ER Stress in ERp57 Knockout Knee Joint Chondrocytes Induces Osteoarthritic Cartilage Degradation and Osteophyte Formation

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.

Chondrocyte Adipogenic Differentiation in Softening Osteoarthritic Cartilage

A chondrocyte-to-osteoblast lineage continuum exists in the growth plate. Adipogenic differentiation of chondrocytes in vivo should be investigated. Here, unilateral anterior crossbite (UAC), which can induce osteoarthritic lesions in the temporomandibular joint (TMJ), was applied to 6-wk-old C57BL/6 mice. Matrix loss in TMJ cartilage was obvious, as demonstrated by safranin O staining, and the condylar cartilage elastic modulus values, detected by using atomic force microscopy (AFM), were reduced, indicating cartilage softening that might be linked with loss of the highly charged proteoglycan. By crossing the Rosa26/tdTomato (TdT) mice with Sox9;CreERT2 mice or with Col10;CreERT2 mice, we obtained the Sox9-TdT and Col10-TdT strains, respectively, in which the Sox9- or Col10-expressing cells, accordingly, were labeled by TdT. A few TdT-labeled cells in both strains expressed AdipoQ or DMP-1. The Sox9-TdT+AdipoQ+ cells were primarily located in the deep zone cartilage and then in the whole cartilage. Col10-TdT+AdipoQ+ cells, Sox9-TdT+DMP-1+ cells, and Col10-TdT+DMP-1+ cells were located in the deep zone region. UAC promoted AdipoQ and DMP-1 expression in cartilage. The percentages of Sox9-TdT+AdipoQ+ and Col10-TdT+AdipoQ+ cells to Sox9-TdT+ and Col10-TdT+ cells, respectively, were increased (both P < 0.05), implying that more chondrocytes were undergoing adipogenic differentiation in the UAC group, the cartilage of which was softened. The percentages of Sox9-TdT+DMP-1+ and Col10-TdT+DMP-1+ cells to Sox9-TdT+ cells and Col10-TdT+ cells, respectively, were increased (both P < 0.05), consistent with our report that UAC enhanced deep zone cartilage calcification, causing stiffening of the deep zone cartilage. Our present data demonstrated that TMJ chondrocyte descendants can become adipogenic in vivo in addition to becoming osteogenic. This potential was promoted in osteoarthritic cartilage, in which deep zone cartilage calcification-associated cartilage stiffening and proteoglycan loss-associated cartilage softening were both stimulated.

Disease-modifying therapeutic strategies in osteoarthritis: current status and future directions

Osteoarthritis (OA) is the most common form of arthritis. It is characterized by progressive destruction of articular cartilage and the development of chronic pain and constitutes a considerable socioeconomic burden. Currently, pharmacological treatments mostly aim to relieve the OA symptoms associated with inflammation and pain. However, with increasing understanding of OA pathology, several potential therapeutic targets have been identified, enabling the development of disease-modifying OA drugs (DMOADs). By targeting inflammatory cytokines, matrix-degrading enzymes, the Wnt pathway, and OA-associated pain, DMOADs successfully modulate the degenerative changes in osteoarthritic cartilage. Moreover, regenerative approaches aim to counterbalance the loss of cartilage matrix by stimulating chondrogenesis in endogenous stem cells and matrix anabolism in chondrocytes. Emerging strategies include the development of senolytic drugs or RNA therapeutics to eliminate the cellular or molecular sources of factors driving OA. This review describes the current developmental status of DMOADs and the corresponding results from preclinical and clinical trials and discusses the potential of emerging therapeutic approaches to treat OA.

Sonic Hedgehog Induces Mesenchymal Stromal Cell Senescence-Associated Secretory Phenotype and Chondrocyte Apoptosis in Human Osteoarthritic Cartilage

Hedgehog (HH) signaling plays a critical role in osteoarthritis (OA) pathogenesis, but the molecular mechanism remains to be elucidated. We show here that Sonic Hedgehog (SHH) gene expression is initiated in human normal cartilage stromal cells (NCSC) and increased in OA cartilage mesenchymal stromal cells (OA-MSCs) during aging. Manifesting a reciprocal cellular distribution pattern, the SHH receptors PTCH1 and SMO and transcription factors GLI2 and GLI3 are expressed by chondrocytes (OAC) in OA cartilage. SHH autocrine treatment of osteoarthritis MSC stimulates proliferation, chondrogenesis, hypertrophy, and replicative senescence with elevated SASP gene expression including IL1B, IL6, CXCL1, and CXCL8. SHH paracrine treatment of OAC suppresses COL2A1, stimulates MMP13, and induces chondrocyte apoptosis. The OA-MSC conditioned medium recapitulates the stimulatory effects of SHH on OAC catabolism and apoptosis. SHH knock-down in OA-MSC not only inhibits catabolic and senescence marker expression in OA-MSC, but also abolishes the effect of the OA-MSC conditioned medium on OAC catabolism and apoptosis. We propose that SHH is a key mediator between OA-MSC and OA chondrocytes interaction in human OA cartilage via two mechanisms: (1) SHH mediates MSC growth and aging by activating not only its proliferation and chondrogenesis, but also low-grade inflammation and replicative senescence, and (2) SHH mediates OA-MSC-induced OAC catabolism and apoptosis by creating a pro-inflammatory microenvironment favoring tissue degeneration during OA pathogenesis.

Senescent Tissue-Resident Mesenchymal Stromal Cells Are an Internal Source of Inflammation in Human Osteoarthritic Cartilage

Human osteoarthritic cartilage contains not only chondrocytes (OACs), but also mesenchymal stromal cells (OA-MSCs), whose abundance increases during osteoarthritis (OA). However, it is not clear how OA-MSC contributes to OA pathogenesis. Here, we show that aging OA-MSC plays an important role in cell senescence, fibrosis, and inflammation in cartilage. Protein array analysis indicates that OA-MSC expresses pro-inflammatory senescence associated secretory phenotype (SASP) including IL-1β, IL-6, IL-8, and CXCL1, 5, and 6, which play key roles in OA pathogenesis. OAC is a main recipient of the inflammatory signals by expressing receptors of cytokines. RNAseq analysis indicates that the transition from normal cartilage stromal cells (NCSCs) to OA-MSC during aging results in activation of SASP gene expression. This cell transition process can be recapitulated by a serial passage of primary OAC in cell culture comprising (1) OAC dedifferentiation into NCSC-like cells, and (2) its subsequent senescence into pro-inflammatory OA-MSC. While OAC dedifferentiation is mediated by transcriptional repression of chondrogenic gene expression, OA-MSC senescence is mediated by transcriptional activation of SASP gene expression. We postulate that, through replication-driven OAC dedifferentiation and mesenchymal stromal cell (MSC) senescence, OA-MSC becomes an internal source of sterile inflammation in human cartilage joint.

Metabolic reprogramming in chondrocytes to promote mitochondrial respiration reduces downstream features of osteoarthritis

Metabolic dysfunction in chondrocytes drives the pro-catabolic phenotype associated with osteoarthritic cartilage. In this study, substitution of galactose for glucose in culture media was used to promote a renewed dependence on mitochondrial respiration and oxidative phosphorylation. Galactose replacement alone blocked enhanced usage of the glycolysis pathway by IL1β-activated chondrocytes as detected by real-time changes in the rates of proton acidification of the medium and changes in oxygen consumption. The change in mitochondrial activity due to galactose was visualized as a rescue of mitochondrial membrane potential but not an alteration in the number of mitochondria. Galactose-replacement reversed other markers of dysfunctional mitochondrial metabolism, including blocking the production of reactive oxygen species, nitric oxide, and the synthesis of inducible nitric oxide synthase. Of more clinical relevance, galactose-substitution blocked downstream functional features associated with osteoarthritis, including enhanced levels of MMP13 mRNA, MMP13 protein, and the degradative loss of proteoglycan from intact cartilage explants. Blocking baseline and IL1β-enhanced MMP13 by galactose-replacement in human osteoarthritic chondrocyte cultures inversely paralleled increases in markers associated with mitochondrial recovery, phospho-AMPK, and PGC1α. Comparisons were made between galactose replacement and the glycolysis inhibitor 2-deoxyglucose. Targeting intermediary metabolism may provide a novel approach to osteoarthritis care.