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Rheumatology Advance Access originally published online on January 31, 2008
Rheumatology 2008 47(3):281-288; doi:10.1093/rheumatology/kem323
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

17β-Oestradiol up-regulates the expression of a functional UDP-glucose dehydrogenase in articular chondrocytes: comparison with effects of cytokines and growth factors

L. Maneix1, G. Beauchef1, A. Servent1, Y. Wegrowski2, F. X. Maquart2, N. Boujrad3, G. Flouriot3, J. P. Pujol1, K. Boumediene1, P. Galéra1 and S. Moslemi1

1Laboratory of Extracellular Matrix and Pathology, Faculty of Medicine, IFR 146 ICORE, University of Caener-Low Normandy, Caen, 2Laboratory of Medical Biochemistry and Molecular Biology, CNRS UMR 6198, Faculty of Medicine, University of Reims, Reims and 3Laboratory of Molecular Endocrinology of the Reproduction, CNRS UMR 6026, University of Rennes I, Rennes, France.

Correspondence to: S. Moslemi, Laboratory of Extracellular Matrix and Pathology, Faculty of Medicine, University of Caen-Low Normandy, 14032 Caen Cedex, France. E-mail: safa.moslemi{at}unicaen.fr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. To investigate the mechanisms by which cytokines and 17β-oestradiol (17β-E2) modulate gene expression and activity of uridine diphosphoglucose dehydrogenase (UGDH), a key enzyme of GAG synthesis in articular chondrocytes.

Methods. Rabbit articular chondrocytes (RAC) from 3-week-old animals were incubated for 24 h with TGF-β, insulin like growth factor-I (IGF-I), IL-1β, IL-6 and 17β-E2. GAG synthesis was measured by [35S]-sulphate labelling and the expression of the UGDH gene was estimated by both real-time polymerase chain reaction and western blotting, whereas the enzyme activity was assayed by a spectrophotometric procedure. In addition, the transcriptional activity of several UGDH gene promoter constructs was determined in RAC transiently transfected with wild-type or deleted human oestrogen receptor-{alpha} gene (hER{alpha}66 or hER{alpha}46, respectively).

Results. 17β-E2 and its receptor hER{alpha}66 enhanced GAG neosynthesis in rabbit articular chondrocytes, as did TGF-β1 whereas IL-1β decreased this synthesis. 17β-E2 was found to exert positive regulatory effects at mRNA, protein and UGDH activity levels. In addition, the receptor hER{alpha}66, but not hER{alpha}46, increased the transcriptional activity of the UGDH gene. In contrast, no clear correlation between transcription, translation and activity of the UGDH was found under the effects of the cytokines studied. However, TGF-β enhanced the enzyme activity, whereas IL-1β, IL-6 and IGF-I were without significant effect.

Conclusions. 17β-E2 enhanced GAG synthesis in chondrocytes via up-regulation of the UGDH gene expression and enzyme activity. These data provide insights into the molecular mechanisms involved in the regulation of the UGDH gene and offer new approaches to investigate its potential alteration in joint diseases.

KEY WORDS: UDP-glucose dehydrogenase, Glycosaminoglycan synthesis, 17β-oestradiol, Oestrogen receptor, Cytokines, Cartilage, Chondrocytes


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The chondrocyte, the unique cell type in mature articular cartilage, is considered as a terminally differentiated cell that maintains the normal cartilage-specific matrix phenotype. Aggrecans are high-molecular weight proteoglycans that confer visco-elasticity to the hyaline cartilage and are responsible for its mechanical properties. A great number of GAG chains (mainly chondroitin sulphate and keratan sulphate) are found in articular cartilage, usually in covalent linkage to proteoglycan core proteins. GAGs, the synthesis of which is modulated by cytokines [1, 2] and growth factors [3], are implicated in several physiological and pathological events such as signal transduction, cell proliferation, migration, cancer progression, metastasis and tumour angiogenesis [4, 5]. The role played by the GAGs in the homeostasis of articular cartilage is critical, as illustrated by the quantitative and qualitative alterations of their composition in joint diseases, including OA and RA, leading to the loss of the physico-mechanical properties of the tissue [6]. However, the mechanisms regulating their synthesis are not fully understood.

UDP-glucose dehydrogenase (UGDH, EC1.1.1.22) catalyses the oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronic acid (UDP-GlcUA), accompanied by the reduction of two molecules of NAD+. UDP-GlcUA is required for the detoxification through glucuronidation of a variety of xeno- and endobiotics including opioids, steroids or thyroid hormones and haem proteins in the liver [7]. In addition, UDP-GlcUA is also the precursor of GlcUA, a critical monosaccharide for the biosynthesis of GAGs such as hyaluronan and aggrecans, all considered as major components of extracellular matrix (ECM). Mutations in the sugarless gene, which encodes a Drosophila protein with homology to UGDH, has revealed that the level of intracellular UDP-GlcUA could influence GAG synthesis [8]. Therefore, the availability of UDP-Glc and its irreversible conversion to UDP-GlcUA by the UGDH enzyme appears as a key regulatory system in GAG synthesis.

Despite the cDNA cloning and promoter identification of the UGDH gene [9, 10], the cis-acting elements and trans-acting factors involved in the regulation of UGDH gene expression in articular chondrocytes are not fully characterized. It has been shown that the 5' region up to –374 bp of the human promoter UGDH gene contains GC-rich boxes and that Sp1 consensus sequences are responsible for the up- or down-regulation of the UGDH gene in MRC5 fibroblasts through Sp1 binding, as a consequence of TGF-β1 stimulation or hypoxic conditions, respectively [11]. Sp1 plays a key role in the basal expression of various ECM genes [12]. Three Sp1 binding elements (GC boxes) were recently found to play a role in the expression of the UGDH large transcript [13]. Moreover, we recently demonstrated that overexpression of c-Krox inhibits GAG synthesis and that this effect is mediated by a Sp1/Sp3 binding cis-sequence located between +18 and +39 bp of the UGDH gene in rabbit articular chondrocytes [14].

In addition to cytokines and growth factors that regulate the metabolism of cartilage-specific macromolecules, including type II collagen and aggrecans, there is now evidence that oestrogens, in particular 17β-E2, influence also the tissue ECM. Indeed, besides their central role in the reproduction processes, these hormones are involved in the pathe physiology of several non-reproductive tissues including bone and cartilage. Oestrogens are important regulators of cartilage homeostasis, by acting directly or indirectly on chondrocyte proliferation, differentiation and matrix protein synthesis [15]. Moreover, they can be synthesized locally from androgens via aromatase suggesting that the intracrine pathway contributes, beside the known endocrine one, to the oestrogen production and this can influence cartilage physiology [16–18]. The regulatory effect of oestrogens on the growth-plate cartilage is not only well established in different models [19, 20] but also in clinical observations of inactivating mutations in the oestrogen receptor-{alpha} (ER{alpha}) gene and human aromatase deficiency [21, 22]. However, the exact role of oestrogens in articular cartilage homeostasis remains controversial, and may depend on several factors such as species, sex, age or dose [23–25]. Nevertheless, there is epidemiological, clinical and experimental evidence suggesting that ovarian oestrogens could potentially modify the metabolic activity of joint tissues [25–27]. Indeed, the prevalence of OA, the major cause of functional impairment and disability in aged populations [28], increases among women over 50 yrs and prescription of oestrogen replacement therapy in this case may be associated with reduced risk of radiographic hip and knee OA. Furthermore, oestrogens and SERMs (selective oestrogen receptor modulators) may act directly on the joint tissue through oestrogen receptors {alpha} and β, which are present in articular cartilage of different species [29, 30].

Although non-genomic pathways were recently demonstrated, oestrogens exert their effect principally through the receptors ER{alpha} and ERβ, which belong to the nuclear receptor superfamily of transcription factors, structurally organized in six distinct domains (A–F). Two transactivation functions, AF-1 and AF-2, located respectively in B and E receptor domains, act cooperatively to increase transcriptional activity of oestrogen-target genes. An isoform of human ER{alpha} (hER{alpha}), hER{alpha}46 (46 kDa in size), was first detected in MCF-7 human breast cancer cells. It lacks the N-terminal A and B domains and is consequently deprived of AF-1 sequence [31]. Depending on the cellular context and hER{alpha} isoform co-expression, hER{alpha}46 is able to efficiently repress the transcription of oestradiol-target genes induced by hER{alpha}66, suppressing AF-1 activity of long-form oestrogen receptor. To date, the exact function of hER{alpha}46 in articular chondrocytes is not known.

The aim of the present study was to further characterize the effect of 17β-E2 on the GAG chain biosynthesis and the UGDH expression in rabbit articular chondrocytes at the mRNA, protein and enzymatic activity levels, and to compare its effects with those of cytokines (IL-1β, IL-6) and growth factors, such as TGF-β1 and insulin like growth factor-1 (IGF-I), all known to modulate cartilage ECM. To determine the role played by the ER{alpha} receptor and the specific cis-sequences of the UGDH promoter, we investigated the effects of this transcription factor on UGDH transcriptional activity by transient co-transfection experiments.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
General materials
Chemical reagents were of the highest grade from Sigma (Saint Quentin-Fallavier, France). Fetal calf serum (FCS), DMEM, di-deoxynucleosidetriphosphate (dNTPs) and phosphate-buffered saline (PBS) were purchased from Invitrogen (Cergy-Pontoise, France). Protein Assay kit was from Bio-Rad (Marnes-la-Coquette, France). Random hexamers, ABI Prism cDNA Archive kit and 2X SYBR Green Master Mix were purchased from Applied Biosystems (Courtabœuf, France). The primers used for real-time PCR were synthesized by Eurogentec Corporation (Seraing, Belgium). UDP-xylose was from CarboSources Services (Athens, GA, USA), IL-6 was from Euromedex (Mundolsheim, France), IGF-I was from Sigma and IL-1β and TGF-β1 were purchased from R&D Systems (Lille, France). [35S]-sulphate was purchased from Perkin Elmer (Courtabœuf, France) and double-charcoal-treated FCS was from Abcys (Paris, France).

Antibodies
Rabbit polyclonal anti-UGDH was prepared by Y.W. Rabbit monoclonal anti-β-tubulin and horseradish peroxidase (HRP)-conjugated goat anti-rabbit were purchased from Tebu-Bio (Le Perray en Yvelines, France).

Chondrocyte culture
Rabbit articular chondrocytes were isolated from articular cartilage slices of shoulders and knees of 3-week-old male rabbits, by sequential digestion with hyaluronidase (Coger, Paris, France), trypsin (Coger) and collagenase (Sigma). The cells were seeded generally at 3.5 x 105 cells per 9.6 cm2 dish or 1.5 x 106 cells per 55 cm2 dish in 2 or 8 ml of DMEM containing 10% FCS, supplemented with glutamine (2 mM) and antibiotics: penicillin (100 UI/ml), erythromycin (100 µg/ml) and fungizone (0.25 µg/ml). They were grown for 8–12 days at 37°C in a 5% CO2 humidified atmosphere, with medium change every 2–3 days. TGF-β1 (3 ng/ml), IGF-I (10 ng/ml), IL-1β (1 ng/ml), IL-6 (25 ng/ml) and 17β-E2 (1 nM) were added in 2% FCS/DMEM to cells at 80% confluency and incubated for 24 h.

GAG synthesis
Chondrocytes in 6-well plates were transiently transfected, at 80% confluency, with 10 µg of the expression vector pCR3.1-hER{alpha}66 or pCR3.1-hER{alpha}46, encoding long or short oestrogen receptor isoform (empty pCR3.1 plasmid was used as control). After 12 h, the medium was changed to 10% FCS/DMEM. Other RAC were incubated for 24 h with 17β-E2 (1 nM) in 2% double-charcoal-treated FCS/DMEM medium. Twenty-four hours after treatment or 48 h post-transfection, the RAC were labelled for 24 h with [35S]-sulphate (2 µCi/ml) in 1.5 ml total volume per well. After overnight extraction at 4°C with 8 M guanidium chloride containing 0.1% Triton X-100 and proteinase inhibitors (10 mM benzamidine, 100 mM N-caproic acid, 50 mM etylenediamine-tetracetic (EDTA), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 mM N-ethyl maleimide), the samples were precipitated for 1 h at 37°C with 0.5% cetylpyridinium chloride monohydrate (CPC) containing 0.25 M Na2SO4, in the presence of 40 µg/ml chondroitin sulphate as a carrier. The precipitates were collected on Whatman glass filters that were washed successively with 0.025 M Na2SO4/0.005% CPC, cold distilled water, cold 10% trichloroacetic acid and cold 95% ethanol. The radioactivity of the filters was then determined by scintillation liquid in a Packard scintillation counter (Perkin Elmer, Courtabœuf, France).

Enzymatic activity of UGDH
Twenty-four hours after incubation with the factors, the RAC cultures were rinsed twice with PBS and scraped in 500 µl of 100 mM sodium-glycine buffer pH 8.7 containing 10% glycerol. Cell lysates were obtained by four cycles of rapid freezing/thawing. After centrifugation (10 000 g, 10 min), the protein content of the supernatant was determined by the Bradford procedure and an aliquot was assayed for UGDH activity in a Hitachi U-2000 spectrophotometer through measurement of the NAD+ reduction as follows: first, the background was recorded at 340 nm by adding 95 µl of 100 mM sodium-glycine buffer in both test and reference cells; next, 30 µl of the supernatant was incubated in the test cell with 5 mM NAD+ in a 95 µl total volume of sodium-glycine buffer for 5 min at room temperature, in order to measure NAD+-consuming total enzyme activities. After stabilization of absorption, 5 µl of 10 mM UDP-Glc, with or without 20 mM UDP-xylose (a specific inhibitor of UGDH activity) [32] and 5 µl of the 100 mM sodium-glycine buffer were then added respectively in each test and reference cell, and absorption was recorded for an additional 5 min, in order to evaluate specific UGDH activity. Total activity and specific UGDH enzymatic activity, corresponding respectively to media before and after adding UDP-Glc, were expressed as the variation of absorption during 1 min of incubation per milligram of protein.

Western blotting
Twenty-four hours after incubation with cytokines, growth factors or 17β-E2, the RAC cultures were washed three times with PBS and scraped in 600 µl of 50 mM Tris–HCl lysis buffer pH 7.5 containing 1% NP-40, 150 mM NaCl, 1 mM EGTA and protease inhibitors: PMSF (1 mM), pepstatin A (1 µg/ml), aprotinin (1 µg/ml) and leupeptin (1 µg/ml). After 30 min on ice, the supernatants were collected by centrifugation. The cell layer-associated proteins (20 µg) were resolved on a 8% polyacrylamide gel, using 1% SDS/Tris glycine buffer. The proteins were then electrotransferred onto polyvinylidene difluoride (PVDF) membrane, using a transfer buffer (25 mM Tris–HCl, pH 8.3, 192 mM glycine and 20% methanol). Free protein-binding sites of the PVDF membranes were blocked in Tris-buffered saline (TBS) containing 10% non-fat dry milk. After overnight incubation at 4°C, the membranes were rinsed three times in TBS containing 0.1% Tween-20 (TBST), and incubated with a 1 : 750 dilution of rabbit polyclonal anti-UGDH antibody in TBST-containing 5% non-fat dry milk. After 2 h incubation, the membranes were rinsed 8 x 5 min in TBST, and incubated for 2 h with a secondary antibody (HRP-conjugated goat anti-rabbit antibody at 1 : 6000 dilution). The blots were then washed 8 x 5 min in TBST, and the membrane-bound secondary antibodies were revealed using a western blot detection kit (Super Signal West Pico Chemiluminescence Substrate, Pierce, Brebières, France). UGDH protein signals were captured with the Fluor-S Multimager video system (Bio-Rad) and quantified with the ImageQuant software (Amersham Biosciences, Orsay, France). The UGDH signal was normalized to that of β-tubulin.

Quantitative RT-PCR
Total RNA was extracted using Trizol® Reagent, according to the manufacturer's instructions (Invitrogen), and the integrity of 28S and 18S ribosomal RNA was verified in denaturating conditions on 1% agarose gel prepared in 10% MOPS 10X and 16% formaldehyde, and visualized by ethidium bromide staining. RNA (2 µg) was first treated with deoxyribonuclease I (DNase I, Invitrogen) and then reverse transcribed into cDNA using an ABI Prism cDNA Archive kit (Applied Biosystems) in the presence of random hexamers, 5 mM of each dNTP, reverse transcriptase buffer 10X and 50 U/µl of Multi Scribe reverse transcriptase in a final volume of 20 µl. The reverse-transcription mixture was incubated at 25°C for 10 min and the reaction was performed by heating at 37°C for 1 h. To test the efficiency of reverse transcription, β-actin housekeeping gene cDNA was submitted to 40 cycles of amplification (1 cycle: 95°C/1 min; 55°C/1 min; 72°C/1 min) in a PTC 100 Programmable Thermal Controller thermocycler (MJ Research) using a PCR kit (Invitrogen) with following primers: 5' GTG GGG CGC CCA GGC ACC A 3' and reverse: 5' CTC CTT AAT GTC ACG CAC 3'. Then, PCR products were separated on a 2% agarose gel and visualized by ethidium bromide staining. Real-time PCR amplifications were performed with 5 µl of cDNA diluted 1 : 100, using UGDH sequence-specific forward: 5' GAT GTT CTG AAT TTG GTT TAT CTC TGT G 3' and reverse: 5' TTC TCT GGT AGT CAT TCA TGT CTA TAA CCT 3' primers (600 nM). UGDH primers were defined with the Primer Express software (Applied Biosystems). Each primer set was used in a final volume of 15 µl, using 2X SYBR Green Master Mix, and PCR asssays were carried out in triplicate on an ABI Prism 7000 sequence detection system (Applied Biosystems). Thermocycling conditions were 95°C/10 min (initial denaturation) followed by 40 cycles of 95°C/10 s (denaturation) and 60°C/1 min (annealing and extension). The threshold was set above the non-template control background and within the linear phase of target gene amplification to calculate the cycle number at which transcription was first detected (denoted CT). Each sample was run in triplicate, and analysis of relative gene expression was done by using the 2–{triangleup}{triangleup}CT method [33]. Briefly, the relative change in gene expression is calculated by subtracting the CT of the target gene (UGDH) from the internal control (18S rRNA). UGDH and 18S primers (forward: 5' CGG CTA CCA CAT CCA AGG AA 3' and reverse: 5' GCT GGA ATT ACC GCG GCT 3') were purchased from Eurogentec.

Transient transfection experiments
Eighty per cent confluent RAC in six-well plates were transiently transfected by the calcium phosphate precipitation method [34]. Luciferase reporter plasmids (10 µg) containing various deletions of UGDH promoter were co-transfected with a pSV40 β-galactosidase expression vector (2 µg) as an internal control of transfection efficiency, and/or with a pCR3.1 vector (10 µg) with or without the cDNA encoding hER{alpha}. Expression vectors used for hER{alpha} encode either long form of ER{alpha} or short mutated inoperative form of the receptor, denoted hER{alpha}66 or hER{alpha}46, respectively [35]. After 12–15 h, the medium was changed to 10% FCS/DMEM. Twenty-four hours later, the samples were harvested and the protein content, luciferase and β-galactosidase activities were assayed. Luciferase activity was measured on whole cell extract (Promega kit, Charbonnières les Bains, France) in a luminometer (Berthold Lumat LB 9501, Bad Wildbad, Germany). β-Galactosidase activity was assayed using a colorimetric assay method (Bio-Rad), whereas the protein amount was determined by the Bradford colorimetric method (Bio-Rad). Luciferase activities were normalized to transfection efficiency and protein content, and expressed in relative luciferase units (RLUs).

Statistical analysis
Results were representative of at least two experiments in triplicate unless otherwise specified. For GAG synthesis and enzymatic activity measurements, data are presented as box plots, respectively, representative of four or three experiments performed in triplicates. Student's t-test was used to analyse differences between control and treated groups. Statistically significant differences were set at ***P < 0.001, **P < 0.01 and *P < 0.05.

The study was approved by the local ethics committee.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
17β-E2 and its receptor hER{alpha}66 stimulate GAG neosynthesis in articular chondrocytes
We first determined whether 17β-E2 and its receptors hER{alpha}66 or hER{alpha}46 were capable of modulating GAG production in RACs. Cells were either treated for 24 h with 17β-E2 (1 nM), TGF-β1 (3 ng/ml), IL-1β (1 ng/ml) or transfected with the expression vectors hER{alpha}66 or hER{alpha}46 (10 µg) and labelled for 24 h with [35S]-sulphate. Our data show that 17β-E2 and its receptor hER{alpha}66 enhanced [35S]-sulphate incorporation into GAG chains by approximately 30% (Fig. 1A and B, P < 0.05). In contrast, hER{alpha}46 did not induce any significant effect. As expected, incubation of cells with TGF-β1 resulted in a marked increase (~100%, P < 0.001) of [35S]-sulphate labelling, whereas IL-1β reduced the incorporation to 55% of the control value (P < 0.001).


Figure 1
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  		FIG. 1. Effect of 17β-E2 (A) and hER{alpha} overexpression (B) on GAG neosynthesis by primary RACs. RACs were transfected with 10 µg of expression vector pCR3.1, containing or not the long (hER{alpha}66) or short isoform (hER{alpha}46) of oestrogen receptor cDNA; or treated for 24 h with 1 nM 17β-E2, 3 ng/ml TGF-β1 or 1 ng/ml IL-1β. Twenty-four hours later, or 48 h post-transfection, the cells were maintained in DMEM + 2% double-charcoal-treated FCS supplemented with 2 µCi/ml of [35S]-sulphate. After 24 h incubation, cells were harvested in guanidium–Triton X-100 medium and radioactivity incorporated into neosynthesized GAGs was measured in a Packard scintillation counter. Values of four experiments in triplicate are given as box plots with the 25th, 50 (median) and 75th percentiles. Means are shown as lozenges. Statistically significant differences with the control were determined by the Student's two-tailed t-test (*P < 0.05 and ***P < 0.001).

 
17β-E2 and TGF-β1 stimulate UGDH activity in articular chondrocytes
Since UGDH appears as a key regulatory enzyme in GAG synthesis, we therefore evaluated the potential role of 17β-E2 and some cytokines/growth factors in the modulation of UGDH activity in RAC, by using a spectrophotometric protocol based on the reduction of NAD+ by whole cell lysates. First, we determined the total enzymatic activities that consume NAD+ as a cofactor and could interfere with the detection of UGDH-specific activity. There was a general increase in total activities in the presence of TGF-β1, IGF-I or 17β-E2 (Fig. 2A). No significant effect was observed with others factors. After stabilization of absorption (5 min), UDP-Glc was added, as the substrate of UGDH, and the variation of the absorption was measured, reflecting the UGDH activity. Results were normalized by calculating the ratio of UGDH activity to total activities, which better reflects the specific activity of UGDH present in the articular chondrocytes. We showed that UGDH activity is significantly elevated, by at least 2-fold in the presence of TGF-β1 (P < 0.01) and 17β-E2 (P < 0.01) (Fig. 2B). To verify the specificity of UGDH activity, a pool of 35 µg of different treatments (control, TGF-β1, IGF-I, IL-1β, IL-6 and 17β-E2) was incubated in the presence of UDP-xylose, a specific inhibitor of UGDH. The results show that the activity of the enzyme was inhibited by 42% (P < 0.001), whereas the total activities were not significantly altered (Fig. 2C).


Figure 2
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  		FIG. 2. Effect of cytokines/growth factors and 17β-E2 treatment on enzymatic activities in RAC. Primary RAC were grown for 10 days in DMEM containing 10% FCS and then treated with either cytokines/growth factors or 17β-E2 for 24 h in the same medium but containing 2% FCS. Cell extracts were harvested and submitted to Bradford's assays and spectrophotometric analysis as described in Materials and methods. Enzymatic activities were expressed as variation of absorbance per minute and per milligram of protein. (A) Total NAD+-consuming enzymatic activities were assayed in RAC extracts. (B) Specific UGDH activity was determined after adding UDP-Glc and normalized to total enzymatic activities. (C) Specificity of UGDH activity was verified by incubation with a specific inhibitor of the enzyme, UDP-xylose. Values of the three experiments in triplicate are given as box plots with the 25th, 50th (median) and 75th percentiles. Means are shown as lozenges. Statistically significant differences with the control were determined by the Student's test (***P < 0.001 and **P < 0.01).

 
17β-E2, IL-1β, IL-6, TGF-β1 and IGF-I stimulate UGDH protein synthesis in articular chondrocytes
We then investigated whether 17β-E2 and cytokines/growth factors could exert a regulatory effect on UGDH protein expression. Western blotting performed on whole cell extracts, using a polyclonal anti-UGDH antibody, revealed the presence of the UGDH protein, whatever the treatment. Figure 3A and B shows that all the factors increased UGDH protein expression by ~30–60%. Correlation between UGDH protein levels and enzymatic activity was only found for TGF-β1 and 17β-E2 treatments. To test the specificity and the sensitivity of the UGDH antibody, increasing concentrations of UGDH-purified protein (Sigma) were run on an SDS–PAGE gel (Fig. 3C). The intensity of electrophoretic bands was proportional to the amounts of proteins loaded in the gel, in agreement with expected results.


Figure 3
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  		FIG. 3. Western blotting analysis of UGDH protein in primary RAC following 24 h treatment with either TGF-β1, IGF-I, IL-1β, IL-6 or 17β-E2. Primary chondrocytes were treated with TGF-β1, IGF-I, IL-1β, IL-6 or 17β-E2 for 24 h. (A) Protein extracts (20 µg per lane) were fractionated on SDS–PAGE and then analysed by western blotting with anti-UGDH and anti-β-tubulin antibodies. (B) Signals were captured with autoradiography films and then quantified with the ImageQuant software. UGDH expression was normalized to the β-tubulin protein signal. (C) Positive control, with a concentration range of commercially purified UGDH (Sigma). Results are representative of three independent experiments. Numbers in bold represent the percentage relative to the control.

 
17β-E2 up-regulates UGDH mRNA steady-state levels in a time-dependent manner
Using real-time quantitative PCR, we next determined the effect of the factors on UGDH mRNA steady-state levels. As shown in the Fig. 4A, the expression of UGDH mRNA was increased (by 1.4- to 2-fold) in the presence of IL-1β (P < 0.05), IL-6 (P < 0.05) and 17β-E2 (P < 0.01). Thus, UGDH stimulation by 17β-E2 was confirmed at the level of mRNA transcription, protein and enzymatic activity levels. In contrast to western blotting and enzymatic activity data, no difference was observed in the UGDH mRNA amounts between control and TGF-β1 treatment. In addition, UGDH stimulation by 17β-E2 was found to be time-dependent, with a 2-fold increase of mRNA found at 6 h (Fig. 4B).


Figure 4
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  		FIG. 4. Effect of cytokines/growth factors and 17β-E2 on UGDH mRNA steady-state levels. Primary RAC were incubated for 24 h with TGF-β1, IGF-I, IL-1β, IL-6 or 17β-E2, as indicated in Fig. 2. Total RNA (2 µg) was extracted and reverse-transcribed into cDNA by using random hexamers and Multi Scribe reverse-transcriptase enzyme. The cDNAs were used in real-time PCR. The data were normalized to the expression level of 18S rRNA as a reference. UGDH mRNA expression levels were assayed for each treatment (A) and effect of 17β-E2 on this expression was determined in a time-dependent manner after incubation of RAC during 0.5, 1, 3, 6 or 24 h with 1 nM of the steroid (B). For each time, results are mean ± S.D. of two experiments performed in triplicate and expressed as percentage of control without 17β-E2. Statistically significant differences relative to the control were obtained by the Student's test (***P < 0.001, **P < 0.01 and *P < 0.05). Numbers in bold represent the percentage ± S.D.

 
A minimal 100-bp region of UGDH gene promoter mediates the up-regulatory effect of hER{alpha}66 in RAC-transient transfection experiments
To better understand the mechanism that mediates the up-regulation of UGDH gene by 17β-E2, we co-transfected the hER{alpha}66 and/or hER{alpha}46 expression vectors with several constructs of the 5'-flanking region of the UGDH gene promoter, coupled with luciferase reporter gene (Fig. 5). The basal expression of the reporter gene was always increased by hER{alpha}66, whatever the construct size, including the shortest –100/+50 bp. These results suggest that a minimal 100 bp region of UGDH gene promoter mediates the hER{alpha}66-induced UGDH gene transactivation. Moreover, hER{alpha}46 isoform did not significantly modify the basal expression of the reporter UGDH gene, indicating that hER{alpha}66 stimulation of UGDH gene is specific to this isoform and probably depends directly on AF-1 transactivation functions of ER{alpha}.


Figure 5
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  		FIG. 5. Effect of hER{alpha}66 and hER{alpha}46 estrogen receptor isoforms on the transcriptional activity of UGDH promoter constructs. Primary RAC were grown to 80% confluency in 10% FCS/DMEM medium and then co-transfected with either 10 µg of pCR3.1-hER{alpha}66 or pCR3.1-hER{alpha}46 expression vectors, together with 10 µg of UGDH reporter plasmid (promoter constructs –644/, –374/, –196/, –165/ and –100/+50 bp, containing various deletions and cloned upstream the luciferase reporter gene). RAC were also co-transfected with a β-galactosidase expression vector, pSV40-β-gal (2 µg), used as an internal control for transfection efficiency. Relative luciferase activities were measured in total cell extracts and normalized for transfection efficiency. Values are expressed as percentages, relative to the empty pCR3.1 plasmid. Results are expressed as the means ± S.D. of three experiments in triplicate. Numbers in bold represent the percentage ± S.D.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
Regulation of UGDH gene expression is a critical step of GAG synthesis in all tissues, including that of articular cartilage. Here, we investigated the potential role of 17β-E2 in the modulation of UGDH gene activity. In fact, it is already known that the sexual hormones, oestrogens and androgens, responsible for gender dimorphism and reproduction, also play a regulatory role in tissues and organs that are not directly involved in procreation. Our data demonstrated that among the factors tested, only 17β-E2 was able to up-regulate both expression and enzymatic activity of UGDH, resulting in an increase of GAG chain synthesis in RACs. Furthermore, 17β-E2-induced UGDH activity was clearly decreased by UDP-xylose, a specific inhibitor of UGDH, indicating that our spectrophotometric protocol used to evaluate UGDH enzymatic activity was valid. In this work, we aimed to investigate 17β-E2-mediated molecular mechanisms of ECM remodelling rather than to propose a model of OA. However, RAC of young males is a well-characterized model to study molecular mechanisms of type II collagen and UGDH expression [14]. It is well known that in this model, RAC preserve their differentiated phenotype and morphology in monolayer cell culture system as proved by the maintenance of a high type II/type I collagen ratio [36].

Estrogens act through both genomic and non-genomic mechanisms. The genomic mechanism involves the diffusion of oestrogens across the plasma membrane and activation of specific intracellular receptors (ER{alpha} and ERβ) that are expressed in a broad range of cell types including articular chondrocytes [30]. In preliminary studies, we showed that ER{alpha} expression profile in RAC was quite similar to that of osteoblasts with the presence of two isoforms, hER{alpha}66 and hER{alpha}46 receptors (unpublished data). The oestrogen/ER complex binds and activates DNA-specific cis-elements in target genes (oestrogen responsive elements, ERE). However, this complex can also affect transcriptional regulation without directly binding to ERE, but through modulation of some trans-factors such as Sp1, Sp3, c-fos or c-jun in the recognition of their respective cis-elements, ‘GC boxes’ and AP-1. UGDH is an ERE-less gene that contains an inverted TATA box element that is essential for basal UGDH promoter activity, as shown in hepatocarcinoma HepG2 cells [11]. However, its 644 bp promoter contains 12 GC characteristic sequences, that bind Sp1, Sp3 or hc-Krox factors. In our cellular model, UGDH transcriptional activity is enhanced by hER{alpha}66 but not by hER{alpha}46. Our results suggest that this effect is mediated by a sequence located between –100 and +50 bp in the UGDH promoter and may depend on the AF-1 transactivation domain of hER{alpha}66. In this proximal region, three potential binding sites for transcription factors Sp1, Sp3 and hc-Krox have been found by database searches: two are located upstream of the transcription start site at –88/–67 and –61/–39, whereas the third was found downstream at the position +18/+39. This last sequence binds the Sp1/Sp3 factors with great affinity. Thus, the control of Sp1/Sp3 ratio and/or binding activity by hER{alpha}66 appears as an important pathway for the regulation of UGDH gene promoter. This hypothesis is supported by a study in which hER{alpha}46 deletion of AF-1 domain antagonized the proliferative influence of hER{alpha}66 and abolished synergic transcriptional effect between ER{alpha} and Sp1 transcription factors [35]. Work is in progress to investigate hER{alpha}66 and Sp1/Sp3 interaction in our model, using gel retardation assays, imunoprecipitation experiments and chromatin immunoprecipitation. The transcriptional modulation of oestrogen-responsive genes involves recruitment of ligand-activated ER{alpha}/β, either directly through interactions with their specific DNA binding cis-sequences (ERE), or the protein/protein interactions with other transcription factors such as AP-1 or nuclear factor-{kappa}B (NF-{kappa}B) [37]. Interestingly, it was shown that ER{alpha} could form complexes with Sp1 or Sp3 to regulate the activity of ERE-dependant retinoic acid receptor-{alpha}1 and adenosine deaminase promoters [38]. Moreover, previous studies showed that hER{alpha} binds to c-Jun but not to c-Fos family members (AP-1 complex), in vitro and in vivo, and that this interaction is likely to be involved in 17β-E2 regulation of AP-1 response elements [39]. In some studies, hER{alpha} was found to interact also directly with the NF-{kappa}B pathway, inhibiting its capacity to bind to DNA: this effect was attributed either to the action of the receptor on I{kappa}B processing, which in turn would prevent nuclear translocation of NF-{kappa}B factor, or to a direct selective inhibition of p65 DNA binding [40]. Moreover, binding activity of NF-{kappa}B transcription factor was enhanced in articular chondrocytes undergoing dedifferentiation process [34]: ER{alpha} inhibition of this nuclear transcription factor could be a part of the mechanisms whereby 17β-E2 would prevent dedifferentiation of chondrocytes.

It is known that Sp1/Sp3 ratio regulates collagen type II gene (COL2A1) expression in chondrocytes [41]. In this regard, it is noteworthy that 17β-E2 was also found to up-regulate COL2A1 expression in male RAC (data not shown). However, it has been reported that 17β-E2 could suppress the insulin-induced increase of type II collagen synthesis in bovine articular chondrocytes [42]. This apparent discrepancy could be due to the fact that chondrocytes were from adult female cows and that hypoxic conditions (5% O2) and serum-free medium were used to culture the cells. In addition, chondrocyte genes, including UGDH and COL2A1, are differentially regulated under normoxia and hypoxia [11] and steroid hormones are known to exert biphasic or sometimes opposite effects in male or female models [43]. Moreover, immunocytochemical results in this study [42] showed a general increase in ER{alpha} expression when cell culture with medium containing serum was continued with serum-free medium. However, one cannot rule out the hypothesis of the expression of an ER{alpha} isoform (like hER{alpha}46) that antagonizes the positive effect of the long isoform hER66 [35]. Nevertheless, our data show that 17β-E2 is a positive modulator of both UGDH and COL2A1 in articular chondrocytes. Its effects may take part in the control of cartilage ECM homeostasis and may be confirmed by animal models such as ovariectomized rats.

The role of IGF-I and TGF-β1 in the metabolism of normal and OA cartilage has been documented [44]. Although normal chondrocytes produce both IGF-I and IGF-II, IGF-I constitutes the main isoform capable of up-regulating proteoglycan synthesis in adult chondrocytes. Our results did not reveal any regulatory role of IGF-I on UGDH activity and mRNA levels. This finding is controversial with previous reports concerning the general increase in synthesis of anabolic constituents of ECM, in particular secretion of proteoglycans, by IGF family members [3]. However, IGF-I seems to enhance UGDH protein levels, as reflected by our western blots. The role of TGF-β1 in articular cartilage is complex and its activity seems to depend on the differentiated state of chondrocytes. TGF-β1 and its main intracellular signal molecules Smad2/Smad3 are essential in preventing hypertrophic differentiation of chondrocytes and in maintaining the differentiated phenotype of articular cartilage [45]. Here we report that TGF-β1 increases UGDH activity and protein but not UGDH mRNA synthesis. This might be due to different kinetics of TGF-β1 effects on UGDH gene transcription and UGDH protein synthesis or to some post-transcriptional effects. However, TGF-β has been recently demonstrated to up-regulate UGDH mRNA expression in MRC5 fibroblasts [11]. This discrepancy may be explained by different transcriptional regulation mechanisms and/or by difference in UGDH mRNA half-lives between fibroblasts and chondrocytes. Moreover, TGF-β effects can be dependent on the age: it has been demonstrated that TGF-β1 treatment of human cartilage increased chondrocyte UGDH activity in a manner inversely correlated with age [46].

The catabolic role of pro-inflammatory cytokines in cartilage homeostasis is well documented. The synovial fluids of patients with OA and RA contain high levels of IL-1β [47].The deleterious effects of IL-1β on the anabolism of proteoglycan chondrocytes could involve the repression of galactose β-1,3-glucuronosyltransferase I [2] a key enzyme catalysing the transfer of Glc-UA to β-linked galactose residue during GAG biosynthesis. In the same way, the role of IL-6 in the pathophysiology of articular cartilage is controversial, as this cytokine is capable, in concert with IL-1β, to increase the matrix metalloproteinases production, to reduce the synthesis of proteoglycans [1], whereas it stimulates the production of tissue inhibitors of metalloproteinases by chondrocytes [48]. In our present work, in contrast with the effects of TGF-β1 and 17β-E2, IL-1β and IL-6 did not induce the activity of UGDH although they increased its protein and mRNA levels. This suggests that specific regulating mechanisms may control the UGDH activity.

In conclusion, among the factors investigated here, 17β-E2 appears as a potent regulator of both expression and activity of UGDH in articular chondrocytes. Its effect is exerted through the receptor ER{alpha} and is likely to involve Sp1 and/or Sp3 transcription factors. This steroid hormone also up-regulates GAG synthesis in cartilage homeostasis and may take part in its repair potentialities in joint diseases. Such mechanisms might be influenced by altered oestrogen synthesis and action on the chondrocytes possibly aggravated by post-menopausal status and by altered local sex steroid synthesis and metabolism.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
Funding: This work was supported by the Regional Council of Low-Normandy (CRBN) and the National Fund for Territorial Development (FNADT) (Program ‘Normal and Pathologic Extracellular Matrix’).

Disclosure statement: The authors have declared no conflicts of interest.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Submitted 3 August 2007; revised version accepted 1 November 2007.
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