Figure 4
Blocking increased histone deacetylase (HDAC) activity in recombinant human transforming growth factor β 1 (rhTGFβ 1 )-treated primary human osteoblasts neutralizes the inhibitory effect of rhTGFβ1 on recombinant human bone morphogenic protein (rhBMP)-2 and rhBMP-7 signaling. (A) HDAC activity in primary human osteoblasts (N = 4, n = 3) treated with 5 ng/ml rhTGFβ1 (gray bars) for 72 h. In order to inhibit HDAC activity lysates were coincubated with two subtoxic doses (100 μM and 200 μM) of valproic acid. Trichostatin A, provided with the kit used, was used as inhibitor control. (B) Primary human osteoblasts (N = 4) infected with Ad5-BRE-Luc adenoviral particles (Smad1/5/8 reporter construct) were stimulated with 50 ng/ml rhBMP-2 (light grey bars) or rhBMP-7 (dark gray bars) ± 5 ng/ml rhTGFβ1 (hatched bars) and 100 μM or 200 μM HDAC inhibitor (valproic acid) for 72 h. Luciferase activity was measured in cell lysates. The single dose of rhBMP-2 or rhBMP-7 induced Smad1/5/8 signaling, which was completely blocked by rhTGFβ1. Blocking HDAC activity with valproic acid abolished the rhTGFβ1-dependent inhibition of rhBMP-2 and rhBMP-7 induced Smad1/5/8 signaling. **P < 0.01; ***P < 0.001.