WNT-3A promotes intracellular Ca²⁺-mediated phosphorylation of CaMKII and its nuclear translocation. (A) WNT-3A caused calcium mobilization in AHACs, particularly at low doses. Primary AHACs were treated with different amounts of recombinant WNT-3A or vehicle control for 5 min and then subjected to fluorometric determination of calcium mobilization (n = 9). (B) WNT3A-induced calcium mobilization assay in primary AHACs is G protein dependent. AHACs were preincubated overnight at 37°C with 1 µg/ml of the G protein inhibitor PTX and then treated for 5 min with 100 ng/ml of WNT-3A or vehicle control. PTX treatment blocked calcium mobilization induced by WNT-3A (n = 6). (C) Q-PCR for AXIN2 mRNA in primary chondrocytes exposed for 24 h to different doses of recombinant WNT-3A. WNT-3A induced dose-dependent up-regulation of AXIN2 in primary AHACs (n = 3). (D) WNT-3A induced nuclear accumulation of the phosphorylated form of CaMKII. Immunofluorescence staining for pCaMKII in P0 AHACs stimulated for 24 h with WNT-3A in combination with either the CaMKII inhibitor KN93 or its inactive analogue KN92 (both 10 µM). Bar, 20 µm. (E–I). Quantification of the pCaMKII fluorescence intensity in the nuclear and in the cytoplasmic fractions of stimulated AHACs. The pixel intensity profile was plotted over a linear section of a whole cell (E and F). The nuclear and cytoplasmic fluorescence intensity was then calculated as the area under the curve (G–I). (J) WNT3A promotes phosphorylation of CaMKII in T286 at 15 min and 1 h from WNT-3A stimulation. RFU, relative fluorescence unit; RFI, relative fluorescence intensity. Data were analyzed with an unpaired t test except in C, where a Kruskal Wallis with a Dunns post-test was used. Error bars indicate mean ± SEM. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.