Regulation of gingival keratinization by laminin332

Objectives: Adequately keratinized gingiva is believed necessary for the maintenance of healthy periodontal tissues by keeping a mechanical barrier resistant to the various adverse conditions. However, it is still unclear which kind of genes regulate gingival keratinization and how keratinized gingiva is maintained. Therefore, the goal of this study was to identify the factors controlling keratinization of gingiva.

Methods: cDNA-microarray was performed to identify specific genes associated with keratinized gingiva comparing total RNA collected from keratinized gingiva and non-keratinized gingiva from Wistar rat (6weeks old, n=1). Oral mucosal epithelial cells (OMECs) derived from human squamous cell carcinoma (DS Pharma Biomedical Co., Osaka, Japan) were treated with recombinant proteins of candidate genes and the mRNA and protein levels of Keratin 1 (K1) and Keratin 10 (K10) were measured by real-time RT-PCR and immunohistochemistry (IHC). OMECs were also co-cultured with fibroblasts harvested from the rat keratinized gingiva or the non-keratinized gingiva, for analysis of the effect of adding the fibroblasts (mesenchymal cells) on epithelial cell keratinization.

Results: From cDNA-microarray analysis, Laminin332 (Lam332) was identified as a gingival keratinization-associated gene, and a high protein expression level of Lam332 in keratinized gingiva was confirmed by IHC. The treatment with recombinant human LAM332 (1000 ng/mL) significantly enhanced the keratinization of OMECs. (K1: 2.1-fold and K10: 2.2-fold expression, p<0.01: one-way ANOVA/Turkey). Expression levels of LAM332 and keratinization of OMECs were enhanced in co-cultures with the fibroblasts harvested from keratinized gingiva, compared with those from non-keratinized gingiva. (Lamα3: 1.2-fold, Lamγ2: 1.4-fold, K1: 2.0-fold, K10: 1.5-fold expression, p<0.05: unpaired t-test).
Conclusions: Laminin332, which was highly expressed in keratinized gingiva, regulated the keratinization of gingival epithelial cells. The data also suggest that mesenchymal tissue might play important roles in gingival keratinization.