Human cartilage model of the precocious osteoarthritis-inducingCOL2A1p.Arg719Cys reveals pathology-driving matrix defects and a failure of the ER proteostasis network to recognize the defective procollagen-II
Authors: Yammine Kathryn M. , Abularach Sophia Mirda , Xiong Michael , Kim Seo-yeon , Bikovtseva Agata A. , Butty Vincent L. , Schiavoni Richard P. , Bateman John F. , Lamandé Shireen R. , Shoulders Matthew D. 
Published: November 2024

ABSTRACTObjectivesMutations in the procollagen-II gene (COL2A1) often cause chondrodysplasias, including the precocious osteoarthritis-inducing p.Arg719Cys. Understanding the molecular basis of such diseases has long been challenging, owing to a lack of models accurately reflecting disease genotypes and phenotypes. To address this challenge, we develop and characterizein vitrohuman cartilage derived from wild-type and disease-causing Arg719CysCOL2A1isogenic induced pluripotent stem cell (iPSC) lines.MethodsUsing directed differentiation of iPSCs to chondrocytes, we generated cartilage from wild-type and Arg719CysCOL2A1lines. We compared the resulting protein, cell, and tissue properties using immunohistochemistry, electron microscopy, SDS-PAGE, RNA-sequencing, and quantitative interactomics.ResultsWhile both wild-type and disease lines deposited a cartilage matrix, the Arg719Cys matrix was deficient. Arg719Cys collagen-II was excessively post-translationally modified and modestly intracellularly retained, leading to endoplasmic reticulum (ER) distention suggestive of an ER storage defect. Interactomic studies indicated that Arg719Cys procollagen-II was not differentially engaged by the ER proteostasis network. RNA-sequencing showed that the ER storage defect engendered by Arg719Cys procollagen-II also did not activate cellular stress responses, including the unfolded protein response. These data suggest that cells fail to properly recognize Arg719Cys-associated procollagen-II defects.ConclusionsA failure to identify and rectify defective procollagen-II folding in cells expressing Arg719Cys procollagen-II leads to the deposition of a sparse and defective collagen-II matrix, culminating in pathology. Combined with the highly expandable human cartilage disease model reported here, this work provides motivation and a platform to discover therapeutic strategies targeting procollagen folding, quality control, and secretion in this collagenopathy and others.KEY MESSAGESWhat is already known on this topicThe p.Arg719Cys substitution in procollagen-II (COL2A1) causes precocious generalized osteoarthritis with mild chondrodysplasia, inherited in an autosomal dominant fashion. Previous studies of recombinant procollagen-II indicate that this pathologic substitution disrupts fibril formationin vitro. In homozygous transgenic mice expressing the human allele, this substitution disrupts growth plate organization and collagen-II fibril density. However, the molecular etiology of pathology remains unknown and has not been explored in relevant human models that properly represent the underlying genetics of this disorder.What this study addsWe compare human iPSC-derived cartilage from Arg719Cys procollagen-II expressing chondrocytes and an isogenic wild-type control to identify defects in procollagen-II, the chondrocytes expressing it, and the matrices they deposit.We observe phenotypes consistent with those described in patients and mouse models, including a sparse collagen-II network in the extracellular matrix and distended ER. We also observe that Arg719Cys procollagen-II is slow to fold and accumulates intracellularly, an ER storage defect that likely propagates to a pathologically insufficient matrix.RNA-sequencing and quantitative interactomic analyses reveal that this ER storage defect does not activate cellular stress responses, such as the unfolded protein response.The ER proteostasis network does not detectably recognize the intracellularly retained Arg719Cys procollagen-II.The cell’s failure to identify the underlying folding defect likely leads to deposition of a defective matrix, and that defective matrix ultimately culminates in disease phenotypes.How this study might affect research, practice, or policyThis study uses human iPSC-derived cartilage to characterize the disease phenotype and test hypotheses regarding the molecular etiology of this disease. The results set the stage to explore collagen proteostasis-associated pathways as novel therapeutic targets. Moreover, the iPSC-derived system provides an ideal platform for pre-clinical testing and development of therapeutic strategies that benefit patients with this collagenopathy, and that could also be more broadly applied to other collagen-related diseases.