Figure 3. Inhibition of Wnt canonical... | Rockefeller University Press

Figure 3.

$Figure 3. Inhibition of Wnt canonical pathway with DKK1 does not rescue the loss of the chondrocyte phenotype. (A–C) WNT-3A activates the Wnt canonical pathway in AHACs. (A) Western blotting for β-catenin in the cytoplasmic fraction of AHACs treated with 100 ng/ml WNT-3A or vehicle control for 24 h. (B) WNT-3A activated the SUPER8TOPFlash reporter assay in P0 AHACs after 24 h of stimulation (n = 3). (C) WNT-3A up-regulated the expression of the endogenous Wnt canonical target gene AXIN2 in AHACs as evaluated by Q-PCR. Values were normalized for the housekeeping gene β-actin. (D–I) Primary AHACs were treated for 24 h with 100 ng/ml of recombinant WNT-3A or DKK1, alone or in combination, and were subjected to gene expression analysis by PCR. Blockade of the Wnt canonical pathway by DKK1 rescued the modulation of AXIN2, PCNA, and MMP13 mRNA (D–F) but not the down-regulation of COL2A1, Aggrecan, and SOX9 (G–I). (J) In the absence of exogenous WNT-3A, treatment with 100 ng/ml DKK1 resulted, as expected, in down-regulation of the mRNA levels of AXIN2 and PCNA, but also in a paradoxical down-regulation of COL2A1 and Aggrecan similar to that induced by WNT-3A. Q-PCR data, n = 6. Gene expression values are reported as a percentage of up- or down-regulation compared with control (100% is indicated by the broken line in the graph). Data were analyzed with an unpaired t test. Error bars indicate mean ± SEM. *, P < 0.05; **, P < 0.005.$

Inhibition of Wnt canonical pathway with DKK1 does not rescue the loss of the chondrocyte phenotype. (A–C) WNT-3A activates the Wnt canonical pathway in AHACs. (A) Western blotting for β-catenin in the cytoplasmic fraction of AHACs treated with 100 ng/ml WNT-3A or vehicle control for 24 h. (B) WNT-3A activated the SUPER8TOPFlash reporter assay in P0 AHACs after 24 h of stimulation (n = 3). (C) WNT-3A up-regulated the expression of the endogenous Wnt canonical target gene AXIN2 in AHACs as evaluated by Q-PCR. Values were normalized for the housekeeping gene β-actin. (D–I) Primary AHACs were treated for 24 h with 100 ng/ml of recombinant WNT-3A or DKK1, alone or in combination, and were subjected to gene expression analysis by PCR. Blockade of the Wnt canonical pathway by DKK1 rescued the modulation of AXIN2, PCNA, and MMP13 mRNA (D–F) but not the down-regulation of COL2A1, Aggrecan, and SOX9 (G–I). (J) In the absence of exogenous WNT-3A, treatment with 100 ng/ml DKK1 resulted, as expected, in down-regulation of the mRNA levels of AXIN2 and PCNA, but also in a paradoxical down-regulation of COL2A1 and Aggrecan similar to that induced by WNT-3A. Q-PCR data, n = 6. Gene expression values are reported as a percentage of up- or down-regulation compared with control (100% is indicated by the broken line in the graph). Data were analyzed with an unpaired t test. Error bars indicate mean ± SEM. *, P < 0.05; **, P < 0.005.

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