Osteoarthritis is characterized by a progressive degradation of articular cartilage leading to loss of joint function. The molecular mechanisms regulating pathogenesis and progression of osteoarthritis are poorly understood. Remarkably, some characteristics of this joint disease resemble chondrocyte differentiation processes during skeletal development by endochondral ossification. In healthy articular cartilage, chondrocytes resist proliferation and terminal differentiation. By contrast, chondrocytes in diseased cartilage progressively proliferate and develop hypertrophy. Moreover, vascularization and focal calcification of joint cartilage are initiated. Signaling molecules that regulate chondrocyte activities in both growth cartilage and permanent articular cartilage during osteoarthritis are thus interesting targets for disease-modifying osteoarthritis therapies.

In cartilage, chondrocytes are responsible for the biogenesis and maintenance of the extracellular matrix (ECM) composed of proteins, glycoproteins and proteoglycans. Various cellular stresses, such as hypoxia, nutrient deprivation, oxidative stress or the accumulation of advanced glycation end products (AGEs) during aging, but also translational errors or mutations in cartilage components or chaperone proteins affect the synthesis and secretion of ECM proteins, causing protein aggregates to accumulate in the endoplasmic reticulum (ER). This condition, referred to as ER stress, interferes with cartilage cell homeostasis and initiates the unfolded protein response (UPR), a rescue mechanism to regain cell viability and function. Chronic or irreversible ER stress, however, triggers UPR-initiated cell death. Due to unresolved ER stress in chondrocytes, diseases of the skeletal system, such as chondrodysplasias, arise. ER stress has also been identified as a contributing factor to the pathogenesis of cartilage degeneration processes such as osteoarthritis (OA). This review provides current knowledge about the biogenesis of ECM components in chondrocytes, describes possible causes for the impairment of involved processes and focuses on the ER stress-induced cell death in articular cartilage during OA. Targeting of the ER stress itself or intervention in UPR signaling to reduce death of chondrocytes may be promising for future osteoarthritis therapy.

Objective: Syndecan 4, a heparan sulfate proteoglycan, has been associated with osteoarthritis. The present study was undertaken to analyze the functional role of syndecan 4 in endochondral ossification of mouse embryos and in adult fracture repair, which, like osteoarthritis, involves an inflammatory component.Methods: Sdc4 promoter activity was analyzed in Sdc4^−/−lacZ‐knockin mice, using β‐galactosidase staining. Endochondral ossification in embryos from embryonic day 16.5 was assessed by histologic and immunohistologic staining. Bone fracture repair was analyzed in femora of adult mice on days 7 and 14 postfracture. To evaluate Sdc2 and Sdc4 gene expression with and without tumor necrosis factor α (TNFα) and Wnt‐3a stimulation, quantitative real‐time polymerase chain reaction was performed.Results: In Sdc4^−/−lacZ‐knockin animals, syndecan 4 promoter activity was detectable at all stages of chondrocyte differentiation, and Sdc4 deficiency inhibited chondrocyte proliferation. Aggrecan turnover in the uncalcified cartilage of the epiphysis was decreased transiently in vivo, but this did not lead to a growth phenotype at birth. In contrast, among adult mice, fracture healing was markedly delayed in Sdc4^−/−animals and was accompanied by increased callus formation. Blocking of inflammation via anti‐TNFα treatment during fracture healing reduced these changes in Sdc4^−/−mice to levels observed in wild‐type controls. We analyzed the differences between the mild embryonic and the severe adult phenotype, and found a compensatory up‐regulation of syndecan 2 in the developing cartilage of Sdc4^−/−mice that was absent in adult tissue. Stimulation of chondrocytes with Wnt‐3a in vitro led to increased expression of syndecan 2, while stimulation with TNFα resulted in up‐regulation of syndecan 4 but decreased expression of syndecan 2. TNFα stimulation reduced syndecan 2 expression and increased syndecan 4 expression even in the presence of Wnt‐3a, suggesting that inflammation has a strong effect on the regulation of syndecan expression.Conclusion: Our results demonstrate that syndecan 4 is functionally involved in endochondral ossification and that its loss impairs fracture healing, due to inhibition of compensatory mechanisms under inflammatory conditions.

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.

Gender differences are a common finding in osteoarthritis (OA). This may result from a differential response of males and females to endoplasmic reticulum (ER) stress in articular chondrocytes. We have previously described that ER stress in cartilage-specific ERp57 KO mice (ERp57 cKO) favors the development of knee OA, since this stress condition cannot be adequately compensated in articular chondrocytes with increasing age leading to the induction of apoptotic cell death and subsequent cartilage degeneration. The aim of this study was to enlighten gender-specific differences in ER stress, apoptosis, and OA development in ERp57 cKO mice. The analyses were extended by in vitro studies on the influence of estradiol in CRISPR/Cas9-generated C28/I2 ERp57 knock out (KO) and WT cells. ER stress was evaluated by immunofluorescence analysis of the ER stress markers calnexin (Cnx) and binding-immunoglobulin protein (BiP), also referred to as glucose-regulating protein 78 (GRP78) in vivo and in vitro. Apoptotic cell death was investigated by a commercially available cell death detection ELISA and TUNEL assay. OA development in mice was analyzed by toluidine blue staining of paraffin-embedded knee cartilage sections and quantified by OARSI-Scoring. Cell culture studies exhibited a reduction of ER stress and ER stress-induced apoptosis in C28/I2 cells in presence of physiological estradiol concentrations. This is consistent with a slower increase in age-related ER stress and a reduced number of apoptotic chondrocytes in female mice compared to male littermates contributing to a reduced osteoarthritic cartilage degeneration in female mice. Taken together, this study demonstrates that the female sex hormone estradiol can reduce ER stress and ER stress-induced apoptosis in articular chondrocytes, thus minimizing critical events favoring osteoarthritic cartilage degeneration. Therefore, the inhibition of ER stress through a modulation of effects induced by female sex hormones appears to be attractive for OA therapy.

: Long-bone growth by endochondral ossification is cooperatively accomplished by chondrocyte proliferation, hypertrophic differentiation, and appropriate secretion of collagens, glycoproteins, and proteoglycans into the extracellular matrix (ECM). Before folding and entering the secretory pathway, ECM macromolecules in general are subject to extensive posttranslational modification, orchestrated by chaperone complexes in the endoplasmic reticulum (ER). ERp57 is a member of the protein disulfide isomerase (PDI) family and facilitates correct folding of newly synthesized glycoproteins by rearrangement of native disulfide bonds. Here, we show that ERp57-dependent PDI activity is essential for postnatal skeletal growth, especially during the pubertal growth spurt characterized by intensive matrix deposition. Loss of ERp57 in growth plates of cartilage-specific ERp57 knockout mice (ERp57 KO) results in ER stress, unfolded protein response (UPR), reduced proliferation, and accelerated apoptotic cell death of chondrocytes. Together this results in a delay of long-bone growth with the following characteristics: (1) enlarged growth plates; (2) expanded hypertrophic zones; (3) retarded osteoclast recruitment; (4) delayed remodeling of the proteoglycan-rich matrix; and (5) reduced numbers of bone trabeculae. All the growth plate and bone abnormalities, however, become attenuated after the pubertal growth spurt, when protein synthesis is decelerated and, hence, ERp57 function is less essential. © 2015 American Society for Bone and Mineral Research.