Glucocorticoids inhibit the transcriptional activity of LEF/TCF in differentiating osteoblasts in a glycogen synthase kinase-3β-dependent and -independent manner

Abstract

Glucocorticoids, widely used as immune suppressors, cause osteoporosis by inhibiting bone formation. In MC3T3-E1 osteoblast-like cultures, dexamethasone (DEX) activates glycogen synthase kinase-3β (GSK3β) and inhibits a differentiation-related cell cycle that occurs at a commitment stage immediately after confluence. Here we show that DEX inhibition of the differentiation- related cell cycle is associated with a decrease in β-catenin levels and inhibition of LEF/TCF-mediated transcription. These inhibitory activities are no longer observed in the presence of lithium, a GSK3β inhibitor. DEX decreased the serum-responsive phosphorylation of protein kinase B/Akt-Ser 473 within minutes, and this inhibition was also observed after 12 h. When the phosphatidylinositol 3-kinase (PI3K)/Akt pathway was inhibited by wortmannin, DEX no longer inhibited β-catenin levels. Furthermore, DEX-mediated inhibition of LEF/TCF transcriptional activity was attenuated in the presence of dominant negative forms of either PI3K or protein kinase B/Akt. These results suggest cross-talk between the PI3K/Akt and Wnt signaling pathways. Consistent with a role for Wnt signaling in the osteoblast differentiation-related cell cycle, wortmannin partially negated the DEX inhibition of this cell cycle. DEX also induced histone deacetylase (HDAC) 1, which is known to inhibit LEF/TCF transcriptional activity. Overexpression of HDAC1 negated the inhibitory effect of DEX on LEF/TCF transcriptional activity. In the presence of trichostatin A, a deacetylase inhibitor, DEX-mediated inhibition of the differentiation-related cell cycle was partially negated. When administered together, wortmannin and trichostatin A completely negated the inhibitory effect of DEX on the differentiation-related cell cycle. These results suggest that inhibition of a PI3K/Akt/GSK3β/β-catenin/LEF axis and stimulation of HDAC1 cooperate to mediate the inhibitory effect of DEX on Wnt signaling and the osteoblast differentiation-related cell cycle.