title: Involvement of smad2 and Erk/Akt cascade in TGF-β1-induced apoptosis in human gingival epithelial cells.
abstract: Periodontitis is the most prevalent infectious disease caused by periodontopathic bacteria and is also a chronic inflammatory disease. Gingival crevicular fluid (GCF) is an inflammatory exudate that seeps into the gingival crevices or periodontal pockets around teeth with inflamed gingiva, and contains various materials including leukocytes and cytokines. Since gingival epithelial cells, which form a barrier against bacterial challenges, are affected by GCF, cytokines or other materials contained within GCF are engaged in the maintenance and disruption of the epithelial barrier. Accordingly, its compositional pattern has been employed as a reliable objective index of local inflammation. Transforming growth factor β1 (TGF-β1) levels in GCF were previously shown to be markedly higher in patients with periodontitis than in healthy subject. However, it currently remains unclear how TGF-β1 affects gingival epithelial cell growth or apoptosis; therefore, elucidating the mechanism responsible may lead to a deeper understanding of pathogenic periodontitis. In the present study, the human gingival epithelial cell line, OBA9 cells were stimulated with recombinant TGF-β1. Apoptosis-related protein levels were determined by Western blotting. Caspase-3/7 activity was measured with a Caspase-Glo assay kit. Surviving and apoptotic cells were detected using an MTS assay and TUNEL staining, respectively. TGF-βRI siRNA and smad2 siRNA were transfected into cells using the lipofectamine RNAiMAX reagent. TGF-β1 elevated caspase-3 activity and the number of TUNEL-positive apoptotic cells in OBA9 cells. Furthermore, while the levels of the pro-apoptotic proteins Bax, Bak, Bim, and Bad were increased in OBA9 cells stimulated with TGF-β1, the TGF-β1 treatment also decreased the levels of anti-apoptotic proteins such as Bcl-2 and Bcl-xL in a time-dependent manner. Additionally, TGF-β1 up-regulated the protein levels of cleaved caspase-9. These results indicated that TGF-β1-induced apoptosis was involved in a mitochondria-related intrinsic pathway. TGF-β1 phosphorylated smad2 in OBA9 cells and this phosphorylation was clearly reduced by SB431542 (a TGF-β type I receptor inhibitor). Consistent with this result, SB431542 or smad2 siRNA-induced reductions in smad2 protein expression levels attenuated TGF-β1-induced apoptosis. On the other hand, the ligation of TGF-β1 on its receptor also stimulated the phosphorylation of Erk and Akt, which are smad2-independent pathways. However, the inhibition of Erk/Akt signaling pathways by U0126, a MEK-Erk inhibitor and LY294002, a PI3Kinase-Akt inhibitor, augmented TGF-β1-induced apoptosis in OBA9 cells. Taken together, the results of present study demonstrated that TGF-β1 activated both the smad2 and Erk/Akt cascades via its receptor on gingival epithelial cells, even though these two pathways have opposite roles in cell death and survival, and the culmination of these signaling events induced mitochondria-dependent apoptosis in gingival epithelial cells. Based on the results of the present study, we herein proposed for the first time, that TGF-β1 is a novel target cytokine for monitoring the progression of periodontal disease.
authors.affiliation: Department of Periodontal Medicine, Division of Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Yoshimoto
authors.affiliation: Department of Periodontal Medicine, Division of Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Electronic address: tfuji@hiroshima-u.ac.jp.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Fujita
authors.affiliation: Department of Periodontal Medicine, Division of Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
authors.forename: Mikihito
authors.initials: M
authors.lastname: Kajiya
authors.affiliation: Department of Periodontal Medicine, Division of Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Matsuda
authors.affiliation: Department of Periodontal Medicine, Division of Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Ouhara
authors.affiliation: Department of Periodontal Medicine, Division of Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Shiba
authors.affiliation: Department of Periodontal Medicine, Division of Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
authors.forename: Hidemi
authors.initials: H
authors.lastname: Kurihara
mesh_terms: D000111:Acetylcysteine / Q000378:metabolism; D017209:Apoptosis; D001549:Benzamides / Q000737:chemistry; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D004149:Dioxoles / Q000737:chemistry; D004847:Epithelial Cells / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005881:Gingiva / Q000378:metabolism*; D006801:Humans; D007249:Inflammation / Q000378:metabolism; D020935:MAP Kinase Signaling System; D010518:Periodontitis / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D051899:Smad2 Protein / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
chemical_list: C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; D004149:Dioxoles; D034741:RNA, Small Interfering; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D000111:Acetylcysteine
keywords: Apoptosis; Erk/Akt cascade; Human gingival epithelial cells; Smad2; TGF-β1