Fibronectin Mutations Impair Chondrogenesis in Spondylometaphyseal Dysplasia
Neha E. H. Dinesh1, Justine Rousseau2, Deane F. Mosher3, Mike Strauss1, Jeannie Mui5, Philippe M. Campeau2*, Dieter P.Reinhardt 1,4*
1. Faculty of Medicine and Health Sciences, Department of Anatomy and Cell Biology, Ï㽶ÊÓƵ, Montreal, QC, Canada 2. Centre Hospitalier Universitaire Sainte-Justine Research Center, Montreal, QC, Canada 3. Departments of Biomolecular Chemistry and Medicine, University of Wisconsin, Madison, WI, USA , 4. Faculty of Dental Medicine and Oral Health Sciences, Ï㽶ÊÓƵ, Montreal, QC, Canada, 5. Facility for Electron Microscopy Research of Ï㽶ÊÓƵ, Montreal, QC, Canada, *Co-senior authors
Introduction: Fibronectin (FN) is a key extracellular matrix glycoprotein that is indispensable for the physiological development and function of major organ systems in vertebrates. FN mutations with an autosomal dominant pattern of inheritance causes corner fracture type spondylometaphyseal dysplasia (SMDCF) in humans; however, the molecular mechanism underlying this pathology is not known. Here, we have employed induced pluripotent stem cells (IPSCs) to develop a cell culture model system for SMDCF to investigate the consequences of FN mutations on the fate of mutant protein in the secretory pathway of patient mesenchymal stem cells (MSCs) and to analyze the differentiation of the mutant stem cells into chondrocytes, the cell types that produce cartilage.
Methods, Results and Discussion: Consistent with our previous findings, FN mutations were identified to impair protein secretion from MSCs with accumulation of FN within the cells and significantly reduced secretion of FN into the culture medium. This was corroborated with analyses of plasma samples from SMDCF patients that showed a similar reduction in circulating FN. FN was observed to accumulate in ribosome-covered intracellular vesicles within the mutant stem cells which originate from the rough endoplasmic reticulum (RER) and become positive for lysosomal markers. Accumulation of intracellular protein within the cells-initiated increase in cellular stress markers and an altered mitochondrial structure. Bulk Ï㽶ÊÓƵ sequencing analysis revealed a wide dysregulation of the transcriptome 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 cellular proliferation. FN mutants displayed alterations in expression of FN1 splice variants under chondrogenic stimulus. Additionally, FN mutants are characterized by significantly downregulated expression of transforming growth factor beta-1 (TGFβ1), a key chondrogenic factor essential for mesenchymal condensation. Exogenous supplementation of both FN and TGFβ1 promoted MSC condensation and improved chondrogenesis in the mutants.
Conclusion: Taken together, our data demonstrate the cellular consequences of FN mutations on stem cells and how it leads to altered chondrogenesis in SMDCF patients.