¥âig-h3 Is Required for Differentiation of Normal Human Oral Keratinocytes

Serial subculture of primary normal human oral keratinocytes (NHOKs) to the post-mitotic stage induces terminal differentiation, which is in part linked to elevated levels of TGF-b that is a potent differentiation inducer of epithelial cells. In addition, the level of big-h3, a secreted protein inducible by TGF-b, was found to progressively increase in NHOKs during subculture-induced differentiation. Objectives: Our main objective was to determine whether big-h3 is involved in keratinocyte differentiation. Methods: To test this hypothesis, the expression of big-h3 in primary NHOKs was blocked by transfecting cells with the antisense big-h3 cDNA construct. Results:These cells demonstrated dramatic reductions in big-h3 and in the differentiation marker involucrin and an increase (60~70%) in in vitro life span versus empty vector-transfected cells. In contrast, opposite observations were made following transfection cells with the big-h3 cDNA construct. In addition, an increased big-h3 level in exponentially replicating NHOKs, by both transfecting cells with the big-h3 cDNA construct and TGF-b1-treatment, induced keratinocyte differentiation by increasing transglutaminase promoter activity but did not change the levels of PLC-g1 and intracellular calcium. Conclusion:These results indicate that the terminal differentiation of NHOKs by serial subculture is associated with big-h3, which is induced by TGF-b, but is not linked to the calcium regulation by mobilizing intracellular calcium. Supported by a grant 02-PJ1-PG3-20507-0038 from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea.