Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Sep;17(9):2128-2143.
doi: 10.1080/15548627.2020.1816342. Epub 2020 Sep 18.

Keratinocyte autophagy enables the activation of keratinocytes and fibroblastsand facilitates wound healing

Lei Qiang  1   2 , Seungwon Yang  1 , Yan-Hong Cui  1 , Yu-Ying He  1
Affiliations

Keratinocyte autophagy enables the activation of keratinocytes and fibroblastsand facilitates wound healing

Lei Qiang et al. Autophagy. 2021 Sep.

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.

Keywords: Autophagy; CCL2/MCP-1; TNF; differentiation; fibroblast; inflammation; keratinocyte; migration; proliferation; wound healing.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.
Epidermal autophagy deficiency inhibits wound healing. (A) Macroscopy view of wound healing on days 0, 2, 4 and 6 in WT, atg5 cKO, and atg7 cKO mice. Scale bar: 1 mm. (B) Quantification of A. (C and E) Wound healing as monitored by histological staining of skin sections at the wound edges at 2, 4, 6 and 8 d after injury in WT, atg5 cKO and atg7 cKO mice. Epi: Epidermis; Der: Dermis; Es: Eschar. GT: Granulation Tissue. Black dotted lines denote dermal-epidermal boundaries (C). Scale bar: 200 μm. (D) Quantification of the length of hyper-proliferative epidermis. (F) Quantification of the granulation tissue areas. All bars represent mean ± SD (n = 6). *, P < 0.05; *, P < 0.01; ***, P < 0.001; compared with WT mice (B) or between comparison groups (D, F); Student’s t-test
Figure 2.
Figure 2.
Keratinocyte autophagy deficiency inhibits cell proliferation and differentiation in vivo. (A) Immunohistochemical (IHC) staining of MKI67 in wound sections on day 6 in WT and atg5 cKO mice. Scale bar: 50 μm. (B) Quantification of the percentage (%) of MKI67-positive cells in newly regenerated epidermis in A (n = 6). (C and E) Immunofluorescence staining of KRT14 (green), KRT10 (red), and nucleus (blue, DAPI) in non-wounded and wounded skin on day 2, 4, and 6 post-wounding in WT mice (C) or in wounded skin on day 4 in WT, atg5 cKO, and atg7 cKO mice (E). Scale bar: 200 (C) and 100 (E) μm. (D and F) Quantification of C (D) and E (F) (n = 3). All bars represent mean ± SD. *, P < 0.05; *, P < 0.01; ***, P < 0.001; Student’s t-test
Figure 3.
Figure 3.
Keratinocyte autophagy deficiency inhibits the activation of fibroblasts and the recruitment of macrophages and neutrophils. (A) Mallory staining for collagen (blue) of wound sections on day 6 in WT, atg5 cKO, and atg7 cKO mice. Scale bar: 50 μm (B) IHC staining of ACTA2 in wound sections on day 6 in WT, atg5 cKO, and atg7 cKO mice. Scale bar: 20 μm (C) Quantification of B (n = 8). (D) IHC staining of ADGRE1 in wound sections on day 6 in WT, atg5 cKO, and atg7 cKO mice. Scale bar: 20 μm (E) Quantification of D (n = 15). (F) IHC staining of MPO in wound sections on day 2 in WT, atg5 cKO, and atg7 cKO mice. Scale bar: 20 μm (G) Quantification of F (n = 10). *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s t-test
Figure 4.
Figure 4.
Epidermal autophagy deficiency inhibits wound healing through inhibiting CCL2. (A) Mouse cytokine array of normal and wounded skin (wound and adjacent area) sections on day 3 in WT and atg5 cKO mice. Dots with alterations were indicated by blue and red line. (B) The relative mean pixel density (fold of WT Non-Wound) of cytokines was listed with a mouse cytokine array assay (n = 6). (C) Real-time PCR for CCL2 and CXCL1 mRNA level in HaCaT cells stably transfected with shCON or shATG5 at 0, 6 and 24 h following TNF treatment. (D) CCL2 production in conditioned medium from HaCaT cells infected with shCON and shATG5 over a time course following TNF treatment. (E, F, and G) Real-time PCR analysis for CCL2 in NHEK cells transfected with siNC, siATG5, and siATG7, treated with or without TNF (100 ng/ml) for 8 h. Data are shown as mean ± S.E. (n ≥ 3). (H and I) Immunofluorescence staining of KRT14 and CCL2 in intact (H) and wound skin (I) on day 2 in WT, atg5 cKO, and atg7 cKO mice. Scale bar: 50 μm. (J) Quantification of the levels of CCL2 in wound healing on day 2 (I) was analyzed by ImageJ (N = 15). The right side of blue dot line is the wound area, and the inside of white dot indicates the area of keratinocyte expressing KRT14. (K) Wound healing as monitored by histological staining at day 8 after injury in WT, atg5 cKO mice with or without CCL2 topical treatment. Epi: epidermis; Der: dermis; Es: eschar. Scale bars: 200 (left panel) and 100 (right penal) µm. (L and M) Quantification of wound gap (L) granulation tissue (M) in K (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001; between comparison groups. #, P < 0.05; ##, P < 0.01 for B and C; compared with their corresponding non-wounded control groups. n.s., not statistically significant. Student’s t-test. Results were obtained from at least three independent experiments (C-G)
Figure 5.
Figure 5.
Autophagy promotes keratinocyte migration and proliferation through CCL2. (A) Representative images of cell migration assay with HaCaT cells with or without knockdown of either ATG5 or ATG7 treated with or without TNF (100 ng/ml) and/or CCL2 (100 ng/ml) for 24 h. Scale bars: 200 µm. (B) Quantification of A (n = 8). (C) Cell proliferation assay in cells as in A (n = 3). ***, P < 0.001; compared with its corresponding group without CCL2 treatment. ###, P < 0.001; compared with the shCON+Vehicle group. (D) Representative images of cell migration assay with NHEK cells, transfected with siNC, siATG5 and siATG7, treated with or without TNF (100 ng/ml) and/or CCL2 (100 ng/ml) for 1 d. Scale bars: 100 µm. (E) Quantification of D (n = 8). (F) Cell proliferation assay in NHEK cells transfected with siNC, siATG5 and siATG7, then treated with or without TNF (100 ng/ml) and/or CCL2 (100 ng/ml) for 2 d (n = 3). All bars represent mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s t-test. Results were obtained from at least three independent experiments (A-F)
Figure 6.
Figure 6.
Autophagy in keratinocytes regulates fibroblast activation. (A) Schematic summary of experimental procedure. (B) Representative phase-contrast image and (C) quantification of collagen-based cell contraction in human dermal fibroblast cells with or without conditioned medium from HaCaT cells infected with shCON or shATG5 and treated with TNF for 48 h. TNF in medium was neutralized before being added to human dermal fibroblast cells. (D and E) Representative phase-contrast image (D) and quantification (E) of collagen-based cell contraction in human dermal fibroblast cells with or without conditioned medium from HaCaT cells stably transfected with shCON or shATG5 treated with TNF for 48 h. TNF and CCL2 in medium were neutralized before being added to human dermal fibroblast cells. *P < 0.05, **P < 0.01, compared with the corresponding vehicle (Veh) group. #, P < 0.05, compared with the shCON TNF group. Student’s t-test. Results were obtained from at least three independent experiments
Figure 7.
Figure 7.
Autophagy is required for TNF mediated CCL2 transcription through MAPK1/3-AP1 signaling, and the MAPK1/3 activity is controlled by the AMPK-BRAF axis. (A) Schematic representation of the (dis)AP1, NFKB, and (pro)AP1/SP1 sites of the human CCL2 promoter. Red nucleotides indicate mutations made in the human CCL2 promoter. (B) Reporter assay for the Human CCL2 promoter with wild-type sequence transfected into HaCaT cells with shCON or shATG5, following treatment with or without TNF for 24 h. (C) Same as B except that HaCaT cells were transfected with the human CCL2 promoter with wild-type sequence or mutation of the (dis)AP1 site, NFKB site, or (pro)AP1/SP1 site. (D) Same as B except that HaCaT cells were transfected with the human CCL2 promoter with mutation of NFKB site, following treatment with or without TNF for 24 h. (E) Same as D except that HaCaT cells were transfected with the human CCL2 promoter with mutation of (pro)AP1/SP1 site. (F) Same as B except that HaCaT cells were transfected with promoters with a repeated sequence of the NFKB-RE (NFKB response element) or AP1-RE (AP1 response element). (G) Same as B except that the cells were treated with PD98059 (20 µM) 2 h prior to and during TNF treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; compared with its corresponding Veh group. #, P < 0.05; ##, P < 0.01; compared with shCON group (B, D-G), or with WT/TNF group (C); Student’s t-test. (H and I) Immunoblot analysis of p-MAPK1/3, MAPK1/3, ATG12–ATG5, and ATG7 in HaCaT cells infected with shCON, shATG5 (H), or shATG7 (I), starved overnight, followed by treatment with or without TNF (100 ng/ml) over a time course. (J and K) Immunoblot analysis of p-MAPK1/3, ATG12–ATG5, and ATG7 in NHEK cells transfected with siNC, siATG5 (J), or siATG7(K), starved with 5 fold-diluted the complete medium for NHEK for 24 h, treated with or without TNF (100 ng/ml) for 30 min. (L) Immunoblot analysis of p-BRAF (S729), BRAF, p-PRKAA1, PRKAA1, p-CRTC1, CRTC1, and MAP1LC3B/LC3B-I/II (microtubule-associated protein 1 light chain 3 beta) in HaCaT cells infected with shCON, shATG5, or shATG7. (M) Immunoblot analysis for p-BRAF, BRAF, p-MAPK1/3, and MAPK1/3 in HaCaT cells infected with shCON, shATG5, or shATG7, starved overnight, and treated with the AMPK inhibitor compound C (AMPKi 1 μM) for 2 h, followed by treatment with TNF (100 ng/ml) for 15 min. (N) Real-time PCR analysis for CCL2 mRNA level in HaCaT cells treated with or without Compound C (1 μM) for 5 h and then treated with TNF (100 ng/ml) for 24 h. Data are shown as mean ± S.E. (n ≥ 3). *P < 0.05; **P < 0.01; compared with the corresponding group without compound C treatment; Student’s t-test (C). (O) Immunoblot analysis of p-PRKAA1, PRKAA1, p-MAPK1/3, MAPK1/3, and GAPDH in HaCaT cells treated with or without TNF (100 ng/ml) or CCL2 (100 ng/ml) for 15 min. Results were obtained from at least three independent experiments
Figure 8.
Figure 8.
TNF induces keratinocyte autophagy through NFKB activation. (A-H) Immunofluorescence analysis of, ATG5 (A-B), ATG7 (C-D), SQSTM1 (E-F), and BECN1 (G-H) in keratinocytes in non-wounded and wounded skin on day 2 post-wounding. Cytokeratin (CK) was used as a keratinocyte marker. The dotted line indicates CK-positive keratinocytes. Scale bar: 50 μm. All bars represent mean ± SD (n = 10). (I) Real-time PCR analysis for ATG5, BECN1, ATG7, GABARAPL2, ATG9, ATG16L1, and SQSTM1 mRNA level in NHEK cells, starved with 5 fold-diluted the defined medium for NHEK for 24 h, then treated with or without TNF (100 ng/ml) for 18 h. Data are shown as mean ± S.E. (n ≥ 3). (J) Immunoblot assay for ATG5, ATG7, BECN1, and SQSTM1 protein level in NHEK cells, starved with 5 fold-diluted the complete medium for NHEK for 24 h, treated with or without TNF (100 ng/ml) for 18 h. (K) Immunoblot assay for MAP1LC3B-I/II, SQSTM1, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) in NHEK cells, starved with 5 fold-diluted the complete medium for NHEK, then treated with or without the lysosome inhibitor bafilomycin A1 (BafA1, 25 nM), and treatment of TNF (100 ng/ml) for 18 h. (L) Luciferase reporter analysis of NFKB response element in NHEK cells treated with or without TNF (100 ng/ml) for 18 h. (M) Real-time PCR analysis for ATG5, BECN1, ATG7, GABARAPL2, ATG9, ATG16L1, and SQSTM1 mRNA level in NHEK cells transfected with siNC or siRELA, starved with 5 fold-diluted the defined medium for NHEK for 24 h, then treated with or without TNF (100 ng/ml) for 18 h. Data are shown as mean ± S.E. (n ≥ 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; Results were obtained from at least three independent experiments
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Klionsky DJ. Autophagy: from phenomenology to molecular understanding in less than a decade. Nat Rev Mol Cell Biol. 2007;8:931–937. - PubMed
    1. Mizushima N, Levine B, Cuervo AM, et al. Autophagy fights disease through cellular self-digestion. Nature. 2008;451:1069–1075. - PMC - PubMed
    1. Mizushima N, Yoshimori T, Ohsumi Y.. The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol. 2011;27:107–132. - PubMed
    1. Ho TT, Warr MR, Adelman ER, et al. Autophagy maintains the metabolism and function of young and old stem cells. Nature. 2017;543:205–210. - PMC - PubMed
    1. Kroemer G, Marino G, Levine B. Autophagy and the integrated stress response. Mol Cell. 2010;40:280–293. - PMC - PubMed

Publication types