Smad7 Regulates Dental Epithelial Proliferation during Tooth Development

Abstract

Tooth morphogenesis involves dynamic changes in shape and size as it proceeds through the bud, cap, and bell stages. This process requires exact regulation of cell proliferation and differentiation. Smad7, a general antagonist against transforming growth factor–β (TGF-β) signaling, is necessary for maintaining homeostasis and proper functionality in many organs. While TGF-β signaling is widely involved in tooth morphogenesis, the precise role of Smad7 in tooth development remains unknown. In this study, we showed that Smad7 is expressed in the developing mouse molars with a high level in the dental epithelium but a moderate to weak level in the dental mesenchyme. Smad7 deficiency led to a profound decrease in tooth size primarily due to a severely compromised cell proliferation capability in the dental epithelium. Consistent with the tooth shrinkage phenotype, RNA sequencing (RNA-seq) analysis revealed that Smad7 ablation downregulated genes referred to epithelial cell proliferation and cell cycle G1/S phase transition, whereas the upregulated genes were involved in responding to TGF-β signaling and cell cycle arrest. Among these genes, the expression of Cdkn1a (encoding p21), a negative cell proliferation regulator, was remarkably elevated in parallel with the diminution of Ccnd1 encoding the crucial cell cycle regulator cyclin D1 in the dental epithelium. Meanwhile, the expression level of p-Smad2/3 was ectopically elevated in the developing tooth germ of Smad7 null mice, indicating the hyperactivation of the canonical TGF-β signaling. These effects were reversed by addition of TGF-β signaling inhibitor in cell cultures of Smad7−/− molar tooth germs, with rescued expression of cyclin D1 and cell proliferation rate. In sum, our studies demonstrate that Smad7 functions primarily as a positive regulator of cell proliferation via inhibition of the canonical TGF-β signaling during dental epithelium development and highlight a crucial role for Smad7 in regulating tooth size.

Differential lncRNA/mRNA Expression Profiling and Functional Network Analyses in Bmp2 Deletion of Mouse Dental Papilla Cells

“…The KEGG analysis showed that the DEGs were largely enriched in the TGF-β/BMP signaling pathway, Hippo signaling pathway, Wnt signaling pathway, and signaling pathways regulating pluripotency of stem cells ( Figure 4A ). These signal pathways are cross talks and are involved in dental and other cell proliferation and differentiation via regulating transcriptional and growth factors including Id2 , Id3 , Fgf2 , Klf10 , Lhx1 , Smad7 , Tcf7 , Wnt1 , and Wnt11 ( Ho et al, 2011 ; Koizumi et al, 2013 ; Sternberg et al, 2013 ; Bakopoulou et al, 2015 ; Chen et al, 2016 ; Zhang et al, 2016 ; Wang and Martin, 2017 ; Neves and Sharpe, 2018 ; Chen et al, 2019 ; Liu et al, 2019 ; Chakka et al, 2020 ; Niki et al, 2021 ).…”

“…In order to verify the transcriptome sequencing results between the iBmp2 fx/fx and iBmp2 ko/ko cells and further analyze the possible signaling network mediated by Bmp2, the 9 representative mRNAs and 3 lncRNAs were selected for validation by using RT-qPCR since the 9 Bmp signal-associated mRNAs are involved in the dental cell proliferation, differentiation, and tooth development ( Ho et al, 2011 ; Koizumi et al, 2013 ; Sternberg et al, 2013 ; Bakopoulou et al, 2015 ; Chen et al, 2016 ; Zhang et al, 2016 ; Chen et al, 2019 ; Liu et al, 2019 ; Chakka et al, 2020 ), and three novel lncRNAs (87,211.1, 87,189.1, and 86,888.1) co-expressed with these mRNAs ( Figure 6B ). The RT-qPCR results showed that the expression of lncRNA87211.1 and lncRNA87189.1 decreased in the iBmp2 ko/ko cells, as same as the results of RNA-seq, but lncRNA86888.1 expression was increased in the iBmp2 ko/ko cells which was opposite to the results of RNA-seq ( Figure 7A ).…”

“…Based on the results of the KEGG, the PPI, and lncRNA–mRNA co-expression, the 9 mRNAs ( Fgf2 , Id2 , Id3 , Lhx1 , Smad7 , Tcf7 , Wnt1 , Wnt11 , and Klf10 ) and 3 lncRNAs (87,211.1, 87,189.1, and 86,888.1) were selected as representative genes for RT-qPCR validation as these genes are involved in the dental cell proliferation and differentiation as well as tooth development and formation induced by Bmp2 signaling ( Tsuboi et al, 2003 ; Koizumi et al, 2013 ; Sternberg et al, 2013 ; Chen et al, 2016 ; Graf et al, 2016 ; Wu et al, 2016 ; Li et al, 2019 ; Liu et al, 2019 ; Qin et al, 2019 ; Nottmeier et al, 2021 ) and three novel lncRNAs co-expressed with those mRNAs in the dental papilla cells. Our study demonstrated that the validated 9 mRNAs and 2 lncRNAs by RT-qPCR were consistent with the results of the RNA-seq, and the PCC method can be used to predict co-expression networks between mRNAs and lncRNAs.…”

“…The selected 9 mRNAs are involved in the dental cell proliferation, differentiation, and homeostasis ( Ho et al, 2011 ; Koizumi et al, 2013 ; Sternberg et al, 2013 ; Bakopoulou et al, 2015 ; Chen et al, 2016 ; Zhang et al, 2016 ; Wang and Martin, 2017 ; Neves and Sharpe, 2018 ; Chen et al, 2019 ; Liu et al, 2019 ; Chakka et al, 2020 ; Niki et al, 2021 ). The loss of Bmp2 resulted in a decrease of these gene expressions detected by RNA-seq and RT-qPCR ( Figures 2 , 7 ).…”

USP34 regulates tooth root morphogenesis by stabilizing NFIC

“…[13][14][15][16] Deficiency of Smad7, a general antagonist against TGF-β signaling, led to reduced tooth size and affected cell proliferation. 17 The question that remains to be answered then, is when and how these molecules achieve their specificity during tooth development.…”