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

RNAi Reduces Expression and Intracellular Retention of Mutant Cartilage Oligomeric Matrix Protein

Mutations in cartilage oligomeric matrix protein (COMP), a large extracellular glycoprotein expressed in musculoskeletal tissues, cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. These mutations lead to massive intracellular retention of COMP, chondrocyte death and loss of growth plate chondrocytes that are necessary for linear growth. In contrast, COMP null mice have only minor growth plate abnormalities, normal growth and longevity. This suggests that reducing mutant and wild-type COMP expression in chondrocytes may prevent the toxic cellular phenotype causing the skeletal dysplasias. We tested this hypothesis using RNA interference to reduce steady state levels of COMP mRNA. A panel of shRNAs directed against COMP was tested. One shRNA (3B) reduced endogenous and recombinant COMP mRNA dramatically, regardless of expression levels. The activity of the shRNA against COMP mRNA was maintained for up to 10 weeks. We also demonstrate that this treatment reduced ER stress. Moreover, we show that reducing steady state levels of COMP mRNA alleviates intracellular retention of other extracellular matrix proteins associated with the pseudoachondroplasia cellular pathology. These findings are a proof of principle and the foundation for the development of a therapeutic intervention based on reduction of COMP expression

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

Antibodies used in studies.

<p>Ab = Abcam, Cambridge, MA, C = Calbiochem, San Diego, CA, K = Kamiya, Thousand Oaks, CA, MP = Molecular Probes, Carlsbad, CA, NM = NeoMarkers, SC = Santa Cruz, Santa Cruz, CA, and R&D = R&D Systems, Minneapolis, MN.</p

shRNAs directed against human COMP reduces steady-state levels of endogenous COMP mRNA from primary chondrocytes.

<p>A population of integrants for each shRNA was isolated using puromycin selection as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010302#s4" target="_blank">Methods</a> section. Primary human chondrocytes, with and without an shRNA, were cultured in monolayer for one week and RNA was collected. COMP is expressed in primary human chondrocytes grown in monolayer culture. qRT-PCR was used to quantify the COMP mRNA levels and β-actin mRNA levels were used to normalize the RNA.</p

Cells expressing MT-COMP with a COMP-directed shRNA 3B show reduction of intracellular COMP and CRT.

<p>COS-7 cells with (<b>I–L</b>) and without (<b>E–H</b>) shRNA 3B were infected with adenoviruses expressing MT-COMP (<b>E–L</b>). Uninfected COS-7 cells with shRNA 3B serves as controls (<b>A–D</b>). Cells were grown on coverslips for 48 hours after adenoviral infection and immunostained with COMP and CRT antibodies as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010302#s4" target="_blank">Methods</a> section. DAPI (blue) staining was used to localize all of the cells in the field. COMP (green in E) co-localizes (merge-yellow in H) with CRT (red in F).</p

shRNAs directed against human COMP effectively reduce steady-state levels of COMP mRNA and protein.

<p>COS-7 cells were stably transfected with shRNAs directed against COMP, and individual clones identified and selected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010302#s4" target="_blank">Methods</a> section. Cells were infected with an adenovirus expressing MT-COMP and collected 48 hours after infection. RNA and protein were purified for subsequent analysis. (<b>A</b>) Northern blot analysis of steady-state COMP mRNA in transfected cells. (<b>B</b>) Quantification of mRNA in a. Absence of a shRNA was set to 100% and all other lanes were compared to the no shRNA reference sample. snU6 mRNA was used to normalize protein loading. (<b>C</b>) Western blot analysis. (<b>D</b>) Quantification of protein in c in which the level of COMP protein from COS-7 cells expressing recombinant COMP protein in the absence of a shRNA was set to 100% and all other protein levels were compared to this reference. β-actin was used to control for protein loading. All experiments were replicated at least three times.</p

shRNA knockdown was maintained in the presence of high levels of COMP.

<p>COS-7 cells with and without 3B shRNA integrant were infected with a DOX-inducible MT-COMP adenovirus. DOX dosage ranging from 1 ng/ml to 10 µg/ml was used. Protein lysate was collected 48 hours after adenovirus infection. (<b>A</b>) Western blot analysis of COMP expression in the absence of shRNA 3B. (<b>B</b>) Western blot analysis of COMP expression in the presence of shRNA 3B.</p

Schematic of human COMP showing shRNA target locations relative to mRNA structure and protein domains.

<p>Solid gray blocks are used to indicate the 5′UTR, coding region and 3′UTR of COMP mRNA. The positions of COMP protein domains including the pentamer, epithelial growth factor (EGF)-like, type 3 calcium repeat and globular domains are shown below the protein structure. The position of the common D469del mutation in exon 17B is designated by arrow. Stem-loop hairpins indicate target positions of the shRNAs.</p