bioRxiv2024
Mutations in the COL2A1 gene, encoding procollagen-II, cause various chondrodysplasias, including precocious osteoarthritis with mild spondyloepiphyseal dysplasia engendered by the p.Arg719Cys substitution. The molecular mechanisms underlying these disorders remain incompletely understood, largely owing to the absence of models faithfully recapitulating the human disease. Here, we developed an in vitro human cartilage model using isogenic induced pluripotent stem cell (iPSC) lines carrying either wild-type or Arg719Cys COL2A1. Directed differentiation into chondrocytes yielded cartilage tissues that were analyzed by immunohistochemistry, electron microscopy, SDS-PAGE, and RNA-sequencing. Tissues derived from Arg719Cys heterozygotes displayed a deficient matrix, closely reflecting the human disease phenotype. Arg719Cys procollagen-II was excessively post-translationally modified and partially retained within the endoplasmic reticulum (ER), leading to ER distention. Notably, despite introduction of an aberrant cysteine residue--expected to engage redox-sensitive folding and quality control pathways--Arg719Cys procollagen-II was not detectably recognized by the ER proteostasis network. The resulting inability to mount a quality control response, including activation of the unfolded protein response, indicates a failure in cellular surveillance. As a result, malformed procollagen-II both accumulates intracellularly and is secreted, contributing to the deposition of a structurally compromised extracellular matrix that drives disease pathology. The iPSC-derived cartilage model presented here provides a genetically defined and expandable, human-based system for dissecting the mechanisms of failed proteostasis in collagenopathies. These findings shed light on the types of substitutions in procollagen that cells can or cannot recognize, and underscore the therapeutic potential of targeting cellular surveillance and collagen quality control pathways in COL2A1-related disorders and beyond. SIGNIFICANCEThe p.Arg719Cys substitution in COL2A1 is known to cause dominantly inherited precocious osteoarthritis with mild spondyloepiphyseal dysplasia, but its molecular pathogenesis remains poorly understood. Prior work suggests disrupted fibrillogenesis as the molecular etiology, yet phenotypically relevant human models are lacking. Here, we use human iPSC-derived cartilage to uncover how the Arg719Cys substitution alters procollagen-II folding, modification, trafficking, and matrix deposition. We observe hallmark disease phenotypes, including a sparse collagen-II matrix and distended endoplasmic reticulum (ER), consistent with intracellular accumulation of procollagen-II. Surprisingly, the mutant procollagen-II does not appear to be differentially engaged by the ER proteostasis network, despite the incorporation of an aberrant and apparently non-disulfide bond-forming Cys residue, suggesting a fundamental failure of cellular surveillance. This unrecognized misfolding leads to secretion of defective collagen culminating in matrix dysfunction. These findings establish a mechanistic basis for disease etiology and highlight collagen quality control as a potentially tractable therapeutic target. The iPSC-based model provides a scalable, human-relevant platform for dissecting disease mechanisms and developing therapies for COL2A1-related disorders and other collagenopathies.