Activation of the TLR4-JNK but not the TLR4-ERK pathway induced by indole-3-acetic acid exerts anti-proliferative effects on Caco-2 cells.

We previously found that indole-3-acetic acid (IAA) produced from tryptophan by gut microbiota decreases the expression of tumor necrosis factor α (TNFα), which is implicated in the pathogenesis of colorectal cancer (CRC). The present study aimed to determine IAA involvement in the proliferation of CRC-derived Caco-2 cells. Cell proliferation was suppressed by IAA, whereas IAA-induced aryl hydrocarbon receptor activation had no impact. IAA activated extracellular signal-related (ERK) and c-Jun N-terminal (JNK) kinases, but not p38. Toll-like receptor 4 (TLR4) may be required to activate ERK and JNK, but only the TLR4-JNK pathway might elicit the anti-proliferative effects of IAA. Thus, IAA may be a ligand for TLR4 that contributes to inhibiting CRC cell proliferation by activating TLR4-mediated JNK. Because IAA did not induce cytotoxicity, inhibiting cell cycle progression might affect the anti-proliferative capacity of IAA. Therefore, colonic IAA accumulation might help to prevent CRC development and progression.

Skatole-induced p38 and JNK activation coordinately upregulates, whereas AhR activation partially attenuates TNFα expression in intestinal epithelial cells.

Increased tumor necrosis factor α (TNFα) expression in intestinal epithelial cells (IECs) plays a major role in the development and progression of inflammatory bowel disease (IBD) and colorectal cancer (CRC). The present study aimed to clarify the relationship between TNFα and skatole, a tryptophan-derived gut microbiota metabolite. The aryl hydrocarbon receptor (AhR) antagonist CH223191 promoted, whereas the p38 inhibitor SB203580 suppressed the increase in TNFα mRNA and protein expression induced by skatole in intestinal epithelial Caco-2 cells. The c-Jun N-terminal kinase (JNK) inhibitor SP600125 repressed only the increased TNFα protein expression, whereas the extracellular signal-regulated kinase (ERK) pathway inhibitor U0126 did not affect increased TNFα expression at any level. A neutralizing antibody against TNFα partially inhibited skatole-induced cell death. Overall, these results suggested that TNFα expression is increased by the concerted actions of skatole-activated p38 and JNK, and that TNFα exerts autocrine/paracrine actions on IECs despite partial suppression by activated AhR. Therefore, skatole might play an important role in the development and progression of IBD and CRC via increased TNFα expression.

Microcystin-LR incorporated into colonic cells through probenecid-sensitive transporters leads to upregulated MCP-1 expression induced by JNK activation.

Harmful algae that inhabit eutrophic lakes produce cyanotoxic microcystins. Therefore, the relationship between chronic exposure to microcystins via drinking water and organ disorders has been investigated. The present study aimed to determine whether representative microcystin-LR is involved in increased monocyte chemoattractant protein-1 (MCP-1) expression in rat colonic mucosa and enterocyte-like differentiated Caco-2 cells. The mRNA expression of MCP-1 was increased in the colons of rats administered with microcystin-LR, compared with controls. Furthermore, mRNA levels of MCP-1 expression significantly and positively correlated with those of Adhesion G Protein-Coupled Receptor E1 (ADGRE1; EMR1; F4/80), an indicator of macrophage infiltration, suggesting that increased MCP-1 expression induced by microcystin-LR promotes macrophage infiltration into the colon. Microcystin-LR increased MCP-1 expression in enterocyte-like differentiated Caco-2 cells, by activating c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase (ERK) or p38. The findings of transporter inhibitors indicated that microcystin-LR is incorporated into cells via ATP Binding Cassette (ABC) or solute carrier (SLC) transporters other than the organic anion transporting polypeptides (OATPs)1B1, 1B3, 2B1, and 1A2, which this leads to increased MCP-1 expression in the colon through activating JNK. Thus, increased MCP-1 expression induced by microcystin-LR might be a trigger for initiating tumorigenesis with inflammation in the colon because increased MCP-1 expression induces inflammation associated with macrophage infiltration into the colon, and chronic inflammation is associated with the initiation of tumorigenesis.

TLR4 may be a novel indole-3-acetic acid receptor that is implicated in the regulation of CYP1A1 and TNFα expression depending on the culture stage of Caco-2 cells.

Most studies of indole derivatives such as IAA produced by intestinal microbiota have been based on the premise that binding to AhR leads to biological responses. We previously revealed that IAA binds to more than one receptor, and thus the present study aimed to identify a new receptor for IAA and analyze its mechanism of action. We found that the TLR4 antagonist TAK-242 did not affect the IAA-induced increase in CYP1A1 expression at 3 h and decreased TNFα expression at 8 days. However, TAK-242 alleviated decreased TNFα expression induced by IAA at 2 days and promoted IAA-induced increased CYP1A1 expression by inhibiting JNK activation at 8 days. Taken together, TLR4 may be a novel IAA receptor with signaling pathways that regulate CYP1A1 and TNFα expression depending on the culture stage of Caco-2 cells. Furthermore, our findings offer important clues for elucidating the action mechanisms of indole derivatives that affect hosts.

Suppression of TNFα expression induced by indole-3-acetic acid is not mediated by AhR activation in Caco-2 cells.

Indole-3-acetic acid (IAA) produced by intestinal bacteria from tryptophan in dietary proteins is considered to suppress the inflammatory response through aryl hydrocarbon receptor (AhR) activation. However, AhR activation was not involved in the downregulation of tumor necrosis factor α (TNFα) expression induced by IAA in Caco-2 cells. The activation of unidentified IAA receptors might attenuate the inflammatory response to TNFα in colorectal cancer cells.