Inhibition of Microtubule Assembly in Osteoblasts Stimulates Bone Morphogenetic Protein 2 Expression and Bone Formation through Transcription Factor Gli2

Abstract

Bone morphogenetic protein 2 (BMP-2) is essential for postnatal bone formation and fracture repair. By screening chemical libraries for BMP-2 mimics using a cell-based assay, we identified inhibitors of microtubule assembly as stimulators of BMP-2 transcription. These microtubule inhibitors increased osteoblast differentiation in vitro, stimulated periosteal bone formation when injected locally over murine calvaria, and enhanced trabecular bone formation when administered systemically in vivo. To explore molecular mechanisms mediating these responses, we examined effects of microtubule inhibitors on the hedgehog (Hh) pathway, since this pathway is known to regulate BMP-2 transcription in osteoblasts and microtubules have been shown to be involved in Hh signaling in Drosophila. Here we show that in osteoblasts, inhibition of microtubule assembly increased cytoplasmic levels and transcriptional activity of Gli2, a transcriptional mediator of Hh signaling that we have previously shown to enhance BMP-2 expression in osteoblasts (M. Zhao et al., Mol. Cell. Biol. 26:6197-6208, 2006). Microtubule inhibition blocked β-TrCP-mediated proteasomal processing of Gli2 in osteoblasts. In summary, inhibition of microtubule assembly enhances BMP-2 gene transcription and subsequent bone formation, in part, through inhibiting proteasomal processing of Gli2 and increasing intracellular Gli2 concentrations.

ACKNOWLEDGMENTS

We thank Alexandra Joyner for kindly providing Gli2^+/− null mice, Hiroshi Sasaki for kindly providing Gli expression vectors and Gli reporter constructs, and Irina Kaverina for suggestions for microtubule experiments. We also thank Gloria Gutierrez, I. Ross Garrett, and Gianni Rossini for their help in the early experiments and Andrew Hart for proofreading.

This study was supported by NIH grants AG024637, AR051165, and AR050605 and grants from the Department of Veterans Affairs.