1,721,033 research outputs found
Transcriptional regulation by thyroid hormone of an mRNA homologous to a protease inhibitor.
No full textWe have previously cloned a cDNA of a rat liver mRNA, designated 4-12B, markedly induced by triiodothyronine (T3) at a pretranslational level [Magnuson, M.A., Dozin, B., & Nikodem, V.M. (1985) J. Biol. Chem. 260, 5906-5912]. Here we show that this hormonal effect is due in part to an increase of the rate of transcription of the 4-12B gene. In addition, the nucleotide sequence of 4-12B cDNA has been determined, revealing significant similarity with the sequences of the superfamily of serine protease inhibitors and a very high homology with contrapsin, a mouse serum trypsin inhibitor, at the level of nucleotide and amino acid sequence (77.9 and 66.8%, respectively). The optimized alignment of the putative reactive center region of 4-12B with four related members of this superfamily revealed that lysine-serine residues are located at the reactive site or adjacent to it, thus suggesting that the triiodothyronine-regulated rat 4-12B mRNA might code for a protease inhibitor with trypsin-like specificity. Although not enough data are presently available to assign definitively antitryptic activity to this protein, the high degree of similarity with members of the superfamily of serine protease inhibitors leaves no doubt that 4-12B is a member of this superfamily
Differentiation-Dependent activation of the extracellular fatty acid binding protein (ex-FABP) gene during chondrogenesis
No full textChicken hypertrophic chondrocytes secrete the extracellular fatty acid binding protein (Ex-FABP), a lipocalin not expressed by their undifferentiated precursors. Genomic clones coding for the full protein are here structurally and functionally analyzed. We first determined that the promoter sequence markedly differs from that reported for the homologous p20K, and we confirmed by genomic DNA Southern analysis the exactness of our sequence. This is of relevance since we have identified another lipocalin gene within the region of discrepancy, indicating thereby the existence of a lipocalin cluster within the same chromosomal locus. By transient transfections with 5'-deletions and the chloramphenicol acetyl transferase (CAT) reporter gene, the region between nt -926 and nt -629 was shown to be strongly active, specifically in hypertrophic chondrocytes and not in dedifferentiated cells
Response of young, aged and osteoarthritic human articular chondrocytes to inflammatory cytokines: molecular and cellular aspects
No full textThe aim of this study was to investigate the metabolic properties of human articular chondrocytes derived from young, aged and osteoarthritic subjects and their genetic adaptation to a catabolic challenge (i.e. the inflammatory cytokines interleukin-1alpha and tumor necrosis factor-alpha), in the absence or presence of diacerein, a drug potentially useful in osteoarthritis. Chondrocytes in primary culture were analyzed for newly secreted proteins, metalloproteinase synthesis and activity, and production of nitric oxide by-products. Results show that chondrocytes from normal but aged subjects present biochemical properties closer to osteoarthritic-derived cartilage than to normal young cartilage, as indicated by cell morphology, cell proliferation rate and pattern of protein secretion (in particular stromelysin-1 and interstitial collagenase). According to patient age and cartilage physiopathology, chondrocytes secrete increasing amounts of a protein identified by micro-sequencing as chitinase-like protein. Upon exposure to the inflammatory cytokines, chondrocytes, regardless the age or the status of the donor, significantly enhance their production of stromelysin-1, interstitial collagenase, interleukin-6 and interleukin-8. By contrast, the chitinase-like protein is not modulated by the cytokines. The pattern of protein secretion and metal loproteinase activity in chondrocytes from aged subjects appeared to be different from that of young patients, but was highly expressed in osteoarthritic chondrocytes. Diacerein, at therapeutically useful concentrations, consistently counteracts the stimulatory effect of cytokines on newly secreted proteins, metal loproteinase activity and nitric oxide production, whereas a selective nitric oxide blocker alone is ineffective. These data demonstrate that a specific gene program is turned on in cytokine-stimulated chondrocytes, which involves production of proteins engaged in remodeling and destruction of cartilage matrix. Part of these mechanisms appears to be operative also in unstimulated aged chondrocytes. Diacerein largely prevents the metabolic alterations caused by cytokine exposure in human chondrocytes, possibly through its ability to block early intracellular mediators after cytokine stimulation, such as oxygen radicals. (C) 2002 Elsevier Science B.V./International Society of Matrix Biology. Published by Elsevier Science B.V. All rights reserved
Changes in the expression of collagen genes show two stages in chondrocyte differentiation 'in vitro'.
No full textA computer-based technique for cell aggregation analysis: cell-condensation in "in vitro" chondrogenesis
No full textResponse of young, aged and osteoarthritic human articular chondrocytes to inflammatory cytokines: molecular and cellular aspects
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Type VI collagen expression is upregulated in the early events of chondrocyte differentiation.
No full textDedifferentiated 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)
Serum-free growth medium sustains commitment of human articular chondrocyte through maintenance of SOX9 expression.
No full textSPECIES VARIABILITY IN THE DIFFERENTATION POTENTIAL OF IN VITRO EXPANDED ARTICULAR CHONDROCYTES RESTRICT PREDICTIVE STUDIES ON CARTILAGE REPAIR USING ANIMAL MODELS
No full textAutologous chondrocyte implantation is currently applied in clinics as an innovative tool for articular cartilage repair. Animal models have been and still are being used to validate and further improve the technique. However, in various species, the outcome varies from hyaline-like cartilage to fibrocartilage. This may be due partly to the spontaneous dedifferentiation of chondrocytes once cultured in vitro. Here we assessed whether the extent of dedifferentiation varies between species and we hypothesized that the level of chondrocyte phenotype stability during expansion may contribute to the maintenance of their chondrogenic commitment and redifferentiation potential. Condyle chondrocytes were harvested from sheep, dog, and human, and expanded for 1, 6, or 12 cell duplications. At each interval, cell phenotype was monitored (morphology and biosynthesis of cartilage markers) and redifferentiation was assessed by an in vitro assay of chondrogenesis in micromass pellet and an in vivo assay of ectopic cartilage formation in immunodeficient mice. Results indicate that, during culture, the sheep chondrocyte phenotype is maintained better than that of human chondrocytes, which in turn dedifferentiate to a lesser extent than dog chondrocytes Accordingly, after expansion, sheep chondrocytes spontaneously reform hyaline-like cartilage; human chondrocytes redifferentiate only under stimulation with chondrogenic inducers whereas, after a few passages, dog chondrocytes lose any capacity to redifferentiate regardless of the presence of inducers. Thus, conditions allowing cartilage formation in one species are not necessarily transposable to other species. Therefore, results with animal models should be cautiously applied to humans. In addition, for tissue-engineering purposes, the number of cell duplications must be, for each species, carefully monitored to remain in the range of amplification allowing redifferentiation and chondrogenesis
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