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 <i>Atg7</i> (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. <i>In vitro</i> 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, <i>sqstm1</i> 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. <b>Abbreviation</b>: 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.