliferation via PRL8a6

Mouse epidermal stem cells (EpSCs) transfected with control siRNA (siControl) or siRNA targeting Prl8a1 (siPrl8a1), Prl8a6 (siPrl8a6) or Prl8a9 (siPrl8a9) were analyzed for cell proliferation via MTT assay (A), and the expression of Prl8a1, Prl8a6, and Prl8a9 was analyzed via RT-qPCR (B). Mouse EpSCs transfected with the control vector or Angptl4-expressing plasmid (Angptl4) in combination with siControl or siPrl8a6 were analyzed for cell proliferation via MTT assay (C) and for the expression of Angptl4 and Prl8a6 via RT-qPCR (D). Data are presented as the mean ± SD, n = 3–5. * P < 0.05, **P < 0.01, ***P < 0.001, compared with siControl-transfected cells (A,B) or vector- and siControl-transfected cells (C, D); ###P < 0.001, compared with EpSCs transfected with siControl in combination with Angptl4 (B) (A, C, D: one-way ANOVA; B: two-tailed Student’s t test).

Figure 7 .

ANGPTL4 facilitates cell cycle progression in epidermal stem cells via PRL8a6

Mouse epidermal stem cells transfected with control vector, Angptl4-expressing plasmid (Angptl4) or Prl8a6-expressing plasmid (Prl8a6) in combination with control siRNA (siControl) or Prl8a6 siRNA (siPrl8a6) were analyzed for cell cycle distribution by flow cytometry (A, B) and for the expression of Angptl4, Prl8a6, cyclin E2, cyclin A2, cyclin B1, Cdk1, Cdkn2a, and Cdkn2b by RT-qPCR. (C‒I). Data are presented as the mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, compared with cells transfected with vector and siControl; #P < 0.05, ###P < 0.001, comparison between cells transfected with Angptl4 and siControl and cells transfected with Angptl4 and siPrl8a6 (one-way ANOVA).

Cell proliferation assay

The proliferation of the EpSCs was assessed via the MTT assay [39]. The cells were cultured in a 96-well plate for the indicated time. Then, 10 μL of 5 mg/mL MTT was added to each well. After incubation for 4 h at 37 °C, the culture medium was discarded and 150 μL of DMSO was added to each well to solubilize the formazan crystals. A Multiskan ^TM Spectrum Microplate Reader (Thermo Fisher Scientific, Waltham, USA) was used to measure the optical density (OD) at 490 nm.

Flow cytometry analysis

Flow cytometry was used to assess the expressions of biomarkers of EpSC. Briefly, mouse EpSCs were incubated with FACS buffer consisting of 5% FBS and 1% goat serum in PBS at 25°C for 1 h. The cells were then incubated with PE-conjugated anti-CD71 and FITC-conjugated anti-CD49f antibodies (BD Biosciences, San Jose, USA) in the dark for 30 min. Additionally, PE- or FITC-conjugated IgG2a (BD Biosciences) served as an isotype control. After washing and resuspension, the cells were analyzed via a flow cytometer (Beckman Coulter, Brea, USA).

The distribution of the cell cycle was assessed via flow cytometry. EpSCs were fixed in 75% ethanol at 4°C overnight. After centrifugation, the cells were incubated with PI/RNase staining buffer (BD Biosciences) for 30 min in the dark. The content of the DNA was quantified via a flow cytometer (Beckman Coulter).

Statistical analysis

Each experiment was repeated at least three times. All the data are expressed as the mean ± SD. GraphPad Prism 9 software was used for statistical analysis. Differences between two groups were analyzed by two-tailed Student’s t test or one-way ANOVA, as indicated in the figure legend. Statistical significance was defined as a P value of less than 0.05.

Results

ANGPTL4 is upregulated in EpSCs in periwound skin tissue during skin wound healing

We previously reported that the expression of ANGPTL4 was increased in cells in the basal layer of the epidermis of periwound skin tissue during wound healing in mice [16]. To determine whether ANGPTL4 is expressed by EpSCs, periwound skin tissues were harvested from mice with full-thickness skin defects at different time points after wounding, and immunohistochemical staining of serial sections was performed to detect ANGPTL4 and the EpSC marker β1 integrin. ANGPTL4 ⁺ cells were localized in and above the basal layer of the epidermis in the periwound tissue. β1 integrin ⁺ cells were exclusively present in the basal layer of the epidermis. The number of these cells increased significantly over time following skin wounding ( Figure 1A,B). Notably, most cells in the basal layer were double positive for ANGPTL4 and β1 integrin ( Figure 1A). These results demonstrate that ANGPTL4 is upregulated in EpSCs during wound healing.

Figure 1 .

Expression of ANGPLT4 and β1 integrin in periwound tissue during wound healing in mice

(A) Representative images of immunohistochemical staining for ANGPTL4 (red) and β1 integrin (brown) in uninjured skin tissue (day 0) and serial sections of periwound tissues at various time points in the wound healing process. (B) Quantification of ANGPTL4- and β1 integrin-positive cells via ImageJ software. Data are presented as the mean ± SD, n = 3 mice/group. *P < 0.05, compared with uninjured skin tissue (one-way ANOVA). Scale bars: 50 μm and 25 μm in the left and right panels, respectively, of the β1 integrin and ANGPTL4 immunohistochemical staining images.

Proinflammatory cytokines and growth factors upregulate ANGPTL4 expression in EpSCs

It has been reported that the expressions of proinflammatory cytokines (TNFα and IL-1β) and growth factors (EGF, NGF, FGF7, and TGF-β1) are increased in periwound skin tissue and contribute to wound healing [40]. To investigate whether these molecules contribute to the upregulation of ANGPTL4 in EpSCs during wound healing, we first collected periwound tissues at different time points after wounding and examined the expressions of Angptl4 and these molecules via RT-qPCR. The mRNA levels of Angptl4, Tnfα, Il-1β , Egf, Ngf, Fgf7, and Tgf-β1 increased in a time-dependent manner in the wound healing process ( Figure 2A–G). Spearman correlation analysis revealed a positive correlation between the increase in Angptl4 mRNA level and the increase in the mRNA levels of Tnfα , Il-1β, Egf, Ngf, Fgf7, and Tgf-β1 ( Figure 2H–M), indicating that the expression of Angptl4 in EpSCs in the periwound tissue may be upregulated by these molecules in the wound healing process.

Figure 2 .

Expressions of Angptl4, proinflammatory cytokines and growth factors in periwound skin tissues during wound healing

(A–G) The mRNA levels of Angptl4, tumor necrosis factor α (Tnfα), interleukin 1β (Il-1β), epidermal growth factor (Egf), nerve growth factor (Ngf), fibroblast growth factor ( Fgf7), and transforming growth factor β1 (Tgf-β1) in uninjured skin (day 0) and the periwound skin tissues at different time points in the wound healing process were detected via RT-qPCR. (H–M) Spearman correlation analysis between the mRNA levels of Angptl4 and those of Tnfα (H), Il-1β (I), Egf (J), Ngf (K), Fgf7 (L), or Tgf-β1 (M). Data are presented as the mean ± SD, n = 3 mice/group. *** P < 0.001, compared with uninjured skin tissue (one-way ANOVA).

We then investigated the effects of TNFα, IL-1β, NGF, EGF, FGF7 and TGF-β1 on Angptl4 expression in EpSCs isolated from neonatal mouse skin tissue and cultured in vitro. These cells exhibited a cobblestone morphology under light microscopy and expressed high levels of the EpSC markers α6 integrin, β1 integrin, and CK19 and low levels of the cell differentiation marker CD71 ( Supplementary Figure S1). We first reviewed the literature to determine the optimal concentration of each of the above proinflammatory cytokines and growth factors to stimulate the expression of Angptl4 or other genes in other cell types. We then performed time-course experiments in which these concentrations were used to treat EpSCs to determine the effects of these molecules on Angptl4 mRNA expression and to determine the optimal time at which maximum Angptl4 mRNA expression was achieved. Time-course studies revealed that all the molecules tested were able to stimulate Angptl4 mRNA expression in mouse EpSCs, and the maximum expression of Angptl4 was achieved by stimulation with 5 ng/mL TNFα or 100 ng/mL IL-1β for 6 h ( Figure 3A,B), 40 ng/mL NGF for 12 h ( Figure 3C), 50 ng/mL EGF or 25 ng/mL FGF7 for 24 h ( Figure 3D,E), and 5 ng/mL TGF-β1 for 36 h ( Figure 3F). We then performed dose-response experiments by stimulating EpSCs with different concentrations of the above proinflammatory cytokines or growth factors for the optimal times obtained in the time course studies to determine the optimal concentration to induce the highest expression of Angptl4 mRNA. The results revealed that Angptl4 mRNA expression in EpSCs peaked after treatment with 5 ng/mL TNFα, 100 ng/mL IL-1β for 6 h, 40 ng/mL NGF for 12 h, 60 ng/mL EGF or 25 ng/mL FGF7 for 24 h, or 10 ng/mL TGF-β1 for 36 h ( Figure 3G–L). Western blot analysis revealed that TNFα, IL-1β, NGF, EGF, FGF7 and TGF-β1 also upregulated ANGPTL4 expression at the protein level ( Figure 3M,N). Taken together, these results indicate that the upregulation of TNFα, IL-1β, EGF, NGF, FGF7, and TGF-β1 in periwound tissues after skin injury induces Angptl4 expression in EpSCs.

Figure 3 .

Effects of proinflammatory cytokines and growth factors on Angptl4 expression in mouse epidermal stem cells

(A–L) Mouse epidermal stem cells (EpSCs) were treated with 5 ng/mL TNFα, 100 ng/mL IL-1β, 40 ng/mL NGF, 50 ng/mL EGF, 25 ng/mL FGF, or 5 ng/mL TGF-β1 for different durations (A-F) or with different concentrations of TNFα or IL-1β for 6 h (G,H), NGF for 12 h (I), EGF or FGF7 for 24 h (J,K), or TGFβ1 for 36 h (L), and ANGPTL4 expression was assessed by RT-qPCR. (M,N) Mouse EpSCs were treated with 5 ng/mL TNFα, 100 ng/mL IL-1β, 40 ng/mL NGF, 60 ng/mL EGF, 25 ng/mL FGF7, or 10 ng/mL TGF-β1 for 24 h, and ANGPTL4 protein expression was assessed via western blot analysis (M) and quantified via ImageJ software (N). Data are presented as the mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, compared with untreated cells (one-way ANOVA). The abbreviations used in this figure are the same as those used in Figure 2.

ANGPTL4 promotes the expressions of prolactin family members and molecules involved in cell cycle progression and regulation

Our previous study revealed that Angptl4 deficiency in mice impaired EpSC proliferation and delayed re-epithelialization of cutaneous wounds [16]. However, the molecular mechanisms involved remain poorly understood. We performed RNA sequencing of EpSCs from Angptl4 ^–/– and WT mice to obtain a global view of the transcriptome profile. GO pathway analysis of biological processes revealed pathways related to cell proliferation and regulation ( Figure 4A) and cell cycle phase transitions and regulation ( Figure 4B). Among the pathways related to epithelial cell proliferation and regulation shown in Figure 4A, the mRNA levels of cyclin-dependent kinase inhibitor 2a ( Cdkn2a) and Cdkn2b, whose translational proteins are inhibitors of cyclin-dependent kinase 4 ( Cdk4) and Cdk6, respectively, were increased in Angptl4-deficient EpSCs ( Figure 4C). In addition, the expression levels of Prl2a1, Prl8a1, Prl8a6, and Prl8a9, which are members of the mouse Prl family that are homologous to human PRL, were decreased in Angptl4-deficient EpSCs. The pathways in Figure 4B revealed a decrease in Ccna2 (cyclin A2), Ccne2 (cyclin E2), Ccnb1 (cyclin B1), and Cdk1, and an increase in Cdkn2A and Cdkn2b in Angptl4-deficient EpSCs ( Figure 4C). The altered expressions of these genes in Angptl4-deficient EpSCs were validated by RT-qPCR ( Figure 4D). Since PRL has been reported to stimulate the proliferation of keratinocytes and other cell types [ 41, 42], the above results suggest that the expressions of the Prl family members Prl2a1 , Prl8a1, Prl8a6, and Prl8a9 and molecules involved in cell cycle progression (cyclin E2, cyclin A2, cyclin B1, and Cdk1) and regulation ( Cdkn2a and Cdkn2b) are regulated by ANGPTL4 and may be involved in ANGPTL4-induced EpSC proliferation.

Figure 4 .

Gene Ontology (GO) enrichment analysis and validation of differentially expressed genes between Angptl4-deficient and wild-type epidermal stem cells

(A-B) The top 12 pathways related to cell proliferation and regulation (A) and the top 16 pathways related to cell cycle phase transitions and regulation (B) were obtained from GO pathway analysis of biological processes based on differentially expressed genes between Angptl4 knockout (KO) and wild-type (WT) epidermal stem cells. (C-D) Differentially expressed genes involved in cell proliferation and cell cycle regulation in the EpSCs from the WT and Angptl4-KO mice were detected by RNA sequencing (C) and confirmed by RT-qPCR (D). Data are presented as the mean ± SD, n = 3. **P < 0.01, ***P < 0.001 (two-tailed Student′s t test).

We then evaluated the effects of ANGPTL4 on the expressions of Prl2a1 , Prl8a1, Prl8a6, Prl8a9, cyclin A2, cyclin B1, Cdk1, Cdkn2a and Cdkn2b by transfecting EpSCs from WT mice and Angptl4-KO mice with a control vector or Angptl4-expressing plasmid. RT-qPCR results revealed that overexpression of ANGPTL4 in WT EpSCs ( Figure 5A) had no significant effect on Prl2a1 mRNA expression ( Figure 5B) but significantly elevated the mRNA levels of Prl8a1, Prl8a6, Prl8a9, cyclin A2, cyclin B1, cyclin E2 and Cdk1 ( Figure 5C–I) and significantly reduced the mRNA levels of Cdkn2A and Cdkn2B ( Figure 5J,K). Transfection of Angptl4-deficient EpSCs with an Angptl4-expressing plasmid resulted in similar changes in these expressions ( Figure 5A–K). These results demonstrated that upregulation of ANGPTL4 in EpSCs stimulated the expressions of Prl8a1, Prl8a6, Prl8a9, cyclin A2, cyclin B1, cyclin E2, and Cdk1 and inhibited the expression of Cdkn2A and Cdkn2B.

Figure 5 .

Effect of ANGPTL4 on the expressions of prolactin family members and molecules involved in cell cycle regulation in epidermal stem cells

Epidermal stem cells from wild-type (WT) and Angptl4-knockout (KO) mice were transfected with control vector or Angptl4-expressing plasmid (Angptl4). The expressions of Angptl4 (A), prolactin family members (B–E), cyclin family members (F–H), cyclin-dependent kinase 1 (Cdk1) (I), cyclin-dependent kinase inhibitor 2a (Cdkn2a) and Cdkn2b (J,K) were evaluated by RT-qPCR. n=3. Data are presented as the mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student′s t test).

ANGPTL4 promotes EpSC proliferation by accelerating PRL8a6-mediated G1 to S and G2 cell cycle transitions

We further investigated whether PRL8a1, PRL8a6, and PRL8a9 could stimulate EpSC proliferation and mediate the proproliferative effect of ANGPTL4 on EpSCs. The expressions of Prl8a1, Prl8a6 or Prl8a9 in mouse EpSCs were inhibited by RNA interference. MTT assays revealed that the knockdown of Prl8a6 but not Prl8a1 or Prl8a9 in EpSCs significantly inhibited cell proliferation ( Figure 6A,B). While Angptl4 overexpression in EpSCs significantly increased Prl8a6 mRNA expression and promoted cell proliferation, Prl8a6 knockdown reversed Angptl4 overexpression-induced Prl8a6 expression and EpSC proliferation ( Figure 6C,D). These results indicate that PRL8A6 is essential for EpSC proliferation under resting conditions and mediates the proproliferative effect of ANGPTL4 on EpSCs.

To elucidate the mechanisms involved in ANGPTL4- and PRL8a6-induced EpSC proliferation, flow cytometry was used to assess the influence of ANGPTL4 and PRL8a6 on the cell cycle distribution of mouse EpSCs. Overexpression of ANGPTL4 or PRL8a6 in EpSCs resulted in a notable reduction in the population of cells in the G1 phase and a significant increase in the populations of cells in the S and G2 phases. Knockdown of Prl8a6 in EpSCs reversed the ANGPTL4-induced changes in the cell cycle distribution ( Figure 7A,B). These results indicate that PRL8a6 not only promotes cell cycle progression from the G1 phase to the S and G2 phases but also mediates ANGPTL4-induced cell cycle progression in EpSCs. Since Angptl4 deletion in EpSCs decreased the mRNA levels of Prl8a6, cyclin A2, cyclin B1, cyclin E2, and Cdk1 and increased the expression of Cdkn2a and Cdkn2b mRNA ( Figure 4C,D), we investigated whether ANGPTL4 regulates these molecules through PRL8a6. The RT-qPCR results demonstrated that overexpression of Angptl4 or PRL8a6 in the EpSCs led to a significant increase in the expression of cyclin A2, cyclin B1, cyclin E2, and Cdk1 and a pronounced reduction in the expression of CDK4 and the CDK6 inhibitors Cdkn2a and Cdkn2b ( Figure 7D–I). These findings indicate that ANGPTL4 and PRL8a6 promote the G1 to S phase transition by increasing the expression of cyclin E2, downregulating the expression of Cdkn2a, and promoting the G2 to M phase transition by increasing the expressions of cyclin A2, cyclin B1, and Cdk1. Further studies revealed that the knockdown of Prl8a6 in EpSCs effectively reversed ANGPTL4-induced alterations in these mRNAs, with the exception of Cdkn2b ( Figure 7C–I). These results indicate that ANGPTL4 facilitates G1 to S and G2 cell cycle progression in EpSCs through PRL8a6, thereby promoting cell proliferation.

ANGPTL4 is upregulated in periwound skin tissues and promotes the proliferation of EpSC and wound re-epithelialization through PRL8a6

To determine whether ANGPTL4 enhances EpSC proliferation during wound healing via PRL8a6, we first examined the expression of Angptl4 and Prl8a6 in periwound tissues in the skin wound healing process in mice. RT-qPCR results demonstrated that the mRNA levels of Angptl4 and Prl8a6 increased over time in the process of wound healing ( Figure 8A,B). Spearman correlation analysis revealed a positive correlation between the mRNA levels of Prl8a6 and Angptl4 ( Figure 8C). We then investigated the contribution of ANGPTL4 and PRL8A6 to the proliferation of EpSCs and the regeneration of epidermis by inhibiting Angptl4 and Prl8a6 expressions in the periwound epidermis through intradermal injection of siRNAs targeting Angptl4 and Prl8a6, respectively. Injection of either Angptl4 siRNA or Prl8a6 siRNA significantly delayed skin wound healing, especially on days 2--6 after wounding ( Figure 8D,E). The periwound skin tissues were harvested on day 4 after the injection. H&E staining of the periwound skin tissue sections revealed a notable reduction in the regenerated epidermis in Angptl4 siRNA- or Prl8a6 siRNA-administered mice compared with that in the control siRNA-administered mice ( Figure 8F,G). Immunohistochemical staining of serial sections of periwound skin tissues revealed that in the control siRNA-injected tissue, β1 integrin-positive EpSCs were predominantly localized in the basal layer of the epidermis, with the majority expressing the cell proliferation marker PCNA. The administration of Angptl4 siRNA or Prl8a6 siRNA resulted in a notable decrease in the proportion of cells that exhibited dual positivity for PCNA and β1 integrin in the regenerated epidermis ( Figure 8H,I). RT-qPCR analysis revealed that the mRNA levels of Angptl4 and Prl8a6 were significantly elevated following skin injury, and the administration of Angptl4 siRNA and Prl8a6 siRNA in the periwound epidermis resulted in a significant reduction in their respective mRNA levels, with Angptl4 siRNA also leading to a notable decrease in Prl8a6 expression ( Figure 8J). These findings indicate that the upregulation of ANGPTL4 in periwound tissue in the wound healing process enhances the expression of Prl8a6. Collectively, these results demonstrate that Angptl4 is upregulated in periwound tissue following skin injury, thereby promoting EpSC proliferation and wound re-epithelialization through the induction of Prl8a6 expression.

Figure 8 .

Knockdown of Angptl4 or Prl8a6 in periwound skin tissue inhibits epidermal stem cell proliferation and delays cutaneous wound healing in mice

(A–C) Detection of Angptl4 and Prl8a6 mRNA expression in uninjured skin (day 0) and periwound tissue after wounding by RT-qPCR (A,B) and Spearman correlation analysis between Angptl4 and Prl8a6 mRNA levels (C). (D–J) Control siRNA (siControl), Angptl4 siRNA (siAngptl4), or Prl8a6 siRNA (siPrl8a6) was injected intradermally into the periwound skin tissue after wounding. The wounds were photographed (D), and the wound area was quantified (E). The periwound skin tissues were harvested on day 4 for H&E staining (F), immunohistochemical staining for β1 integrin and PCNA in serial sections (H,I), and RT-qPCR detection of Angptl4 and Prl8a6 mRNA expression (J). D, F, and H are representative images. The wound area (E), regenerated epidermis (G), and β1 integrin- and PCNA-positive cells (I) were quantified via ImageJ software. Data are presented as the mean ± SD. A, B, G, I and J: n = 3 mice/group. E: n = 6 mice/group. *P < 0.05, **P < 0.05, *** P < 0.001, compared with uninjured skin (A, B, J) or periwound skin tissue injected with siControl (E, G, I); ###P < 0.001, compared with periwound skin tissue injected with siControl (J) (one-way ANOVA). Scale bars: 50 μm in F and 50 μm and 25 μm in the left and right panels of H, respectively.

Discussion

The present study sought to elucidate the microenvironmental factors that regulate Angptl4 expression during skin wound healing in mice. Our findings revealed a positive correlation between Angptl4 mRNA expression and the mRNA levels of proinflammatory cytokines (Tnfα, Il-1β) or growth factors (Ngf, Egf, Fgf7 and Tgf-β1) in periwound tissue. These factors augment Angptl4 expression in EpSCs. Furthermore, the mechanisms underlying ANGPTL4-induced EpSC proliferation were investigated, revealing that ANGPTL4 promotes EpSC proliferation by upregulating Prl8a6 expression. Our in vivo studies demonstrated that ANGPTL4 facilitated skin wound healing by enhancing the proliferation of EpSCs at the wound edge epidermis through the upregulation of Prl8a6 expression.

Skin wound healing is a complex process involving a diverse array of cell types, including neutrophils, macrophages, EpSCs, endothelial cells and fibroblasts. The migration, proliferation, and differentiation of these cells are regulated by a range of cytokines and growth factors that are produced at the site of the wound [19]. TNFα, IL-1β, FGF-7, EGF, NGF, and TGF-β play a role in wound re-epithelialization by stimulating keratinocyte migration and/or proliferation [ 17– 19]. Our study revealed that in the skin wound healing process, ANGPTL4 was upregulated in EpSCs located in the periwound skin tissue, and the expressions of the aforementioned proinflammatory cytokines and growth factors were increased in the periwound tissue and could stimulate Angptl4 expression in EpSCs, indicating that Angptl4 is upregulated by these microenvironmental factors in the wound healing process. As ANGPTL4 promotes EpSC proliferation during wound healing, our studies identified not only microenvironmental regulators of Angptl4 but also novel mechanisms involved in wound re-epithelialization induced by TNFα and IL-1β, FGF-7, EGF, NGF, and TGF-β in the skin wound healing process.

TNFα stimulates ANGPTL4 expression in adipocytes via a FOXO1-dependent signaling pathway [20], IL-1β induces ANGPTL4 expression in adipocytes by activating the NF-κB and JNK signaling pathways [21], EGF upregulates the expression of ANGPTL4 in head and neck squamous cell carcinoma by inducing COX-2 expression [23], and TGF-β induces ANGPTL4 expression in breast cancer cells through the Smad signaling pathway [24]. It is possible that TNFα, IL-1β, EGF and TGF-β stimulate Angptl4 expression in mouse EpSCs through the same signaling pathways as those in other cells. However, additional research is necessary to substantiate this hypothesis. The mechanisms involved in NGF- and FGF7-induced Angptl4 expression in EpSCs require further investigation.

The mechanisms underlying ANGPTL4-induced EpSC proliferation were investigated through the analysis of differentially expressed genes between Angptl4-deficient EpSCs and WT EpSCs via RNA sequencing. Bioinformatics analysis of RNA sequencing data and gain-of-function studies revealed that overexpression of Angptl4 in EpSCs resulted in the upregulation of Prl8a1, Prl8a6 , Prl8a9, cyclin E2, cyclinA2, cyclinB1, and Cdk1, whereas the expressions of Cdkn2a and Cdkn2b were downregulated. Among the Prl family members, only PRL8a6 was identified as a key regulator of EpSC proliferation under resting conditions. PRL8a6 mediates ANGPTL4-induced EpSC proliferation by regulating cell cycle, specifically by decreasing the cell population in the G1 phase and increasing the cell populations in the S and G2 phases. Moreover, PRL8a6 was identified as a mediator of ANGPTL4-induced upregulation of cyclins E2, A2, and B1, as well as Cdk1 expression, and downregulation of Cdkn2A expression. Cyclins and CDKs play pivotal roles in regulating the progression of the cell cycle [43]. CDKN2A functions as a negative regulator of CDK4, which is critical for the transition from quiescence to the G1 phase and subsequent progression throughout the G1 phase. Cyclin E2 plays a role in the transition from the G1 phase to the S phase by activating CDK2. Cyclin A2 is involved in the replication of DNA during S phase and the transition from G2 phase to M phase through the activation of CDK2 and CDK1, respectively. Cyclin B controls the transition from the G2 phase to the M phase by activating CDK1 [43]. Therefore, our findings indicate that PRL8a6 accelerates cell cycle progression by modulating the expression of CDK1 and the regulators of CDK1/2/4, thereby promoting EpSC proliferation.

Finally, we investigated whether ANGPTL4 induces EpSC proliferation via PRL8a6 in the wound healing process. We found that knockdown of either Angptl4 or Prl8a6 in periwound tissues impaired EpSC proliferation in the periwound epidermis, decreased epidermal regeneration, and delayed wound healing. Since Prl8a6 mRNA levels were positively correlated with Angptl4 mRNA levels in periwound tissues during wound healing, Angptl4 knockdown in periwound tissues significantly decreased Prl8a6 mRNA level, and our in vitro study demonstrated that ANGPTL4 stimulated EpSC proliferation by upregulating Prl8a6 in EpSCs. These in vivo results strongly support that ANGPTL4 facilitates the proliferation of EpSCs during wound healing by increasing Prl8a6 expression. To the best of our knowledge, this is the first investigation revealing the expression of PRL8a6 in EpSCs and its significant role in the proliferation of EpSCs under both resting conditions and in the process of skin wound re-epithelialization. PRL interacts with PRLR, resulting in the recruitment of JAK2, which subsequently activates various downstream signaling pathways, including the STAT3, STAT5, ERK, PI3K, and AKT pathways [44]. PRL has been reported to stimulate the proliferation of pancreatic β cells by facilitating the transition of the cell cycle from the G1 phase to the S and G2/M phases through the activation of the STAT5 signaling pathway [41]. Additionally, PRL has been shown to regulate cell cycle progression in testicular cells via the JAK2/STAT5 pathway [42]. As a homolog of PRL, PRL8a6 may also stimulate EpSC proliferation through the JAK2/STAT5 pathway. However, this possibility requires further investigation.

To our knowledge, this is the first report demonstrating that PRL8a6 is upregulated in periwound tissue and contributes to wound re-epithelialization and healing. PRL8a6 and its upstream stimulator ANGPTL4 are potential therapeutic targets for the treatment of skin wounds, such as the development of recombinant proteins and gene expression activators.

In summary, our study demonstrated that following skin injury, elevated levels of proinflammatory cytokines and growth factors within the periwound tissue stimulate the expression of Angptl4 in EpSCs, which in turn stimulates the proliferation of EpSCs by inducing Prl8a6 expression, thereby facilitating skin wound re-epithelialization and healing. ANGPTL4 and PRL8a6 are potential therapeutic targets for the treatment of skin wounds.

Supplementary Data

Supplementary data is available at Acta Biochimica et Biophysica Sinica online.

COMPETING INTERESTS

The authors declare that they have no conflict of interest.

Funding Statement

This work was supported by grants from the Suzhou Science and Technology Development Program (Nos. SKY2023108 and SKY2023107), Suzhou Municipal Science and Technology Bureau (No. SKYD2022074), and Wuzhong District Science and Technology Program (Nos. WZYW2022034 and WZYW2021019).