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Homeodomain gene  Distal-less-3  ( Dlx3 ) plays an important role during tooth development. Our previous studies indicate that DLX3 inhibits proliferation of human dental pulp cells (hDPCs). However, the mechanism of DLX3 regulating proliferation of hDPCs and maintaining the quiescence of the cells remain unknown. Given the importance of canonical Wnt signaling in the proliferation of dental pulp cell and tooth development, we hypothesized that DLX3 inhibited proliferation of hDPCs through inactivation of canonical Wnt signaling. With overexpression or knock-down of DLX3 in primary hDPCs, we found DLX3 down regulated canonical Wnt signaling and its downstream target genes. And when the DLX3 overexpressed-cells were treated with lithium chloride, the proliferation inhibition by DLX3 was reversed. We also found that DLX3 enhanced the expression of DKK1 and the reduced proliferation of hDPCs by DLX3 was reversed with knock-down of DKK1. Furthermore, luciferase reporter assay and chromatin immunoprecipitation assay showed DLX3 was able to bind to  Dkk1  promoter region from nucleotides (nt) -1656 to -1245, and stimulated  Dkk1  promoter activity. Mutagenesis studies further revealed two DLX3 responsive elements in  Dkk1  promoter. Taken together, our data indicate that DLX3 inhibits proliferation of hDPCs via inactivation of Wnt/β-catenin signaling pathway by directly binding to  Dkk1  promoter and increasing its expression.

DLX3 Inhibits the Proliferation of Human Dental Pulp Cells Through Inactivation of Canonical Wnt/β-Catenin Signaling Pathway