Normal structure and function of the lung is maintained in homeostasis and repaired/regenerated following diverse injuries by regionally defined stem/progenitor cells. Stem cells reside in unique tissue microenvironments, known as the stem cell “niche”, which constitutes stem cell progeny, other niche-support cells including mesenchymal cells (MCs), and the surrounding extracellular matrix (ECM). The stem cell niche provides instructive cues for stem cell self-renewal and differentiation. Fibrotic lungs undergo substantial changes in the tissue biomechanical properties, manifested by stiffening of the ECM. Cells residing in the stem cell niche sense and respond to alterations in the stiffness of the microenvironment, highlighting matrix stiffness as an important mechanical component of the stem cell niche. In preliminary studies, we have characterized the stiffness of alveolar type 2 epithelial stem cell (AT2) niche associated with Pdgfrα+ lung MCs. Alveolar organoid culture in newly developed, stiffness-tunable 3D hydrogels demonstrated that matrix stiffness constitutes an AT2 niche. We recently identified that α6-integrin is a mechanosensitive integrin subunit; stiff matrix-induced α6 expression, primarily an α6 isoform with a shorter cytoplasmic domain (α6S), mediates lung fibroblast invasion into the basement membrane. New preliminary data now show that in addition to α6 expression, matrix stiffness regulates alternative splicing of α6 pre-mRNA in Pdgfrα+ lung MCs, resulting in differential expression of a distinct α6 isoform with a longer cytoplasmic domain (α6L) under soft /homeostatic matrix conditions and a switch from α6L to α6S predominance under stiff/fibrotic matrix conditions. We found that α6L expression promotes lipogenic differentiation of lung MCs and confers the AT2- niche function, facilitating reinstatement of lung homeostasis. In contrast, α6S expression impairs the AT2- niche function and promotes fibrogenic/invasive differentiation of lung MCs, contributing to lung fibrosis. In this proposal, we hypothesize that matrix stiffness-dependent alternative splicing of α6-integrin regulates the repair of injured lungs by controlling alveolotrophic vs. fibrogenic differentiation of lung mesenchymal cells. Specific aims in the proposed study are: (1) determination of the mechanisms by which matrix stiffness regulates alternative splicing of α6-integrin; (2) determination of the mechanisms by which distinct α6-integrin cytoplasmic variants mediate alveolotrophic vs. fibrogenic differentiation of lung mesenchymal cells; and (3) testing the potential of targeting matrix stiffness-dependent alternative splicing of α6-integrin for the reversal of sustained pulmonary fibrosis in mice. Understanding the mechanisms by which lung epithelial stem cells interact with their niches in normal vs. pathological repair of the injured lung will provide novel therapeutic approaches to prevent, treat, and potentially reverse pulmonary fibrosis.