Tongue epithelial KT-1 cell-cycle arrest by TGF-beta associated with induction of p21(Cip1) and p15 (Ink4b)

Abstract

Tongue epithelium continuously turns over in adults. Our previous study showed that epidermal growth factor and fibroblast growth factor-2 stimulated proliferation of KT-1 cells derived from tongue epithelium, suggesting that these signals serve as positive regulators for tongue epithelial proliferation. To investigate a negative regulation of tongue epithelial cell proliferation, we studied effects of transforming growth factor-beta (TGF-beta) on KT-1 cells. Proliferation assays showed that TGF-beta inhibited proliferation of KT-1 cells in a dose dependent manner. Cell-cycle analysis showed that TGF-beta induced G(0)/G(1) cell cycle arrest in KT-1 cells. We also examined expressions of Ink4 and Cip/Kip family mRNA by quantitative reverse transcription-polymerase chain reaction. We found that TGF-beta induced p15(Ink4b) and p21(Cip1) mRNA expressions. These results strongly suggest that G(0)/G(1) cell cycle arrest is associated with increased p15(Ink4b) and p21(Cip1) expressions. Moreover, p21(Cip1) mRNA was localized in suprabasal cells of tongue epithelium, suggesting that p21(Cip1) play a role in cell-cycle exit along with tongue epithelial differentiation. Taken together, our results suggest that TGF-beta signaling serves as negative regulator of tongue epithelial cell proliferation, and may control tongue epithelial cell differentiation through modulating expression of p21(Cip1).

Fig. 1

Expression of TGF-β signaling component mRNA. RT-PCR analysis was performed using tongue epithelia (TE) and KT-1 cells. Gapdh was used as a loading control. H₂O was used as a negative control

Fig. 2

Effects of TGF-β on KT-1 cell proliferation and cell-cycle profile. a Cell growth was measured with the WST-8 assay in the presence of TGF-β3. Growth of KT-1 cells was inhibited in a dose-dependent manner by treatment with TGF-β3. The absorbance at 590 nm was normalized to the cell treatment with 0 ng/mL of TGF-β3. b Percentage of BrdU-labeled cells (BrdU⁺). TGF-β3 (1 ng/mL) reduced the percentage of BrdU⁺ cells. This experiment was repeated three times. The Asterik indicates p < 0.01, compared with 0 ng/mL of TGF-β3 (control). c Cell-cycle distribution of KT-1 cells with 0 ng/mL (left) or 1 ng/mL of TGF-β3 (right). TGF-β3 increased the ratio of G₀/G₁ phase and reduced the ratio of S phase. This experiment was repeated three times with similar results

Fig. 3

TGF-β3 regulates CDKIs mRNA expression in KT-1 cells. a CDKIs mRNA levels in KT-1 cells after TGF-β3 treatment. KT-1 cells were treated with 0 ng/mL (Control) or 1 ng/mL of TGF-β3 for 6 h. Total RNA from each sample was used in qRT-PCR analysis. Expression levels of each gene were normalized with control samples. Asterisks indicate p < 0.01, compared with control samples. b Time course of p15^Ink4b and p21^Cip1 mRNA levels. RNA was obtained from 0 h (untreated), 2 h, 6 h, and 24 h after TGF-β3 treatment. Expression levels of each time point were normalized with 0 h samples

Fig. 4

Expression of p21^Cip1 mRNA in tongue epithelium. In situ hybridization was performed with digoxigenin-labeled anti-sense RNA probes. The signals for p21^Cip1 mRNA were observed in suprabasal cells of tongue epithelium. Dotted lines indicate the epithelial-mesenchymal boundary, and dashed lines, the basal-suprabasal cell layer boundary. Scale bars indicate 20 μm