Indirubin and Indigo Are Potent Aryl Hydrocarbon Receptor Ligands Present in Human Urine

Abstract

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates genes involved in xenobiotic metabolism, cellular proliferation, and differentiation. Numerous xenobiotic and biological compounds are known to interact with AhR, but it remains an orphan receptor, because its physiological ligand is unknown. We identified AhR ligands in human urine using a yeast AhR signaling assay and then characterized their properties. Two ligands, indirubin and indigo, were both present at average concentrations of ϳ0.2 nM in the urine of normal donors. Indirubin was also detected in fetal bovine serum and contributed half of the total AhR ligand activity. The activities of indirubin and indigo were comparable with or more potent than that of the archetypal ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, in yeast AhR activation assays. We suggest that the endogenous levels and potencies of indirubin and indigo are such that they activate AhR-mediated signaling mechanisms in vivo. AhR,1 also called the dioxin receptor, is a ligand-activated transcription factor that is present in most cell and tissue types of the body (1). AhR-mediated signaling is required for potent xenobiotic ligands such as TCDD and polychlorinated biphenyls to produce toxic responses (2, 3). Toxic effects that are linked to xenobiotic AhR ligand exposures in animals include cancers, reproductive impairment, endometriosis, birth defects, and immunological impairment (4 -6). The toxic potential of xenobiotic AhR ligands is currently a major concern for regulatory agencies that are responsible for protecting public and environmental health. Although numerous xenobiotic ligands for AhR have been identified, the AhR is considered to be an orphan receptor, because its physiological ligand(s) and its function are not known. Tryptophan and other indole-containing compounds (7-9), bilirubin (10), 7-ketocholesterol (11), lipoxin A4 (12), flavones, and related compounds (13) interact with AhR to produce activation or inhibition of signal transduction. In general, the low levels, lack of potency, and restricted distribution of these compounds make them unlikely candidates as major regulators of AhR signaling in most tissue types. We reasoned that human urine might be a good place to search for endogenous AhR ligands, and we developed a methodology to detect and isolate such compounds. EXPERIMENTAL PROCEDURESMaterials-Blue rayon was kindly provided by Dr. Hayatsu (Okayama University, Okayama City, Japan). General chemicals, essentially analytical grade, were purchased from Wako (Kyoto, Japan). TCDD was purchased from CIL (Andover, MA, USA). Indigo and ␤-glucuronidase were purchased from Sigma. Indirubin was synthesized as described in Hoessel et al. (14). Indigo and indirubin were further purified by HPLC before use.Yeast Assay for AhR Ligand Activity-The assay procedure was essentially as described by Miller (15). The yeast strain YCM3 was grown overnight at 30°C in synthetic glucose medium lacking tryptophan. Test chemicals (dissolved in Me 2 SO...

Indirubin-3′-monoxime inhibits autophosphorylation of FGFR1 and stimulates ERK1/2 activity via p38 MAPK

“…Consistent with several previous reports that indirubin-3 0 -monoxime inhibits intracellular targets other than FGFR1 (Hoessel et al, 1999;Adachi et al, 2001;Leclerc et al, 2001;Xie et al, 2004;Sethi et al, 2006), we found that in NIH/3T3 cells indirubin-3 0 -monoxime inhibited the phosphorylation of CDK2 and Rb, a CDK-regulated protein involved in regulation of cellcycle progression. However, the concentration of indirubin-3 0 -monoxime required to inhibit Rb and CDK2 phosphorylation was higher for FCS-stimulated cells than for FGF-1-stimulated cells, that is, 20 and 5 mM, respectively.…”

“…Furthermore, indirubin-3 0 -monoxime, but not indirubin, was able to inhibit CDK9 in a cell-free kinase assay and to inhibit replication of HIV-1 in blood mononuclear cells and macrophages (Heredia et al, 2005). Additionally, it was recently reported that indirubin derivatives were able to block Stat3 signaling by inhibiting the Src kinase activity and in this way induce apoptosis in human cancer cells (Nam et al, 2005), and also, bind to and inhibit glycogen synthetase kinase-3 (Leclerc et al, 2001;Polychronopoulos et al, 2004), aryl hydrocarbon receptor (Adachi et al, 2001), muscle glycogen phosphorylase b (Kosmopoulou et al, 2004) and c-Jun NH2-terminal kinase (JNK) (Xie et al, 2004). More recently, indirubin-3 0 -monoxime was found to inhibit the activation of nuclear factor-kB resulting in enhancement of apoptosis induced by tumor necrosis factors in human leukemic cells (Sethi et al, 2006).…”

The Aryl Hydrocarbon Receptor Ligand ITE Inhibits TGFβ1-Induced Human Myofibroblast Differentiation

“…23,25 Candidate endogenous ligands of the AhR include ketocholesterol, bilirubin, indigo, lipoxin A 4 , and tryptophan derivatives such as ITE. 22,[27][28][29][30][31][32] However, the AhR is betterknown for its ligation by a diverse group of xenobiotics, the most potent of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). 22 Agonist binding induces nuclear translocation of the AhR, which allows it to regulate expression of a variety of genes, including those of the cytochrome P450 family of drug-metabolizing enzymes.…”

Exploiting the Versatility of Human Cytochrome P450 Enzymes: The Promise of Blue Roses From Biotechnology

“…Recently, this story took a new twist with the report that indigo and indirubin may be potent physiological ligands of the Ah receptor (AhR), with similar potency to that of the prototypical xenobiotic ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (24). The AhR is a ligand-activated transcription factor responsible for the coordinated regulation of a battery of xenobioticmetabolizing enzymes including P450s 1A1, 1A2, and 1B1, certain UDP-glucuronosy l transferases, NADPH-quinon e reductase, and an aldehyde dehydrogenase (25).…”