TM14 Is a New Member of the Fibulin Family (Fibulin-7) That Interacts with Extracellular Matrix Molecules and Is Active for Cell Binding

Abstract

We identified a new extracellular protein, TM14, by differential hybridization using mouse tooth germ cDNA microarrays. TM14 cDNA encodes 440 amino acids containing a signal peptide. The protein contains 3 EGF modules at the center, a C-terminal domain homologous to the fibulin module, and a unique Sushi domain at the N terminus. In situ hybridization revealed that TM14 mRNA was expressed by preodontoblasts and odontoblasts in developing teeth. TM14 mRNA was also expressed in cartilage, hair follicles, and extraembryonic tissues of the placenta. Immunostaining revealed that TM14 was localized at the apical pericellular regions of preodontoblasts. When the dentin matrix was fully formed and dentin mineralization occurred, TM14 was present in the predentin matrix and along the dentinal tubules. We found that the recombinant TM14 protein was glycosylated with N-linked oligosaccharides and interacted with heparin, fibronectin, fibulin-1, and dentin sialophosphoprotein. We also found that TM14 preferentially bound dental mesenchyme cells and odontoblasts but not dental epithelial cells or nondental cells such as HeLa, COS7, or NIH3T3 cells. Heparin, EDTA, and anti-integrin ␤1 antibody inhibited TM14 binding to dental mesenchyme cells, suggesting that both a heparan sulfate-containing cell surface receptor and an integrin are involved in TM14 cell binding. Our findings indicate that TM14 is a cell adhesion molecule that interacts with extracellular matrix molecules in teeth and suggest that TM14 plays important roles in both the differentiation and maintenance of odontoblasts as well as in dentin formation. Because of its protein characteristics, TM14 can be classified as a new member of the fibulin family: fibulin-7. The extracellular matrix (ECM)4 plays active roles during organ development and in mature tissue functions. Effects on cell behavior and gene expression are often mediated through interactions between ECM molecules and cell surface receptors, leading to signal transduction across the plasma membrane. It is well known that many ECM proteins, including collagens, elastin, as well as other glycoproteins and proteoglycans are crucial for morphogenesis during embryonic development, and dysfunctions of these molecules cause congenital defects in humans. We have been interested in ECM molecules important in both tooth development and diseases. Previously, we identified the enamel matrix-specific protein ameloblastin that is essential for maintaining differentiated dental epithelial cells (ameloblasts) and for enamel formation (1-3). In this report, we characterized a new dentin matrix protein that we named TM14, which we identified in mouse tooth germ cDNA microarrays by differential hybridization (4).Mature teeth consist of two major mineralized tissues, dentin and enamel, the hardest tissue in the body. The development of these tissues is initiated by reciprocal interactions between the dental epithelium and mesenchyme, leading to the terminal differentiation of matrix-producing ameloblasts and odontoblasts, ...

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