Ras-p21 Activation by Nicotine in Periodontal Ligament Fibroblasts

Tobacco smoking is one of the major factors in the development of a variety of cancers including those of the oral mucosa. At the molecular level, cancer causes disturbances in protein function leading to uncontrolled activation of specific cellular signaling pathways. A protein central to the regulation of cell proliferation is the Ras-p21 (H-, K- and N-) family of G-proteins. Ras-p21 protein is activated upon binding to GTP. This leads to the activation of the MAP kinase pathway resulting in cell proliferation. Improper activation of Ras-p21 has been implicated in colon, pancreatic, lung and oral cancers. Nicotine is a major constituent in tobacco smoke. Objective: the present study was designed to investigate the short-term effects of nicotine on Ras-p21 activation in Rat Periodontal Ligament Fibroblasts (RPDLs). Methods: pull-down experiments with GST-Raf (binds to the GTP-bound form of Ras-p21) were carried out. Results: it was shown that H-Ras and not K-Ras or N-Ras was activated upon stimulation of RPDLs with nicotine. Twenty-four hour serum starved RPDLs were subjected to various nicotine concentrations for 10 min. The optimum nicotine concentration (10^-4M) that caused maximal activation of Ras-p21 was carried forward for a time course experiment which demonstrated that Ras-p21 was optimally activated after 1 hr stimulation with nicotine. The pharmacological agents BAPTA-AM (a calcium chelator) and U73122 (phospholipase C inhibitor) were used to study the role of calcium in Ras-p21 activation. Results demonstrated that the nicotine-induced activation of Ras-p21 pathway showed partial calcium dependency. In summary, we have shown that Ras-p21 is maximally activated at a concentration of 10^-4M nicotine and that this pathway is partially regulated by calcium. Conclusion: the current study provides a possible molecular mechanism for nicotine induced activation of Ras-p21. This information may be of help in developing approaches to control the development/progression of oral cancer.