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Fig. 1. TGF-ß regulates Wnt5a expression in mice. (A)
Semiquantitative RT-PCR of Wnt5a cDNA from wild-type and DNIIR
mammary glands, illustrating reduced expression of Wnt5a mRNA in
DNIIR glands relative to wild-type control glands. 18S was used to normalize
for the amount of total RNA. PCR product is shown in the linear range (22
cycles for Wnt5a and 15 cycles for 18S). Wild-type and DNIIR labels represent
mammary glands from individual mice administered ZnSO4 for 2 weeks
to induce DNIIR transgene expression. (B) Wnt5a and HGF protein levels
in wild-type and DNIIR mammary glands depicted by western blot show reduced
Wnt5a protein levels in DNIIR glands and elevated HGF levels. Wild-type and
DNIIR labels represent mammary glands from separate mice. ß-Tubulin was
used to normalize for the amount of protein loaded. (C-F) Primary
epithelial cells and fibroblasts isolated from 8-week-old adult virgin Balb/c
mice were cultured and treated with TGF-ß1. Treatment resulted in an
induction of Wnt5a mRNA as measured by semiquantitative RT-PCR in
both primary epithelial cells (C) and primary fibroblasts (D). Induction of
Wnt5a was also seen at the protein level in both cell types (E,F). Vimentin
expression was used as a marker for fibroblasts. (G,H) Mammary
epithelial cells (G) and fibroblasts (H) were untreated (-) or pretreated (+)
with cycloheximide followed by treatment with vehicle (-) or TGF-ß1 for
12 hours (+), at which time RNA was extracted from the cells. The level of
Wnt5a mRNA was determined by semi-quantitative RT-PCR. Treatment with
TGF-ß in the presence of cycloheximide resulted in an increase in
Wnt5a mRNA, suggesting that TGF-ß stimulates Wnt5a expression
even in the absence of new protein synthesis.
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