(A–D) TOPFLASH assay. Mouse and human serum (10%) and Wnt3A protein (10 ng/ml) activated canonical Wnt signaling to the same degree (A). Activation of Wnt signaling by human serum was suppressed by Fz8/Fc (500 ng/ml). ∗p < 0.05 versus human serum (B). Serum-induced Wnt signaling activity was higher in aged mice (C) and in mice with heart failure (D). Data are presented as mean ±SD. PO, mice with pressure overload; DCM, mice with dilated cardiomyopathy.

(E) Silver staining of SDS-PAGE gel. Serum obtained from control mice and mice with heart failure were incubated with Fz8/Fc and precipitated by protein G. SDS-PAGE of the precipitates revealed that the amount of a protein of ∼26 kDa (arrowhead) was increased in the serum from mice with heart failure. PO, mice with pressure overload; DCM, mice with dilated cardiomyopathy.

(F and G) Pull-down assay. C1q was precipitated by Fz8/Fc, but not by IgG/Fc (F). C1q was precipitated by Fz8 CRD-AP, but not by AP (G).

(H and I) Binding kinetics of C1q to Fz8 CRD. A binding curve (H) and a Scatchard plot (I) are shown.

(J) TOPFLASH assay. C1q dose dependently activated canonical Wnt signaling, which was blocked by Fz8/Fc (20 μg/ml) or Dkk-1 (20 ng/ml). Data are presented as mean ±SD. ∗p < 0.01 versus of C1q (100 μg/ml).

(K) β-catenin stabilization assay. β-catenin stabilization assay was performed in HEK293 cells 1 hr after C1q stimulation (200 μg/ml).

(L) Axin2 mRNA levels. C1q (100 μg/ml) and Wnt3A (10 ng/ml) activate canonical Wnt signaling to the same degree as assessed by Axin2 mRNA induction in HEK293 cells. Axin2 mRNA was assessed 24 hr after stimulation. Data are presented as mean ±SD.

(M and N) Dose-response curves of C1q and Wnt3A on TOPFLASH activity. Fz8 overexpression induced marked leftward shift of the response curve of C1q-induced TOPFLASH activity (M) but had minimal effects on that of Wnt3A-induced TOPFLASH activity (N).