Dentin sialophosphoprotein (DSPP) consists of dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). The spatial-temporal expression of DSPP is largely restricted during differentiational stages of dental cells. DSPP plays a vital role in tooth development. It is known that an osteoblast-specific transcription factor, Runx2, is essential for osteoblast differentiation. However, effects of Runx2 on DSPP transcription remain unknown. Here, we studied different roles of Runx2 in controlling DSPP expression in mouse preodontoblast (MD10-F2) and odontoblast (MO6-G3) cells. Two Runx2 isoforms were expressed in preodontoblast and odontoblast cells, and in situ hybridization assay showed that DSPP expression increased, whereas Runx2 was downregulated during odontoblast differentiation and maturation. Three potential Runx2 sites are present in promoters of mouse and rat DSPP genes. Runx2 binds to these sites as demonstrated by electrophoretic mobility shift assay and supershift experiments. Mutations of Runx2 sites in mouse DSPP promoter resulted in a decline of promoter activity in MD10-F2 cells compared with an increase of its activity in MO6-G3 cells. Multiple Runx2 sites were more active than a single site in regulating the DSPP promoter. Furthermore, forced overexpression of Runx2 isoforms induced increases of endogenous DSPP protein levels in MD10-F2 cells but reduced its expression in MO6-G3 cells consistent with the DSPP promoter analysis. Thus, our results suggest that differential positive and negative regulation of DSPP by Runx2 is dependent on use of cytodifferentiation of dental ectomesenchymal-derived cells that may contribute to the spatial-temporal expression of DSPP during tooth development.Tooth organogenesis is the result of reciprocal interactions between epithelial-mesenchymal cells leading to the terminal differentiation of matrix-producing cells (1-2). Dental papilla mesenchymal cells give rise to dental pulp cells, which maintain the homeostasis of dental mineralized tissues and support dentin, and odontoblasts, which synthesize dentin extracellular matrix. Recently, several in vitro and in vivo studies have demonstrated that dental pulp cells are capable of differentiating into odontoblasts and producing a mineralizing matrix, particularly during reparative dentinogenesis associated with injury and disease (3, 4).Odontoblast and dental pulp cells synthesize and secrete several collagenous and non-collagenous proteins (NCPs) 1 to form a unique dentin extracellular matrix. Dentin sialophosphoprotein (DSPP) is a phosphorylated parent protein that is cleaved post-translationally into two dentin NCPs: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) (5-6). DSPP gene is encoded by five exons and four introns (7) with the DSP sequences located at the NH 2 terminus (exons 1-4 and the 5Ј region of exon 5) and the DPP domain found at the COOH region (remainder of exon 5). DSP and DPP contain high levels of carbohydrate and sialic acid as well as aspartic acid and phosphoserine, suggesting a fu...

BackgroundSynucleinopathy is any of a group of age-related neurodegenerative disorders including Parkinson's disease, multiple system atrophy, and dementia with Lewy Bodies, which is characterized by α-synuclein inclusions and parkinsonian motor deficits affecting millions of patients worldwide. But there is no cure at present for synucleinopathy. Rapamycin has been shown to be neuroprotective in several in vitro and in vivo synucleinopathy models. However, there are no reports on the long-term effects of RAPA on motor function or measures of neurodegeneration in models of synucleinopathy.MethodsWe determined whether long-term feeding a rapamycin diet (14 ppm in diet; 2.25 mg/kg body weight/day) improves motor function in neuronal A53T α-synuclein transgenic mice (TG) and explored underlying mechanisms using a variety of behavioral and biochemical approaches.ResultsAfter 24 weeks of treatment, rapamycin improved performance on the forepaw stepping adjustment test, accelerating rotarod and pole test. Rapamycin did not alter A53T α-synuclein content. There was no effect of rapamycin treatment on midbrain or striatal monoamines or their metabolites. Proteins adducted to the lipid peroxidation product 4-hydroxynonenal were decreased in brain regions of both wild-type and TG mice treated with rapamycin. Reduced levels of the presynaptic marker synaptophysin were found in several brain regions of TG mice. Rapamycin attenuated the loss of synaptophysin protein in the affected brain regions. Rapamycin also attenuated the loss of synaptophysin protein and prevented the decrease of neurite length in SH-SY5Y cells treated with 4-hydroxynonenal.ConclusionTaken together, these data suggest that rapamycin, an FDA approved drug, may prove useful in the treatment of synucleinopathy.

The proto-oncogene molecule c-Crk plays a role in growth factor-induced activation of Ras. Sphingosine 1-phosphate (SPP), a metabolite of cellular sphingolipids, has previously been shown to play a role in growth factor receptor signaling (Olivera, A., and Spiegel, S. (1993) Nature 365, 557-560). SPP was found to strongly induce tyrosine phosphorylation of Crk, but not Shc, in NIH-3T3 parental, insulin-like growth factor-I receptoroverexpressing and Crk-overexpressing (3T3-Crk) fibroblasts. Sphingosine, a metabolic precursor of SPP, also produced a slight increase in tyrosine phosphorylation of Crk. In contrast, other sphingolipid metabolites including ceramide did not alter Crk tyrosine phosphorylation. Furthermore, Crk enhanced SPP-induced mitogenesis, as measured by SPP-stimulated [ 3 H]thymidine incorporation in a manner proportional to the level of Crk expression in 3T3-Crk cells. This stimulation appears to be Ras-dependent, whereas SPP stimulation of MAP kinase activity is Ras-independent. These data indicate that SPP activates a tyrosine kinase that phosphorylates Crk and that Crk is a positive effector of SPP-induced mitogenesis.Increasing evidence suggests that the sphingolipid ceramide and its metabolites sphingosine and sphingosine 1-phosphate (SPP) 1 represent a new class of intracellular second messengers that mediate a variety of cellular functions (1-5). Sphingosine and SPP have been shown to induce mitogenesis in a wide range of cell types (5-7). Platelet-derived growth factor (PDGF), a potent mitogen, increases cellular levels of sphingosine and SPP (8, 9). Moreover, inhibition of the PDGF-induced increase in SPP levels markedly decreased PDGF-induced cellular proliferation (8).The downstream signaling pathways utilized by these sphingolipids have not been fully elucidated. Sphingosine and SPP are known to increase intracellular calcium (6, 10 -12) and phosphatidic acid levels (13, 14) and decrease cAMP levels (6, 15). SPP has also been shown to stimulate the Raf/MEK/MAP kinase signaling pathway (16). In an attempt to understand the molecular mechanisms underlying sphingosine and SPP-induced mitogenesis, we investigated the effect of these sphingolipids on intracellular signaling molecules upstream of the MAP kinase cascade, and then we examined the role of the MAP kinase signaling pathway.It has previously been shown that activation of either Shc-or Crk-related pathways leads to activation of the MAP kinase cascade. Shc is an SH2 domain-containing protein that becomes tyrosine-phosphorylated and associates with Grb2 in response to growth factor receptor stimulation (17-19). Signaling through Shc appears to be a common pathway by which both tyrosine kinase growth factor receptors and certain G-protein-coupled receptors lead to activation of Ras (20). Crk is a noncatalytic SH2 and SH3 domain-containing adaptor molecule that shares structural homology with . Like Grb2, Crk associates with the guanine nucleotide exchange factor mSos and a related molecule called C3G (23, 24). Thus, Crk provides an...

Tooth formation is the result of reciprocal instructive interactions between oral epithelium and cranial neural-crest-derived ectomesenchymal tissues. These interactions lead to the cytodifferentiation of highly specialized matrix-forming cell types, the ameloblast, odontoblast, and cementoblast, that produce the mineralized tissues enamel, dentin, and cementum, respectively. Our laboratory has been developing immortalized dental cell lines representative of these various cell types to facilitate studies on gene regulation, cell differentiation, matrix formation, and mineralization. Odontoblasts are solely responsible for the synthesis and secretion of the dentin extracellular matrix bilayer that consists of non-mineralized predentin and mineralized dentin. The mouse immortalized MO6-G3 cell line expresses the major matrix proteins associated with the odontoblast phenotype, producing a matrix that is capable of mineralization. This cell line serves as a useful tool in studies designed to explore the various processes of dentinogenesis. In this paper, we present studies using the mouse odontoblast cell line MO6-G3 as examples of the various research applications. Studies highlighted are: in vitro promoter studies investigating the tooth-specific gene regulation of the major non-collagenous dentin matrix protein, dentin sialophosphoprotein; regulation of tertiary dentin formation by cytokines, such as transforming growth factor-Beta 1; and the utilization of dentally relevant cells in dental material biocompatibility testing.