(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).
See also .

(A) TOPFLASH assay. Wnt signaling activation by serum was partially blocked by Fz8/Fc (10 μg/ml) or C1q depletion, but not by C3 or C5 depletion. Combination of Fz8/Fc and C1q depletion did not further decrease TOPFLASH activity. Data are presented as mean ±SD. *p < 0.01 versus normal serum.
(B) TOPFLASH assay. In wild-type (WT) mice, serum from aged mice showed higher TOPFLASH activity than serum from young mice. Serum from young C1qadeficient mice showed lower TOPFLASH activity compared with serum from young WT mice, and the elevation of TOPFLASH activity during aging was not observed in C1qa-deficient mice. Data are presented as mean ±SD. *p < 0.01 versus serum obtained from young WT mice. **p < 0.01 versus aged serum obtained from WT mice.
(C–F) β-catenin stabilization assay. Human serum activated Wnt signaling, which was abolished by Fz8/Fc (10 μg/ml) (C). Wnt signaling activation by serum was also abolished by C1q depletion, but not by C3 or C5 depletion (D). Reduced Wnt signaling activation by C1q depletion was fully restored by C1q (10 μg/ml) application (E). The results were quantified by measuring the relative amount of β-catenin over actin (F). Data are presented as mean ±SD. *p < 0.05 versus normal human serum (n = 5).
(G) Expression levels of Axin2 mRNA in various tissues of 3-month-old wild-type (n = 8), C1qa-deficient (n = 8), and C3-deficient (n = 4) mice. Expression levels of Axin2 gene expression were lower in various tissues of C1qa-deficient mice, but not in those of C3-deficient mice. Data are presented as mean ±SD. *p < 0.05 compared with wild-type mice. L.N., lymph node; SkM, skeletal muscle.
See also .

(A and B) Serum C1q concentration of mice at different ages was assessed by ELISA (A) and western blot (B). Serum C1q concentration was increased with aging. Data in (A) are presented as mean ±SD.
(C and D) Western blot analysis of C1q in peritoneal macrophages (C) and in various tissues (D) derived from wild-type mice at different ages. C1q expression in macrophages and skeletal muscle was increased at 1 year of age, whereas a robust increase in C1q expression in other tissues was observed at 2 years of age.
(E) Expression levels of Axin2 mRNA in various tissues from young (3 months old) and aged (2 years old) wild-type (young, n = 8; aged, n = 4) and C1qa-deficient mice (young, n = 8; aged, n = 3). Axin2 gene expression was increased with aging in multiple tissues of wild-type mice (WT), but not in those of C1qa-deficient mice (C1qKO). L.N., lymph node; SkM, skeletal muscle. Data are presented as mean ±SD. *p < 0.05 compared with young wild-type mice.

(A) HepG2 cells were cultured and stimulated with or without C1q (100 μg/ml) in a serum-free condition for 24 hr. Culture media and total cell lysate were analyzed by western blotting. Both C1r and C1s protein were observed in the culture media under serum-free condition.
(B) 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) did not.
(C) TOPFLASH assay. HEK293 cells transfected with control siRNA (siCon) responded to both C1q (100 μg/ml) and Wnt3A (10 ng/ml), but those transfected with siRNAs against C1r and C1s (siC1r/s) responded to Wnt3A, but not to C1q. Data are presented as mean ±SD.
(D) TOPFLASH assay. Activation of Wnt signaling by C1q (100 μg/ml) was inhibited by an endogenous C1-inhibitor (C1-INH: 100 μg/ml) or by a neutralizing antibody against C1s (M241: 100 μg/ml). Data are presented as mean ±SD. *p < 0.01 versus C1q alone.
(E and F) C4 cleavage assay. C4b deposition on the cell surface was assessed by immunostaining (E) or ELISA (F). C4b deposition was increased after Fz8 overexpression. Data are presented as mean ±SD. *p < 0.05 versus control vector (n = 5).
(G) Coomassie staining of SDS-PAGE gel. LRP6 extracellular domain (ECD)-IgG/Fc fusion protein (4 μg) was incubated with active-C1s (176 ng) with or without a neutralizing antibody against C1s (M241). Proteins were fractionated by SDS-PAGE and visualized by Coomassie staining. C1s treatment of LRP6 ECD resulted in the appearance of two major bands (indicated by * and **). Amino acid sequencing revealed that * represented LRP6 ECD (amino acids 793–1368) + IgG/Fc, and ** represented LRP6 ECD (amino acids 20–792).
(H) Amino acid sequence alignment of potential C1s cleavage site in the third β-propeller domain of LRP5 and LRP6. C1s cleavage site is predicted to be between arginine (R) and alanine (A). Cleavage site of C1s is highly conserved among species.

(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 .

(A and B) β-catenin stabilization assay. Satellite cells and fibroblasts were stimulated with C1q (100 μg/ml) or Wnt3A (10 ng/ml). Both C1q and Wnt3A activated Wnt signaling in these cells (A). Cells were also stimulated with serum derived from young (2 months old) or aged mice (2 years old). The extent of Wnt signaling activation by serum from aged mice was greater than that by serum from young mice, and activation of Wnt signaling by serum from aged mice was attenuated by M241.
(C) X-gal staining of skeletal muscle after injury. Skeletal muscle of young (2 months old) TOPGAL mice was cryoinjured and treated with PBS or C1q (50 μg/ml). X-gal staining showed that β-gal activity was slightly increased 2 days after cryoinjury, which was enhanced by C1q.
(D) Quantitative analysis of β-gal activity. TOPGAL mice were treated as in (C), and tissue β-gal activity was measured and corrected with tissue weight. Data are presented as mean ± SD. *p < 0.01 versus sham-operated mice treated with PBS (n = 10).
(E) Real-time PCR analysis. Mice were treated as in (C), and the expressions of lrp5, lrp6, C1r, and C1s were analyzed by real-time PCR. Data are presented as mean ± SD. *p < 0.01 versus sham-operated mice (n = 6).
(F and G) BrdU incorporation assay in satellite cells (F) and fibroblasts (G). Satellite cells and fibroblasts were stimulated with C1q (100 μg/ml) or Wnt3A (10 ng/ml) for 24 hr. BrdU incorporation during the last 12 hr (satellite cells) or 4 hr (fibroblasts) was assayed by ELISA. C1q and Wnt3A inhibited satellite cell proliferation and stimulated fibroblast proliferation. Data are presented as mean ± SD. *p < 0.01 versus control (n = 4).
(H) Collagen concentration in the culture media. After stimulation with C1q (100 μg/ml) or Wnt3A (10 ng/ml) for 24 hr, medium was changed to serum-free medium, and soluble collagen released to the medium was quantified 6 hr later. C1q and Wnt3A increased collagen production in fibroblasts. Data are presented as mean ±SD. *p < 0.01 compared with control (n = 4).
(I and J) BrdU incorporation assay in satellite cells (I) and fibroblasts (J). Satellite cells and fibroblasts were cultured and stimulated with serum (5%) for 24 hr. BrdU incorporation was assayed as in (F) and (G). Serum from aged mice reduced satellite cell proliferation and stimulated fibroblast proliferation, which was attenuated by M241. Data are presented as mean ±SD. *p < 0.01 versus serum from young mice. **p < 0.01 versus serum from aged mice (n = 4).
(K) Collagen concentration in the culture media. After stimulation with serum for 24 hr, soluble collagen in the medium was assayed as in (H). Serum from aged mice increased collagen production in fibroblasts, which was attenuated by M241 treatment. *p < 0.01 versus serum from young mice. Data are presented as mean ±SD. **p < 0.01 versus serum from aged mice (n = 4).
(L and M) Number of proliferating satellite cells (L) and fibroblasts (M) in cryoinjured skeletal muscle of young mice (2 months old) in vivo. Sections were immunostained with M-cadherin (a satellite cell marker), Vimentin (a fibroblast marker), and phospho-histone H3 (pH3) (a mitotic marker). Proliferating satellite cells and fibroblasts were identified as M-cadherin/pH3 double-positive cells and Vimentin/pH3 double-positive cells, respectively. C1q treatment reduced satellite cell proliferation and stimulated fibroblast proliferation in cryoinjured skeletal muscle. Data are presented as mean ±SD. *p < 0.05 versus control (n = 5).
See also .

(A) Axin2 mRNA expression. Skeletal muscle of young (2 months old) wild-type (WT) and C3-deficient (C3KO) mice was cryoinjured and treated with PBS or C1q (50 μg/ml). RNA was extracted 3 days later. C1q treatment increased Axin2 gene expression in injured skeletal muscle of both wild-type and C3-deficient mice. Data are presented as mean ±SD. *p < 0.01 versus PBS (n = 4).
(B) Immunostaining of skeletal muscle after cryoinjury. Tissue samples were harvested 5 days after injury and immunostained with embryonic myosin heavy-chain (Red) and type I/III Collagen (green). Four wild-type mice (eight samples) and three C3-deficient mice (six samples) were used for each group, and representative figures are shown. C1q treatment impaired muscle regeneration and increased fibrosis in both wild-type and C3-deficient mice. Scale bar, 150 μm.
(C) Expression of Col3a1 gene. RNA was harvested 3 days after injury. C1q treatment increased Col3a1 expression in injured skeletal muscle of both wild-type and C3-deficient mice. Data are presented as mean ±SD. *p < 0.01 versus PBS (n = 4).
(D) Soluble collagen content in skeletal muscle. Samples were harvested 5 days after injury. C1q treatment increased soluble collagen content in skeletal muscle after cryoinjury of both wild-type and C3-deficient mice. Data are presented as mean ±SD. *p < 0.01 versus PBS (n = 4).
(E) Axin2 mRNA expression. Skeletal muscle of aged (2 years old) wild-type (WT) mice or aged C1qa-deficient mice (C1qKO) was cryoinjured and treated with M241 or BB5.1 (500 μg/ml each). RNA was extracted 3 days after cryoinjury. The expression of Axin2 was suppressed by M241 treatment or in C1qa-deficient mice, but not by BB5.1 treatment. Data are presented as mean ±SD. *p < 0.01 versus aged WT PBS (n = 4).
(F) Immunostaining of skeletal muscle after cryoinjury. Tissue samples were harvested 5 days after injury and immunostained as in (B). Three wild-type mice (six samples) and two C1qa-deficient mice (four samples) were used, and representative figures are shown. Impaired skeletal muscle regeneration in aged mice was restored by M241 treatment, but not by BB5.1 treatment, and was not observed in C1qa-deficient mice. Scale bar, 150 μm.
(G) Expression of Col3a1 gene. RNA was extracted 3 days after cryoinjury. The expression of Col3a1 gene was reduced by M241 treatment or in C1qa-deficient mice, but not by BB5.1 treatment. Data are presented as mean ±SD. *p < 0.01 versus aged WT PBS (n = 4).
(H) Soluble collagen content in skeletal muscle. Samples were harvested 5 days after cryoinjury. Soluble collagen content was attenuated by M241 treatment or in C1qa-deficient mice, but not by BB5.1 treatment. Data are presented as mean ±SD. *p < 0.01 versus aged WT PBS (n = 6).