M6A demethylase ALKBH5 mediated Igfbp4 mRNA m6A modification drives fibroblast activation and pathological upper...

Laryngotracheal stenosis (LTS) is a fibroproliferative disease of the upper airway characterised by dysregulated extracellular matrix deposition and fibroblast activation. AlkB Homologue 5 (ALKBH5) has emerged as a key regulator of disease pathogenesis by modulating mRNA stability, yet its role in LTS remains unclear.

We established a LTS rat model and observed mRNA m6A methylation and m6A demethylase ALKBH5 in stenotic tissues. Using single-cell RNA sequencing, in vivo ALKBH5 knockout models, and in vitro gain- and loss-of-function assays, we explored the role of ALKBH5 in fibroblast activation and fibrosis. The downstream target of ALKBH5 and its underlying molecular mechanism were identified through integrating RNA-seq and MeRIP-seq analyses, and further validated by RNA immunoprecipitation (RIP)-quantitative PCR (qPCR), dual-luciferase reporter assays, and in vitro gain- and loss-of-function experiments. In addition, the therapeutic potential of exogenous modulation of ALKBH5 target was evaluated in LTS models.

ALKBH5 acted as a key pro-fibrotic regulator by enhancing the expression of COL1A1, COL3A1 and alpha-smooth muscle actin (α-SMA), increasing fibroblast contractility, and promoting airway fibrosis progression in LTS. RNA-seq and MeRIP-seq analyses identified Igfbp4 as a direct target of ALKBH5. RIP-qPCR and luciferase reporter assay confirmed ALKBH5 binding to the 3'-untranslated region of Igfbp4. Functional studies revealed that IGFBP4 inhibited β-catenin signalling and attenuated fibroblast activation. Overexpression of IGFBP4 partly reversed the profibrotic effects of ALKBH5, both in vitro and in vivo, significantly reducing collagen deposition and airway narrowing in LTS rats.

Our findings identify a novel ALKBH5-IGFBP4 regulatory axis that drives fibroblast activation and airway fibrosis in LTS. Targeting ALKBH5 or supplementing IGFBP4 may become novel therapeutic strategies for LTS.