An 18-base-pair sequence in the mouse proalpha1(II) collagen gene is sufficient for expression in cartilage and binds nuclear proteins that are selectively expressed in chondrocytes.

The molecular mechanisms by which mesenchymal cells differentiate into chondrocytes are still poorly understood. We have used the gene for a chondrocyte marker, the proalpha1(II) collagen gene (Col2a1), as a model to delineate a minimal sequence needed for chondrocyte expression and identify chondrocyte-specific proteins binding to this sequence. We previously localized a cartilage-specific enhancer to 156 bp of the mouse Col2a1 intron 1. We show here that four copies of a 48-bp subsegment strongly increased promoter activity in transiently transfected rat chondrosarcoma (RCS) cells and mouse primary chondrocytes but not in 10T1/2 fibroblasts. They also directed cartilage specificity in transgenic mouse embryos. These 48 bp include two 11-bp inverted repeats with only one mismatch. Tandem copies of an 18-bp element containing the 3' repeat strongly enhanced promoter activity in RCS cells and chondrocytes but not in fibroblasts. Transgenic mice harboring 12 copies of this 18-mer expressed luciferase in ribs and vertebrae and in isolated chondrocytes but not in noncartilaginous tissues except skin and brain. In gel retardation assays, an RCS cell-specific protein and another closely related protein expressed only in RCS cells and primary chondrocytes bound to a 10-bp sequence within the 18-mer. Mutations in these 10 bp abolished activity of the multimerized 18-bp enhancer, and deletion of these 10 bp abolished enhancer activity of 465- and 231-bp intron 1 segments. This sequence contains a low-affinity binding site for POU domain proteins, and competition experiments with a high-affinity POU domain binding site strongly suggested that the chondrocyte proteins belong to this family. Together, our results indicate that an 18-bp sequence in Col2a1 intron 1 controls chondrocyte expression and suggest that RCS cells and chondrocytes contain specific POU domain proteins involved in enhancer activity.     

Identification and characterization of arginase II as a chondrocyte phenotype-specific gene

We have previously shown that activation of extracellular signal-regulated protein kinase-1 and -2 (ERK1/2) causes chondrocyte dedifferentiation, which contributes to the destruction of arthritic cartilage. In the present study, we identified genes involved in the ERK1/2 regulation of chondrocyte dedifferentiation. Several genes were identified by subtractive hybridization, and, of these, arginase II was selected for further functional characterization. Similar to the pattern of type II collagen expression, which is a hallmark of chondrocyte differentiation, arginase II expression was increased during chondrogenesis of mesenchymal cells. The high expression level of arginase II was decreased during dedifferentiation of chondrocytes, whereas its expression was restored during redifferentiation of the dedifferentiated chondrocytes. Inhibition of ERK1/2 signaling in chondrocytes enhanced type II collagen expression with a concomitant increase in expression and activity of arginase II. However, ectopic expression of arginase II or inhibition of its activity did not affect chondrocyte differentiation. The results collectively indicate that expression of arginase II is specific to the chondrocyte phenotype, although the expression of arginase II alone is not sufficient for articular chondrocytes to maintain a differentiated phenotype.     

SV40-immortalization of rabbit articular chondrocytes: alteration of differentiated functions.

Cell lines were established from rabbit articular chondrocytes following transfection with a plasmid encoding SV40 early function genes. This resulted in cell immortalization (130 passages have been completed for the oldest cell line) with acquisition of characteristics of partial transformation such as reduced serum requirements for normal and clonal growth. The immortalized chondrocytes, called SVRAC, did not form multilayer foci when maintained in postconfluent culture. Their ability to form colonies in soft agar was not increased in comparison with normal chondrocytes, but they were weakly tumorigenic in nude mice. SVRAC lost the ability to synthesize type II collagen and Alcian blue-stainable matrix, which are markers of the differentiated chondrocyte phenotype, and synthesized predominantly type I collagen. Studies of collagen gene expression showed that pro alpha 1 (II) mRNA was undetectable, whereas pro alpha 1 (I) collagen mRNA was expressed even in late passage cultures. Unlike normal dedifferentiated chondrocytes, SVRAC were unable to re-express the differentiated phenotype in response to tridimensional culture or microfilament depolymerization. Cell lines obtained from chondrocytes transfected either in primary culture or just after release of cells from cartilage displayed the same behaviour. Thus SV40 early genes were able to immortalize rabbit articular chondrocytes, but the resulting cell lines displayed an apparently irreversibly dedifferentiated phenotype. These cell lines can be used as models to identify regulatory pathways that are required for the maintenance or reexpression of differentiated function in chondrocytes.     

Proteoglycan and collagen synthesis are correlated with actin organization in dedifferentiating chondrocytes.

The dedifferentiation of chondrocytes in culture is classically associated with a transition from a rounded to a spread morphology. However, the loss of chondroitin sulfate proteoglycan (CSPG) and type II collagen gene expression (markers of the differentiated chondrocyte) does not occur for all polygonal or fibroblast-like cells at the same stage of culture. Furthermore, it has been demonstrated that retinoic acid-dedifferentiated chondrocytes can reexpress type II collagen if treated by the microfilament disruptive drug dihydrocytochalasin B, without a return to the spherical shape. In the present study, we have investigated by fluorescent double-staining whether the synthesis of both CSPG and type II collagen by dedifferentiating chick chondrocytes in low density cultures is dependent on a type of actin organization. We report that the synthesis of CSPG and type II collagen synthesis is coincident with the presence of a faint microfibrillar actin architecture but is absent in chondrocytes showing well defined actin cables. This correlation was observed independently of the shapes exhibited by the cells. Moreover, type I collagen (marker of the dedifferentiated chondrocyte) is synthesized mainly in cells showing large actin cables. This study, performed in the absence of drugs, suggests that actin organization, rather than changes in cell shape, is involved in modulating the chondrogenic phenotype in vitro.     

Involvement of fibroblast growth factor 18 in dedifferentiation of cultured human chondrocytes

Objective:  Chondrocytes inevitably decrease production of cartilaginous matrices during long-term cultures with repeated passaging; this is termed dedifferentiation. To learn more concerning prevention of dedifferentiation, we have focused here on the fibroblast growth factor (FGF) family that influences chondrocyte proliferation or differentiation.
Materials and methods:  We have compared gene expression between differentiated cells in passage 3 (P3) and dedifferentiated ones in P8 of human cultured chondrocytes. We also performed ligand administration of the responsive factor or its gene silencing, using small interfering RNA (siRNA).
Results:  FGFs 1, 5, 10, 13 and 18 were higher at P8 compared to P3, while FGFs 9 and 14 were lower. Especially, FGF18 showed a 10-fold increase by P8. Ligand administration of FGF18 in the P3 cells, or its gene silencing using siRNA in the P8 cells, revealed dose-dependent increase and decrease respectively in type II collagen/type I collagen ratio. Exogenous FGF18 also upregulated expression of transforming growth factor beta (TGF-β), the anabolic factor of chondrocytes, in P3 chondrocytes, but P8 cells maintained a low level of TGF-β expression, suggesting a decrease in responsiveness of TGF-β to FGF18 stimulation in the dedifferentiated chondrocytes.
Conclusion:  FGF18 seems to play a role in maintenance of chondrocyte properties, although its expression was rather high in dedifferentiated chondrocytes. Upregulation of FGF18 in dedifferentiated chondrocytes implied that it may be a marker of dedifferentiation.     

SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene.

The identification of mutations in the SRY-related SOX9 gene in patients with campomelic dysplasia, a severe skeletal malformation syndrome, and the abundant expression of Sox9 in mouse chondroprogenitor cells and fully differentiated chondrocytes during embryonic development have suggested the hypothesis that SOX9 might play a role in chondrogenesis. Our previous experiments with the gene (Col2a1) for collagen II, an early and abundant marker of chondrocyte differentiation, identified a minimal DNA element in intron 1 which directs chondrocyte-specific expression in transgenic mice. This element is also a strong chondrocyte-specific enhancer in transient transfection experiments. We show here that Col2a1 expression is closely correlated with high levels of SOX9 RNA and protein in chondrocytes. Our experiments indicate that the minimal Col2a1 enhancer is a direct target for Sox9. Indeed, SOX9 binds to a sequence of the minimal Col2a1 enhancer that is essential for activity in chondrocytes, and SOX9 acts as a potent activator of this enhancer in cotransfection experiments in nonchondrocytic cells. Mutations in the enhancer that prevent binding of SOX9 abolish enhancer activity in chondrocytes and suppress enhancer activation by SOX9 in nonchondrocytic cells. Other SOX family members are ineffective. Expression of a truncated SOX9 protein lacking the transactivation domain but retaining DNA-binding activity interferes with enhancer activation by full-length SOX9 in fibroblasts and inhibits enhancer activity in chondrocytes. Our results strongly suggest a model whereby SOX9 is involved in the control of the cell-specific activation of COL2A1 in chondrocytes, an essential component of the differentiation program of these cells. We speculate that in campomelic dysplasia a decrease in SOX9 activity would inhibit production of collagen II, and eventually other cartilage matrix proteins, leading to major skeletal anomalies.     

Development and phenotypic characterization of a high density in vitro model of auricular chondrocytes with applications in reconstructive plastic surgery

Cultivation of phenotypically stable auricular chondrocytes will have applications in autologous chondrocyte transplantation and reconstructive surgery of cartilage. Chondrocytes grown in monolayer culture rapidly dedifferentiate assuming a fibroblast-like morphology and lose their cartilage-specific pattern of gene expression. Three-dimensional high-density culture models mimic more closely the in vivo conditions of cartilage. Therefore, this study was undertaken to test whether the high-density cultures might serve as a suitable model system to acquire phenotypically and functionally differentiated auricular chondrocytes from porcine cartilage. Freshly isolated porcine auricular chondrocytes were cultured for 7 passages in monolayer culture. From each passage (passage 0 and 1-7) cells were introduced to high-density cultures and examined by transmission electron microscopy. Western blotting was used to analyse the expression of cartilage-specific markers, such as collagen type II and cartilage specific proteoglycan, fibronectin, cell adhesion and signal transduction receptor beta1-integrin, matrix metalloproteinases (MMP-9, MMP-13), cyclo-oxygenase (COX)-2 and the apoptosis commitment marker, activated caspase-3. When dedifferentiated auricular chondrocytes from monolayer passages 0-4 were cultured in high-density culture, they recovered their chondrocytic phenotype and formed cartilage nodules surrounded by fibroblast-like cells and synthesised collagen type II, proteoglycans, fibronectin and beta1-integrins. However, chondrocytes from monolayer passages 5-7 did not redifferentiate to chondrocytes even when transferred to high-density culture, and did not synthesize a chondrocyte-specific extracellular matrix. Instead, they produced increasing amounts of MMP-9, MMP-13, COX-2, activated caspase-3 and underwent apoptosis. Three-dimensional high-density cultures may therefore be used to obtain sufficient quantities of fully differentiated auricular chondrocytes for autologous chondrocyte transplantation and reconstructive plastic surgery.     

Expression of mRNAs for collagens and other matrix components in dedifferentiating and redifferentiating human chondrocytes in culture

Cell cultures were initiated from epiphyseal cartilages, diaphyseal periosteum, and muscle of 16-week human fetuses. Total RNAs isolated from these cultures were analyzed for the levels of mRNAs for major fibrillar collagens, two proteoglycan core proteins and osteonectin. In standard monolayer cultures the differentiated chondrocyte phenotype was replaced by a dedifferentiated one: the mRNA levels of cartilage-specific type II collagen decreased upon subculturing, while those of types I and III collagen, and the core proteins increased. When the cells were transferred to grow in agarose, redifferentiation (reappearance of type II collagen mRNA) occurred. Fibroblasts grown from periosteum and muscle were found to contain mRNAs for types I and III collagen and proteoglycan cores. When these cells were transferred to agarose they acquired a shape indistinguishable from chondrocytes, but no type II collagen mRNA was observed.     

Monoclonal antibody 11-fibrau: a useful marker to characterize chondrocyte differentiation stage

The aim of this study was to determine the feasibility of discriminating between differentiated and dedifferentiated chondrocytes by using the Mab 11-fibrau. Mab 11-fibrau did not bind to differentiated chondrocytes in cartilage of human knee joint, auricle, or nasal septum. During monolayer culture, when cells dedifferentiate, the number of 11-fibrau positive cells gradually increased and reached up to 100% after 4 passages. When differentiated chondrocytes were cultured in alginate, most (90--95%) of the cells remained 11-fibrau negative, in accordance with previous studies demonstrating that differentiated chondrocytes cultured in alginate keep their phenotype. Dedifferentiated (11-fibrau positive) cells were subjected to different redifferentiation regimes. As a well-known fact, cultures in alginate in medium where FCS was replaced by IGF1 and TGF beta 2 results in increased collagen type II formation, indicative for redifferentiation. However, the cells remained 11-fibrau positive, suggesting they are not (yet) fully redifferentiated. On the other hand, when dedifferentiated cells (after 4 passages in monolayer culture) were seeded in a biomaterial and implanted subcutaneously in a nude mouse, the newly formed cartilage matrix contained collagen type II and the 11-fibrau staining on the cells had disappeared. Our results indicate that 11-fibrau may be a reliable and sensitive marker of chondrocyte phenotype.     

Redifferentiation of dedifferentiated chondrocytes on chitosan membranes and involvement of PKCalpha and P38 MAP kinase

To investigate the effects of chitosan on the redifferentiation of dedifferentiated chondrocytes, we used chondrocytes obtained from a micromass culture system. Micromass cultures of chick wing bud mesenchymal cells yielded differentiated chondrocytes, but these dedifferentiated during serial monolayer subculture. When the dedifferentiated chondrocytes were cultured on chitosan membranes they regained the phenotype of differentiated chondrocytes. Expression of protein kinase C (PKC) increased during chondrogenesis, decreased during dedifferentiation, and increased again during redifferentiation. Treatment of the cultures with phorbol 12-myristate 13-acetate (PMA) inhibited redifferentiation and down-regulated PKC. In addition, the expression of p38 mitogen-activated protein (MAP) kinase increased during redifferentiation, and its inhibition suppressed redifferentiation. These findings establish a culture system for producing chondrocytes, point to a new role of chitosan in the redifferentiation of dedifferentiated chondrocytes, and show that PKC and p38 MAP kinase activities are required for chondrocyte redifferentiation in this model system.     

Targeting of focal adhesion kinase by small interfering RNAs reduces chondrocyte redifferentiation capacity in alginate beads culture with type II collagen

Type II collagen is a major protein that maintains biological and mechanical characteristics in articular cartilage. Focal adhesion kinase (FAK) is known to play a central role in integrin signaling of cell–extracellular matrix (ECM) interactions, and chondrocyte–type II collagen interactions are very important for cartilage homeostasis. In this study, we focused on phosphorylation of FAK and MAP kinase in chondrocyte–type II collagen interaction and dedifferentiation, and the effects of FAK knockdown on chondrocyte‐specific gene expression and cell proliferation were determined. The addition of exogenous type II collagen to chondrocytes increased levels of tyrosine phosphorylation, p‐FAK_Y397, and p‐ERK1/2. In contrast, expression levels of p‐FAK_Y397 and p‐ERK1/2, but not p‐Smad2/3, were decreased in dedifferentiated chondrocytes with loss of type II collagen expression. Type II collagen expression was significantly increased when dedifferentiated chondrocytes were transferred to alginate beads with TGF‐β1 or type II collagen, but transfected cells with small interfering RNA for FAK (FAK‐siRNA) inhibited mRNA expression of type II collagen and SOX‐6 compared to the control. These FAK‐siRNA‐transfected cells could not recover type II collagen even in the presence of TGF‐β1 or type II collagen in alginate beads culture. We also found that FAK‐siRNA‐transfected cells decreased cell proliferation rate, but there was no effect on glycosaminoglycans (GAGs) secretion. We suggest that FAK is essentially required in chondrocyte communication with type II collagen by regulating type II collagen expression and cell proliferation. J. Cell. Physiol. 218: 623–630, 2009. © 2008 Wiley‐Liss, Inc.