Inhibition of chondrocyte differentiation in vitro by constitutive and inducible overexpression of the c-fos proto-oncogene

We have investigated the role of c-Fos in chondrocyte differentiation in vitro using both constitutive and inducible overexpression approaches in ATDC5 chondrogenic cells, which undergo a well-defined sequence of differentiation from chondroprogenitors to fully differentiated hypertrophic chondrocytes. Initially, we constitutively overexpressed exogenous c-fos in ATDC5 cells. Several stable clones expressing high levels of exogenous c-fos were isolated and those also expressing the cartilage marker type II collagen showed a marked decrease in cartilage nodule formation. To investigate further whether c-Fos directly regulates cartilage differentiation independently of potential clonal variation, we generated additional clones in which exogenous c-fos expression was tightly controlled by a tetracycline-regulatable promoter. Two clones, DT7.1 and DT12.4 were capable of nodule formation in the absence of c-fos. However, upon induction of exogenous c-fos, differentiation was markedly reduced in DT7.1 cells and was virtually abolished in clone DT12.4. Pulse experiments indicated that induction of c-fos only at early stages of proliferation/differentiation inhibited nodule formation, and limiting dilution studies suggested that overexpression of c-fos decreased the frequency of chondroprogenitor cells within the clonal population. Interestingly, rates of proliferation and apoptosis were unaffected by c-fos overexpression under standard conditions, suggesting that these processes do not contribute to the observed inhibition of differentiation. Finally, gene expression analyses demonstrated that the expression of the cartilage markers type II collagen and PTH/PTHrP receptor were down-regulated in the presence of exogenous c-Fos and correlated well with the differentiation status. Moreover, induction of c-fos resulted in the concomitant increase in the expression of fra-1 and c-jun, further highlighting the importance of AP-1 transcription factors in chondrocyte differentiation. These data demonstrate that c-fos overexpression directly inhibits chondrocyte differentiation in vitro, and therefore these cell lines provide very useful tools for identifying novel c-Fos-responsive genes that regulate the differentiation and activity of chondrocytes.

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Human Umbilical Cord Mesenchymal Stem Cells Induce into Chondrocytes in 3D Cultured System

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.749.198 ◽

2013 ◽

Vol 749 ◽

pp. 198-205

Author(s):

Li Yu ◽

Jing Liu ◽

Chao Xu ◽

Er Mei Luo ◽

Ming Qiao Tang

Keyword(s):

Real Time ◽

Alcian Blue ◽

Culture System ◽

Type Ii Collagen ◽

Human Umbilical Cord ◽

Type Ii ◽

Pellet Culture ◽

Hla Dr

Objective: To investigate a better method of inducing hUC-MSCs into chondrocytes in different culture system in vitro. Method: hUC-MSCs were isolated and cultured by tissue block culture, and the cells surface antigens were identified by flow cytometry, hUC-MSCs were cultured with chondrogenic media and stained with Alcian Blue. The production of matrix was estimated from the determination of hydroxyproline content and Alcian Blue method. Expressions of glycosaminoglycan (GAG), type II collagen and Sox-9 were assayed by real-time fluorescence quantitative PCR. Results: The cultured hUC-MSCs phenotype was CD105+/CD29+/CD44+/ CD31-/CD34-/ CD40-/CD45-/HLA-DR-. hUC-MSCs weakly expressed chondrocyte marker, which strongly expressed GAG and type II collagen after chondrogenic induction, and the cells were incubated in pellet culture with higher expression. Real-time PCR results demonstrated that chondrogenic induction cells were expressed GAG, type II collagen and Sox-9, and the cells were incubated in pellet culture with higher expression. Conclusion: hUC-MSCs incubated in pellet culture is more conducive to differentiate into chondrocytes than those cultured in monolayer culture system.

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Type VI collagen expression is upregulated in the early events of chondrocyte differentiation

Development ◽

10.1242/dev.117.1.245 ◽

1993 ◽

Vol 117 (1) ◽

pp. 245-251

Author(s):

R. Quarto ◽

B. Dozin ◽

P. Bonaldo ◽

R. Cancedda ◽

A. Colombatti

Keyword(s):

Suspension Culture ◽

Type I Collagen ◽

Type Ii Collagen ◽

Type I ◽

Type Ii ◽

Type Vi Collagen ◽

Full Spectrum ◽

Collagen Expression ◽

Hypertrophic Chondrocyte

Dedifferentiated chondrocytes cultured adherent to the substratum proliferate and synthesize large amounts of type I collagen but when transferred to suspension culture they decrease proliferation, resume the chondrogenic phenotype and the synthesis of type II collagen, and continue their maturation to hypertrophic chondrocyte (Castagnola et al., 1986, J. Cell Biol. 102, 2310–2317). In this report, we describe the developmentally regulated expression of type VI collagen in vitro in differentiating avian chondrocytes. Type VI collagen mRNA is barely detectable in dedifferentiated chondrocytes as long as the attachment to the substratum is maintained, but increases very rapidly upon passage of the cells into suspension culture reaching a peak after 48 hours and declining after 5–6 days of suspension culture. The first evidence of a rise in the mRNA steady-state levels is obtained already at 6 hours for the alpha 3(VI) chain. Immunoprecipitation of metabolically labeled cells with type VI collagen antibodies reveals that the early mRNA rise is paralleled by an increased secretion of type VI collagen in cell media. Induction of type VI collagen is not the consequence of trypsin treatment of dedifferentiated cells since exposure to the actin-disrupting drug cytochalasin or detachment of the cells by mechanical procedures has similar effects. In 13-day-old chicken embryo tibiae, where the full spectrum of the chondrogenic differentiation process is represented, expression of type VI collagen is restricted to the articular cartilage where chondrocytes developmental stage is comparable to stage I (high levels of type II collagen expression).(ABSTRACT TRUNCATED AT 250 WORDS)

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Discovery and development of a type II collagen neoepitope (TIINE) biomarker for matrix metalloproteinase activity: From in vitro to in vivo

Analytical Biochemistry ◽

10.1016/j.ab.2006.10.034 ◽

2007 ◽

Vol 361 (1) ◽

pp. 93-101 ◽

Cited By ~ 59

Author(s):

O.V. Nemirovskiy ◽

D.R. Dufield ◽

T. Sunyer ◽

P. Aggarwal ◽

D.J. Welsch ◽

...

Keyword(s):

Matrix Metalloproteinase ◽

Type Ii Collagen ◽

Type Ii ◽

Metalloproteinase Activity

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Effects of Extracellular Matrix on the Morphology and Behaviour of Rabbit Auricular Chondrocytes in Culture

Journal of Biomedicine and Biotechnology ◽

10.1155/jbb.2005.364 ◽

2005 ◽

Vol 2005 (4) ◽

pp. 364-373 ◽

Cited By ~ 7

Author(s):

Vega Villar-Suárez ◽

B. Colaço ◽

I. Calles-Venal ◽

I. G. Bravo ◽

J. G. Fernández-Álvarez ◽

...

Keyword(s):

Extracellular Matrix ◽

Type Ii Collagen ◽

Cell Phenotype ◽

Nodule Formation ◽

Type Ii ◽

Protein Extract ◽

Positive Effects ◽

Reticular Pattern ◽

Chondrocyte Cultures ◽

Plastic Surfaces

Isolated chondrocytes dedifferentiate to a fibroblast-like shape on plastic substrata and proliferate extensively, but rarely form nodules. However, when dissociation is not complete and some cartilage remnants are included in the culture, proliferation decreases and cells grow in a reticular pattern with numerous nodules, which occasionally form small cartilage-like fragments. In an attempt to reproduce this stable chondrogenic state, we added a cartilage protein extract, a sugar extract, and hyaluronan to the medium of previously dedifferentiated chondrocytes. When protein extract was added, many cartilaginous nodules appeared. Hyaluronan produced changes in cell phenotype and behaviour, but not nodule formation. Protein extract has positive effects on the differentiation of previously proliferated chondrocytes and permits nodule formation and the extensive production of type-II collagen. A comparison with incompletely dissociated chondrocyte cultures suggests that the presence of some living cells anchored to their natural extracellular matrix provides some important additional factors for the phenotypical stability of chondrocytes on plastic surfaces. In order to elucidate if it is possible that the incidence of apoptosis is related to the results, we also characterized the molecular traits of apoptosis.

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Extracellular Nucleotides Selectively Induce Migration of Chondrocytes and Expression of Type II Collagen

International Journal of Molecular Sciences ◽

10.3390/ijms21155227 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5227

Author(s):

Marcin Szustak ◽

Edyta Gendaszewska-Darmach

Keyword(s):

Migration Rate ◽

Monolayer Culture ◽

Type Ii Collagen ◽

Extracellular Nucleotides ◽

Nonbridging Oxygen ◽

Nucleotide Receptors ◽

Type Ii ◽

Mrna Transcripts ◽

Chondrogenic Phenotype

The migration of chondrocytes from healthy to injured tissues is one of the most important challenges during cartilage repair. Additionally, maintenance of the chondrogenic phenotype remains another limitation, especially during monolayer culture in vitro. Using both the differentiated and undifferentiated chondrogenic ATDC5 cell line, we showed that extracellular nucleotides are able to increase the migration rate of chondrocytes without affecting their chondrogenic phenotype. We checked the potency of natural nucleotides (ATP, ADP, UTP, and UDP) as well as their stable phosphorothioate analogs, containing a sulfur atom in the place of one nonbridging oxygen atom in a phosphate group. We also detected P2y1, P2y2, P2y4, P2y6, P2y12, P2y13, and P2y14 mRNA transcripts for nucleotide receptors, demonstrating that P2y1 and P2y13 are highly upregulated in differentiated ATDC5 cells. We showed that ADPβS, UDPβS, and ADP are the best stimulators of migration of differentiated chondrocytes. Additionally, ADP and ADPβS positively affected the expression of type II collagen, a structural component of the cartilage matrix.

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Levels of matrix metalloproteinase-2 in committed differentiation of bone marrow mesenchymal stem cells induced by kartogenin

Journal of International Medical Research ◽

10.1177/0300060519853399 ◽

2019 ◽

Vol 47 (7) ◽

pp. 3261-3270

Author(s):

Cheng Wang ◽

Qiaohui Liu ◽

Xiaoyuan Ma ◽

Guofeng Dai

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Matrix Metalloproteinase ◽

Type Ii Collagen ◽

Matrix Metalloproteinase 2 ◽

Type Ii ◽

Proliferation And Differentiation ◽

Marrow Mesenchymal Stem Cells

Objective To measure the inductive effect of kartogenin on matrix metalloproteinase-2 levels during the differentiation of human bone marrow mesenchymal stem cells (hMSCs) into chondrocytes in vitro. Methods In vitro cultured bone marrow hMSCs were grown to the logarithmic phase and then divided into three groups: control group (0 µM kartogenin), 1 µM kartogenin group and 10 µM kartogenin group. After 72 h of culture, cell proliferation and differentiation were observed microscopically. Matrix metalloproteinase-2 (MMP-2) in the cell supernatant and type II collagen levels in the cells were detected by enzyme linked immunosorbent assay and immunofluorescence staining, respectively. Results Kartogenin induced the proliferation and differentiation of hMSCs. With the increase of kartogenin concentration, the level of type II collagen was increased, while the level of MMP-2 decreased. Conclusion These findings indicate that kartogenin can induce hMSCs to differentiate into chondrocytes, and with the increase of kartogenin concentration, degeneration of the cartilage extracellular matrix may be inhibited.

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Effects of bio-mimic collagen II-g-hyaluronic acid copolymers on chondrocyte maintenance

Journal of Bioactive and Compatible Polymers ◽

10.1177/0883911519876069 ◽

2019 ◽

Vol 34 (4-5) ◽

pp. 373-385

Author(s):

Kuan Wei Lee ◽

Tang-Ching Kuan ◽

Ming Wei Lee ◽

Chen Show Yang ◽

Lain-Chyr Hwang ◽

...

Keyword(s):

Extracellular Matrix ◽

Cell Proliferation ◽

Hyaluronic Acid ◽

Specific Binding ◽

Type Ii Collagen ◽

Polymerase Chain Reaction Analysis ◽

Collagen Fibrils ◽

Type Ii ◽

Human Cartilage

Extracellular matrix has an important part of the role in tissue engineering and regenerative medicine, so it is necessary to understand the various interactions between cells and extracellular matrix. Type II collagen and hyaluronic acid are the major structural components of the extracellular matrix of articular cartilage, and they are involved in fibril formation, entanglement and binding. The aim of this study was to prepare type II collagen fibrils with surface grafted with hyaluronic acid modified at the reducing end. The topographic pattern of type II collagen fibrils showed a significant change after the surface coupling of hyaluronic acid according to atomic force microscopy scanning. The presence of hyaluronic acid on the type II collagen fibrillar surface was confirmed by the specific binding of nanogold labelled with lectin. No significant increase in cell proliferation was detected by a WST-1 assay. According to histochemical examination, the maintenance of the round shape of chondrocytes and increased glycosaminoglycan secretion revealed that these cell pellets with Col II- g-hyaluronic acid molecules contained un-dedifferentiated chondrocytes in vitro. In the mixture with the 220-kDa Col II- g-hyaluronic acid copolymer, the expression of type II collagen and aggrecan genes in chondrocytes increased as demonstrated by real-time polymerase chain reaction analysis. Experimental results show that the amount of hyaluronic acid added during culturing of chondrocytes can maintain the functionality of chondrocytes and thus allow for increased cell proliferation that is suitable for tissue repair of human cartilage.

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