Jamie Fitzgerald

Three Novel Collagen VI Chains, α4(VI), α5(VI), and α6(VI)

We report the identification of three new collagen VI genes at a single locus on human chromosome 3q22.1. The three new genes are COL6A4, COL6A5, and COL6A6 that encode the α4(VI), α5(VI), and α6(VI) chains. In humans, the COL6A4 gene has been disrupted by a chromosome break. Each of the three new collagen chains contains a 336-amino acid triple helix flanked by seven N-terminal von Willebrand factor A-like domains and two (α4 and α6 chains) or three (α5 chain) C-terminal von Willebrand factor A-like domains. In humans, mRNA expression of COL6A5 is restricted to a few tissues, including lung, testis, and colon. In contrast, the COL6A6 gene is expressed in a wide range of fetal and adult tissues, including lung, kidney, liver, spleen, thymus, heart, and skeletal muscle. Antibodies to the α6(VI) chain stained the extracellular matrix of human skeletal and cardiac muscle, lung, and the territorial matrix of articular cartilage. In cell transfection and immunoprecipitation experiments, mouse α4(VI)N6-C2 chain co-assembled with endogenous α1(VI) and α2(VI) chains to form trimeric collagen VI molecules that were secreted from the cell. In contrast, α5(VI)N5-C1 and α6(VI)N6-C2 chains did not assemble with α1(VI) and α2(VI) chains and accumulated intracellularly. We conclude that the α4(VI)N6-C2 chain contains all the elements necessary for trimerization with α1(VI) and α2(VI). In summary, the discovery of three additional collagen VI chains doubles the collagen VI family and adds a layer of complexity to collagen VI assembly and function in the extracellular matrix.

A new FACIT of the collagen family: COL21A1

Interrogation of the Human Genome data for sequences related to the von Willebrand factor A‐domain module identified a previously unreported 4.1 kb full‐length cDNA that is predicted to encode a new member of the collagen superfamily of extracellular matrix proteins, collagen XXI. The domain organization of collagen XXI comprised an N‐terminal signal sequence, followed by single von Willebrand factor A‐domain and thrombospondin domains, and an interrupted collagen triple helix. The organization of these motifs predict that collagen XXI is a new member of the FACIT collagen sub‐family. Expression analysis indicated that COL21A1 mRNA is present in many tissues including heart, stomach, kidney, skeletal muscle and placenta, and radiation hybrid mapping localized the COL21A1 gene to 6p11‐12.

Proteasomal Degradation of Unassembled Mutant Type I Collagen Pro-α1(I) Chains

We have previously shown that type I procollagen pro-α1(I) chains from an osteogenesis imperfecta patient (OI26) with a frameshift mutation resulting in a truncated C-propeptide, have impaired assembly, and are degraded by an endoplasmic reticulum-associated pathway (Lamandé, S. R., Chessler, S. D., Golub, S. B., Byers, P. H., Chan, D., Cole, W. G., Sillence, D. O. and Bateman, J. F. (1995)J. Biol. Chem. 270, 8642–8649). To further explore the degradation of procollagen chains with mutant C-propeptides, mouse Mov13 cells, which produce no endogenous pro-α1(I), were stably transfected with a pro-α1(I) expression construct containing a frameshift mutation that predicts the synthesis of a protein 85 residues longer than normal. Despite high levels of mutant mRNA in transfected Mov13 cells, only minute amounts of mutant pro-α1(I) could be detected indicating that the majority of the mutant pro-α1(I) chains synthesized are targeted for rapid intracellular degradation. Degradation was not prevented by brefeldin A, monensin, or NH4Cl, agents that interfere with intracellular transport or lysosomal function. However, mutant pro-α1(I) chains in both transfected Mov13 cells and OI26 cells were protected from proteolysis by specific proteasome inhibitors. Together these data demonstrate for the first time that procollagen chains containing C-propeptide mutations that impair assembly are degraded by the cytoplasmic proteasome complex, and that the previously identified endoplasmic reticulum-associated degradation of mutant pro-α1(I) in OI26 is mediated by proteasomes.

Heritability of articular cartilage regeneration and its association with ear wound healing in mice.

OBJECTIVE Emerging evidence suggests that genetic components contribute significantly to cartilage degeneration in osteoarthritis pathophysiology, but little information is available on the genetics of cartilage regeneration. Therefore, this study was undertaken to investigate cartilage regeneration in genetic murine models using common inbred strains and a set of recombinant inbred (RI) lines generated from LG/J (healer of ear wounds) and SM/J (nonhealer) inbred mouse strains. METHODS An acute full-thickness cartilage injury was introduced in the trochlear groove of 8-week-old mice (n=265) through microsurgery. Mouse knee joints were sagittally sectioned and stained with toluidine blue to evaluate regeneration. For the ear wound phenotype, a bilateral 2-mm through-and-through puncture was created in 6-week-old mice (n=229), and healing outcomes were measured after 30 days. Broad-sense heritability and genetic correlations were calculated for both phenotypes. RESULTS Time-course analysis of the RI mouse lines showed no significant regeneration until 16 weeks after surgery; at that time, the strains could be segregated into 3 categories: good, intermediate, and poor healers. Analysis of heritability (H2) showed that both cartilage regeneration (H2=26%; P=0.006) and ear wound closure (H2=53%; P<0.00001) were significantly heritable. The genetic correlations between the two healing phenotypes for common inbred mouse strains (r=0.92) and RI mouse lines (r=0.86) were found to be extremely high. CONCLUSION Our findings indicate that articular cartilage regeneration in mice is heritable, the differences between the mouse lines are due to genetic differences, and a strong genetic correlation between the two phenotypes exists, indicating that they plausibly share a common genetic basis. We therefore surmise that LG/J by SM/J intercross mice can be used to dissect the genetic basis of variation in cartilage regeneration.

Mechanically induced c-fos expression is mediated by cAMP in MC3T3-E1 osteoblasts

In serum‐deprived MC3T3‐E1 osteoblasts, mechanical stimulation caused by mild (287 × g) centrifugation induced a 10‐fold increase in mRNA levels of the proto‐oncogene, c‐fos. Induction of c‐fos was abolished by the cAMP‐dependent protein kinase inhibitor H‐89, suggesting that the transient c‐fos mRNA increase is mediated by cAMP. Down‐regulation of protein kinase C (PKC) activity by chronic TPA treatment failed to significantly reduce c‐fos induction, suggesting that TPA‐sensitive isoforms of PKC are not responsible for c‐fos up‐regulation. In addition, 287 × g centrifugation increased intracellular prostaglandin E2 (PGE2) levels 2.8‐fold (P<0.005). Since we have previously shown that prostaglandin E2 (PGE2) can induce c‐fos expression via a cAMP‐mediated mechanism, we asked whether the increase in c‐fos mRNA was due to centrifugation‐induced PGE2 release. Pretreatment with the cyclooxygenase inhibitors indomethacin and flurbiprofen did not hinder the early induction of c‐fos by mechanical stimulation. We conclude that c‐fos expression induced by mild mechanical loading is dependent primarily on cAMP, not PKC, and initial induction of c‐fos is not necessarily dependent on the action of newly synthesized PGE2.—Fitzgerald, J., Hughes‐Fulford, M. Mechanically induced c‐fos expression is mediated by cAMP in MC3T3‐E1 osteoblasts. FASEB J. 13, 553–557 (1999)

The N-terminal N5 subdomain of the alpha 3(VI) chain is important for collagen VI microfibril formation.

Collagen VI assembly is unique within the collagen superfamily in that the α1(VI), α2(VI), and α3(VI) chains associate intracellularly to form triple helical monomers, and then dimers and tetramers, which are secreted from the cell. Secreted tetramers associate end-to-end to form the distinctive extracellular microfibrils that are found in virtually all connective tissues. Although the precise protein interactions involved in this process are unknown, the N-terminal globular regions, which are composed of multiple copies of von Willebrand factor type A-like domains, are likely to play a critical role in microfibril formation, because they are exposed at both ends of the tetramers. To explore the role of these subdomains in collagen VI intracellular and extracellular assembly, α3(VI) cDNA expression constructs with sequential N-terminal deletions were stably transfected into SaOS-2 cells, producing cell lines that express α3(VI) chains with N-terminal globular domains containing modules N9-N1, N6-N1, N5-N1, N4-N1, N3-N1, or N1, as well as the complete triple helix and C-terminal globular domain (C1-C5). All of these transfected α3(VI) chains were able to associate with endogenous α1(VI) and α2(VI) to form collagen VI monomers, dimers, and tetramers, which were secreted. Importantly, cells that expressed α3(VI) chains containing the N5 subdomain, α3(VI) N9-C5, N6-C5, and N5-C5, formed microfibrils and deposited a collagen VI matrix. In contrast, cells that expressed the shorter α3(VI) chains, N4-C5, N3-C5, and N1-C5, were severely compromised in their ability to form end-to-end tetramer assemblies and failed to deposit a collagen VI matrix. These data demonstrate that the α3(VI) N5 module is critical for microfibril formation, thus identifying a functional role for a specific type A subdomain in collagen VI assembly.

The expanded collagen VI family: new chains and new questions

Abstract Collagen VI is a component of the extracellular matrix of almost all connective tissues, including cartilage, bone, tendon, muscles and cornea, where it forms abundant and structurally unique microfibrils organized into different suprastructural assemblies. The precise role of collagen VI is not clearly defined although it is most abundant in the interstitial matrix of tissues and often found in close association with basement membranes. Three genetically distinct collagen VI chains, α1(VI), α2(VI) and α3(VI), encoded by the COL6A1. COL6A2 and COL6A3 genes, were first described more than 20 years ago. Their molecular assembly and role in congenital muscular dystrophy has been broadly characterized. In 2008, three additional collagen VI genes arrayed in tandem at a single gene locus on chromosome 3q in humans, and chromosome 9 in mice, were described. Following the naming scheme for collagens the new genes were designated COL6A4. COL6A5 and COL6A6 encoding the α4(VI), α5(VI) and α6(VI) chains, respectively. This review will focus on the current state of knowledge of the three new chains.

WARP Is a Novel Multimeric Component of the Chondrocyte Pericellular Matrix That Interacts with Perlecan

WARP is a novel member of the von Willebrand factor A domain superfamily of extracellular matrix proteins that is expressed by chondrocytes. WARP is restricted to the presumptive articular cartilage zone prior to joint cavitation and to the articular cartilage and fibrocartilaginous elements in the joint, spine, and sternum during mouse embryonic development. In mature articular cartilage, WARP is highly specific for the chondrocyte pericellular microenvironment and co-localizes with perlecan, a prominent component of the chondrocyte pericellular region. WARP is present in the guanidine-soluble fraction of cartilage matrix extracts as a disulfide-bonded multimer, indicating that WARP is a strongly interacting component of the cartilage matrix. To investigate how WARP is integrated with the pericellular environment, we studied WARP binding to mouse perlecan using solid phase and surface plasmon resonance analysis. WARP interacts with domain III-2 of the perlecan core protein and the heparan sulfate chains of the perlecan domain I with KD values in the low nanomolar range. We conclude that WARP forms macromolecular structures that interact with perlecan to contribute to the assembly and/or maintenance of “permanent” cartilage structures during development and in mature cartilages.

WARP is a new member of the von Willebrand factor A-domain superfamily of extracellular matrix proteins.

We report a new member of the von illebrand factor A‐domain protein superfamily, WARP (for von illebrand factor ‐domain‐ elated rotein). The full‐length mouse WARP cDNA is 2.3 kb in size and predicts a protein of 415 amino acids which contains a signal sequence, a VA‐like domain, two fibronectin type III‐like repeats, and a short proline‐ and arginine‐rich segment. WARP mRNA was expressed predominantly in chondrocytes and in vitro expression experiments in transfected 293 cells indicated that WARP is a secreted glycoprotein that forms disulphide‐bonded oligomers. We conclude that WARP is a new member of the von Willebrand factor A‐domain (VA‐domain) superfamily of extracellular matrix proteins which may play a role in cartilage structure and function.

A eutherian X-linked gene, PDHA1, is autosomal in marsupials: A model for the evolution of a second, testis-specific variant in eutherian mammals

We report the cloning and mapping of a gene (PDHA) for the pyruvate dehydrogenase E1 alpha subunit in marsupials. In situ hybridization and Southern blot analysis show that PDHA is autosomal in marsupials, mapping to chromosome 3q in Sminthopsis macroura and 5p in Macropus eugenii. Since these locations represent a region that was translocated to the p arm of the human X chromosome following marsupial/eutherian divergence, we suggest that the marsupial PDHA gene is homologous to PDHA1, the somatic eutherian isoform located on human Xp and mouse X. Only one copy of PDHA is found in marsupials, whereas a second, testis-specific, intronless form is observed in eutherian mammals. We also suggest that translocation of PDHA to the eutherian X chromosome, which is inactivated during spermatogenesis, led to the evolution of a second testis-specific locus by retroposition to an autosome.