User:Kinkreet/SCRA/Complement C1q Activates Canonical Wnt Signaling and Promotes Aging-Related Phenotypes

Wnt is a large family of secreted proteins involved in development and the self-renewal and differentiation of stem cells. Canonical Wnt signalling molecules binds to Wnt receptors Frizzled family of serpentine proteins, and low-densitiy lipoprotein receptor related protein 5/6 (LRP5/6); once activated, cytoslic beta-catenin is no longer degraded but stabilized, and translocates into the nucleaus where it binds to T cell factor/Lymphoid enhancer factor (Tcf/Lef) and induces Tcf/Lef transcription.

Complement C1q (in the serum) activates the canonical Wnt pathway by binding to the extracellular cysteine-rich domain (CRD) of the Wnt receptor Frizzled (Fz), inducing C1s-dependent cleavage of the ectoderm of Wnt coreceptor low-densitiy lipoprotein receptor-related protein 6 (Lrp6). Activation of Wnt decrease the ability of cells to regenerate (sign of aging). Further evidence to suggest this includes:


 * Serum C1q expression and concentration increases with aging (as determined using ELISA and Western blot analysis ), this coincides with increased Wnt signalling during aging.
 * During aging in wild-type mice, Wnt signalling is augmented. Wnt signalling is not augmented in Cq1-deficient mice.
 * Administration of exogenous Cq1 inhibited muscle regeneration. Disruption of the C1qa gene or inhibition of C1s leads to retoration of regeneration.
 * Canonical Wnt signalling is augmented in mouse model of accelerated aging
 * Inhibition of Wnt signalling reverse age-associated impairment of skeletal muscle regeneration..

Method for measuring canonical Wnt signalling
A TOPFLASH reporter gene assay is used to determine the level of canonical Wnt activation. The version used here involves a vector containing a single luciferase gene regulated by 8 Tcf/Lef binding sites (AGATCAAAGG), with each site separated by a spacer (GGGTA). Stabilized β-catenin translocate to the nucleus and binds to and activate the Tcf/Lef transcription factor, which in turn binds to Tcf/Lef binding sites and induce the expression of the gene it regulates, which in this case is the luciferase gene. Thus, the higher the activation of Wnt, the higher the population of cytosolic stabilized β-catenin, the higher the level of Tcf/Lef transcription, and the high expression and thus concentration of luciferase in Wnt-active cells.

Cells would have been plated and transfected with the TOPFLASH vector. 2 days after transfection, the cells are lysed and the levels of luciferase is measured by luciferase reporter assay kit, which makes use of the fact that luciferase is bioluminescent, thus increase in Wnt signalling can be monitored by the light intensity emitted from the cells.

(need to criticize this method - not precise because transfection have a low success rate, background bioluminescence. The transfection process itself may induce the cell to respond. The group said so themselves, that TOPFLASH is carried out a late time points, and therefore be affected by other factors which indirectly modulate Tcf/Lef.

C1q binds specifically to Frizzled
It has already been previously described that Wnt signalling is increased in aging samples, and thus the level of Wnt ligands associated with aging is likely to increase in aging samples compared to young samples. The Komuro group used the TOPFLASH reporter gene assay and found that canonical Wnt signalling is also increased in mice with heart disease (specifically pressure overload and dilated cardiomyopathy).

Frizzled (Fz) is part of the Fz/low density lipoprotein (LDL) receptor-related protein (LRP) complex to which Wnt proteins bind to; thus, any Wnt ligands is likely to bind to Fz (more specifically, the cysteine-rich domain, or CRD). Using a Fz8CRD-IgG/Fc fusion proteins to precipitate Fz-binding proteins (using protein G; IgG/Fc was used as a control), the Komuro group was able to identify a 26kDa protein upregulated in aging mice, and even more so in mice with heart disease. Mass spectrometry identified the band as C1qa, a major constituent of the complement C1q. The experiment was repeated for Fz1, -2, -4 and -7, and the results was the same.

C1q is a Wnt ligand which binds specifically to Frizzled
Because there is a strong correlation between the levels of C1qa and Wnt signalling, it has led to the hypothesis that C1qa is a Wnt ligand particularly involved in aging. To confirm this hypothesis, Komuro and group administered C1qa to mice and observed an increased level of Tcf/Lef transcription (as determined by TOPFLASH) and a corresponding increased level of stabilized cytosolic β-catenin (the population which induces Tcf/Lef transcription. This binding of C1qa is thought to be specific and in competition with Wnt (as determined by a heterologous competition assay), with similar binding affinities but with a 200-fold effective dose.

C1qa is unique within the complements as C3 or C5 depleted serum did not reduce Wnt activity nor reduce β-catenin levels, only C1q depleted serum reduced Wnt activity and β-catenin levels. Adding Fz8/Fc into C3 or C5 depleted serum also reduced Wnt activity, but Fz8/Fc addition into C1q depleted serum did not reduce Wnt activity further.

C1q is a Wnt ligand which binds specifically to Frizzled and increases Wnt signalling by C1s-dependent cleavage of the extracellular domain of LRP6
The complement system is part of the innate immunity; the classical pathway is triggered by the activation of C1, which consists of C1q and two proenzymes C1r and C1s. C1q binds to aggregated immunoglobulins which alters its conformation and activates it; this causes the autoactivation of C1r and consequently C1s. C1s then cleaves C2 and C4 to activates subsequent steps of the complement pathway. C1r and C1s is expressed and secreted by target cells, and is involved in C1qa-dependent canonical Wnt activation. Knockdown of C1r and C1s using siRNA or inhibition of C1r/C1s using antibodies and inhibitors led to a decrease in C1qa-dependent canonical Wnt activation. Induction of C1qa led to the appearance of cleaved LRP6 N-terminal fragments in the media, where the LRP6 is cleaved between residues Arg792-Ala793. The C-terminal domain seems be be autophagocytosed after cleavage as chloroquine treatment (which prevents autophagosome-lysosome fusion and thus inhibits autophagy) saw an accumulation of undegraded LRP6 ICD.

The cleavage of LRP6 is C1qa-dependent, N-terminally myc-tagged LRP6 was transfected into HepG2 cells (a human liver carcinoma cell line). Immunoblotting using anti-myc antibody showed that upon normal serum treatment, the N-terminal (containing the myc tag) is cleaved and found in large numbers in the culture medium; while with C1qa-depleted serum, the N-terminal remains with the cell. This is observed in mice models also.

It is likely that the C-terminal domain is responsible for Wnt signalling transduction. Transfection of DEL-LRP6 (a deletion mutant lacking the N-terminus prior to the cleavage point) into the cell saw a 47-fold increase in Wnt signalling compared to WT-LRP6. Phosphorylation of the cleaved fragment was also increased after C1qa treatment of the wildtype or transfected mutant. A mutant C1qa which has glycine residues substituted for Arg792-Ala793 saw a lower Wnt activation rate compared to wildtype after C1qa stimulation. These suggest that the cleaved C-terminus of LRP6 is sufficient for Wnt signalling activation, and phosphorylation may have a role in this process.

C1qa activates Wnt signalling in skeletal muscle and decrease regenerative capacity
Treatment of satellite cells and fibroblasts, derived from young mice, with C1q and Wnt3a resulted in the stabilization of beta-catenin and thus increased Wnt signalling; this is marked by an increase in Axin2 mRNA expression, which has previously been linked to increased Wnt signalling. They repeated this experiment in aged mice, and found that the increase in Wnt signalling is even more prominent in both cell populations. Thus, C1qa induces Wnt activation in both satellite cells and fibroblasts in vitro.

In vivo, using hydrogel containing C1q for the treatment, it was shown that C1q alone did not induce Wnt activation; it is only when the tissue is cryoinjured did Wnt signalling activity dramatically increased. This is probably because after injury, the expression of the C1r and C1s gene is upregulated (as determined by RT-PCR), providing a higher sensitivity to C1q-mediated Wnt activation.

The physiological effect of C1q treatment in vivo after cryoinjury, presumably through increased Wnt signalling, is increase fibrosis and decreased satellite cell proliferation. Thus, C1q impairs skeletal muscle regeneration. To confirm this, the level of collagen, a marker of fibroblasts, is increased after C1q treatment, or in aged mice. These effects were quenched using M241, an antibody against C1q.

C1qa-mediated promotion of age-related phenotypes are independent of the classical complement pathway
Canonical Wnt signalling was activated when the gastrocnemius muscle of young mice were cryoinjured and then treated with C1qa. The administration of C1qa or the activation of canonical Wnt impaired regeneration and resulted in more fibroblasts in the tissue. In C3-deficient mice, whose classical complement pathway is nulled, Wnt activation and levels of regeneration in control and C1qa treated mice saw no difference with wildtype mice. This means that the impairment of regeneration mediated by C1qa acts independently of the classical complement pathway.

The gastrocnemius muscle of aged wildtype and C1qa-deficient (C1qaKO) mice were cryoinjured, a hydrogen containing M241 or BB5.1 was inserted. BB5.1 is an antibody against C5 and inhibits the classical complement pathway without affecting C1; whereas M241 inhibits the classical complement pathway by inhibiting C1s and C1q. In M241 treated mice, the activation of Wnt was at comparable levels to C1qaKO mice, further indicating C1qa/Wnt-mediated impairment of regeneration is independent of the classical complement pathway.