No abstract

Transcription factors bind to cell‐specific cis‐regulatory elements, such as enhancers and promoters, to initiate much of the gene expression program of different biological process. Odontoblast differentiation is a necessary step for tooth formation and is also governed by a complex gene regulatory network. Our previous in vitro experiments showed that Krüppel‐like factor 4 (KLF4) can promote odontoblastic differentiation of both mouse dental papillary cells (mDPCs) and human dental pulp cells; however, its mechanism remains unclear. We first used Wnt1‐Cre; KLF4fx/fx (Klf4 cKO) mice to examine the role of KLF4 during odontoblast differentiation in vivo and demonstrated significantly impaired dentin mineralization and enlarged pulp/root canals. Additionally, combinatory analysis using RNA‐seq and ATAC‐seq revealed genomewide direct regulatory targets of KLF4 in mouse odontoblasts. We found that KLF4 can directly activate the TGF‐β signaling pathway at the beginning of odontoblast differentiation with Runx2 as a cofactor. Furthermore, we found that KLF4 can directly upregulate the expression levels of Dmp1 and Sp7, which are markers of odontoblastic differentiation, through binding to their promoters. Interestingly, as a transcription factor, KLF4 can also recruit histone acetylase as a regulatory companion to the downstream target genes to positively or negatively regulate transcription. To further investigate other regulatory companions of KLF4, we chose histone acetylase HDAC3 and P300. Immunoprecipitation demonstrated that KLF4 interacted with P300 and HDAC3. Next, ChIP analysis detected P300 and HDAC3 enrichment on the promoter region of KLF4 target genes Dmp1 and Sp7. HDAC3 mainly interacted with KLF4 on day 0 of odontoblastic induction, whereas P300 interacted on day 7 of induction. These temporal‐specific interactions regulated Dmp1 and Sp7 transcription, thus regulating dentinogenesis. Taken together, these results demonstrated that KLF4 regulates Dmp1 and Sp7 transcription via the modulation of histone acetylation and is vital to dentinogenesis. © 2019 American Society for Bone and Mineral Research.

The clinical translation of regenerative endodontics demands further development of suitable scaffolds. Here, we assessed the possibility of using silk fibroin scaffold for pulp regeneration with dental pulp stem cells (DPSCs) and basic fibroblast growth factor (bFGF) in ectopic root canal transplantation model. Porous silk fibroin scaffolds were fabricated using freeze-drying technique (with or without bFGF incorporation), and characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy. DPSCs were isolated, characterized, seeded onto scaffolds, and inserted into the tooth root fragments. Cell viability and morphology were tested in the 3D model in vitro using CCK8 assay and SEM. Furthermore, the ectopic transplantation model was used to verify the generation of pulp-like tissue in DPSCs seeded silk fibroin scaffold with bFGF, as examined by histological analysis. DPSCs seeded in silk fibroin scaffold survived, exhibited cytoplasmic elongation in scaffolds at least 4 weeks in culture. bFGF promoted DPSCs viability in tooth fragments/scaffolds (TSS) between 7 and 28 days. Pulp-like tissue was generated in the bFGF-incorporated TSS with DPSCs. Histologically, the generated tissue was shown to be with well vascularity, have new matrix deposition and dentin-like tissue formation, and consist of both the transplanted and host-derived cells. Collectively, these data support the use of bFGF-incorporated silk fibroin scaffold as a highly promising scaffold candidate for future treatment concepts in regenerative endodontics to save teeth.

Purpose Dental caries is a multifactorial infectious disease. In this study, we investigated whether single nucleotide polymorphisms (SNPs) in vitamin D receptor (VDR) gene were associated with susceptibility to permanent tooth caries in Chinese adolescents. Method A total of 200 dental caries patients and 200 healthy controls aged 12 years were genotyped for VDR gene polymorphisms using the PCR-restriction fragment length polymorphism (PCR-RFLP) assay. All of them were examined for their oral and dental status with the WHO criteria, and clinical information such as the Decayed Missing Filled Teeth Index (DMFT) was evaluated. Genomic DNA was extracted from the buccal epithelial cells. The four polymorphic SNPs (Bsm I, Taq I, Apa I, and Fok I) in VDR were assessed for both genotypic and phenotypic susceptibilities. Results Among the four examined VDR gene polymorphisms, the increased frequency of the CT and CC genotype of the Fok I VDR gene polymorphism was associated with dental caries in 12-year-old adolescent, compared with the controls (X2 = 17.813, p ≤ 0.001). Moreover, Fok I polymorphic allele C frequency was significantly increased in the dental caries cases, compared to the controls (X2 = 14.144, p ≤ 0.001, OR = 1.730, 95% CI = 1.299–2.303). However, the other three VDR gene polymorphisms (Bsm I, Taq I, and Apa I) showed no statistically significant differences in the caries groups compared with the controls. Conclusion VDR-Fok I gene polymorphisms may be associated with susceptibility to permanent tooth caries in Chinese adolescent.

Transcription factors (TFs) regulate the expression of target genes, inducing changes in cell morphology or activities needed for cell fate determination and differentiation. The BMP signaling pathway is widely regarded as one of the most important pathways in vertebrate skeletal biology, of which BMP2 is a potent inducer, governing the osteoblast differentiation of bone marrow stromal cells (BMSCs). However, the mechanism by which BMP2 initiates its downstream transcription factor cascade and determines the direction of differentiation remains largely unknown. In this study, we used RNA-seq, ATAC-seq, and animal models to characterize the BMP2-dependent gene regulatory network governing osteoblast lineage commitment. Sp7-Cre; Bmp2fx/fx mice (BMP2-cKO) were generated and exhibited decreased bone density and lower osteoblast number (n > 6). In vitro experiments showed that BMP2-cKO mouse bone marrow stromal cells (mBMSCs) had an impact on osteoblast differentiation and deficient cell proliferation. Osteogenic medium induced mBMSCs from BMP2-cKO mice and control were subjected to RNA-seq and ATAC-seq analysis to reveal differentially expressed TFs, along with their target open chromatin regions. Combined with H3K27Ac CUT&Tag during osteoblast differentiation, we identified 2338 BMP2-dependent osteoblast-specific active enhancers. Motif enrichment assay revealed that over 80% of these elements were directly targeted by RUNX2, DLX5, MEF2C, OASIS, and KLF4. We deactivated Klf4 in the Sp7 + lineage to validate the role of KLF4 in osteoblast differentiation of mBMSCs. Compared to the wild-type, Sp7-Cre; Klf4fx/+ mice (KLF4-Het) were smaller in size and had abnormal incisors resembling BMP2-cKO mice. Additionally, KLF4-Het mice had fewer osteoblasts and decreased osteogenic ability. RNA-seq and ATAC-seq revealed that KLF4 mainly “co-bound” with RUNX2 to regulate downstream genes. Given the significant overlap between KLF4- and BMP2-dependent NFRs and enriched motifs, our findings outline a comprehensive BMP2-dependent gene regulatory network specifically governing osteoblast differentiation of the Sp7 + lineage, in which Klf4 is a novel transcription factor.