ATF7 drives diabetic wound healing via NOTCH1 repression and N1ICD-dependent macrophage polarization control
Pengcheng Xu, Yuan Xue, Linlin Feng, Jingwen Kuang, Xiaochen Hu, Huiyi Tang, Biao Cheng, Limin Wei
Pengcheng Xu, Yuan Xue, Linlin Feng, Jingwen Kuang, Xiaochen Hu, Huiyi Tang, Biao Cheng, Limin Wei
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Research Article Dermatology Inflammation

ATF7 drives diabetic wound healing via NOTCH1 repression and N1ICD-dependent macrophage polarization control

Abstract

Chronic, non-healing wounds are a severe diabetic complication. The underlying mechanisms are not fully understood, and the role of ATF7 in this context has not been well characterized. In our study, we utilized db/db diabetic mice and AAV-mediated keratinocyte-specific Atf7 overexpression in vivo. HaCaT keratinocyte/THP-1 macrophage cocultures under high glucose were used in vitro. Our results showed that ATF7 was upregulated in diabetic wounds. Keratinocyte-specific Atf7 overexpression accelerated diabetic wound closure, enhanced re-epithelialization, granulation tissue formation, and keratinocyte proliferation, while suppressing macrophage M1 polarization and inflammation. Multiomics screening identified NOTCH1 as a key ATF7 target. ATF7 transcriptionally repressed NOTCH1 by recruiting Suv39h1, increasing H3K9me3 at the NOTCH1 promoter. This reduced NOTCH1 protein and its active intracellular domain (N1ICD) within keratinocyte-derived exosomes. ATF7-overexpressing keratinocyte exosomes carried less N1ICD, leading to decreased N1ICD transfer to macrophages and subsequent inhibition of M1 polarization. Notably, local injection of exosomes from ATF7-overexpressing keratinocytes accelerated wound healing in db/db mice. In summary, ATF7 promotes diabetic wound healing by repressing NOTCH1 transcription via H3K9me3, thereby reducing exosomal N1ICD secretion from keratinocytes and inhibiting macrophage M1 polarization. This identifies the ATF7/NOTCH1/exosome axis as a therapeutic target.

Authors

Pengcheng Xu, Yuan Xue, Linlin Feng, Jingwen Kuang, Xiaochen Hu, Huiyi Tang, Biao Cheng, Limin Wei

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Figure 5

ATF7 overexpression inhibits macrophage M1 polarization.

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ATF7 overexpression inhibits macrophage M1 polarization. (A) Experiment...
(A) Experimental workflow: Human monocytic THP-1 cells were differentiated into M0 macrophages (THP-1-M0) using 20 nM PMA for 36 hours. HaCaT cells were infected with lentivirus for 48 hours, followed by coculture with THP-1-M0 macrophages in a Transwell system for 24 hours (upper chamber, HaCaT; lower chamber, macrophages). (B) mRNA expression of M1 markers (NOS2, CD86, and MHCII) in cocultured macrophages was measured by qRT-PCR. (C) Flow cytometric analysis of MHCII+ cells in cocultured macrophages. (D) Flow cytometric analysis of CD86+ cells in cocultured macrophages. (E) mRNA expression of proinflammatory cytokines (TNFA, IL6, and IL1B) in cocultured macrophages were measured by qRT-PCR. HaCaT cells were infected with LV-NC or LV-ATF7 and cocultured with THP-1–derived macrophages under HG conditions. In the rescue group (LV-ATF7 + LPS/IFN-γ), macrophages were additionally treated with LPS and IFN-γ during coculture to force M1 polarization. (F) mRNA expression of M1 markers (CD86 and MHCII) in macrophages was measured by qRT-PCR. (G) Flow cytometric analysis of CD86+ cells in cocultured macrophages. (H) Cell viability was assessed by the CCK-8 assay. (I and J) Cell migration ability was analyzed by the scratch wound healing assay. Scale bar: 200 μm. (K) Representative immunofluorescence images of Ki67 expression and quantitative analysis for Ki67+ cells. Nuclei counterstained with DAPI. Scale bar: 50 μm (original magnification: ×400). Results are expressed as mean ± SD. **P < 0.01; ***P < 0.001 by 2-tailed, unpaired Student’s t test (B–E) or 1-way ANOVA with Bonferroni’s post hoc test (F–H, J, and K). All cell experiments were performed with n = 3 replicates.