Characterization of primary cultures of chondrocytes from type II collagen/β-galactosidase transgenic mice

Lefebvre, Véronique; Garofalo, Silvio; Zhou, Guang; Metsäranta, Marjo; Vuorio, Eero; Crombrugghe, Benoít de

doi:10.1016/0945-053x(94)90199-6

Cited by 147 publications

(137 citation statements)

References 13 publications

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“…At the time of infection with the retrovirus coding for large T, our primary chondrocytes expressed high levels of type II collagen and cartilage-specific proteoglycans (23), typical markers of differentiated chondrocytes. These initial populations of primary chondrocytes also contained mRNAs for type X collagen, MGP, OP, and ALP, indicating that an important proportion of the ceils were hypertrophic.…”

Section: Discussionmentioning

confidence: 95%

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Type X collagen gene expression in mouse chondrocytes immortalized by a temperature-sensitive simian virus 40 large tumor antigen.

Lefebvre

Garofalo

Crombrugghe

1995

The Journal of Cell Biology

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Abstract. Mouse endochondral chondrocytes were immortalized with a temperature-sensitive simian virus 40 large tumor antigen. Several clonal isolates as well as pools of immortalized cells were characterized. In monolayer cultures at the temperature permissive for the activity of the large tumor antigen (32°C), the cells grew continuously with a doubling time of ,~2 d, whereas they stopped growing at nonpermissive temperatures (37°C-39°C). The cells from all pools and from most clones expressed the genes for several markers of hypertrophic chondrocytes, such as type X collagen, matrix Gla protein, and osteopontin, but had lost expression of type lI collagen mRNA and failed to be stained by alcian blue which detects cartilagespecific proteoglycans. The cells also contained mRNAs for type I collagen and bone Gla protein, consistent with acquisition of osteoblastic-like properties. Higher levels of mRNAs for type X collagen, bone Gla protein, and osteopontin were found at nonpermissive temperatures, suggesting that the expression of these genes was upregulated upon growth arrest, as is the case in vivo during chondrocyte hypertrophy. Cells also retained their ability to respond to retinoic acid, as indicated by retinoic acid dose-dependent and timedependent increases in type X collagen mRNA levels. These cell lines, the first to express characteristic features of hypertrophic chondrocytes, should be very useful to study the regulation of the type X collagen gene and other genes activated during the last stages of chondrocyte differentiation. C HONDROCYTES are highly specialized cells that are derived from mesenchymal cells during embryonic development to form the various cartilages of vertebrates. Chondrocytes are characterized by the production of cartHage-specific extracellular macromolecules including the collagen types H, IX, and XI, and the proteoglycan aggrecan (16). In permanent cartilages, the phenotype of chondrocytes is stably maintained, whereas in endochondral cartilages, chondrocytes undergo a further differentiation process (16,34). During this process, chondrocytes mature into hypertrophic ceils around which the cartilage matrix becomes calcified, and subsequently undergo apoptosis, making way for invading osteoblasts. Hypertrophic chondrocytes still contain type II collagen and aggrecan RNAs, although at reduced levels (21, 38), they are characterized by the synthesis of type X collagen, which is uniquely expressed by these cells (10,21,39), and by the expression of markers typically associated with osteoblasts. Both hypertrophic chondrocytes and osteoblasts have the ability to induce mineralization of the extracellular matrix and to produce alka-

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Section: Discussionmentioning

confidence: 95%

“…Rib chondrocytes and skin fibroblasts were prepared from transgenic and nontransgenic 1-3-d-old mice (BtD2FI) as described previously (23). Chondrocytes, fibroblasts, and ROS cells were cultured in standard medium at 37°C under 8% CO2.…”

Section: Other Cellsmentioning

confidence: 99%

Type X collagen gene expression in mouse chondrocytes immortalized by a temperature-sensitive simian virus 40 large tumor antigen.

Lefebvre

Garofalo

Crombrugghe

1995

The Journal of Cell Biology

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“…For this study, we avoided using the conventional chondrocyte culture system derived from the rib cartilage of neonatal mice (32), because most of the cartilage belongs to the permanent cartilage that does not undergo endochondral ossification but maintains cartilage phenotypes. Instead, we succeeded in isolating chondrocytes from the growth plate with more than 99% purity, as confirmed by the X-gal staining of cultured cells from transgenic mice expressing osteoblast-or chondrocyte-specific marker genes (Fig.…”

Section: Discussionmentioning

confidence: 99%

Impairment of Bone Healing by Insulin Receptor Substrate-1 Deficiency

Shimoaka

Kamekura

Chikuda

et al. 2004

Journal of Biological Chemistry

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Insulin receptor substrate-1 (IRS-1) is an essential molecule for intracellular signaling of insulin-like growth factor (IGF)-I and insulin, both of which are potent anabolic regulators of bone and cartilage metabolism. To investigate the role of IRS-1 in bone regeneration, fracture was introduced in the tibia, and its healing was compared between wild-type (WT) mice and mice lacking the IRS-1 gene (IRS-1 ؊/؊ mice). Among 15 IRS-1 ؊/؊ mice, 12 remained in a non-union state even at 10 weeks after the operation, whereas all 15 WT mice showed a rigid bone union at 3 weeks. This impairment was because of the suppression of callus formation with a decrease in chondrocyte proliferation and increases in hypertrophic differentiation and apoptosis. Reintroduction of IRS-1 to the IRS-1 ؊/؊ fractured site using an adenovirus vector significantly restored the callus formation. In the culture of chondrocytes isolated from the mouse growth plate, IRS-1 ؊/؊ chondrocytes showed less mitogenic ability and Akt phosphorylation than WT chondrocytes. An Akt inhibitor decreased the IGF-Istimulated DNA synthesis of chondrocytes more potently in the WT culture than in the IRS-1 ؊/؊ culture. We therefore conclude that IRS-1 deficiency impairs bone healing at least partly by inhibiting chondrocyte proliferation through the phosphatidylinositol 3-kinase/Akt pathway, and we propose that IRS-1 can be a target molecule for bone regenerative medicine.

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“…Rat chondrosarcoma (RCS) cells (49) were cultured as described previously (49,50). Primary chondrocytes were isolated from the ribs of newborn mice as described previously (50,51) and used within the first passage.…”

Section: Methodsmentioning

confidence: 99%

RhoA/ROCK Signaling Suppresses Hypertrophic Chondrocyte Differentiation

Wang

Woods

Sabari

et al. 2004

Journal of Biological Chemistry

117

118

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Coordinated proliferation and differentiation of growth plate chondrocytes is required for normal growth and development of the endochondral skeleton, but little is known about the intracellular signal transduction pathways regulating these processes. We have investigated the roles of the GTPase RhoA and its effector kinases ROCK1/2 in hypertrophic chondrocyte differentiation. RhoA, ROCK1, and ROCK2 are expressed throughout chondrogenic differentiation. RhoA overexpression in chondrogenic ATDC5 cells results in increased proliferation and a marked delay of hypertrophic differentiation, as shown by decreased induction of alkaline phosphatase activity, mineralization, and expression of the hypertrophic markers collagen X, bone sialoprotein, and matrix metalloproteinase 13. These effects are accompanied by activation of cyclin D1 transcription and repression of the collagen X promoter by RhoA. In contrast, inhibition of Rho/ROCK signaling by the pharmacological inhibitor Y27632 inhibits chondrocyte proliferation and accelerates hypertrophic differentiation. Dominant-negative RhoA also inhibits induction of the cyclin D1 promoter by parathyroid hormone-related peptide. Finally, Y27632 treatment partially rescues the effects of RhoA overexpression. In summary, we identify the RhoA/ROCK signaling pathway as a novel and important regulator of chondrocyte proliferation and differentiation.The development and growth of endochondral bones (such as ribs, vertebrae, and the long bones of vertebrate limbs) are regulated through the highly controlled rates of proliferation and hypertrophic differentiation of growth plate chondrocytes (1-3). In the growth plate, chondrocytes first undergo a series of cell divisions along the longitudinal axis of the growing bone, thereby forming characteristic columns of clonal cells. Chondrocytes then withdraw from the cell cycle and begin to increase their cell volume until reaching the fully differentiated state of hypertrophic chondrocytes. Transition from a proliferating to a hypertrophic phenotype involves numerous changes in gene expression, for example the induction of the collagen X (4, 5), matrix metalloproteinase 13 (6, 7), and bone sialoprotein (BSP) 1 (8 -10) genes. The latter two genes are also expressed by osteoblasts, suggesting similar biological properties of hypertrophic chondrocytes and osteoblasts. The fate of hypertrophic chondrocytes is still debated, but it appears that the majority of these cells undergo apoptosis and are replaced by bone tissue (11). Longitudinal growth of endochondral bones therefore requires both proliferation and differentiation-associated hypertrophy of growth plate chondrocytes.Disruption of chondrocyte proliferation and/or differentiation by gene mutations commonly results in chondrodysplasias that are characterized by skeletal deformities and reduced growth (12)(13)(14). Mutations in genes encoding extracellular matrix molecules, growth factors, receptors, and transcription factors have been identified as causes of several chondrodysplasias. Fo...

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Characterization of primary cultures of chondrocytes from type II collagen/β-galactosidase transgenic mice

Cited by 147 publications

References 13 publications

Type X collagen gene expression in mouse chondrocytes immortalized by a temperature-sensitive simian virus 40 large tumor antigen.

Type X collagen gene expression in mouse chondrocytes immortalized by a temperature-sensitive simian virus 40 large tumor antigen.

Impairment of Bone Healing by Insulin Receptor Substrate-1 Deficiency

RhoA/ROCK Signaling Suppresses Hypertrophic Chondrocyte Differentiation

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