Wit 3.0, a novel gene derived from edentulous oral mucosa, encodes cytoplasmic molecules facilitating oral mucosa wound contraction

Oral mucosal tissue adapts to tooth extraction, often resulting in generation of distinct epithelial and connective tissues. It has been postulated that oral mucosal wounds heal with minimum scarring compare to adult wounds. Therefore, oral mucosa wound may provide a novel model for studies establishing a treatment for minimizing scarring during adult tissue repair. We hypothesize that the differential expression of genes in response to wound healing due to tooth extraction contribute in part to the structure and physiology unique of edentulous oral mucosal and the scarless wound healing formation. The objective of this study is to identify and characterize such candidate genes. Edentulous oral mucosa was experimentally created in male Sprague-Dawley rats by the extraction of maxillary molars. The overlapping cDNAs encoding a 3.0 kilobase long mRNA were cloned using differential display polymerase chain reaction comparing the rat edentulous oral mucosa and the untreated gingiva. In situ hybridization revealed that fibroblasts within the scar-like hyaline connective tissue adjacent to the tooth extraction site were the cellular source of this mRNA. The candidate gene, wit 3.0 sequence partially matched with uncharacterized human cDNAs deposited in the GenBank database. Wit 3.0 has been found to generate two transcripts, wit 3.0alpha and beta. Western blot of transfected NIH3T3 fibroblasts showed that the translated fusion peptides of wit 3.0alpha and beta, 40 KD and 43 KD respectively, were identified as single bands with or without a reducing treatment. Immunocytology and cell-fraction Western blot indicated that wit 3.0 peptides terminally localized in cytoplasmic region and appeared to associate with cytoskeletal structures. Collagen gel contraction assay was performed using NIH3T3 fibroblasts transfected with expression vector containing wit 3.0alpha, wit 3.0beta, BAP (transfection control) and without the vector (untreated control). During the initial 24 hours, wit 3.0alpha and beta transfection significantly accelerated the rate of collagen gel contraction (p<0.05) as compared with transfection and untreated controls. In conclusion, these data suggest that wit 3.0 may be involved in the fibroblast derived tissue contraction that primarily contributes to a rapid wound closure of the oral wound healing. The findings in this study may provide a novel clue to ameliorate the scar formation in adult wound healing and to delineate the molecular mechanism of edentulous mucosa formation.