Cox-2 is regulated by Toll-like receptor-4 (TLR4) signaling: Role in proliferation and apoptosis in the intestine

Abstract

Background & aims: We recently showed that mice deficient in Toll-like receptor 4 (TLR4) or its adapter molecule MyD88 have increased signs of colitis compared with wild-type (WT) mice after dextran sodium sulfate (DSS)-induced injury. We wished to test the hypothesis that cyclooxygenase 2 (Cox-2)-derived prostaglandin E2 (PGE2) is important in TLR4-related mucosal repair.

Methods: Cox-2 expression was analyzed by real-time polymerase chain reaction, immunohistochemistry, Western blotting, and luciferase reporter constructs. Small interfering RNA was used to inhibit expression of MyD88. TLR4-/- or WT mice were given 2.5% DSS for 7 days. Proliferation and apoptosis were assessed using bromodeoxyuridine staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays, respectively. PGE2 was given orally to DSS-treated mice.

Results: Intestinal epithelial cell lines up-regulated Cox-2 expression in a TLR4- and MyD88-dependent fashion. Lipopolysaccharide-mediated stimulation of PGE2 production was blocked by a selective Cox-2 inhibitor or small interfering RNA against MyD88. After DSS injury, Cox-2 expression increased only in WT mice. TLR4-/- mice have significantly reduced proliferation and increased apoptosis after DSS injury compared with WT mice. PGE2 supplementation of TLR4-/- mice resulted in improvement in clinical signs of colitis and restoration of proliferation and apoptosis to WT values. The mechanism for improved epithelial repair may be through PGE2-dependent activation of the epidermal growth factor receptor.

Conclusions: We describe an important link between TLR4 signaling and Cox-2 expression in the gut. TLR4 and MyD88 signaling are required for optimal proliferation and protection against apoptosis in the injured intestine. Although TLR4 signaling is beneficial in the short term, chronic signaling through TLR4 may lower the threshold for colitis-associated cancer.

Figure 1. LPS induces Cox-2 expression in human intestinal epithelial cell lines in a TLR4- and MyD88-dependent fashion

A. Cox-2 expression after stimulation with LPS (2μg/ml) in T84 (LPS unresponsive) and SW480 (LPS responsive) human intestinal epithelial cell lines. TaqMan real-time PCR demonstrated inducible Cox-2 expression stimulated by LPS in SW480 cells. LPS unresponsive T84 cells showed no change in Cox-2 expression in response to LPS stimulation. Data are represented as mean ± SEM of relative values of expression in 3 individual experiments of triplicate samples (NS: non significant, *P < 0.05). B. LPS responsive SW480 cells were stimulated with LPS (2μg/ml) for indicated times. TaqMan real-time PCR demonstrates LPS-induced expression of Cox-2 mRNA with a peak at 4 hours of stimulation. Data are represented as mean ± SEM of relative values of expression in 3 individual experiments of triplicate samples (*P < 0.05, **P < 0.001). C. Western Blot analysis of Cox-2 protein expression in SW480. Cells were stimulated with LPS for indicated periods in top panel. Lower panel demonstrates stimulation of cells with LPS (2μg/ml) or PGN (2μg/ml). Blots of whole cell lysates (25 μg/lane) were probed with Cox-2 antibody. Data are one representative experiment of three with similar results. β-actin was used as an internal control for protein loading. D. Extent of MyD88 suppression by siRNA. SW480 cells were transiently transfected with siRNA against MyD88, or GAPDH. Negative siRNA which has no significant homology to any gene sequences was applied as a control. The knockdown efficiency of siRNA against MyD88 was assessed by real-time PCR and Western blot. The siRNA was decreased both MyD88 mRNA and protein expression. Negative siRNA as well as siRNA against GAPDH did not affect MyD88 mRNA or protein expression. E. MyD88-dependent induction of Cox-2 in response to LPS. SW480 cells were stimulated with LPS (5μg/ml) for 4 hrs and co-transfected with either MyD88 siRNA or negative control siRNA. Untransfected control samples were not LPS treated. TaqMan real-time PCR demonstrated LPS induced expression of Cox-2 in negative control siRNA samples. This induction of Cox-2 by LPS was largely abolished in the cells in which MyD88 was blocked with siRNA, indicating a MyD88 dependent pathway. Data are represented as mean ± SEM of relative values of expression in 3 individual experiments of triplicate samples (**P < 0.001). F. LPS regulation of Cox-2 gene promoter activity. The intestinal epithelial cell line SW480 was cotransfected with the Cox-2 (−1432/+59) luciferase reporter construct, MyD88 siRNA, or the empty pGL3 vector control, together with an internal control pRL-KT (Renilla luciferase) plasmid. Cells were stimulated with LPS (5μg/ml) for 4 hours. Reporter gene activation was significantly higher in cells stimulated with LPS than non-stimulated cells. MyD88 siRNA abrogated promoter activation in response to LPS. Data are represented as mean ± SEM of relative light units in 3 individual experiments with triplicate samples (*P < 0.05). G. LPS induced PGE₂ production in the intestinal epithelial cell line SW480. Cells were stimulated with LPS (2μg/ml) for 30 min. PGE₂ concentration in supernatant was measured by monoclonal EIA. LPS stimulation resulted in PGE₂ production within 30 min. MyD88 siRNA inhibited LPS induced PGE₂ production as did a selective Cox-2 inhibitor (NS398 5μM) or a Cox-1/Cox-2 inhibitor (indomethacin 5μM). Data are represented as mean ± SEM of 2 individual measurements of duplicate samples taken from 3 individual experiments (*P < 0.05). H. Effect of TLR2 ligands on production of PGE₂ compared with the TLR4 ligand LPS in the intestinal epithelial cell line SW480. SW480 was stimulated with a TLR4 ligand LPS (2μg/ml), or TLR2 ligands PGN (2μg/ml) or Pam3CSK4 (500ng/ml) for indicated periods of time. Concentration of PGE₂was examined by monoclonal EIA. There were significant differences in the stimulation of PGE₂ between LPS versus TLR2 ligands. Data are represented as mean ± SD of triplicate samples taken from 2 individual experiments (P < 0.05, between LPS and PGN or Pam3CSK4 for each time period).

Figure 2. Cox-2 expression is decreased in TLR4−/− mice following DSS-induced colitis

A. TaqMan real-time PCR demonstrated up regulation of Cox-2 expression in the colon of WT mice but not in TLR4−/− mice after 7 days of DSS treatment (n=3 for each group on day 0, n=12 for the other groups). Data are represented as mean ± SEM of relative values of expression in 4 individual experiments (*P < 0.05). B. Western Blot analysis for Cox-2 in the colon. Immunoblots of tissue lysate proteins (25 μg/lane) prepared from colonic samples of TLR4−/− and MyD88−/−, and WT control mice prior to and after 7 days of DSS treatment. Membranes were probed with Cox-2 antibody. Positive control consists of cell lysate from LPS (2μg/ml) stimulated RAW 264.7 cells (right lane). Cox-2 protein expression was greater in WT colon. Data are one representative experiment of three independent studies. β-actin was used as an internal control for protein loading. C. Immunofluorescent staining for Cox-2 in the colon before and after 7 days of DSS treatment. Prior to DSS treatment, colonic tissue does not express detectable levels of Cox-2 in either TLR4−/− mice (C) or WT controls (A). Immunofluorescent signal (red color of rhodamine) of Cox-2 was strongly detected in the colonic epithelial cytoplasm (arrow) and lamina propria cells (arrow head) in WT mice (B) but is very low in TLR4−/− mice (D) after DSS treatment. Insets show phase contrast images identifying the orientation of colonic sections. D. Expression of Cox-2 by lamina propria macrophages using double staining of Cox-2 (FITC green) and CD68 (TRITC red). Most Cox-2 positive lamina propria cells were double-stained with CD68 (macrophage marker). Representative data are from WT mice treated with 7 days of DSS. Arrows indicates double positive cells showing yellow cytoplasmic staining. TLR4−/− mice do not have Cox-2 positive LP macrophages (see Figure 2C). E. PGE₂ production in DSS-induced colitis. Colonic tissues from TLR4−/− and WT controls before and after 7 days of DSS were cultured in media for 24 hours and concentration of PGE₂ in the supernatants were analyzed by EIA (n= 4 for each group). Data are represented as mean ± SD of duplicate samples taken from 3 individual experiments. There was a significant difference in PGE₂ production in TLR4−/− mice following DSS treatment compared with WT mice (*P < 0.05).

Figure 3. TLR4−/− mice have a persistent decrease in epithelial proliferation and increased apoptosis following 7 days of DSS treatment

A. TLR4−/− and WT control mice were treated with 2.5% DSS for 7 days (left panels) followed by 7 days of recovery (right panels). Animals were injected with BrdU 90minutes prior to sacrifice. Colonic sections taken from day 0 (A; WT, D; TLR4−/−), day 7 (B: WT, E: TLR4−/−) and day 14 (C: WT, F: TLR4−/−) were stained with anti-BrdU. BrdU positive cells are identified by black staining of nuclei. TLR4−/− mice have fewer BrdU positive cells in the crypts on day 7 (E panel) as well as on day 14 (F panel). Tissues were counterstained with methyl green (original magnification 200x). B. BrdU positive cells were counted in 3 crypts of each colon segment per HPF (9 crypts/mouse). Bars show mean ± SEM proliferating cells / crypts (n=3 for each group on day 0 (i.e. untreated), n=5 for the other groups). The proliferating cells in TLR4−/− mice were significantly fewer than in WT controls after 7 days of DSS treatment (day 7) as well as during recovery (day 14) from DSS-induced colitis (**P < 0.001). C. Apoptotic cells in the colonic crypt epithelial cells of TLR4−/− and WT controls were determined by TUNEL assay. Representative sections were taken on day 7 of DSS treatment. Red staining of nuclei indicates apoptotic cells, which was observed mainly in the surface epithelium. Sections were counterstained with DAPI (blue) to identify the orientation of nuclei. TLR4−/− mice showed increased apoptotic cells compared with WT controls (original magnification 200X). D. Numbers of apoptotic cells were counted in 300 total epithelial cells in 3 areas of each colon segment (n=3 for each group on day 0 (untreated), n=6–7 for the other groups). Bars show mean ± SEM of the number of apoptotic cells to 100 total nuclei of the epithelial cells. Significant increases of apoptotic cells in TLR4−/− mice compared with WT controls after 7 days of DSS treatment (*P < 0.05). The apoptotic cells were still increased in TLR4−/− mice even after recovery (day 14, *P < 0.05).

Figure 4. PGE₂ supplementation improves signs of colitis and restores epithelial healing after DSS-induced injury in TLR4 −/− mice

A. Weight change was examined daily during the 7 days of DSS treatment. Vehicle (PBS) treated TLR4−/− mice have significantly more weight loss than WT control mice (*P < 0.05). PGE₂ treated TLR4−/− mice and have significantly less weight loss than vehicle treated TLR4−/− mice and are comparable to WT mice. The data represents the average (± SEM) of three independent experiments with a total of 19 mice (TLR4−/− Vehicle (n=6), TLR4−/− PGE₂ (n=7) and WT controls (n=6)). B. PGE₂ treated TLR4−/− mice had a significant reduction in bleeding on days 2 through 6 compared with vehicle (PBS) treated TLR4−/− mice (*p<0.05). Stool blood was calculated as follows: 0=no blood, 1=trace occult blood positive, 2= strongly occult blood positive, and 4=bloody diarrhea. Standard error is shown. C. BrdU labeling of intestinal epithelial cells was performed for vehicle treated TLR4−/−, PGE₂ treated TLR4−/− and WT controls at the end of 7 days of DSS treatment. (Original magnification 200X) D. BrdU positive cells were counted per HPF in 3 crypts of each colon segment (9 areas / mouse). Bars show mean ± SEM of proliferating cells / crypts (n=5 in each group). There is a significant increase in BrdU positive cells in PGE₂ -treated TLR4−/− mice compared with vehicle treated TLR4−/− mice (**P < 0.001). PGE₂-treated TLR4−/− mice are not significantly different than WT mice. E. Apoptotic cells in the crypt epithelial cells were determined by TUNEL assay. Representative sections were taken from vehicle treated TLR4−/−, PGE₂-treated TLR4−/− mice and WT controls as indicated. Red staining of nuclei indicates apoptotic cells. Sections were counterstained with DAPI (blue) to identify the nuclei of epithelial cells. PGE₂ treated TLR4−/− mice showed a marked decrease of apoptotic cells compared with vehicle treated TLR−/− mice. The frequency of TUNEL positive epithelial cells in PGE₂ treated TLR4−/− mice was similar to WT controls (original magnification 200X). F. Number of apoptotic cells was counted in 300 total epithelial cells in triplicate in every 3 areas for each colon segment (n=5 in each group). Bars show mean ± SEM of the number of apoptotic cells per 100 total nuclei in the epithelial cells counted. There is a significant decrease of apoptotic cells in PGE₂ treated TLR4−/− mice compared with vehicle treated TLR4−/− mice (**P < 0.001).

Figure 5. LPS induces EGFR phosphorylation in a MyD88-dependent fashion

A. Western Blot analysis of EGFR and its phosphorylation in human intestinal epithelial cell line SW480. Cells were stimulated with LPS for indicated periods. Blots of whole cell lysates (25 μg/lane) were probed with either EGFR (top panel) or phosphorylated EGFR (bottom panel). LPS stimulated EGFR phosphorylation. Data are one representative experiment of three independent experiments with similar results. B. Immunofluorescent staining for EGFR and phospho-EGFR in SW480. Cells were cultured on glass slides and treated with LPS (2μg/ml) for indicated periods. Nuclei were stained with DAPI. After 30 minutes of stimulation, confocal images show an increased intensity of staining of phospho-EGFR but not EGFR. Insets show negative control in which primary antibodies were omitted. Original magnification 400x. C. Flow cytometric analysis of phospho EGFR (Tyr1068) after stimulation with LPS for indicated periods of time. Histograms show an increase in log fluorescence intensity. Bars represent geometric mean of fluorescence intensity of phospho EGFR positive cells ± SD based on 3 individual experiments (LPS 2μg/ml) with triplicate samples (*P < 0.05). D. LPS-induced EGFR phosphorylation is MyD88-dependent. Western Blot analysis of EGFR and its phosphorylated form are shown. Cells were stimulated with LPS for indicated periods. Blots of whole cell lysates (25 μg/lane) were probed with EGFR or phospho-EGFR antibody. Cells transfected with MyD88 siRNA had no phosphorylation of EGFR in response to LPS. Data are one representative experiment of three independent experiments with similar results. E. LPS induced EGFR phosphorylation is Cox-2 dependent. Cells were stimulated with LPS (2μg/ml) for 30 min with or without Cox inhibitors as indicated, and whole cell lysates (22 μg/lane) probed with EGFR or phospho EGFR antibodies. Western blot analysis demonstrates that EGFR phosphorylation is inhibited by a non-selective Cox-2 inhibitor, (NS398 5μM), or a Cox-1 and Cox-2 inhibitor (indomethacin selective (5μM). As a control, EGF (10nMol=6ng/ml) was added to cells for 30mins. EGF-mediated phosphorylation of the EGFR was not inhibited by Cox inhibitors (last three lanes). Data are one representative experiment of five independent experiments with similar results. F. LPS induced cell proliferation via EGFR activation. SW480 cells were stimulated with LPS (2μg/ml) for indicated periods with or without EGFR specific tyrosine kinase inhibitor AG1478. Data are shown as the means ± SD of percentage of absorbance in comparison to non treated control cells from three independent experiments.

Figure 6. TLR4−/− mice have decreased EGFR phosphorylation following DSS-induced colitis due to defective production of mucosal PGE₂

A. Immunofluorescent staining for phospho EGFR in the colon after 7 days of DSS treatment. Phosphorylated EGFR was strongly detected in surface epithelial cells in WT mice. PBS treated TLR4−/− mice had decreased epithelial cell EGFR phosphorylation following DSS colitis. PGE₂ treatment partly restored the expression of phosphorylated EGFR. The pictures show two individual mouse tissue samples of each genotype with or without PGE₂ treatment. B. Quantification of the expression levels of phospho EGFR. Staining intensity of 10 randomly selected areas of epithelial cells per slide was analyzed using MetaMorph software. Staining intensity of phospho EGFR was significantly decreased in TLR4−/− mice compared with WT mice and PGE₂ treated TLR4−/− mice after 7 days of DSS treatment.

Figure 7. Model of TLR4-mediated Cox-2 regulation

In the setting of intestinal injury, LPS exposure of intestinal epithelial cells (possibly basolaterally) and lamina propria macrophages results in TLR4 activation and signaling via MyD88. This activates a variety of signaling pathways culminating in transcription factor translocation and engagement of the Cox-2 promoter. Cox-2 is transcribed and translated; it acts on arachidonic acid to generate PGG₂ which is rapidly converted to PGH₂ and then microsomal PG E synthase-1 converts it to PGE₂. PGE₂ through its receptors EP2 or EP4 can activate downstream signaling molecules such as the tyrosine kinase Src or the lipid kinase PI’3 kinase which can lead to transactivation of the EGF receptor. EGFR signaling is associated with proliferation and protection against apoptosis in intestinal epithelial cells. PGE₂ produced by macrophages may also act in trans on intestinal epithelial cells. In the absence of TLR4 signaling, Cox-2 expression is greatly decreased.