Mutations in Fibronectin Dysregulate Chondrogenesis in Corner Fracture Type Spondylometaphyseal Dysplasia

Abstract

AbstractFibronectin (FN) is a key extracellular matrix glycoprotein indispensable for the development and function of major vertebrate organ systems. Autosomal dominant FN mutations cause corner fracture-type spondylometaphyseal dysplasia (SMDCF). However, the molecular pathomechanisms underlying this pathology are not known. Here, we have employed patient-derived induced pluripotent stem cells (iPSCs) as an SMDCF cell culture model to investigate the consequences of FN mutations in mesenchymal stem cells (MSCs) and differentiated cartilage-producing chondrocytes. The FN mutations impaired protein secretion from MSCs leading to significant accumulation of FN within the cells and strong reduction of FN in the extracellular matrix. Analyses of plasma samples from SMDCF patients showed a similar reduction in circulating FN. In mutant MSCs, FN accumulated in ribosome-covered intracellular vesicles originating from the rough endoplasmic reticulum and transitioning into lysosomes. These lysosomal vesicles were not cleared from the cytosol. Accumulation of intracellular FN elevated cellular stress markers and altered mitochondrial structure. Bulk RNA sequencing analysis revealed a specific transcriptomic dysregulation of the patient cells relative to controls. Analysis of MSC differentiation into chondrocytes showed impaired mesenchymal condensation for the mutant cells with significant reduction of key chondrogenic markers and impaired cell proliferation. FN mutant cells also displayed alterations in FN splice variants under chondrogenic stimuli. Additionally, FN mutant cells showed significantly downregulated expression of transforming growth factor beta-1 (TGFβ1), a key chondrogenic factor essential for mesenchymal condensation. Exogenous supplementation of FN or TGFβ1 promoted MSC condensation in the mutant cells. Together, the data demonstrate the cellular consequences of SMDCF-causing FN mutations on stem cells and the molecular pathogenic pathways leading to altered chondrogenesis.Significance /HighlightsSMDCF-causing mutations in fibronectin impair protein secretion in iPSC-derived mesenchymal stem cells.Mutant fibronectin and ER protein folding chaperones are directly exported from the rough endoplasmic reticulum into vesicles covered with ribosomes, which transition into lysosomes.These vesicles massively accumulate within the cytosol.Mutations in fibronectin impair mesenchymal condensation of MSCs and their differentiation into chondrocytes.Exogenous addition of purified fibronectin or TGFβ-1 improves mesenchymal condensation of the mutant stem cells.