ABSTRACT

Macroautophagy/autophagy is a cellular catabolic process that is implicated in several physiological and pathological processes. However, the role of epidermal autophagy in wound healing remains unknown. Here, using mice with genetic ablation of the essential Atg5 (autophagy related 5) or Atg7 (autophagy related 7) in their epidermis to inhibit autophagy, we show that keratinocyte autophagy regulates wound healing in mice. Wounding induces the expression of autophagy genes in mouse skin. Epidermis-specific autophagy deficiency inhibits wound closure, re-epithelialization, keratinocyte proliferation and differentiation, dermal granulation tissue formation, and infiltration of immune cells including macrophages, neutrophils, and mast cells, while it does not affect angiogenesis. Using cytokine array screening, we found that autophagy deficiency inhibits the transcription and production of the cytokine CCL2/MCP-1 by TNF. At the molecular level, TNF induces autophagic flux and the expression of autophagy genes through NFKB in epidermal keratinocytes. TNF promotes CCL2 transcription through the autophagy-AMPK-BRAF-MAPK1/3/ERK-activator protein 1 (AP1) pathway. Indeed, treating mice with recombinant CCL2 can reverse the effect of autophagy deficiency in keratinocytes. At the cellular level, we found that CCL2 induction via autophagy in keratinocytes is required not only for keratinocyte migration and proliferation but also for dermal fibroblast activation. Our findings demonstrate a critical role of epidermal autophagy in wound healing in vivo and elucidate a critical molecular machinery coordinating keratinocyte-fibroblast interaction in skin repair.

Abbreviations: ACTA2/α-SMA: actin alpha 2, smooth muscle; ACTB: β-actin; ADGRE1: adhesion G protein-coupled receptor E1; AMPK: AMP-activated protein kinase; AP1: activator protein 1; AP1-RE: AP1 response element; ATG: autophagy-related; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; BRAF: B-Raf proto-oncogene, serine/threonine kinase; C5: complement C5; CCL2/MCP-1: C-C motif chemokine ligand 2; CCL3: C-C motif chemokine ligand 3; CK: cytokeratin; cKO: conditional knockout; CRTC1: CREB-regulated transcription coactivator 1; CXCL1: C-X-C motif chemokine ligand 1; CXCL2: C-X-C motif chemokine ligand 2; ECM: extracellular matrix; EGF: epidermal growth factor; FGF7: fibroblast growth factor 7; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HBEGF: heparin binding EGF like growth factor; HPRT1: hypoxanthine phosphoribosyltransferase 1; IHC: immunohistochemical; IL1B: interleukin 1 beta; KRT10: keratin 10; KRT14: keratin 14; MAP1LC3B/LC3B-I/II: microtubule-associated protein 1 light chain 3 beta; MAPK1/3/ERK: mitogen-activated protein kinase 1/3; MKI67/Ki-67: marker of proliferation; MPO: myeloperoxidase; NFKB: NF-kappa B, nuclear factor kappa-light-chain-enhancer of activated B cells; NFKB-RE: NFKB response element; PDGF: platelet-derived growth factor; PECAM1: platelet and endothelial cell adhesion molecule 1; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; RELA/p65: RELA proto-oncogene, NFKB subunit; shCON: small hairpin negative control; siNC: negative control; siRNA: small interfering RNA; SP1: sp1 transcription factor; SQSTM1/p62: sequestosome 1; TGFA: transforming growth factor alpha; TGFB1: transforming growth factor beta 1; TIMP1: TIMP metallopeptidase inhibitor 1; TNF/TNF-alpha: tumor necrosis factor; TREM1: triggering receptor expressed on myeloid cells 1; WT: wild-type

摘要

巨自噬/自噬是一种细胞分解代谢过程，涉及多种生理和病理过程。然而，表皮自噬在伤口愈合中的作用仍然未知。在这里，使用对必需Atg5（自噬相关 5）或Atg7进行基因消融的小鼠（自噬相关 7）在它们的表皮中抑制自噬，我们表明角质形成细胞自噬调节小鼠的伤口愈合。伤口诱导小鼠皮肤中自噬基因的表达。表皮特异性自噬缺乏抑制伤口闭合、上皮再形成、角质形成细胞增殖和分化、真皮肉芽组织形成以及包括巨噬细胞、中性粒细胞和肥大细胞在内的免疫细胞浸润，但不影响血管生成。使用细胞因子阵列筛选，我们发现自噬缺陷抑制了 TNF 对细胞因子 CCL2/MCP-1 的转录和产生。在分子水平上，TNF通过NFKB在表皮角质形成细胞中诱导自噬通量和自噬基因的表达。TNF促进CCL2通过自噬-AMPK-BRAF-MAPK1/3/ERK-激活蛋白 1 (AP1) 通路进行转录。事实上，用重组 CCL2 治疗小鼠可以逆转角质形成细胞自噬缺陷的影响。在细胞水平上，我们发现通过角质形成细胞中的自噬诱导CCL2不仅是角质形成细胞迁移和增殖所必需的，也是真皮成纤维细胞活化所必需的。我们的研究结果证明了表皮自噬在体内伤口愈合中的关键作用，并阐明了在皮肤修复中协调角质形成细胞-成纤维细胞相互作用的关键分子机制。

缩写：ACTA2/α-SMA：肌动蛋白α2，平滑肌；ACTB：β-肌动蛋白；ADGRE1：粘附 G 蛋白偶联受体 E1；AMPK：AMP激活的蛋白激酶；AP1：激活蛋白 1；AP1-RE：AP1响应元件；ATG：自噬相关；ATG16L1：自噬相关 16 like 1；BECN1：beclin 1；BRAF：B-Raf 原癌基因，丝氨酸/苏氨酸激酶；C5：补C5；CCL2/MCP-1：CC基序趋化因子配体2；CCL3：CC基序趋化因子配体3；CK：细胞角蛋白；cKO：有条件淘汰赛；CRTC1：CREB ​​调节的转录共激活因子 1；CXCL1：CXC 基序趋化因子配体 1；CXCL2：CXC基序趋化因子配体2；ECM：细胞外基质；EGF：表皮生长因子；FGF7：成纤维细胞生长因子 7；GABARAPL2：GABA A 型受体相关蛋白样 2；GAPDH：3-磷酸甘油醛脱氢酶；HBEGF：肝素结合EGF样生长因子；HPRT1：次黄嘌呤磷酸核糖基转移酶 1；IHC：免疫组化；IL1B：白细胞介素 1 β；KRT10：角蛋白 10；KRT14：角蛋白 14；MAP1LC3B/LC3B-I/II：微管相关蛋白1轻链3β；MAPK1/3/ERK：丝裂原活化蛋白激酶 1/3；MKI67/Ki-67：增殖标志物；MPO：髓过氧化物酶；NFKB：NF-kappa B，活化 B 细胞的核因子 kappa-轻链增强子；NFKB-RE：NFKB反应元件；PDGF：血小板衍生生长因子；PECAM1：血小板和内皮细胞粘附分子1；PRKAA1：蛋白激酶 AMP 激活的催化亚基 α 1；RELA/p65：RELA 原癌基因，NFKB 亚基；shCON：小发夹 MAPK1/3/ERK：丝裂原活化蛋白激酶 1/3；MKI67/Ki-67：增殖标志物；MPO：髓过氧化物酶；NFKB：NF-kappa B，活化 B 细胞的核因子 kappa-轻链增强子；NFKB-RE：NFKB反应元件；PDGF：血小板衍生生长因子；PECAM1：血小板和内皮细胞粘附分子1；PRKAA1：蛋白激酶 AMP 激活的催化亚基 α 1；RELA/p65：RELA 原癌基因，NFKB 亚基；shCON：小发夹 MAPK1/3/ERK：丝裂原活化蛋白激酶 1/3；MKI67/Ki-67：增殖标志物；MPO：髓过氧化物酶；NFKB：NF-kappa B，活化 B 细胞的核因子 kappa-轻链增强子；NFKB-RE：NFKB反应元件；PDGF：血小板衍生生长因子；PECAM1：血小板和内皮细胞粘附分子1；PRKAA1：蛋白激酶 AMP 激活的催化亚基 α 1；RELA/p65：RELA 原癌基因，NFKB 亚基；shCON：小发夹 RELA 原癌基因，NFKB 亚基；shCON：小发夹 RELA 原癌基因，NFKB 亚基；shCON：小发夹阴性对照；siNC：阴性对照；siRNA：小干扰RNA；SP1：sp1转录因子；SQSTM1/p62：隔离体 1；TGFA：转化生长因子α；TGFB1：转化生长因子β1；TIMP1：TIMP金属肽酶抑制剂1；TNF/TNF-α：肿瘤坏死因子；TREM1：在骨髓细胞1上表达的触发受体；WT：野生型