Fig. 5.

ERα activation does not modify the basal or immediate Wnt dependent effects on osteoblast differentiation. Fetal rat osteoblasts were transfected for 24 hours to express native ERα. The cells were treated for 24 hours with vehicle (0), 20 mM LiCl and/or 10 μM WAg alone or in combination with 10 nM of 17βE as indicated. DNA synthesis measured by pulse labeling with [³H]thymidine during the last 2 hours of treatment, and assaying acid insoluble [³H]thymidine incorporation (A). Collagen and noncellagen protein synthesis was measured by pulse labeling with [³H]proline during the last 2 hours of treatment, and assaying acid insoluble [³H]proline incorporation into collagenase digestible and collagenase insensitive cell extracts. Collagen/total protein synthesis was calculated by correcting for the 5.4 fold relative enrichment of proline in collagen (Peterkofsky and Diegelmann, 1971; Centrella et al., 1992) (B). Total protein content was measured with Bradford reagent (Bradford, 1976) (C). Alkaline phosphatase specific activity was measured by p-nitrophenol phosphate hydrolysis and correcting for protein content (Centrella et al., 1995) (D). Osteoprotegerin secretion was determined in conditioned medium extracts (McCarthy et al., 1994), corrected for total protein content, by Western blot (E). WAg significantly increased DNA synthesis rate (A), and suppressed percent collagen synthesis (B) and alkaline phosphatase activity (D) in vehicle or 17βE treated cells, and significantly increased total protein content in 17βE treated cells (C); (* = P <0.05).