(A) Western blot analysis of LRP6 fragment in the culture media from HepG2 cells treated with C1q (100 μg/ml). N-terminal cleaved fragment of endogenous LRP6 was detected in the culture media. ECD, extracellular domain; ICD, intracellular domain.
(B) Western blot analysis of LRP6 in the membrane/organelle fraction of HepG2 cells treated with C1q (100 μg/ml) or Wnt3A (10 ng/ml). C-terminal cleaved fragment of LRP6 (~140 kDa) was detected by anti-LRP6 ICD Ab only in the cells treated with C1q plus lysosomal inhibitor Chloroquine (50 μM).
(C) Western blot analysis of the N-terminal cleaved form of LRP6 in culture media. N-terminal cleaved form of LRP6 was detected in culture media conditioned by cells treated with normal human serum, but not with C1q-depleted serum. Addition of purified C1q protein (100 μg/ml) to C1q-depleted serum restored the activity to cleave LRP6. IgHC, immunoglobulin heavy chain.
(D and E) N-terminal cleaved fragment of LRP6 in the serum was analyzed by western blotting (D) and ELISA (E). In wild-type (WT) mice, the amount of cleaved form of endogenous LRP6 ectodomain was increased by 2-fold in serum from aged mice (2 years old: 130 ng/ml) compared with serum from young mice (2 months old: 60 ng/ml). Cleaved LRP6 was not detected in the serum from young C1qa-deficient mice. Data are presented as mean ±SD.
(F–H) TOPFLASH assay. Overexpression of N-terminal truncated LRP6 (Del-LRP6) resulted in enhanced activation of Wnt signaling compared with wild-type LRP6 (WT-LRP6) (F). Cells transfected with WT-LRP6 responded to both C1q (100 μg/ml) and Wnt3A (10 ng/ml) (G), whereas those transfected with C1s-resistant LRP6 (Mt-LRP6) responded to Wnt3A, but not to C1q (H). Data are presented as mean ±SD.
(I) Western blot analysis of C-terminal LRP6 fragment in the membrane/organelle fraction and N-terminal LRP6 fragment in the culture media after treatment of HepG2 cells with C1q (100 μg/ml). Both C-terminal and N-terminal LRP5/6 fragments were not detected in cells transfected with siRNAs against C1r and C1s (C1r/s), LRP5 and LRP6 (LRP5/6), or cells transfected with Shisa (Shisa O/E). An arrow indicates C-terminal LRP6 fragment, and an arrowhead indicates N-terminal LRP6 fragment.
(J) (Top) β-catenin stabilization assay. HepG2 cells transfected with control siRNA (Con) responded to C1q (100 μg/ml), but those transfected with siRNAs against C1r and C1s (C1r/s), LRP5 and LRP6 (LRP5/6) or cells transfected with Shisa (Shisa O/E) did not. (Bottom) TOPFLASH assay. HEK293 cells transfected with control siRNA responded to both C1q (100 μg/ml) and Wnt3A (10 ng/ml), whereas those transfected with siRNAs against C1r and C1s (C1r/s) responded to Wnt3A, but not to C1q. Data are presented as mean ±SD.
(K) Schematic diagram of C1q-induced activation of Wnt signaling. Upon binding to Fz receptors, C1q activates C1r/C1s, which results in LRP5/6 cleavage and activation of Wnt signaling.
See also .