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Figure 4. Shisa Cell-Autonomously Inhibits Wnt Signaling by Retaining Fz within the ER (A) Schematic drawing of Shisa, fused with the ER retention KDEL signal, and deletion constructs employed. Shisa-KDEL, 5H-Shisa, and 3H-Shisa were generated by fusing a cassette containing a heterologous signal peptide followed by a Flag sequence to shisa cDNA fragments. Numbers in parentheses indicate corresponding Shisa amino acid residues. (A′) Western blotting with α-Flag mAb shows equivalent protein productions of each construct. Cells were transfected with each construct (100 ng) in 12-well plate. (A′) KDEL signal suppressed secretion of Shisa. Cells were transfected as in (A′). (B) Shisa and Shisa-KDEL, but not 5H- and 3H-shisa, inhibited TOPFLASH reporter activation induced by XWnt8-Fz8-Lrp6 expression. Luciferase activities are indicated as fold activation/repression compared with the activity obtained from cells transfected with empty-vector and reporter (lane 1). Each experiment was carried out at least in triplicate, and error bars represent the standard deviation. Transfection was carried out in a 96-well plate with DNAs (per well): TOPFLASH reporter, 10 ng; nlacZ, 1 ng; Xwnt8, 5 ng; fz8, 1 ng; lrp6, 1 ng; shisa, shisa-KDEL, 5H-shisa, and 3H-shisa, 5 ng (+) or 25 ng (++). (C) Shisa failed to inhibit the signaling induced by a high dose of Lrp6 alone. DNA used: lrp6, 20 ng; shisa 5 ng for lane 3; and 25 ng for lane 4. (D) Shisa cell-autonomously inhibited Wnt signaling in the cells receiving the signal. Stimulator and receptor cells were transfected separately and mixed in the combination presented at the bottom of the figure. Co: Cells transfected with the empty-vector alone. DNA used: mwnt3a, 500 ng; fz8, 8 ng; lrp6, 8 ng; TOPFLASH reporter, 80 ng; nlacZ, 8 ng; shisa, 200 ng. (E–I) Shisa suppressed Wnt8-AP and Fz8 interaction. Live cells were stained with 1 nM of Wnt8-AP. Cells in an 8-well chamber slide were transiently transfected with DNAs: fz8, 2 ng; shisa, 50 ng. CM, condition media used. T, construct used for transfection. (J–O′) Confocal immunofluorescent images of HEK 293T cells. Phase contrast images were (J), (K), (L), (M), and (N). ER was marked by DsRedER in (J′), (K′), (L′), (M′), and (N′). (J–J‴) Transfected with fz8-GFP (green) (cell surface expression of Fz, n = 100, 98%). (K–K‴) Transfected with fz8-GFP and shisa (ER retention of Fz, n = 100, 90%). (L–L‴) Transfected with fz8-GFP and shisa-KDEL (ER retention of Fz, n = 100, 84%). (M–M‴) Transfected with fz8-GFP and 5H-shisa (cell surface expression of Fz, n = 100, 92%). (N–N‴) Transfected with lrp6-GFP and shisa (cell surface expression of Lrp6, n = 100, 86%). (O–O′) Transfected with fz8-HA and shisa-myc (colocalization, n = 100, 84%). Cells were transfected in an 8-well glass chamber with DNAs: fz8-GFP, 2 ng; Lrp6-GFP, 2 ng; fz8-HA, 2 ng; shisa, 50 ng; shisa-KDEL, 50 ng; 5H-shisa, 50 ng; shisa-Myc, 50 ng; pDsRed-ER, 10 ng. Image published in: Yamamoto A et al. (2005) Copyright © 2005. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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