RUNX2 Regulation of LEF-1 in CCD Cells with Different Defects

Cleidocranial Dysplasia (CCD) is an autosomal dominant disorder causing skeletal and dental abnormalities including delayed eruption and supernumerary teeth. RUNX2 has been shown to be a critical transcriptional factor in bone and tooth formation and mutations in RUNX2 are responsible for CCD. Lymphoid enhancer factor 1 (LEF-1) belongs to the High Mobility Group (HMG)-box family and is an important nuclear effector of the canonical Wnt signaling cascade during tooth formation. Studies have shown that RUNX2 regulates LEF1 during osteoblast maturation. Our hypothesis is that LEF-1 is also regulated by RUNX2 during tooth formation.  Objectives: To compare LEF-1 gene expression in normal control cells and CCD cells with different types of genetic defects. Methods: Human dental pulp (DP) cells extracted from control and CCD patients were cultured and harvested at 90% confluence; total RNA and proteins were extracted from the harvested cells according to manufactures’ protocols. qRT-PCR and immunuocytochemical (ICC) staining were used to detect the LEF1 mRNA and protein expression, respectively. Results: QRT-PCR data demonstrated the differential LEF1 gene expression among cells carrying different RUNX2 mutations in dental cells.  The LEF-1 gene expression at mRNA level was significant down regulated (p=2.2E^-06) in pulp cells carrying RUNX2 R225Q mutation in comparison to the cells having a contiguous heterozygous deletion on 6p21 including RUNX2.  The similar findings were also seen at the protein level by ICC staining. Other dental cell types (PDL & EOE) did not shown dysregulation of LEF1. Conclusion: Our data suggest that different types of RUNX2 mutations may regulate LEF1 through different mechanisms such as dominant negative versus haploinsufficiency and that regulation is tissue/cell type specific. These studies may provide new insights in our understanding of the pathogenesis of CCD as well as the relationship between RUNX2 and LEF1 during tooth formation. Support: NIDCR 5T32DE017607DART/NIDCR-R03DE019516(JD).