Transcriptional activity of vitamin D receptor in human periodontal ligament cells is diminished under inflammatory conditions

Abstract

Background: Although vitamin D₃ deficiency is considered as a risk factor for periodontitis, supplementation during periodontal treatment has not been shown to be beneficial to date. Human periodontal ligament cells (hPDLCs) are regulated by vitamin D₃ and play a fundamental role in periodontal tissue homeostasis and inflammatory response in periodontitis. The aim of this study is to investigate possible alterations of the vitamin D₃ activity in hPDLCs under inflammatory conditions.

Methods: Cells isolated from six different donors were treated with either 1,25(OH)₂ D₃ (0 to 10 nM) or 25(OH)D₃ (0 to 100 nM) in the presence and absence of ultrapure or standard Porphyromonas gingivalis lipopolysaccharide (PgLPS), Pam3CSK4, or interferon-γ for 48 hours. Additionally, nuclear factor (NF)-κB inhibition was performed with BAY 11-7082. The bioactivity of vitamin D in hPDLCs was assessed based on the gene expression levels of vitamin D receptor (VDR)-regulated genes osteocalcin and osteopontin. Additionally, VDR and CYP27B1 expression levels were measured.

Results: The vitamin D₃ -induced increase of osteocalcin and osteopontin expression was significantly decreased in the presence of standard PgLPS and Pam3CSK4, which was not observed by ultrapure PgLPS. Interferon-y had diverse effects on the response of hPDLCs to vitamin D₃ metabolites. NF-kB inhibition abolished the effects of standard PgLPS and Pam3CSK4. Standard PgLPS and Pam3CSK4 increased VDR expression in the presence of vitamin D₃ . CYP27B1 expression was not affected by vitamin D₃ and inflammatory conditions.

Conclusions: This study indicates that the transcriptional activity of VDR is diminished under inflammatory conditions, which might mitigate the effectiveness of vitamin D₃ supplementation during periodontal treatment.

Keywords: VDR; inflammation; mesenchymal stem cells; periodontal ligament; vitamin D.

FIGURE 1

Gene expression of osteocalcin in hPDLCs treated with 1,25(OH)₂D₃ under physiological and inflammatory conditions hPDLCs of six healthy donors were treated with 1,25(OH)₂D₃ (1 nM, 10 nM) in the presence and absence of standard PgLPS (1 µg/mL; A) + sCD14 (0.2 µg/mL), Pam3CSK4 (1 µg/mL; B) or IFN‐γ (0.1 µg/mL; C) for 48 hours. Osteocalcin gene expression levels were measured with qPCR. Y‐axes show the n‐fold expression of osteocalcin expression compared with untreated cells ( = 1). GAPDH served as endogenous control. Data are presented as mean ± SEM of six different donors. *Significant difference between groups, P <0.05

FIGURE 2

Gene expression of osteocalcin in hPDLCs treated with 25(OH)D₃ under physiological and inflammatory conditions hPDLCs of six healthy donors were treated with 25(OH)D3 (10 nM, 100 nM) in the presence and absence of standard PgLPS (1 µg/mL; A) + sCD14 (0.2 µg/mL), Pam3CSK4 (1 µg/mL; B) or IFN‐γ (0.1 µg/mL; C) for 48 hours. Osteocalcin gene expression levels were measured with qPCR. Y‐axes show the n‐fold expression of osteocalcin expression compared with untreated cells ( = 1). GAPDH served as endogenous control. Data are presented as mean ± SEM of six different donors. *Significant difference between groups, P <0.05

FIGURE 3

Gene expression of osteopontin in hPDLCs treated with 1,25(OH)₂D₃ under physiological and inflammatory conditions. hPDLCs of six healthy donors were treated with 1,25(OH)2D3 (1 nM, 10 nM) in the presence and absence of standard PgLPS (1 µg/mL; A) + sCD14 (0.2 µg/mL), Pam3CSK4 (1 µg/mL; B) or IFN‐γ (0.1 µg/mL; C) for 48 hours. Osteopontin gene expression levels were measured with qPCR. Y‐axes show the n‐fold expression of osteopontin expression compared with untreated cells ( = 1). GAPDH served as internal reference. Data are presented as mean ± SEM of six different donors. *Significant difference between groups, P <0.05

FIGURE 4

Gene expression of osteopontin in hPDLCs treated with 25(OH)D₃ under physiological and inflammatory conditions. hPDLCs of six healthy donors were treated with 25(OH)D3 (10 nM, 100 nM) in the presence and absence of standard PgLPS (1 µg/mL; A) + sCD14 (0.2 µg/mL), Pam3CSK4 (1 µg/mL; B), or IFN‐γ (0.1 µg/mL; C) for 48 hours. Osteopontin gene expression levels were measured with qPCR. Y‐axes show the n‐fold expression of osteopontin expression compared with untreated cells ( = 1). GAPDH served as endogenous control. Data are presented as mean ± SEM of six different donors. *Significant difference between groups, P <0.05

FIGURE 5

Gene expression levels of osteocalcin and osteopontin in hPDLCs treated with 1,25(OH)₂D₃ or 25(OH)D₃ under physiological and inflammatory conditions and NF‐κB inhibition. hPDLCs of six healthy donors were treated with 1,25(OH)₂D₃ (10 nM; A and C) or 25(OH)D₃ (100 nM; B and D) in the presence and absence of standard PgLPS (1 µg/mL) + sCD14 (0.2 µg/mL) or Pam3CSK4 (1 µg/mL;) for 48 hours. In addition, experiments were performed in the presence and absence of NF‐κB inhibitor BAY 11‐7082 (0.3 µg/mL). Osteocalcin (A and B) and osteopontin (C and D) gene expression levels were measured with qPCR. Y‐axes show the n‐fold expression of osteocalcin and osteopontin expression, respectively, compared with untreated cells ( = 1). GAPDH served as endogenous control. Data are presented as mean ± SEM of six different donors

FIGURE 6

Gene expression of VDR in hPDLCs treated with 1,25(OH)₂D₃ or 25(OH)D₃ under physiological and inflammatory conditions. hPDLCs of six healthy donors were treated with 1,25(OH)₂D₃ (1 nM, 10 nM; A through C) or 25(OH)D₃ (10 nM, 100 nM; D through F) in the presence and absence of standard PgLPS (1 µg/mL; A and D) + sCD14 (0.2 µg/mL), Pam3CSK4 (1 µg/mL; B and E), or IFN‐γ (0.1 µg/mL; C and F) for 48 hours. VDR gene expression levels were measured with qPCR. Y‐axes show the n‐fold expression of VDR expression compared with untreated cells ( = 1). GAPDH served as endogenous control. Data are presented as mean ± SEM of six different donors. *Significant difference between groups, P <0.05