Biomedical subjects

B Dozin

Publications and source records attributed to B Dozin.

At least 19 recordsLinked to original sources

PubMed2005

Autologous chondrocyte implantation (ACI) for aged patients: development of the proper cell expansion conditions for possible therapeutic applications.

OBJECTIVE: Proliferation and chondrogenic commitment of cultured articular chondrocytes are impaired when cells derive from aged donors. In those subjects the feasibility of cell-based therapies for articular surface repair is reduced. Moreover, the use of serum as medium supplement elicits non-physiological responses in cultured chondrocytes. This study was therefore undertaken to identify the expansion culture conditions needed to sustain growth and chondrogenic commitment of chondrocytes harvested from aged human subjects. DESIGN: Articular cartilage was obtained from aged (69-75 years) and from young adult subjects (27-35 years). Chondrocytes were isolated and cultured in serum-free (SF) or in serum-supplemented [fetal calf serum (FCS)] conditions. Chondrocytes were expanded in monolayer for five duplications and processed for RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The differentiation potential was assessed by micromass pellet cultures before and after expansion in either culture medium, or after a prolonged exposure to serum followed by a period in SF condition. RESULTS: Only SF-cultured chondrocytes reached five duplications within 25-35 days, maintaining the expression of some chondrogenic markers and without altering the levels of active matrix metalloproteinase 3 (MMP-3). Only the pellets derived from SF-expanded cultures positively stained for cartilage matrix deposition. On the contrary, exposure to serum diminished the proliferation capacities, abolished the differentiation potential in the same cells and elicited transcription of the MMP-3 gene. Shifting culture conditions from FCS to SF resumed growth rates but proper extracellular matrix deposition was only partially restored. CONCLUSIONS: The SF conditions have proven valuable to prime cell proliferation and to sustain proper commitment in chondrocytes from aged patients. This culturing approach may represent a therapeutic chance extendable to a range of patients normally excluded from clinical protocols based on autologous chondrocyte implantation (ACI).

Adult↗

PubMed2001

Extracellular fatty acid binding protein (ex-FABP) is a stress protein expressed during chondrocyte and myoblast differentiation.

OBJECTIVE: We have isolated and characterized in our laboratory a lipocalin specifically binding unsaturated long chain fatty acids (Ex-FABP). In developing chicken embryo long bones, Ex-FABP first appears at the boundary of the cone of hypertrophic cartilage. 'In vitro' EX-FABP is highly expressed by differentiating hypertrophic chondrocytes. Ex-FABP is expressed also in the forming myotubes both 'in vivo' and 'in vitro'. In cultured chondrocytes, Ex-FABP expression is strongly induced by treatment with inflammatory agents such as the bacterial liposaccharide LPS or interleukin-6. The possible mechanism for this induction was investigated. Expression of Ex-FABP was studied in other stress conditions. DESIGN: To investigate a possible mechanism for Ex-FABP induction by LPS or interleukin-6, we have cultured the cells in the presence of either hydrogen peroxide or the NO donor SNAP (S-nitrosil-acetil-D, L-penicillamine), two agents known to produce cellular stresses through the activation of specific signalling pathways. To investigate Ex-FABP expression in other stress conditions, chondrocytes were cultured for 3 days in the presence of alpha,alpha-dipyridyl, an agent inhibiting prolyl hydroxylase activity and collagen secretion. Supplement of this agent to the culture medium results in an impairment of collagen secretion and assembly and the consequent altered interaction of the cell with the surrounding extracellular matrix. In addition Ex-FABP expression was studied also in chondrocytes cultured in the absence of serum, a stress condition activating cell defence mechanisms. RESULTS: We have excluded that induction of Ex-FABP expression by inflammatory agents is mediated by oxidative stress or NO production. Ex-FABP expression was induced also by changes in the hypertrophic chondrocyte microenvironment, considered either as extracellular matrix surrounding the cell in culture or as nature and concentration of growth factor in the culture medium. CONCLUSIONS: No definitive data are so far available on the possible role of Ex-FABP when induced by cellular stresses. The capacity of the protein to specifically bind and transport unsaturated long chain fatty acids suggests that lipid metabolism and fatty acid utilization by the cells may be involved. Based on literature data the NRL/N-GAL (neu-related lipocalin/neutrophil gelatinase-associated lipocalin) protein was proposed as a possible mammal counterpart of the chick Ex-FABP. We have suggested that Ex-FABP and NRL/NGAL expression in forming bones and muscles is part of a 'physiological' acute phase response. Interestingly the expression of Ex-FABP and NRL/NGAL is also activated in osteoarthritic cartilage and in the case of NRL/N-GAL during neoplastic transformation of chondrogenic lineage cells.

2,2'-Dipyridyl↗

PubMed2000

Ex-FABP: a fatty acid binding lipocalin developmentally regulated in chicken endochondral bone formation and myogenesis.

Extracellular fatty acid binding protein (Ex-FABP) is a 21 kDa lipocalin specifically binding fatty acids, expressed during chicken embryo development in hypertrophic cartilage, in muscle fibers and in blood granulocytes. In chondrocyte and myoblast cultures Ex-FABP expression is increased by inflammatory agents and repressed by anti-inflammatory agents. In adult cartilage Ex-FABP is expressed only in pathological conditions such as in dyschondroplastic and osteoarthritic chickens. The possible mammalian counterpart is the Neu-related lipocalin (NRL), a lipocalin overexpressed in rat mammary cancer; NRL is homologous to the human neutrophil gelatinase associated lipocalin (NGAL) expressed in granulocytes and in epithelial cells in inflammation and malignancy and to the Sip24 (super-inducible protein 24), an acute phase lipocalin expressed in mouse after turpentine injection. Immunolocalization and in situ hybridization showed that NRL/NGAL is expressed in hypertrophic cartilage, in forming skeletal muscle fibers and in developing heart. In adult cartilage NRL/NGAL was expressed in articular cartilage from osteoarthritic patients and in chondrosarcoma. Moreover, NRL was induced in chondrocyte and myoblast cultures by an inflammatory agent. We propose that these lipocalins (Ex-FABP, NRL/NGAL, Sip24) represent stress proteins physiologically expressed in tissues where active remodeling is taking place during development and also present in tissues characterized by an acute phase response due to pathological conditions.

Acute-Phase Reaction↗

PubMed2000

Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy.

Bone marrow stromal cells (BMSC) are an attractive target for novel strategies in the gene/cell therapy of hematologic and skeletal pathologies, involving BMSC in vitro expansion/transfection and reinfusion. We investigated the effects of in vitro expansion on BMSC pluripotentiality, proliferative ability, and bone-forming efficiency in vivo. BMSC from three marrow donors were cultured to determine their growth kinetics. At each passage, their differentiation potential was verified by culture in inductive media and staining with alizarin red, alcian blue, or Sudan black, and by immunostaining for osteocalcin or collagen II. First passage cells were compared to fresh marrow for their bone-forming efficiency in vivo. Stromal cell clones were isolated from five donors and characterized for their multidifferentiation ability. The lifespan and differentiation kinetics of five of these clones were determined. After the first passage, BMSC had a markedly diminish proliferation rate and gradually lost their multiple differentiation potential. Their bone-forming efficiency in vivo was reduced by about 36 times at first confluence as compared to fresh bone marrow. Experiments on the clones yielded comparable results. Culture expansion causes BMSC to gradually lose their early progenitor properties. Both the duration and the conditions of culture could be crucial to successful clinical use of these cells and must be considered when designing novel therapeutic strategies involving stromal mesenchymal progenitor manipulation and reinfusion.

Adult↗

PubMed2000

Developmental control of chondrogenesis and osteogenesis.

During vertebrate embryogenesis, bones of the vertebral column, pelvis, and upper and lower limbs, are formed on an initial cartilaginous model. This process, called endochondral ossification, is characterized by a precise series of events such as aggregation and differentiation of mesenchymal cells, and proliferation, hypertrophy and death of chondrocytes. Bone formation initiates in the collar surrounding the hypertrophic cartilage core that is eventually invaded by blood vessels and replaced by bone tissue and bone marrow. Over the last years we have extensively investigated cellular and molecular events leading to cartilage and bone formation. This has been partially accomplished by using a cell culture model developed in our laboratory. In several cases observations have been confirmed or directly made in the developing embryonic bone of normal and genetically modified chick and mouse embryos. In this article we will review our work in this field.

Animals↗

PubMed1998

A divergent role of COOH-terminal domains in Nurr1 and Nur77 transactivation.

Orphan nuclear receptors such as Nurr1 and Nur77 have conserved amino acid sequences in the zinc finger DNA binding domains and similar COOH-terminal regions, but have no known ligands. These receptors can bind DNA sequences (response elements) as monomers and can also heterodimerize with the retinoid X receptor to activate transcription. We report here the identification and initial characterization of a novel COOH-terminal truncated isoform of Nurr1, Nurr1a. Internal splicing of Nurr1 generates a frameshift such that a stop codon is prematurely encoded resulting in a naturally occurring COOH-terminal truncation. Embryonic and postnatal mouse brain showed both Nurr1 and Nurr1a mRNAs expressed during development. To characterize essential COOH-terminal elements that may be deleted from Nurr1a and determine function in putative ligand binding, we created COOH-terminal deletion mutants. Nurr1, Nur77, and 3'-truncated mutants bind in gel mobility shift assays to the monomeric Nur77 response element (B1A-RE). However, in transient transfection assays, a truncation of as little as 15 Nurr1 COOH-terminal amino acids diminished transcriptional activation of B1A-thymidine kinase-chloramphenicol acetyltransferase reporter. This result was not seen for a similar Nur77 deletion mutant, Nur77-586. Unlike full-length Nurr1 and Nur77, transactivation by Nur77-586 was not augmented in response to the presence of retinoid-like receptor and 9-cis-retinoic acid. Thus, the interaction of putative ligand binding and transactivation for Nurr1 and Nur77 may function differently.

3T3 Cells↗

PubMed1997

Computer-based technique for cell aggregation analysis and cell aggregation in in vitro chondrogenesis.

No quantitative methods are currently available to measure different aggregation parameters in cell cultures. In this paper we describe a computer-based technique for the automatic and reliable analysis of cellular aggregates, starting from optical microscopy images of living cells grown in suspension. The method allows determination, on the same sample at different time intervals, of quantitative parameters, including aggregation percentage, average number of cells in aggregates, and aggregate size statistical distribution. To determine the number of cells in an aggregate starting from its two-dimensional microscopic profile, a model has been proposed and verified, using sphere packing theory. Algorithms have been tested on chondrocyte suspension cultures, where cell aggregation is a very early and critical event leading to cell differentiation. Using this technique for the analysis of chick embryo chondrocyte cultures, we observed that aggregate size and development kinetics depend on the culture conditions used. The method, with minor adaptations, is of potential use also in other cell systems to evaluate aggregation indexes or to study aggregation kinetics.

Animals↗

PubMed1997

Modulation of commitment, proliferation, and differentiation of chondrogenic cells in defined culture medium.

The factors regulating the growth and development of mesenchymal precursor cells toward chondrogenesis are not well identified. We have developed a defined serum-free culture system that allows the proliferation of chick embryo chondrogenic cells and their maturation toward hypertrophic chondrocytes. Proliferation is obtained in adhesion in medium supplemented with insulin (Ins), Dexamethasone (Dex), and either basic fibroblast growth factor (FGF-2), platelet-derived growth factor bb, epithelial growth factor, or GH; the highest mitogenic response is induced by FGF-2 in synergy with Ins. Ins can be substituted by Ins-like growth factor I. When these cells are transferred into suspension culture in Ins/Dex and T3 without growth factor supplement, they undergo the complete chondrogenic development characterized by type X collagen synthesis and cellular hypertrophy. During differentiation, Ins cannot be substituted by Ins-like growth factor I. Chondrogenesis is also evidenced by the formation of hypertrophic cartilage when the medium is supplemented with ascorbic acid. If T3 is introduced in the proliferation phase, the cells fail to differentiate to hypertrophy in suspension unless bone morphogenetic protein-2 is added. Assays of ectopic tissue formation in nude mice, with cells implanted sc after adsorption on collagen sponge or porous hydroxyapatite ceramics, indicate that cells grown in Ins/FGF-2 reform mainly cartilage in vivo, whereas expansion in Ins/T3/Dex/FGF-2 leads to the formation of cartilage, bone, and adipose tissue.

Adipocytes↗

PubMed1997

Identification of a nuclear protein from rat developing brain as heterodimerization partner with thyroid hormone receptor-beta.

Thyroid hormone receptors (TR) are ligand-activated transcription factors that modulate the expression of certain target genes in a developmental and tissue-specific manner. These specificities are determined by the tissue distribution of the TR isoforms alpha1 and beta1, the structure of the thyroid hormone response element (TRE) bound by the receptor, and heterodimerization partners. Among these, retinoid X receptors (RXR) have been recognized as the principal partners for TR. The present work reports the identification of a novel nuclear protein from 19-day-old embryonic rat brain that displays a distinct interaction pattern with TR isoforms at the level of the TRE of two genes known to be differentially expressed and regulated by thyroid hormone (T3): the ubiquitous malic enzyme and the brain-specific myelin basic protein. Electrophoretic gel mobility shift assays demonstrate that only TRbeta1 forms a specific complex with the rat brain nuclear factor on the myelin basic protein-TRE, but not on the malic enzyme-TRE. Thus, the interaction is selectively determined by both the receptor isoform and the structure of the TRE. The expression of this brain nuclear factor is restricted to the perinatal period, when myelination is sensitive to T3. Gel supershift assays with RXR-specific antibodies indicate that this factor is not one of the known RXR isoforms. However, it is most likely a new member of the RXR subfamily because it could be supershifted with an antibody raised against the highly conserved DNA-binding domain of RXRs.

Animals↗

PubMed1995

Expression of the cartilage matrix protein gene at different chondrocyte developmental stages.

Cartilage matrix protein (CMP), a major noncollagenous component of certain types of hyaline cartilage, is synthesized by chondrocytes in a developmentally regulated manner. In this study, we monitored the accumulation of CMP in the developing chicken limb and sternum by immunostaining. In older embryos, the specific extracellular staining was restricted to the resting/proliferative zone of metaphyseal cartilage and to the immediately adjacent hypertrophic cartilage. A lack of staining was observed in the peripheral layers of articular cartilage. Data were compared with the accumulation of CMP mRNA measured by Northern analysis relative to other cartilage-specific messages in cell cultures representing different stages of chondrocyte differentiation, as well as with the steady state mRNA levels in tissue samples. We found a correlation between the gene expression pattern of the in vitro cultures and the one observed in certain in vivo differentiation stages. The high-density mesenchyme culture was utilized as a model for studying the events at early stage I (stage Ia) of chondrogenesis. This culture was characterized by relatively low steady state mRNA levels for cartilage proteins, including the later activation of the CMP gene as compared to type II collagen or link protein genes, and relatively high steady state mRNA levels for type VI collagen and beta-actin. Chicken embryo chondrocyte cultures obtained from sterna of 14-day-old embryos, however, consisted predominantly of stage Ib chondrocytes, and showed high steady state levels for cartilage proteins, but relatively lower levels for type VI collagen and beta-actin mRNAs. In accordance with the in vivo data, a relatively high steady state level was detected for CMP mRNA in cultures of hypertrophic (stage II) chondrocytes. We also performed transient expression assays in the various culture systems to study the role of the promoter upstream and intronic control regions in the tissue- and developmental stage-specific regulation of the CMP gene. We showed that the enhancer worked in a lineage-specific manner, by further stimulating the minimal promoter activity independent of the developmental stage of chondrocytes, while it did not in other tissues. The promoter upstream control regions, however, seemed to play a role in restricting the promoter activity to a certain chondrocyte developmental stage.

Animals↗

PubMed1993

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

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)

Animals↗

PubMed1992

Expression, regulation, and tissue distribution of the Ch21 protein during chicken embryogenesis.

The Ch21 protein is one of the marker proteins whose synthesis and secretion by differentiating tibia chondrocytes is progressively increased during chicken embryogenesis (Descalzi-Cancedda, F., Manduca, P., Tacchetti, C., Fossa, P., Quarto, R. and Cancedda, R. (1988) J. Cell Biol. 107, 2455-2463). We report the corresponding full-length cDNA sequence and the complete primary structure of the protein, which contains a 20-residue signal peptide. The synthesis of the protein is directed by a 1450-base mRNA characterized by an unusually long 5'-untranslated leader sequence and is regulated only at the transcriptional level as shown by run-off transcription assays. During differentiation, maximal expression of the protein characterizes stage II hypertrophic chondrocytes. In situ hybridization on embryo sections reveals that the protein is expressed by several structures derived from the chondrogenic lineage and that, in addition, it is a major translational product in granulocytes. High cell density largely influences the expression of the Ch21 protein in chondrocyte cultures. When embryonic avian cells of different origin are grown to confluency, the expression of the Ch21 protein is observed in only some of the cell lines. Thus quiescence per se is not the primary factor determining the expression of the protein.

Amino Acid Sequence↗

PubMed1992

Constitutive myc expression impairs hypertrophy and calcification in cartilage.

The myc oncogene is expressed by proliferating quail embryo chondrocytes (QEC) grown as adherent cells and is repressed in QEC maintained in suspension culture. To investigate the interference of myc expression during chondrocyte differentiation, QEC were infected with a retrovirus carrying the v-myc oncogene (QEC-v-myc). Uninfected or helper virus-infected QEC were used as control. In adherent culture, QEC-v-myc displayed a chondrocytic phenotype and synthesized type II collagen and Ch21 protein, while control chondrocytes synthesized type I and type II collagen with no Ch21 protein detected as long as the attachment to the plastic was kept. In suspension culture, QEC-v-myc readily aggregated and within 1 week the cell aggregates released small single cells; still they secreted only type II collagen and Ch21 protein. In the same conditions control cell aggregates released hypertrophic chondrocytes producing type II and type X collagens and Ch21 protein. In the appropriate culture conditions, QEC-v-myc reconstituted a tissue defined as nonhypertrophic, noncalcifying cartilage by the high cellularity, the low levels of alkaline phosphatase enzymatic activity, and the absence of type X collagen synthesis and of calcium deposition. We conclude that the constitutive expression of the v-myc oncogene keeps chondrocytes in stage I (active proliferation and synthesis of type II collagen) and prevents these cells from reconstituting hypertrophic calcifying cartilage.

Animals↗

PubMed1992

Cell condensation in chondrogenic differentiation.

Reduction of intercellular spaces in the areas of prospective cartilage and bone formation (precartilage condensation) precedes chondrogenesis and may represent an important step in the process of cartilage differentiation during limb skeletogenesis. We have attempted to clarify the role of the microenvironment established during cell condensation, taking advantage of a tissue culture model system that allows condensation (i.e., increased cell density due to cell aggregation) and chondrogenic differentiation (i.e., synthesis of cartilage-specific extracellular matrix proteins, such as type II collagen and acquisition of a chondrocyte morphology) of chick embryo cartilage-derived undifferentiated cells. To prevent condensation cells were grown in carboxymethylcellulose and changes in the differentiation pathway were evaluated. In another series of experiments, we have separated single cells from the aggregated cells and analyzed their differentiation properties. Morphological analyses and the evaluation of type II collagen expression, at both the protein and the mRNA level, show that a reduced rate of cell clustering and cell to cell contact parallels a reduction of cell recruitment into the differentiation program. On the basis of our results, we suggest that the following cascade of events regulates the early stages of chondrocyte differentiation: (a) the acquisition of the ability to establish cell to cell contacts, (b) the formation of a permissive environment capable of activating the differentiation program, and (c) the expression of differentiation markers.

Animals↗

PubMed1992

Thyroid hormone, insulin, and glucocorticoids are sufficient to support chondrocyte differentiation to hypertrophy: a serum-free analysis.

Chondrocytes from chicken embryo tibia can be maintained in culture as adherent cells in Coon's modified Ham's F-12 medium supplemented with 10% FCS. In this condition, they dedifferentiate, losing type II collagen expression in favor of type I collagen synthesis. Their differentiation to hypertrophy can be obtained by transferring them to suspension culture. Differentiation is evidenced by the shift from type I to type II and type IX collagen synthesis and the following predominant expression of type X collagen, all markers of specific stages of the differentiation process. To identify the factors required for differentiation, we developed a serum-free culture system where only the addition of triiodothyronine (T3; 10(-11) M), insulin (60 ng/ml), and dexamethasone (10(-9) M) to the F-12 medium was sufficient to obtain hypertrophic chondrocytes. In this hormonal context, chondrocytes display the same changes in the pattern of protein synthesis as described above. For proper and complete cell maturation, T3 and insulin concentrations cannot be modified. Insulin cannot be substituted by insulin-like growth factor-I, but dexamethasone concentration can be decreased to 10(-12) M without chondrogenesis being impaired. In the latter case, the expression of type X collagen and its mRNA are inversely proportional to dexamethasone concentration. When ascorbic acid is added to the hormone-supplemented medium, differentiating chondrocytes organize their matrix leading to a cartilage-like structure with hypertrophic chondrocytes embedded in lacunae. However, this structure does not present detectable calcification, at variance with control cultures maintained in FCS. Accordingly, in the presence of the hormone mixture, the differentiating chondrocytes have low levels of alkaline phosphatase activity. This report indicates that T3 and insulin are primary factors involved in the onset and progression of chondrogenesis, while dexamethasone supports cell viability and modulates some differentiated functions.

Alkaline Phosphatase↗

PubMed1992

In vitro differentiation of mouse embryo chondrocytes: requirement for ascorbic acid.

Chondrocytes enzymatically dissociated from 13-day-old mouse embryo tibia grow in monolayer culture with a fibroblast-like phenotype and express high levels of type I collagen. Chondrogenesis can be induced by transferring the adherent cells in suspension culture and maintaining them in the constant presence of mouse embryo extract. Round shaping of the cells and formation of multicellular aggregates rapidly follow the passage in anchorage-independent conditions. Cell differentiation is evidenced by a marked decrease in the level of type I collagen and by the induction of type II collagen which accumulates when ascorbic acid is included in the culture medium. The addition of the vitamin also triggers the aggregated chondrocytes to organize their extracellular matrix giving rise to a structure closely resembling the in vivo developing cartilage.

Animals↗

PubMed1990

The Ch21 protein, developmentally regulated in chick embryo, belongs to the superfamily of lipophilic molecule carrier proteins.

Ch21, a developmentally regulated low molecular weight protein observed in chick embryo skeletal tissues, is expressed "in vitro" by differentiating chondrocytes at a late stage of development. Here we report the complete amino acid sequence of the protein. 86% of the total amino acid sequence was deduced by sequences of 17 high performance liquid chromatography-separated proteolytic fragments and 33 amino acid residues at the amino-terminal end of protein purified from spent culture medium of hypertrophic chondrocytes. Furthermore we isolated by molecular cloning the corresponding cDNA and determined its nucleotide sequence. By combining protein and nucleotide sequence data we determined the primary structure of the entire Ch21. It consists of 158 amino acids and has a molecular mass of 18.065 kDa. Computer-assisted analysis showed that the Ch21 belongs to the superfamily of low molecular weight proteins sharing a basic framework for binding and transport of small hydrophobic molecules.

Amino Acid Sequence↗

PubMed1990

Stabilization of the mRNA follows transcriptional activation of type II collagen gene in differentiating chicken chondrocyte.

The differentiation of chicken endochondral chondrocyte can be induced in vitro by transferring to suspension culture dedifferentiated chondrocytes grown as adherent cells. During the differentiation process, the synthesis of type I collagen is repressed while the genes encoding type II, IX, and X collagens are gradually activated. The changes in the steady-state levels of the mRNAs for type I, IX, and X collagens were shown to be fully accounted for by proportional modifications in the rates of specific gene transcription (Castagnola, P., Dozin, B., Moro, G., and Cancedda, R. (1988) J. Cell Biol. 106, 461-467). In the present report, we demonstrate that the cellular accumulation of type II collagen mRNA in differentiating chondrocyte is regulated not only at the transcription level but also through a stabilization of the RNA transcript as shown by the enhanced half-life of the mRNA in differentiated cells treated with actinomycin D. We also give evidence that this additional mechanism: 1) is specific for type II collagen, 2) does not occur at an early stage of differentiation when type II collagen mRNA starts accumulating, 3) affects only the cytoplasmic form of the RNA transcript.

Animals↗