Glucocorticoids Inhibit the Transcriptional Activity of LEF/TCF in Differentiating Osteoblasts in a Glycogen Synthase Kinase-3β-dependent and -independent Manner

Elisheva Smith,Baruch Frenkel

doi:10.1074/jbc.m406294200

Elisheva Smith, Baruch Frenkel

Open Access

https://doi.org/10.1074/jbc.m406294200

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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-3beta (GSK3beta) 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 beta-catenin levels and inhibition of LEF/TCF-mediated transcription. These inhibitory activities are no longer observed in the presence of lithium, a GSK3beta 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 beta-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/GSK3beta/beta-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.

Highlights

Glucocorticoids, widely used as immune suppressors, cause osteoporosis by inhibiting bone formation
These results suggest that inhibition of a phosphatidylinositol 3-kinase (PI3K)/Akt/GSK3␤/␤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
GCs Inhibit LEF/TCF-mediated Transcription via GSK3␤— We have shown previously that GC-mediated inhibition of the osteoblast differentiation-related cell cycle occurs via activation of GSK3␤ and proteasomal degradation of c-myc [13]

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Three culture models were employed in this study. A highly osteoblastic MC3T3-E1 subclone was isolated previously [7] and used between passage 7 and 11. Human osteoblast cultures were obtained previously from vertebrae of subjects undergoing neurosurgical procedures [27], and frozen cell stocks were thawed for the present study. Cells were suspended in buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 10% glycerol, 10 mM EDTA, and protease and phosphatase inhibitors as described above. Suspensions were homogenized, and extracts were centrifuged at 20,000 ϫ g for 30 min to remove cell debris. Cell pellets were suspended in two packed cell volumes of hypotonic buffer containing 10 mM HEPES (pH 7.5), 10 mM KCl, 3 mM MgCl2, 1 mM EDTA (pH 8.0), and protease and phosphatase inhibitors as described above. The nuclear suspension was homogenized and centrifuged at 20,000 ϫ g for 1 h to remove cell debris.

RESULTS

DISCUSSION

Elisheva Smith and Baruch Frenkel