Function of AMPK/mTOR Signaling in TGF-β1-Induced Pterygium Fibroblast Proliferation and Transdifferentiation.

Abstract

Purpose: This study aimed to investigate the regulatory role of the AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR) signaling pathway in mediating transforming growth factor-beta 1 (TGF-β1)-induced cellular proliferation and transdifferentiation processes in human pterygium fibroblasts (HPFs).

Methods: HPFs were stimulated with TGF-β1 in vitro. Cell viability was assessed using the CCK-8 assay at 12/24/48-h post-stimulation, while migratory capacity was evaluated through standardized wound healing assays. Quantitative real-time PCR (qPCR) and western blotting analyses were employed to evaluate the expression of proliferation marker proliferating cell nuclear antigen (PCNA) and myofibroblast transdifferentiation biomarker α-smooth muscle actin (α-SMA). Western blotting further characterized the activation status of AMPK/mTOR signaling by quantifying phosphorylated AMPK (p-AMPK) and phosphorylated mTOR (p-mTOR), with total AMPK and mTOR levels serving as loading controls. To establish mechanistic causality, TGF-β1-primed HPFs were modulated using the AMPK inhibitor Compound C and activator AICAR for 24 h. Functional consequences were analyzed through CCK-8 viability assays and wound healing assays, while molecular correlates were assessed via qPCR and western blotting for PCNA, α-SMA, and pathway components. This comprehensive approach delineated the AMPK/mTOR axis as a critical regulator of TGF-β1-driven fibrotic phenotype acquisition in HPFs.

Results: Following TGF-β1 pretreatment-induced activation of human HPFs, both cell viability and migratory capacity were markedly enhanced, with concomitant upregulation of PCNA and α-SMA. Compound C-mediated AMPK inhibition potentiated the TGF-β1-induced enhancements in HPFs viability and migration rate, concomitant with reduced p-AMPK/AMPK ratio and elevated expression of PCNA, α-SMA, and p-mTOR/mTOR ratio. Conversely, AICAR-driven AMPK activation attenuated TGF-β1-stimulated effects, demonstrating diminished viability, suppressed migratory capacity, increased p-AMPK/AMPK ratio, and decreased expression of PCNA, α-SMA, and p-mTOR/mTOR ratio.

Conclusions: This study demonstrates the critical regulatory role of the AMPK/mTOR signaling pathway in controlling TGF-β1-induced proliferation and transdifferentiation in HPFs, thereby providing a potential mechanistic framework for developing novel therapeutic interventions targeting fibrotic ocular surface disorders.