S. Sebillaud

The inorganic pyrophosphate transporter ANK preserves the differentiated phenotype of articular chondrocyte.

The differentiated phenotype of chondrocyte is lost in pathological situations and after interleukin (IL)-1β challenge. Wnt proteins and the inorganic pyrophosphate (PPi) transporter Ank regulate the differentiation process in many cell types. We investigated the possible contribution of Ank and/or PPi to the maintenance of the differentiated chondrocyte phenotype with special care to Wnt signaling. Primary articular chondrocytes lost their phenotype upon IL-1β challenge, with cessation of type II collagen and Sox-9 expression. Ank expression and PPi transport were strongly reduced by IL-1β, whereas Wnt-5a was the only Wnt protein increased. Transient overexpression of Ank counteracted most of IL-1β effects on Type II collagen, Sox-9, and Wnt-5a expression. When resting chondrocytes were transfected with a siRNA against Ank, this reproduced the phenotype induced by IL-1β. In both cases, no markers for hypertrophic chondrocytes were detected. The conditioned supernatant from chondrocytes knocked-down for Ank contained Wnt-5a, which activated Tcf/Lef reporter plasmids and promoted translocation of β-catenin into the nucleus without activating the c-Jun N-terminal kinase (JNK) pathway. Supplementation with PPi compensated for most effects of Ank deficiency on Type II collagen, Sox-9, and Wnt-5 expression, both in IL-1β and Ank knock-down conditions. Phenotype changes induced by IL-1β were also supported by activation of the JNK pathway, but this latter was not sensitive to PPi supplementation. Altogether our data demonstrate that the transport of PPi by ANK contributed to the maintenance of the differentiated phenotype of chondrocyte by controlling the canonical Wnt pathway in a Wnt-5a-dependent manner.

Calcium Input Potentiates the Transforming Growth Factor (TGF)-β1-dependent Signaling to Promote the Export of Inorganic Pyrophosphate by Articular Chondrocyte

Transforming growth factor (TGF)-β1 stimulates extracellular PPi (ePPi) generation and promotes chondrocalcinosis, which also occurs secondary to hyperparathyroidism-induced hypercalcemia. We previously demonstrated that ANK was up-regulated by TGF-β1 activation of ERK1/2 and Ca2+-dependent protein kinase C (PKCα). Thus, we investigated mechanisms by which calcium could affect ePPi metabolism, especially its main regulating proteins ANK and PC-1 (plasma cell membrane glycoprotein-1). We stimulated articular chondrocytes with TGF-β1 under extracellular (eCa2+) or cytosolic Ca2+ (cCa2+) modulations. We studied ANK, PC-1 expression (quantitative RT-PCR, Western blotting), ePPi levels (radiometric assay), and cCa2+ input (fluorescent probe). Voltage-operated Ca2+-channels (VOC) and signaling pathways involved were investigated with selective inhibitors. Finally, Ank promoter activity was evaluated (gene reporter). TGF-β1 elevated cCa2+ and ePPi levels (by up-regulating Ank and PC-1 mRNA/proteins) in an eCa2+ dose-dependent manner. TGF-β1 effects were suppressed by cCa2+ chelation or L- and T-VOC blockade while being mostly reproduced by ionomycin. In the same experimental conditions, the activation of Ras, the phosphorylation of ERK1/2 and PKCα, and the stimulation of Ank promoter activity were affected similarly. Activation of SP1 (specific protein 1) and ELK-1 (Ets-like protein-1) transcription factors supported the regulatory role of Ca2+. SP1 or ELK-1 overexpression or blockade experiments demonstrated a major contribution of ELK-1, which acted synergistically with SP1 to activate Ank promoter in response to TGF-β1. TGF-β1 promotes input of eCa2+ through opening of L- and T-VOCs, to potentiate ERK1/2 and PKCα signaling cascades, resulting in an enhanced activation of Ank promoter and ePPi production in chondrocyte.

Evidence for species differences in the regulation of MMPs by all-trans retinoic acid in cytokine-stimulated chondrocytes

In inflammatory conditions, chondrocytes produce large amounts of matrix metalloproteases (MMP) and nitric oxide (NO) thought to contribute to joint degradation. We tested the ability of all-trans retinoic acid (ATRA, a retinoic acid receptor (RAR) agonist) to modulate these inflammatory genes in chondrocytes from humans or rats, chosen as representative of animal models of arthritis. All RAR subtypes and RXR-alpha or -beta were expressed at the mRNA level in both species, although IL-1beta (10 ng/ml) inhibited RAR subtypes more markedly in rat than in human cells. ATRA (300 or 1000 nM) inhibited IL-1-induced expression of iNOS and nitrites level in both species, although the NO pathway was induced maximally in rat cells. ATRA displayed controversial effects on MMPs between rat and human chondrocytes, especially for MMP-9 expression. The effects of ATRA were irrelevant to the nuclear translocation of AP-1. The present data underlines that retinoids have a species-dependent impact on IL-1-induced responses in chondrocytes, suggesting that extrapolation of their pharmacological properties from animal cells has a poor relevance to clinical situation.

Inorganic phosphate (Pi) modulates the expression of key regulatory proteins of the inorganic pyrophosphate (PPi) metabolism in TGF-β1-stimulated chondrocytes.

The balance between extracellular inorganic phosphate (ePi) and extracellular inorganic pyrophosphate (ePPi) is controlled by four membrane proteins: the transporters ANK (exporting PPi outside the cells) and PiT-1 (importing ePi into the cells), and the enzymes PC-1 (generating ePPi from nucleotides) and Tissue Non-specific Alkaline Phosphatase (TNAP, hydrolyzing ePPi into ePi). TGF-β1 was shown to stimulate ANK and PC-1 expression in articular chondrocytes, and subsequent ePPi level, as well as to increase ePi uptake by inducing PiT-1 expression in a chondrogenic cell line. Thus, we investigated the ability of ePi to modulate the effect of TGF-β1 on the regulatory proteins of the ePi/ePPi balance in chondrocytes. In the pathophysiological range of 0.01-1 mM, ePi was inactive by itself but potentiated the stimulatory effects of TGF-β1 on ANK, PC-1 or PiT-1 mRNA (RT-qPCR) and protein (Western blot) levels. PC-1 activity was also increased by TGF-β1 and further potentiated by ePi supplementation. TNAP mRNA and activity became undetectable in response to TGF-β1. These data suggest that ePi could increase ePPi level by changing the control of ANK and PC-1 expression by TGF-β1, further highlighting an adaptative regulation of the Pi/PPi balance to prevent basic calcium phosphate deposition into the joints.

207 INFLUENCE OF CALCIUM LEVEL ON THE EFFECT OF TRANSFORMING GROWTH FACTOR BETA-1 IN THE GENERATION OF INORGANIC PYROPHOSPHATE BY ARTICULAR CHONDROCYTE

Results: Cyclic loading for 1 and 4 hours significantly increased GAG release when compared to the control explants (P<0.05 and P<0.01, respectively). Neither nitric oxide nor VEGF expression could be detected in the medium from any of the loaded or unloaded controls. A significant increase in total protein (normalised to mass) was found after 4 hours loading compared to the equivalent control (P=0.003), an increase was also apparent after only 1 hour of loading but did not reach significance. After loading, quantification of FGF2 and FGF18, detected by western blot analysis after normalisation to actin, suggested a similar level of expression to the unloaded controls. At both time points, cyclic loading induced expression of pERK compared to total ERK, when compared to the control explants, with the 4 hour period showing a significant difference (P<0.001). No expression of MMP13 or WNT16 was detected by western blotting in the loaded or the unloaded controls. Conclusions: This study demonstrates that in response to cyclic loading, a significant increased amount of GAGs are released into the medium together with an activation of pERK. In addition, an increase in total protein was detected in loaded explants. This study also suggests that FGF2, FGF18, MMP13, VEGF, FGFR3 and NO do not play an immediate role in cyclic loading. Further investigation is needed to explore longer durations of cyclic loading.

199 ARTICULAR LEVELS OF ADIPONECTIN ARE NOT CHONDROPROTECTIVE IN 3D CULTURES OF HUMAN CHONDROCYTES

Purpose: Adipokines are found in synovial fluid of OA patients and circulating levels of adiponectin (Apn) are reduced in obesity. As obese patients are at increasing risk of developing osteoarthritis (OA), even in non-weight bearing joints, we tested the hypothesis that low (physiological) concentrations of Apn could impair chondrocytes functions towards an “OA-like” phenotype. Methods: Firstly, the expression of Apn and Apn receptors (AdipoR) was characterized by quantitative RT-PCR and western blotting. Secondly, a dose-ranging comparative study of physiological concentrations (0.2, 1 or 5μg/ml) of globular (gApn) and full length (flApn) forms of adiponectin was performed on chondrocytes cultured in alginate beads. The parameters measured were: proteoglycans (PGs) synthesis (by radiolabelled sulfate incorporation), pro-inflammatory mediators release (NOx by Griess method, PGE2 and TNFα by ELISA) and MMP-13 activity (by fluorimetric assay). Thirdly, in co-stimulation experiments, the ability of gApn or flApn to modify chondrocytes responses to the pro-catabolic factor, IL-1beta (10ng/ml) or the pro-anabolic factor, IGF-1 (50ng/ml) was studied. Results: We demonstrated that chondrocytes expressed Apn (mRNA & protein) in monolayers or alginate beads as well as AdipoR1 (mRNA & Protein) and AdipoR2 (mRNA). We found that, in the concentration range tested, gApn was inactive on all parameters except a weak inhibitory effect on MMP-13 at 5μg/mL. In contrast, the highest concentration (5μg/ml) of flApn was able to decrease PGs synthesis by 27%, to induce NO and PGE2 release, and to increase MMP-13 activity. A weak effect was seen for lower concentrations of flApn on PGs synthesis and MMP-13 activity. Neither gApn nor flApn reduced the stimulating effect of IGF-1 on PGs synthesis, but flApn weakly potentiated the inducing effect of IL-1beta on PGE2 release and MMP-13 activity. Conclusions: This preliminary study shows that, in human chondrocytes: i) gApn was less active than flApn; ii) flApn was active in the upper range of Apn joint levels found in OA patients; iii) flApn failed to protect from the deleterious effect of IL-1. Our data do neither support the meaning that Apn could be chondroprotective in OA nor that a reduced articular level could be deleterious for cartilage.