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Smad2 overexpression reduces the proliferation of the junctional epithelium
M K Alotaibi1, Y Kitase2, C F Shuler3
1Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, BC, Canada Advanced Education in General Dentistry, Riyadh Military Hospital, Kingdom of Saudi Arabia.
Abstract:
The overexpression of the intracellular signaling molecule of the transforming growth factor-beta family (TGF-β) Smad2 was found to induce apoptosis and inhibit the proliferation rate of oral epithelial cells. Therefore, the aim of this study was to investigate in vivo the effect of Smad2 overexpression on the proliferation rate of the junctional epithelium (JE). Smad2 overexpression was driven by the cytokeratin 14 promoter (K14-Smad2) in transgenic mice. The K14-Smad2 mice were compared with wild-type (WT) mice selected as the control group. Samples were stained with hematoxylin and eosin stains and analyzed by image analysis. Immunohistochemistry was conducted for proliferating cell nuclear antigen (PCNA) and c-Myc as markers of cell proliferation. The expression of cyclin-dependent kinase inhibitors (P15, P21, and P27) was determined by real-time polymerase chain-reaction (RT-PCR). The quantity of phosphorylated retinoblastoma (pRB) was determined with Western blots. The overexpression of Smad2 altered the area of the junctional epithelial cells in one-year-old K14-Smad2 mice. The area was 32,768 (± 3,473) μm(2) for the WT and 24,937.25 (± 1,965) μm(2) for the K14-Smad2 mice. There was a significant difference in the proliferation rates of the JE (PCNA-positive cells) between the WT and K14-Smad2 mice, 20.7% (± 1.1) and 2.1% (± 0.5), respectively. A significant difference in c-Myc expression occurred between experimental and control samples. The K14-Smad2 mice had a mean of 2.3% (± 0.6), and the WT mice had a mean of 20.1% (± 3.6). Smad2 overexpression up-regulated the mRNA expression of P15 by 2.3-fold and that of P27 by 5.5-fold in the K14-Smad2 mice. Finally, the pRB protein showed a 2.3 (± 0.5)-fold increase in K14-Smad2 mice when compared with WT mice. Smad2 overexpression inhibits the proliferation of JE cells by down-regulating c-Myc and up-regulating P15 and P27, which resulted in an increase in pRB, leading to cell-cycle arrest.
Insights
Smad2 overexpression in transgenic mice significantly inhibited junctional epithelium (JE) cell proliferation. This involved down-regulating c-Myc and up-regulating cell cycle inhibitors P15 and P27, leading to cell-cycle arrest.
Area of Science:
- Cell Biology
- Molecular Biology
- Oral Epithelial Cell Research
Background:
- Transforming growth factor-beta (TGF-β) signaling molecule Smad2 overexpression can induce apoptosis and inhibit oral epithelial cell proliferation.
- The role of Smad2 in the proliferation of the junctional epithelium (JE) in vivo remains largely unexplored.
Purpose of the Study:
- To investigate the in vivo effect of Smad2 overexpression on the proliferation rate of the junctional epithelium (JE).
- To elucidate the molecular mechanisms underlying Smad2-mediated regulation of JE cell proliferation.
Main Methods:
- Generation of transgenic mice with Smad2 overexpression in the JE driven by the cytokeratin 14 promoter (K14-Smad2).
- Analysis of JE morphology and proliferation markers (PCNA, c-Myc) using immunohistochemistry and image analysis.
- Quantification of cyclin-dependent kinase inhibitors (P15, P21, P27) mRNA expression via RT-PCR and phosphorylated retinoblastoma (pRB) protein levels via Western blot.
Main Results:
- Smad2 overexpression significantly reduced JE cell proliferation, evidenced by decreased PCNA-positive cells (2.1% vs. 20.7% in WT mice).
- K14-Smad2 mice exhibited reduced c-Myc expression and increased mRNA levels of P15 (2.3-fold) and P27 (5.5-fold) compared to wild-type (WT) controls.
- Western blot analysis revealed a 2.3-fold increase in phosphorylated retinoblastoma (pRB) protein in K14-Smad2 mice, indicating cell-cycle arrest.
Conclusions:
- Smad2 overexpression inhibits JE cell proliferation through the downregulation of c-Myc and upregulation of cell cycle inhibitors P15 and P27.
- The observed increase in pRB levels suggests that Smad2-induced cell-cycle arrest is a key mechanism in regulating JE cell proliferation.
- These findings provide novel insights into the role of TGF-β/Smad2 signaling in maintaining periodontal homeostasis.
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