FaceBase: A Community-Driven Hub for Data-Intensive Research.

The FaceBase Consortium, funded by the National Institute of Dental and Craniofacial Research of the National Institutes of Health, was established in 2009 with the recognition that dental and craniofacial research are increasingly data-intensive disciplines. Data sharing is critical for the validation and reproducibility of results as well as to enable reuse of data. In service of these goals, data ought to be FAIR: Findable, Accessible, Interoperable, and Reusable. The FaceBase data repository and educational resources exemplify the FAIR principles and support a broad user community including researchers in craniofacial development, molecular genetics, and genomics. FaceBase demonstrates that a model in which researchers "self-curate" their data can be successful and scalable. We present the results of the first 2.5 y of FaceBase's operations as an open community and summarize the data sets published during this period. We then describe a research highlight from work on the identification of regulatory networks and noncoding RNAs involved in cleft lip with/without cleft palate that both used and in turn contributed new findings to publicly available FaceBase resources. Collectively, FaceBase serves as a dynamic and continuously evolving resource to facilitate data-intensive research, enhance data reproducibility, and perform deep phenotyping across multiple species in dental and craniofacial research.

Inhibiting Hh Signaling in Gli1+ Osteogenic Progenitors Alleviates TMJOA.

The increased prevalence of temporomandibular joint osteoarthritis (TMJOA) in children and adolescents has drawn considerable attention as it may interfere with mandibular condyle growth, resulting in dento-maxillofacial deformities. However, treatments for osteoarthritis have been ineffective at restoring the damaged bone and cartilage structures due to poor understanding of the underlying degenerative mechanism. In this study, we demonstrate that Gli1+ cells residing in the subchondral bone contribute to bone formation and homeostasis in the mandibular condyle, identifying them as osteogenic progenitors in vivo. Furthermore, we show that, in a TMJOA mouse model, derivatives of Gli1+ cells undergo excessive expansion along with increased but uneven distribution of osteogenic differentiation in the subchondral bone, which leads to abnormal subchondral bone remodeling via Hedgehog (Hh) signaling activation and to the development of TMJOA. The selective pharmacological inhibition and specific genetic inhibition of Hh signaling in Gli1+ osteogenic progenitors result in improved subchondral bone microstructure, attenuated local immune inflammatory response in the subchondral bone, and reduced degeneration of the articular cartilage, providing in vivo functional evidence that targeting Hh signaling in Gli1+ osteogenic progenitors can modulate bone homeostasis in osteoarthritis and provide a potential approach for treating TMJOA.

BMP Signaling in Regulating Mesenchymal Stem Cells in Incisor Homeostasis.

Bone morphogenetic protein (BMP) signaling performs multiple essential functions during craniofacial development. In this study, we used the adult mouse incisor as a model to uncover how BMP signaling maintains tissue homeostasis and regulates mesenchymal stem cell (MSC) fate by mediating WNT and FGF signaling. We observed a severe defect in the proximal region of the adult mouse incisor after loss of BMP signaling in the Gli1+ cell lineage, indicating that BMP signaling is required for cell proliferation and odontoblast differentiation. Our study demonstrates that BMP signaling serves as a key regulator that antagonizes WNT and FGF signaling to regulate MSC lineage commitment. In addition, BMP signaling in the Gli1+ cell lineage is also required for the maintenance of quiescent MSCs, suggesting that BMP signaling not only is important for odontoblast differentiation but also plays a crucial role in providing feedback to the MSC population. This study highlights multiple important roles of BMP signaling in regulating tissue homeostasis.

Protein Arginine Methyltransferase PRMT1 Is Essential for Palatogenesis.

Cleft palate is among the most common birth defects. Currently, only 30% of cases have identified genetic causes, whereas the etiology of the majority remains to be discovered. We identified a new regulator of palate development, protein arginine methyltransferase 1 (PRMT1), and demonstrated that disruption of PRMT1 function in neural crest cells caused complete cleft palate and craniofacial malformations. PRMT1 is the most highly expressed of the protein arginine methyltransferases, enzymes responsible for methylation of arginine motifs on histone and nonhistone proteins. PRMT1 regulates signal transduction and transcriptional activity that affect multiple signal pathways crucial in craniofacial development, such as the BMP, TGFß, and WNT pathways. We demonstrated that Wnt1-Cre;Prmt1 fl/fl mice displayed a decrease in palatal mesenchymal cell proliferation and failure of palatal shelves to reach the midline. Further analysis in signal pathways revealed that loss of Prmt1 in mutant mice decreased BMP signaling activation and reduced the deposition of H4R3me2a mark. Collectively, our study demonstrates that Prmt1 is crucial in palate development. Our study may facilitate the development of a better strategy to interrupt the formation of cleft palate through manipulation of PRMT1 activity.

Smad6 Methylation Represses NFκB Activation and Periodontal Inflammation.

The balance between pro- and anti-inflammatory signals maintains tissue homeostasis and defines the outcome of chronic inflammatory diseases such as periodontitis, a condition that afflicts the tooth-supporting tissues and exerts an impact on systemic health. The induction of tissue inflammation relies heavily on Toll-like receptor (TLR) signaling, which drives a proinflammatory pathway through recruiting myeloid differentiation primary response gene 88 (MyD88) and activating nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). TLR-induced production of proinflammatory cytokines and chemokines is reined in by anti-inflammatory cytokines, including the transforming growth factor ß (TGFß) family of cytokines. Although Smad6 is a key mediator of TGFß-induced anti-inflammatory signaling, the exact mechanism by which TGFß regulates TLR proinflammatory signaling in the periodontal tissue has not been addressed to date. In this study, we demonstrate for the first time that the ability of TGFß to inhibit TLR-NFκB signaling is mediated by protein arginine methyltransferase 1 (PRMT1)-induced Smad6 methylation. Upon methylation, Smad6 recruited MyD88 and promoted MyD88 degradation, thereby inhibiting NFκB activation. Most important, Smad6 is expressed and methylated in the gingival epithelium, and PRMT1-Smad6 signaling promotes tissue homeostasis by limiting inflammation. Consistent with this, disturbance of Smad6 methylation exacerbates inflammation and bone loss in experimental periodontitis. The dissected mechanism is therapeutically important, as it highlights the manipulation of PRMT1-Smad6 signaling as a novel promising strategy to modulate the host immune response in periodontitis.

Processing of a cellular polypeptide by 3CD proteinase is required for poliovirus ribonucleoprotein complex formation.

Poliovirus interactions with host cells were investigated by studying the formation of ribonucleoprotein complexes at the 3' end of poliovirus negative-strand RNA which are presumed to be involved in viral RNA synthesis. It was previously shown that two host cell proteins with molecular masses of 36 and 38 kDa bind to the 3' end of viral negative-strand RNA at approximately 3 to 4 h after infection. We tested the hypothesis that preexisting cellular proteins are modified during the course of infection and are subsequently recruited to play a role in viral replication. It was demonstrated that the 38-kDa protein, either directly or indirectly, is the product of processing by poliovirus 3CD/3C proteinase. Only the modified 38-kDa protein, not its precursor protein, has a high affinity for binding to the 3' end of viral negative-strand RNA. This modification depends on proteolytically active proteinase, and a direct correlation between the levels of 3CD proteinase and the 38-kDa protein was demonstrated in infected tissue culture cells. The nucleotide (nt) 5-10 region (positive-strand numbers) of poliovirus negative-strand RNA is important for binding of the 38-kDa protein. Deletion of the nt 5-10 region in full-length, positive-strand RNA renders the RNA noninfectious in transfection experiments. These results suggest that poliovirus 3CD/3C proteinase processes a cellular protein which then plays an essential role during the viral life cycle.

Determinants of membrane association for poliovirus protein 3AB.

Poliovirus protein 3AB may serve as the lipophilic carrier of a protein primer (VPg or 3B) used for the initiation of genomic viral RNA synthesis. In order to study the membrane-protein interactions of 3AB required for its role in poliovirus RNA replication, we have developed an in vitro membrane association assay capable of distinguishing membrane-bound from non-membrane-bound proteins that are cotranslated together in the presence of canine microsomal membranes. This assay utilizes equilibrium sedimentation analysis in high density sucrose gradients to measure membrane association of both wild type and mutated forms of 3AB. Using this assay and other biochemical assays, we have identified the following properties of the 3AB-membrane interaction: (a) 3AB is able to post-translationally associate with microsomal membranes, (b) 3AB is able to associate with membranes in a manner consistent with that of an integral membrane protein, (c) 3AB contains a critical hydrophobic sequence within the carboxyl-terminal half of the protein that is required for membrane association, and (d) the introduction of charged residues into this hydrophobic sequence disrupts the 3AB membrane-protein interaction. Taken together, these studies indicate that poliovirus protein 3AB associates tightly with biological membranes de novo in a manner that would allow it to serve as a lipophilic anchor for the assembly of the poliovirus RNA replication complex.