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1.  Distribution of the amelogenin protein in developing, injured and carious human teeth 
Amelogenin is the major enamel matrix protein with key roles in amelogenesis. Although for many decades amelogenin was considered to be exclusively expressed by ameloblasts, more recent studies have shown that amelogenin is also expressed in other dental and no-dental cells. However, amelogenin expression in human tissues remains unclear. Here, we show that amelogenin protein is not only expressed during human embryonic development but also in pathological conditions such as carious lesions and injuries after dental cavity preparation. In developing embryonic teeth, amelogenin stage-specific expression is found in all dental epithelia cell populations but with different intensities. In the different layers of enamel matrix, waves of positive vs. negative immunostaining for amelogenin are detected suggesting that the secretion of amelogenin protein is orchestrated by a biological clock. Amelogenin is also expressed transiently in differentiating odontoblasts during predentin formation, but was absent in mature functional odontoblasts. In intact adult teeth, amelogenin was not present in dental pulp, odontoblasts, and dentin. However, in injured and carious adult human teeth amelogenin is strongly re-expressed in newly differentiated odontoblasts and is distributed in the dentinal tubuli under the lesion site. In an in vitro culture system, amelogenin is expressed preferentially in human dental pulp cells that start differentiating into odontoblast-like cells and form mineralization nodules. These data suggest that amelogenin plays important roles not only during cytodifferentiation, but also during tooth repair processes in humans.
doi:10.3389/fphys.2014.00477
PMCID: PMC4261713  PMID: 25540624
amelogenin; ameloblasts; tooth; odontoblast; enamel; carious; dental injury; dental pulp
2.  Oral epithelial stem cells – implications in normal development and cancer metastasis 
Experimental cell research  2014;325(2):111-129.
Oral mucosa is continuously exposed to environmental forces and has to be constantly renewed. Accordingly, the oral mucosa epithelium contains a large reservoir of epithelial stem cells necessary for tissue homeostasis. Despite considerable scientific advances in stem cell behavior in a number of tissues, fewer studies have been devoted to the stem cells in the oral epithelium. Most of oral mucosa stem cells studies are focused on identifying cancer stem cells (CSC) in oral squamous cell carcinomas (OSCCs) among other head and neck cancers. OSCCs are the most prevalent epithelial tumors of the head and neck region, marked by their aggressiveness and invasiveness. Due to their highly tumorigenic properties, it has been suggested that CSC may be the critical population of cancer cells in the development of OSCC metastasis. This review presents a brief overview of epithelium stem cells with implications in oral health, and the clinical implications of the CSC concept in OSCC metastatic dissemination.
doi:10.1016/j.yexcr.2014.04.021
PMCID: PMC4157336  PMID: 24803391
Oral squamous cell carcinoma; epithelial stem cells; invasion; metastasis; cancer stem cells; oral mucosa
3.  In vitro cytokines release profile: Predictive value for metastatic potential in head and neck squamous cell carcinomas 
Head & neck  2013;35(11):1542-1550.
Background
Head and neck squamous carcinomas (HNSCC) have devastating morbidity rates with mortality mainly because of metastasis.
Methods
Multiplex Enzyme-linked-immunosorbent-assay (ELISA) to assay a variety of cytokine levels secreted by a panel of stage- and anatomic site-specific primary, recurrent and metastatic University of Michigan-HNSCC cell lines over a 72-hour time-course.
Results
Conditioned medium from metastatic or recurrent HNSCC showed significantly higher amounts of interleukin (IL)-6, IL-6 receptor, Tumor Growth Factor-beta (TGF-β) and Vascular Endothelial Growth Factor (VEGF) than nonmetastatic cells or normal oral keratinocytes. Tumor Necrosis Factor was only secreted by the stage IV, metastatic, or recurrence-derived cell lines.
Conclusion
The cytokine profile of cultured HNSCC cells suggests that high levels of IL-6 and IL-6R, TGF-β, and VEGF are significantly related with their metastatogenic potential and provide rationale for determining if serum testing for a combination of these four soluble factors could be of predictive value for the HNSCC tumor progression and clinical outcome.
doi:10.1002/hed.23191
PMCID: PMC4157347  PMID: 23322448
Head and neck squamous carcinoma (HNSCC); cell lines; cytokines profiling; multiplex analysis; clinical outcome; metastasis
4.  In vitro evaluation of Sialyl Lewis X relationship with head and neck cancer stem cells 
Objective
To evaluate in vitro the potential links between sialyl Lewis X (sLeX) and cancer stem cells (CSC) in head and neck squamous cell carcinoma (HNSCC). HNSCC is an aggressive malignancy with high mortality mainly due to metastasis. CSC have emerged as important players in HNSCC metastasis. sLeX is a tetrasaccharide carbohydrate known to play a key-role in metastatic dissemination by promoting binding of the tumor cells to the endothelium.
Study Design
Experimental, in vitro.
Setting
Laboratory of Head and Neck Cancer Metastasis, University of Michigan.
Subjects and Methods
A panel of stage- and anatomic-site specific primary and metastatic HNSCC cell lines was assessed by flow cytometry to quantify sLeX relative expression levels. Serum-free conditioned media from the same HNSCC lines was collected over a time-course of 72 hours and assessed by Western-blot for secreted sLeX expression. Representative HNSCC cell lines were cultured as floating orospheres (condition that enhance CSC growth) or under normal adherent conditions and characterized by flow cytometry for CSC markers (CD44, aldehyde dehydrogenase [ALDH]) comparatively with sLeX expression.
Results
sLeX is predominantly expressed in carcinomas originating from the oral cavity. Secreted sLeX is also found to be high in oral carcinomas and increased over the analyzed time course. Floating orospheres were strongly positive for CD44 and ALDH confirming CSC enrichment of the orospheres. Tumor cells grown as orospheres are 95-100% positive for sLeX compared to 10-40% of adherent counterpart.
Conclusion
These studies provide the first evidence of sLeX relationship with CSC in HNSCC.
doi:10.1177/0194599813482879
PMCID: PMC4157351  PMID: 23558285
head and neck squamous cell carcinomas; sLeX (sialyl Lewis X); CD44; stem cells; in vitro analysis
5.  Circadian Rhythms Regulate Amelogenesis 
Bone  2013;55(1):158-165.
Ameloblasts, the cells responsible for making enamel, modify their morphological features in response to specialized functions necessary for synchronized ameloblast differentiation and enamel formation. Secretory and maturation ameloblasts are characterized by the expression of stage-specific genes which follows strictly controlled repetitive patterns. Circadian rhythms are recognized as key regulators of development and diseases of many tissues including bone. Our aim was to gain novel insights on the role of clock genes in enamel formation and to explore the potential links between circadian rhythms and amelogenesis. Our data shows definitive evidence that the main clock genes (Bmal1, Clock, Per1 and Per2) oscillate in ameloblasts at regular circadian (24h) intervals both at RNA and protein levels. This study also reveals that two markers of ameloblast differentiation i.e. amelogenin (Amelx; a marker of secretory ameloblasts) and kallikrein-related peptidase 4 (Klk4, a marker of maturation ameloblasts) are downstream targets of clock genes. Both, Amelx and Klk4 show 24h oscillatory expression patterns and their expression levels are up-regulated after Bmal1 over-expression in HAT-7 ameloblast cells. Taken together, these data suggest that both the secretory and the maturation stage of amelogenesis might be under circadian control. Changes in clock genes expression patterns might result in significant alterations of enamel apposition and mineralization.
doi:10.1016/j.bone.2013.02.011
PMCID: PMC3650122  PMID: 23486183
Clock genes; enamel; amelogenin; kallikrein-related peptidase 4; odontoblasts
6.  Molecular and circadian controls of ameloblasts 
European journal of oral sciences  2011;119(Suppl 1):35-40.
Stage-specific expression of ameloblast-specific genes is controlled by differential expression of transcription factors. In addition, ameloblasts follow daily rhythms in their main activities i.e. enamel protein secretion and enamel mineralization. This time related control is orchestrated by oscillations of clock proteins involved in circadian rhythms regulation. Our aim was to identify the potential links between daily rhythms and developmental controls of ameloblast differentiation. The effects of selected transcriptional factors Distal-less homeobox 3 (Dlx3) and Runt related transcription factor 2 (Runx2) and clock gene Nuclear receptor subfamily 1, group D, member 1 (Nr1d1) on secretory and maturation ameloblasts [using stage-specific markers amelogenin (Amel), enamelin (Enam) and kallikrein related-peptidase 4 (Klk4)] were evaluated in HAT-7 ameloblast cell line. Amel and Enam steady-state RNA expression levels were down-regulated in Runx2 over-expressing cells and up-regulated in Dlx3 over-expressing cells. In contrast, Klk4 was up-regulated by both Dlx3 and Runx2. Furthermore, a temporal and spatial relationship between clock genes and ameloblast differentiation markers was detected. Of interest, clock genes not only affected rhythmic expression of ameloblast specific genes but also influenced the expression of Runx2. Multi-scale mathematical modeling is being explored to further understand the temporal and developmental controls of ameloblast differentiation. Our study provides novel insights into the regulatory mechanisms sustaining ameloblast differentiation.
doi:10.1111/j.1600-0722.2011.00918.x
PMCID: PMC3516856  PMID: 22243224
Ameloblast gene regulation; circadian rhythms; clock genes; multi-scale modeling; enamel
7.  Tissue Regeneration in Dentistry 
doi:10.1155/2012/586701
PMCID: PMC3362032  PMID: 22666252
8.  Innovative Approaches to Regenerate Enamel and Dentin 
The process of tooth mineralization and the role of molecular control of cellular behavior during embryonic tooth development have attracted much attention the last few years. The knowledge gained from the research in these fields has improved the general understanding about the formation of dental tissues and the entire tooth and set the basis for teeth regeneration. Tissue engineering using scaffold and cell aggregate methods has been considered to produce bioengineered dental tissues, while dental stem/progenitor cells, which can differentiate into dental cell lineages, have been also introduced into the field of tooth mineralization and regeneration. Some of the main strategies for making enamel, dentin, and complex tooth-like structures are presented in this paper. However, there are still significant barriers that obstruct such strategies to move into the regular clinic practice, and these should be overcome in order to have the regenerative dentistry as the important mean that can treat the consequences of tooth-related diseases.
doi:10.1155/2012/856470
PMCID: PMC3359805  PMID: 22666253
9.  Expression of Clock Proteins in Developing Tooth 
Gene expression patterns : GEP  2010;11(3-4):202-206.
Morphological and functional changes during ameloblast and odontoblast differentiation suggest that enamel and dentin formation is under circadian control. Circadian rhythms are endogenous self-sustained oscillations with periods of 24 hours that control diverse physiological and metabolic processes. Mammalian clock genes play a key role in synchronizing circadian functions in many organs. However, close to nothing is known on clock genes expression during tooth development. In this work, we investigated the expression of four clock genes during tooth development. Our results showed that circadian clock genes Bmal1, clock, per1, and per2 mRNAs were detected in teeth by RT-PCR. Immunohistochemistry showed that clock protein expression was first detected in teeth at the bell stage (E17), being expressed in EOE and dental papilla cells. At post-natal day four (PN4), all four clock proteins continued to be expressed in teeth but with different intensities, being strongly expressed within the nucleus of ameloblasts and odontoblasts and down-regulated in dental pulp cells. Interestingly, at PN21 incisor, expression of clock proteins was down-regulated in odontoblasts of the crown-analogue side but expression was persisting in root-analogue side odontoblasts. In contrast, both crown and root odontoblasts were strongly stained for all four clock proteins in first molars at PN21. Within the periodontal ligament (PDL) space, epithelial rests of Malassez (ERM) showed the strongest expression among other PDL cells. Our data suggests that clock genes might be involved in the regulation of ameloblast and odontoblast functions, such as enamel and dentin protein secretion and matrix mineralization.
doi:10.1016/j.gep.2010.12.002
PMCID: PMC3073654  PMID: 21156215
Clock genes; Tooth development; Bmal1; Clock; Per1; Per2; expression pattern; immunohistochemistry
10.  Regulation of Dental Enamel Shape and Hardness 
Journal of dental research  2010;89(10):1024-1038.
Epithelial-mesenchymal interactions guide tooth development through its early stages and establish the morphology of the dentin surface upon which enamel will be deposited. Starting with the onset of amelogenesis beneath the future cusp tip, the shape of the enamel layer covering the crown is determined by five growth parameters. Appositional growth occurs at a mineralization front along the ameloblast distal membrane in which amorphous calcium phosphate (ACP) ribbons form and lengthen. The ACP ribbons convert to calcium hydroxyapatite as the ribbons elongate. Appositional growth involves a secretory cycle that leaves an imprint of incremental lines. A potentially important function of enamel proteins is to ensure alignment of successive mineral increments on the tips of enamel ribbons deposited in the previous cycle so the crystallites lengthen with each cycle. Enamel crystallites harden in a maturation process that involves mineral deposition on the sides of existing crystallites until they interlock with adjacent crystallites. Neutralization of acidity generated by hydroxyapatite formation is a key part of the mechanism. Here we review the growth parameters that determine the shape of the enamel crown as well as the mechanisms of enamel appositional growth and maturation.
doi:10.1177/0022034510375829
PMCID: PMC3086535  PMID: 20675598
appositional growth; amelogenin; enamelin; ameloblastin; tooth
11.  Identifying Promoter Elements Necessary for Enamelin Tissue-Specific Expression 
Cells, tissues, organs  2008;189(1-4):98-104.
Enamel development requires the strictly regulated spatiotemporal expression of genes encoding enamel matrix proteins. The mechanisms orchestrating the initiation and termination of gene transcription at each specific stage of amelogenesis are unknown. In this study, we identify cis-regulatory regions necessary for normal enamelin (Enam) expression. Sequence analysis of the Enam promoter 5′-noncoding region identified potentially important cis-regulatory elements located within 5.2 kb upstream of the Enam translation initiation site. DNA constructs containing 5.2 or 3.9 kb upstream of the Enam translation initiation site were linked to an LacZ reporter gene and used to generate transgenic mice. The 3.9-kb Enam-LacZ transgenic lines showed no expression in ameloblasts, but ectopic LacZ staining was detected in osteoblasts. In contrast, the 5.2-kb Enam-LacZ construct was sufficient to mimic the endogenous Enam ameloblast-specific expression pattern. Our study provides new insights into the molecular control of Enam cell- and stage-specific expression.
doi:10.1159/000151429
PMCID: PMC2754408  PMID: 18703866
Enam; cis-regulatory elements; Ameloblasts; Enamel proteins; Osteoblasts; Tissue-specific expression
12.  Distal cis-regulatory elements are required for tissue-specific expression of enamelin (Enam) 
European journal of oral sciences  2008;116(2):113-123.
Enamel formation is orchestrated by the sequential expression of genes encoding enamel matrix proteins; however, the mechanisms sustaining the spatio–temporal order of gene transcription during amelogenesis are poorly understood. The aim of this study was to characterize the cis-regulatory sequences necessary for normal expression of enamelin (Enam). Several enamelin transcription regulatory regions, showing high sequence homology among species, were identified. DNA constructs containing 5.2 or 3.9 kb regions upstream of the enamelin translation initiation site were linked to a LacZ reporter and used to generate transgenic mice. Only the 5.2-Enam–LacZ construct was sufficient to recapitulate the endogenous pattern of enamelin tooth-specific expression. The 3.9-Enam–LacZ transgenic lines showed no expression in dental cells, but ectopic β-galactosidase activity was detected in osteoblasts. Potential transcription factor-binding sites were identified that may be important in controlling enamelin basal promoter activity and in conferring enamelin tissue-specific expression. Our study provides new insights into regulatory mechanisms governing enamelin expression.
doi:10.1111/j.1600-0722.2007.00519.x
PMCID: PMC2701970  PMID: 18353004
ameloblasts; enamel proteins; enamelin; osteoblasts; tissue-specific expression
13.  Functions of KLK4 and MMP-20 in dental enamel formation 
Biological chemistry  2008;389(6):695-700.
Two proteases are secreted into the enamel matrix of developing teeth. The early protease is enamelysin (MMP-20). The late protease is kallikrein 4 (KLK4). Mutations in MMP20 and KLK4 both cause autosomal recessive amelogenesis imperfecta, a condition featuring soft, porous enamel containing residual protein. MMP-20 is secreted along with enamel proteins by secretory stage ameloblasts. Enamel protein cleavage products accumulate in the space between the crystal ribbons, helping to support them. MMP-20 steadily cleaves accumulated enamel proteins, so their concentration decreases with depth. Kallikrein 4 is secreted by transition and maturation stage ameloblasts. KLK4 aggressively degrades the retained organic matrix following the termination of enamel protein secretion. The principle functions of MMP-20 and KLK4 in dental enamel formation are to facilitate the orderly replacement of organic matrix with mineral, generating an enamel layer that is harder, less porous, and unstained by retained enamel proteins.
doi:10.1515/BC.2008.080
PMCID: PMC2688471  PMID: 18627287
amelogenesis imperfecta; EMSP1; enamelysin; kallikrein; matrix metalloproteinase; tooth enamel
14.  Identifying Promoter Elements Necessary for Enamelin Tissue-Specific Expression 
Cells, Tissues, Organs  2008;189(1-4):98-104.
Enamel development requires the strictly regulated spatiotemporal expression of genes encoding enamel matrix proteins. The mechanisms orchestrating the initiation and termination of gene transcription at each specific stage of amelogenesis are unknown. In this study, we identify cis- regulatory regions necessary for normal enamelin (Enam) expression. Sequence analysis of the Enam promoter 5′-noncoding region identified potentially important cis-regulatory elements located within 5.2 kb upstream of the Enam translation initiation site. DNA constructs containing 5.2 or 3.9 kb upstream of the Enam translation initiation site were linked to an LacZ reporter gene and used to generate transgenic mice. The 3.9-kb Enam-LacZ transgenic lines showed no expression in ameloblasts, but ectopic LacZ staining was detected in osteoblasts. In contrast, the 5.2-kb Enam-LacZ construct was sufficient to mimic the endogenous Enam ameloblast-specific expression pattern. Our study provides new insights into the molecular control of Enam cell- and stage-specific expression.
doi:10.1159/000151429
PMCID: PMC2754408  PMID: 18703866
Enam; cis-regulatory elements; Ameloblasts; Enamel proteins; Osteoblasts; Tissue-specific expression

Results 1-14 (14)