Bleomycin: A novel osteogenesis inhibitor of dental follicle cells via a TGF‐β1/SMAD7/RUNX2 pathway

Abstract

Background and Purpose: Tooth eruption is a complicated process regulated by the dental follicles (DF). Our recent study discovered that tooth eruption was inhibited upon injection of bleomycin into DF. However, the mechanisms were unknown. Experimental Approach: Human dental follicle cells (hDFCs) were treated by bleomycin or exogenous TGF-β1 or transfected by plasmids loading SMAD7 or shRNA targeting SMAD7, followed by osteogenesis induction assay and signalling analysis. Human fresh DF tissues and Wistar rats were used to further confirm bleomycin function. Key Results: Bleomycin decreased expression of RUNX2 and osteogenic genes in hDFCs, reducing osteogenic capacity. TGF-β1 expression was up-regulated in bleomycin-treated hDFCs. The effects of exogenous TGF-β1 were similar to those of bleomycin in hDFCs. Additionally, compared to SMAD2/3, SMAD7 expression increased more in bleomycin-or TGF-β1-treated hDFCs. Overexpression of SMAD7 likewise significantly decreased RUNX2 expression and osteogenic capacity of hDFCs. Knockdown of SMAD7 markedly attenuated the inhibitory effects of bleomycin and TGF-β1 on osteogenic capacity and RUNX2 expression of hDFCs. Most importantly, changes in TGF-β1, SMAD7, and RUNX2 expressions were similar in the DF of rats and humans treated with bleomycin. Conclusion and Implications: SMAD7 was a negative regulator of osteogenic differentiation in DFCs through suppressing RUNX2 expression. Bleomycin or TGF-β1 inhibited osteogenic differentiation of DFCs via a TGF-β1/SMAD7/RUNX2 pathway. Our findings might be beneficial for enhancing the osteogenic activity of DFCs or inhibiting the eruption of undesirable teeth.

Oral cancer cell to endothelial cell communication via exosomal miR-21/RMND5A pathway

“…According to our previous study [ 15 ], 20 µg of total protein was loaded for SDS-PAGE electrophoresis and transferred to the PVDF membrane at a voltage of 110 V (90 min). After that, the membrane was blocked in 5% skimmed milk for 1 h, and incubated with primary antibodies (anti Flag: #2064, Dia-An Biotechnology, 1:3000; anti-NF-κB: #8242, Cell Signaling Technology, 1:1000; anti-phos NF-κB: #3033, Cell Signaling Technology, 1:1000; anti-ERK: #4695, Cell Signaling Technology, 1:2000; anti-phos ERK: #4370, Cell Signaling Technology, 1:3000; anti-GAPDH: sc-365,062, Santa Cruz Biotechnology, 1:2000) at 4 o C overnight, followed by incubation with HRP-conjugated secondary antibodies at room temperature for 1 h. The protein signals were detected with ECL kit.…”

M6A Demethylase Inhibits Osteogenesis of Dental Follicle Stem Cells via Regulating miR-7974/FKBP15 Pathway

“…All participants signed an informed consent document before tissue collection. DFSCs were cultured in an α-MEM medium consisting of 10% fetal bovine serum (Gibco, Erie County, NY, USA) and 1% penicillin-streptomycin solution (Sigma-Aldrich LLC., Saint Louis, MO, USA) and were incubated in a humidified atmosphere containing 5% CO 2 at 37 • C. The cell culture medium was refreshed every 2 days, and primary cells from passages 3-5 were used for experiments as previously described [2].…”

“…Dental follicle stem cells (DFSCs) are multipotent stem cells capable of differentiation into osteoblasts, adipocytes, fibroblasts, and cementoblasts. The inhibition of osteogenesis in DFSCs results in abnormal tooth development and eruption [1,2]. The further elucidation of the mechanism of DFSC osteogenic differentiation is of great significance for the treatment of dental developmental diseases and the advancement of bone tissue engineering.…”

Mechanisms during Osteogenic Differentiation in Human Dental Follicle Cells

“…The BMP signaling pathway belongs to the family of the TGF-β signaling pathway, which is down-regulated in DFC sub-clones with reduced osteogenic differentiation potentials [ 45 ]. However, the impact of the TGF-β signaling pathway on the differentiation potential is complex because supplementation of the osteogenic differentiation medium with TGF-β inhibits the osteogenic differentiation of DFCs [ 46 ]. In contrast, BMPs are well-known osteogenic growth factors and in particular, BMP2, BMP7 and BMP9 offer high potential for stimulating osteogenic differentiation or cementogenic differentiation in DFCs [ 47 , 48 ].…”