Gingival Fibroblast in vitro Wounds Treated with Nicotine, and/or TGFb1

Objectives: Nicotine treated gingival fibroblast cells have inhibited wound response that results in a delay of cell migration rates in vitro, and slowed healing rates clinically. We have also shown that gingival fibroblast cells decreased their response to TGFb1 in the presence of nicotine and several signal transduction proteins increased or decreased phosphorylation. In this study, we screened over 600 signaling proteins for changes in phosphorylation when treated with nicotine and/or TGFb1 during human gingival wounds in vitro.

Methods: Human gingival cells were cultured and grown to confluence in cell culture medium (low glucose DMEM, 10% fetal bovine serum, 1% antimycotic/antibiotic). Cells were starved for 24 hours without serum, and then treated with nicotine (1 mg/ml) for 2 hrs before TGFb1 (0.5ng/ml) treatment and in vitro wounding. Four treatment groups: controls (C), nicotine (N), TGFb1 (TGF), and N+TGF were divided into wounded and unwounded groups for a total of eight treatments. To analyze protein phosphorylation, cells were lysed in ice cold lysis buffer with phosphatase inhibitors recommended by Kinexus, frozen and transported to Kinexus for screening of over 600 phosphorylated proteins.

Results: Wounded vs unwounded groups were compared in the four treatments. In addition, N treated was compared to C, TGF and TGF+N. Many proteins involved in cell migration including p38 MAP kinase, MEK1, and PCTK1 were increased in C and TGF, but decreased in N and TGF treated groups. Nicotine treatment increased phosphorylation of CDK1/2 (cell cycle protein) and PKCd (an inducer of apoptosis). Some results reinforced previous Western blot data from similar experiments.

Conclusions: This phospho-protein screening will help investigators design experiments to establish important signal transduction pathways changed by gingival fibroblast exposure to nicotine and/or growth factors.

Supported by Baylor Oral Health Foundation and the Texas A&M Health Science Center Vice President for Research Development Grant.