The progeny of rabbit articular chondrocytes synthesize collagen types I and III and type I trimer, but not type II. Verifications by cyanogen bromide peptide analysis

Benya, Paul D.; Padilla, Silvia R.; Nimni, Marcel E.

doi:10.1021/bi00624a009

Cited by 294 publications

(100 citation statements)

References 33 publications

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“…Also, like other procollagens, this material could be converted to faster migrating forms by limited enzymatic digestions, suggesting that, in the native form, there are large, nonhelical regions in addition to the helical ones. After pepsin or chymotrypsin digestion of this procollagen, two chains were produced, frequently in a 2:1 ratio, which migrated on NaDodSO4 gels near the positions of the A and B chains recently isolated from human fetal tissues (22,29), and possibly the X and Y chains isolated from senescent chondrocyte cultures (30 Fig. 5.…”

Section: Discussionmentioning

confidence: 99%

Embryonic neural retina collagen: In vitro synthesis of high molecular weight forms of type II plus a new genetic type

Linsenmayer

Little

1978

Proc. Natl. Acad. Sci. U.S.A.

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Chicken neural retina cells from 6-to 7-day embryos were labeled with [3Hjproline for 24 and 72 hr and the collagenous proteins of the medium were analyzed. Ninety percent of the collagenous proteins eluted from DEAE-celiulose columns as a peak near the middle of the gradient. Upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis, this material consisted predominantly of a slowly migrating procollagen, a smaller amount of an intermediate migrating form, and some a-chain material. Limited enzymatic digestion of this DEAE peak material plus mapping of its CNBr peptides identified this material as a type II precursor. The remaining 10% of the collagenous proteins eluted from DEAE-cellulose at the end of the gradient. Electrophoresis of this fraction showed four major bands. Two migrated near the type II precursors, a third migrated somewhat more slowly, and the fourth was near,& chain dimers. In addition, there were two minor bands. Limited pepsin digestion of this DEAE peak material produced two bands: one migrated slightly more slowly than al, and the second, considerably more slowly. The CNBr peptide pattern of this material appears to be different from any previously de-scribed. Thus, neural retina cells in culture synthesize at least two genetically distinct classes of collagenous proteins. One represents precursors to type II. The second is composed of multiple, very high molecular weight forms which may represent precursors (procollagens) of a new genetic type(s) of collagen.During development, the assembly of those embryonic structures formed largely of extracellular material requires the coordinated synthesis, transport, and deposition of the macromolecular components. These processes can be readily studied in epithelial tissues that exhibit polarized secretion of their products. In the developing eye, for example, the corneal epithelium produces the collagen of the primary corneal stroma (1-4) and the neural retina epithelium synthesizes the collagen that is deposited in the vitreous body (5, 6). A major portion of the collagen synthesized by both of these epithelia is type II (4, 6, 7), which was previously thought to be synthesized only in cartilage (8-10) and notochord (11)(12)(13).To examine further the production of the collagens produced by the neural retina epithelium, we have studied the collagenous molecules obtained from the medium of primary cultures of embryonic neural retina cells. The predominant procollagen forms synthesized by these cultures are precursors of type II collagen (about 90%) which are only slowly converted to completely processed material the size of a chains. In addition, about 10% of the collagenous proteins are not type II and may represent a different genetic type(s) of collagen. Some components of this material migrate considerably more slowly on sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis than the highest molecular weight form of the type II procollagen. After 24 hr in culture, fresh medium was added containing [2,3-3H]proline (...

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Section: Discussionmentioning

confidence: 99%

Embryonic neural retina collagen: In vitro synthesis of high molecular weight forms of type II plus a new genetic type

Linsenmayer

Little

1978

Proc. Natl. Acad. Sci. U.S.A.

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“…Cell Cultures-RAC were prepared from the shoulders and knees of 3-week old rabbits, as previously described (46,50). Cells were seeded at 2 ϫ 10 4 cells/cm 2 in either 6-well plates, 100-mm dishes, or 75-, 150-, and 175-cm 2 flasks and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat inactivated fetal calf serum (FCS), glutamine (2 mM), penicillin (100 IU/ml), streptomycin (100 g/ml), and Fungizone (0,25 g/ml) in a 5% CO 2 atmosphere.…”

Section: Methodsmentioning

confidence: 99%

Down-regulation of Human Type II Collagen Gene Expression by Transforming Growth Factor-β1 (TGF-β1) in Articular Chondrocytes Involves SP3/SP1 Ratio

Chadjichristos¹,

Ghayor²,

Herrouin³

et al. 2002

Journal of Biological Chemistry

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Although transforming growth factor ␤1 (TGF-␤1) is generally considered as a stimulator of type I collagen production in smooth organs, we found that it can inhibit type II collagen biosynthesis in primary rabbit articular chondrocytes (RAC) at transcriptional levels. Constructs of promoter and first intron sequences associated with the luciferase reporter gene were used to delineate the gene sequences involved in TGF-␤1 control of human COL2A1 gene transcription. Cotransfection of these DNA fragments with a T␤RII/I cDNA hybrid receptor, capable of inducing a TGF-␤1 dominant negative effect, showed that TGF-␤1 inhibits specifically COL2A1 gene transcription in RAC by a 63-bp proximal promoter. Footprint and gel retardation analyses revealed that the TGF-␤1-induced inhibition effect exerted through the 63-bp promoter sequence implies a multimeric complex that binds to the ؊41/؊33 sequence and involves Sp1 and Sp3 transcription factors. Transfection of decoy Sp-binding oligonucleotides corroborated the implication of the proximal promoter in the TGF-␤1-induced inhibition of COL2A1 gene transcription. In addition, TGF-␤1 was found to increase the expression of Sp3 without significant changes to its binding level, but repressed both the biosynthesis and binding activity of Sp1. In functional assays, Sp3 inhibited the 63-bp promoter activity and prevented Sp1 induction of transcription. These findings suggest that TGF-␤1 inhibition of COL2A1 gene transcription in RAC is mediated by an increase of the Sp3/Sp1 ratio and by the repression of Sp1 transactivating effects on that gene.

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“…Cells grown both in monolayer as well as in suspension in alginate were studied. Chondrocyte culture in alginate has been shown to preserve the normal chondrocyte phenotype [14], which can be lost after prolonged culture in monolayer [15]. The results show that key members of both the PI3K and ERK/MAPK pathways are activated by IGF-I treatment of chondrocytes, but that only activity of the PI3K pathway is required for the stimulation of proteoglycan synthesis.…”

Section: Introductionmentioning

confidence: 95%

IGF-I stimulation of proteoglycan synthesis by chondrocytes requires activation of the PI 3-kinase pathway but not ERK MAPK

Starkman

Cravero

DelCarlo

et al. 2005

Biochemical Journal

155

161

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The IGF-I (insulin-like growth factor-I) signalling pathway responsible for regulation of proteoglycan synthesis in chondrocytes has not been defined and is the focus of the present study. Chondrocytes isolated from normal human articular cartilage were stimulated with IGF-I in monolayer culture or in suspension in alginate. IGF-I activated members of both the PI3K (phosphoinositide 3-kinase) pathway and the ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) pathway. The PI3K inhibitors LY294002 and wortmannin blocked IGF-I-stimulated Akt phosphorylation without blocking ERK phosphorylation and this was associated with complete inhibition of proteoglycan synthesis. A decrease in IGF-I-stimulated proteoglycan synthesis was also observed upon inhibition of mTOR (mammalian target of rapamycin) and p70S6 kinase, both of which are downstream of Akt. The MEK (MAPK/ERK kinase) inhibitors PD98059 and U0126 blocked IGF-I-stimulated ERK phosphorylation but did not block the phosphorylation of Akt and did not decrease proteoglycan synthesis. Instead, in alginate- cultured chondrocytes, the MEK inhibitors increased IGF-I-stimulated proteoglycan synthesis when compared with cells treated with IGF-I alone. This is the first study to demonstrate that IGF-I stimulation of the PI3K signalling pathway is responsible for the ability of IGF-I to increase proteoglycan synthesis. Although IGF-I also activates the ERK/MAPK pathway, ERK activity is not required for proteoglycan synthesis and may serve as a negative regulator

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The progeny of rabbit articular chondrocytes synthesize collagen types I and III and type I trimer, but not type II. Verifications by cyanogen bromide peptide analysis

Cited by 294 publications

References 33 publications

Embryonic neural retina collagen: In vitro synthesis of high molecular weight forms of type II plus a new genetic type

Embryonic neural retina collagen: In vitro synthesis of high molecular weight forms of type II plus a new genetic type

Down-regulation of Human Type II Collagen Gene Expression by Transforming Growth Factor-β1 (TGF-β1) in Articular Chondrocytes Involves SP3/SP1 Ratio

IGF-I stimulation of proteoglycan synthesis by chondrocytes requires activation of the PI 3-kinase pathway but not ERK MAPK

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