Autophagic degradation of SQSTM1 enables fibroblast activation to accelerate wound healing

Wound healing is a meticulously coordinated and intricate progression that necessitates precise regulation of fibroblast behavior. Macroautophagy/autophagy is a degradation system for clearing damaged cellular components. SQSTM1/p62 (sequestosome 1), a well-established autophagy receptor, also functions as a signaling hub beyond autophagy. Here, we observed a significant upregulation of autophagy in fibroblasts after wounding. Using mice with fibroblast-specific deletion of Atg7 (autophagy related 7), we found that fibroblast autophagy governed wound healing. Fibroblast autophagy deficiency delayed proper dermal repair that was mired in insufficient fibroblast proliferation, migration, and myofibroblast transition. In vitro experiments further revealed that autophagy deficiency disrupted TGFB1 (transforming growth factor beta 1)-induced fibroblast proliferation, migration, and myofibroblast differentiation. Mechanistically, autophagy deficiency led to SMAD2 (SMAD family member 2) and SMAD3 sequestration within SQSTM1 bodies and attenuated TGFB1-induced receptor-regulated SMAD (R-SMAD) phosphorylation in an SQSTM1-dependent manner. Furthermore, sqstm1 deletion rescued the delayed skin wound healing caused by autophagy deficiency, and autophagy inducers promoted wound healing in an SQSTM1-dependent manner. Our findings highlight the critical role of fibroblast autophagy in wound healing and elucidate the underlying mechanisms by which autophagy regulates fibroblast behavior.Abbreviation: 3-MA: 3-methyladenine; ACTA2/α-SMA: actin alpha 2, smooth muscle; ACTB: actin beta; AMPK: AMP-activated protein kinase; ATG: autophagy related; BiFC: bimolecular fluorescence complementation; COL1A2: collagen type I alpha 2 chain; ECM: extracellular matrix; FGF: fibroblast growth factor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HDF: human dermal fibroblast; HVGs: highly variable genes; KO: knockout; LMNB1: lamin B1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MKI67/Ki-67: marker of proliferation Ki-67; MTOR/mTOR: mechanistic target of rapamycin kinase; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; NFKB: nuclear factor kappa B; NLRP3: NLR family pyrin domain containing 3; PCA: principal component analysis; PI3K: phosphoinositide 3-kinase; R-SMAD: receptor-regulated SMAD; SBE: SMAD binding element; shCON: small hairpin negative control; siNC: negative control; siRNA: small interfering RNA; SMAD: SMAD family member; SQSTM1/p62: sequestosome 1; ssGSEA: single-sample gene set enrichment analysis; TGFB/TGF-β: transforming growth factor beta; TGFBR1: transforming growth factor beta receptor 1; TGFBR2: transforming growth factor beta receptor 2; VIM: vimentin; WT: wild-type; ZFYVE9/SARA: zinc finger FYVE-type containing 9.