Chop (Ddit3) Is Essential for D469del-COMP Retention and Cell Death in Chondrocytes in an Inducible Transgenic Mouse Model of Pseudoachondroplasia

Cartilage oligomeric matrix protein (COMP), a secreted glycoprotein synthesized by chondrocytes, regulates proliferation and type II collagen assembly. Mutations in the COMP gene cause pseudoachondroplasia and multiple epiphyseal dysplasia. Previously, we have shown that expression of D469del-COMP in transgenic mice causes intracellular retention of D469del-COMP, thereby recapitulating pseudoachondroplasia chondrocyte pathology. This inducible transgenic D469del-COMP mouse is the only in vivo model to replicate the critical cellular and clinical features of pseudoachondroplasia. Here, we report developmental studies of D469del-COMP-induced chondrocyte pathology from the prenatal period to adolescence. D469del-COMP retention was limited prenatally and did not negatively affect the growth plate until 3 weeks after birth. Results of immunostaining, transcriptome analysis, and qRT-PCR suggest a molecular model in which D469del-COMP triggers apoptosis during the first postnatal week. By 3 weeks (when most chondrocytes are retaining D469del-COMP), inflammation, oxidative stress, and DNA damage contribute to chondrocyte cell death by necroptosis. Importantly, by crossing the D469del-COMP mouse onto a Chop null background (Ddit3 null), thereby eliminating Chop, the unfolded protein response was disrupted, thus alleviating both D469del-COMP intracellular retention and premature chondrocyte cell death. Chop therefore plays a significant role in processes that mediate D469del-COMP retention. Taken together, these results suggest that there may be an optimal window before the induction of significant D469del-COMP retention during which endoplasmic reticulum stress could be targeted

Joint degeneration in a mouse model of pseudoachondroplasia: ER stress, inflammation, and block of autophagy

Pseudoachondroplasia (PSACH), a short limb skeletal dysplasia associated with premature joint degeneration, is caused by misfolding mutations in cartilage oligomeric matrix protein (COMP). Here, we define mutant-COMP-induced stress mechanisms that occur in articular chondrocytes of MT-COMP mice, a murine model of PSACH. The accumulation of mutant-COMP in the ER occurred early in MT-COMP articular chondrocytes and stimulated inflammation (TNFα) at 4 weeks, and articular chondrocyte death increased at 8 weeks while ER stress through CHOP was elevated by 12 weeks. Importantly, blockage of autophagy (pS6), the major mechanism that clears the ER, sustained cellular stress in MT-COMP articular chondrocytes. Degeneration of MT-COMP articular cartilage was similar to that observed in PSACH and was associated with increased MMPs, a family of degradative enzymes. Moreover, chronic cellular stresses stimulated senescence. Senescence-associated secretory phenotype (SASP) may play a role in generating and propagating a pro-degradative environment in the MT-COMP murine joint. The loss of CHOP or resveratrol treatment from birth preserved joint health in MT-COMP mice. Taken together, these results indicate that ER stress/CHOP signaling and autophagy blockage are central to mutant-COMP joint degeneration, and MT-COMP mice joint health can be preserved by decreasing articular chondrocyte stress. Future joint sparing therapeutics for PSACH may include resveratrol

Primary Osteoarthritis Early Joint Degeneration Induced by Endoplasmic Reticulum Stress Is Mitigated by Resveratrol

Increasing numbers of people are living with osteoarthritis (OA) due to aging and obesity, creating an urgent need for effective treatment and preventions. Two top risk factors for OA, age and obesity, are associated with endoplasmic reticulum (ER) stress. The I-ERS mouse, an ER stress-driven model of primary OA, was developed to study the role of ER stress in primary OA susceptibility. The I-ERS mouse has the unique ability to induce ER stress in healthy adult articular chondrocytes and cartilage, driving joint degeneration that mimics early primary OA. In this study, ER stress-induced damage occurred gradually and stimulated joint degeneration with OA characteristics including increased matrix metalloproteinase activity, inflammation, senescence, chondrocyte death, decreased proteoglycans, autophagy block, and gait dysfunction. Consistent with human OA, intense exercise hastened and increased the level of ER stress-induced joint damage. Notably, loss of a critical ER stress response protein (CHOP) largely ameliorated ER stress-stimulated OA outcomes including preserving proteoglycan content, reducing inflammation, and relieving autophagy block. Resveratrol diminished ER stress-induced joint degeneration by decreasing CHOP, TNFα, IL-1β, MMP-13, pS6, number of TUNEL-positive chondrocytes, and senescence marker p16 INK4a. The finding, that a dietary supplement can prevent ER stressed-induced joint degeneration in mice, provides a preclinical foundation to potentially develop a prevention strategy for those at high risk to develop OA

Identification of SOX9 Interaction Sites in the Genome of Chondrocytes

Our previous work has provided strong evidence that the transcription factor SOX9 is completely needed for chondrogenic differentiation and cartilage formation acting as a "master switch" in this differentiation. Heterozygous mutations in SOX9 cause campomelic dysplasia, a severe skeletal dysmorphology syndrome in humans characterized by a generalized hypoplasia of endochondral bones. To obtain insights into the logic used by SOX9 to control a network of target genes in chondrocytes, we performed a ChIP-on-chip experiment using SOX9 antibodies.The ChIP DNA was hybridized to a microarray, which covered 80 genes, many of which are involved in chondrocyte differentiation. Hybridization peaks were detected in a series of cartilage extracellular matrix (ECM) genes including Col2a1, Col11a2, Aggrecan and Cdrap as well as in genes for specific transcription factors and signaling molecules. Our results also showed SOX9 interaction sites in genes that code for proteins that enhance the transcriptional activity of SOX9. Interestingly, a strong SOX9 signal was also observed in genes such as Col1a1 and Osx, whose expression is strongly down regulated in chondrocytes but is high in osteoblasts. In the Col2a1 gene, in addition to an interaction site on a previously identified enhancer in intron 1, another strong interaction site was seen in intron 6. This site is free of nucleosomes specifically in chondrocytes suggesting an important role of this site on Col2a1 transcription regulation by SOX9.Our results provide a broad understanding of the strategies used by a "master" transcription factor of differentiation in control of the genetic program of chondrocytes

Transcriptional regulation of chondrogenesis by coactivator Tip60 via chromatin association with Sox9 and Sox5

Sox9 is a transcription factor of the SRY family required for several steps of chondrogenesis. It activates the expression of various chondrocyte-specific genes, but the mechanisms and role of cofactors involved in Sox9-regulated gene transcription are not fully understood. Here, we report on the characterization of a Tat interactive protein-60 (Tip60) as Sox9-associated protein identified in a yeast two-hybrid screen. Both in vitro and in vivo assays confirmed the specificity of interactions between Sox9 and Tip60 including the existence of an endogenous complex containing both polypeptides in chondrocytes. Gel shift assays showed the presence of a complex containing Sox9, Tip60 and the DNA of an enhancer region of the Col2a1 promoter. Reporter assays using a Col2a1 promoter with multimerized Col2a1 Sox9-binding sites indicated that Tip60 enhanced the transcriptional activity of Sox9. A larger Col2a1 promoter showed that Tip60 increased the activity of this promoter in the presence of both Sox9 and Sox5. Ectopic expression of Sox9 and transient-cotransfection with Tip60 in COS7 cells showed a more diffuse subnuclear colocalization, suggesting changes in the chromatin structure. Chromatin immunoprecipitation assays showed that Tip60, Sox9 and Sox5 associated with the same Col2a1 enhancer region. Consistent with a role of Tip60 in chondrogenesis, addition of Tip60 siRNA to limb-bud micromass cultures delayed chondrocyte differention. Tip60 enhances acetylation of Sox9 mainly through K61, 253, 398 residues; however, the K61/253/398A mutant of Sox9 still exhibited enhanced transcriptional activity by Tip60. Our results support the hypothesis that Tip60 is a coactivator of Sox9 in chondrocytes

Influence des facteurs de croissance sur les cellules cultivees en lattis

SIGLEINIST T 74383 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Resveratrol Reduces COMPopathy in Mice Through Activation of Autophagy

ABSTRACT Misfolding mutations in cartilage oligomeric matrix protein (COMP) cause it to be retained within the endoplasmic reticulum (ER) of chondrocytes, stimulating a multitude of damaging cellular responses including ER stress, inflammation, and oxidative stress, which ultimately culminates in the death of growth plate chondrocytes and pseudoachondroplasia (PSACH). Previously, we demonstrated that an antioxidant, resveratrol, substantially reduces the intracellular accumulation of mutant‐COMP, dampens cellular stress, and lowers the level of growth plate chondrocyte death. In addition, we showed that resveratrol reduces mammalian target of rapamycin complex 1 (mTORC1) signaling, suggesting a potential mechanism. In this work, we investigate the role of autophagy in treatment of COMPopathies. In cultured chondrocytes expressing wild‐type COMP or mutant‐COMP, resveratrol significantly increased the number of Microtubule‐associated protein 1A/1B‐light chain 3 (LC3) vesicles, directly demonstrating that resveratrol‐stimulated autophagy is an important component of the resveratrol‐driven mechanism responsible for the degradation of mutant‐COMP. Moreover, pharmacological inhibitors of autophagy suppressed degradation of mutant‐COMP in our established mouse model of PSACH. In contrast, blockage of the proteasome did not substantially alter resveratrol clearance of mutant‐COMP from growth plate chondrocytes. Mechanistically, resveratrol increased SIRT1 and PP2A expression and reduced MID1 expression and activation of phosphorylated protein kinase B (pAKT) and mTORC1 signaling in growth plate chondrocytes, allowing clearance of mutant‐COMP by autophagy. Importantly, we show that optimal reduction in growth plate pathology, including decreased mutant‐COMP retention, decreased mTORC1 signaling, and restoration of chondrocyte proliferation was attained when treatment was initiated between birth to 1 week of age in MT‐COMP mice, translating to birth to approximately 2 years of age in children with PSACH. These results clearly demonstrate that resveratrol stimulates clearance of mutant‐COMP by an autophagy‐centric mechanism. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research