Huojun Cao

MicroRNAs Play a Critical Role in Tooth Development

MicroRNAs are known to regulate gene function in many tissues and organs, but their expression and function, if any, in tooth development are elusive. We sought to identify them by microRNA screening analyses and reveal their overall roles by inactivating Dicer1 in the dental epithelium and mesenchyme. Discrete sets of microRNAs are expressed in molars compared with incisors as well as epithelium compared with mesenchyme. Conditional knockout (cKO) of Dicer1 (mature microRNAs) in the dental epithelium of the Pitx2-Cre mouse results in multiple and branched enamel-free incisors and cuspless molars, and change in incisor patterning and in incisor and molar size and shape. Analyses of differentiating dental epithelial markers reveal a defect in ameloblast differentiation. Conversely, the cervical loop (stem cell niche) is expanded in Dicer1 cKO. These results demonstrate that tooth development is tightly controlled by microRNAs and that specific microRNAs regulate tooth epithelial stem cell differentiation.

Aryl Hydrocarbon Receptor Agonists Induce MicroRNA-335 Expression and Inhibit Lung Metastasis of Estrogen Receptor Negative Breast Cancer Cells

The aryl hydrocarbon receptor (AHR) was initially identified as a receptor that bound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related environmental toxicants; however, there is increasing evidence that the AHR is an important new drug target for treating multiple diseases including breast cancer. Treatment of estrogen receptor (ER)-negative MDA-MB-231 and BT474 breast cancer cells with TCDD or the selective AHR modulator 6-methyl-1,3,-trichlorodibenzofuran (MCDF) inhibited breast cancer cell invasion in a Boyden chamber assay. These results were similar to those previously reported for the antimetastic microRNA-335 (miR-335). Both TCDD and MCDF induced miR-335 in MDA-MB-231 and BT474 cells and this was accompanied by downregulation of SOX4, a miR-335-regulated (inhibited) gene. The effects of TCDD and MCDF on miR-335 and SOX4 expression and interactions of miR-335 with the 3′-UTR target sequence in the SOX4 gene were all inhibited in cells transfected with an oligonucleotide (iAHR) that knocks down the AHR, thus confirming AHR-miR-335 interactions. MCDF (40 mg/kg/d) also inhibited lung metastasis of MDA-MB-231 cells in a tail vein injection model, showing that the AHR is a potential new target for treating patients with ER-negative breast cancer, a disease where treatment options and their effectiveness are limited. Mol Cancer Ther; 11(1); 108–18. ©2011 AACR.

The Pitx2:miR-200c/141:noggin pathway regulates Bmp signaling and ameloblast differentiation

The mouse incisor is a remarkable tooth that grows throughout the animal’s lifetime. This continuous renewal is fueled by adult epithelial stem cells that give rise to ameloblasts, which generate enamel, and little is known about the function of microRNAs in this process. Here, we describe the role of a novel Pitx2:miR-200c/141:noggin regulatory pathway in dental epithelial cell differentiation. miR-200c repressed noggin, an antagonist of Bmp signaling. Pitx2 expression caused an upregulation of miR-200c and chromatin immunoprecipitation assays revealed endogenous Pitx2 binding to the miR-200c/141 promoter. A positive-feedback loop was discovered between miR-200c and Bmp signaling. miR-200c/141 induced expression of E-cadherin and the dental epithelial cell differentiation marker amelogenin. In addition, miR-203 expression was activated by endogenous Pitx2 and targeted the Bmp antagonist Bmper to further regulate Bmp signaling. miR-200c/141 knockout mice showed defects in enamel formation, with decreased E-cadherin and amelogenin expression and increased noggin expression. Our in vivo and in vitro studies reveal a multistep transcriptional program involving the Pitx2:miR-200c/141:noggin regulatory pathway that is important in epithelial cell differentiation and tooth development.

Differentiation of human dental stem cells reveals a role for microRNA‐218

BACKGROUND Regeneration of lost periodontium is the ultimate goal of periodontal therapy. Advances in tissue engineering have demonstrated the multilineage potential and plasticity of adult stem cells located in periodontal apparatus. However, it remains unclear how epigenetic mechanisms controlling signals determine tissue specification and cell lineage decisions. To date, no data are available on micro-RNA (miRNA) activity behind human-derived dental stem cells (DSCs). MATERIAL AND METHODS In this study, we isolated periodontal ligament stem cells, dental pulp stem cells and gingival stem cells from extracted third molars; human bone marrow stem cells were used as a positive control. The expression of OCT4A and NANOG was confirmed in these undifferentiated cells. All cells were cultured under osteogenic inductive conditions and RUNX2 expression was analyzed as a marker of mineralized tissue differentiation. The miRNA expression profile was obtained at baseline and after osteogenic induction in all cell types. RESULTS The expression of RUNX2 demonstrated successful osteogenic induction of all cell types, which was confirmed by alizarin red stain. The analysis of 765 miRNAs demonstrated a shift in miRNA expression that occurred in all four stem cell types, including a decrease in hsa-mir-218 across all differentiated cell populations. Hsa-mir-218 targets RUNX2 and decreases RUNX2 expression in undifferentiated human DSCs. DSC mineralized tissue type differentiation is associated with a decrease in hsa-mir-218 expression. CONCLUSION These data reveal a miRNA-regulated pathway for the differentiation of human DSCs and a select network of human miRNAs that control DSC osteogenic differentiation.

Tbx1 Regulates Progenitor Cell Proliferation in the Dental Epithelium by Modulating PITX2 Activation of p21

Tbx1(-/-) mice present with phenotypic effects observed in DiGeorge syndrome patients however, the molecular mechanisms of Tbx1 regulating craniofacial and tooth development are unclear. Analyses of the Tbx1 null mice reveal incisor microdontia, small cervical loops and BrdU labeling reveals a defect in epithelial cell proliferation. Furthermore, Tbx1 null mice molars are lacking normal cusp morphology. Interestingly, p21 (associated with cell cycle arrest) is up regulated in the dental epithelium of Tbx1(-/-) embryos. These data suggest that Tbx1 inhibits p21 expression to allow for cell proliferation in the dental epithelial cervical loop, however Tbx1 does not directly regulate p21 expression. A new molecular mechanism has been identified where Tbx1 inhibits Pitx2 transcriptional activity and decreases the expression of Pitx2 target genes, p21, Lef-1 and Pitx2c. p21 protein is increased in PITX2C transgenic mouse embryo fibroblasts (MEF) and chromatin immunoprecipitation assays demonstrate endogenous Pitx2 binding to the p21 promoter. Tbx1 attenuates PITX2 activation of endogenous p21 expression and Tbx1 null MEFs reveal increased Pitx2a and activation of Pitx2c isoform expression. Tbx1 physically interacts with the PITX2 C-terminus and represses PITX2 transcriptional activation of the p21, LEF-1, and Pitx2c promoters. Tbx1(-/+)/Pitx2(-/+) double heterozygous mice present with an extra premolar-like tooth revealing a genetic interaction between these factors. The ability of Tbx1 to repress PITX2 activation of p21 may promote cell proliferation. In addition, PITX2 regulation of p21 reveals a new role for PITX2 in repressing cell proliferation. These data demonstrate new functional mechanisms for Tbx1 in tooth morphogenesis and provide a molecular basis for craniofacial defects in DiGeorge syndrome patients.

Hierarchical Interactions of Homeodomain and Forkhead Transcription Factors in Regulating Odontogenic Gene Expression

FoxJ1 is a forkhead transcription factor expressed in multiple tissues during development and a major regulator of cilia development. FoxJ1−/− mice present with defects in odontogenesis, and we correlate these defects to hierarchical interactions between homeodomain factors Pitx2 and Dlx2 with FoxJ1 in regulating their expression through direct physical interactions. Chromatin immunoprecipitation assays reveal endogenous Pitx2 and Dlx2 binding to the Dlx2 promoter and Dlx2 binding to the FoxJ1 promoter as well as Dlx2 and FoxJ1 binding to the amelogenin promoter. PITX2 activation of the Dlx2 promoter is attenuated by a direct Dlx2 physical interaction with PITX2. Dlx2 autoregulates its promoter, and Dlx2 transcriptionally activates the downstream gene FoxJ1. Dlx2 and FoxJ1 physically interact and synergistically regulate both Dlx2 and FoxJ1 promoters. Dlx2 and FoxJ1 also activate the amelogenin promoter, and amelogenin is required for enamel formation and late stage tooth development. FoxJ1−/− mice maxillary and mandibular incisors are reduced in length and width and have reduced amelogenin expression. FoxJ1−/− mice show a reduced and defective ameloblast layer, revealing a biological effect of these transcription factor hierarchies during tooth morphogenesis. These transcriptional mechanisms may contribute to other developmental processes such as neuronal, pituitary, and heart development.

TBX1 protein interactions and microRNA-96-5p regulation controls cell proliferation during craniofacial and dental development: implications for 22q11.2 deletion syndrome

T-box transcription factor TBX1 is the major candidate gene for 22q11.2 deletion syndrome (22q11.2DS, DiGeorge syndrome/Velo-cardio-facial syndrome), whose phenotypes include craniofacial malformations such as dental defects and cleft palate. In this study, Tbx1 was conditionally deleted or over-expressed in the oral and dental epithelium to establish its role in odontogenesis and craniofacial developmental. Tbx1 lineage tracing experiments demonstrated a specific region of Tbx1-positive cells in the labial cervical loop (LaCL, stem cell niche). We found that Tbx1 conditional knockout (Tbx1(cKO)) mice featured microdontia, which coincides with decreased stem cell proliferation in the LaCL of Tbx1(cKO) mice. In contrast, Tbx1 over-expression increased dental epithelial progenitor cells in the LaCL. Furthermore, microRNA-96 (miR-96) repressed Tbx1 expression and Tbx1 repressed miR-96 expression, suggesting that miR-96 and Tbx1 work in a regulatory loop to maintain the correct levels of Tbx1. Cleft palate was observed in both conditional knockout and over-expression mice, consistent with the craniofacial/tooth defects associated with TBX1 deletion and the gene duplication that leads to 22q11.2DS. The biochemical analyses of TBX1 human mutations demonstrate functional differences in their transcriptional regulation of miR-96 and co-regulation of PITX2 activity. TBX1 interacts with PITX2 to negatively regulate PITX2 transcriptional activity and the TBX1 N-terminus is required for its repressive activity. Overall, our results indicate that Tbx1 regulates the proliferation of dental progenitor cells and craniofacial development through miR-96-5p and PITX2. Together, these data suggest a new molecular mechanism controlling pathogenesis of dental anomalies in human 22q11.2DS.

The LIM Homeodomain Transcription Factor LHX6 A TRANSCRIPTIONAL REPRESSOR THAT INTERACTS WITH PITUITARY HOMEOBOX 2 (PITX2) TO REGULATE ODONTOGENESIS

Background: Odontogenesis is tightly controlled by the interactions of transcription factors. Results: LHX6 acts as a transcriptional repressor and interacts with PITX2 to regulate craniofacial development. Conclusion: LHX6 is required for mandible and dental morphogenesis through repression of gene expression. Significance: This is the first report of LHX6 transcriptional repressor activity and its ability to inhibit the activity of other transcription factors. LHX6 is a LIM-homeobox transcription factor expressed during embryogenesis; however, the molecular mechanisms regulating LHX6 transcriptional activities are unknown. LHX6 and the PITX2 homeodomain transcription factor have overlapping expression patterns during tooth and craniofacial development, and in this report, we demonstrate new transcriptional mechanisms for these factors. PITX2 and LHX6 are co-expressed in the oral and dental epithelium and epithelial cell lines. Lhx6 expression is increased in Pitx2c transgenic mice and decreased in Pitx2 null mice. PITX2 activates endogenous Lhx6 expression and the Lhx6 promoter, whereas LHX6 represses its promoter activity. Chromatin immunoprecipitation experiments reveal endogenous PITX2 binding to the Lhx6 promoter. LHX6 directly interacts with PITX2 to inhibit PITX2 transcriptional activities and activation of multiple promoters. Bimolecular fluorescence complementation assays reveal an LHX6·PITX2 nuclear interaction in living cells. LHX6 has a dominant repressive effect on the PITX2 synergistic activation with LEF-1 and β-catenin co-factors. Thus, LHX6 acts as a transcriptional repressor and represses the expression of several genes involved in odontogenesis. We have identified specific defects in incisor, molar, mandible, bone, and root development and late stage enamel formation in Lhx6 null mice. Amelogenin and ameloblastin expression is reduced and/or delayed in the Lhx6 null mice, potentially resulting from defects in dentin deposition and ameloblast differentiation. Our results demonstrate that LHX6 regulates cell proliferation in the cervical loop and promotes cell differentiation in the anterior region of the incisor. We demonstrate new molecular mechanisms for LHX6 and an interaction with PITX2 for normal craniofacial and tooth development.

A Pituitary Homeobox 2 (Pitx2):microRNA-200a-3p:β-catenin Pathway Converts Mesenchymal Cells to Amelogenin-expressing Dental Epithelial Cells

Background: An efficient method is required for converting somatic cells to specific cell fates. Results: A combination of Pitx2 and miR-200a-3p promotes dental epithelial gene expression in cells. Conclusion: A two-step method efficiently converts mesenchymal cells to dental epithelial cells. Significance: A new method to generate dental epithelial cells, which are difficult to isolate, is identified for use in regeneration and repair. Pitx2, Wnt/β-catenin signaling, and microRNAs (miRs) play a critical role in the regulation of dental stem cells during embryonic development. In this report, we have identified a Pitx2:β-catenin regulatory pathway involved in epithelial cell differentiation and conversion of mesenchymal cells to amelogenin expressing epithelial cells via miR-200a. Pitx2 and β-catenin are expressed in the labial incisor cervical loop or epithelial stem cell niche, with decreased expression in the differentiating ameloblast cells of the mouse lower incisor. Bioinformatics analyses reveal that miR-200a-3p expression is activated in the pre-ameloblast cells to enhance epithelial cell differentiation. We demonstrate that Pitx2 activates miR-200a-3p expression and miR-200a-3p reciprocally represses Pitx2 and β-catenin expression. Pitx2 and β-catenin interact to synergistically activate gene expression during odontogenesis and miR-200a-3p attenuates their expression and directs differentiation. To understand how this mechanism controls cell differentiation and cell fate, oral epithelial and odontoblast mesenchymal cells were reprogrammed by a two-step induction method using Pitx2 and miR-200a-3p. Conversion to amelogenin expressing dental epithelial cells involved an up-regulation of the stem cell marker Sox2 and proliferation genes and decreased expression of mesenchymal markers. E-cadherin expression was increased as well as ameloblast specific factors. The combination of Pitx2, a regulator of dental stem cells and miR-200a converts mesenchymal cells to a fully differentiated dental epithelial cell type. This pathway and reprogramming can be used to reprogram mesenchymal or oral epithelial cells to dental epithelial (ameloblast) cells, which can be used in tissue repair and regeneration studies.

Dact2 Represses PITX2 Transcriptional Activation and Cell Proliferation through Wnt/beta-Catenin Signaling during Odontogenesis

Dact proteins belong to the Dapper/Frodo protein family and function as cytoplasmic attenuators in Wnt and TGFβ signaling. Previous studies show that Dact1 is a potent Wnt signaling inhibitor by promoting degradation of β-catenin. We report a new mechanism for Dact2 function as an inhibitor of the canonical Wnt signaling pathway by interacting with PITX2. PITX2 is a downstream transcription factor in Wnt/β-catenin signaling, and PITX2 synergizes with Lef-1 to activate downstream genes. Immunohistochemistry verified the expression of Dact2 in the tooth epithelium, which correlated with Pitx2 epithelial expression. Dact2 loss of function and PITX2 gain of function studies reveal a feedback mechanism for controlling Dact2 expression. Pitx2 endogenously activates Dact2 expression and Dact2 feeds back to repress Pitx2 transcriptional activity. A Topflash reporter system was employed showing PITX2 activation of Wnt signaling, which is attenuated by Dact2. Transient transfections demonstrate the inhibitory effect of Dact2 on critical dental epithelial differentiation factors during tooth development. Dact2 significantly inhibits PITX2 activation of the Dlx2 and amelogenin promoters. Multiple lines of evidence conclude the inhibition is achieved by the physical interaction between Dact2 and Pitx2 proteins. The loss of function of Dact2 also reveals increased cell proliferation due to up-regulated Wnt downstream genes, cyclinD1 and cyclinD2. In summary, we have identified a novel role for Dact2 as an inhibitor of the canonical Wnt pathway in embryonic tooth development through its regulation of cell proliferation and differentiation.