Oxidative stress caused by a low concentration of hydrogen peroxide induces senescence-like changes in mouse gingival fibroblasts

Periodontal tissue deteriorates under persistent oxidative stress induced by inflammatory reactions in the microflora of the oral cavity. This study aimed to evaluate the cellular properties of mouse gingival fibroblasts (MGFs) in the presence of oxidative stress. MGFs from 10-, 30- and 52-week-old mice were used to evaluate the changes in the cellular properties with aging. The study investigated the effects of oxidative stress on the cellular properties of MGFs from 10-week-old mice. The expression of p53, p21 and murine double minute 2 (Mdm2) in the MGFs in response to oxidative stress was also examined. By day 8, the number of MGFs increased in culture. However, the increase was markedly lower in MGFs derived from aged mice. Oxidative stress due to hydrogen peroxide (H2O2)-induced morphological changes characterized by a round shape with enlarged nuclei and expanded cytoplasm. The cell number of MGFs was decreased subsequent to treatment with 50 μM or a higher concentration of H2O2. MGFs treated with H2O2 at 20 μM showed a similar cell growth curve as the one seen in 52-week-old mice. Phosphorylated p53 protein was increased in MGFs subsequent to treatment with 20 μM H2O2, along with an upregulated transcription of p21 and Mdm2 mRNAs. These results suggest that treatment with a lower concentration of H2O2 in MGFs induces cell cycle arrest, resulting in stress-induced premature senescence, possibly correlated with the development of periodontal diseases.


Introduction
Aging is a physiological phenomenon, which commonly occurs in various organs and tissues (1). Age-dependent morphological and cell kinetic changes in the organs and tissues are associated with the development of various diseases in the elderly. A decline in the function of organs and tissues is a normal phenomenon associated with aging, and is also considered to reduce the quality of life (1).
Several reports are available regarding the relationship between aging and periodontitis (2)(3)(4)(5). The aging of the periodontal tissue is involved in the development of periodontitis in elderly individuals (6).
The age-dependent morphological and cell kinetic changes of the gingival tissue were delineated, using both the 5-bromo-2'-deoxyuridine (BrdU) incorporation and the terminal deoxynucleotidyl transferase-mediated deoxyuridine-5'-triphosphate (dUTP)-biotin nick end-labeling (TUNEL) methods (7,8). Those studies demonstrated that with aging there is a significant apoptosis-induced decrease in the cellular component of the subepithelial connective tissue of both the gingival and junctional epithelial layer. Furthermore, an age-dependent increase in the number of TUNEL-positive cells occurred only in the subepithelial connective tissue, although gingival tissue, buccal mucosa, tongue dorsal, ventral mucosae and skin have similar histological structures (9).
Oxidative stress is one of the most important causative factors for the induction of cell apoptosis (10,11). Incubationinduced subcytotoxic stress with potentially harmful molecules, such as hydrogen peroxide (H 2 O 2 ) brought the cells into a state similar to senescence, termed stress-induced premature senescence (SIPS) (12)(13)(14). Therefore, in this study, the agedependent changes in the cell number in cultured mouse gingival fibroblasts (MGFs), as well as the changes in the biological behavior subsequent to treatment with H 2 O 2 in the MGFs were investigated.

Materials and methods
Animals. BALB/c mice were used to investigate the age-related changes in the cell number in MGFs in 10-, 15-, 30-and 52-week-old mice (n=3 per group). In addition, 10-weekold mice were used for the oxidative stress experiment (n=3 per group). The animals were bred in a strictly monitored air-conditioned clean room and were fed standard laboratory pellets and water ad libitum. The experiment was performed according to the guidelines of the Animal Center at the Kyushu University.
Gingival fibroblast cultures. The animals were sacrificed using an excessive amount of ether. The gingival tissues were removed, immediately washed in phosphate-buffered saline (PBS) with gentamicin (10 µg/ml; Invitrogen, Carlsbad, CA, USA) and transferred into a culture dish. The gingival fibroblasts were grown in α-MEM, supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 µg/ ml streptomycin in a humidified atmosphere with 5% CO 2 at 37˚C. The medium was changed every other day. The cells were grown to semi-confluence, harvested by trypsinization at 37˚C for 5 min, then subcultivated with culture medium in a new dish. The experiments were performed using earlypassage fibroblasts before the fourth passage.
Cell growth assay. MGFs (1.0x10 4 cells/well) were seeded onto 12-well plates in the α-MEM with serum. The cell numbers in three-wells of each group were counted chronologically at 2, 4, 6 and 8 days after cultivation. The results were expressed as the mean ± SD.
Oxidative stress. At 48 h after seeding, MGFs were exposed to oxidative stress for 2 h. Various concentrations of H 2 O 2 were diluted in α-MEM with 10% FBS. Subsequent to treatment with H 2 O 2 , the cultures were rinsed twice with PBS and incubated in α-MEM with 10% FBS. Some samples were stained with nuclear fast red to observe cellular and nuclear morphological changes in the MGFs after the oxidative stress.
Senescence-associated β-galactosidase (SA-β-Gal) activity. MGFs were cultured for 14 days subsequent to treatment with H 2 O 2 . The SA-β-Gal-positive cell ratios were determined in three wells, using a senescence detection kit, according to the manufacturer's instructions. To avoid any non-specific staining due to confluence, SA-β-Gal cytochemical staining was performed on non-confluent cells.
Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). A semi-quantitative RT-PCR analysis was carried out. Total-RNA extracted from cultured mouse gingival cells was isolated using the SV total-RNA isolation system, according to the manufacturer's instructions. cDNA was generated from isolated total-RNA by reverse transcription (RT) with SuperScript III and subjected to PCR amplification with the specific primer sets (Table I). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal RNA control for the comparison of RNA levels in each sample. The PCR products were separated by electrophoresis on agarose gels and then stained with ethidium bromide.
Immunoblotting for the p53 phosphorylation status. An immunoblot analysis for p53 was carried out using the proteins isolated from the cultured MGFs after the treatment with 20 µM H 2 O 2 for 2 h. These cells were lysed in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA pH 8.0, 0.1% SDS), supplemented with protease inhibitor cocktail (50 µM), lactacystin (20 µM) and PMSF. The protein samples were separated using SDS-PAGE, transferred to an Immun-Blot ® PVDF Membrane, and immunoblotted with anti-p53 antibody (sc-6243; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-phospho-p53 antibody (S15; R&D Systems, Inc., Minneapolis, MN, USA). The membrane was incubated with suitable secondary antibody conjugated with horseradish peroxidase. Immunoreactive proteins were visualized using an ECL detection system. Any emitted light was detected using a cooled CCD-camera (LAS-1000; Fujifilm, Tokyo, Japan).
Statistical analysis. The experiments were repeated three times for the independent MGF samples. A statistical analysis was Table I. Primer sets used for semi-quantitative RT-PCR. Target  Sequence   p53  F: 5'-GGA AAT TTG TAT CCC GAG TAT CTG-3'  R: 5'-GTC TTC CAG TGT GAT GAT GGT AA-3'   p21  F: 5'-TGT CCA ATC CTG GTG ATG TC-3  performed combining one-way ANOVA with the Tukey-Kramer comparison test or Student's t-test. P-values <0.05 or 0.01 were considered to indicate statistically significant differences.

Results
Age-dependent change in the cell number in MGFs. MGFs isolated from 10-, 30-and 52-week-old mice were cultured for 2, 4, 6 and 8 days. Although the MGFs showed cell proliferation while cultured, the number of MGFs was different on each culture day, depending on the age of the mouse, from which the sample was derived. Marked differences were detected in the cell number in 10-and 30-week-old, and 30-and 52-weekold mice (P<0.05) at day 8 of the culturing. A statistically significant difference was also observed between 10-and 52-week-old mice (P<0.01), indicating that the increase in the number of cultured MGFs was apparently lower in samples from older mice (Fig. 1). In the subsequent oxidative stress experiments MGFs derived from 10-week-old mice were used.  (Fig. 2) (13,14).    (Fig. 3). Statistically significant differences were demonstrated in each sample at day 8 of culturing (P<0.05) (Fig. 4).    ( Fig. 5). Furthermore, H 2 O 2 -treated cells had a round shape with enlarged nucleus and expanded cytoplasm, as observed in the senescent fibroblasts. Therefore, MGFs treated with 20 µM H 2 O 2 seemed to mimic the age-dependent decrease of the proliferative activity in the 52-week-old mice.

Detection of SA-β-Gal in the MGFs using a 20 µM H 2 O 2 treatment.
The ratio of SA-β-Gal (a senescence marker)-positive cells was examined in H 2 O 2 -treated and non-treated samples, since oxidative stress after H 2 O 2 treatment induced senescence-like morphological and functional changes. Two days after the treatment, the ratio of SA-β-Gal-positive cells was higher in H 2 O 2 -treated compared to non-treated samples (Fig. 6). Significant differences were noted in the number of SA-β-Gal-positive cells in 20 µM H 2 O 2 -treated and nontreated samples (P<0.05), subsequent to culture for 6, 8, 11 and 14 days.
Expression of p53, p21 and Mdm2 mRNA, and the phosphorylation of p53 protein in the culture MGFs. Oxidative stress induced senescence-like changes in the MGFs, and therefore the expression of p53 having the potential to initiate cell cycle arrest or cell death (15,16) was further examined. In addition, we examined the expression of p53 downstream genes, cyclin-dependent kinase inhibitor p21 and Mdm2 (17) was examined. The phosphorylated p53 protein was detected using an antibody specifically recognizing human, monkey and rat p53 phosphorylated at the serine-15 and the comparable phosphorylated site in mouse p53 (serine-18  (Fig. 7).

Discussion
The present study demonstrated that oxidative stress generated by treatment with a lower concentration of H 2 O 2 induced a decrease in the cell number in the cultured MGFs with an increased ratio of SA-β-Gal-positive cells, a marker of cellular senescence. Oxidative stress generated by externally added H 2 O 2 induces SIPS in variety of cell types (13,14,18). This study demonstrated, for the first time, that SIPS in gingival fibroblasts was induced by a lower-concentration H 2 O 2 -treatment than previously reported. Previous reports have proven a low concentration of H 2 O 2 treatment to induce a higher proliferative activity in human dorsal fibroblasts, rabbit lens epithelial cells, baby hamster kidney fibroblasts and embryonic Chinese hamster ovary fibroblasts (19)(20)(21). Therefore, it is reasonable to assume that the gingival fibroblast is more sensitive to H 2 O 2 oxidative stress.
Oxidative stress is one of the most important causative factors for the induction of pathological states, including periodontitis (22)(23)(24)(25)(26). Periodontal tissue tends to deteriorate under persistent oxidative stress induced by inflammatory reactions in the microflora of the oral cavity (27). A previous report demonstrated that gingival fibroblast and dorsal fibroblast had different cellular properties, such as the expression of integrin and extracellular matrix receptors. Consequently, experiments using dorsal fibroblasts are not necessarily valid for oral tissues as well (28). These different cellular properties appear to be correlated with higher sensitivity to oxidative stress, thus resulting in the induction of SIPS.
The p53 gene expression was examined since gingival fibroblasts appeared to be more sensitive to oxidative stress. p53 activation is closely correlated with cell cycle arrest and apoptosis when cells deteriorate due to pathogenic stress. There was no change in the level of p53 mRNA and protein expression shown by the semi-quantitative RT-PCR analysis and western blotting, respectively. However, western blotting demonstrated an increase of phosphorylated p53 protein. Therefore, an increased p21 expression level, suppressing the CDK4-and CDK6-activation, is considered to be induced by the increase of phosphorylated p53 protein, resulting in a decrease of cell number in the MGFs treated with a lower concentration of H 2 O 2 . The Mdm2 expression was also activated at the transcription level. These results suggest that Mdm2 functions as a negative feedback regulator to maintain p53 at a low level under oxidative stress, since the major function of Mdm2 is to interact with p53 and thereby induce the ubiquitination and degradation of p53 (29).
In this study, H 2 O 2 -induced oxidative stress generated an increase in the phosphorylated p53 protein-level at serine-18 in the MGFs. The phosphorylation at this site in mouse p53 is comparable to that of human p53 at serine-15. Phosphorylation of human p53 at serine-15 has a significant role in its reduced interaction with the Mdm2, its negative regulator, and is involved in the impairment of the Mdm2 function by inhibiting p53-dependent transactivation (30). Therefore, the serine-18 of mouse p53 protein may be one of the critical phosphorylation sites that lead to cell cycle arrest and SIPS under oxidative stress. However, the H 2 O 2 -mediated protein kinases involved in p53 phosphorylation remain unknown. Nevertheless, these results do not exclude the possibility that phosphorylation at other sites on p53 may be associated with the occurrence of these phenomena.
Therefore, gingival fibroblasts are more sensitive to oxidative stress, resulting in cell cycle arrest due to an increase of phosphorylated p53 protein. This characteristic of the gingival fibroblasts may be associated with the marked agedependent decrease of cellular components in the periodontal tissue described in previous studies (7)(8)(9), as well as with the development of periodontal diseases. In order to develop new preventive methods against various periodontal diseases, additional investigations regarding the molecular interactions within the gingival fibroblasts in association with oxidative stress are required.