mNkd inhibits the canonical Wnt pathway and mNkd-mRNA level is
up-regulated by Wnt. (A) HEK293 cells were transfected
with expression constructs that included 0.02 μg of LEF-1, 0.2 μg
of luciferase reporter (25), 0.02 μg of pRL-TK (Promega), 0.1 μg of
pCGWnt-1, and 0.2 μg of GFP, mNkd, or mNkd-mutant derivatives. LEF-1
luciferase reporter activities were determined. (B) mNkd
did not inhibit the β-catenin-activated Lef-1 reporter. HEK293 cells
were transfected with expression constructs that included 0.02 μg of
LEF-1, 0.2 μg of luciferase reporter (25), 0.02 μg of pRL-TK
(Promega), 0.1 μg of β-catenin, and 0.2 μg of vector or mNkd.
(C) mNkd inhibited Xwnt-8-induced secondary axes
formation in Xenopus. Control Xenopus
embryos are shown at Upper Left. Embryos were injected
ventrally with 5–10 pg of Xwnt-8 RNA and developed with secondary axes
(Upper Right). Secondary axes were scored as complete
(arrows) or partial (arrowheads) based on development of anterior
structures such as eyes and cement glands. Coexpression of mNkd with
Xwnt-8 decreased the frequency of secondary axes formation as well as
the percentage of secondary axes that contained anterior structures
(Lower Left and Lower Right). This effect
was dose-dependent. (D) BALB/c LI mouse liver
epithelial cells were treated with either Wnt-3a or Neo-conditioned
medium (34) for indicated hours. PCR was carried out by using primer
pairs 5′-TGTGAACCATTCCCCCACATCAA-3′ and 5′-AAATGGGGTGTCAAGGAGGTGGAA-3′.
The PCR products were separated by agarose gel electrophoresis. GAPDH,
glyceraldehyde-3-phosphate control.