Kynurenine promotes the goblet cell differentiation of HT-29 colon carcinoma cells by modulating Wnt, Notch and AhR signals

Abstract

Abstract.Various amino acids regulate cell growth and differentiation. In the present study, we examined the ability of HT-29 cells to differentiate into goblet cells in RPMI and DMEM which are largely different in the amounts of numerous amino acids. Most of the HT-29 cells differentiated into goblet cells downregulating the stem cell marker Lgr5 when cultured in DMEM, but remained undifferentiated in RPMI. The goblet cell differentiation in DMEM was inhibited by 1-methyl-tryptophan (1-MT), an inhibitor of indoleamine 2,3 dioxygenase-1 which is the initial enzyme in tryptophan metabolism along the kynurenine (KN) pathway, whereas tryptophan and KN induced goblet cell differentiation in RPMI. The levels of Notch1 and its activation product Notch intracytoplasmic domain in HT-29 cells were lower in DMEM than those in RPMI and were increased by 1-MT in both media. HT-29 cells grown in both media expressed β-catenin at the same level on day 2 when goblet cell differentiation was not observed. β-catenin expression, which was increased by 1-MT in both media, was decreased by KN. DMEM reduced Hes1 expression while enhancing Hath1 expression. Finally, aryl hydrocarbon receptor (AhR) activation moderately induced goblet cell differentiation. Our results suggest that KN promotes goblet cell differentiation by regulating Wnt, Notch, and AhR signals and expression of Hes1 and Hath1. IntroductionCell lines are usually maintained in defined media that commonly contain amino acids, vitamins, glucose and inorganic salts for normal cell metabolism. However, the composition of medium formulations vary widely in concentrations of glucose and other metabolic precursors, including amino acids. Thus, many types of cells showed different responses to culture media in regards to proliferation and differentiation. Th17 differentiation was induced more efficiently in Iscove's modified Dulbecco's medium (IMDM) than in RPMI (1). Bone marrow-derived dendritic cells in IMDM expressed higher levels of co-stimulatory and MHC II molecules, compared to the cells generated in RPMI (2). In addition, hepatocyte differentiation and propagation and phenotype of corneal stroma-derived stem cells were modulated by culture media (3,4). Subsequently, the underlying mechanisms of the medium-induced changes in cell proliferation and differentiation have been partially elucidated. Differentiation of Th17 cells was increased by endogenous aryl hydrocarbon receptor (AhR) agonists derived from aromatic amino acids such as tryptophan, which is underrepresented in RPMI compared with IMDM (1). Mesenchymal stem cells co-cultured with AML12 liver cells were able to differentiate into hepatocytelike cells expressing hepatocyte-specific markers including albumin, α-fetoprotein and cytokeratin 18 mRNAs, which were expressed at the initial 7-day culture in DMEM while being expressed during the 7-to 14-day culture in DMEM/ F12 (3). In a study using MDA-MB-231 breast cancer cells, ~25.6% of genes were expressed at significantly different levels in cells grown in MEM, DM...

Insight into the relationship between aryl-hydrocarbon receptor and β-catenin in human colon cancer cells

“…Administration of kynurenine, which is a tryptophan metabolite and an endogenous AhR agonist, activated β-catenin and proliferation of human colon cancer cell HCT116 and increased tumor growth in mice [23]. In another colon carcinoma cell line, HT-29, β-catenin expression was decreased by kynurenine, while it was increased by 1-methyl-tryptophan, an inhibitor of indoleamine 2, 3 dioxygenase-1, a key enzyme of kynurenine production [24]. When these previous studies are compared, there are inconsistencies and conflicting data as to whether AhR agonists enhance or attenuate β-catenin and its signal.…”

The metabolic network model of primed/naive human embryonic stem cells underlines the importance of oxidation-reduction potential and tryptophan metabolism in primed pluripotency

“…Using mass spectrometry, kynurenine levels has been reported to be significantly increased (by 27 folds) in primed human embryonic cells in comparison to embryonal carcinoma cells [39]. Interestingly, recent investigations on tumors, have reported kynurenine’s impact on signaling cascades such as Wnt, Notch and PI3K, which are fundamental signaling pathways for pluripotency as well [40, 41]. We also observed that IDO1, a key enzyme in tryptophan degradation through kynurenine, was downregulated in all the naive cells (Additional file 1: Table S2), which further underlines the importance of kynurenine pathway in primed pluripotency.…”

Regulation of Intestinal Stem Cell Stemness by the Aryl Hydrocarbon Receptor and Its Ligands

“…Interestingly, KN may regulate epithelial cell fate through the AhR. Of note are recent findings demonstrating that both tryptophan and KN promote GC differentiation in HT-29 cells as determined by Muc2 gene expression (59). Analyses confirmed that both inhibition of KN synthesis by 1-Methlytryptophan (1-MT) and inhibition of AhR signaling by its antagonist α-naphthoflavone suppresses Muc2 gene expression, suggesting a loose connection between AhR activation and KN synthesis in the production of GCs.…”

“…Importantly however, while KN was shown to increase the protein expression of β-catenin relative to NICD (Wnt vs. Notch signals), these effects were dependent on the media in which the cells were grown (i.e., DMEM vs. RPMI) which can vary in amino acid and glucose content (59). Interestingly, an early report by Park et al has indicated that AhR is highly expressed in…”