Bili inhibits Wnt/beta-catenin signaling by regulating the recruitment of axin to LRP6

Abstract

Background: Insights into how the Frizzled/LRP6 receptor complex receives, transduces and terminates Wnt signals will enhance our understanding of the control of the Wnt/ss-catenin pathway.

Methodology/principal findings: In pursuit of such insights, we performed a genome-wide RNAi screen in Drosophila cells expressing an activated form of LRP6 and a beta-catenin-responsive reporter. This screen resulted in the identification of Bili, a Band4.1-domain containing protein, as a negative regulator of Wnt/beta-catenin signaling. We found that the expression of Bili in Drosophila embryos and larval imaginal discs significantly overlaps with the expression of Wingless (Wg), the Drosophila Wnt ortholog, which is consistent with a potential function for Bili in the Wg pathway. We then tested the functions of Bili in both invertebrate and vertebrate animal model systems. Loss-of-function studies in Drosophila and zebrafish embryos, as well as human cultured cells, demonstrate that Bili is an evolutionarily conserved antagonist of Wnt/beta-catenin signaling. Mechanistically, we found that Bili exerts its antagonistic effects by inhibiting the recruitment of AXIN to LRP6 required during pathway activation.

Conclusions: These studies identify Bili as an evolutionarily conserved negative regulator of the Wnt/beta-catenin pathway.

Figure 1. dBili (cg11848) is a negative regulator of Wg signaling.

(A) Secondary reporter assay in clone 8 cells showing gfp, armadillo (arm), ran and dBili dsRNA effects on wingless (wg) (white bars) and ΔNLRP6 (gray bars) activation of β-catenin reporter, dTF12. Individual dsRNA effects on reporter activity in the absence of wg or ΔNLRP6 (inset). (B) Expression pattern of dBili mRNA by in situ hybridization. Embryos are oriented with anterior on the left and posterior on the right. (a & b) Stages 5–6 showing dBili expression in the neuroectoderm and cells adjacent to the ventral midline (black arrows) with high levels of expression in cells adjacent to the ventral furrow (white arrowhead). (c–e). At stages 13–17 the expression of dBili in the developing central nervous system (panel c & e) and the ventral epidermis in stripes (e & e'). At the end of embryogenesis (stage 16/17), the majority of dBili expression is restricted to the CNS (panel d). (C) (a & b) Wing imaginal discs. Panel a, shows dBili expression in the wing pouch (black arows) and in the notum/hinge area (red arrow). Panel b, staining for Wg protein in wing pouch and notum/hinge (blue arrow). (b & c) Leg imaginal discs. Panel c shows dBili staining in the ventral wedge (black arrows), identical to Wg expression (white arrow, panel b) (d & e) Expression in the eye disc of dBili (black arrows, d) and Wg (white arrows, e). Expression in the antenna disc of dBili (d, red arrow) and Wg (e, blue arrow).

Figure 2. Expansion of wg signaling activity in embryos (ventral view) with diminished levels of dBili.

(A) Cuticle preparation of WT control embryo displaying wild type patterning of the embryonic epidermis with uniformly spaced denticle belts. (B) Cuticle preparation of embryo expressing shRNA targeting dBili (shdBili). (B') WT and shdBili expressing embryos showing a weaker phenotype (black arrows). (C & D) High magnification view of A & B, respectively. D' & E show boxed region in D. (E–I) Engrailed (En) protein expression. (F & G) Expression of engrailed in WT embryos is restricted to one or two cell-layers in the epidermis during early stage 12 (germband retraction) and in the ventral epidermis at stage 14 (onset of head involution and dorsal closure). (H & I) En expression in shdBili stage-matched embryos (compare with F & G).

Figure 3. Bili's effect on Wnt signaling is conserved in zebrafish.

(A) Wnt8 overexpression phenotypes with increasing severity. Embryos were scored as wt (green), small eyes (yellow), no eyes (orange) or severe (red). (B) Morpholino effect on in vitro expression of human Bili (hBili) or zfBili protein (control (como) or zfBili morpholino (bilimo)). (C) In the top panel, the effect of bilimo (loss of function) on Wnt8 GOF phenotype compared to como, is shown (n = 73 low Wnt dose, n = 147 medium Wnt dose, n = 57 high Wnt dose). Bili GOF (bili rna) rescues Wnt8 overexpression phenotype (control Renilla (co rna)). (n = 125 per dose of Wnt8) (bottom panel).

Figure 4. Bili inhibits Wnt/β-catenin transcriptional reporters in human cell-lines.

(A) Pretreatment with Bili or Axin1 siRNA leads to a 2-fold greater activation of a β-catenin responsive reporter after treatment with WNT3A conditioned media (black bars) in RKO-BAR cells compared to control siRNA. β-catenin (β-cat) siRNA ablated Wnt 3A mediated reporter activation. Error bars represent STDEV. (B) hBili overexpression inhibits Wnt3A mediated β-catenin responsive reporter activation in HEK293T cells. (C) hBili overexpression inhibits WNT1, ΔNLRP6, and DVl, but not BIO, or β-catenin mediated β-catenin responsive reporter activation in HEK293T cells. HEK293T cells stably expressing a β-catenin responsive luciferase reporter were transfected or treated with WNT1, ΔNLRP6, DVL, BIO, or β-catenin along with GFP or Bili. Error bars represent STDEV. (D) Bili colocalizes with LRP6. HEK293T cells were transfected with flag-hBili and hLRP6-eGFP and imaged. (*p<0.05, **p<0.005, ***p<0.0005)

Figure 5. Bili associates with LRP6 and attenuates Axin recruitment to LRP6 in HEK293T cells.

(A) hBili interacts with LRP6. HA-LRP6 was cotransfected with flag-GFP or flag-hBili in HEK293T cells. Cell lysates were immunoprecipitated with anti-flag antibody and immunoblotted with anti-HA antibody. (B) dBili interacts with LRP6. dBili-HA and ΔNLrp6-myc were cotransfected in S2R+ cells. Cell lysates were immunoprecipitated with anti-myc antibody and immunoblotted with anti-HA antibody. (C) Overexpression of Bili attenuates GSK3 induced AXIN/LRP6 association. HEK293T cells stably expressing LRP6-eGFP were cotransfected with AXIN-myc, GSK3B and Bili-flag or vector control. Cell lysates were immunoprecipitated with anti-eGFP antibody and immunoblotted with anti-myc. Data shown are representative of three independent experiments. (D) Bili knockdown enhances GSK3B induced AXIN/LRP6 association. HEK293T cells stably expressing LRP6-eGFP were transfected with Bili siRNA or control siRNA. 48 hours later BilisiRNA or control siRNA treated cells were transfected with AXIN-myc and GSKB. Cell lysates were immunoprecipitated with anti-eGFP antibody and immunoblotted with anti-myc. Data shown are representative of three independent experiments.