Cells Expressing Partially Unfolded R789C/p.R989C Type II Procollagen Mutant Associated With Spondyloepiphyseal Dysplasia Undergo Apoptosis

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We investigated the effects of the presence of R75C (p.R275C), R519C (p.719C), R789C (p.R989C), and G853E (p.G1053E) type II collagen (COL2A1) mutants, associated with distinct forms of spondyloepiphyseal dysplasia (SED), on the biological processes occurring in chondrocytic cells harboring those mutants. Mutant-specific biological responses of cells were initiated by activating tetracycline (Tet)-dependent expression of type II collagen mutants. Employing microscopic and biochemical assays, we determined that cells expressing the thermolabile R789C (p.R989C) type II collagen mutant undergo apoptosis. In contrast, in cells expressing the thermostable R75C (p.R275C), R519C (p.719C), and G853E (p.G1053E) mutants, apoptotic markers were not apparent. We also demonstrated that the R789C (p.R989C) mutant formed atypical complexes with endoplasmic reticulum (ER)-resident chaperones, thereby indicating an “unfolded protein response” (UPR) of cells harboring this specific mutant. Apoptotic changes were also demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and cleaved caspase 3 assays in the growth plates of mice harboring the R992C (p.R1147C) substitution in type II collagen. Based on these results, we propose that the intracellular presence of structurally altered type II collagen mutants could activate an apoptotic response, thereby limiting cell survival. By analyzing the response of cells to the altered structure of collagen mutants, our study contributes to better understanding the molecular basis of the pathological changes seen in vivo at the tissue level. Hum Mutat 29(6), 841-851, 2008.

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