Skeletal Abnormalities in Mice Lacking Extracellular Matrix Proteins, Thrombospondin-1, Thrombospondin-3, Thrombospondin-5, and Type IX Collagen

Abstract

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,

Comparative Proteomic Analysis of Normal and Collagen IX Null Mouse Cartilage Reveals Altered Extracellular Matrix Composition and Novel Components of the Collagen IX Interactome

“…Although COMP is a known collagen IX-binding protein, the effect of COMP ablation is more subtle than the collagen IX knock-out, with only a mild disruption in chondrocyte columnar organization and no effect on the distribution of matrilin-3 (33). One question that arises from our data is whether any of the perturbations in the collagen IX knock-out ECM were caused by reduced COMP levels.…”

“…Fractions-Collagen IX ablation causes age-dependent pathological changes in chondrocyte organization in growth plate cartilage and the redistribution/loss of ECM components (8,9,33). To further characterize these defects we investigated the global effect of collagen IX ablation on protein expression in femoral head cartilage at two postnatal developmental time points.…”

Antisense Reduction of Mutant COMP Reduces Growth Plate Chondrocyte Pathology

“…Because absence of COMP has no effect on fertility and longevity in mice, COMP ASOs are a potential innovative and important treatment approach for PSACH. 27,28 ASO technology is a promising therapeutic approach for suppressing disease-associated genes, but delivery to tissues that do not have a rich blood supply can be challenging. Newer generations of chemically modified ASOs have enhanced RNA-binding affinity, nuclease stability, and pharmacokinetic properties that enable broad distribution to peripheral tissues in animals.…”

“…Support for this approach comes from COMP knockout mice that are normal in all growth parameters, with only mild growth plate disturbances and flattening of the articular cartilage surface after exercise. 27 This suggests that COMP is not necessary for normal skeletal development, longevity, or fertility in mice, 28 whereas the presence of mutant COMP is detrimental to skeletal growth. Therefore, decreasing COMP expression should limit the pathology.…”

Regulation of Cellular Redox Signaling by Matricellular Proteins in Vascular Biology, Immunology, and Cancer

“…Gene deletion studies indicate relatively little overlap in functions of the five TSPs (3,196), although some receptor interactions are conserved between family members. Integrins are the only known receptor class interacting with the primordial TSP in insects that remains functional for the modern proteins (26).…”