Immunochemical and immunocytochemical studies of the C-propeptide of type II procollagen in chondrocytes of the growth plate.

The C-propeptide of type II procollagen has previously been implicated in cartilage calcification. To further characterize this propeptide, we have investigated its molecular status and intracellular distribution in bovine fetal growth plate chondrocytes, particularly within the calcifying zone, using cell isolation, Western blotting, and localization with immunofluorescence and immunogold techniques. We found that in all cells freshly isolated by collagenase digestion the C-propeptide was a component of type II pro-alpha chains. No free C-propeptide was detected intracellularly. In situ localization of the C-propeptide by immunostaining employing immunofluorescence revealed the presence of procollagen in most growth plate cells, staining being most intense in hypertrophic cells. In the latter, large dilations of the rough endoplasmic reticulum were observed. These were not found in proliferating cells and had an approximate diameter of 5 microns. With immunogold localization these, together with Golgi-derived secretory granules, stained for the C-propeptide. These combined results suggest that in all cells of the growth plate the C-propeptide is a constituent part of type II collagen pro-alpha chains, and that it is usually segregated in the rough endoplasmic reticulum at a time when, according to other studies, collagen synthesis ceases in the lower hypertrophic zone and calcification of the extracellular matrix ensues. This suggests that the intracellular translocation of type II collagen pro-alpha chains may change in hypertrophic cells at this time.     

Expression, in cartilage, of a 7-amino-acid deletion in type II collagen from two unrelated individuals with Kniest dysplasia.

Kniest dysplasia is a heritable chondrodysplasia that severely affects skeletal growth. Recent evidence suggests that the etiology is based on mutations in COL2A1, the gene for collagen type II. We report the detection and partial characterization of an identical defect in type II collagen in two unrelated patients with Kniest dysplasia. Analysis of cyanogen bromide (CB)-digested cartilage samples from both probands by SDS-PAGE revealed an abnormal band for peptide alpha 1(II)CB12. The peptide was purified and digested with endoproteinase Asp-N. Fragments unique to the Kniest tissues were identified by reverse-phase high-pressure liquid chromatography and by sequence analysis. The results established a deletion of amino acids 102-108 of the alpha 1(II) triple-helical domain, which disrupted the (gly-X-Y)n repeat needed for helix formation. This was confirmed by sequence analysis of DNA amplified from both probands, revealing the molecular basis to be a single nucleotide mutation at a CpG dinucleotide (GCG-->GTG) in the codon for alanine 102. The mutation created a new splice donor site, which would account for the absence of the last seven amino acids from the 3' end of exon 12 in alpha 1(II)CB12. Light and electron micrographs of the probands' cartilage showed the perilacunar foamy matrix ("Swiss cheese") characteristic of Kniest dysplasia and chondrocytes containing dilated rough endoplasmic reticulum, which earlier studies had shown were filled with type II procollagen. These two cases strengthen the concept that Kniest dysplasia is based on mutations of COL2A1 and belongs within the broad spectrum of chondrodysplasias caused by type II collagenopathies.     

Morphological modifications of knee articular cartilage in bullfrogs (Lithobates catesbeianus) (Anura: Ranidae) during postmetamorphic maturation

The organization of knee articular cartilage of the bullfrog (Lithobates catesbeianus) differs in relation to morphofunctional adaptation in many aspects from similar structures in mammals. Thus, we investigated the structural organization and distribution of the extracellular matrix components in three articular cartilage regions in the distal epiphysis of the femur and proximal epiphysis of the tibia in male bullfrogs at 7, 540 and 1,080 days after metamorphosis. Cartilage thickness and cell density decreased in all regions with age. The basophilia differed among cartilage sites during aging. Calcium deposits were detected in growth cartilage of the femur and tibia in older animals. Immunohistochemical staining for chondroitin-6-sulfate was positive in the pericellular and territorial matrix in all samples. Positive immunostaining for type I collagen was observed in the superficial layer at all ages and in ossification centers of older animals. Reactivity to type II collagen was intense and was found throughout the stroma at all ages. Ultrastructural analysis of the epiphyseal region, in young animals, showed that the cytoplasm of chondrocytes was rich in rough endoplasmic reticulum, Golgi complex and mitochondria. In old animals, were observed a reduction in the size and number of mitochondria, disintegration of rough endoplasmic reticulum, and vacuolization of the Golgi complex. The bullfrog articular cartilage presented structural and organizational changes during aging which may contribute to the functional cartilage deterioration in old animals.     

An amino acid substitution (Gly853-->Glu) in the collagen alpha 1(II) chain produces hypochondrogenesis.

The spondyloepiphyseal dysplasia subclassification of bone dysplasias includes achondrogenesis, hypochondrogenesis, and spondyloepiphyseal dysplasia congenita. The phenotypic expression of these disorders ranges from mild to perinatal lethal forms. We report the detection and partial characterization of a defect in type II collagen in a perinatal lethal form of hypochondrogenesis. Electrophoresis in sodium dodecyl sulfate-polyacrylamide of CB peptides (where CB represents cyanogen bromide) from type II collagen of the diseased cartilage showed a doublet band for peptide alpha 1(II)CB10 and evidence for post-translational overmodification of the major peptides (CB8, CB10, and CB11) seen as a retarded electrophoretic mobility. Peptide CB10 was digested by endoproteinase Asp-N; and on reverse-phase high pressure liquid chromatography, fragments of abnormal mobility were noted. Sequence analysis of a unique peptide D12 revealed a single amino acid substitution (Gly-->Glu) at position 853 of the triple helical domain. This was confirmed by sequence analysis of amplified COL2A1 cDNA, which revealed a single nucleotide substitution (GGA-->GAA) in 5 of 10 clones. Electron micrographs of the diseased cartilage showed a sparse extracellular matrix and chondrocytes containing dilated rough endoplasmic reticulum, which suggested impaired assembly and secretion of the mutant protein. This case further documents the molecular basis of the spondyloepiphyseal dysplasia spectrum of chondrodysplasias as mutations in COL2A1.     

Type II achondrogenesis-hypochondrogenesis: identification of abnormal type II collagen.

We have extended the study of a mild case of type II achondrogenesis-hypochondrogenesis to include biochemical analyses of cartilage, bone, and the collagens produced by dermal fibroblasts. Type I collagen extracted from bone and types I and III collagen produced by dermal fibroblasts were normal, as was the hexosamine ratio of cartilage proteoglycans. Hyaline cartilage, however, contained approximately equal amounts of types I and II collagen and decreased amounts of type XI collagen. Unlike the normal SDS-PAGE mobility. Two-dimensional SDS-PAGE revealed extensive overmodification of all type II cyanogen bromide peptides in a pattern consistent with heterozygosity for an abnormal pro alpha 1(II) chain which impaired the assembly and/or folding of type II collagen. This interpretation implies that dominant mutations of the COL2A1 gene may cause type II achondrogenesis-hypochondrogenesis. More generally, emerging data implicating defects of type II collagen in the type II achondrogenesis-hypochondrogenesis-spondyloepiphyseal dysplasia congenita spectrum and in the Kniest-Stickler syndrome spectrum suggest that diverse mutations of this gene may be associated with widely differing phenotypic outcome.     

Multiple signals induce endoplasmic reticulum stress in both primary and immortalized chondrocytes resulting in loss of differentiation, impaired cell growth, and apoptosis

The endoplasmic reticulum is the site of synthesis and folding of secretory proteins and is sensitive to changes in the internal and external environment of the cell. Both physiological and pathological conditions may perturb the function of the endoplasmic reticulum, resulting in endoplasmic reticulum stress. The chondrocyte is the only resident cell found in cartilage and is responsible for synthesis and turnover of the abundant extracellular matrix and may be sensitive to endoplasmic reticulum stress. Here we report that glucose withdrawal, tunicamycin, and thapsigargin induce up-regulation of GADD153 and caspase-12, two markers of endoplasmic reticulum stress, in both primary chondrocytes and a chondrocyte cell line. Other agents such as interleukin-1beta or tumor necrosis factor alpha induced a minimal or no induction of GADD153, respectively. The endoplasmic reticulum stress resulted in decreased chondrocyte growth based on cell counts, up-regulation of p21, and decreased PCNA expression. In addition, perturbation of endoplasmic reticulum function resulted in decreased accumulation of an Alcian Blue positive matrix by chondrocytes and decreased expression of type II collagen at the protein level. Further, quantitative real-time PCR was used to demonstrate a down-regulation of steady state mRNA levels coding for aggrecan, collagen II, and link protein in chondrocytes exposed to endoplasmic reticulum stress-inducing conditions. Ultimately, endoplasmic reticulum stress resulted in chondrocyte apoptosis, as evidenced by DNA fragmentation and annexin V staining. These findings have potentially important implications regarding consequences of endoplasmic reticulum stress in cartilage biology.     

Areas of asbestoid (amianthoid) fibers in human thyroid cartilage characterized by immunolocalization of collagen types I,II, IX,XI and X

The distribution of type I, II, IX, XI and X collagens in and close to areas of asbestoid (amianthoid) fibers in thyroid cartilages of various ages was investigated in this study. Asbestoid fibers were first detected in thyroid cartilage from a 3-year-old male child. Areas of asbestoid fibers functionally appear to serve as guide rails for vascularization of thyroid cartilage. Alcian blue staining in the presence of 0.3 M MgCl₂ revealed a loss of glycosaminoglycans in areas of asbestoid fibers. In addition, the fibers reacted positively with antibodies against collagen types II, IX and XI, but showed no staining with antibodies to collagen types I and X. Territorial matrix of adjacent chondrocytes showed the same staining pattern. In addition to staining for type II, IX and XI collagens, asbestoid fibers showed strong immunostaining for type I collagen after puberty but not for type X collagen. However, groups of chondrocytes within areas of asbestoid fibers reacted strongly with antibodies to type X collagen, suggesting that this collagen plays an important role in matrix of highly differentiated chondrocytes. The finding that these type X collagen-positive chondrocytes also revealed immunostaining for type I collagen confirms previous studies showing that hypertrophic chondrocytes can further differentiate into cells that are characterized by the synthesis of type X and I collagens.     

COMP mutations, chondrocyte function and cartilage matrix

Cartilage oligomeric matrix protein (COMP) is a large extracellular pentameric glycoprotein found in the territorial matrix surrounding chondrocytes. More than 60 unique COMP mutations have been identified as causing two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED/EDM1). Recent studies demonstrate that calcium-binding and calcium induced protein folding differ between wild type and mutant COMP proteins and abnormal processing of the mutant COMP protein causes the characteristic large lamellar appearing rough endoplasimic reticulum (rER) cisternae phenotype observed in PSACH and EDMI growth plate chondrocytes. To understand the cellular events leading to this intracellular phenotype, PSACH chondrocytes with a G427E, D469del and D511Y mutations were grown in 3-D culture to produce cartilage nodules. Each nodule was assessed for the appearance and accumulation of cartilage-specific proteins within the rER and for matrix protein synthesis. All three COMP mutations were associated with accumulation of COMP in the rER cisternae by 4 weeks in culture, and by 8 weeks the majority of chondrocytes had the characteristic cellular phenotype. Mutations in COMP also affect the secretion of type IX collagen and matrilin-3 (MATN3) but not the secretion of aggrecan and type II collagen. COMP, type IX collagen and MATN3 were dramatically reduced in the PSACH matrices, and the distribution of these proteins in the matrix was diffuse. Ultrastructural analysis shows that the type II collagen present in the PSACH matrix does not form organized fibril bundles and, overall, the matrix is disorganized. The combined absence of COMP, type IX collagen and MATN3 causes dramatic changes in the matrix and suggests that these proteins play important roles in matrix assembly.     

Comparison of the Ruthenium hexammine trichloride method to other methods of chemical fixation for preservation of avian physeal cartilage

Summary Several methods of chemical fixation of avian physeal cartilage were compared. The Ruthenium hexammine trichloride method was compared to isotonic glutaraldehyde and neutral buffered formalin for light microscopy and paraffin embedment, and to two osmium-ferrocyanide methods and a combination of 1% glutaraldehyde and 4% formaldehyde for electron microscopy. Only the Ruthenium hexammine trichloride method prevented the loss of matrix proteoglycans and shrinkage of chondrocytes. In undecalcified paraffin-embedded cartilage, preservation of matrix and cellular detail was excellent, but Ruthenium hexammine trichloride interfered with Haematoxylin and Eosin staining. Glutaraldehyde gave more intense eosinophilia than neutral buffered formalin. Ultrastructurally, the Ruthenium hexammine trichloride method was the most consistent and gave the best overall fixation. Matrix elements and cellular and nuclear membranes were well preserved. It did result in vacuolation of the cytoplasm and mitochondria, and it increased granularity of the cytoplasm, chromatin, and rough endoplasmic reticulum. Other fixatives produced minimal vacuolation and finer granularity, but preservation was less consistent, cell/matrix contrast was often excessive, and they caused shrinkage of all chondrocytes. Large dilatations of the rough endoplasmic reticulum that appear to be cytoplasmic inclusions by light microscopy are described for the first time in avian cartilage.     

Localization of collagens and alkaline phosphatase activity during mineralization and ossification of human first rib cartilage

The localization of type X collagen and alkaline phosphatase activity was examined in order to gain a better understanding of tissue remodelling during development of human first rib cartilage. First rib cartilages from children and adolescents showed no staining for type X collagen and alkaline phosphatase activity. After onset of mineralization in the late second decade, a peripheral ossification process preceded by mineralized fibrocartilage could be distinguished from a more central one preceded by mineralized hyaline cartilage. No immunostaining for type X collagen was found in either type of cartilage. However, strong staining for alkaline phosphatase activity was detected around chondrocyte-like cells within fibrocartilage adjacent to the peripheral mineralization front, while a weaker staining pattern was observed around chondrocytes of hyaline cartilage near the central mineralization front. In addition, the territorial matrix of some chondrocytes within the hyaline cartilage revealed staining for type I collagen, suggesting that these cells undergo a dedifferentiation process, which leads to a switch from type II to type I collagen synthesis. The study provides evidence that mineralization of the hyaline cartilage areas in human first rib cartilage occurs in the absence of type X collagen synthesis but in the presence of alkaline phosphatase. Thus, mineralization of first rib cartilage seems to follow a different pattern from endochondral ossification in epiphyseal discs.     

Human polymer-based cartilage grafts for the regeneration of articular cartilage defects

The use of autologous chondrocyte implantation (ACI) and its further development combining autologous chondrocytes with bioresorbable matrices may represent a promising new technology for cartilage regeneration in orthopaedic research. Aim of our study was to evaluate the applicability of a resorbable three-dimensional polymer of pure polyglycolic acid (PGA) for the use in human cartilage tissue engineering under autologous conditions. Adult human chondrocytes were expanded in vitro using human serum and were rearranged three-dimensionally in human fibrin and PGA. The capacity of dedifferentiated chondrocytes to re-differentiate was evaluated after two weeks of tissue culture in vitro and after subcutaneous transplantation into nude mice by propidium iodide/fluorescein diacetate (PI/FDA) staining, scanning electron microscopy (SEM), gene expression analysis of typical chondrocyte marker genes and histological staining of proteoglycans and type II collagen. PI/FDA staining and SEM documented that vital human chondrocytes are evenly distributed within the polymer-based cartilage tissue engineering graft. The induction of the typical chondrocyte marker genes including cartilage oligomeric matrix protein (COMP) and cartilage link protein after two weeks of tissue culture indicates the initiation of chondrocyte re-differentiation by three-dimensional assembly in fibrin and PGA. Histological analysis of human cartilage tissue engineering grafts after 6 weeks of subcutaneous transplantation demonstrates the development of the graft towards hyaline cartilage with formation of a cartilaginous matrix comprising type II collagen and proteoglycan. These results suggest that human polymer-based cartilage tissue engineering grafts made of human chondrocytes, human fibrin and PGA are clinically suited for the regeneration of articular cartilage defects.     

sec24d encoding a component of COPII is essential for vertebra formation, revealed by the analysis of the medaka mutant, vbi

We characterized a medaka mutant, vertebra imperfecta (vbi), that displays skeletal defects such as craniofacial malformation and delay of vertebra formation. Positional cloning analysis revealed a nonsense mutation in sec24d encoding a component of the COPII coat that plays a role in anterograde protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus. Immunofluorescence analysis revealed the accumulation of type II collagen in the cytoplasm of craniofacial chondrocytes, notochord cells, and the cells on the myoseptal boundary in vbi mutants. Electron microscopy analysis revealed dilation of the ER and defective secretion of ECM components from cells in both the craniofacial cartilage and notochord in vbi. The higher vertebrates have at least 4 sec24 paralogs; however, the function of each paralog in development remains unknown. sec24d is highly expressed in the tissues that are rich in extracellular matrix and is essential for the secretion of ECM component molecules leading to the formation of craniofacial cartilage and vertebra.     

Considerations on the use of ear chondrocytes as donor chondrocytes for cartilage tissue engineering

Articular cartilage is often used for research on cartilage tissue engineering. However, ear cartilage is easier to harvest, with less donor-site morbidity. The aim of this study was to evaluate whether adult human ear chondrocytes were capable of producing cartilage after expansion in monolayer culture. Cell yield per gram of cartilage was twice as high for ear than for articular cartilage. Moreover, ear chondrocytes proliferated faster. Cell proliferation could be further stimulated by the use of serum-free medium with Fibroblast Growth Factor 2 (FGF2) in stead of medium with 10% serum. To evaluate chondrogenic capacity, multiplied chondrocytes were suspended in alginate and implanted subcutaneously in athymic mice. After 8 weeks the constructs demonstrated a proteoglycan-rich matrix that contained collagen type II. Constructs of ear chondrocytes showed a faint staining for elastin. Quantitative RT-PCR revealed that expression of collagen type II was 2-fold upregulated whereas expression of collagen type I was 2-fold down regulated in ear chondrocytes expanded in serum-free medium with FGF2 compared to serum-containing medium. Expression of alkaline phosphatase and collagen type X were low indicating the absence of terminal differentiation. We conclude that ear chondrocytes can be used as donor chondrocytes for cartilage tissue engineering. Furthermore, it may proof to be a promising alternative cell source to engineer cartilage for articular repair.     

Tissue engineering strategies for cartilage generation--micromass and three dimensional cultures using human chondrocytes and a continuous cell line

Utilizing ATDC5 murine chondrogenic cells and human articular chondrocytes, this study sought to develop facile, reproducible three-dimensional models of cartilage generation with the application of tissue engineering strategies, involving biodegradable poly(glycolic acid) scaffolds and rotating wall bioreactors, and micromass pellet cultures. Chondrogenic differentiation, assessed by histology, immunohistochemistry, and gene expression analysis, in ATDC5 and articular chondrocyte pellets was evident by the presence of distinct chondrocytes, expressing Sox-9, aggrecan, and type II collagen, in lacunae embedded in a cartilaginous matrix of type II collagen and proteoglycans. Tissue engineered explants of ATDC5 cells were reminiscent of cartilaginous structures composed of numerous chondrocytes, staining for typical chondrocytic proteins, in lacunae embedded in a matrix of type II collagen and proteoglycans. In comparison, articular chondrocyte explants exhibited areas of Sox-9, aggrecan, and type II collagen-expressing cells growing on fleece, and discrete islands of chondrocytic cells embedded in a cartilaginous matrix.     

A novel role for Bcl-2 associated-athanogene-1 (Bag-1) in regulation of the endoplasmic reticulum stress response in mammalian chondrocytes

BAG-1 (Bcl-2 associated athanogene-1) is a multifunctional protein, linking cell proliferation, cell death, protein folding, and cell stress. In vivo, BAG-1 is expressed in growth plate and articular cartilage, and the expression of BAG-1 is decreased with aging. Chondrocytes respond to endoplasmic reticulum (ER) stress with decreased expression of extracellular matrix proteins, and prolonged ER stress leads to chondrocyte apoptosis. Here we demonstrate for the first time that BAG-1 is involved in ER stress-induced apoptosis in chondrocytes. Induction of ER stress through multiple mechanisms all resulted in downregulation of BAG-1 expression. In addition, direct suppression of BAG-1 expression resulted in chondrocyte growth arrest and apoptosis, while stable overexpression of BAG-1 delayed the onset of ER stress-mediated apoptosis. In addition to regulating apoptosis, we also observed decreased expression of collagen type II in BAG-1 deficient chondrocytes. In contrast, overexpression of BAG-1 resulted in increased expression of collagen type II. Moreover, under ER stress conditions, the reduced expression of collagen type II was delayed in chondrocytes overexpressing BAG-1. These results suggest a novel role for BAG-1 in supporting viability and matrix expression of chondrocytes.     

Type II and VI collagen in nasal and articular cartilage and the effect of IL-1α on the distribution of these collagens

The distribution of type II and VI collagen was immunocytochemically investigated in bovine articular and nasal cartilage. Cartilage explants were used either fresh or cultured for up to 4 weeks with or without interleukin 1α (IL-1α). Sections of the explants were incubated with antibodies for both types of collagen. Microscopic analyses revealed that type II collagen was preferentially localized in the interchondron matrix whereas type VI collagen was primarily found in the direct vicinity of the chondrocytes. Treatment of the sections with hyaluronidase greatly enhanced the signal for both types of collagen. Also in sections of explants cultured with IL-1α a higher level of labeling of the collagens was found. This was apparent without any pre-treatment with hyaluronidase. Under the influence of IL-1α the area positive for type VI collagen that surrounded the chondrocytes broadened. Although the two collagens in both types of cartilage were distributed similarly, a remarkable difference was the higher degree of staining of type VI collagen in articular cartilage. Concomitantly we noted that digestion of this type of cartilage hardly occurred in the presence of IL-1α whereas nasal cartilage was almost completely degraded within 18 days of culture. Since type VI collagen is known to be relatively resistant to proteolysis we speculate that the higher level of type VI collagen in articular cartilage is important in protecting cartilage from digestion.     

Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix

Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro.     

Selective intracellular retention of extracellular matrix proteins and chaperones associated with pseudoachondroplasia.

Mutations in the cartilage oligomeric matrix protein (COMP) gene result in pseudoachondroplasia (PSACH), which is a chondrodysplasia characterized by early-onset osteoarthritis and short stature. COMP is a secreted pentameric glycoprotein that belongs to the thrombospondin family of proteins. We have identified a novel missense mutation which substitutes a glycine for an aspartic acid residue in the thrombospondin (TSP) type 3 calcium-binding domain of COMP in a patient diagnosed with PSACH. Immunohistochemistry and immunoelectron microscopy both show abnormal retention of COMP within characteristically enlarged rER inclusions of PSACH chondrocytes, as well as retention of fibromodulin, decorin and types IX, XI and XII collagen. Aggrecan and types II and VI collagen were not retained intracellularly within the same cells. In addition to selective extracellular matrix components, the chaperones HSP47, protein disulfide isomerase (PDI) and calnexin were localized at elevated levels within the rER vesicles of PSACH chondrocytes, suggesting that they may play a role in the cellular retention of mutant COMP molecules. Whether the aberrant rER inclusions in PSACH chondrocytes are a direct consequence of chaperone-mediated retention of mutant COMP or are otherwise due to selective intracellular protein interactions, which may in turn lead to aggregation within the rER, is unclear. However, our data demonstrate that retention of mutant COMP molecules results in the selective retention of ECM molecules and molecular chaperones, indicating the existence of distinct secretory pathways or ER-sorting mechanisms for matrix molecules, a process mediated by their association with various molecular chaperones.     

Matrilin-1 Is Essential for Zebrafish Development by Facilitating Collagen II Secretion

Matrilin-1 is the prototypical member of the matrilin protein family and is highly expressed in cartilage. However, gene targeting of matrilin-1 in mouse did not lead to pronounced phenotypes. Here we used the zebrafish as an alternative model to study matrilin function in vivo. Matrilin-1 displays a multiphasic expression during zebrafish development. In an early phase, with peak expression at about 15 h post-fertilization, matrilin-1 is present throughout the zebrafish embryo with exception of the notochord. Later, when the skeleton develops, matrilin-1 is expressed mainly in cartilage. Morpholino knockdown of matrilin-1 results both in overall growth defects and in disturbances in the formation of the craniofacial cartilage, most prominently loss of collagen II deposition. In fish with mild phenotypes, certain cartilage extracellular matrix components were present, but the tissue did not show features characteristic for cartilage. The cells showed endoplasmic reticulum aberrations but no activation of XBP-1, a marker for endoplasmic reticulum stress. In severe phenotypes nearly all chondrocytes died. During the early expression phase the matrilin-1 knockdown had no effects on cell morphology, but increased cell death was observed. In addition, the broad deposition of collagen II was largely abolished. Interestingly, the early phenotype could be rescued by the co-injection of mRNA coding for the von Willebrand factor C domain of collagen IIα1a, indicating that the functional loss of this domain occurs as a consequence of matrilin-1 deficiency. The results show that matrilin-1 is indispensible for zebrafish cartilage formation and plays a role in the early collagen II-dependent developmental events.