Heparin and LPS-induced COX-2 expression in airway cells: a link between its anti-inflammatory effects and GAG sulfation

Abstract

Purpose/aim: Previous studies have indicated that the sulfated polysaccharide heparin has anti-inflammatory effects. However, the mechanistic basis for these effects has not been fully elucidated.

Materials and methods: NCI-H292 (mucoepidermoid) and HBE-1 (normal) human bronchial epithelial cells were treated with LPS alone or in the presence of high-molecular-weight (HMW) fully sulfated heparin or desulfated HMW heparin. Cells were harvested to examine the phosphorylation levels of ERK1/2, p38, and NF-kB p65 and COX-2 protein expression by Western blot and gene expression of both COX-2 and CXCL-8 by TaqMan qRT-PCR.

Results: Heparin is known to exert an influence on receptor-mediated signaling through its ability to both potentiate and inhibit the receptor-ligand interaction, depending upon its concentration. In H292 cells, fully-sulfated HMW heparin significantly reduced LPS-induced gene expression of both COX-2 and CXCL-8 for up to 48 hours, while desulfated heparin had little to no significant suppressive effect on signaling or on COX-2 gene or protein expression. Desulfated heparin, initially ineffective at preventing LPS-induced CXCL8 up-regulation, reduced CXCL8 transcription at 24 hours. In contrast, in normal HBE-1 cells, fully sulfated heparin significantly suppressed only ERK signaling, COX-2 gene expression at 12 hours, and CXCL-8 gene expression at 6 and 12 hours, while desulfated heparin had no significant effects on LPS-stimulated signaling or on gene or protein expression. Sulfation determines heparin's influence and may reflect the moderating role of GAG sulfation in lung injury and health.

Conclusions: Heparin's anti-inflammatory effects result from its nonspecific suppression of signaling and gene expression and are determined by its sulfation.

Keywords: COX-2; LPS; heparin; lung; sulfation.

Fig. 1

The effect of HMW heparin on phosphorylation of ERK1/2 (A), p38 (B), and NF-κB p65 (C) in LPS-treated H292 cells. Levels of phosphorylation were determined 30 mins after treatment. HMW heparin at 500 µg/mL significantly prevented LPS-induced phosphorylation of these signaling pathways. * P < 0.05 and ** P < 0.01. Values represent mean ± SE for triplicate analyses, normalized to the “LPS only” group. A representative Western blot for each signaling pathway is shown in (D).

Fig. 2

The effect of HMW heparin on COX-2 protein expression in LPS-treated H292 cells at 12 hrs (A), 24 hrs (B), and 48 hrs (C) after treatment. 500 µg/mL of HMW heparin prevented LPS-induced COX-2 expression at 24 and 48 hrs after treatment. (D) Representative Western blots for each time point are shown. * P < 0.05 and ** P < 0.01. Values represent mean ± SE for triplicate analyses, normalized to the “LPS only” group.

Fig. 3

Fold change in gene expression in H292 cells at 6 hrs, 12 hrs, and 24 hrs following heparin treatment. Quantitative real-time PCR (TaqMan assay) was performed and GAPDH was used as the housekeeping gene. (A) 500 µg/mL of HMW heparin significantly inhibited mRNA expression of COX-2 in H292 stimulated with LPS at all time points. (B) HMW heparin at 50 µg/mL significantly prevented LPS-induced CXCL8 mRNA expression at 12 and 24 hrs, and 500 µg/mL of HMW heparin significantly prevented LPS-induced CXCL8 mRNA expression at 6 hrs after treatment. * P < 0.05 and ** P < 0.01. Values represent mean ± SE for triplicate analyses, normalized to the “LPS only” group of that time point.

Fig. 4

The effect of sulfation levels of heparin on phosphorylation of ERK1/2 (A), p38 (B), and NF-κB p65 (C). H292 cells were treated with either HMW heparin (500 µg/mL) or desulfated HMW heparin (500 µg/mL) in the presence of 10 µg/mL of LPS for 30 mins. HMW heparin significantly prevented LPS-induced phosphorylation of these signaling pathways. However, desulfated HMW heparin (DS-Heparin) had no significant effect on phosphorylation levels increased by LPS. * P < 0.05 and ** P < 0.01. Values represent mean ± SE for triplicate analyses, normalized to the “LPS only” group. (D) Representative Western blots for each signaling pathway are shown.

Fig. 5

The effect of the sulfation levels of heparin on COX-2 protein expression in LPS-treated H292 cells at 12hrs (A), 24 hrs (B), and 48 hrs (C) after treatment. HMW heparin significantly inhibited protein expression of COX-2 in H292 cells stimulated with LPS at all time points. Desulfated HMW heparin (DS-Heparin) significantly enhanced up-regulation of COX-2 protein at 48 hrs following treatment with LPS. * P < 0.05 and ** P < 0.01. Values represent mean ± SE for triplicate analyses, normalized to the “LPS only” group. (D) Representative Western blots for each time point are shown.

Fig. 6

Fold change in gene expression in H292 cells at 6 hrs, 12 hrs, and 24 hrs following treatment with either HMW heparin (500 µg/mL) or desulfated HMW heparin (DS-Heparin at 500 µg/mL) in the presence of LPS. (A) HMW heparin significantly inhibited mRNA expression of COX-2 in H292 cells stimulated with 10 µg/mL LPS at all time points. However, DS-Heparin significantly enhanced up-regulation of COX-2 mRNA expression by LPS at 24 hrs after treatment. (B) Both HMW heparin and DS-Heparin, at the same concentration of 500 µg/mL, significantly prevented LPS-induced CXCL8 mRNA expression at 24 hrs after treatment. * P < 0.05 and ** P < 0.01. Values represent mean ± SE for triplicate analyses, normalized to the “LPS only” group of that time point.

Fig. 7

The effect of HMW heparin on phosphorylation of ERK1/2 (A), p38 (B), and NF-κB p65 (C) in LPS-treated HBE-1 normal human bronchial epithelial cells. Levels of phosphorylation were determined 30 mins after treatment. HMW heparin at 50 and 500 µg/mL significantly suppressed phosphorylation of the ERK signaling pathway. ** P < 0.01. Values represent mean ± SE for triplicate analyses, normalized to the “LPS only” group. (D) A representative Western blot for each signaling pathway is shown.

Fig. 8

The effect of the sulfation levels of heparin on COX-2 protein expression in LPS-treated HBE-1 human bronchial epithelial cells at 12hrs (A), 24 hrs (B), and 48 hrs (C) after treatment. HMW heparin did not significantly inhibit protein expression of COX-2 in HBE-1 cells stimulated with LPS. Desulfated HMW heparin (DS-Heparin) apparent up-regulation of COX-2 protein at 24 hrs was not significant. * P < 0.05 and ** P < 0.01. Values represent mean ± SE for triplicate analyses, normalized to the “LPS only” group. (D) Representative Western blots for each time point from the same experiment are shown.

Fig. 9

Fold change in gene expression in HBE-1 cells at 6 hrs, 12 hrs, and 24 hrs following treatment with either HMW heparin (500 µg/mL) or desulfated HMW heparin (500 µg/mL) in the presence of LPS at 1 µg/mL. (A) HMW heparin significantly inhibited mRNA expression of COX-2 in HBE-1 cells stimulated with LPS only at 12 hours after treatment. DS-Heparin did not significantly affect regulation of COX-2 mRNA by LPS at any time point. (B) HMW heparin at 500 µg/mL significantly prevented LPS-induced CXCL8 mRNA expression at 12 and 24 hrs after treatment; 50 µg/mL HMW Heparin was effective only at 12 hrs. Desulfated HMW Heparin (DS-Heparin) had no significant effects on CXCL8 gene expression. * P < 0.05 and ** P < 0.01. Values represent mean ± SE for triplicate analyses, normalized to “LPS only” group of that time point.