JUN promotes hypertrophic skin scarring via CD36 in preclinical in vitro and in vivo models

Abstract

Pathologic skin scarring presents a vast economic and medical burden. Unfortunately, the molecular mechanisms underlying scar formation remain to be elucidated. We used a hypertrophic scarring (HTS) mouse model in which Jun is overexpressed globally or specifically in α-smooth muscle or collagen type I–expressing cells to cause excessive extracellular matrix deposition by skin fibroblasts in the skin after wounding. Jun overexpression triggered dermal fibrosis by modulating distinct fibroblast subpopulations within the wound, enhancing reticular fibroblast numbers, and decreasing lipofibroblasts. Analysis of human scars further revealed that JUN is highly expressed across the wide spectrum of scars, including HTS and keloids. CRISPR-Cas9–mediated JUN deletion in human HTS fibroblasts combined with epigenomic and transcriptomic analysis of both human and mouse HTS fibroblasts revealed that JUN initiates fibrosis by regulating CD36. Blocking CD36 with salvianolic acid B or CD36 knockout model counteracted JUN-mediated fibrosis efficacy in both human fibroblasts and mouse wounds. In summary, JUN is a critical regulator of pathological skin scarring, and targeting its downstream effector CD36 may represent a therapeutic strategy against scarring.

Fig. 1.. Jun overexpression induces scar formation.

(A) Targeting construct of JUN doxycycline (dox)–inducible mice. In JUN mice, the construct at the Rosa 26 locus (rTtA) is coupled with the tetracycline operator minimal promoter (tetO) and leads to Jun overexpression in the presence of dox (top right) but no change to Jun expression without dox (top middle). In the Control mice, Jun is physiologically expressed due to the lack of the rtTA with (bottom right) and without dox (bottom middle). (B) Schematic showing experimental approach: Six-millimeter stented excisional dorsal wounds were created in JUN and Control mice. Dox (20 μl of 2 mg/ml) was administered on the day of surgery and on alternate postoperative days (POD) until complete wound closure (POD 14). Wounds were harvested for fluorescence-activated cell sorting (FACS) and histology (n = 18 mice per group per time point). (C) Representative gross photographs of healed (POD 14) wounds from JUN and Control mice receiving dox (2 mg/ml). White dotted line, healed scar. Scale bar, 0.25 cm. (D) Representative hematoxylin and eosin (H&E)–stained wounds of JUN and Control mice. Scale bars, 75 μm (top) and 25 μm (bottom). Yellow dotted lines show the whorl pattern of hypertrophic scars (HTSs). (E) Comparison of dermal thickness in wounds of JUN and Control mice at POD 14. (F) Representative Masson’s trichrome–stained wounds of JUN and Control mice at POD 14. Comparison of total collagen content (defined as relative mean gray density) from Masson trichrome staining. Scale bar, 100 μm. (G) Immunofluorescently labeled collagen type I (COL1) (red), COL3 (red), and CD31 (green) in JUN and Control mice on POD 14. Scale bars, 100 μm. DAPI, 4′,6-diamidino-2-phenylindole. (H) Picrosirius red–stained wounds of JUN and Control mice on POD 14. Scale bars, 25 μm. Computational quantification of collagen fiber networks evaluating length, branching, brightness, width, and number of fibers in JUN mice. All data are presented as means ± SEM. n = 3 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 2.. Jun drives fibrosis through a specific reticular fibroblast subpopulation.

(A) Schematic showing experimental approach for immune and fibroblast cell contribution (top): Six-millimeter stented excisional dorsal wounds were created in the dorsum of JUN and Control mice. Dox (20 μl of 2 mg/ml) was administered on the day of surgery and on alternative days until complete wound closure on POD 14. Wounds were harvested for FACS to isolate fibroblasts and immune cell populations and to compare wounds histologically. Schematic showing experimental approach for parabiosis experiment and bone marrow transplantation (BMT) with 1 million whole bone marrow cells isolated from JUN mice and transduced to express GFP and luciferase. Six-millimeter stented excisional dorsal wounds were then created on the dorsum of Control parabionts. Dox (20 μl of 2 mg/ml) was administered on the day of surgery and on alternative days until POD 14. Wounds were harvested on POD 14 for FACS and histological assessment of healing wounds. (B) Bar graphs (left) showing the numbers of lipofibroblasts and reticular fibroblasts in JUN and Control mice throughout wound healing (POD 0, 4, 7, and 14). Bar graphs (right) showing % change in numbers of lipofibroblasts and reticular fibroblasts in JUN (compared to Control mice equivalent time point) and Control mice (compared to Control baseline). (C) Immunofluorescent staining for adiponectin (labeling lipofibroblasts and extracellular adiponectin) and α-smooth muscle actin (α-SMA; labeling myofibroblasts and vascular smooth muscle cells) in the wounds of JUN and Control mice on POD 14. Scale bars, 25 μm. (D) Bar graphs (left) showing the total percent of immune cells (CD45⁺), lymphocytes, granulocytes, and monocytes in JUN (compared to Control mice equivalent time point) and Control mice (compared to Control baseline) throughout wound healing (POD 0, 1, 7, and 14). Immunofluorescent images (right) illustrating the different immune cells in JUN and Control mice on POD 14: CD1a (Langerhans’ cells), CD3 (T cells), B220 (B cells), CD11b (or “MAC-1,” monocytes, and macrophages), and CD45 (all hematopoietic cells). Scale bar, 30 μm. Bar graph showing % change in numbers of immune cells in JUN and Control mice (bottom). (E) JUN/Control parabionts 3 weeks post-parabiosis surgery with representative FACS plot showing successful chimerism with 36.7% GFP⁺ CD45⁺ LIVE single cells in the peripheral circulation of Control parabiont 4 weeks post-parabiosis surgery. (F) Representative immunostaining showing GFP⁺ cells in wounds of the Control parabiont at POD 14 within blood vessels and the dermis. (G) A representative wound from a Control parabiont on POD 14, which resembles the wounds of Control mice in Fig. 1C. *P < 0.05, **P < 0.01, and ***P < 0.001. All data are presented as means ± SEM. n = 3 independent experiments. *P < 0.05, **P < 0.01, and ****P < 0.0001.

Fig. 3.. Jun up-regulates fibrotic signaling pathways in reticular fibroblasts and PPARγ signaling in lipofibroblasts.

(A) Schematic showing experimental approach: Lipofibroblast and reticular fibroblasts were isolated by FACS on POD 7 for ATAC- and RNA-seq analysis. (B) Heatmap showing hierarchical clustering of differentially expressed genes (FDR < 0.01, fold change > 2) between JUN and Control in lipofibroblasts (top left) and reticular fibroblasts (top right). Heatmap showing chromatin regions with changes in accessibility (FDR < 0.01, fold change > 2) in JUN versus Control mice in lipofibroblasts (bottom left) and reticular fibroblasts (bottom right). (C) Gene set enrichment analysis showing the most up-regulated GO (top) and Kyoto Encyclopedia of Genes and Genomes (bottom) pathways (based on RNA-seq data) in JUN versus Control mice in lipofibroblasts (yellow) and reticular fibroblasts (red) on POD 7. (D) Schematic showing the up-regulated genes identified from the PPARγ signaling pathway in lipofibroblasts in JUN versus Control mice on POD 7; red stars show significantly up-regulated genes (P < 0.05). Gene expression track analysis of individual genes in the PPARγ signaling pathway up-regulated in lipofibroblasts in JUN versus Control mice on POD 7. Yellow arrow indicates the promoter. (E) Schematic showing significantly up-regulated genes identified from multiple converging fibrotic signaling pathways [Hippo, transforming growth factor–β (TGFβ), bone morphogenic protein (BMP), and wingless (WNT)] in reticular fibroblasts in JUN versus Control mice on POD 7; red stars show significantly up-regulated genes (P < 0.05). Gene expression track analysis of individual genes found in the fibrotic signaling pathways up-regulated in reticular fibroblasts in JUN versus Control mice on POD 7. Yellow arrow indicates the promoter. (F) Genes with significantly increased expression and opening promoter chromatin (distance from TSS <100 kb) (left, red) or decreased expression and closing promoter chromatin (right, blue) in lipofibroblasts (top) and reticular fibroblasts (bottom) in JUN versus Control mice on POD 7 (P < 0.05). Significant genes found to be both up-regulated and with opening chromatin in JUN versus Control mice in reticular (bottom, n = 26) and lipofibroblasts (top, n = 3) (*P < 0.05). All data are presented as means ± SEM.

Fig. 4.. CD36 antagonism minimizes JUN-dependent fibroproliferative activity.

(A) Schematic of experimental approach. Left: Human normal skin (NS), scar, HTS, and keloid specimens were assessed for expression of JUN and fibrogenic fibroblast markers using tissue protein arrays and immunofluorescence (IF). Primary cultures of fibroblasts were derived from skin specimens, and CRISPR-Cas9 was used to delete JUN expression. Human fibroblasts derived from HTSs (hHTSs) with (KO) and without (non-KO) JUN deletion were compared for proliferation, apoptosis, and by RNA- and ATAC-seq. Right: hHTS-derived human fibroblasts were treated with salvianolic acid (SAB; 100 μM) for 48 hours before analysis of COL1 and TGFβ protein secretion, proliferation, apoptosis, and production of reactive oxygen species (ROS). (B) Tissue array showing JUN expression in hNS, non-keloid scar (hSc), and hHTS. Scale bar, 10 μm. (C) Apoptosis and proliferation analysis of HTS fibroblasts with and without KO of JUN over 4 days, assessed using annexin V and 5-Ethynyl-2′-deoxyuridine (EdU) labeling, respectively. (D) Heatmaps from RNA- and ATAC-seq comparative analysis showing differently expressed genes (FDR < 0.01, fold change > 2) and chromatin accessibility regions in HTS and HTS-KO fibroblasts. (E) Bar chart demonstrating the genes that were significantly down-regulated (90), exhibited a decrease in chromatin accessibility (5451), and exhibited both a decrease in chromatin accessibility and were down-regulated (106) in KO versus non-KO HTS fibroblasts (P < 0.05). (F) Bar graph showing the FPKM (fragments per kilobase of exon model per million reads mapped) of CD36 in HTS versus HTS-KO fibroblasts. (G) RNA-seq expression and ATAC-seq tracks for CD36 in HTS fibroblasts. Yellow arrow indicates the promoter. (H) Plated hHTS fibroblasts immunostained with (left) fibroblast-specific protein 1 (FSP-1, purple)/CD36 (red)/DAPI (white) and (right) JUN (green)/CD36 (red). Scale bar, 10 μm. (I) Plated hHTS fibroblasts immunostained with CD36 (green)/JUN (red)/TGF-β (orange, top row) and CD36 (green)/COL1 (orange, bottom row) without (left) and with (right) SAB treatment. Scale bars, 10 μm. (J) RT-qPCR analysis (top); JUN and CD36 expression in hHTS fibroblasts at baseline, after SAB treatment, and in hHTS-KO fibroblasts. Bottom: Adipogenic-associated genes [Adipoq (adiponectin), peroxisome proliferator–activated receptor-γ (Ppar-γ), Perilipin, Fatty acid–binding protein 4 (Fabp4), and Fabp5] in hHTS fibroblasts at baseline, after SAB treatment, and in hHTS-KO fibroblasts (*P < 0.05 and **P < 0.01). (K) hHTS fibroblasts with and without 48 hours of SAB treatment compared for proliferation by EdU staining (top), COL1 secretion (middle), and TGFβ (bottom) secretion. All data are presented as means ± SEM. n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 5.. CD36 antagonism reverses hypertrophic dermal scarring.

(A) Schematic of the experimental approach: Six-millimeter stented excisional dorsal wounds were created in JUN and Control mice. Dox (20 μl of 2 mg/ml) and SAB (100 μM) were administered on the day of surgery and on alternate PODs until complete wound closure (POD 14). Wounds were harvested for FACS and histology (n = 18 mice). Representative gross photographs of healed (POD 14) wounds of JUN (B) and Control (H) mice. Scale bars, 0.25 cm. Representative H&E- and Masson trichrome–stained wounds of JUN (C) and Control (I) mice. Scale bars, 150 μm. Picrosirius red–stained wounds of JUN (D) and Control (J) mice on POD 14. Scale bars, 25 μm. Sections are not adjacent slides but are from the same experimental group. Immunofluorescently labeled Col1 (red) and Col3 (purple) in JUN (F) and Control (L) mice on POD 14. Scale bars, 100 μm. Bar graph showing the numbers of lipofibroblasts and reticular fibroblasts in JUN (E) and Control (K) mice at POD 14. Bar graph showing the total percent of immune cells in JUN (G) and Control (M) mice at POD 14. All data are presented as means ± SEM. n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.