Yaping Lei

The murine amelogenin promoter: developmentally regulated expression in transgenic animals.

We are interested in understanding hierarchical regulation pathways that control gene expression in developing teeth. In pursuit of the molecular basis for the regulated expression of amelogenin by developing ameloblasts during tooth formation, we isolated the murine amelogenin promoter. Analysis of this promoter will provide additional details towards the identification of signals generated through instructive-, dissimilar-germ layer interactions that are for responsible for temporal- and spatial-regulation for amelogenin gene expression. Using transgenic mice we demonstrate that a 2263 nucleotide stretch of the murine amelogenin promoter conveys appropriate temporal- and spatial-regulation for amelogenin gene expression in response to instructive-signals. These transgenic animals are useful reagents to further dissect signaling pathways responsible for regulated gene expression by terminally differentiated ameloblasts.

The circadian clock modulates enamel development.

Fully mature enamel is about 98% mineral by weight. While mineral crystals appear very early during its formative phase, the newly secreted enamel is a soft gel-like matrix containing several enamel matrix proteins of which the most abundant is amelogenin (Amelx). Histological analysis of mineralized dental enamel reveals markings called cross-striations associated with daily increments of enamel formation, as evidenced by injections of labeling dyes at known time intervals. The daily incremental growth of enamel has led to the hypothesis that the circadian clock might be involved in the regulation of enamel development. To identify daily rhythms of clock genes and Amelx, we subjected murine ameloblast cells to serum synchronization to analyze the expression of the circadian transcription factors Per2 and Bmal1 by real-time PCR. Results indicate that these key genetic regulators of the circadian clock are expressed in synchronized murine ameloblast cell cultures and that their expression profile follows a circadian pattern with acrophase and bathyphase for both gene transcripts in antiphase. Immunohistological analysis confirms the protein expression of Bmal and Cry in enamel cells. Amelx expression in 2-day postnatal mouse molars dissected every 4 hours for a duration of 48 hours oscillated with an approximately 24-hour period, with a significant approximately 2-fold decrease in expression during the dark period compared to the light period. The expression of genes involved in bicarbonate production (Car2) and transport (Slc4a4), as well as in enamel matrix endocytosis (Lamp1), was greater during the dark period, indicating that ameloblasts express these proteins when Amelx expression is at the nadir. The human and mouse Amelx genes each contain a single nonconserved E-box element within 10 kb upstream of their respective transcription start sites. We also found that within 2 kb of the transcription start site of the human NFYA gene, which encodes a positive regulator of amelogenin, there is an E-box element that is conserved in rodents and other mammals. Moreover, we found that Nfya expression in serum-synchronized murine ameloblasts oscillated with a strong 24-hour rhythm. Taken together, our data support the hypothesis that the circadian clock temporally regulates enamel development.

Altered Amelogenin Self-assembly Based on Mutations Observed in Human X-linked Amelogenesis Imperfecta (AIH1)

A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit. The principle protein of enamel is amelogenin, a 180-amino acid hydrophobic protein that self-assembles to form nanospheres. We have used independent technical methods, consisting of the yeast two-hybrid (Y2H) assay and surface plasmon resonance (SPR), to demonstrate the importance of amelogenin self-assembly domains. In addition, we have analyzed mutations in amelogenin observed in patients with amelogenesis imperfecta who demonstrate defects in enamel formation. Assessments of self-assembly of these mutant amelogenins by either SPR or Y2H assay yield concordant data. These data support the conclusion that the amelogenin amino-terminal self-assembly domain is essential to the creation of an enamel extracellular organic matrix capable of directing mineral formation. It also suggests that a pathway through which point mutations in the amelogenin protein can adversely impact on the formation of the enamel organ is by disturbing self-assembly of the organic matrix. These data support the utilization of the Y2H assay to search for protein interactions among extracellular matrix proteins that contribute to biomineralization and provide functional information on protein-protein and protein-mineral interactions.

Structural organization and cellular localization of tuftelin-interacting protein 11 (TFIP11)

Abstract.Tuftelin-interacting protein (TFIP11) was first identified in a yeast two-hybrid screening as a protein interacting with tuftelin. The ubiquitous expression of TFIP11 suggested that it might have other functions in non-dental tissues. TFIP11 contains a G-patch, a protein domain believed to be involved in RNA binding. Using a green fluorescence protein tag, TFIP11 was found to locate in a novel subnuclear structure that we refer to as the TFIP body. An in vivo splicing assay demonstrated that TFIP11 is a novel splicing factor. TFIP11 diffuses from the TFIP body following RNase A treatment, suggesting that the retention of TFIP11 is RNA dependent. RNA polymerase II inhibitor (-amanitin and actinomycin D) treatment causes enlargement in size and decrease in number of TFIP bodies, suggesting that TFIP bodies perform a storage function rather than an active splicing function. The TFIP body may therefore represent a new subnuclear storage compartment for splicing components.

Perturbed amelogenin secondary structure leads to uncontrolled aggregation in amelogenesis imperfecta mutant proteins.

Mutations in amelogenin sequence result in defective enamel, and the diverse group of genetically altered conditions is collectively known as amelogenesis imperfecta (AI). Despite numerous studies, the detailed molecular mechanism of defective enamel formation is still unknown. In this study, we have examined the biophysical properties of a recombinant murine amelogenin (rM180) and two point mutations identified from human DNA sequences in two cases of AI (T21I and P41T). At pH 5.8 and 25 °C, wild type (WT) rM180 and mutant P41T existed as monomers, and mutant T21I formed lower order oligomers. CD, dynamic light scattering, and fluorescence studies indicated that rM180 and P41T can be classified as a premolten globule-like subclass protein at 25 °C. Thermal denaturation and refolding monitored by CD ellipticity at 224 nm indicated the presence of a strong hysteresis in mutants compared with WT. Variable temperature tryptophan fluorescence and dynamic light scattering studies showed that WT transformed to a partially folded conformation upon heating and remained stable. The partially folded conformation formed by P41T, however, readily converted into a heterogeneous population of aggregates. T21I existed in an oligomeric state at room temperature and, upon heating, rapidly formed large aggregates over a very narrow temperature range. Thermal denaturation and refolding studies indicated that the mutants are less stable and exhibit poor refolding ability compared with WT rM180. Our results suggest that alterations in self-assembly of amelogenin are a consequence of destabilization of the intrinsic disorder. Therefore, we propose that, like a number of other human diseases, AI appears to be due to the destabilization of the secondary structure as a result of amelogenin mutations.

Transgene Animal Model for Protein Expression and Accumulation into Forming Enamel

Understanding the cellular and molecular events that regulate the formation of enamel is a major driving force in efforts to characterize critical events during amelogenesis. It is anticipated that through such an understanding, improvements in prevention, diagnosis and treatment-intervention into heritable and acquired diseases of enamel could be achieved. While knowledge of the precise role of an enamel-specific protein in directing the formation of inorganic crystallites remains refractory, progress has been made with other aspects of amelogenesis that can be brought to bear on the subject. One such area of progress has been with the identification of an ameloblast-lineage specific amelogenin gene promoter. This promoter can be used to direct the expression of enamel-specific proteins, as well as the expression of proteins foreign to amelogenesis, into the enamel extracellular matrix where their effect on biomineralization can be ascertained in a prospective manner. The resulting enamel from such animals can be examined by morphologic and biochemical modalities in order to identify the effect of the transgene protein on enamel crystallite formation and subsequent biomineralization. This manuscript outlines such a strategy with the potential for enhancing our understanding of amelogenesis.

The role of bioactive nanofibers in enamel regeneration mediated through integrin signals acting upon C/EBPα and c-Jun

Enamel formation involves highly orchestrated intracellular and extracellular events; following development, the tissue is unable to regenerate, making it a challenging target for tissue engineering. We previously demonstrated the ability to trigger enamel differentiation and regeneration in the embryonic mouse incisor using a self-assembling matrix that displayed the integrin-binding epitope RGDS (Arg-Gly-Asp-Ser). To further elucidate the intracellular signaling pathways responsible for this phenomenon, we explore here the coupling response of integrin receptors to the biomaterial and subsequent downstream gene expression profiles. We demonstrate that the artificial matrix activates focal adhesion kinase (FAK) to increase phosphorylation of both c-Jun N-terminal kinase (JNK) and its downstream transcription factor c-Jun (c-Jun). Inhibition of FAK blocked activation of the identified matrix-mediated pathways, while independent inhibition of JNK nearly abolished phosphorylated-c-Jun (p-c-Jun) and attenuated the pathways identified to promote enamel regeneration. Cognate binding sites in the amelogenin promoter were identified to be transcriptionally up-regulated in response to p-c-Jun. Furthermore, the artificial matrix induced gene expression as evidenced by an increased abundance of amelogenin, the main protein expressed during enamel formation, and the CCAAT enhancer binding protein alpha (C/EBPα), which is the known activator of amelogenin expression. Elucidating these cues not only provides guidelines for the design of synthetic regenerative strategies and opportunities to manipulate pathways to regulate enamel regeneration, but can provide insight into the molecular mechanisms involved in tissue formation.

A simplified genetic design for mammalian enamel

A biomimetic replacement for tooth enamel is urgently needed because dental caries is the most prevalent infectious disease to affect man. Here, design specifications for an enamel replacement material inspired by Nature are deployed for testing in an animal model. Using genetic engineering we created a simplified enamel protein matrix precursor where only one, rather than dozens of amelogenin isoforms, contributed to enamel formation. Enamel function and architecture were unaltered, but the balance between the competing materials properties of hardness and toughness was modulated. While the other amelogenin isoforms make a modest contribution to optimal biomechanical design, the enamel made with only one amelogenin isoform served as a functional substitute. Where enamel has been lost to caries or trauma a suitable biomimetic replacement material could be fabricated using only one amelogenin isoform, thereby simplifying the protein matrix parameters by one order of magnitude.

Folding, assembly, and aggregation of recombinant murine amelogenins with T21I and P41T point mutations.

Two point mutations (T21I and P40T) within amelogenin have been identified from human DNA sequences in 2 instances of amelogenesis imperfecta. We studied the folding and self-assembly of recombinant amelogenin (rM180) compared to the T21I and P40T mutants analogs. At pH 5.8 and 25°C, rM180 and the P41T mutant existed as monomers, whereas the T21I mutant formed small oligomers. At pH 8 and 25°C, all of the amelogenin samples formed nanospheres with hydrodynamic radii (RH) of around 15–16 nm. Upon heating to 37°C, particles of P41T increased in size (RH = 18 nm). During thermal denaturation at pH 5.8, both of the mutant proteins refolded more slowly than the wild-type (WT) rM180. Variable temperature tryptophan fluorescence and dynamic light scattering studies showed that the WT transformed to a partially folded conformation upon heating and remained stable. Thermal denaturation and refolding studies indicated that the mutants were less stable and exhibit a greater ability to prematurely aggregate compared to the WT. Our data suggest that in the case of P41T, alterations in the self-assembly of amelogenin are a consequence of destabilization of the secondary structure, while in the case of T21I they are a consequence of change in the overall hydrophobicity at the N-terminal region. We propose that alterations in the assembly (i.e. premature aggregation) of mutant amelogenins may have a profound effect on intra- and extracellular processes such as amelogenin secretion, proteolysis, and its interactions with nonamelogenins as well as with the forming mineral.

Perturbed Amelogenin Protein Self-assembly Alters Nanosphere Properties Resulting in Defective Enamel Formation

Dental enamel is a unique composite bioceramic material that is the hardest tissue in the vertebrate body, containing long-, thin-crystallites of substituted hydroxyapatite. Enamel functions under immense loads in a bacterial-laden environment, and generally without catastrophic failure over a lifetime for the organism. Unlike all other biogenerated hard tissues of mesodermal origin, such as bone and dentin, enamel is produced by ectoderm-derived cells called ameloblasts. Recent investigations on the formation of enamel using cell and molecular approaches have been coupled to biomechanical investigations at the nanoscale and mesoscale levels. For amelogenin, the principle protein of forming enamel, two domains have been identified that are required for the proper assembly of multimeric units of amelogenin to form nanospheres. One domain is at the amino-terminus and the other domain in the carboxyl-terminal region. Amelogenin nanospheres are believed to influence the hydroxyapatite crystal habit. Both the yeast two-hybrid assay and surface plasmon resonance have been used to examine the assembly properties of engineered amelogenin proteins. Amelogenin protein was engineered using recombinant DNA techniques to contain deletions to either of the two self-assembly domains. Amelogenin protein was also engineered to contain single amino-acid mutations/substitutions in the amino-terminal self-assembly domain; and these amino-acid changes are based upon point mutations observed in humans affected with a hereditary disturbance of enamel formation. All of these alterations reveal significant defects in amelogenin self-assembly into nanospheres in vitro . Transgenic animals containing these same amelogenin deletions illustrate the importance of a physiologically correct bio-fabrication of the enamel protein extracellular matrix to allow for the organization of the enamel prismatic structure.