The role of prostaglandin E2 (PGE 2) in toll-like receptor 4 (TLR4)-mediated colitis-associated neoplasia

Abstract

Background: We have previously found that TLR4-deficient (TLR4-/-) mice demonstrate decreased expression of mucosal PGE 2 and are protected against colitis-associated neoplasia. However, it is still unclear whether PGE 2 is the central factor downstream of TLR4 signaling that promotes intestinal tumorigenesis. To further elucidate critical downstream pathways involving TLR4-mediated intestinal tumorigenesis, we examined the effects of exogenously administered PGE 2 in TLR4-/- mice to see if PGE 2 bypasses the protection from colitis-associated tumorigenesis.

Method: Mouse colitis-associated neoplasia was induced by azoxymethane (AOM) injection followed by two cycles of dextran sodium sulfate (DSS) treatment. Two different doses of PGE 2 (high dose group, 200 microg, n = 8; and low dose group, 100 microg, n = 6) were administered daily during recovery period of colitis by gavage feeding. Another group was given PGE 2 during DSS treatment (200 microg, n = 5). Inflammation and dysplasia were assessed histologically. Mucosal Cox-2 and amphiregulin (AR) expression, prostanoid synthesis, and EGFR activation were analyzed.

Results: In control mice treated with PBS, the average number of tumors was greater in WT mice (n = 13) than in TLR4-/- mice (n = 7). High dose but not low dose PGE 2 treatment caused an increase in epithelial proliferation. 28.6% of PBS-treated TLR4-/- mice developed dysplasia (tumors/animal: 0.4 +/- 0.2). By contrast, 75.0% (tumors/animal: 1.5 +/- 1.2, P < 0.05) of the high dose group and 33.3% (tumors/animal: 0.3 +/- 0.5) of the low dose group developed dysplasia in TLR4-/- mice. Tumor size was also increased by high dose PGE 2 treatment. Endogenous prostanoid synthesis was differentially affected by PGE 2 treatment during acute and recovery phases of colitis. Exogenous administration of PGE 2 increased colitis-associated tumorigenesis but this only occurred during the recovery phase. Lastly, PGE 2 treatment increased mucosal expression of AR and Cox-2, thus inducing EGFR activation and forming a positive feedback mechanism to amplify mucosal Cox-2.

Conclusions: These results highlight the importance of PGE 2 as a central downstream molecule involving TLR4-mediated intestinal tumorigenesis.

Figure 1

Experimental protocol. PGE ₂ was administered in two different doses (high dose group 200 μg/mouse, and low dose group 100 μg/mouse) and phases (recovery period and during DSS treatment). Control mice were given the same volume of PBS. AOM and DSS (2.5%) were administered as shown. Mice were sacrificed on day 56.

Figure 2

Administration of PGE ₂ promotes development of colitis-associated neoplasia in TLR4-/- mice. A. Incidence of dysplasia. The number of dysplastic lesions was counted per mouse under the microscope. Data are expressed as mean (± SD) (*P < 0.05). B. Microscopic pictures of H & E staining in colon sections (Original magnification ×100, Bars indicate 200 μm).

Figure 3

PGE ₂ administration did not alter severity of chronic colitis. A. Cumulative disease activity index (DAI) score defined as body weight change, hemocult blood, and stool consistency. Score was measured three times per week. B. Histological score of colitis. There are no statistical differences in severity of colitis between the mice that received different PGE ₂ treatment doses and timing (n = 13 WT; n = 7 PBS group; n = 8 high dose PGE ₂ 200 μg group; and n = 6 low dose PGE ₂ 100 μg group; n = 5 PGE ₂ 200 μg during DSS treatment; NS: no significance). C. Mucosal 15d-PGJ2 (left panel) and endogenous PGE ₂ (right panel) synthesis during acute colitis. Mice received AOM and after 14 days, DSS was given for 7 days. PGE ₂ treatment (200 μg/day) significantly up-regulates mucosal 15d-PGJ2 synthesis but not endogenous PGE ₂ in TLR4-/- mice at day 7 of DSS colitis (n = 5 each). WT mice received only AOM and DSS. Data are expressed as mean (± SD) (*P < 0.05). D. 15d-PGJ2 (left panel) and endogenous PGE ₂ (right panel) synthesis at 14 day recovery from DSS colitis. Mice were examined at the end of the first cycle of AOM-DSS treatment. There is no difference of 15d-PGJ2 synthesis between TLR4-/- mice that received PGE ₂ (200 μg/day) during DSS colitis and during the recovery from DSS colitis (n = 5 each, P = 0.441). By contrast, significant increase of endogenous PGE ₂ is shown in the mice that received PGE ₂ during the recovery phase. This increase of endogenous PGE ₂ is not seen in the mice that received PGE ₂ during DSS treatment (acute phase). Data are expressed as mean (± SD).

Figure 4

High dose PGE ₂ administration drives epithelial cell proliferation in the setting of chronic colitis. A. Immunohistochemical staining of incorporated BrdU. Cells under S-phase (proliferative) are labeled with BrdU as brown nuclei. Original magnification 200 ×, Bars indicate 100 μm. B Proliferative index (Number of BrdU positive cells per crypt) was counted in intestinal epithelial cells. BrdU positive cells were counted in 10 crypts of each colon segment per high power field (HPF) (30 crypts/mouse). The high dose group (n = 8) has significantly greater number of BrdU positive cells per crypt than PBS treated controls (n = 7). Data are expressed as mean (± SD) (*P < 0.05).

Figure 5

High dose PGE ₂ may promote a positive feedback loop in EGFR signal-mediated cell proliferation by inducing Cox-2 expression. A. Mucosal expression of AR in TLR4-/- mice treated with PGE ₂; day 56 in AOM-DSS model (Left graph: mRNA analyzed by real-time PCR, Right graph: protein measured by ELISA). Data are represented as mean (± SD) of relative values of expression (n = 7 PBS group, n = 8 high dose and n = 6 low dose groups, *P < 0.05). B. Western blot analysis of phosphorylated EGFR and EGFR in the colon. Results from three representative samples obtained from TLR4-/- mice under each treatment in AOM-DSS model. An equal amount of protein (25 μg/lane) was loaded per lane. The membrane was sequentially probed for phospho-EGFR and EGFR.

Figure 6

PGE ₂-mediated up-regulation of Cox-2 expression. A. Real-time PCR results for mucosal Cox-2 mRNA expression in TLR4-/- mice treated with PGE ₂; day 56 in AOM-DSS model. Data are represented as mean (± SD) of relative values of expression (n = 7 PBS group; n = 8 high dose group, PGE ₂ 200 μg; and n = 6 low dose group, PGE ₂ 100 μg; n = 5 PGE ₂ during DSS treatment, PGE ₂ 200 μg; *P < 0.05). B. Immunofluorescent staining for Cox-2 in the colon. Representative pictures show Cox-2 positive cells (green - FITC) in lamina propria cells. High dose PGE ₂ treated mice show many Cox-2 positive cells in the lamina propria. Low dose PGE ₂ treated mice and PBS treated mice have very few Cox-2 positive cells in lamina propria. Double staining demonstrates most Cox-2 positive cells (green - FITC) are also positive for CD68 (red - TRITC) both in the non-tumor area (middle panel) and tumor area (bottom) (serial pictures in same area are shown). Original magnification: 400 ×. Bars indicate 50 μm. C. Western blot analysis of Cox-2 production in murine macrophage cell line RAW246.7 and TLR4-/- peritoneal macrophages. Representative results from three independent experiments. Cells were treated with 10 μM PGE ₂ for 18 hours. An equal amount of protein was loaded per lane. The membrane was sequentially probed for Cox-2 and β-actin.

Figure 7

Role of PGE2 in bridging TLR4 signaling and intestinal tumorigenesis. A. In WT intestine, TLR4 expression is increased in chronic inflammation and PGE ₂ synthesis is induced by Cox-2 induction. Produced PGE ₂ stimulates the expression and release of AR and then EGFR activation either directly or through AR release. Sustained activation of this pathway results in aberrant cell proliferation and tumorigenesis. B. In the absence of TLR4, chronic intestinal inflammation does not induce tumorigenesis. C. When PGE ₂ is exogenously administered to TLR4-/- mice ①, PGE ₂ activates EGFR either directly ④ or through AR ②③. This may be not enough to induce tumorigenesis. However, exogenous PGE ₂ induces Cox-2 production in macrophages ⑤ that causes the release of endogenous PGE ₂, forming a positive feedback loop, which induces tumorigenesis ⑥.