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Transcription factor epiprofin is essential for tooth morphogenesis by regulating epithelial cell fate and tooth number

In tooth morphogenesis, the dental epithelium and mesenchyme interact reciprocally for growth and differentiation to form the proper number and shapes of teeth. We previously identified epiprofin (Epfn), a gene preferentially expressed in dental epithelia, differentiated ameloblasts, and certain ectodermal organs. To identify the role of Epfn in tooth development, we created Epfn-deficient mice (Epfn-/-). Epfn-/- mice developed an excess number of teeth, enamel deficiency, defects in cusp and root formation, and abnormal dentin structure. Mutant tooth germs formed multiple dental epithelial buds into the mesenchyme. In Epfn-/- molars, rapid proliferation and differentiation of the inner dental epithelium were inhibited, and the dental epithelium retained the progenitor phenotype. Formation of the enamel knot, a signaling center for cusps, whose cells differentiate from the dental epithelium, was also inhibited. However, multiple premature nonproliferating enamel knot-like structures were formed ectopically. These dental epithelial abnormalities were accompanied by dysregulation of Lef-1, which is required for the normal transition from the bud to cap stage. Transfection of an Epfn vector promoted dental epithelial cell differentiation into ameloblasts and activated promoter activity of the enamel matrix ameloblastin gene. Our results suggest that in Epfn-deficient teeth, ectopic nonproliferating regions likely bud off from the self-renewable dental epithelium, form multiple branches, and eventually develop into supernumerary teeth. Thus, Epfn has multiple functions for cell fate determination of the dental epithelium by regulating both proliferation and differentiation, preventing continuous tooth budding and generation.

The Kruppel-like Factor Epiprofin Is Expressed by Epithelium of Developing Teeth, Hair Follicles, and Limb Buds and Promotes Cell Proliferation*

We identified a cDNA clone for epiprofin, which is preferentially expressed in teeth, by differential hybridization using DNA microarrays from an embryonic day 19.5 mouse molar cDNA library. Sequence analysis revealed that this cDNA encodes a member of the Krüppel-like factor family containing three characteristic C2H2-type zinc finger motifs. The full-length cDNA was obtained by the 5′ Cap capture method. Except for its 5′-terminal sequence, the epiprofin mRNA sequence is almost identical to the predicted sequence of Krüppel-like factor 14/Sp6 (specificity protein 6), which was previously identified in expressed sequence tag data bases and GenBank™ by an Sp1 zinc finger DNA-binding domain search (Scohy, S., Gabant, P., Van Reeth, T., Hertveldt, V., Dreze, P. L., Van Vooren, P., Riviere, M., Szpirer, J., and Szpirer, C. (2000) Genomics 70, 93-101). This sequence difference is due to differences in the assignment of the location of exon 1. In situ hybridization revealed that epiprofin mRNA is expressed by proliferating dental epithelium, differentiated odontoblast, and also hair follicle matrix epithelium. In addition, whole mount in situ hybridization showed transient expression of epiprofin mRNA in cells of the apical ectodermal ridge in developing limbs and the posterior neuropore. Transfection of an epiprofin expression vector revealed that this molecule is localized in the nucleus and promotes cell proliferation. Thus, epiprofin is a highly cell- and tissue-specific nuclear protein expressed primarily by proliferating epithelial cells of teeth, hair follicles, and limbs that may function in the development of these tissues by regulating cell growth.

Neural Crest Stem Cells from Dental Tissues: A New Hope for Dental and Neural Regeneration

Several stem cell sources persist in the adult human body, which opens the doors to both allogeneic and autologous cell therapies. Tooth tissues have proven to be a surprisingly rich and accessible source of neural crest-derived ectomesenchymal stem cells (EMSCs), which may be employed to repair disease-affected oral tissues in advanced regenerative dentistry. Additionally, one area of medicine that demands intensive research on new sources of stem cells is nervous system regeneration, since this constitutes a therapeutic hope for patients affected by highly invalidating conditions such as spinal cord injury, stroke, or neurodegenerative diseases. However, endogenous adult sources of neural stem cells present major drawbacks, such as their scarcity and complicated obtention. In this context, EMSCs from dental tissues emerge as good alternative candidates, since they are preserved in adult human individuals, and retain both high proliferation ability and a neural-like phenotype in vitro. In this paper, we discuss some important aspects of tissue regeneration by cell therapy and point out some advantages that EMSCs provide for dental and neural regeneration. We will finally review some of the latest research featuring experimental approaches and benefits of dental stem cell therapy.

Dental pulp stem cells as a multifaceted tool for bioengineering and the regeneration of craniomaxillofacial tissues

Dental pulp stem cells, or DPSC, are neural crest-derived cells with an outstanding capacity to differentiate along multiple cell lineages of interest for cell therapy. In particular, highly efficient osteo/dentinogenic differentiation of DPSC can be achieved using simple in vitro protocols, making these cells a very attractive and promising tool for the future treatment of dental and periodontal diseases. Among craniomaxillofacial organs, the tooth and salivary gland are two such cases in which complete regeneration by tissue engineering using DPSC appears to be possible, as research over the last decade has made substantial progress in experimental models of partial or total regeneration of both organs, by cell recombination technology. Moreover, DPSC seem to be a particularly good choice for the regeneration of nerve tissues, including injured or transected cranial nerves. In this context, the oral cavity appears to be an excellent testing ground for new regenerative therapies using DPSC. However, many issues and challenges need yet to be addressed before these cells can be employed in clinical therapy. In this review, we point out some important aspects on the biology of DPSC with regard to their use for the reconstruction of different craniomaxillofacial tissues and organs, with special emphasis on cranial bones, nerves, teeth, and salivary glands. We suggest new ideas and strategies to fully exploit the capacities of DPSC for bioengineering of the aforementioned tissues.

Role of Epiprofin, a zinc-finger transcription factor, in limb development

The formation and maintenance of the apical ectodermal ridge (AER) is critical for the outgrowth and patterning of the vertebrate limb. In the present work, we have investigated the role of Epiprofin (Epfn/Sp6), a member of the SP/KLF transcription factor family that is expressed in the limb ectoderm and the AER, during limb development. Epfn mutant mice have a defective autopod that shows mesoaxial syndactyly in the forelimb and synostosis (bony fusion) in the hindlimb and partial bidorsal digital tips. Epfn mutants also show a defect in the maturation of the AER that appears flat and broad, with a double ridge phenotype. By genetic analysis, we also show that Epfn is controlled by WNT/b-CATENIN signaling in the limb ectoderm. Since the less severe phenotypes of the conditional removal of b-catenin in the limb ectoderm strongly resemble the limb phenotype of Epfn mutants, we propose that EPFN very likely functions as a modulator of WNT signaling in the limb ectoderm.

Epiprofin/Sp6 regulates Wnt-BMP signaling and the establishment of cellular junctions during the bell stage of tooth development

Epiprofin/Specificity Protein 6 (Epfn) is a Krüppel-like family (KLF) transcription factor that is critically involved in tooth morphogenesis and dental cell differentiation. However, its mechanism of action is still not fully understood. We have employed both loss-of-function and gain-of-function approaches to address the role of Epfn in the formation of cell junctions in dental cells and in the regulation of junction-associated signal transduction pathways. We have evaluated the expression of junction proteins in bell-stage incisor and molar tooth sections from Epfn(−/−) mice and in dental pulp MDPC-23 cells overexpressing Epfn. In Epfn(−/−) mice, a dramatic reduction occurs in the expression of tight junction and adherens junction proteins and of the adherens-junction-associated β-catenin protein, a major effector of canonical Wnt signaling. Loss of cell junctions and β-catenin in Epfn(−/−) mice is correlated with a clear decrease in bone morphogenetic protein 4 (BMP-4) expression, a decrease in nestin in the tooth mesenchyme, altered cell proliferation, and failure of ameloblast cell differentiation. Overexpression of Epfn in MDPC-23 cells results in an increased cellular accumulation of β-catenin protein, indicative of upregulation of canonical Wnt signaling. Together, these results suggest that Epfn enhances canonical Wnt/β-catenin signaling in the developing dental pulp mesenchyme, a condition that promotes the activity of other downstream signaling pathways, such as BMP, which are fundamental for cellular induction and ameloblast differentiation. These altered signaling events might underlie some of the most prominent dental defects observed in Epfn(−/−) mice, such as the absence of ameloblasts and enamel, and might throw light on developmental malformations of the tooth, including hyperdontia.

Enhanced Wnt/β-catenin signalling during tooth morphogenesis impedes cell differentiation and leads to alterations in the structure and mineralisation of the adult tooth

Previous studies have indicated that over‐activation of the wingless interaction site (Wnt)/β‐catenin signalling pathway has important implications for tooth development, at the level of cell differentiation and morphology, as well as for the production of supernumerary teeth. Here, we provide evidence for a crucial role of this signalling pathway during the stage of tooth morphogenesis. We have developed an in vitro model consisting of 14.5‐day‐old mouse embryo first molars, in which the Wnt pathway is overactivated by the glycogen synthase kinase‐3 inhibitor 6‐bromoindirubin‐3′‐oxime (BIO; 20 µM).

Laminin surface binding sites and metastatic potential of 3LL tumor cells, increased by indomethacin

The level of laminin receptor expression on tumor cell surface has been correlated with the capacity of tumor cells to metastasize. In the present work we show that indomethacin treatment of a low metastatic 3LL tumor cells increases the ability of these cells to form lung metastasis and the binding of [125I] laminin on their cell surface. Scatchard analysis showed that the incubation with indomethacin (10(-7) M) for 48 h induced a specific increase of laminin binding sites on 3LL cell surface (1.5 fold per cell), presenting both a high and low affinity class of binding sites. On the other hand, indomethacin treatment (2 mg/kg weight) of tumor bearing mice increased the number of spontaneous metastatic nodules on the lung surface. Likewise, when 3LL tumor cells were incubated with indomethacin (10(-7) M) for 48 h, we observed an enhancement of lung metastatic nodules after intravenous injection of tumor cells. This last effect was partially reversed by peptides DPGYIGSR or YIGSR, corresponding to the active site at the B1 chain of laminin, with ability to bind the 67-kD laminin cell surface receptors. In summary, our results show that the increased attachment of 3LL tumor cells to laminin mediated by indomethacin is directly correlated with the metastatic activity of these cells, and suggests that the indomethacin effect on the metastatic potential could involve a modulation of laminin receptors on tumor cell surface.

DLK1 regulates branching morphogenesis and parasympathetic innervation of salivary glands through inhibition of NOTCH signalling

Delta‐like proteins 1 and 2 (DLK1, 2) are NOTCH receptor ligands containing epidermal growth factor‐like repeats, which regulate NOTCH signalling. We investigated the role of DLK and the NOTCH pathway in the morphogenesis of the submandibular salivary glands (SMGs), using in vitro organotypic cultures.

Stem Cell and Regenerative Medicine

Stem cells have been identified and isolated in many adult tissues. They exhibit a great plasticity and the ability to give rise to differentiated cells of several lineages. The possibility of transplantation of these stem cells into an adult to develop, integrate and rebuild destroyed tissues or organs has encouraged the study of the mechanisms of the differentiation of stem cells. These cells are nowadays being called a panacea in numerous diseases and, although their functional role is not well known, they are present in several areas of the human therapy, increasing the clinical applications. They represent the future of the transplant in medicine, and open, moreover, new perspectives in the treatment of diseases, as it is the case of the regenerative medicine. Here we review the current literature examining several aspect of medical therapy such as the applicability of experimental models to clinical practice.