Luke Hoeppner

E-mail: hoepp005@umn.edu

Thesis Advisor: Jennifer Westendorf

Year entered: 2004

Degrees received:
B.S., Genetics, University of Wisconsin, Madison, WI 2004

Thesis research:
Lymphoid Enhancer Binding Factor (Lef) 1 is a transcription factor linked with the Wnt/LRP5/ß-catenin signaling cascade, which regulates osteoblast proliferation and survival, bone density and skeletal strength. We previously demonstrated that suppression of Lef1 promotes osteoblast mineralization while Lef1 overexpression blocks osteoblast maturation. We also showed that Lef1 blocks Runx2-dependent activation of the osteocalcin promoter. In this study we demonstrate that Lef1 blocks activation from the neighboring Runx2 binding site, even when the sequence is changed from a Runx binding element to a GAL4 DNA binding element. A truncated form of Lef1 (Lef1ΔN) that lacks the ß-catenin binding domain does not repress osteocalcin expression as well as full-length Lef1, even though it retains a functional DNA binding domain. Lef1ΔN is transcribed from a promoter in the intron between exons 2 and 3 in humans (exons 3 and 4 in mice). Runx2 binds a consensus Runx binding element in this promoter and activates transcription of the Lef1ΔN transcript. Northern blot analysis indicates that the 2.3 kB Lef1ΔN transcript is not present in undifferentiated MC3T3-E1 preosteoblasts but becomes detectable after six days of osteogenic stimulation. These data suggest that premature expression of Lef1ΔN in osteoblast progenitors might block osteoblast differentiation. To test this hypothesis, we stably overexpressed Lef1ΔN in the murine myo-osteoblast progenitor C2C12 cell line and induced differentiation in osteogenic medium containing ascorbic acid, ß-glycerol phosphate, and BMP2. Lef1ΔN overexpression decreased the rate of alkaline phosphatase production compared to control cells. Together these data suggest that Lef1ΔN is a Runx2 target gene and an important regulator of osteoblast differentiation. Our model predicts that Lef1ΔN is normally expressed during the maturation process where it competes with endogenous Lef1 for binding elements and thereby mitigates the Wnt signaling pathway. If expressed earlier, Lef1ΔN blocks preosteoblast maturation.