Prior studies have demonstrated that the pineal hormone, melatonin, can stimulate chloramphenicol acetyltransferase activity in Drosophila SL-3 cells transfected with a chloramphenicol acetyltransferase reporter construct containing the response element of rat bone sialoprotein (BSP). Based on these findings, studies were performed to determine whether melatonin could similarly modulate the expression of BSP in two cell lines, the MC3T3-E1(MC3T3) pre-osteoblast and rat osteoblast-like osteosarcoma 17/2.8 cell. Initial studies demonstrated that MC3T3 cells grown in the presence of 50 nM melatonin underwent cell differentiation and mineralization by day 12 instead of the 21-day period normally required for cells grown in untreated media. Melatonin increased gene expression of BSP and the other bone marker proteins, including alkaline phosphatase (ALP); osteopontin; secreted protein, acidic and rich in cysteine; and osteocalcin in MC3T3 cells in a concentrationdependent manner. Levels of melatonin as low as 10 nM were capable of stimulating transcription of these genes when cells were grown in the presence of ␤-glycerophosphate and ascorbic acid. Under these conditions, melatonin induced gene expression of the bone marker proteins; however, this does not occur until the 5th day after seeding the culture dishes. Thereafter, MC3T3 cells responded to melatonin within 2 h of treatment. The fully differentiated rat osteoblast-like osteosarcoma 17/ 2.8 cells responded rapidly to melatonin and displayed an increase in the expression of BSP, ALP, and osteocalcin genes within 1 h of exposure to the hormone. To determine whether melatonin-induced osteoblast differentiation and bone formation are mediated via the transmembrane receptor, MC3T3 cells were treated in the presence and absence of melatonin with either luzindole, a competitive inhibitor of the binding of melatonin to the transmembrane receptors, or pertussis toxin, an uncoupler of G i from adenylate cyclase. Both luzindole and pertussis toxin were shown to reduce melatonin-induced expression of BSP and ALP. These results demonstrate, for the first time, that the pineal hormone, melatonin, is capable of promoting osteoblast differentiation and mineralization of matrix in culture and suggest that this hormone may play an essential role in regulating bone growth.Melatonin is the major hormone released from the pineal gland, and its levels are synchronized by environmental light with nightly plasma concentrations reaching approximately 50 times higher than that reached during daytime (1-3). Melatonin regulates a variety of physiological and pathophysiological processes including hypothalamic control of circadian rhythms (4 -6), regulation of reproductive function in seasonally breeding species (7), and regulation of temperature (8), sexual development (9, 10), the immune system (11), and the cardiovasculature (12). It has also been shown to influence cell differentiation where it can either stimulate or suppress cell division depending on its concentration or the cell ...

The mammalian tooth forms by a series of reciprocal epithelial-mesenchymal interactions. Although several signaling pathways and transcription factors have been implicated in regulating molar crown development, relatively little is known about the regulation of root development. Four genes encoding nuclear factor I (NFI) transcription-replication proteins are present in the mouse genome: Nfia, Nfib, Nfic, and Nfix. In order to elucidate its physiological role(s), we disrupted the Nfic gene in mice. Heterozygous animals appear normal, whereas Nfic ؊/؊ mice have unique tooth pathologies: molars lacking roots, thin and brittle mandibular incisors, and weakened abnormal maxillary incisors. Feeding in Nfic ؊/؊ mice is impaired, resulting in severe runting and premature death of mice reared on standard laboratory chow. However, a soft-dough diet mitigates the feeding impairment and maintains viability. Although Nfic is expressed in many organ systems, including the developing tooth, the tooth root development defects were the prominent phenotype. Indeed, molar crown development is normal, and well-nourished Nfic ؊/؊ animals are fertile and can live as long as their wild-type littermates. The Nfic mutation is the first mutation described that affects primarily tooth root formation and should greatly aid our understanding of postnatal tooth development.Tooth formation is a complex developmental process that is mediated through a series of epithelial-mesenchymal interactions (29). Several signaling pathways required for early molar tooth development have been identified, including the BMP (31), FGF (13, 27), SHH (5, 10), and WNT (25, 33) pathways. In addition, targeted disruption of a number of transcription factors, including Msx1 and -2 (1), Dlx1 and -2 (22), Pax-9 (21) and others, severely disrupts early tooth development. In contrast, only one pathway regulating late tooth developement has been identified. The Tabby (Ta), Downless (Dl), and Crinkled (Cr) mouse mutations each cause similar defects of postnatal development (32) by disrupting different components of the Ectodysplasin-A hormone signaling system.The nuclear factor I (NFI) family of transcription-replication factors is encoded by four genes in mammals (NFI-A, -B, -C, and -X) and a single gene in Drosophila melanogaster and Caenorhabditis elegans (8,14,23). Prokaryotic homologues of the NFI genes have not been identified. NFI was discovered as a protein required for adenovirus DNA replication in vitro (19,20), but it is now clear that NFI proteins play an important role in the expression of many cellular genes (reviewed in reference 8). NFI-C/CTF was the initial NFI gene cloned (24) and contains a prototypical proline-rich transcriptional activation domain, as well as a heptamer repeat homologous to the C-terminal domain of RNA polymerase II (18). Because products of the four NFI genes are often coexpressed (3) and bind to similar DNA sequences (15), it has been difficult to determine the roles of individual NFI family members in gene expression during deve...

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Our previous studies have demonstrated that nuclear factor I-C (NFI-C) null mice developed short molar roots that contain aberrant odontoblasts and abnormal dentin formation. Based on these findings, we performed studies to elucidate the function of NFI-C in odontoblasts. Initial studies demonstrated that aberrant odontoblasts become dissociated and trapped in an osteodentin-like mineralized tissue. Abnormal odontoblasts exhibit strong bone sialoprotein expression but a decreased level of dentin sialophosphoprotein expression when compared with wild type odontoblasts. Loss of Nfic results in an increase in p-Smad2/3 expression in aberrant odontoblasts and pulp cells in the subodontoblastic layer in vivo and primary pulp cells from Nfic-deficient mice in vitro. Cell proliferation analysis of both cervical loop and ectomesenchymal cells of the Nfic-deficient mice revealed significantly decreased proliferative activity compared with wild type mice. In addition, Nfic-deficient primary pulp cells showed increased expression of p21 and p16 but decreased expression of cyclin D1 and cyclin B1, strongly suggesting cell growth arrest caused by a lack of Nfic activity. Analysis of the pulp and abnormal dentin in Nfic-deficient mice revealed an increase in apoptotic activity. Further, Nfic-deficient primary pulp cells exhibited an increase in caspase-8 and -3 activation, whereas the cleaved form of Bid was hardly detected. These results indicate that the loss of Nfic leads to the suppression of odontogenic cell proliferation and differentiation and induces apoptosis of aberrant odontoblasts during root formation, thereby contributing to the formation of short roots.Tooth development is a complex and well coordinated developmental process that is achieved through a series of reciprocal interactions between dental epithelium and neural crest-derived ectomesenchyme (EM).2 The dental epithelium gives rise to the outer and inner enamel epithelium from which ameloblasts differentiate, whereas EM cells differentiate into odontoblasts. The critical roles of several transcription factors and growth factors in crown formation have been well documented (1, 2). After completion of crown formation, the inner and outer enamel epithelial cells proliferate and form Hertwig's epithelial root sheath that plays a key role in root formation. It is believed, based on information derived from crown development, that Hertwig's epithelial root sheath induces the differentiation of EM cells from the radicular pulp area into odontoblasts that are responsible for root dentin formation. However, the molecular mechanisms responsible for root development are not well understood (3-5).The nuclear factor I (NFI) family of transcription/replication factors was first discovered as a family of proteins required for the replication of adenovirus DNA in vitro (6). The NFI gene family encodes site-specific transcription factors essential for the development of a number of organ systems (7). There are four NFI gene family members in vertebrates (Nfia, Nfib, Nfic...

These results indicate that PDL Fbs after tooth extraction actively proliferative, migrate into the coagulum, form dense connective tissue, and differentiate into Obs which form new bone during socket healing.