Cartilage oligomeric matrix protein (COMP) is a pentameric extracellular protein expressed in cartilage and other musculoskeletal tissues. Mutations in the COMP gene cause pseudoachondroplasia (PSACH), a severe dwarfing condition that has a growth plate chondrocyte pathology. PSACH is characterized by intracellular retention of COMP and other extracellular matrix (ECM) proteins, which form an ordered matrix within large rough endoplasmic reticulum cisternae. This accumulation is cytotoxic and causes premature chondrocyte cell death, thereby depleting chondrocytes needed for normal long bone growth. Research to define the underlying molecular mechanisms of PSACH has been hampered by the lack of a suitable model system. In this study, we achieved robust expression of human mutant (MT) or wild-type (WT) COMP in mice by using a tetracycline-inducible promoter. Normal growth plate distribution of ECM proteins was observed in 1-month-old WT-COMP and C57BL\6 control mice. In contrast, the structure of the MT-COMP growth plate recapitulated the findings of human PSACH growth plate morphology, including (1) retention of ECM proteins, (2) intracellular matrix formation in the rER cisternae, and (3) increased chondrocyte apoptosis. Therefore, we have generated the first mouse model to show extensive intracellular retention of ECM proteins recapitulating the human PSACH disease process at the cellular level.
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.
Pseudoachondroplasia (PSACH), a severe short-limb dwarfing condition, results from mutations that cause misfolding of the cartilage oligomeric matrix protein (COMP). Accumulated COMP in growth plate chondrocytes activates endoplasmic reticulum stress, leading to inflammation and chondrocyte death. Using a MT-COMP mouse model of PSACH that recapitulates the molecular and clinical PSACH phenotype, we previously reported that oxidative stress and inflammation play important and unappreciated roles in PSACH pathology. In this study, we assessed the ability of antioxidant and anti-inflammatory agents to affect skeletal and cellular pathology in our mouse model of PSACH. Treatment of MT-COMP mice with aspirin or resveratrol from birth to P28 decreased mutant COMP intracellular retention and chondrocyte cell death, and restored chondrocyte proliferation. Inflammatory markers associated with cartilage degradation and eosinophils were present in the joints of untreated juvenile MT-COMP mice, but were undetectable in treated mice. Most importantly, these treatments resulted in significantly increased femur length. This is the first and only therapeutic approach shown to mitigate both the chondrocyte and long-bone pathology of PSACH in a mouse model and suggests that reducing inflammation and oxidative stress early in the disease process may be a novel approach to treat this disorder.
Thrombospondin-5 (TSP5) is a large extracellular matrix glycoprotein found in musculoskeletal tissues. TSP5 mutations cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia; both show a characteristic growth plate phenotype with retention of TSP5, type IX collagen (Col9), and matrillin-3 in the rough endoplasmic reticulum. Whereas most studies focus on defining the disease process, few functional studies have been performed. TSP5 knockout mice have no obvious skeletal abnormalities, suggesting that TSP5 is not essential in the growth plate and/or that other TSPs may compensate. In contrast, Col9 knockout mice have diminished matrillin-3 levels in the extracellular matrix and earlyonset osteoarthritis. To define the roles of TSP1, TSP3, TSP5, and Col9 in the growth plate, all knockout and combinatorial strains were analyzed using histomorphometric techniques. While significant alterations in growth plate organization were found in certain single knockout mouse strains, skeletal growth was only mildly disturbed. In contrast, dramatic changes in growth plate organization in TSP3/5/Col9 knockout mice resulted in a 20% reduction in limb length, corresponding to similar short stature in humans. These studies show that type IX collagen may regulate growth plate width; TSP3, TSP5, and Col9 appear to contribute to growth plate organization; and TSP1 may help define the Thrombospondin-5 (TSP5), also known as cartilage oligomeric matrix protein, is the fifth member of the TSP gene family that includes trimeric (TSP1 and TSP2) and pentameric (TSP3, TSP4, and TSP5) protein members. 1First identified in cartilage and considered to be cartilage specific, TSP5 was subsequently found in other musculoskeletal tissues and is used as a marker for osteoarthritis and joint injury.2-9 TSP5 has been intensely studied, because mutations in this gene were shown to cause pseudoachondroplasia and multiple epiphyseal dysplasia (MED/EDM1). 10 -17 Mutations result in a dominantnegative effect on TSP5 causing massive retention of mutant TSP5 and other extracellular matrix (ECM) proteins, including types II and IX collagen and matrillin-3 (MATN3), in large rough endoplasmic reticulum cis-ternae. 15,18 -20 Recent studies show that these ECM proteins associate with TSP5 to form a structured intracellular matrix network. 20 Interestingly, mutations in MATN3 (MED/EDM5) and all three type IX collagen (Col9) genes also cause MED phenotypes (MED/EDM2, -3, and -6) that are typically milder than the MED condition caused by TSP5 mutations. 21Different approaches have attempted to define the role of TSP5. Recent work suggests that TSP5 stimulates chondrocyte proliferation through the binding of granulinepithelin precursor, an autocrine growth factor. 22 Binding experiments demonstrate that TSP5 interacts with types I,
Mutations in the gene encoding cartilage oligomeric matrix protein (COMP) cause pseudoachondroplasia (PSACH), a severe dwarfing condition. Pain, a significant complication, has generally been attributed to joint abnormalities and erosion and early onset osteoarthritis. Previously, we found that the Inflammatory-related transcripts were elevated In growth plate and articular cartilages, Indicating that Inflammation plays an important role in the chondrocyte disease pathology and may contribute to the overall pain sequelae. Here, we describe the effects of D469-delCOMP expression on the skeleton and growth plate chondrocytes with the aim to define a treatment window and thereby reduce pain. Consistent with the human PSACH phenotype, skeletal development of D469del-COMP mice was normal and similar to controls at birth. By postnatal day 7 (P7), the D469del-COMP skeleton, limbs, skull and snout were reduced and this reduction was progressive during postnatal growth, resulting in a short-limbed dwarfed mouse. Modulation of prenatal and postnatal expression of D469del-COMP showed minimal retention/cell death at P7 with some retention/cell death by P14, suggesting that earlier treatment intervention at the time of PSACH diagnosis may produce optimal results. Important and novel findings were an increase In Inflammatory proteins generally starting at P21 and that exercise exacerbates Inflammation. These observations suggest that pain in PSACH may be related to an intrinsic inflammatory process that can be treated symptomatically and is not related to early joint erosion. We also show that genetic ablation of CHOP dampens the inflammatory response. observed in mice expressing D469del-COMP. Toward identifying potential treatments, drugs known to decrease cellular stress (lithlum, phenyl butyric add, and valproate) were assessed. Interestingly, all diminished the chondrocyte pathology but had untoward outcomes on mouse growth, development, and longevity. Collectively, these results define an early treatment window in which chondrocytes can be salvaged, thereby potentially Increasing skeletal growth and decreasing pain.
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