Changes in the Nuclear and Cytoplasmic Levels of Type I and Type II Collagen RNAs during Growth of Chondrocytes in 5‐Bromo‐2′‐Deoxyuridine

Saxe, Stephen A.; Lukens, Lewis; Pawlowski, Philip J.

doi:10.1111/j.1749-6632.1985.tb51220.x

Cited by 11 publications

(10 citation statements)

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“…Still the cells synthesized only type I collagen. Our data, in agreement with findings of other groups (12,14,21,23), might suggest a translational regulation of the type I collagen expression during the in vitro differentiation process of chondrocytes from dedifferentiated cells.…”

Section: Discussionsupporting

confidence: 83%

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

Quarto

Dozin

Tacchetti

et al. 1990

The Journal of Cell Biology

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Abstract. Single cells from enzymatically dissociated chick embryo tibiae have been cloned and expanded in fresh or conditioned culture media. A cloning efficiency of ,,o13% 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 ,x,50% of them showed positivity for type II collagen. (6,19,24). Chondrocytes from Hamburger and Hamilton stage 28-30 (17) chick embryo tibiae dedifferentiate when cultured on plastic dishes. These cells, as well as dedifferentiated chondrocytes from another source (4), maintain the dedifferentiated phenotype as long as the conditions for the cell attachment to the plastic are kept. When the same cells are transferred into suspension culture, they resume the chondrocytic phenotype and mature to hypertrophic chondrocytes through a transient stage of cell aggregation (7). This process is accompanied by an increase in the length of the cell cycle and in the number of quiescent and degenerating cells (16), as well as by a modulation in the transcriptional activity of the collagen genes (9). We have recently reported the identification, purification, and characterization of a novel, low molecular weight protein, named Ch21, expressed and secreted by the in vitro differentiating chondrocytes at a late stage of development (11). When dedifferentiated cells are cultured in suspension in the constant presence of ascorbic acid, they organize their own extracellular matrix and develop into a tissue closely resembling calcifying hypertrophic cartilage (28,29).The heterogeneity of the dedifferentiated cell population did not allow us to definitely state whether the cells followed successive stages of differentiation or the culture conditions made a selection of cell subpopulations. To answer this important question we selected clones of dedifferentiated cells to follow the respective behavior of each clone in terms of morphology and collagen phenotype in the different culture conditions. We show that up to 45 % of the cell populations obtained from single cloned dedifferentiated cells are still able to differentiate to hypertrophic chondrocytes and to reconstitute hyp...

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Section: Discussionsupporting

confidence: 83%

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

Quarto

Dozin

Tacchetti

et al. 1990

The Journal of Cell Biology

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“…An unexpected finding of considerable interest was the presence of low levels of type II collagen mRNA in the cytoplasm of prechondrogenic mesenchymal cells at the earliest stages of limb development, indicating the type II collagen gene is being transcribed well before the onset of overt cartilage differentiation and the accumulation of detectable amounts of type II collagen. Furthermore, our results are consistent with other recent studies (1,9,31) indicating that type I collagen gene expression during chondrogenesis is regulated, at least in part, at the translational level, since substantial amounts of type I collagen mRNAs are present in the cytoplasm of well-differentiated chondrocytes which do not synthesize detectable amounts of type I collagen.…”

Section: T He Onset Of Cartilage Differentiation In the Developingsupporting

confidence: 82%

Collagen gene expression during limb cartilage differentiation.

Kosher¹,

Kulyk²,

Gay³

1986

The Journal of Cell Biology

227

128

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Abstract. As limb mesenchymal cells differentiate into chondrocytes, they initiate the synthesis of type II collagen and cease synthesizing type I collagen. Changes in the cytoplasmic levels of type I and type II collagen mRNAs during the course of limb chondrogenesis in vivo and in vitro were examined using cloned cDNA probes. A striking increase in cytoplasmic type II collagen m R N A occurs coincident with the crucial condensation stage of chondrogenesis in vitro, in which prechondrogenic mesenchymal cells become closely juxtaposed before depositing a cartilage matrix. Thereafter, a continuous and progressive increase in the accumulation of cytoplasmic type II coUagen m R N A occurs which parallels the progressive accumulation of cartilage matrix by ceils. The onset of overt chondrogenesis, however, does not involve activation of the transcription of the type II collagen gene. Low levels of type II collagen m R N A are present in the cytoplasm of prechondrogenic mesenchymal cells at the earliest stages of limb development, well before the accumulation of detectable levels of type II collagen. Type I collagen gene expression during chondrogenesis is regulated, at least in part, at the translational level. Type I collagen mRNAs are present in the cytoplasm of differentiated chondrocytes, which have ceased synthesizing detectable amounts of type I collagen. T HE onset of cartilage differentiation in the developinglimb is characterized by a transient cellular condensation or aggregation process in which prechondrogenic mesenchymal cells become closely juxtaposed to one another before initiating cartilage matrix deposition. During this process, a cell-cell interaction, cell shape change, or some other event occurs which is necessary to trigger the chondrogenic differentiation of the cells (15). The critical condensation process may be initiated, at least in part, by a progressive decrease in the accumulation of extracellular hyaluronate (20,35). Fibronectin and type I collagen have been implicated in the cell-cell interaction occurring during condensation (7, 21, 34). Prostaglandin-mediated elevations in cAMP levels during condensation are involved in regulating chondrogenesis (3, 6, 11, 14, 16-18, 32, 33). A change in the shape of the cells from a flattened mesenchymal morphology to a rounded configuration also plays an important role in the process (2, 41).Although considerable insight has been gained into the extracellular influences and intracellular regulatory molecules that are involved in regulating limb chondrogenesis, virtually nothing is known about the molecular mechanisms by which these factors directly influence the changes in gene activity that occur during cartilage differentiation. Prechondrogenic limb mesenchymal cells synthesize type I collagen (36). As the cells differentiate into chondrocytes, they initiate the synthesis of cartilage-characteristic type II collagen and cease synthesizing type I collagen (36). Thus the conversion of mesenchymal chondrogenic progenitor cells into chondrocytes i...

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“…Another fibrillar collagen found in both the corneal primary and secondary stromas, as well as in mesenchymal tissues in general, is type I. Collagen I is a heterotrimer, composed of two al(1) chains and an a2(I) chain. In hyaline cartilages, mRNAs for both of the collagen I a-chains are present, but type I collagen is not made (Focht and Adams, 1984;Saxe et al, 1985). One reason for this is that the 012(I) gene in cartilage is transcribed primarily from a downstream promotor located within the second intron (between exons 2 and 3; see Bennett and Adams, 1990;Beck et al, 1991).…”

Section: Molecular Isoformsmentioning

confidence: 99%

Analysis of transcriptional isoforms of collagen types IX, II, and I in the developing avian cornea by competitive polymerase chain reaction

Fitch

Gordon

Gibney

et al. 1995

Developmental Dynamics

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The genes for the al(IX), al(II), and a2(I) collagen chains can give rise to different isoforms of mRNA, generated by alternative promotor usage [for al(1X) and &?(I)] or alternative splicing [for cyl(II)]. In this study, we employed competitive reverse transcriptase PCR to quantitate the amounts of transcriptional isoforms for these genes in the embryonic avian cornea from its inception (about 3 1/2 days of development) to 11 days. In order to compare values at different time points, the results were normalized to those obtained for the "housekeeping" enzyme, glycerol-3-phosphate dehydrogenase (G3PDH). These values were compared to those obtained from other tissues (anterior optic cup and cartilage) that synthesize different combinations of the collagen isoforms. We found that, in the cornea, transcripts from the upstream promotor of al(1X) collagen (termed "long IX") were predominant at stage 18-20 (about 3 1/2 days), but then fell rapidly, and remained at a low level. By 5 days (just before stromal swelling) the major mRNA isoform of al(1X) was from the downstream promotor (termed "short IX"). The relative amount of transcript for the short form of type IX collagen rose to a peak at about 6 days of development, and then declined. Throughout this period, the predominant transcriptional isoform of the collagen type I1 gene was IIA (i.e., containing the alternatively spliced exon 2). This indicates that the molecules of type I1 collagen that are assembled into heterotypic fibrils with type I collagen possess, at least transiently, an amino-terminal globular domain similar to that found in collagen types I, 111, and V. For type I, the "bone/tendon" mRNA isoform of the cy2(I) collagen gene was predominant; transcripts from the downstream promotor were at basal levels. In other tissues expressing collagen types IX and 11, long IX was expressed predominantly with the IIA form in the anterior optic cup at stage 22/23; in 14 112 day cartilage, long IX was expressed predominantly along with the IIB form of al(1I). The downstream transcript of the a2(I) gene (Icart) was found at high levels only in cartilage.

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Changes in the Nuclear and Cytoplasmic Levels of Type I and Type II Collagen RNAs during Growth of Chondrocytes in 5‐Bromo‐2′‐Deoxyuridine

Cited by 11 publications

References 0 publications

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

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

Collagen gene expression during limb cartilage differentiation.

Analysis of transcriptional isoforms of collagen types IX, II, and I in the developing avian cornea by competitive polymerase chain reaction

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