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, DVL-2 is essential for Wnt-3a-induced down-regulation of COL2A1. Knockdown of DVL-2 reversed the inhibition by Wnt-3a of COL2A1 as evaluated by PCR (n=3). (D) Knock down of DVL-2 but not of DVL-1 or DVL-3 inhibited Wnt-3a
, Primary chondrocytes were transfected with siRNA specific to DVL-1, DVL-2, DVL-3 or siRNA unpaired Student's t-test (*, p.0
After being washed three times with PBS, cartilage explants were treated with Wnt-3a, IL-1? or both in the DMEM-F12 medium supplemented with 10% FBS, 1% glutamine and 1% penicillin/ streptomycin for 48 h. 35 S-labeled proteoglycans in the medium were digested with papain (1 mg/ml), precipitated by cetylpyridinium chloride then dissolved in scintillation fluid, The radioactivity associated with GAGs was measured by liquid scintillation counting ,
, After the treatment, cells were washed with PBS and fixed with 4% (w/v) para-formaldehyde solution and permeabilized with 0.1% Triton-X100 then blocked in PBS containing 0.2% fish skin gelatin. Next, cells were incubated with anti-NF?B/p65 antibodies (1/400, Cell signaling), then washed with PBS and incubated with fluorophore-conjugated secondary antibodies. The nuclei were stained with DAPI. Digital images were captured with an inverted microscope, Leica DMI3000 B (Leica Microsystems, Germany), Immunofluorescence Chondrocytes were seeded onto the coverslips in 24-well plates and allowed to grow for 24 h, then treated or not with Wnt-3a and/or IL-1? for 20 min
, Given that loss of ?-catenin abolished the effect of Wnt-3a on ADAMTS-4 (Figure 3G), these data support the notion that inhibition of NF-?B pathway by Wnt-3a is ?-catenin-dependent, NF-?B/p65 into the nucleus
, Wnt-3a but even promoted its expression (Figure 3G). Altogether, these results demonstrated that Wnt-3a down regulates the expression of ADAMTS-4 by attenuating NF-?B signaling through ?-catenin-dependent pathway
, Wnt-3a counteracts IL-1?-induced up-regulation of ADAMTS-4 by reducing nuclear translocation of NF?B/p65
, therefore suggesting that induction of ADAMTS-4 is involved in the pathogenesis of OA. In line with these studies, treatment of human primary chondrocytes with IL-1? strongly increased the expression of ADAMTS-4 and did not significantly affect that of ADAMTS-5 (Figure. 4A). In addition, the knockdown of NF-?B/p65/RELA reduced by 80% the expression of ADAMTS-4 induced by IL-1? in primary chondrocytes (Figure 4B). Given that Wnt-3a reduced the activation of NF?B signaling pathway, the ability of Wnt-3a to counteract IL-1?-induced up-regulation of ADAMTS-4 was tested. As expected, co-treatment with Wnt-3a attenuated by about 60% the induction of ADAMTS-4 by IL-1? (Figure 4C), indicating that Wnt-3a is able to antagonize the effect of IL-1? on ADAMTS-4 expression. Similarly, analysis of ADAMTS-4 activity indicated co-treatment with Wnt-3a reduced by 30% the IL-1?-induced activity of ADAMTS-4 (Figure 4D). Altogether, these results clearly demonstrate that Wnt, human chondrocytes, the aggrecanase ADAMTS-4 but not ADAMTS-5 is induced by IL-1?, the main cytokine incriminated in OA pathogenesis, through activation of NF?B signaling pathway
, To gain insight into the Discussion WNT signaling was considered to be associated with arthritic cartilage destruction, since the level of ?-catenin was found to be, SAPK/JNK and NF?B signaling pathways have been suggested to be involved in the regulation of the expression of ADAMTSs by IL-1?
we provide evidence that Wnt/?-catenin signaling is a potent inhibitor of ADAMTS-4 in human primary chondrocytes and cartilage explants, remarkable contrast to what has been observed in animal models ,
, The presence of four putative NF-?B motifs in human ADAMTS4 gene promoter [24] suggests that it may be regulated by NF-?B and that Wnt-3a may inhibit ADAMTS-4 by suppressing the activation of NF-?B signaling. In agreement with this hypothesis, the knockdown of NF-?B/p65/RelA strongly reduced basal and IL-1?-induced expression of ADAMTS-4. Investigation of the mechanism involved in Wnt-3a down regulation of ADAMTS4 showed that Wnt3a impaired the activation of NF-?B signaling by reducing nuclear translocation of NF-?B/p65. In line with this, Wnt-3a inhibited the expression of IL6 and IL-1?, two established target genes of NF-?B pathway in human chondrocytes. It is well known that activation of NF-?B signaling by proinflammatory cytokines such as IL-1? potently induced cartilage matrix degradation. Investigation of the effect of Wnt/?-catenin signaling on the stimulatory effect of IL-1? on ADAMTS-4 expression and activity revealed that Wnt/?-catenin signaling counteracts these effects by reducing the nuclear translocation of NF-?B/p65 induced by the cytokine. This suggests that Wnt/?-catenin signaling counteracts NF-?B signaling in cartilage and may safeguard cartilage from degradation, These observations strongly argue for differential regulation of ADAMTS-4 by Wnt/?-catenin signaling in human and animal chondrocytes
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, Supporting Information
, Wnt/?-catenin signaling counteracts interleukine-1-induced ADAMTS4 si-RNA
, Human chondrocytes were pretreated with vehicle or DKK1 for 30 minutes then exposed to 100ng/ml Wnt-3a for 12 hours, the Western blot showed the Wnt-induced accumulation of ?-catenin was abolished by DKK1 (n=3), the ?-actin was used as loading control. (E) si-CTNNB1 was transfected into chondrocytes for 48 hours, Western blot revealed the ?-catenin level was lower comparing to the si-control group (n=3). (F) Silencing of ?-catenin by si-CTNNB1 suppressed the Wnt-3a-induced-C) Primary chondrocytes were transfected with siRNA specific to (A) DVL-1 (SiDVL-1), (B) DVL-2
, for 48 h and the level of DVL-1, DVL-2 and DVL-3 was determined by RT-PCR (n?3)
, Data are expressed as mean ± S.D. Statistical analysis was performed with an unpaired Student's t-test (*, p < 0
, Specific DNA segment for each of the ten Fz receptors (standards) was amplified by RT-PCR, purified on agarose gel, quantified and used as template for Q-PCR. Absolute quantification was determined compared to standards that are run in parallel
, Human chondrocytes were transfected with si-control, si-Fz-6 or si-Fz-8 for 48 h, knockdown efficiency of si-Fz-6 (B, n=6) and si-Fz-8 (C, n=5) was determined by RTPCR. Data are expressed as mean ± S.D. of 3 separate experiments. Statistical analysis was performed with an unpaired Student