Mingdong Yang

Aryl Hydrocarbon Receptor Is a Transcriptional Activator of the Human Breast Cancer Resistance Protein (BCRP/ABCG2)

Breast cancer resistance protein (BCRP/ABCG2) is a membrane-bound efflux transporter important in cellular detoxification and multidrug resistance. Some aryl hydrocarbon receptor (AHR) agonists were reported to induce BCRP expression in human colon carcinoma cells. However, a direct involvement of AHR transcriptional regulation remains unexplored. In this study, we show that BCRP induction by AHR ligands occurs in human intestinal, liver, and mammary carcinoma cells and in primary colonocytes and hepatocytes. Increased BCRP transporter activity consistent with gene induction was also evident in the Caco2 subclone C2bbe1 cells. Using RNA interference and ectopic expression techniques to manipulate cellular AHR status, we confirmed AHR dependence of ABCG2 gene regulation. By gene promoter analysis, chromatin immunoprecipitation, and electrophoretic mobility shift assays, an active, proximal dioxin-response element at −194/−190 base pairs upstream of the transcription start site of the human ABCG2 gene was identified. Despite a common observation in human-derived cells, our in vitro and in vivo studies supported by phylogenetic footprinting analysis did not find that mouse Abcg2 is subject to AHR regulation. We conclude that AHR is a direct transcriptional regulator of human BCRP and provide an unprecedented role of AHR in cellular adaptive response and cytoprotection by up-regulating an important ATP-binding cassette efflux transporter.

Activation of Nuclear Factor (Erythroid-2 Like) Factor 2 by Toxic Bile Acids Provokes Adaptive Defense Responses to Enhance Cell Survival at the Emergence of Oxidative Stress

Oxidative stress, causing necrotic and apoptotic cell death, is associated with bile acid toxicity. Using liver (HepG2, Hepa1c1c7, and primary human hepatocytes) and intestinal (C2bbe1, a Caco-2 subclone) cells, we demonstrated that toxic bile acids, such as lithocholic acid (LCA) and chenodeoxycholic acid, induced the nuclear factor (erythroid-2 like) factor 2 (Nrf2) target genes, especially the rate-limiting enzyme in glutathione (GSH) biosynthesis [glutamate cysteine ligase modulatory subunit (GCLM) and glutamate cysteine ligase catalytic subunit (GCLC)] and thioredoxin reductase 1. Nrf2 activation and induction of Nrf2 target genes were also evident in vivo in the liver of CD-1 mice treated 7 to 8 h or 4 days with LCA. Silencing of Nrf2 via small-interfering RNA suppressed basal and bile acid-induced mRNA levels of the above-mentioned genes. Consistent with this, overexpression of Nrf2 enhanced, but dominant-negative Nrf2 attenuated, Nrf2 target gene induction by bile acids. The activation of Nrf2-antioxidant responsive element (ARE) transcription machinery by bile acids was confirmed by increased nuclear accumulation of Nrf2, enhanced ARE-reporter activity, and increased Nrf2 binding to ARE. It is noteworthy that Nrf2 silencing increased cell susceptibility to LCA toxicity, as evidenced by reduced cell viability and increased necrosis and apoptosis. Concomitant with GCLC/GCLM induction, cellular GSH was significantly increased in bile acid-treated cells. Cotreatment with N-acetyl-l-cysteine, a GSH precursor, ameliorated LCA toxicity, whereas cotreatment with buthionine sulfoximine, a GSH synthesis blocker, exacerbated it. In summary, this study provides molecular evidence linking bile acid toxicity to oxidative stress. Nrf2 is centrally involved in counteracting such oxidative stress by enhancing adaptive antioxidative response, particularly GSH biosynthesis, and hence cell survival.

Participation of nuclear factor (erythroid 2-related), factor 2 in ameliorating lithocholic acid-induced cholestatic liver injury in mice

BACKGROUND AND PURPOSE Lithocholic acid (LCA), the most toxic bile acid, induces cholestatic liver injury in rodents. We previously showed that LCA activates the oxidative stress‐responsive nuclear factor (erythroid‐2 like), factor 2 (Nrf2) in cultured liver cells, triggering adaptive responses that reduce cell injury. In this study, we determined whether Nrf2 protects the liver against LCA‐induced toxicity in vivo.

Induction of cytochrome P450 1A by cow milk-based formula: a comparative study between human milk and formula.

During the treatment of neonatal apnea, formula‐fed infants, compared to breastfed infants, show nearly three‐fold increase in clearance of caffeine, a substrate of cytochrome P450 1A (CYP1A) and in part CYP3A4. However, human milk is known to contain higher concentrations of environmental pollutants than infant formula, which are potent CYP1A inducers. To gain insight into the mechanism underlying this apparent contradiction, we characterized CYP1A and CYP3A4 induction by human milk and cow milk‐based infant formula. The mRNA and protein expression of CYP1A1/1A2 were significantly induced by cow milk‐based formula, but not by human milk, in HepG2 cells. Luciferase reporter assay demonstrated that cow milk‐based formula but not human milk activated aryl hydrocarbon receptor (AhR) significantly. The cotreatment of 3,4‐dimethoxyflavone, an AhR antagonist, abolished the formula‐induced CYP1A expression. In addition, AhR activation by dibenzo[a,h]anthracene, a potent AhR agonist, was significantly suppressed by infant formula and even more by human milk. In contrast, CYP3A4 mRNA expression was only mildly induced by formula and human milk. Consistently, neither formula nor human milk substantially activated pregnane X receptor (PXR). Effects of whey and soy protein‐based formulas on the AhR–CYP1A and the PXR–CYP3A4 pathways were similar to those of cow milk‐based formula. In conclusion, infant formula, but not human milk, enhances in vitro CYP1A expression via an AhR‐mediated pathway, providing a potential mechanistic basis for the increased caffeine elimination in formula‐fed infants.

Induction of multidrug resistance transporter ABCG2 by prolactin in human breast cancer cells.

The multidrug transporter, breast cancer resistance protein, ABCG2, is up-regulated in certain chemoresistant cancer cells and in the mammary gland during lactation. We investigated the role of the lactogenic hormone prolactin (PRL) in the regulation of ABCG2. PRL dose-dependently induced ABCG2 expression in T-47D human breast cancer cells. This induction was significantly reduced by short-interfering RNA–mediated knockdown of Janus kinase 2 (JAK2). Knockdown or pharmacologic inhibition of the down-stream signal transducer and activator of transcription-5 (STAT5) also blunted the induction of ABCG2 by PRL, suggesting a role for the JAK2/STAT5 pathway in PRL-induced ABCG2 expression. Corroborating these findings, we observed PRL-stimulated STAT5 recruitment to a region containing a putative γ-interferon activation sequence (GAS) element at −434 base pairs upstream of the ABCG2 transcription start site. Introduction of a single mutation to the −434 GAS element significantly attenuated PRL-stimulated activity of a luciferase reporter driven by the ABCG2 gene promoter and 5′-flanking region containing the −434 GAS motif. In addition, this GAS element showed strong copy number dependency in its response to PRL treatment. Interestingly, inhibitors against the mitogen-activated protein kinase and phosphoinositide-3-kinase signaling pathways significantly decreased the induction of ABCG2 by PRL without altering STAT5 recruitment to the GAS element. We conclude that the JAK2/STAT5 pathway is required but not sufficient for the induction of ABCG2 by PRL.

Role of STAT5 and epigenetics in lactation-associated upregulation of multidrug transporter ABCG2 in the mammary gland

The multidrug resistance efflux transporter ATP-binding cassette subfamily G member 2 (ABCG2) is not only overexpressed in certain drug-resistant cancers but is also highly expressed in the mammary gland during lactation, carrying xenobiotics and nutrients into milk. We sought to investigate the molecular mechanisms involved in the upregulation of ABCG2 during lactation. Expression profiling of different mouse Abcg2 mRNA isoforms (E1a, E1b, and E1c) revealed that E1b is predominantly expressed and induced in the lactating mouse mammary gland. Despite this induction, analyses of CpG methylation status and published ChIP-seq datasets reveal that E1b promoter sequences in the virgin gland are already hypomethylated and marked with the open chromatin histone mark H3K4me2. Using a forced-weaning model to shut down lactation, we found that within 24 h there was a significant reduction in Abcg2 mRNA expression and a loss of signal transducer and activator of transcription-5 (STAT5) occupancy at the mouse Abcg2 gene. Luciferase reporter assays further showed that some of these STAT5-binding regions that contained interferon-γ-activated sequence (GAS) motifs function as an enhancer after prolactin treatment. We conclude that Abcg2 is already poised for expression in the virgin mammary gland and that STAT5 plays an important role in Abcg2 expression during lactation.

Riboflavin uptake transporter Slc52a2 (RFVT2) is upregulated in the mouse mammary gland during lactation

While it is well recognized that riboflavin accumulates in breast milk as an essential vitamin for neonates, transport mechanisms for its milk excretion are not well characterized. The multidrug efflux transporter ABCG2 in the apical membrane of milk-producing mammary epithelial cells (MECs) is involved with riboflavin excretion. However, it is not clear whether MECs possess other riboflavin transport systems, which may facilitate its basolateral uptake into MECs. We report here that transcripts encoding the second (SLC52A2) and third (SLC52A3) member of the recently discovered family of SLC52A riboflavin uptake transporters are expressed in milk fat globules from human breast milk. Furthermore, Slc52a2 and Slc52a3 mRNA are upregulated in the mouse mammary gland during lactation. Importantly, the induction ofSlc52a2, which was the major Slc52a riboflavin transporter in the lactating mammary gland, was also observed at the protein level. Subcellular localization studies showed that green fluorescent protein-tagged mouse SLC52A2 mainly localized to the cell membrane, with no preferential distribution to the apical or basolateral membrane in polarized kidney MDCK cells. These results strongly implicate a potential role for SLC52A2 in riboflavin uptake by milk-producing MECs, a critical step in the transfer of riboflavin into breast milk.