Hypoxia-regulated human periodontal ligament cells via Wnt/β-catenin signaling pathway

Abstract

Background: The aim of this study is to investigate the effects of hypoxia on the proliferation, morphology, migration ability, hypoxia inducible factor (HIF) 1 (HIF-1) expression, and the relationship with Wnt/β-catenin signaling of human periodontal ligament cells (hPDLCs) in vitro.

Methods: hPDLCs (4th passage) cultured by the tissue culture method were randomly assigned to slight (5% O2), severe hypoxia (1% O2) groups, and the control (21% O2) group, respectively. From 1st to 7th day, the optical density values were detected, and the growth curve was described. Wound healing assay was done to observe the migration ability of hPDLCs under various O2 conditions. Then reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expression of cementum-related genes and Wnt signaling pathway-related genes. Further, RT-qPCR, Western blot, and immunofluorescence staining method were constructed to show HIF expressions under different O2 concentrations in hPDLCs.

Results: The growth rate of hPDLCs decreased with the reduction of O2 content by degree, and the morphology of hPDLCs changed in different O2 contents. Besides, hPDLCs migrate faster in 21% and 5% O2 than in 1% O2, and both the expressions of cementum-related genes and Wnt signaling pathway-related genes were raised under hypoxic conditions. In addition, with the reduction of O2 concentration, the messenger RNA and protein level expression of HIF were all increased, and HIF was gradually transported from cytoplasm into the nucleus and in 1% O2 concentration, it was mainly expressed in the nucleus.

Conclusion: This finding demonstrated that hypoxia was capable of suppressing the proliferation and migration ability, changing the morphology of hPDLCs, and stabilizing HIF-1α against degradation and promoting its translocation to the nucleus. Meanwhile, hypoxia may regulate hPDLCs proliferation and cementogenic differentiation via Wnt/β-catenin signaling pathway, which may potentially provide a novel insight into the etiology and treatment of periodontal diseases.

Figure 1

Cultivation and identification of human periodontal ligament cells (hPDLCs). (A) 4th passage of hPDLCs. Cells were fusiform in shape and were arranged in a sarciniform or swirl pattern. Original magnification, ×200. (B) Vimentin expression in hPDLCs. Vimentin was found in the cytoplasm with a yellow-brown color. Original magnification, ×200. (B) Keratin expression in hPDLCs. Keratin was not found in hPDLCs, indicating hPDLCs are mesenchymal cells derived from the embryonic mesoderm. Original magnification, ×200. (D) Growth curve of hPDLCs under various hypoxia conditions from 1st to 7th day. The growth rate of hPDLCs decreased by degree with the reduction of O₂ content, but the growth curves were alike in 3 groups, which were similar to an ‘S’, and all these groups had the highest optical density value at the 6th day. (D6^∗∗, P < .01, D7^∗, P = .03).

Figure 2

The morphology of human periodontal ligament cells (hPDLCs) changed in different O₂ contents. (A) In the 1% O₂ concentration, the number of hPDLCs in 1 microscopy significantly reduced, and hPDLCs were arranged sparser compared with normal conditions. Besides, cell bodies shrank, and processes were much shorter and slender. The morphology of hPDLCs in 5% O₂ conditions was between the control and 1% O₂ groups. The columns represented original magnification ×100, ×200, ×400, respectively. (B) The relative alkaline phosphatase activity. hPDLCs showed a significant increase with the reduction of O₂ concentration at 3rd and 7th day. (D3^∗, P = .03, D7^∗, P = .03).

Figure 3

Migration ability of the human periodontal ligament cells (hPDLCs) on different O₂ conditions. (A) Wound-healing assay showed that in the first 24 hours, the scratched area of hPDLCs preconditioned in 1% O₂ was larger than the control group, but the scratched area of hPDLCs preconditioned in 5% O₂ had no significant difference with the control group. After 48 hours, the difference was much more obvious between the 1% O₂ group and the control group, whereas the scratched area of hPDLCs preconditioned in 5% O₂ still had no significant difference with the control group, suggesting that hPDLCs migrated faster in 21% and 5% than in 1% O₂. (B) Migration area cells on different O₂ conditions. The number of cells in the migration area was much less in 1% O₂ compared with the control group. (^∗P = .02).

Figure 4

The relative gene expression of bone-related genes. Reverse transcription quantitative real-time polymerase chain reaction analysis showed that hypoxic conditions enhanced the cementogenic differentiation of human periodontal ligament cells (hPDLCs). The promotion of cementogenic differentiation of hPDLCs is greater in the severe 1% O₂ concentration group than the slight 5% O₂ group. Mineralization-related gene expression of osteopontin (OPN) and alkaline phosphatase for hPDLCs and the cementum-related cementum protein1 (CEMP1) and cementum attached protein (CAP) genes were all significantly upregulated in 1% O₂ conditions compared to other 2 groups at both 3rd and 7th day. (A) OPN. (D3^∗, P = .04, D7^∗, P = .06). (B) Alkaline phosphatase. (D3^∗, P = .02, D7^∗, P = .03). (C) Cementum-related genes of CEMP1 (D3^∗, P = .02, D7^∗, P = .03). (D) CAP. (D3, P = .07, D7^∗, P = .04).

Figure 5

Wnt signaling pathway-related gene expression of human periodontal ligament cells on different O₂ conditions. (A–C) The expression of Wnt-related genes AXIN2 (^∗∗P < 0.01), β-catenin (D3^∗, P = .03, D7^∗, P = .03), and MYC (D3^∗, P = .03, D7^∗, P = .02) increased slightly in hypoxic groups at both 3rd and 7th day. (D) The relative protein expression of Wnt-related genes increased in 1% O₂ and 5% O₂ concentration groups at both 3rd and 7th day.

Figure 6

Hypoxia inducible factor (HIF) expression of human periodontal ligament cells on different O₂ conditions. (A) Immunofluorescence staining results showed that under normal O₂ conditions, HIF-1α degraded rapidly and mainly expressed in cytoplasm, but with the concentration of O₂ reduced, HIF was gradually transported into the nucleus and mainly expressed in the nucleus, suggesting that hypoxia stabilizes HIF-1α against degradation and forms functional complexes in the nucleus. (B) Immumohistochemical staining results of HIF in periodontitis tissues. (C) Western blot results showed an increase of HIF-1α expression in hypoxic conditions. (^∗P = .02).