Korean J Physiol Pharmacol.  2015 Jan;19(1):51-57. 10.4196/kjpp.2015.19.1.51.

Peptidoglycan Induces the Production of Interleukin-8 via Calcium Signaling in Human Gingival Epithelium

• Son A
• Shin DM
• Hong JH

• KMID: 2071819
• DOI: http://doi.org/10.4196/kjpp.2015.19.1.51

Abstract

The etiology of periodontal disease is multifactorial. Exogenous stimuli such as bacterial pathogens can interact with toll-like receptors to activate intracellular calcium signaling in gingival epithelium and other tissues. The triggering of calcium signaling induces the secretion of pro-inflammatory cytokines such as interleukin-8 as part of the inflammatory response; however, the exact mechanism of calcium signaling induced by bacterial toxins when gingival epithelial cells are exposed to pathogens is unclear. Here, we investigate calcium signaling induced by bacteria and expression of inflammatory cytokines in human gingival epithelial cells. We found that peptidoglycan, a constituent of gram-positive bacteria and an agonist of toll-like receptor 2, increases intracellular calcium in a concentration-dependent manner. Peptidoglycan-induced calcium signaling was abolished by treatment with blockers of phospholipase C (U73122), inositol 1,4,5-trisphosphate receptors, indicating the release of calcium from intracellular calcium stores. Peptidoglycan-mediated interleukin-8 expression was blocked by U73122 and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester). Moreover, interleukin-8 expression was induced by thapsigargin, a selective inhibitor of the sarco/endoplasmic reticulum calcium ATPase, when thapsigargin was treated alone or co-treated with peptidoglycan. These results suggest that the gram-positive bacterial toxin peptidoglycan induces calcium signaling via the phospholipase C/inositol 1,4,5-trisphosphate pathway, and that increased interleukin-8 expression is mediated by intracellular calcium levels in human gingival epithelial cells.

Keyword

Ca2+ signaling; Cytokine; Human oral epithelial cells; Inflammation; Toll like receptors

MeSH Terms

Bacteria
Bacterial Toxins
Calcium
Calcium Signaling*
Calcium-Transporting ATPases
Cytokines
Epithelial Cells
Epithelium*
Gram-Positive Bacteria
Humans
Inflammation
Inositol 1,4,5-Trisphosphate Receptors
Interleukin-8*
Peptidoglycan*
Periodontal Diseases
Phospholipases
Reticulum
Thapsigargin
Toll-Like Receptor 2
Toll-Like Receptors
Type C Phospholipases
Bacterial Toxins
Calcium
Calcium-Transporting ATPases
Cytokines
Inositol 1,4,5-Trisphosphate Receptors
Interleukin-8
Peptidoglycan
Phospholipases
Thapsigargin
Toll-Like Receptor 2
Toll-Like Receptors
Type C Phospholipases

Figure

• 

  Fig. 1 PGN-induced Ca2+ responses in human gingival epithelial cells. (A) Changes in [Ca2+]i induced by concentrations of PGN ranging from 1 to 125 µg/ml (n=3). (B) Analysis of concentration-dependent changes in the peak of fluorescence based on the F340/F380 ratio. (C) Human gingival epithelial cells were stimulated with LPS (100 µg/ml) and PGN (50 µg/ml). Total mRNA was extracted from the cells, and IL-8 mRNA expression was measured (n=3). Data are presented as mean±SE. *p<0.05; **p<0.01 versus control.

• 

  Fig. 2 Characterization of PGN-induced Ca2+ signaling. (A) Effect of removal of extracellular Ca2+ on PGN-induced increase in [Ca2+]i (n=3). (B) Effect of Tg (1 µM) on PGN-induced [Ca2+]i increases in the absence of extracellular Ca2+ (n=3). (C) Effect of U73122 (10 µM) and U73343 (10 µM) on PGN-induced increase in [Ca2+]i (n=3). Ionomycin was used as a positive control. (D) Effect of 2APB (75 µM) on PGN-induced increase in [Ca2+]i (n=3). (E) Oral gingival epithelial cells were treated with U73122, U73343, or BAPTA-AM for 20 min and stimulated with PGN for 1 h and then total RNA was extracted from the cells. Data are presented as mean±SE. *p<0.05; **p<0.01 versus control.

• 

  Fig. 3 Effect of Ca2+ signaling on IL-8 mRNA expression. Oral epithelial cells were stimulated with Tg and PGN for 1 h and then total RNA was extracted from the cells. Data are presented as mean±SE of values from four independent experiments. Data are represented as mean±SE. **p<0.05; ***p<0.01 versus control.

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