The human tooth is the hardest organ of the body, and is composed of enamel, dentin, and dental pulp. Dentin provides the basis of the tooth shape by lining the inner parts of the root and crown. Odontoblasts deposit dentin, an organic matrix that contains collagen, noncollagenous proteins, phospholipids, and growth factors. In this study, we sought to reveal the proteins in human dentin by using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteomic approaches. Human third molar dentins were cut, isolated, and demineralized, and the extracted proteins were separated on SDS-PAGE. In-gel digested peptides were analyzed using reverse-phase LC-MS/MS. We identified 233 total and 68 common proteins from 3 individuals with high confidence, including a variety of collagenous and noncollagenous proteins such as DSPP, biglycan, osteoglycin, osteopontin, and osteocalcin. In addition to known proteins, we also identified various matrix and serum proteins deposited in the dentin, including asporin, lumican, mimecan, and SOD3. This study provides the first list of proteomes that are detected in human dentin. This proteome list is useful in that it defines the organic matrix of dentin and helps to characterize odontoblasts.

In bone remodeling, various transcriptional factors are involved, and the deficiency or overexpression of some of these factors results in bone defects. Myeloid elf-1-like factor (MEF) is one of the Ets transcription factors that control the expression of genes that are critical for biologic processes such as cell proliferation, differentiation, and death. Previously, we reported that MEF promotes cell proliferation and functions as a negative regulator of osteogenic differentiation by interacting directly with Runx2 and suppressing its transcriptional activity. To investigate the in vivo function of MEF in bone formation and bone remodeling in vivo, we generated transgenic mice that overexpress MEF in osteoblasts under the control of the 2.3-kb Col1a1 promoter, named Col1a1-MEF. Femoral bone in Col1a1-MEF transgenic mice exhibited low bone mass with fewer trabecular bones and thinner and less developed cortical bones. The mineralized volume fraction (BV/TV) and bone-forming rate (BFR) were remarkably decreased to about 63% and 40%, respectively, in 6-week-old MEF transgenic mice compared with wild-type mice. In addition, reduced bone mineral density was observed in lumbar vertebrae of Col1a1-MEF transgenic mice. The number of TRACP þ osteoclasts was increased in Col1a1-MEF transgenic mice and MEF-overexpressing MC3T3-E1 cells. All these in vivo results suggest that MEF suppresses bone formation by osteoblasts and facilitates bone resorption by activating osteoclasts indirectly. ß

Glypican 3 (GPC3), a member of heparin sulfate proteoglycans, is attached to the cell surface by a glycosylphosphatidylinositol anchor and is reported to be overexpressed in liver cancers. In order to identify GPC3 binding proteins on the cell surface, we constructed a cDNA containing the C-terminal cell surface-attached form of GPC3 (GPC3c) in a baculoviral vector. The GPC3c bait protein was produced by expressing the construct in Sf21 insect cells and double purified using a His column and Flag immunoprecipitation. Purified GPC3c was used to uncover GPC3c-interacting proteins. Using an LC-MS/MS proteomics strategy, we identified glucose transporter 1 (GLUT1) as a novel GPC3 interacting protein from the HepG2 hepatoma cell lysates. The interaction was confirmed by immunoprecipitation (IP)-WB analysis and surface plasmon resonance (SPR). SPR result showed the interaction of GLUT1 to GPC3c with equilibrium dissociation constants (K(D) ) of 1.61 nM. Moreover, both incubation with GPC3c protein and transfection of Gpc3c cDNA into HepG2 cells resulted in reduced glucose uptake activity. Our results indicate that GPC3 plays a role in glucose transport by interacting with GLUT1.