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J P Pujol

Publications and source records attributed to J P Pujol.

At least 19 recordsLinked to original sources
PubMed2000

Semiquantitative reverse transcription-polymerase chain reaction analysis of syndecan-1 and -4 messages in cartilage and cultured chondrocytes from osteoarthritic joints.

OBJECTIVE: To determine the steady-state of messenger RNA (mRNA) levels of syndecan-1 and syndecan-4 in cartilage samples and chondrocytes derived from human osteoarthritic knee joints. METHODS: Steady-state levels of gene-specific mRNA (relative to beta-actin) were measured by semiquantitative polymerase chain reaction (PCR). RESULTS: RT-PCR allowed detection of syndecan-1 (for the first time) and syndecan-4 in both cartilage samples and articular chondrocytes cultured as primary monolayers. The mRNA levels of syndecan-1 were reduced in cartilage tissue from heavily damaged compared to normal-looking areas whereas those of syndecan-4 were significantly increased. In contrast, the expression of syndecan-1 was higher in cultured chondrocytes derived from the fibrillated osteoarthritic cartilage than in cells obtained from intact cartilage, while the syndecan-4 message levels did not differ between the two sites. CONCLUSION: The expression of the cell-surface syndecans 1 and 4 is altered during the osteoarthritic degradative process of the knee joint. The discoordinate syndecan gene expression, which is probably related to the chondrocyte proliferation and clustering, may contribute to the disorganization of the cartilage and the development of OA processes. Isolation and culturing the chondrocytes as monolayers dramatically change the expression of these genes and cannot reflect the in situ condition.

Actins↗
PubMed1999

Avocado/soya unsaponifiables enhance the expression of transforming growth factor beta1 and beta2 in cultured articular chondrocytes.

OBJECTIVE: Avocado and soya unsaponifiables (ASU) have been reported to exert beneficial effects in the treatment of periodontal and osteoarticular diseases. They are supposed to stimulate deposition and repair of extracellular matrix components, but the mechanisms underlying their action are not well understood. In view of the repair potential of osteoarthritic (OA) cartilage and the role that the transforming growth factor beta (TGFbeta) system could play in that process, we carried out in vitro studies to determine the mechanism of action of ASU on articular chondrocytes that may account for the beneficial effects on cartilage metabolism. METHODS: Cultured bovine articular chondrocytes were treated with various concentrations of ASU, and the expression of both TGFbeta isoforms, 1 and 2, and their receptors (TGFbetaRI and TGFbetaRII) was determined by Northern blot and reverse transcriptase-polymerase chain reaction. Cell transfection with TGFbeta1 promoter constructs was also used to delineate the cis-acting sequences mediating ASU responsiveness in chondrocytes. The level of plasminogen activator inhibitor 1 (PAI-1) was also evaluated by Northern blotting and protein radiolabeling. RESULTS: The data indicated that ASU stimulate the expression of TGFbeta1, TGFbeta2, and PAI-1 by articular chondrocytes. In contrast, the levels of TGFbetaRI and TGFbetaRII were not significantly affected by the compound. Treatment of bovine articular chondrocytes transiently transfected with TGFbeta1 promoter constructs suggested that the effect on TGFbeta1 expression is mediated by the region located between -732 and -1132 bp. CONCLUSION: The results indicate that the ASU-induced stimulation of matrix synthesis previously reported in cultured articular chondrocytes could be explained by the ability to enhance TGFbeta expression in these cells. Further, ASU increase the production of PAI-1, an effect that could help in blocking the plasmin cascade that leads to metalloprotease activation. These data suggest that the compound has properties that might promote TGFbeta-induced matrix repair mechanisms in articular cartilage.

5' Untranslated Regions↗
PubMed1999

Differential effects of interleukin-1 and transforming growth factor beta on the synthesis of small proteoglycans by rabbit articular chondrocytes cultured in alginate beads as compared to monolayers.

Small proteoglycans (PGs) are supposed to play great roles in the assembly of cartilage matrix but the influence of cytokines and growth factors on their synthesis by articular chondrocytes is largely unknown. We investigated whether IL-1 and TGFbeta1 influence the production of small leucine-rich proteoglycans by chondrocytes cultured in a three-dimensional gel, as compared to the common monolayer system. Rabbit articular chondrocytes were cultured in alginate beads for 14 days or as monolayers for 7 days. The effect of 2 ng/ml IL-1beta or TGFbeta1 during the last two days in culture was determined, after [35S]methionine labeling over the last 24 h. Cell-associated and further-removed matrix compartments were separated by centrifugation after sodium citrate/EDTA treatment of alginate beads whereas medium and cell-layer fractions were isolated from monolayer cultures. Total newly synthesized PGs were first isolated by anion-exchange chromatography and the small PGs were further separated from aggrecans by gel-filtration (Sepharose CL-4B) and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Addition of TGFbeta1 resulted in an overall rise in neosynthesized small PG content in both culture systems. However, TGFbeta1 significantly increased to the same extent the percentage of small PGs laid down in the cell-associated and the further-removed matrix compartments of the beads culture (+60%) whereas it augmented the content of small PGs in the medium (+40%) and reduced that of the cell fraction (-35%) in the monolayer culture. By adding IL-1, the amount of total newly synthesized small PGs was decreased in monolayers while it increased in alginate beads. IL-1 was also shown to change the relative distribution of these molecules in the monolayer system in contrast to the alginate beads culture where the proportions were not significantly altered. Electrophoretic analysis of the 35S-labeled small PGs-containing fractions confirmed these effects at the level of the 45-50 kDa-related core proteins. This study demonstrates that TGFbeta and IL-1 differently influence small PG synthesis of rabbit articular chondrocytes depending on whether they are cultured in alginate beads or in monolayers. Moreover, the regulation of small PG expression appears to be different from that of high-molecular weight aggrecans. As these small molecules are playing major roles in matrix assembly and growth factor regulation, the data may have great relevance to the pathogenesis of osteoarthritis and repair of articular cartilage lesions.

Alginates↗
PubMed1999

Stimulating effect of diacerein on TGF-beta1 and beta2 expression in articular chondrocytes cultured with and without interleukin-1.

OBJECTIVE: Diacetylrhein or diacerein has shown efficacy in the treatment of both major forms of osteoarthritis (OA), coxarthrosis as well as gonarthrosis, improving clinical symptoms of the disease (pain reduction and algo-functional index). Both in-vitro and animal models studies suggest that diacerein may have also disease-modifying effects. The drug exerts inhibitory effects on interleukin-1-induced expression of cartilage degrading enzymes. However, its mechanism of action is not completely understood. In view of the role that could play the transforming growth factor (TGF)-beta system in the repair potentialities of OA cartilage, we studied the effect of diacerein on the expression of TGF-beta isoforms 1, 2 and 3 and that of their receptor types I and II in cultured bovine chondrocytes. METHODS: Cultured bovine articular chondrocytes were treated with 10(-5) m diacerein, 10 ng/ml IL-1beta or the combination diacerein+interleukin (IL)-1, and the expression of both TGF-beta isoforms 1, 2 and 3 and that of their receptors TbetaR-I and TbetaR-II was determined by Northern-blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Cell transfections of cDNA constructs containing sequences of the 5'-upstream region of TGF-beta1 promoter were also performed to determine their transcriptional activity in diacerein-treated cultures. RESULTS: The data indicated that diacerein enhances the expression of TGF-beta1 and TGF-beta2. This effect was also found in the presence of IL-1, albeit with smaller intensity. In contrast, the levels of TGF-beta3 and receptors I and II remained unaffected or slighty modified by the compound. Treatment of cells transiently transfected with TGF-beta1 promoter constructs suggested that the stimulating effect on TGF-beta1 expression is mediated by the region -1038 to -1132 base pars. CONCLUSION: The results suggest that diacerein effects on matrix synthesis and turn-over previously reported in cultured articular chondrocytes might be explained in part by the ability of the drug to enhance TGF-beta1 and TGF-beta2 expression in these cells. This mechanism of action may account for the potential disease-modifying properties of diacerein and might give clues as to how future anti-osteoarthritic drugs should be designed.

Animals↗
PubMed1998

Cell-cycle-dependent expression of transforming growth factor beta type I receptor correlates with differential proliferative effects of TGFbeta1 in articular chondrocytes.

We previously found that transforming growth factor beta type 1 (TGFbeta1) had bifunctional effects on articular chondrocytes in culture depending on the proliferative state of the cells. Here, TGFbeta1 responses and the expression of TGFbeta receptors I and II were investigated as a function of growth state in rabbit articular chondrocytes (RAC) and Mv1Lu cells, a cell line which is growth inhibited by TGFbeta1. In contrast to these latter cells, in which DNA synthesis was decreased by TGFbeta1 independently of the cell cycle phases, exponentially growing RAC responded with a stimulation of DNA synthesis while confluent or quiescent cells were growth inhibited. Using synchronized RAC cultures, we showed that inhibitory responses were associated with the G0/G1 phase, whereas proliferative effects were S-phase dependent. Type I receptor mRNA level was severalfold greater in quiescent and slowly proliferating than in exponentially growing cells. In contrast, the expression of type II mRNA did not change. 125I-TGFbeta1 binding to RI in G0/G1-arrested cells was greater than in S-phase, suggesting a correlation with the growth-inhibitory effect of TGFbeta1. Transfection of an RI expression vector in exponentially growing RAC, which normally are growth stimulated by TGFbeta1, induced an inhibitory response, supporting the idea that this effect was due to increased RI expression. These results indicate that the ratio of type I to type II levels is cell cycle dependent and could lead to either negative or positive proliferative responses. In contrast, no influence on the TGFbeta1-induced stimulation of matrix gene transcriptional activity was seen, confirming that TGFbeta cell growth and matrix effects are controlled by separate pathways.

Activin Receptors, Type I↗
PubMed1998

Expression of decorin and biglycan by rabbit articular chondrocytes. Effects of cytokines and phenotypic modulation.

In this study, the levels of mRNAs coding for aggrecan, decorin and biglycan in rabbit articular chondrocytes were investigated, using both monolayer and 3D-alginate cultures treated with TGF-beta 1 and IL-1 beta. The cells were shown to express higher amounts of proteoglycan messages, specially the aggrecan, in gels than in monolayers. TGF-beta 1 increased aggrecan mRNA in both systems, whereas biglycan message was elevated only in alginate. It markedly decreased decorin expression in monolayer, either in primary or passaged cultures. In contrast, IL-1 beta had a weak inhibitory effect on both decorin and biglycan expression. Subculturing induced a dramatic decrease of aggrecan mRNA, while that of decorin augmented. Biglycan expression transiently increased after two passages, whereas it declined in further subcultures. Passaged chondrocytes transferred to alginate re-expressed high levels of aggrecan, decorin and biglycan. The data point to the influence of morphology, proliferative state and environment of the articular chondrocytes on their biosynthetic responses to cytokines. Although these immature cells do not fully reflect the adult chondrocytes present in the cartilage, this study may help in understanding the behaviour of these cells in osteoarticular diseases, where the surrounding extracellular matrix is profoundly altered.

Aggrecans↗
PubMed1997

Differential expression of membrane-anchored proteoglycans in rabbit articular chondrocytes cultured in monolayers and in alginate beads. Effect of transforming growth factor-beta 1.

Cell-surface proteoglycans (PGs) were extracted with Triton X-100 from rabbit articular chondrocytes cultured in monolayers and in alginate beads. They were first purified on DEAE-Trisacryl columns and the proportion of hydrophobic PGs was determined by both Octyl-Sepharose chromatography and partitioning in Triton X-114. These two methods revealed that the proportion of hydrophobic PGs was higher in monolayer culture system as compared to alginate beads (24 and 15%, respectively). Characterization of the PGs by Sepharose CL 6B gel filtration followed by electrophoresis indicated that the PGs isolated from monolayers were composed of three chondroitin sulfate (CS) PGs (core proteins of 180, 100 and 50 kDa) and a heparan sulfate (HS) PG (core protein of 60 kDa). In the alginate system. CSPGs with core proteins of 180, 45 and 32 kDa were observed, but no HSPG was present. In parallel, the effect of TGF-beta on the distribution of membrane-associated PGs was studied. The results showed that the synthesis of cell-surface PGs was stimulated by TGF-beta in monolayers whereas it was inhibited in alginate beads, but the amount of hydrophobic PGs was not altered by the growth factor. These data clearly indicate that TGF-beta induces a differential expression of the PG families present at the cell surface. Taken together, the results reveal the complex regulation of cell-surface PG distribution, which obviously depends on the culture method used and suggest that rabbit articular chondrocytes may differentially respond to extracellular ligands according to their morphological state and environment.

Alginates↗
PubMed1997

Tumour necrosis factor-alpha selectivity modulates expression of collagen genes in rat granulation tissue.

OBJECTIVE: To study the effects of tumour necrosis factor-alpha (TNF-alpha) on granulation tissue in rats. DESIGN: Animal experiment. SETTING: University Hospital, Finland. MATERIAL: 48 rats in 6 groups. INTERVENTIONS: The test implants were treated with either a single or daily injections of 200 ng of TNF-alpha. The samples were collected 4 and/or 7 days postoperatively. MAIN OUTCOME MEASURES: Pro alpha 1 (I) and pro alpha 1 (III) collagen mRNA concentrations, hydroxyproline, nitrogen, uronic acids, and hexosamine content of the sponge; and ingrowth of new granulation tissue into the sponge. RESULTS: A single application of TNF-alpha resulted in no significant differences between the groups. After daily applications of TNF-alpha the accumulation of collagen hydroxyproline and volume of ingrowth of granulation tissue were significantly lower than in the controls. After 7 days there were significant reductions in the concentrations of RNA-ribose, nitrogen, and uronic acids. Daily injections of TNF-alpha reduced pro alpha 1 (I) collagen mRNA concentrations but those of pro alpha 1 (III) collagen mRNA levels were not lowered to the same extent. CONCLUSION: Repeated application of TNF-alpha was required to reduce the synthesis of collagen. This down-regulation was selective as pro alpha 1 (I) collagen mRNA concentrations were lowered more than those of pro alpha 1 (III) collagen mRNA.

Animals↗
PubMed1996

An inositolphosphate glycan released by TGF-beta mimics the proliferative but not the transcriptional effects of the factor and requires functional receptors.

Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional polypeptide that regulates a number of cellular processes including cell growth and deposition of extracellular matrix protein. Despite the fact that the signal transduction by TGF-beta has been intensively studied, the molecular mechanisms of that pathway are not clear. We have studied the possibility that an inositolphosphate glycan (IPG) is involved in transmission of the TGF-beta 1 signal. We show that TGF-beta 1 induces IPG release in both rabbit articular chondrocytes (RAC), which are growth stimulated by the factor and Mv1Lu cell line, which is growth inhibited. This release requires functional TGF-beta heteromeric receptors in these two cell types. We also demonstrate that IPG mimics TGF-beta 1-induced growth stimulation in mesenchymal cells (+100%) and growth inhibition in epithelial cells (-80%). Moreover TGF-beta receptor I (T beta R-I) is not required for inhibition of proliferation induced by IPG since derivated mutants of the Mv1Lu cell line lacking T beta R-I intracellular domain (R-1B) are significantly inhibited (-65%). Additionally, we show that IPG does not take part in the signalling pathway that leads to activation of matrix gene transcription. These results suggest that TGF-beta effects on growth regulation and extracellular matrix synthesis implicate two different signalling pathways, IPG being only involved in growth regulation.

Animals↗
PubMed1995

Modulation of interleukin-1 receptor expression by transforming growth factor-beta in cultured rabbit articular chondrocytes: analysis by reverse transcription-polymerase chain reaction.

Interleukin-1 receptor type I (IL-1RI) expression in cultured rabbit articular chondrocytes (RAC) was studied by reverse transcription-polymerase chain reaction (RT-PCR). A cDNA probe specific for the rabbit IL-1RI gene was constructed using primers derived from the sequence data of the human, murine and chick receptors. Transforming growth factor-beta 1 (TGF beta-1) was shown to transiently increase the level of expected 900-bp PCR product at 1 h of incubation and decrease the expression at 48 and 72 h with no effect at 24 h. In receptor binding assays using [125I]-IL-1 alpha, TGF beta decreased IL-1R bioactivity at all time points. These results suggest that TGF beta-induced down-regulation of IL-1 RI could be responsible for its ability to antagonize the effect of IL-1 and that TGF beta may have a role in the repair of articular cartilage.

Animals↗
PubMed1995

Modulation of rabbit articular chondrocyte (RAC) proliferation by TGF-beta isoforms.

We have previously shown that TGF-beta 1 exerts a bifunctional effect on RAC proliferation. Added to quiescent cultures, it inhibits the entry of G0/G1 cells into S phase whereas in S phase synchronized populations, it stimulates the DNA replication rate with a delayed G2 + M phase and a subsequent transient increase of cell number. As TGF-beta 2 and beta 3 isoforms are also expressed in bone and cartilage tissues, it was of interest to study their effect on RAC proliferation, in comparison to that of TGF-beta 1. Using cell counting and tritiated thymidine incorporation, we found that all the TGF-beta s used here induced an increase of RAC proliferation rate occurring between 24 and 48 h of exposure. TGF-beta 2 appeared as the most efficient form as judged from the maximum of thymidine labelling. However, TGF-beta 3 induced an increase of cell number slightly higher than both TGF-beta 1 and TGF-beta 2 (+30% versus 20% for TGF-beta 1 and beta 2). TGF-beta 2 and beta 3 were able to stimulate the DNA replication rate as previously demonstrated for TGF-beta 1. However, the effect occurred later for TGF-beta 2 and beta 3 (12 h) than for TGF-beta 1 (6 h). This was confirmed by flow cytometric analysis of DNA content. In addition, immunodetection by flow cytometry demonstrated that all TGF-beta isoforms enhanced endogenous expression of TGF-beta-related peptides. The effect was shown to be associated with the cell cycle S phase and was greater for TGF-beta 3 than for TGF-beta 1 and beta 2. These findings suggest that TGF-beta s could act on RAC functions via autocrine and paracrine ways. Taken together, these data indicate that TGF-beta s may modulate proliferation of articular chondrocytes and therefore could play a role in the activation of these cells in the early stages of osteoarthritis.

Animals↗
PubMed1994

Different phosphorylated forms of inositolphosphate glycan could be involved in the transforming growth factor-beta 1 (TGF-beta 1) signalling pathway.

Labelling with [3H]glucosamine was used to prepare a transforming growth factor-beta 1 (TGF-beta 1)-sensitive glycosylphosphatidylinositol (GPI) from monolayer cultures of rabbit articular chondrocytes (RAC), which may be involved in control of the cell cycle. The polar headgroup of this glycosylphosphatidylinositol was generated by both phosphatidylinositol-specific phospholipase C (PI-PLC) and pronase E digestion. The molecule emerged in only one peak on a Dowex AG1-X8 chromatogram, eluted at 0.1 N ammonium formate. In contrast, similar experiments performed on cellular extract from cultures previously labelled with [3H]glucosamine displayed four radioactive peaks eluting at 0.1, 0.2, 0.5 and 1 N ammonium formate, respectively. Evidence that the eluting position of these peaks was dependent on the number of phosphate residues present in each fraction was demonstrated by both [32P]phosphorus labelling and change in the position of alkaline phosphatase-induced shift in elution volume. We also demonstrated that the GPI-derived inositolphosphate glycan (IPG) could be hyperphosphorylated into the cell under the action of a kinase whose activity was enhanced by TGF-beta 1 itself. We have also shown that all of these IPG forms could mimic the TGF-beta-induced increase of DNA replication rate of RAC, with a higher activity for peaks III and IV than peaks I and II.

Animals↗
PubMed1994

Transforming growth factor-beta (TGF-beta) and articular chondrocytes.

Transforming growth factor-beta (TGF-beta) has a dual effect on the proliferation of joint chondrocytes. In medium with a low serum concentration, it inhibits cell growth, while in medium supplemented with 10% fetal calf serum it stimulates cell growth. This stimulation leads to a higher replication rate an a larger number of cells in the G2/M phase of the cell cycle. Since these cells have already replicated their DNA, they can begin mitosis when stimulated by a EGF type factor. This mechanism involves the systems of the TGF-beta receptors which appear to vary with the cell cycle. In addition, a glycane inositophosphate may play a role as a second messenger for TGG-beta in this action. Finally, TGF-beta cannot restore the chondrocyte phenotype in dedifferentiated cells nor limit the dedifferentiation process. It exerts a opposing effect to the deleterious effects of interleukin-1 by inhibiting the expression of the receptors of this cytokine at the level of transcription. These in vitro effects would suggest that TGF-beta plays an important role in the repair potentiality of joint cartilage especially in arthrosis. In vivo studies are however necessary to verify this hypothesis.

Animals↗
PubMed1993

Transforming growth factor beta exerts opposite effects from interleukin-1 beta on cultured rabbit articular chondrocytes through reduction of interleukin-1 receptor expression.

OBJECTIVE: The aim of the study was to determine whether transforming growth factor beta (TGF beta) modulates the effects and the receptor expression of interleukin-1 (IL-1) in rabbit articular chondrocytes (RAC). METHODS: Collagen, glycosaminoglycan, and collagenase production, together with 125I-labeled IL-1 binding, were analyzed in RAC cultures. RESULTS: TGF beta reduced both IL-1 effects on matrix metabolism and IL-1 receptor expression. CONCLUSION: TGF beta acts as an antagonist of the effects of IL-1 through down-regulation of its receptor expression.

Animals↗