C99-05 E3 Ligase Rnf145 Modulates AMPK-dependent Fibrotic Signaling and Remodeling in Airway Smooth Muscle Cells

Abstract Rationale Airway smooth muscle (ASM) contributes critically to airway remodeling and fibrosis in asthma and COPD. Excessive ASM proliferation and extracellular matrix (ECM) deposition lead to airway stiffening and reduced lung function. While transcriptional and post-transcriptional mechanisms regulating ASM remodeling are well characterized, its post-translational regulation remains poorly understood. Ubiquitination, mediated by E3 ubiquitin ligases, tightly regulates protein stability and fibrotic signaling. RNF145, a novel RING E3 ubiquitin ligase, has been associated with abnormal lung function and respiratory symptoms, but its role in regulating airway cellular functions under physiological and pathological conditions remains unknown. This study investigated the role of RNF145 in regulating fibrotic signaling and remodeling responses to profibrotic stimulation in ASM, focusing on its modulation of AMPK activity, collagen synthesis, and migration. Methods RNF145 expression in human airway tissue was determined by immunohistochemical analysis. Primary human ASM cells were subjected to lentiviral RNF145 overexpression, shRNA-mediated knockdown, or pharmacological inhibition using SMER3, followed by TGFβ1 (2.5 ng/mL) stimulation. Cell migration, fibrotic signaling, and ECM expression were assessed by wound-healing assays and immunoblotting, respectively. Mouse precision-cut lung slices (PCLS) were exposed to a fibrotic cocktail (TGFβ1, 5 ng/mL; TNFα, 10 ng/mL; PDGF-AB, 10 ng/mL; and LPA, 5 µM) with or without SMER3 for 72 h, and ECM deposition was assessed. Results Immunohistochemistry of airway tissues from asthmatic donors showed significantly increased RNF145 expression in ASM bundles compared to non-asthmatic controls. In human ASM cells, TGFβ1 stimulation induced a robust, time-dependent increase in RNF145, collagen, and fibronectin expression. RNF145 expression positively correlated with ECM protein accumulation. Both RNF145 knockdown and SMER3 treatment suppressed TGFβ1-induced cell migration and reduced collagen, fibronectin, and α-smooth muscle actin expression in a dose-dependent manner. In mouse PCLS, SMER3 co-treatment attenuated fibrotic cocktail-induced ECM accumulation. Mechanistically, RNF145 overexpression and TGFβ1 stimulation reduced phospho-AMPK levels. Conversely, RNF145 knockdown or inhibition increased AMPK phosphorylation, suggesting that RNF145 represses AMPK-dependent antifibrotic signaling. Conclusion RNF145 functions as a negative regulator of AMPK-mediated antifibrotic signaling in ASM cells. Pharmacological inhibition of RNF145 with SMER3 restores AMPK activation, suppresses ECM production, and mitigates fibrotic remodeling. These findings identify RNF145 as a novel modulator of ASM fibrosis and a potential therapeutic target for airway remodeling in obstructive airway diseases. This abstract is funded by: NIH- RO1HL161205