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

Abstract

Transforming growth factor-β (TGFβ) signaling through SMAD2/3 is an important driver of pathological fibrosis in multiple organ systems. TGFβ signaling and extracellular matrix (ECM) stiffness form an unvirtuous pathological circuit in which matrix stiffness drives activation of latent TGFβ, and TGFβ signaling then drives cellular stress and ECM synthesis. Moreover, ECM stiffness also appears to sensitize cells to exogenously activated TGFβ 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 TGFβ signaling. We showed that LEMD3-SMAD2/3 interactions are inversely correlated with ECM stiffness and TGFβ-driven luciferase activity and that LEMD3 expression is correlated with the mechanical response of the TGFβ-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 Our work reveals novel LEMD3 biology and stiffness-dependent regulation of TGFβ by LEMD3, providing a novel target to antagonize pathological TGFβ signaling.

Idiopathic Pulmonary Fibrosis: An Update on Pathogenesis

“…Moreover, ECM stiffness is sensitive to exogenous TGF-β stimulation through inhibiting the interaction of inner nuclear membrane protein LEM domain-containing protein 3 (LEMD3) and SMAD2/3. LEMD3 is physically connected to the actin cytoskeleton of cells and inhibits TGF-β signaling ( Chambers et al, 2018 ). At the metabolic level, increased matrix stiffness impairs the synthesis of anti-fibrotic lipid mediator PGE2 and reduces expression of rate-limiting prostaglandin biosynthetic enzyme cyclooxygenase 2 (COX-2) and prostaglandin E synthesis (PTGES) through p38/MAPK signaling pathway ( Berhan et al, 2020 ).…”

A Report of a Novel Pathogenic Variant in a Family with Buschke–Ollendorf Syndrome

“…Pathogenic alterations in LEMD3 cause loss of the function of MAN1 protein resulting in abnormal regulation of the TGF-β/BMP signaling, thus augmenting the elastin production in the fibroblasts of the skin. [3][4][5][6] Although the proband and his father harbored the same pathogenic variant, they expressed different phenotypes suggestive of pleiotropy, a well-observed event in BOS. We further noted from previous studies that dermatofibrosis lenticularis disseminata present in the proband was the most prevalent manifestation among BOS patients.…”

Actin Cytoskeleton and Regulation of TGFβ Signaling: Exploring Their Links

“…The lamin-binding protein LEMD3 (MAN-1) inhibits TGFβ signaling by binding SMAD2/3 and promoting their dephosphorylation and nuclear export [ 121 , 122 ]. An interesting study by Chambers et al has shown that the interaction of LEMD3 with SMAD2/3 is negatively regulated by ECM stiffness and antagonized by actin polymerization, highlighting that a stiffened ECM regulates cell response to TGFβ [ 123 ].…”