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

Abstract

Background: 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.

Results: 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".

Conclusion: 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.

Figure 1

Detection of SLUG expression by quantitative RT-PCR. The level of SLUG, RUNX2 Lef-1, SNAIL1 and SNAIL3 expression was examined by quantitative RT-PCR in three hMSC samples cultured up to 28 days in osteogenic medium (A) and in five hOB samples (B). The cDNA obtained from total RNA was subjected to quantitative TaqMan RT-PCR for SLUG, RUNX2, Lef-1, SNAIL1 and SNAIL3 transcript analysis. The experiments were carried out in triplicate, the expression levels were normalized on the basis of GAPDH expression and results of the experiments are reported as relative mRNA expression levels. ΔΔCt method was used to value the gene expression; standard error of the mean (SEM) was calculated. The commitment to osteoblastic lineage of hMSCs was evaluated by Alizarin Red staining for extracellular calcium deposition. The authentic osteoblast phenotype was confirmed in hOBs by staining for alkaline phosphatase (ALP) activity and mineralized matrix deposition (AR, Alizarin Red staining). * = p < 0.05 (respect to day 0).

Figure 2

Effect of Lef-1 overexpression on SLUG expression in hOBs. The effect of Lef-1 overexpression was examined at mRNA (A) and protein (B) level. (A) SLUG mRNA was evaluated by quantitative RT-PCR in hOBs and SaOS-2 osteoblast-like cells transfected with 2.5 μg of hLef-1 (K14-myc-hLEF1) expression plasmid. The cDNA obtained from total RNA was subjected to quantitative TaqMan RT-PCR for SLUG transcript analysis. The expression levels were normalized on the basis of GAPDH expression and results of the experiments are reported as relative mRNA expression levels. Results are representative of three independent experiments carried out in triplicate. ΔΔCt method was used to compare gene expression data; standard error of the mean (SEM) was calculated. * = p < 0.05. (B) SLUG protein levels were examined by Western blot analysis in hOBs and SaOS-2 osteoblast-like cells transfected with 2.5 μg of hLef-1 expression plasmid. Whole cell lysates were prepared and 25 μg of protein run on a 12% SDS-polyacrylamide gel. The proteins were visualized using Supersignal West Femto Substrate (Pierce). The quantitative presentation of the protein levels were performed by densitometric analysis using Anti-IP(3)K as control. D.U. = densitometric units. This experiment was repeated three times with similar results. A representative SLUG and Lef-1 Western blot analysis with size markers (KDa) is reported. * = p < 0.05.

Figure 3

Lef-1 affects the activity of human SLUG promoter "in vitro" and binds it "in vivo". (A) The SLUG promoter region under investigation is reported. The positions of putative Lef/Tcf binding sites are enclosed by rectangles and are compared with those recently investigated by others. Positions of PCR primers used in ChIP experiments are also reported. (B) The DNA construct containing the human SLUG promoter region was cloned into upstream of the firefly luciferase (LUC) reporter gene. hOBs and SaOS-2 osteoblast-like cells were transfected with the pGL3-SLUG Luc reporter vector containing the sequence from +1 to -982 of the human SLUG promoter (pGL3-SLUG 982 bp), in the absence (-) or presence (+) of 2.5 μg of hLef-1 expression plasmid. The results of reporter gene assays were normalized with protein concentration and β-gal activity for transfection efficiency and the data are represented as ratios of luciferase units to β-galactosidase units. MCF7 breast cancer cell line was used as negative control. All experiments were performed in triplicate and the average of the ratio of the reporter activity + SEM is shown. * = p < 0.05. (C) Recruitment of Lef1/TCF transcription factors to the human SLUG promoter is demonstrated by "in vivo" chromatin immunoprecipitation (ChIP) binding assays. Soluble chromatin was prepared from hOBs and immunoprecipitated with the indicated specific antibodies against Lef-1, TCF-1, and TCF-4. The associations of the transcription factors to bound precipitated DNA were monitored on the human SLUG promoter regions 1, 2 and 3 by PCR with the primers indicated in the scheme. Input represents a positive control using the starting material (0.2%) prior to immunoprecipitation. Representative agarose gels are shown.

Figure 4

Treatment of hOBs with the glycogen synthase kinase (GSK-3β) inhibitor, SB216763. (A) A scheme of SB216763 action mechanism is reported (see the text for details). (B) Effect of SB216763 on the TOPflash reporter system. 24 h after transient transfection with the TOPflash plasmid, the cells were treated (+) or not (-) with SB216763 (10 μM) for 24 h prior to harvest. Luciferase activity was normalized to β-galactosidase activity in the same sample. The bars represent mean ± SEM. * = p < 0.05. (C) The levels of β-catenin expression, SLUG and RUNX2 were examined by Western blot in hOBs treated with SB216763 (10 μM) or with the only vehicle (-). The quantitative presentation of the protein levels was performed by densitometric analysis using Anti-IP(3)K as control. D.U. = densitometric units. A representative Western blot analysis with size markers (KDa) is reported. * = p < 0.05.