Pin1 as a Central Modulator of Wnt/β-Catenin Signaling in Pulmonary Fibrosis: Interplay with EBV-LMP1 and Therapeutic Implications.

Background: Pulmonary fibrosis (PF) is characterized by dysregulated signaling, with the Wnt/β-catenin pathway playing a critical role. Pin1, a peptidyl-prolyl isomerase, is implicated in post-translational modifications and cellular signaling. Objectives: This study explores the expression, localization, and functional role of Pin1 in regulating Wnt/β-catenin signaling in human lung fibroblasts (MRC-5 cells). These cells, derived from normal lung tissue, are commonly used to model fibrotic processes due to their ability to mimic fibroblast behavior in PF. Importantly, we report the first demonstration of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1)-mediated Pin1 activation in the context of PF. Notably, we demonstrate that EBV-LMP1 activates Pin1 and amplifies Wnt/β-catenin signaling in fibroblasts. Methods: We employed a combination of Pin1 overexpression and siRNA-mediated knockdown in MRC-5 cells to assess pathway modulation. Subcellular localization analysis was performed, and pathway output was evaluated by quantifying β-catenin, cyclin D1, and Axin2 via Western blotting. Co-immunoprecipitation (Co-IP) was used to examine the Pin1-β-catenin interaction. To examine viral contributions, LMP1 overexpression was carried out, and pharmacological inhibition of Pin1 was achieved using Juglone and PiB. Results: Pin1 expression was significantly higher in MRC-5 cells compared to alveolar epithelial cells, with a 2.5-fold increase in protein levels (P < 0.05). Pin1 was localized to both the cytoplasm and nucleus. Overexpression of Pin1 led to an approximately two-fold increase in β-catenin (192%), cyclin D1 (178%), and Axin2 (165%) expression compared to controls (P < 0.01), while knockdown reduced their levels by 60%, 55%, and 63%, respectively (P < 0.01). The LMP1 overexpression increased Pin1 by 1.8-fold, strengthened its interaction with β-catenin, and amplified Wnt/β-catenin signaling. Treatment with Wnt3a further enhanced β-catenin expression by 2.4-fold, while XAV939 reduced it by 66% (P < 0.01). Pharmacological inhibition of Pin1 using Juglone and PiB significantly suppressed pathway activation, including LMP1-induced enhancement, with reductions in β-catenin levels by 68% and 72%, respectively (P < 0.01). Conclusions: Pin1 is a critical regulator of the Wnt/β-catenin pathway in PF, integrating signals from viral and cellular modulators. This study provides novel evidence of EBV-LMP1's role in activating Pin1 in lung fibroblasts, reinforcing its value as a therapeutic target. Pin1 inhibitors effectively downregulate this signaling cascade, even under hyperactive conditions, highlighting their therapeutic potential for PF treatment. While Pin1 inhibitors effectively downregulate this signaling cascade even under hyperactive conditions, their therapeutic potential remains to be validated in preclinical models.