Fig. 2.

Canonical Wnt pathway activation co-stimulates ERα activity. Fetal rat osteoblasts were transfected for 24 hours to express reporter plasmid TOP-Flash, or ERE driven reporter plasmid, or the oxytocin gene promoter in combination with expression vectors encoding native ERα and Wnt1 (A,B) or with ERα alone (C). The cells were treated with vehicle (0), 10 nM 17βE, 20 mM LiCl, and/or 0.6 to 6 μM of the GSK3 inhibitor SB 415286 (SB) (bottom left panel, B) or 6 μM of SB (bottom right panel, B; C) as indicated, and reporter gene enzyme activity was measured after 24 hours (A,B), or nuclear and cytoplasmic extracts, corrected for total protein content, were probed by Western blot analysis with antibody to β-catenin or cyclophilin B (loading controls) (C). LiCl (A,B) and Wnt 1 expression significantly (A) increased TOP-Flash activity alone, and 17βE significantly increased TOP-Flash activity alone and enhanced the effect of LiCl (A,B). 17βE at 10 nM significantly increased the ERE (A,B) or oxytocin promoter (A) driven reporters alone, and LiCl (A,B) or Wnt1 expression (A) enhanced the effect of 17βE. SB at 6 μM significantly increased the effect of 17βE through ERE in control and LiCl treated cells (B); (* = P <0.05).