SLUG: a new target of lymphoid enhancer factor-1 in human osteoblasts.

Lymphoid Enhancer Factor-1 (Lef-1) is a member of a transcription factor family that acts as downstream mediator of the Wnt/beta-catenin signalling pathway which plays a critical role in osteoblast proliferation and differentiation. In a search for Lef-1 responsive genes in human osteoblasts, we focused on the transcriptional regulation of the SLUG, a zinc finger transcription factor belonging to the Snail family of developmental proteins. Although the role of SLUG in epithelial-mesenchymal transition and cell motility during embryogenesis is well documented, the functions of this factor in most normal adult human tissues are largely unknown. In this study we investigated SLUG expression in normal human osteoblasts and their mesenchymal precursors, and its possible correlation with Lef-1 and Wnt/beta-catenin signalling. The experiments were performed on normal human primary osteoblasts obtained from bone fragments, cultured in osteogenic conditions in presence of Lef-1 expression vector or GSK-3beta inhibitor, SB216763. We demonstrated that the transcription factor SLUG is present in osteoblasts as well as in their mesenchymal precursors obtained from Wharton's Jelly of human umbilical cord and induced to osteoblastic differentiation. We found that SLUG is positively correlated with RUNX2 expression and deposition of mineralized matrix, and is regulated by Lef-1 and beta-catenin. Consistently, Chromatin Immunoprecipitation (ChIP) assay, used to detect the direct Lef/Tcf factors that are responsible for the promoter activity of SLUG gene, demonstrated that Lef-1, TCF-1 and TCF4 are recruited to the SLUG gene promoter "in vivo". These studies provide, for the first time, the evidence that SLUG expression is correlated with osteogenic commitment, and is positively regulated by Lef-1 signal in normal human osteoblasts. These findings will help to further understand the regulation of the human SLUG gene and reveal the biological functions of SLUG in the context of bone tissue.