Abstract Rationale Lung stem cells reside in regionally specialized microenvironments, known as niches, that provide essential biochemical and biomechanical cues to maintain stem cell function. Disruption of the alveolar niche impairs epithelial repair and contributes to pulmonary fibrosis. Fibrotic remodeling is characterized by extracellular matrix (ECM) stiffening, yet the impact of this altered biomechanical environment on mesenchymal niche cell (MC) fate and signaling remains poorly defined. This study investigates how alveolar niche stiffness controls the fate of Pdgfrα+ MCs and delineates the mechanosensitive pathways linking mechanical cues to RNA splicing regulation and cell phenotype transitions. Methods Alveolar niche stiffness was quantified by atomic force microscopy (AFM) in normal and fibrotic lungs of PdgfrαEGFP;Sftpc-CreERT2;R26-LSL-tdT dual lineage-labeled mice. Alveolar epithelium-mesenchyme organoids were cultured in stiffness-tunable 3D PEG hydrogels. Transcriptomic profiling of α6-integrin long (α6L) and short (α6S) isoform-overexpressing Pdgfrα+ MCs was performed by bulk RNA-seq. Minigene assays assessed Rbfox2-dependent α6-integrin splicing. Pdgfra-CreERT2;Rbfox2fl/fl mice were used to test functional roles in vivo. Results AFM revealed a marked increase in alveolar niche stiffness in fibrotic lungs. Using alveolar organoid cultures embedded in stiffness-tunable hydrogels, we identified the mechanical properties of the microenvironment as a critical component of the stem cell niche. Alveolar niche stiffness regulated α6-integrin splicing in Pdgfrα+ MCs, generating α6L and α6S isoforms with distinct cytoplasmic domain and signaling function. The soft, homeostatic niche favored α6L, which mediated basement membrane laminin signaling to sustain lipogenic differentiation and niche support via AMPK/PPARγ activation. In contrast, the stiff, fibrotic niche induced α6S predominance, which recruited tyrosine phosphatase PTPN4 to suppress AMPK/PPARγ signaling and promote a fibrogenic, migratory phenotype through Rab21-dependent integrin trafficking. RNA-seq analyses showed that α6L overexpression upregulated niche-supportive gene signatures and enhanced lipid and surfactant metabolism consistent with lipofibroblast traits, whereas α6S activated fibrogenic and motility programs, driving mesenchymal cells away from lipofibroblast-like lipid storage toward an oxidative, energy-consuming state typical of activated matrix-producing fibroblasts. RBFOX2 was identified as a stiffness-sensitive splicing regulator driving the α6L to α6S switch. Conditional deletion of Rbfox2 in Pdgfrα+ MCs blocked α6-integrin splicing and markedly reduced persistent fibrosis in a repetitive lung injury model. Conclusions Alveolar niche stiffness acts as a biomechanical determinant of mesenchymal niche cell fate through RBFOX2-mediated alternative splicing of α6-integrin. This mechanosensitive splicing switch orchestrates the balance between regenerative and fibrogenic mesenchymal programs. Targeting the RBFOX2-α6-integrin axis may restore regenerative niche functions and provide a therapeutic strategy to counteract progressive pulmonary fibrosis. This abstract is funded by: HL139584, HL156973, and HL174994
Zheng et al. (Fri,) studied this question.