Objectives: Periodontal disease is a chronic inflammation resulting in periodontal attachment loss. Keratinocyte Growth Factor-1 (KGF-1) is upregulated in chronic inflammation and specifically stimulates epithelial cell proliferation by signaling through the epithelial-specific Keratinocyte Growth Factor Receptor (KGFR). First, we examined KGF-1 and KGFR expression and localization in human periodontal tissues. Second, we extended these studies by developing an in vitro mechanical wound model to mimic trauma to the periodontal pocket epithelium and examined ligand independent KGFR activation and cell migration.
Methods: In our study of human gingival tissues, we used immunohistochemistry and laser capture microdissection with RT-PCR to analyze KGF-1 and KGFR expression and localization. To study ligand independent KGFR phosphorylation, KGFR internalization along the wound edge was imaged using immunohistochemical staining and KGFR phosphorylation confirmed using immunoprecipitation with western blotting. Wounding induced oxidative stress was detected using DCFH-DA (2',7'-dichlorofluorescin diacetate) and modulated by pretreatment with an antioxidant. Changes in migration were examined in the presence or absence of pathway specific inhibitors.
Results: KGF-1 protein localized to areas of junctional and basal oral epithelial cells was significantly increased in periodontal pocket epithelium (p<0.01) and oral epithelium (p<0.05) of disease-associated tissues. KGFR localized to the junctional and the parabasal cells of oral epithelium, and was increased in disease-associated pocket epithelium (p<0.05). Laser capture microdissection with RT-PCR confirmedKGF-1 and KGFR were specifically expressed by connective tissue and epithelium, respectively. In our cell culture model, mechanical wounding induced ligand independent KGFR activation. ROS (Reactive Oxygen Species) generation along the wound edge was associated with KGFR activation and scavenging of ROS reduced KGFR phosphorylation. The c-Src family inhibitor, PP1, significantly inhibited KGFR phosphorylation. Functionally cell migration was reduced by PP1 (82.7%), SU5402(70%) and PD98059 (57%).
Conclusions: KGF-1 and KGFR proteins are expressed in health but significantly induced in human diseased periodontal tissues. Microwounding associated generation of ROS mediates KGFR phosphorylation via c-Src kinase signaling and induced wound edge cell migration. Therefore, regulation of epithelial cell behavior associated with the onset and progression of periodontal disease may possibly be mediated by two related but distinct mechanisms. (1) Ligand-dependent activation of KGFR due to upregulation of KGF-1. (2) Ligand-independent activation of KGFR due to chronic microwounding.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:BVAU.2429/418 |
| Date | 05 1900 |
| Creators | Li, Min |
| Publisher | University of British Columbia |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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