Objective. The integrity of cartilage depends on the correct synthesis of extracellular matrix (ECM) components. In case of insufficient folding of proteins in the endoplasmic reticulum (ER) of chondrocytes, ECM proteins aggregate, ER stress evolves, and the unfolded protein response (UPR) is initiated. By this mechanism, chondrocytes relieve the stress condition or initiate cell death by apoptosis. Especially persistent ER stress has emerged as a pathogenic mechanism in cartilage diseases, such as chondrodysplasias and osteoarthritis. As pharmacological intervention is not available yet, it is of great interest to understand cartilage ER stress in detail and to develop therapeutics to intervene. Methods. ERp57-deficient chondrocytes were generated by CRISPR/Cas9-induced KO. ER stress and autophagy were studied on mRNA and protein level as well as by transmission electron microscopy (TEM) in chondrocyte micromass or cartilage explant cultures of ERp57 KO mice. Thapsigargin (Tg), an inhibitor of the ER-residing Ca²⁺-ATPase, and 4-Phenylbutyric acid (4-PBA), a small molecular chemical chaperone, were applied to induce or inhibit ER stress. Results. Our data reveal that the loss of the protein disulfide isomerase ERp57 is sufficient to induce ER stress in chondrocytes. 4-PBA efficiently diffuses into cartilage explant cultures and diminishes excessive ER stress in chondrocytes dose dependently, no matter if it is induced by ERp57 KO or stimulation with Tg. Conclusion. ER-stress-related diseases have different sources; therefore, various targets for therapeutic treatment exist. In the future, 4-PBA may be used alone or in combination with other drugs for the treatment of ER-stress-related skeletal disorders in patients.

Cartilage is essential for skeletal development by endochondral ossification. The only cell type within the tissue, the chondrocyte, is responsible for the production of macromolecules for the extracellular matrix (ECM). Before proteins and proteoglycans are secreted, they undergo posttranslational modification and folding in the endoplasmic reticulum (ER). However, the ER folding capacity in the chondrocytes has to be balanced with physiological parameters like energy and oxygen levels. Specific cellular conditions, e.g., a high protein demand, or pathologic situations disrupt ER homeostasis and lead to the accumulation of poorly folded or misfolded proteins. This state is called ER stress and induces a cellular quality control system, the unfolded protein response (UPR), to restore homeostasis. Different mouse models with ER stress in chondrocytes display comparable skeletal phenotypes representing chondrodysplasias. Therefore, ER stress itself seems to be involved in the pathogenesis of these diseases. It is remarkable that chondrodysplasias with a comparable phenotype arise independent from the sources of ER stress, which are as follows: (1) mutations in ECM proteins leading to aggregation, (2) deficiencies in ER chaperones, (3) mutations in UPR signaling factors, or (4) deficiencies in the degradation of aggregated proteins. In any case, the resulting UPR substantially impairs ECM protein synthesis, chondrocyte proliferation, and/or differentiation or regulation of autophagy and apoptosis. Notably, chondrodysplasias arise no matter if single or multiple events are affected. We analyzed cartilage‐specific ERp57 knockout mice and demonstrated that the deficiency of this single protein disulfide isomerase, which is responsible for formation of disulfide bridges in ECM glycoproteins, is sufficient to induce ER stress and to cause an ER stress‐related bone phenotype. These mice therefore qualify as a novel model for the analysis of ER stress in chondrocytes. They give new insights in ER stress‐related short stature disorders and enable the analysis of ER stress in other cartilage diseases, such as osteoarthritis.

Ageing or obesity are risk factors for protein aggregation in the endoplasmic reticulum (ER) of chondrocytes. This condition is called ER stress and leads to induction of the unfolded protein response (UPR), which, depending on the stress level, restores normal cell function or initiates apoptotic cell death. Here the role of ER stress in knee osteoarthritis (OA) was evaluated. It was first tested in vitro and in vivo whether a knockout (KO) of the protein disulfide isomerase ERp57 in chondrocytes induces sufficient ER stress for such analyses. ER stress in ERp57 KO chondrocytes was confirmed by immunofluorescence, immunohistochemistry, and transmission electron microscopy. Knee joints of wildtype (WT) and cartilage-specific ERp57 KO mice (ERp57 cKO) were analyzed by indentation-type atomic force microscopy (IT-AFM), toluidine blue, and immunofluorescence/-histochemical staining. Apoptotic cell death was investigated by a TUNEL assay. Additionally, OA was induced via forced exercise on a treadmill. ER stress in chondrocytes resulted in a reduced compressive stiffness of knee cartilage. With ER stress, 18-month-old mice developed osteoarthritic cartilage degeneration with osteophyte formation in knee joints. These degenerative changes were preceded by apoptotic death in articular chondrocytes. Young mice were not susceptible to OA, even when subjected to forced exercise. This study demonstrates that ER stress induces the development of age-related knee osteoarthritis owing to a decreased protective function of the UPR in chondrocytes with increasing age, while apoptosis increases. Therefore, inhibition of ER stress appears to be an attractive therapeutic target for OA.