Bmi-1 overexpression mitigates vitamin D deficiency-induced pulmonary fibrosis via TIME pathway

Vitamin D deficiency is increasingly linked to senescence-associated pulmonary fibrosis (SAPF), yet the underlying mechanisms remain incompletely understood. Here, we demonstrate that mesenchymal cell-specific overexpression of Bmi-1 significantly ameliorates SAPF induced by 1,25(OH)₂D deficiency in a mouse model. In Cyp27b1 heterozygous mice, which exhibit 1,25(OH)₂D deficiency, we observed reduced expression of both Bmi-1 and VDR with concurrent elevation of TGF-β1 in lung tissue. Transgenic Bmi-1 overexpression in mesenchymal cells markedly improved respiratory parameters, including inspiratory time, respiratory frequency, tidal volume, and minute ventilation, while reducing pathological collagen deposition and fibrotic markers. Histological and molecular analyses revealed that Bmi-1 overexpression prevented excessive extracellular matrix accumulation and preserved alveolar architecture. Mechanistically, Bmi-1 overexpression suppressed the TGF-β1/IL-11/MEK/ERK (TIME) signaling pathway, reduced cellular senescence markers (p53, p21, p16) and senescence-associated secretory phenotype factors, and inhibited myofibroblast differentiation. Furthermore, 1,25(OH)₂D deficiency-induced DNA damage and inflammatory cell infiltration were significantly attenuated by Bmi-1 overexpression. Through CUT&RUN and luciferase reporter assays, we established that 1,25(OH)₂D₃ directly regulates Tgf-β1 transcription via VDR binding to specific promoter regions, providing a molecular link between vitamin D signaling and fibrotic pathways. In primary pulmonary fibroblasts, 1,25(OH)₂D₃ treatment inhibited Tgf-β1 expression in a time-dependent manner. These findings reveal a novel protective role of Bmi-1 against vitamin D deficiency-induced SAPF and suggest that targeting the Bmi-1/VDR/TGF-β1 axis may represent a promising therapeutic strategy for fibrotic lung diseases.