Identification and characterization of the human type II collagen gene (COL2A1).

The gene contained in the human cosmid clone CosHcol1, previously designated an alpha 1(I) collagen-like gene, has now been identified. CosHcol1 hybridizes strongly to a single 5.9-kilobase mRNA species present only in tissue in which type II collagen is expressed. DNA sequence analysis shows that this clone is highly homologous to the chicken alpha 1(II) collagen gene. These data together suggest that CosHcol1 contains the human alpha 1(II) collagen gene COL2A1. The clone appears to contain the whole gene (30 kilobases in length) and will be extremely useful in the study of cartilage development and for identifying those inherited chondrodystrophies in which defects occur in this gene.     

Asynchronous DNA replication within the human beta-globin gene locus.

The timing of DNA replication of the human beta-globin gene locus has been studied by blot hybridization of newly synthesized BrdUrd-substituted DNA from cells in different stages of the S phase. Using probes that span greater than 120 kilobases across the human beta-globin gene locus, we show that the majority of this domain replicates in early S phase in the human erythroleukemia cell line K562 and in middle-to-late S phase in the lymphoid cell line Manca. However, in K562 cells three small regions display a strikingly different replication pattern than adjacent sequences. These islands, located in the inter-gamma-globin gene region and approximately 20 kilobases 5' to the epsilon-globin gene and 20 kilobases 3' to the beta-globin gene, replicate later and throughout S phase. A similar area is also present in the alpha-globin gene region in K562 cells. We suggest that these regions may represent sites of termination of replication forks.     

Inhibition of cartilage degradation: a combined tissue engineering and gene therapy approach

OBJECTIVE: To determine if tissue-engineered cartilage can be protected from cytokine-induced degradation using a gene therapy approach. METHODS: Chemical and pantropic retroviral gene transfer methodologies were compared for their ability to introduce a luciferase reporter gene into adult bovine cartilage chondrocytes grown in monolayer. Pantropic retrovirus was then used to transduce these cells with human tissue inhibitor of metalloproteinases 1 (TIMP-1), and the stability of expression in monolayer or pellet culture was monitored for 6 weeks. Untransduced and TIMP-1-transduced cells were also used to tissue engineer 3-dimensional cartilage constructs that were then challenged with interleukin-1 (IL-1) for 4 weeks. Conditioned media and residual cartilage were collected for analysis of matrix components, including type II collagen and proteoglycans, and for TIMP-1 production and matrix metalloproteinase (MMP) activity. RESULTS: Chemical transfection of adult bovine chondrocytes gave rise to short-lived reporter expression that was virtually undetectable after 4 weeks of culture. In contrast, pantropic retroviral transduction gave rise to stable expression that persisted at a high level for at least 6 weeks. Pantropic transduction of the cells with TIMP-1 gave rise to similar long-term expression, both in monolayer and pellet cultures. TIMP-1-transduced tissue-engineered cartilage also retained TIMP-1 expression for an additional 4 weeks of culture in the presence of IL-1. Compared with control samples, TIMP-1-transgenic cartilage resisted the catabolic effects of IL-1, with MMP activity reduced to basal levels and a decreased loss of type II collagen. CONCLUSION: Pantropic retroviral transduction permits long-term expression of potentially therapeutic transgenes in adult tissue-engineered cartilage. While TIMP-1 transduction could be used to prevent collagen breakdown, alternative transgenes may be necessary to protect cartilage proteoglycans.     

Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9

OBJECTIVE: Human osteoarthritis (OA) is characterized by a pathologic shift in articular cartilage homeostasis toward the progressive loss of extracellular matrix (ECM). The purpose of this study was to investigate the ability of rAAV-mediated SOX9 overexpression to restore major ECM components in human OA articular cartilage. METHODS: We monitored the synthesis and content of proteoglycans and type II collagen in 3-dimensional cultures of human normal and OA articular chondrocytes and in explant cultures of human normal and OA articular cartilage following direct application of a recombinant adeno-associated virus (rAAV) SOX9 vector in vitro and in situ. We also analyzed the effects of this treatment on cell proliferation in these systems. RESULTS: Following SOX9 gene transfer, expression levels of proteoglycans and type II collagen increased over time in normal and OA articular chondrocytes in vitro. In situ, overexpression of SOX9 in normal and OA articular cartilage stimulated proteoglycan and type II collagen synthesis in a dose-dependent manner. These effects were not associated with changes in chondrocyte proliferation. Notably, expression of the 2 principal matrix components could be restored in OA articular cartilage to levels similar to those in normal cartilage. CONCLUSION: These data support the concept of using direct, rAAV-mediated transfer of chondrogenic genes to articular cartilage for the treatment of OA in humans.     

In situ zymographic localisation of type II collagen degrading activity in osteoarthritic human articular cartilage

OBJECTIVES: Chondrocytic matrix metalloproteinases (MMPs) are believed to be important in osteoarthritic cartilage degradation. The cartilage lesion of osteoarthritis (OA) is focal and often progressive. During its development chondrocytes differentially up and down regulate production of mRNA for individual MMPs. This observation has potential implications for understanding the disease processes that lead to progressive cartilage loss in OA and designing appropriate targeted treatment. The complex regulation of MMP mediated effects means there is a pressing need to establish whether visualisation of MMP mRNA or protein equates to enzyme activity. The technique of in situ zymography (ISZ) offers a way of examining diseased human tissue for in vivo production of an excess of degrading enzyme over inhibitor. The primary objective of this study was to assess, and if positive follow, collagen II degrading activity in cartilage during development of the OA lesion. A secondary objective was to assess whether there was any correlation between sites of collagen II degrading activity and expression of the collagenase (MMP-13), recently implicated in type II collagen degredation in this lesion. METHODS: Biopsied human normal and osteoarthritic cartilage, showing various degrees of damage, was examined by in situ zymography, with and without enzyme inhibitors, to establish sites of type II collagenase activity. Paired samples were probed for MMP-13 mRNA using 35S-labelled oligonucleotide probes. Comparative analyses were performed. RESULTS: In situ zymography showed collagen II degrading activity over chondrocytes only in osteoarthritic cartilage. Distribution and amount varied with the extent of cartilage damage and position of chondrocytes, being greatest in deep cartilage and in cartilage lesions where fissuring was occurring. The enzyme causing the degradation behaved as a matrix metalloproteinase. MMP-13 mRNA expression codistributed with the type II collagenase activity. CONCLUSION: In OA, chondrocytes can degrade type II collagen. The type II collagen degrading activity varies in site and amount as the cartilage lesion progresses and throughout codistributes with MMP-13 mRNA expression.     

Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells

The development of regenerative therapies for cartilage injury has been greatly aided by recent advances in stem cell biology. Induced pluripotent stem cells (iPSCs) have the potential to provide an abundant cell source for tissue engineering, as well as generating patient-matched in vitro models to study genetic and environmental factors in cartilage repair and osteoarthritis. However, both cell therapy and modeling approaches require a purified and uniformly differentiated cell population to predictably recapitulate the physiological characteristics of cartilage. Here, iPSCs derived from adult mouse fibroblasts were chondrogenically differentiated and purified by type II collagen (Col2)-driven green fluorescent protein (GFP) expression. Col2 and aggrecan gene expression levels were significantly up-regulated in GFP+ cells compared with GFP− cells and decreased with monolayer expansion. An in vitro cartilage defect model was used to demonstrate integrative repair by GFP+ cells seeded in agarose, supporting their potential use in cartilage therapies. In chondrogenic pellet culture, cells synthesized cartilage-specific matrix as indicated by high levels of glycosaminoglycans and type II collagen and low levels of type I and type X collagen. The feasibility of cell expansion after initial differentiation was illustrated by homogenous matrix deposition in pellets from twice-passaged GFP+ cells. Finally, atomic force microscopy analysis showed increased microscale elastic moduli associated with collagen alignment at the periphery of pellets, mimicking zonal variation in native cartilage. This study demonstrates the potential use of iPSCs for cartilage defect repair and for creating tissue models of cartilage that can be matched to specific genetic backgrounds.     

Formation of nodular structures resembling mature articular cartilage in long–term primary cultures of human fetal epiphyseal chondrocytes on a hydrogel substrate

Objective. To establish long–term cultures of human fetal epiphyseal chondrocytes under conditions that allow the preservation of a cartilage–specific phenotype. Methods. Chondrocytes isolated from 20—24–week human fetal epiphyseal cartilage were cultured for up to 180 days on plastic dishes previously coated with the hydrogel, poly–(2–hydroxyethyl methacrylate). Morphologic, ultrastructural, and biochemical characteristics of the cultures were examined at various intervals, and the expression of genes encoding types I, II, and IX collagen and aggrecan core protein was determined by Northern hybridizations of total cellular RNA with human–specific complementary DNAs. Results. Human fetal epiphyseal chondrocytes cultured for 180 days under conditions that prevented their attachment to the underlying substratum formed nodular structures with morphologic and structural characteristics resembling mature articular cartilage. The cells in the center of the nodules remained spherical and were surrounded by an abundant cartilaginous extracellular matrix, as evidenced by histochemical and ultrastructural examinations. The cells in the periphery of the nodules acquired a discoid morphology and were surrounded by a sparse extracellular matrix. Biosyn–thetic studies demonstrated the maintenance of a cartilagespecific phenotype throughout the 180 days of culture, with the production of aggrecan and types II, IX, and XI collagens but not type I collagen. Northern hybridizations showed high levels of messenger RNAs (mRNAs) for aggrecan core protein, type II procollagen, and type IX collagen, but type I procollagen mRNA was not detectable even at 180 days of culture. Conclusion. The human chondrocyte culture system described here allows the maintenance of a chondrocyte–specific phenotype for prolonged periods (up to 180 days). The long–term chondrocyte cultures formed nodular structures that resemble mature articular cartilage morphologically, ultrastructurally, biosynthetically, and in the pattern of cartilage–specific gene expression.     

Increased expression of discoidin domain receptor 2 is linked to the degree of cartilage damage in human knee joints: a potential role in osteoarthritis pathogenesis

OBJECTIVE: To investigate the relationship between increased discoidin domain receptor 2 (DDR-2) expression and cartilage damage in osteoarthritis (OA). METHODS: Full-thickness cartilage tissue samples from 16 human knee joints were obtained and the grade of cartilage damage was evaluated according to the Mankin scale. Expression of DDR-2, matrix metalloproteinase 13 (MMP-13), and MMP-derived type II collagen fragments was visualized immunohistochemically. Moreover, upon stimulation with either type II collagen or gelatin, levels of DDR-2 and MMP-13 messenger RNA (mRNA) in primary human articular chondrocytes were assessed by real-time polymerase chain reaction. RESULTS: Immunohistochemical analysis showed an increase in DDR-2 expression in human articular cartilage, which was correlated with the degree of tissue damage. In parallel, the extent of MMP-13 and type II collagen breakdown products was elevated as a function of increased DDR-2 expression and cartilage damage. Furthermore, in vitro experiments revealed an up-regulation of both DDR-2 and MMP-13 mRNA in human articular chondrocytes after stimulation with type II collagen. CONCLUSION: Our data indicate that 3 factors, DDR-2 expression, MMP-13 expression, and the degree of cartilage damage, are linked, such that DDR-2 promotes tissue catabolism, and tissue degradation promotes DDR-2 up-regulation and activation. Thus, the perpetuation of DDR-2 expression and activation can be seen as a vicious circle that ultimately leads to cartilage destruction in OA.     

The human beta-globin locus activation region alters the developmental fate of a human fetal globin gene in transgenic mice.

We linked a 3.3-kilobase fragment containing the entire A gamma-globin gene together with 1.3 kilobases of 5' flanking and 0.37 kilobase of 3' flanking DNA to a 2.5-kilobase fragment containing four of the developmentally stable hypersensitive sites normally located in the 5' region of the human beta-globin locus. This construct was injected into fertilized mouse eggs, and its expression was analyzed in the primitive and definitive erythroid cells, as well as the brain of 14-day embryos. All six transgenic individuals that contained intact copies of the construct expressed the transgene in an erythroid-specific fashion. Expression was observed in both primitive and definitive erythroid cells. This is in marked contrast to previous transgenic mice experiments using the same A gamma-globin gene fragment in isolation, where expression was restricted to primitive erythroid cells. Our results show that the region containing the developmentally stable globin locus hypersensitive sites changes the developmental stage specificity of a human fetal globin gene in transgenic mice. These observations imply that sequences additional to those used here are involved in the developmental control of fetal globin gene expression in vivo. The ability to express fetal globin in adult erythroid cells allows one to consider using fetal globin genes for gene therapy of sickle cell disease.     

Analysis for the major contributor of collagenase to the primary cleavage of type II collagens in cartilage degradation

Degradation of type II collagen is a central process in cartilage destruction seen in osteoarthritis and rheumatoid arthritis. Primary cleavage of type II collagen at the collagenase site is rate-limiting and is, therefore, a critical step for its degradation. The major contributor to this cleavage was identified in three isozymes of collagenase in human cartilage. Primary cultured human chondrocytes were used for the study. The production of collagenase-1 was major in total production for three isozymes of collagenase after stimulations with any concentration of tumor necrosis factor-α and/or interleukin-1 at 48 and 72 h, comprising 98% or greater of the total collagenase. When the production of collagenase-1 was specifically suppressed by the transfection with duplexes of 21-nucleotide small interfering ribonucleic acid into the cells, the activity of type II collagen cleavage was linearly decreased at neutral pH after activation. The relative contribution of collagenase-1 to the primary cleavage of type II collagen was determined to be 85%–93%. These findings suggest that collagenase-1 is a major contributor to the primary cleavage of type II collagens in human cartilage and is a potential therapeutic target for osteoarthritis and rheumatoid arthritis.     

Molecular analysis of the human beta-globin locus activation region.

Recently, DNA sequences containing four erythroid-specific DNase I hypersensitive sites within 20 kilobases 5' of the human epsilon-globin gene have been identified as an important cis-acting regulatory element, the locus activation region (LAR). Subfragments of the LAR, containing either all or only the two 5' or two 3' hypersensitive sites were linked to the human beta-globin gene and analyzed for their effect on globin gene expression in stably transformed mouse erythroleukemia (MEL) cells. Constructs containing all four of the hypersensitive sites increase beta-globin mRNA levels 8- to 13-fold, while constructs with only the 5' or 3' sites increase globin expression to a lesser extent. No effect was seen when the constructs were assayed in 3T3 fibroblasts. All of the LAR derivatives form hypersensitive sites at the corresponding sequence position in MEL cells prior to and after induction of MEL cell differentiation. However, in 3T3 fibroblasts only the hypersensitive site corresponding to the previously described erythroid-specific -10.9 site was formed.     

Analysis for the major contributor of collagenase to the primary cleavage of type II collagens in cartilage degradation

Abstract

Degradation of type II collagen is a central process in cartilage destruction seen in osteoarthritis and rheumatoid arthritis. Primary cleavage of type II collagen at the collagenase site is rate-limiting and is, therefore, a critical step for its degradation. The major contributor to this cleavage was identified in three isozymes of collagenase in human cartilage. Primary cultured human chondrocytes were used for the study. The production of collagenase-1 was major in total production for three isozymes of collagenase after stimulations with any concentration of tumor necrosis factor-α and/or interleukin-1 at 48 and 72?h, comprising 98% or greater of the total collagenase. When the production of collagenase-1 was specifically suppressed by the transfection with duplexes of 21-nucleotide small interfering ribonucleic acid into the cells, the activity of type II collagen cleavage was linearly decreased at neutral pH after activation. The relative contribution of collagenase-1 to the primary cleavage of type II collagen was determined to be 85%–93%. These findings suggest that collagenase-1 is a major contributor to the primary cleavage of type II collagens in human cartilage and is a potential therapeutic target for osteoarthritis and rheumatoid arthritis.     

Reduced amounts of cartilage collagen fibrils and growth plate anomalies in transgenic mice harboring a glycine-to-cysteine mutation in the mouse type II procollagen alpha 1-chain gene.

We have generated transgenic mice harboring a glycine-to-cysteine mutation in residue 85 of the triple helical domain of mouse type II collagen. The offspring of different founders displayed a phenotype of severe chondrodysplasia characterized by short limbs and trunk, cranio-facial deformities, and cleft palate. The affected pups died of acute respiratory distress caused by an inability to inflate lungs at birth. Staining of the skeleton showed a severe retardation of growth for practically all bones. Light microscopic examination indicated a decrease in cartilage matrix density, a severe disorganization of growth plate architecture, and the presence of streaks of fibrillar material in the cartilage matrix. Electron microscopic analysis showed a pronounced decrease in the number of typical thin cartilage collagen fibrils, distension of the rough endoplasmic reticulum of chondrocytes, and the presence of abnormally large banded collagen fibril bundles. The level of expression of the mutant type II procollagen alpha 1 chain transgene in cartilage tissues was approximately equal to that of the endogenous gene in two of the strains. We propose that the principal consequence of the mutation is a considerable reduction in density of the typical thin cartilage collagen fibrils and that this phenomenon causes the severe disorganization of the growth plate. We also postulate that the abnormal thick collagen fibrils are probably related to a defect in crosslinking between the collagen molecules. The cartilage anomalies displayed by these transgenic mice are remarkably similar to those of certain human chondrodysplasias.     

Stable transfer and expression of exogenous human globin genes in human erythroleukemia (K562) cells.

To study the expression of globin genes in human cells, human epsilon-globin genes were transferred into a K562 cell line, Bos, which synthesizes very low amounts of epsilon-globin mRNA. A plasmid (pSV2neo-epsilon) containing a complete epsilon-globin gene and 2 kilobases (kb) of 5' flanking DNA as well as a neomycin-resistance gene and a simian virus 40 origin of replication was transfected into Bos cells; the compound G418, a neomycin analogue, was used to select transformed cells. The presence of unique bands by DNA restriction analysis shows that 11 of 14 of the G418-resistant clones have at least one copy of an integrated epsilon-globin gene. RNA expression measured by RNA blotting shows significantly more epsilon-globin mRNA sequences than in untransfected Bos cells in 10 of 11 lines; in most lines, epsilon-globin mRNA was additionally increased in the presence of hemin. In two lines, epsilon-globin mRNA expression with hemin was comparable to that of a high epsilon-globin producing cell line, K562 clone 2. The one G418-resistant line without epsilon-globin genes had no epsilon-mRNA expression. The high epsilon-mRNA expression in several of the lines suggests that exogenous epsilon-globin genes with only 2-kb 5' flanking DNA may be sufficient to be appropriately expressed in these homologous erythroid cells. These results have implications for the potential success of transfer of normal human genes to human bone marrow cells as an approach to the treatment of inherited anemias.     

Action of collagenase and elastase from human polymorphonuclear leukocytes on human articular cartilage

Summary Collagenase from human polymorphonuclear leukocytes (neutrophil collagenase) attacks collagen type II in solution at a rate intermediate to those of type I and III collagens. This enzyme alone is not able to initiate degradation of native human articular cartilage. If the cartilage is first treated with leukocyte elastase, collagenase slowly degrades collagen.Confirming earlier findings by other investigators, elastase has a dual action on cartilage: The enzyme removes proteoglycans, thus demasking collagen fibers and giving collagenase access to them, and solubilizes collagen at a sizable rate.Although neutrophil collagenase cleaves collagen type II in solution at a high rate, the native, cross-linked status of collagen in cartilage makes it a relatively poor substrate for this enzyme. On a weight by weight scale, elastase and collagenase display about the same collagenolytic potential on human articular cartilage.The elastase/collagenase system from human polymorphonuclear leukocytes could represent a cooperative proteolytic complex in the destruction of cartilage in rheumatoid arthritis.     

Anabolic and catabolic gene expression pattern analysis in normal versus osteoarthritic cartilage using complementary DNA-array technology.

OBJECTIVE: To understand changes in gene expression levels that occur during osteoarthritic (OA) cartilage degeneration, using complementary DNA (cDNA)-array technology. METHODS: Nine normal, 6 early degenerated, and 6 late-stage OA cartilage samples of human knee joints were analyzed using the Human Cancer 1.2 cDNA array and TaqMan analysis. RESULTS: In addition to a large variability of expression levels between different patients, significant expression patterns were detectable for many genes. Cartilage types II and VI collagen were strongly expressed in late-stage specimens, reflecting the high matrix-remodeling activity of advanced OA cartilage. The increase in fibronectin expression in early degeneration suggests that fibronectin is a crucial regulator of matrix turnover activity of chondrocytes during early disease development. Of the matrix metalloproteinases (MMPs), MMP-3 appeared to be strongly expressed in normal and early degenerative cartilage and down-regulated in the late stages of disease. This indicates that other degradation pathways might be more important in late stages of cartilage degeneration, involving other enzymes, such as MMP-2 and MMP-11, both of which were up-regulated in late-stage disease. MMP-11 was up-regulated in OA chondrocytes and, interestingly, also in the early-stage samples. Neither MMP-1 nor MMP-8 was detectable, and MMP-13 and MMP-2 were significantly detectable only in late-stage specimens, suggesting that early stages are characterized more by degradation of other matrix components, such as aggrecan and other noncollagenous molecules, than by degradation of type II collagen fibers. CONCLUSION: This investigation allowed us to identify gene expression profiles of the disease process and to get new insights into disease mechanisms, for example, to develop a picture of matrix proteinases that are differentially involved in different phases of the disease process.     

Molecular cloning and cell cycle-specific regulation of a functional human thymidine kinase gene.

A functional thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene has been molecularly cloned from human DNA. The gene was rescued from a genomic library of TK-deficient mouse L cells transformed to the TK+ phenotype with total HeLa cell DNA. Of 14 overlapping clones, only one contained the intact human TK gene. The cloned recombinant bacteriophage carries a 16-kilobase insert derived entirely from human DNA and is capable of transforming LTK- cells to TK+ with an efficiency of 10 TK+ colonies per ng of DNA per 10(6) cells. Restriction endonuclease mapping shows that the functional human TK gene is at least twice as long as that reported for chicken. A 1.6-kilobase Xho I/EcoRI fragment was subcloned and found to hybridize to a human mRNA of 1.5 kilobases. When introduced into LTK- cells, the cloned human TK gene is regulated in the cell cycle-specific manner characteristic of TK+ mammalian cells. That is, TK activity in synchronized cells increases markedly with the onset of DNA synthesis. The signals governing the S-phase induction of TK activity reside within 16 kilobases of human DNA and are correctly interpreted by mouse cells.