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.