Endoplasmic reticulum stress causes long bone shortening in P4hbC402R/+ mice: A mouse model exhibiting significant features of cole-carpenter syndrome driven by P4HB mutations

Cole-Carpenter syndrome (CCS) is a rare autosomal-dominant genetic disease characterized by craniosynostosis, ocular proptosis, hydrocephalus, distinctive facial features, and bone fragility. Previous cases of CCS are associated with genetic variations in P4HB, which encodes the protein disulfide isomerase (PDI), a key enzyme in protein folding. Patients with CCS caused by P4HB mutations often present with short stature, limb deformities, and abnormal epiphyseal plates. However, the underlying mechanisms are largely unknown. To investigate this, a mouse model expressing the P4hb^C402R mutation (corresponding to P4HB^C400R in humans) was generated. Although the mouse model did not exhibit craniofacial bone defects or brittle bone phenotypes, it did show significantly shortened long bones—a prominent characteristic of P4HB-induced CCS. This was due to impaired proliferation and delayed hypertrophy of growth plate chondrocytes. Mutant PDI was found to accumulate abnormally in the endoplasmic reticulum (ER), and in vitro experiments revealed defects in both the catalytic and chaperone activities of mutant PDI. In addition, we observed enhanced ER stress and activation of the PKR-like ER kinase (PERK) pathway in P4hb^C402R/+ chondrocytes. Inhibition of ER stress mitigated PERK activation, alleviated defective chondrocyte proliferation and differentiation, thereby rescuing bone length. Taken together, enhanced ER stress and the activation of the PERK, potentially initiated by the malfunctioning of PDI^C402R or its abnormal accumulation within the ER, or both, lead to compromised chondrocyte proliferation and differentiation in mice, and ultimately stunts mice growth. This provides new insights into the pathogenesis of P4HB-dominated CCS and offers potential therapeutic targets.