Analysis of chondroprogenitor frequency and cartilage differentiation in a novel family of clonal chondrogenic rat cell lines

Grigoriadis, Agamemnon E.; Heersche, J. N. M.; Aubin, Jane E.

doi:10.1046/j.1432-0436.1996.6050299.x

Cited by 49 publications

(32 citation statements)

References 44 publications

(91 reference statements)

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“…Ap 4 A, FGF9, inhibitors of FGFR3 activity (PD173074) and mitogen-activated protein kinase pathway (U0126) were purchased from Sigma (St. Louis, MO, USA). Cells RCJ3.1C5.18-cells are a sub-clonal mesenchymal cell line that differentiate into chondrocytes and become chondrogenic [15]. They can be stably transfected with full-length human wild-type or the G380R mutant FGFR3 [1,2] that is the characteristic of the most common achondroplasia.…”

Section: Reagentsmentioning

confidence: 99%

Effects of diadenosine tetraphosphate on FGF9-induced chloride flux changes in achondroplastic chondrocytes

Huete

Guzmán-Aránguez

Ortín

et al. 2011

Purinergic Signalling

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Achondroplasia, the most common type of dwarfism, is characterized by a mutation in the fibroblast growth factor receptor 3 (FGFR3). Achondroplasia is an orphan pathology with no pharmacological treatment so far. However, the possibility of using the dinucleotide diadenosine tetraphosphate (Ap 4 A) with therapeutic purposes in achondroplasia has been previously suggested. The pathogenesis involves the constitutive activation of FGFR3, resulting in altered biochemical and physiological processes in chondrocytes. Some of these altered processes can be influenced by changes in cell volume and ionic currents. In this study, the action of mutant FGFR3 on chondrocyte size and chloride flux in achondroplastic chondrocytes was investigated as well as the effect of the Ap 4 A on these processes triggered by mutant FGFR3. Stimulation with the fibroblast growth factor 9 (FGF9), the preferred ligand for FGFR3, induced an enlarged achondroplastic chondrocyte size and an increase in the intracellular chloride concentration, suggesting the blockade of chloride efflux. Treatment with the Ap 4 A reversed the morphological changes triggered by FGF9 and restored the chloride efflux. These data provide further evidence for the therapeutic potential of this dinucleotide in achondroplasia treatment.

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Section: Reagentsmentioning

confidence: 99%

Effects of diadenosine tetraphosphate on FGF9-induced chloride flux changes in achondroplastic chondrocytes

Huete

Guzmán-Aránguez

Ortín

et al. 2011

Purinergic Signalling

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“…The mesenchymal RCJ cell line spontaneously differentiates from displayed polygonal-shaped isolated chondrocytes to cartilage nodules over 4-14 days of culture in differentiating medium, in the presence of dexamethasone (Grigoriadis et al 1996). (A) Cells were cultured in differentiating medium until day 7 (left panel) and day 14 (right panel), serum-starved for 12 h and incubated for 1 h with IGF-I (100 ng/ml) in the presence or absence of 25 mM LY294002.…”

Section: Igf-i Enhances Differentiation Of Rcj Cellsmentioning

confidence: 99%

“…We used the mesenchymal RCJ3.1C5.18 (RCJ) cell line as a cell culture model, which is widely used for growth plate chondrocyte research (McEwen et al 1999, Cohen et al 2006. RCJ cells derive from fetal rat calvaria (Grigoriadis et al 1996, McDougall et al 1996. They undergo over 2 weeks of culture in the presence of dexamethasone a reproducible, time-dependent progression from chondroprogenitors to hypertrophic chondrocytes, accompanied by an upregulation of collagen type II and deposition of cartilage-specific proteoglycans in a sequence that mimics the phenotype of chondrocytes of the growth plate.…”

Section: Introductionmentioning

confidence: 99%

Signaling mechanisms leading to regulation of proliferation and differentiation of the mesenchymal chondrogenic cell line RCJ3.1C5.18 in response to IGF-I

Ciarmatori¹,

Kiepe²,

Haarmann³

et al. 2007

Journal of Molecular Endocrinology

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Since IGF-I is an important chondrocyte growth factor, we sought to examine the intracellular mechanisms by which it exerts two of its pivotal effects, stimulation of proliferation and differentiation. We used the mesenchymal chondrogenic cell line RCJ3.1C5.18, which progresses spontaneously to differentiated growth plate chondrocytes. This differentiation process could be enhanced by exogenous IGF-I. Pharmacological inhibition of the phosphatidylinositol-3 (PI-3) kinase by LY294002, mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)1/2 by U0126, the protein kinase (PK) A pathway by H-89 or KT5720, and the PKC pathway by bisindolylmaleimide suppressed IGF-I-stimulated cell proliferation. In contrast, IGF-I-enhanced early cell differentiation, as assessed by collagen type II and aggrecan gene expression, was not affected by MAPK/ERK1/2 pathway inhibition, but significantly diminished by inhibition of the PI-3 kinase, the PKC and the PKA pathway. Moreover, terminal differentiation of chondrocytes in response to IGF-I, as assessed by gene expression of alkaline phosphatase, Indian hedgehog, and collagen type X, were only interrupted by PI-3 kinase pathway inhibition. In conclusion, IGF-I exerts its differential effect on chondrocyte proliferation vs differentiation through the use of at least four partially interacting intracellular signaling pathways, whose activity is temporarily regulated. When chondrocytes progress from proliferating cells to early and terminal differentiating cells, they progressively inactivate IGF-I-related intracellular signaling pathways. This mechanism might be essential for the complex and cell stage-specific anabolic action of IGF-I in the growth plate.

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“…Colonies negative for expression of any of the mesenchymal lineage markers but with expression of the housekeeping gene (ribosomal protein L32) comparable to that in all other colonies were designated CFC-F. Positive controls for each lineage were: for osteoblasts/ CFC-O, mRNA isolated from microscopically defined mineralized bone nodules present in RC cell cultures under osteogenic conditions (Bellows et al 1986); for adipocytes/CFC-A, mRNA from colonies with patent lipid droplets after 1,25(OH) 2 D 3 treatment of RC cells (Bellows et al 1986); for myocytes/CFC-M, mRNA isolated from rat skeletal muscle; for chondrocytes/ CFC-C, mRNA isolated from the chondrocyte cell line C5·18 (Grigoriadis et al 1996). Neural cell mRNA isolated from neurospheres formed from adult and embryonic neural tissue (kindly provided by Dr C M Morshead, University of Toronto; Kim & Morshead 2003) served as a positive control for nestin and 3 tubulin.…”

Section: Rna Isolation Reverse Transcription and Real Time Pcrmentioning

confidence: 99%

Pleiotropic effects of the steroid hormone 1,25-dihydroxyvitamin D3 on the recruitment of mesenchymal lineage progenitors in fetal rat calvaria cell populations

Zhang¹,

Chan²,

Aubin³

2006

Journal of Molecular Endocrinology

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The steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH) 2 D 3 ) inhibits osteogenesis while stimulating adipogenesis in vitro. We hypothesized that 1,25(OH) 2 D 3 redirects the fate of osteoblast/adipocyte bipotential progenitors and other potential progenitors towards adipogenesis, a process possibly underlying the pathogenesis of osteopenic diseases such as osteoporosis. We therefore tested the global effects of 1,25(OH) 2 D 3 on the recruitment of mesenchymal progenitors including osteogenic, chondrogenic, adipogenic and myogenic lineages (colony forming cell (CFC)-osteoblast (CFC-O), CFC-chondrocyte (CFC-C), CFC-adipocyte (CFC-A), and CFC-myoblast (CFC-M) respectively) in rat calvaria (RC) cell populations using gene expression profiling of single cell-derived colonies. Based on expression of lineage specific transcripts, 86% of single cell-derived colonies in untreated cultures simultaneously co-expressed transcripts of two, three, or four of the mesenchymal lineages tested. The distribution of mesenchymal progenitors in 1,25(OH) 2 D 3 -treated cultures was significantly changed compared with the control group, i.e. CFC-O were reduced (from 6 to 0%) and CFC-O/A bipotential (0 to 8·2%), CFC-C (4 to 10·2%) and CFC-Fibroblast (CFC-F) (4 to 16%) were increased. 1,25(OH) 2 D 3 did not affect the frequency of tri-or tetra-lineage colonies. Single lineage CFC-A colonies were not detected in either the control or 1,25(OH) 2 D 3 treatment group under the conditions tested. Since the parietal bones used for cell isolation derive from neuroectoderm, we also analyzed for expression of the neural markers nestin and 3 tubulin in these colonies. Surprisingly, 90% (45 of 50) of the colonies in the control group expressed neural markers, a frequency not changed by 1,25(OH) 2 D 3 treatment. The current studies demonstrate the global and developmental stage-specific effects of 1,25(OH) 2 D 3 on mesenchymal lineage progenitors, and suggest that the effects of 1,25(OH) 2 D 3 on osteogenesis and adipogenesis in RC populations are mediated, at least in part, by increased recruitment of CFC-O/A, but not CFC-A type precursors.

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Analysis of chondroprogenitor frequency and cartilage differentiation in a novel family of clonal chondrogenic rat cell lines

Cited by 49 publications

References 44 publications

Effects of diadenosine tetraphosphate on FGF9-induced chloride flux changes in achondroplastic chondrocytes

Effects of diadenosine tetraphosphate on FGF9-induced chloride flux changes in achondroplastic chondrocytes

Signaling mechanisms leading to regulation of proliferation and differentiation of the mesenchymal chondrogenic cell line RCJ3.1C5.18 in response to IGF-I

Pleiotropic effects of the steroid hormone 1,25-dihydroxyvitamin D3 on the recruitment of mesenchymal lineage progenitors in fetal rat calvaria cell populations

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