Abstract Rationale Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by persistent fibroblast activation, excessive collagen-rich extracellular matrix (ECM) deposition, and the progressive replacement of normal distal airway and alveolar epithelial structures by areas of bronchiolization and basal-like and/or basaloid cells. Collectively, these events lead to irreversible loss of lung function. Targeting collagen biosynthesis is a relevant therapeutic strategy, and we previously reported upregulation of prolyl-3-hydroxylase 1 (P3H1), a collagen-modifying enzyme and chaperone, in IPF. Its role in IPF pathogenesis, however, has remained undefined. Objectives (a) To carefully map P3H1 expression in control and fibrotic lung tissue. (b) To define how targeting P3H1 affects gene expression in primary lung cells and identify the underlying molecular mechanisms. Methods Multiplex immunofluorescence was used to localize P3H1 in control and fibrotic lung. P3H1 was silenced via siRNA in primary human bronchial epithelial cells (phBECs) and primary human lung fibroblasts (phLFs). Downstream effects were analyzed using mass spectrometry-based proteomics, bulk RNA sequencing, pathway enrichment analysis, immunoblotting, immunocytochemistry, and BrdU incorporation assay to assess proliferation. Results In normal lungs, P3H1 was detected in airway epithelial cells, type II alveolar epithelial cells, and interstitial fibroblasts. In IPF lungs, P3H1 localized additionally to basaloid cells, bronchiolized areas, and αSMA-negative fibroblasts within fibroblast foci, colocalizing with type XVII but not type I collagen. SiRNA-mediated silencing of P3H1 confirmed that P3H1 is essential for type XVII but not type I collagen biosynthesis in primary lung cells. Instead, P3H1 depletion enhanced type I collagen and fibronectin production and induced myofibroblast markers. Pathway enrichment analysis indicated c-Myc suppression and TGF-β activation as key regulatory events. Consistently, P3H1 knockdown upregulated TGF-β2 expression and secretion, activated non-canonical TGF-β/ERK signaling, and reduced c-Myc expression and proliferation in phBECs. Inhibiting TGF-β2 restored c-Myc and normalized fibronectin levels, confirming TGF-β2 as a mediator of these effects. Conclusions P3H1 is widely expressed in lung epithelial cells and fibroblasts and regulates type XVII collagen biosynthesis. P3H1 deficiency in primary lung cells activates TGF-β2 signaling, promotes profibrotic gene expression and ECM secretion, and suppresses epithelial proliferation. Elevated P3H1 in IPF thus likely fulfills a protective role that restrains pathological TGF-β2 activation and controls ECM quality and epithelial viability. These findings argue against its suitability as a therapeutic target in IPF but increase our understanding of intrinsic mechanisms that protect from fibrotic progression. This abstract is funded by: None
Kandhari et al. (Fri,) studied this question.