LEM domain-containing protein 3 antagonizes TGFβ-SMAD2/3 signaling in a stiffness-dependent manner in both the nucleus and cytosol

Transforming growth factor-beta (TGF beta) signaling through SMAD2/3 is an important driver of pathological fibrosis in multiple organ systems. TGF signaling and extracellular matrix (ECM) stiffness form an unvirtuous pathological circuit in which matrix stiffness drives activation of latent TGF beta, and TGF beta signaling then drives cellular stress and ECM synthesis. Moreover, ECM stiffness also appears to sensitize cells to exogenously activated TGF beta through unknown mechanisms. Here, using human fibroblasts, we explored the effect of ECM stiffness on a putative inner nuclear membrane protein, LEM domain-containing protein 3 (LEMD3), which is physically connected to the cell's actin cytoskeleton and inhibits TGF beta signaling. We showed that LEMD3-SMAD2/3 interactions are inversely correlated with ECM stiffness and TGF beta-driven luciferase activity and that LEMD3 expression is correlated with the mechanical response of the TGF beta-driven luciferase reporter. We found that actin polymerization but not cellular stress or LEMD3-nuclear-cytoplasmic couplings were necessary for LEMD3-SMAD2/3 interactions. Intriguingly, LEMD3 and SMAD2/3 frequently interacted in the cytosol, and we discovered LEMD3 was proteolytically cleaved into protein fragments. We confirmed that a consensus C-terminal LEMD3 fragment binds SMAD2/3 in a stiffness-dependent manner throughout the cell and is sufficient for antagonizing SMAD2/3 signaling. Using human lung biopsies, we observed that these nuclear and cytosolic interactions are also present in tissue and found that fibrotic tissues exhibit locally diminished and cytoplasmically shifted LEMD3-SMAD2/3 interactions, as noted in vitro. Our work reveals novel LEMD3 biology and stiffness-dependent regulation of TGF beta by LEMD3, providing a novel target to antagonize pathological TGF beta signaling.