FZC18 reduces cell sensitivity to soluble Wnt3a.

<p>HEK293T cell batches stably expressing FZC18 (1; 4; 5) or empty vector (V) were incubated with either 50% control or Wnt3a conditioned medium (CM) for 16 hr before lysis. CRT (β-catenin-T-Cell factor Regulated Transcription) assays using Super8•Topflash or the negative control Super8•Fopflash reporters are representative of three independent experiments performed in triplicate and normalized to Renilla luciferase activity (mean±SD).</p

Partially purified FZC18 inhibits Wnt3a-induced Wnt/β-catenin signaling.

<p>CRT assay using the β-catenin reporters Super8•Topflash and Super8•Fopflash, as indicated. (A) HEK293-EBNA cells incubated for 16 hr with either 50% control CM or 50% Wnt3a CM. Wnt3a induces an 80-fold increase in CRT. (B and C) Partially purified, Fc tagged human FZC18_CRD (hFZC18_CRD-Fc) dose-dependently inhibits Wnt3a-induced CRT in HEK293-EBNA cells, as shown with Super8•Topflash <i>(B)</i> and Super8•Fopflash <i>(C)</i> CRT reporters. Cells were incubated for 16 hr with 50% Wnt3a CM that had been pre-incubated overnight on a rotary wheel at +4°C with the indicated concentrations of hFc tag alone (recombinant human Fc from IgG, negative control) or hFZC18_CRD-Fc. Results are shown as mean±SD of hFZC18_CRD-Fc/hFc tag ratios. R² indicates 2^nd degree polynomial regression coefficient. Curve fitting is shown by a red line. Super8•Fopflash <i>(C)</i> negative control CRT reporters (C) are shown for the highest concentrations of hFZC18_CRD-Fc/hFc. (D) Immunoblots show hFc and hFZC18_CRD-Fc from each sample using anti-Fc tag antibody.</p

Stable expression of FZC18 in HEK293T cells.

<p>(A) Schematic structure showing the variant 3 of collagen XVIII containing DUF-959, FZC18, Tsp-1 (thrombospondin-1) and ES (endostatin) domains and the FZC18 expression vector. <i>Interrupted collagenous</i> indicates multiple triple helices (collagenous sequences) interrupted by globular domains. Thick horizontal lines indicate the antibodies used. <i>SP</i>, signal peptide; <i>CRD</i>, Cysteine-Rich Domain; <i>myc</i>, myc epitope tag. (B) HEK293T cells stably expressing FZC18 (batches 1; 4; 5) or empty vector (V) were fixed, permeabilized and immunostained with anti-myc, followed by peroxidase-conjugated antibodies (brown). Cells were counterstained with hematoxylin (blue). Original magnification: ×100. Images were acquired on an Olympus BX60 microscope. (C) Immunoblot with anti-FZC18 <i>(red)</i> and anti-myc <i>(yellow)</i> antibodies in HEK293T cell batches (1; 4; 5) stably expressing FZC18 or empty vector (V). α-tubulin is a loading standard.</p

Hypothetical model for the mode of action of FZC18.

<p><i>High:</i> in the absence of FZC18, Wnt3a increases β-catenin signaling. <i>Low:</i> FZC18 binds Wnt3a and FZD receptors, blocking Wnt/β-catenin pathway activation in an SFRP-like mode of action. <i>Rescued:</i> FZC18 inhibitory effects can be partially rescued by increasing the number of FZD receptors at the cell surface.</p

Frizzled 1 and 8 receptors partially rescue the inhibition of Wnt3a-induced CRT by FZC18.

<p>CRT reporter gene assays using the β-catenin-TCF responsive reporter Super8•Topflash in HEK293T cells stably expressing FZC18 (A, B and C) and vector (A, B). Twenty-four hours after transfection with the CRT reporter and increasing amounts of either FZD1 receptor (A), FZD8 receptor (B), FZD8 receptor, FZD8_CRD or FZC8_CRD-GPI (C) cDNAs, cells were incubated either with 50% control CM (L) or with 50% Wnt3a CM for 16 hr. Results are representative of three independent experiments performed in triplicate and normalized to Renilla luciferase activity. Error bars represent standard deviations.</p