J Periodontal Implant Sci.  2019 Jun;49(3):138-147. 10.5051/jpis.2019.49.3.138.

Helicobacter pylori inhibited cell proliferation in human periodontal ligament fibroblasts through the Cdc25C/CDK1/cyclinB1 signaling cascade

Affiliations
  • 1Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou, China. zhaowh@smu.edu.cn

Abstract

PURPOSE
Several studies have shown that the oral cavity is a secondary location for Helicobacter pylori colonization and that H. pylori is associated with the severity of periodontitis. This study investigated whether H. pylori had an effect on the periodontium. We established an invasion model of a standard strain of H. pylori in human periodontal ligament fibroblasts (hPDLFs), and evaluated the effects of H. pylori on cell proliferation and cell cycle progression.
METHODS
Different concentrations of H. pylori were used to infect hPDLFs, with 6 hours of co-culture. The multiplicity of infection in the low- and high-concentration groups was 10:1 and 100:1, respectively. The Cell Counting Kit-8 method and Ki-67 immunofluorescence were used to detect cell proliferation. Flow cytometry, quantitative real-time polymerase chain reaction, and western blots were used to detect cell cycle progression. In the high-concentration group, the invasion of H. pylori was observed by transmission electron microscopy.
RESULTS
It was found that H. pylori invaded the fibroblasts, with cytoplasmic localization. Analyses of cell proliferation and flow cytometry showed that H. pylori inhibited the proliferation of periodontal fibroblasts by causing G2 phase arrest. The inhibition of proliferation and G2 phase arrest were more obvious in the high-concentration group. In the low-concentration group, the G2 phase regulatory factors cyclin dependent kinase 1 (CDK1) and cell division cycle 25C (Cdc25C) were upregulated, while cyclin B1 was inhibited. However, in the high-concentration group, cyclin B1 was upregulated and CDK1 was inhibited. Furthermore, the deactivated states of tyrosine phosphorylation of CDK1 (CDK1-Y15) and serine phosphorylation of Cdc25C (Cdc25C-S216) were upregulated after H. pylori infection.
CONCLUSIONS
In our model, H. pylori inhibited the proliferation of hPDLFs and exerted an invasive effect, causing G2 phase arrest via the Cdc25C/CDK1/cyclin B1 signaling cascade. Its inhibitory effect on proliferation was stronger in the high-concentration group.

Keyword

Cell cycle; Cell proliferation; Helicobacter pylori; Periodontal ligament; Periodontitis

MeSH Terms

Blotting, Western
CDC2 Protein Kinase
Cell Count
Cell Cycle
Cell Proliferation*
Coculture Techniques
Colon
Cyclin B1
Cytoplasm
Fibroblasts*
Flow Cytometry
Fluorescent Antibody Technique
G2 Phase
Helicobacter pylori*
Helicobacter*
Humans*
Methods
Microscopy, Electron, Transmission
Mouth
Periodontal Ligament*
Periodontitis
Periodontium
Phosphorylation
Real-Time Polymerase Chain Reaction
Serine
Tyrosine
CDC2 Protein Kinase
Cyclin B1
Serine
Tyrosine

Figure

  • Figure 1 The ultrastructure of hPDLFs was observed by transmission electron microscopy. (A) Control at ×5,000 magnification. (B) Invasive group at ×6,000 magnification. The black arrow indicates that H. pylori is surrounded by cytoplasmic vacuoles. (C) Invasive group at ×20,000 magnification. The black arrow indicates that H. pylori is surrounded by cytoplasmic vacuoles. The yellow arrow indicates the proliferation and division of H. pylori in cytoplasmic vacuoles. hPDLFs: human periodontal ligament fibroblasts.

  • Figure 2 Effects of H. pylori infection on the proliferation of hPDLFs. (A) OD values of hPDLFs infected by H. pylori for 7 consecutive days. (B) Immunofluorescence staining for the Ki-67 protein in hPDLFs. hPDLFs: human periodontal ligament fibroblasts, OD: optical density; MOI: multiplicity of infection. a)Statistically significant difference (P<0.05); b)Statistically significant difference (P<0.01).

  • Figure 3 The proportion of DNA content in cell cycle stages in hPDLFs was measured by flow cytometry. (A-C) The proportion of DNA content in various stages of the cell cycle. (D) Change in the proportion of cells in various stages of the cell cycle. (E) Change in the proportion of cells in the G2 phase. hPDLFs: human periodontal ligament fibroblasts; MOI: multiplicity of infection. a)Statistically significant difference (P<0.01).

  • Figure 4 Changes in the levels of mRNA transcription and protein expression of cell cycle-related factors in G2 phase. (A) mRNA expression levels of cyclin B1, CDK1, and Cdc25C. (B) Protein expression levels of cyclin B1, CDK1, CDK1-Y15, Cdc25C, and Cdc25C-S216. CDK1: cyclin dependent kinase 1, Cdc25C: cell division cycle 25C, CDK1-Y15: tyrosine phosphorylation of CDK1, Cdc25C-S216: serine phosphorylation of Cdc25C, MOI: multiplicity of infection, GAPDH: glyceraldehyde-3-phosphate dehydrogenase. a)Statistically significant difference (P<0.05); b)Statistically significant difference (P<0.01).


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