METTL3-dependent epigenetic regulation of ULK2 autophagy in hypertrophic scarring.

Abstract

Increased autophagy in fibroblasts drives their differentiation into myofibroblasts, a key process in dermal fibrosis during hypertrophic scar (HS) progression. While N6-methyladenosine (m6A) modification is implicated in fibrosis and autophagy, its mechanistic role in HS remains unclear. In this study, we investigated the involvement of fibroblast autophagy in HS progression and the regulatory mechanisms underlying this process. Our findings demonstrated that HS development is associated with significant autophagy in both human patients and rabbit models, as evidenced by the activation of fibroblast-associated alpha-smooth muscle actin (α-SMA) and type I collagen. Pharmacological inhibition of autophagy using 3-methyladenine effectively suppressed fibroblast-to-myofibroblast differentiation. We further discovered that excessive m6A modifications enhanced autophagy in fibroblasts derived from HS tissues. Mechanistically, we elucidated that methyltransferase-like 3 (METTL3)-mediated m6A modification upregulated unc-51-like kinase 2 (ULK2), a key regulator of autophagy initiation, through techniques such as m6A RNA immunoprecipitation sequencing (MeRIP-seq), qRT-PCR, and Western blotting. Silencing METTL3 impaired autophagic flux, as confirmed by transmission electron microscopy and LC3-II/I ratio analysis, thereby inhibiting fibroblast-to-myofibroblast differentiation. Notably, subcutaneous injection of METTL3 small interfering RNA (siRNA) attenuated cellular autophagy in HS tissues and mitigated HS formation in rabbit ears. These results clarify the causal relationship between METTL3-mediated m6A modification, autophagy, and fibroblast-to-myofibroblast differentiation, providing a mechanistic basis for the therapeutic potential of targeting METTL3 in HS treatment.