The cleavage of N-cadherin is essential for chondrocyte differentiation

The aggregation of chondroprogenitor mesenchymal cells into precartilage condensation represents one of the earliest events in chondrogenesis. N-cadherin is a key cell adhesion molecule implicated in chondrogenic differentiation. Recently, ADAM10-mediated cleavage of N-cadherin has been reported to play an important role in cell adhesion, migration, development and signaling. However, the significance of N-cadherin cleavage in chondrocyte differentiation has not been determined. In the present study, we found that the protein turnover of N-cadherin is accelerated during the early phase of chondrogenic differentiation in ATDC5 cells. Therefore, we generated the subclones of ATDC5 cells overexpressing wild-type N-cadherin, and two types of subclones overexpressing a cleavage-defective N-cadherin mutant, and examined the response of these cells to insulin stimulation. The ATDC5 cells overexpressing cleavage-defective mutants severely prevented the formation of cartilage aggregates, proteoglycan production and the induction of chondrocyte marker gene expression, such as type II collagen, aggrecan and type X collagen. These results suggested that the cleavage of N-cadherin is essential for chondrocyte differentiation.     

P38 mitogen‐activated protein kinase promotes dedifferentiation of primary articular chondrocytes in monolayer culture

Articular cartilage is an avascular tissue with poor regenerative capacity following injury, a contributing factor to joint degenerative disease. Cell‐based therapies for cartilage tissue regeneration have rapidly advanced; however, expansion of autologous chondrocytes in vitro using standard methods causes ‘dedifferentiation’ into fibroblastic cells. Mitogen‐activated protein kinase (MAPK) signalling is crucial for chondrocyte metabolism and matrix production, and changes in MAPK signals can affect the phenotype of cultured cells. We investigated the effects of inhibition of MAPK signalling on chondrocyte dedifferentiation during monolayer culture. Blockade of extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) signalling caused a significant increase in cartilage gene expression, however, also caused up‐regulation of fibrotic gene expression. Inhibition of p38 MAPK (p38) caused a significant up‐regulation of collagen type II while suppressing collagen type I expression. P38 inhibition also resulted in consistently more organized secretion of collagen type II protein deposits on cell culture surfaces. Follow‐on pellet culture of treated cells revealed that MAPK inhibition reduced cell migration from the pellet. ERK and JNK inhibition caused more collagen type I accumulation in pellets versus controls while p38 inhibition strongly promoted collagen type II accumulation with no effect on collagen type I. Blockade of all three MAPKs caused increased GAG content in pellets. These results indicate a role for MAPK signalling in chondrocyte phenotype loss during monolayer culture, with a strong contribution from p38 signalling. Thus, blockade of p38 enhances chondrocyte phenotype in monolayer culture and may promote more efficient cartilage tissue regeneration for cell‐based therapies.     

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.     

Extent of cell differentiation and capacity for cartilage synthesis in human adult adipose‐derived stem cells: Comparison with fetal chondrocytes

This study evaluated the extent of differentiation and cartilage biosynthetic capacity of human adult adipose‐derived stem cells relative to human fetal chondrocytes. Both types of cell were seeded into nonwoven‐mesh polyglycolic acid (PGA) scaffolds and cultured under dynamic conditions with and without addition of TGF‐β1 and insulin. Gene expression for aggrecan and collagen type II was upregulated in the stem cells in the presence of growth factors, and key components of articular cartilage such as glycosaminoglycan (GAG) and collagen type II were synthesized in cultured tissue constructs. However, on a per cell basis and in the presence of growth factors, accumulation of GAG and collagen type II were, respectively, 3.4‐ and 6.1‐fold lower in the stem cell cultures than in the chondrocyte cultures. Although the stem cells synthesized significantly higher levels of total collagen than the chondrocytes, only about 2.4% of this collagen was collagen type II. Relative to cultures without added growth factors, treatment of the stem cells with TGF‐β1 and insulin resulted in a 59% increase in GAG synthesis, but there was no significant change in collagen production even though collagen type II gene expression was upregulated 530‐fold. In contrast, in the chondrocyte cultures, synthesis of collagen type II and levels of collagen type II as a percentage of total collagen more than doubled after growth factors were applied. Although considerable progress has been achieved to develop differentiation strategies and scaffold‐based culture techniques for adult mesenchymal stem cells, the extent of differentiation of human adipose‐derived stem cells in this study and their capacity for cartilage synthesis fell considerably short of those of fetal chondrocytes. Biotechnol. Bioeng. 2010;107: 393–401. © 2010 Wiley Periodicals, Inc.     

The role of hydrogel structure and dynamic loading on chondrocyte gene expression and matrix formation

Crosslinked poly(ethylene glycol) (PEG) hydrogels are attractive scaffolds for cartilage tissue engineering because of their ability to mimic the aqueous environment and mechanical properties of native cartilage. In this study, hydrogel crosslinking density was varied to study the influence of gel structure and the application of dynamic loading (continuous, 1 Hz, 15% amplitude strain) on chondrocyte gene expression over 1 week culture. Gene expression was quantified using real-time RT-PCR for collagen II and aggrecan, the major cartilage extracellular matrix (ECM) components, and collagen I, an indicator of chondrocyte de-differentiation. When chondrocytes were encapsulated in PEG gels with low or high crosslinking, a high collagen II expression compared to collagen I expression (1000 or 100,000:1, respectively) indicated the native chondrocyte phenotype was retained. In the absence of loading, relative gene expression for collagen II and aggrecan was significantly higher (e.g., 2-fold and 4-fold, respectively, day 7) in the low crosslinked gels compared to gels with higher crosslinking. Dynamic loading, however, showed little effect on ECM gene expression in both crosslinked systems. To better understand the cellular environment, ECM production was qualitatively assessed using an in situ immunofluorescent technique and standard histology. A pericellular matrix (PCM) was observed as early as day 3 post-encapsulation and the degree of formation was dependent on gel crosslinking. These results suggest the PCM may protect the cells from sensing the applied loads. This study demonstrates that gel structure has a profound effect on chondrocyte gene expression, while dynamic loading has much less of an effect at early culture times.     

In vitro development of hypertrophic chondrocytes starting from selected clones of dedifferentiated cells

Single cells from enzymatically dissociated chick embryo tibiae have been cloned and expanded in fresh or conditioned culture media. A cloning efficiency of approximately 13% was obtained using medium conditioned by dedifferentiated chondrocytes. A cloning efficiency of only 1.4% was obtained when conditioned medium from hypertrophic chondrocytes was used, and efficiencies of essentially 0 were found with fresh medium or medium conditioned by J2-3T3 mouse fibroblasts. Cell clones were selected by morphological criteria and clones showing a dedifferentiated phenotype (fibroblast-like) were further characterized. Out of 38 clones analyzed, 17 were able to differentiate to the hypertrophic chondrocyte stage and reconstitute hypertrophic cartilage when placed in the appropriate culture conditions. Cells from these clones expressed the typical markers of chondrocyte differentiation, i.e., type II and type X collagens. Clones not undergoing differentiation continued to express only type I collagen. Hypertrophic chondrocytes from differentiating clones were analyzed at the single cell level by immunofluorescence; all the cells were positive for type X collagen, while approximately 50% of them showed positivity for type II collagen.     

Constitutive myc expression impairs hypertrophy and calcification in cartilage.

The myc oncogene is expressed by proliferating quail embryo chondrocytes (QEC) grown as adherent cells and is repressed in QEC maintained in suspension culture. To investigate the interference of myc expression during chondrocyte differentiation, QEC were infected with a retrovirus carrying the v-myc oncogene (QEC-v-myc). Uninfected or helper virus-infected QEC were used as control. In adherent culture, QEC-v-myc displayed a chondrocytic phenotype and synthesized type II collagen and Ch21 protein, while control chondrocytes synthesized type I and type II collagen with no Ch21 protein detected as long as the attachment to the plastic was kept. In suspension culture, QEC-v-myc readily aggregated and within 1 week the cell aggregates released small single cells; still they secreted only type II collagen and Ch21 protein. In the same conditions control cell aggregates released hypertrophic chondrocytes producing type II and type X collagens and Ch21 protein. In the appropriate culture conditions, QEC-v-myc reconstituted a tissue defined as nonhypertrophic, noncalcifying cartilage by the high cellularity, the low levels of alkaline phosphatase enzymatic activity, and the absence of type X collagen synthesis and of calcium deposition. We conclude that the constitutive expression of the v-myc oncogene keeps chondrocytes in stage I (active proliferation and synthesis of type II collagen) and prevents these cells from reconstituting hypertrophic calcifying cartilage.     

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.     

Retinoic acid inhibits chondrogenesis of mesenchymal cells by sustaining expression of N-cadherin and its associated proteins

Retinoic acid (RA) is a well-known regulator of chondrocyte phenotype. RA inhibits chondrogenic differentiation of mesenchymal cells and also causes loss of differentiated chondrocyte phenotype. The present study investigated the mechanisms underlying RA regulation of chondrogenesis. RA treatment in chondrifying mesenchymal cells did not affect precartilage condensation, but blocked progression from precartilage condensation to cartilage nodule formation. This inhibitory effect of RA was independent of protein kinase C and extracellular signal-regulated protein kinase, which are positive and negative regulators of cartilage nodule formation, respectively. The progression from precartilage condensation to cartilage nodule requires downregulation of N-cadherin expression. However, RA treatment caused sustained expression of N-cadherin and its associated proteins including alpha- and beta-catenin suggesting that modulation of expression of these molecules is associated with RA-induced inhibition of chondrogenesis. This hypothesis was supported by the observation that disruption of the actin cytoskeleton by cytochalasin D (CD) blocks RA-induced sustained expression of cell adhesion molecules and overcomes RA-induced inhibition of chondrogenesis. Taken together, our results suggest RA inhibits chondrogenesis by stabilizing cell-to-cell interactions at the post-precartilage condensation stage.     

Establishment and characterization of chondrocyte cell lines from the costal cartilage of SV40 large T antigen transgenic mice

Complete understanding of the physiology and pathology of the cartilage is essential to establish treatments for a variety of cartilage disorders and defects such as rheumatoid arthritis, congenital malformations, and tumors of cartilage. Although synthetic materials have been used in many cases, they possess inherent problems including wear of the materials and low mechanical strength. Autograft has been considered very effective to overcome these problems. However, the limitation of the transplant volume is a major problem in autograft to be overcome. The costal cartilage is the most serious candidate for donor site transplantation, since it is the largest permanent hyaline cartilage in the body. To investigate the possibility using the costal cartilage as a transplant source, we have established and characterized three mouse chondrocyte cell lines (MCC-2, MCC-5, and MCC-35) derived from the costal cartilage of 8-week-old male SV40 large T-antigen transgenic mice. At confluence, all the cell lines formed nodules that could be positively stained with alcian blue (pH 2.5). The size of nodules gradually increased during culturing time. After 2 and 6 weeks of culture, RT-PCR analysis demonstrated that all three cell lines expressed mRNA from the cartilage-specific genes for type II collagen, type XI collagen, aggrecan, and link protein. Furthermore, type X collagen expression was detected in MCC-5 and MCC-35 but not in MCC-2. Any phenotypic changes were not observed over 31 cell divisions. Immunocytochemistry showed further that MCC-2, MCC-5, and MCC-35 produced cartilage-specific proteins type II collagen and type XI collagen, while in addition MCC-5 and MCC-35 produced type X collagen. Treatment with 1alpha, 25-dihydroxyvitamin D(3) inhibited cell proliferation and differentiation of the three cell lines in a dose-dependent manner. These phenotypic characteristics have been found consistent with chondrocyte cell lines established from cartilage tissues other than costal cartilage. In conclusion, costal cartilage shows phenotypic similarities to other cartilages, i.e., articular cartilage and embryonic limbs, suggesting that costal cartilage may be very useful as the donor transplantation site for the treatment of cartilage disorders. Furthermore, the cell lines established in this study are also beneficial in basic research of cartilage physiology and pathology.     

An established rat cell line expressing chondrocyte properties

Chondrocytes express a well-characterized set of marker proteins making these cells useful for studies on differentiation and regulation of gene expression. Because of the inherent instability of primary rat chondrocytes in culture, and because several rat chondrocyte genes have been cloned and characterized (including the collagen II promoter and enhancer), a rat chondrocyte cell line would be especially useful. To obtain this line we infected primary fetal rat costal chondrocytes with a recombinant retrovirus (NIH/J-2) carrying the myc and raf oncogenes, which have been shown to have an "immortalizing" function. Following infection, a rapidly proliferating clonal line was isolated that maintained a stable phenotype through 45 passages (11/2 year in culture). This line, termed IRC, grows in suspension culture as multicellular aggregates and in monolayer culture as polygonal cells which accumulate an alcian blue-stainable matrix. IRC cells synthesize high levels of cartilage proteoglycan core protein, and link protein, but show reduced collagen II expression. In addition, the cells express virally derived myc mRNA and protein, but do not express v-raf. Retinoic acid, which is a known modulator of chondrocyte phenotype, down-regulates expression of chondrocyte marker proteins, while stimulating v-myc expression by IRC cells. These data suggest that v-myc expression by chondrocytes results in rapid cell division and maintenance of many aspects of the differentiated phenotype. These "immortalized" cells, however, remain responsive to agents such as retinoic acid which modulate cell phenotype. The potential exists for development of chondrocyte cell lines from diseased cartilage, as well as from human cartilage.     

Retinoic acid, hemin and hexamethylen bisacetamide interference with "in vitro" differentiation of chick embryo chondrocytes.

We have investigated the effect of all-trans Retinoic acid, and of substances (Hemine and Hexamethylene bisacetamide) which interfere with "in vitro" differentiation of mesenchyme derived cell lineages on the expression of specific markers of hyperthrophy in "in vitro" differentiating chick embryo chondrocytes. (Castagnola P., et al., 1986). Continuous treatment of chondrogenic cells in conditions allowing differentiation "in vitro" with Retinoic acid resulted in persistence of type I collagen synthesis and in lack of type X collagen and Ch 21 protein expression. Hemin treated cells secreted a reduced amount of type X collagen. HMBA treatment inhibited type X collagen expression and caused reduction of the ratio between type II collagen and Ch 21 synthesized. The data indicate an independent regulation of these markers during chondrocyte differentiation.     

N-CAM is not required for initiation of secondary chondrogenesis: the role of N-CAM in skeletal condensation and differentiation.

Condensation precedes chondrogenic differentiation during development of primary cartilage. While neural cell adhesion molecule (N-CAM) enhances condensation, it is unclear whether N-CAM is also required for initiation of chondrogenic differentiation. In this study, the role of N-CAM in secondary chondrogenesis from periosteal cells of the quadratojugal (QJ) from embryonic chicks was studied using several in vitro approaches. The QJ is a membrane bone and so is not preceded by cartilage formation during development. However, QJ periosteal cells can differentiate into chondrocytes to form secondary cartilage in vivo. When QJ periosteal cells were enzymatically released and plated in low density monolayer, clonal or agarose cultures, chondrogenesis was initiated in the absence of N-CAM expression. Furthermore, overexpression of the N-CAM gene in periosteal cells in monolayer culture significantly reduced the number of chondrocyte colonies, suggesting that N-CAM inhibits secondary chondrogenesis. In contrast, and consistent with expression in vivo, N-CAM is expressed during osteogenesis from QJ periosteal cells and mandibular mesenchyme in vitro. These results are discussed in relation to the role of N-CAM in osteogenesis and in primary and secondary condensation.     

Constitutive E2F1 overexpression delays endochondral bone formation by inhibiting chondrocyte differentiation

Longitudinal bone growth results from endochondral ossification, a process that requires proliferation and differentiation of chondrocytes. It has been shown that proper endochondral bone formation is critically dependent on the retinoblastoma family members p107 and p130. However, the precise functional roles played by individual E2F proteins remain poorly understood. Using both constitutive and conditional E2F1 transgenic mice, we show that ubiquitous transgene-driven expression of E2F1 during embryonic development results in a dwarf phenotype and significantly reduced postnatal viability. Overexpression of E2F1 disturbs chondrocyte maturation, resulting in delayed endochondral ossification, which is characterized by reduced hypertrophic zones and disorganized growth plates. Employing the chondrogenic cell line ATDC5, we investigated the effects of enforced E2F expression on the different phases of chondrocyte maturation that are normally required for endochondral ossification. Ectopic E2F1 expression strongly inhibits early- and late-phase differentiation of ATDC5 cells, accompanied by diminished cartilage nodule formation as well as decreased type II collagen, type X collagen, and aggrecan gene expression. In contrast, overexpression of E2F2 or E2F3a results in only a marginal delay of chondrocyte maturation, and increased E2F4 levels have no effect. These data are consistent with the notion that E2F1 is a regulator of chondrocyte differentiation.     

Cartilage proteoglycan core protein gene expression during limb cartilage differentiation

Changes in the steady-state cytoplasmic levels of mRNA for the core protein of the major sulfated proteoglycan of cartilage were examined during the course of limb chondrogenesis in vitro using cloned cDNA probes. Cytoplasmic core protein mRNA begins to accumulate at the onset of overt chondrogenesis in micromass culture coincident with the crucial condensation phase of the process, in which prechondrogenic mesenchymal cells become closely juxtaposed prior to depositing a cartilage matrix. The initiation of core protein mRNA accumulation coincides with a dramatic increase in the accumulation of mRNA for type II collagen, the other major constituent of hyaline cartilage matrix. Following condensation, there is a concomitant progressive increase in cytoplasmic core protein and type II collagen mRNA accumulation which parallels the progressive accumulation of cartilage matrix by the cells. The relative rate of accumulation of cytoplasmic type II collagen mRNA is greater than twice that of core protein mRNA during chondrogenesis in micromass culture. Cyclic AMP, an agent implicated in the regulation of chondrogenesis elicits a concomitant two- to fourfold increase in both cartilage core protein and type II collagen mRNA levels by limb mesenchymal cells. Core protein gene expression is more sensitive to cAMP than type II collagen gene expression. These results suggest that the cartilage proteoglycan core protein and type II collagen genes are coordinately regulated during the course of limb cartilage differentiation, although there are quantitative differences in the extent of expression of the two genes.     

Changes in the chondrocyte and extracellular matrix proteome during post-natal mouse cartilage development

Skeletal growth by endochondral ossification involves tightly coordinated chondrocyte differentiation that creates reserve, proliferating, prehypertrophic, and hypertrophic cartilage zones in the growth plate. Many human skeletal disorders result from mutations in cartilage extracellular matrix (ECM) components that compromise both ECM architecture and chondrocyte function. Understanding normal cartilage development, composition, and structure is therefore vital to unravel these disease mechanisms. To study this intricate process in vivo by proteomics, we analyzed mouse femoral head cartilage at developmental stages enriched in either immature chondrocytes or maturing/hypertrophic chondrocytes (post-natal days 3 and 21, respectively). Using LTQ-Orbitrap tandem mass spectrometry, we identified 703 cartilage proteins. Differentially abundant proteins (q < 0.01) included prototypic markers for both early and late chondrocyte differentiation (epiphycan and collagen X, respectively) and novel ECM and cell adhesion proteins with no previously described roles in cartilage development (tenascin X, vitrin, Urb, emilin-1, and the sushi repeat-containing proteins SRPX and SRPX2). Meta-analysis of cartilage development in vivo and an in vitro chondrocyte culture model (Wilson, R., Diseberg, A. F., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E. J., Gorman, J. J., and Bateman, J. F. (2010) Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics. Mol. Cell. Proteomics 9, 1296-1313) identified components involved in both systems, such as Urb, and components with specific roles in vivo, including vitrin and CILP-2 (cartilage intermediate layer protein-2). Immunolocalization of Urb, vitrin, and CILP-2 indicated specific roles at different maturation stages. In addition to ECM-related changes, we provide the first biochemical evidence of changing endoplasmic reticulum function during cartilage development. Although the multifunctional chaperone BiP was not differentially expressed, enzymes and chaperones required specifically for collagen biosynthesis, such as the prolyl 3-hydroxylase 1, cartilage-associated protein, and peptidyl prolyl cis-trans isomerase B complex, were down-regulated during maturation. Conversely, the lumenal proteins calumenin, reticulocalbin-1, and reticulocalbin-2 were significantly increased, signifying a shift toward calcium binding functions. This first proteomic analysis of cartilage development in vivo reveals the breadth of protein expression changes during chondrocyte maturation and ECM remodeling in the mouse femoral head.