Disrupted type II collagenolysis impairs angiogenesis, delays endochondral ossification and initiates aberrant ossification in mouse limbs.

Cartilage remodelling and chondrocyte differentiation are tightly linked to angiogenesis during bone development and endochondral ossification. To investigate whether collagenase-mediated cleavage of the major cartilage collagen (collagen II) plays a role in this process, we generated a knockin mouse in which the mandatory collagenase cleavage site at PQG775↓776LAG, was mutated to PPG775↓776MPG (Col2a1Bailey). This approach blocked collagen II cleavage, and the production of putative collagen II matrikines derived from this site, without modifying matrix metalloproteinase expression or activity. We report here that this mouse (Bailey) is viable. It has a significantly expanded growth plate and exhibits delayed and abnormal angiogenic invasion into the growth plate. Deeper electron microscopy analyses revealed that, at around five weeks of age, a small number of blood vessel(s) penetrate into the growth plate, leading to its abrupt shrinking and the formation of a bony bridge. Our results from in vitro and ex vivo studies suggest that collagen II matrikines stimulate the normal branching of endothelial cells and promote blood vessel invasion at the chondro-osseous junction. The results further suggest that failed collagenolysis in Bailey leads to expansion of the hypertrophic zone and formation of a unique post-hypertrophic zone populated with chondrocytes that re-enter the cell cycle and proliferate. The biological rescue of this in vivo phenotype features the loss of a substantial portion of the growth plate through aberrant ossification, and narrowing of the remaining portion that leads to limb deformation. Together, these data suggest that collagen II matrikines stimulate angiogenesis in skeletal growth and development, revealing novel strategies for stimulating angiogenesis in other contexts such as fracture healing and surgical applications.

Abundant LacZ activity in the absence of Cre expression in the normal and inflamed synovium of adult Col2a1-Cre; ROSA26RLacZ reporter mice.

Recent analyses of Col2a1-Cre; ROSA26R reporter mice showed that synovial fibroblasts in 7-day mice were LacZ positive, due to a history of Col2a1-Cre expression conferred by their origin in the interzone of the developing joint. We have examined LacZ staining in adult Col2a1-Cre(+/0); ROSA26R(LacZ) mice, with and without inflammatory arthritis, and found that synovial fibroblasts in normal and inflamed synovium are LacZ positive, but Cre negative. Our results suggest that Cre-mediated recombination in joint interzone cells during development endure in adult synovial cells despite the absence of ongoing Cre expression. These findings have important implications and applications for the study of synovial inflammation in models of experimental arthritis.

Hyaluronan synthesis and degradation in cartilage and bone.

Hyaluronan (HA) is a large but simple glycosaminoglycan composed of repeating D-glucuronic acid, beta1-3 linked to N-acetyl-D-glucosamine beta1-4, found in body fluids and tissues, in both intra- and extracellular compartments. Despite its structural simplicity, HA has diverse functions in skeletal biology. In development, HA-rich matrices facilitate migration and condensation of mesenchymal cells, and HA participates in joint cavity formation and longitudinal bone growth. In adult cartilage, HA binding to aggrecan immobilises aggrecan, retaining it at the high concentrations required for compressive resilience. HA also appears to regulate bone remodelling by controlling osteoclast, osteoblast and osteocyte behaviour. The functions of HA depend on its intrinsic properties, which in turn rely on the degree of polymerisation by HA synthases, depolymerisation by hyaluronidases, and interactions with HA-binding proteins. HA synthesis and degradation are closely regulated in skeletal tissues and aberrant synthetic or degradative activity causes disease. The role and regulation of HA synthesis and degradation in cartilage, bone and skeletal development is discussed.

Endoplasmic reticulum-mediated quality control of type I collagen production by cells from osteogenesis imperfecta patients with mutations in the pro alpha 1 (I) chain carboxyl-terminal propeptide which impair subunit assembly.

A heterozygous single base change in exon 49 of COL1A1, which converted the codon for pro alpha 1(I) carboxyl-terminal propeptide residue 94 from tryptophan (TGG) to cysteine (TGT) was identified in a baby with lethal osteogenesis imperfecta (OI64). The C-propeptide mutations in OI64 and in another lethal osteogenesis imperfecta cell strain (OI26), which has a frameshift mutation altering the sequence of the carboxyl-terminal half of the propeptide (Bateman, J. F., Lamande, S. R., Dahl, H.-H. M., Chan, D., Mascara, T. and Cole, W. G. (1989) J. Biol. Chem. 264, 10960-10964), disturbed procollagen folding and retarded the formation of disulfide-linked trimers. Although assembly was delayed, the presence of slowly migrating, overmodified alpha 1(I) and alpha 2(I) chains indicated that mutant pro alpha 1(I) could associate with normal pro alpha 1(I) and pro alpha 2(I) to form pepsin-resistant triple-helical molecules, a proportion of which were secreted. Further evidence of the aberrant folding of mutant procollagen in OI64 and OI26 was provided by experiments demonstrating that the endoplasmic reticulum resident molecular chaperone BiP, which binds to malfolded proteins, was specifically bound to type I procollagen and was coimmunoprecipitated in the osteogenesis imperfecta cells but not control cells. Experiments with brefeldin A, which inhibits protein export from the endoplasmic reticulum, demonstrated that unassembled mutant pro alpha 1(I) chains were selectively degraded within the endoplasmic reticulum resulting in reduced collagen production by the osteogenesis imperfecta cells. This biosynthetic deficiency was reflected in the inability of OI64 and OI26 cells to produce a substantial in vitro collagenous matrix when grown in the continuous presence of ascorbic acid to allow collagen matrix formation. Both these carboxyl-terminal propeptide mutants showed a marked reduction in collagen accumulation to 20% (or less) of control cultures, comparable to the reduced collagen content of tissues from OI26.

Deposition and selective degradation of structurally-abnormal type I collagen in a collagen matrix produced by osteogenesis imperfecta fibroblasts in vitro.

Collagen matrix deposition and turnover were studied in skin fibroblasts from a control and from a patient with lethal perinatal osteogenesis imperfecta (OI) identified as a Gly667 to Arg substitution in the alpha 1(I) chain. A culture system where ascorbic acid was included to stimulate collagen matrix formation over extended culture periods was used. Serial extraction of the control cell collagen matrix confirmed that a substantial mature crosslinked collagen matrix was formed in the control fibroblast cell layer. In contrast, total collagen deposition by the OI fibroblasts was poor, with the quantity of collagen deposited only about a quarter of that of the control cells. Detailed analysis of the OI fibroblast matrix revealed that the mutant collagen chains were incorporated into the collagenous matrix. These data indicate that, when grown with ascorbate in long-term culture, OI fibroblasts reproduced the abnormal matrix deposition pattern of OI tissues in vivo. The overall dramatic reduction in collagen matrix formation was not accounted for by reduced collagen production, since during the period of matrix deposition (days 8-12) the rate of production by the OI cells was only slightly less than that of the control cells. The incorporation of the newly-synthesized OI collagen into the matrix was less efficient than in control cells, reflecting the cooperative nature of matrix deposition. The fate of this mutant collagen containing the Gly to Arg charge-change was followed in the matrix by a pulse-chase experiment and two-dimensional electrophoresis. These data demonstrated that the mutant incorporated into the matrix was unstable, with the proportion of mutant declining during the chase. The deposition of the mutant monomers into a pool more accessible to proteolytic degradation indicated that the mutant and normal collagens did not copolymerize to form collagen fibers of even collagen distribution, but rather the mutant collagen was either enriched on the exposed surfaces of mixed-composition fibers, or was unable to form copolymers efficiently and polymerized into mutant-only fibrillar assemblies more prone to proteolytic attack.

Assessment of procollagen processing defects by fibroblasts cultured in the presence of dextran sulphate.

The culture of skin fibroblasts in the presence of 0.01% (w/v) dextran sulphate results in complete proteolytic processing of procollagen to collagen. Processing occurs predominantly via a pN-collagen intermediate, suggesting that C-propeptide cleavage occurs early during the processing pathway. The processed collagen is associated with the cell-layer fraction. This method of inducing procollagen processing was evaluated for use in detecting procollagen processing abnormalities in heritable connective-tissue diseases. Abnormal type I procollagen processing was clearly demonstrated in two cases with known defects of pN-propeptide cleavage. In one, the cleavage deficiency was due to diminished N-proteinase activity (dermatosparaxis) and in the other case (Ehler's-Danlos syndrome type VIIA) the cleavage site was deleted. In a case of osteogenesis imperfecta (type II) the slow electrophoretic migration of type I collagen alpha-chains due to over-modification of lysine was readily demonstrated. Inefficient procollagen processing was also evident in this patient, as had been previously reported [de Wet, Pihlanjaniemi, Myers, Kelly & Prockop (1983) J. Biol. Chem. 258, 7721-7728]. Thus this method of culture in the presence of dextran sulphate provides a simple and rapid procedure for the detection of procollagen processing defects and electrophoretic abnormalities.