Cloning of the Genes Specifically Expressed in Teeth

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We constructed cDNA library of bovine periodontal ligament (PDL) and then tried to clone cDNA of the genes specifically expressed in PDL not expressed in calvaria, using the gene subtraction technique, however this experiment was unsuccessful. PDL is a unique tissue because it maintains its function under extent mechanical stress caused by occlusion. We speculate that Ca signaling and cytoskeletal elements in PDL cells play important roles in this uniqueness. Since S100A4 is a Ca-binding protein and interacts with cytoskeletal elements, we focused our research on this protein in PDL.We cloned bovine S100A4 cDNA from PDL cDNA library. The highest level of S100A4 mRNA expression was ovserved among the oral tissues examined. PDL of erupted teeth expressed this gene higher than OPDL of unerupted teeth and application of mechanical stress to cultured PDL cells increased the expression level of this gene, indicating stimulative effect of mechanical stress on S100A4 expression. Immunohistological study demonstrated intercellular and extracellular localization of S100A4 in PDL.Western blotting of the culture medium of PDL cells and analysis of S^<35>-methionine labeled culture of PDL cells demonstrated the existence of S100A4 in the medium. Thus, it is likely that PDL cells secrete S100A4 extracellularly. Osteogenic cells produce mineralized-tissue in culture and addition of recombinant S100A4 protein in this culture system inhibited mineralization. In calvaria development, S100A4 mRNA expression was detected in early stage, however its expression was undetectable when mineralization started. Furthermore, S100A4 expression of osteoblasts was not detected in situ Hybridization experiment. Thus, it is possible that S100A4 is a inhibitory factor for mineralization. We can conclude that mechanical stress stimulates S100A4 expression in PDL cells and PDL cells secrete S100A4, which acts as an inhibitor for mineralization although further study is necessary to prove this story.