Microbiota-Dependent and -Independent Production of l-Dopa in the Gut of Daphnia magna.

Host-microbiome interactions are essential for the physiological and ecological performance of the host, yet these interactions are challenging to identify. Neurotransmitters are commonly implicated in these interactions, but we know very little about the mechanisms of their involvement, especially in invertebrates. Here, we report a peripheral catecholamine (CA) pathway involving the gut microbiome of the model species Daphnia magna. We demonstrate the following: (i) tyrosine hydroxylase and Dopa (3,4-dihydroxyphenylalanine) decarboxylase enzymes are present in the gut wall; (ii) Dopa decarboxylase gene is expressed in the gut by the host, and its expression follows the molt cycle peaking after ecdysis; (iii) biologically active l-Dopa, but not dopamine, is present in the gut lumen; (iv) gut bacteria produce l-Dopa in a concentration-dependent manner when provided l-tyrosine as a substrate. Impinging on gut bacteria involvement in host physiology and ecologically relevant traits, we suggest l-Dopa as a communication agent in the host-microbiome interactions in daphnids and, possibly, other crustaceans. IMPORTANCE Neurotransmitters are commonly implicated in host-microbiome communication, yet the molecular mechanisms of this communication remain largely elusive. We present novel evidence linking the gut microbiome to host development and growth via neurotransmitter l-Dopa in Daphnia, the established model species in ecology and evolution. We found that both Daphnia and its gut microbiome contribute to the synthesis of the l-Dopa in the gut. We also identified a peripheral pathway in the gut wall, with a molt stage-dependent dopamine synthesis, linking the gut microbiome to the daphnid development and growth. These findings suggest a central role of l-Dopa in the bidirectional communication between the animal host and its gut bacteria and translating into the ecologically important host traits suitable for subsequent testing of causality by experimental studies.

A Notch-independent mechanism contributes to the induction of Hes1 gene expression in response to hypoxia in P19 cells.

Hes1 is a Notch target gene that plays a major role during embryonic development. Previous studies have shown that HIF-1α can interact with the Notch intracellular domain and enhance Notch target gene expression. In this study, we have identified a Notch-independent mechanism that regulates the responsiveness of the Hes1 gene to hypoxia. Using P19 cells we show that silencing the Notch DNA binding partner CSL does not prevent hypoxia-dependent upregulation of Hes1 expression. In contrast to CSL, knockdown of HIF-1α or Arnt expression prevents Hes1 induction in hypoxia. Deletion analysis of the Hes1 promoter identified a minimal region near the transcription start site that is still responsive to hypoxia. In addition, we show that mutating the GA-binding protein (GABP) motif significantly reduced Hes1 promoter-responsiveness to hypoxia or to HIF-1 overexpression whereas mutation of the hypoxia-responsive element (HRE) present in this region had no effect. Chromatin immunoprecipitation assays demonstrated that HIF-1α binds to the proximal region of the Hes1 promoter in a Notch-independent manner. Using the same experimental approach, the presence of GABPα and GABPß1 was also observed in the same region of the promoter. Loss- and gain-of-function studies demonstrated that Hes1 gene expression is upregulated by hypoxia in a GABP-dependent manner. Finally, co-immunoprecipitation assays demonstrated that HIF-1α but not HIF-2α is able to interact with either GABPα or GABPß1. These results suggest a Notch-independent mechanism where HIF-1 and GABP contribute to the upregulation of Hes1 gene expression in response to hypoxia.

A Molecular Mechanism To Switch the Aryl Hydrocarbon Receptor from a Transcription Factor to an E3 Ubiquitin Ligase.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is known as a mediator of toxic responses. Recently, it was shown that the AhR has dual functions. Besides being a transcription factor, it also possesses an intrinsic E3 ubiquitin ligase function that targets, e.g., the steroid receptors for proteasomal degradation. The aim of this study was to identify the molecular switch that determines whether the AhR acts as a transcription factor or an E3 ubiquitin ligase. To do this, we used the breast cancer cell line MCF7, which expresses a functional estrogen receptor alpha (ERα) signaling pathway. Our data suggest that aryl hydrocarbon receptor nuclear translocator (ARNT) plays an important role in the modulation of the dual functions of the AhR. ARNT knockdown dramatically impaired the transcriptional activation properties of the ligand-activated AhR but did not affect its E3 ubiquitin ligase function. The availability of ARNT itself is modulated by another basic helix-loop-helix (bHLH)-Per-ARNT-SIM (PAS) protein, the repressor of AhR function (AhRR). MCF7 cells overexpressing the AhRR showed lower ERα protein levels, reduced responsiveness to estradiol, and reduced growth rates. Importantly, when these cells were used to produce estrogen-dependent xenograft tumors in SCID mice, we also observed lower ERα protein levels and a reduced tumor mass, implying a tumor-suppressive-like function of the AhR in MCF7 xenograft tumors.

Consensus toxicity factors for polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls combining in silico models and extensive in vitro screening of AhR-mediated effects in human and rodent cells.

Consensus toxicity factors (CTFs) were developed as a novel approach to establish toxicity factors for risk assessment of dioxin-like compounds (DLCs). Eighteen polychlorinated dibenzo-p-dioxins, dibenzofurans (PCDD/Fs), and biphenyls (PCBs) with assigned World Health Organization toxic equivalency factors (WHO-TEFs) and two additional PCBs were screened in 17 human and rodent bioassays to assess their induction of aryl hydrocarbon receptor-related responses. For each bioassay and compound, relative effect potency values (REPs) compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin were calculated and analyzed. The responses in the human and rodent cell bioassays generally differed. Most notably, the human cell models responded only weakly to PCBs, with 3,3',4,4',5-pentachlorobiphenyl (PCB126) being the only PCB that frequently evoked sufficiently strong responses in human cells to permit us to calculate REP values. Calculated REPs for PCB126 were more than 30 times lower than the WHO-TEF value for PCB126. CTFs were calculated using score and loading vectors from a principal component analysis to establish the ranking of the compounds and, by rescaling, also to provide numerical differences between the different congeners corresponding to the TEF scheme. The CTFs were based on rat and human bioassay data and indicated a significant deviation for PCBs but also for certain PCDD/Fs from the WHO-TEF values. The human CTFs for 2,3,4,7,8-pentachlorodibenzofuran, 1,2,3,4,7,8-hexachlorodibenzofuran, 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin, and 1,2,3,4,7,8,9-heptachlorodibenzofuran were up to 10 times greater than their WHO-TEF values. Quantitative structure-activity relationship models were used to predict CTFs for untested WHO-TEF compounds, suggesting that the WHO-TEF value for 1,2,3,7,8-pentachlorodibenzofuran could be underestimated by an order of magnitude for both human and rodent models. Our results indicate that the CTF approach provides a powerful tool for condensing data from batteries of screening tests using compounds with similar mechanisms of action, which can be used to improve risk assessment of DLCs.