This month we are excited to feature an article from the lab of Ravinder Gill and others at the University of Illinois at Chicago. This study focuses on the relationship between serotonin (5-HT) and aryl hydrocarbon receptor (AhR), a heavily studied, evolutionary conserved nuclear receptor expressed throughout mammalian organ systems.The article was published 17 April 2018 in the journal Scientific Reports and includes gene expression data generated on the Mouse OneArray Whole Genome Microarray platform.
The classic AhR signaling cascade involves the activation of xenobiotic metabolism genes, such as cytochrome P450s. The authors focused on AhR activity in the intestinal epithelia, where it was recently established that AhR is involved in immune cell maintenance, the formation of lymphoid follicles, and induction of stem cell proliferation in the colon. Despite these recent discoveries, investigators are still unsure what activates AhR in these non-xenobiotic contexts within the gut. Much focus has been paid to tryptophan metabolites as endogenous activators of AhR; for this reason, the authors decided to focus on serotonin (5-HT). In the gut, 5-HT has diverse functions, and interestingly, 95% of all 5-HT in the body is synthesized in the gut.
5-HT Induces CYP1A1 Activity in the Gut
The authors first showed that 5-HT induces transcription of CYP1A1 mRNA in a dose-dependent manner. They went a step further showing that CYP1A1 was up-regulated via a transcriptional mechanism; 5-HT-induced CYP1A1 transcription was abrogated following treatment with a transcription inhibitor. They also showed that 5-HT increased the activity of the CYP1A1 enzyme in Caco-2 cells.
Next, the authors sought to determine which receptor transports 5-HT into Caco-2 cells to activate AhR. They ruled out the possibility of 5-HT receptors (5-HTR) transporting 5-HT into Caco-2 cells because an inhibitor of 5-HTR failed to block the induction of CYP1A1 by 5-HT. Taking a similar approach, they found that SERT receptors are responsible for 5-HT uptake into Caco-2 cells. Inhibition by a SERT inhibitor blocked the induction of CYP1A1 by 5-HT, and overexpression of SERT caused a more pronounced induction of CYP1A1 by 5-HT.
Interestingly, the authors found similar results when these experiments were repeated with a different gut epithelial cell line, T84, a colonic crypt-derived cell line. While CYP1A1 induction by 5-HT in T84 cells was less than in Caco-2 cells, the authors determined this was due to differences in SERT function in these two cell types.
The Role of AhR in 5-HT Induction of CYP1A1 Expression
The authors embarked on this study to show a connection between 5-HT and AhR, so the next logical step in the study was to show that 5-HT induction of CYP1A1 expression was dependent on AhR signaling. Using AhR antagonists, they showed that 5-HT induction of CYP1A1 was completely eliminated in Caco-2 cells. They confirmed this observation by knocking down AhR and ARNT (a cofactor of AhR) with siRNA constructs. Following knockdown of AhR and ARNT, the induction of CYP1A1 by 5-HT was markedly reduced. The authors also specifically showed that 5-HT was activating AhR and causing it to translocate to the nucleus.
Microarray Analysis of SERT Knockout (KO) Ileal Mucosa
Using Mouse OneArray Whole Genome Microarrays, the authors sought to validate what they had found in Caco-2 and T84 cells. To do so, they compared the transcriptome of ileal mucosa in SERT KO mice versus ileal mucosa in wildtype mice. Interestingly, Cyp1a1 was the most down-regulated gene in SERT KO mice with a 14-fold decrease in expression relative to wildtype. This result confirms the findings mentioned above – without SERT, 5-HT is not transported into the epithelia, and thus Cyp1a1 transcription is not induced. Other AhR target genes were also down-regulated in the SERT KO samples. Cyp1a1 down-regulation in SERT KO mice was confirmed with qPCR, and on the protein level via Western blotting.
Conclusion
In summary, the authors present a comprehensive mechanism whereby 5-HT activates AhR to enter the nucleus and induce expression of CYP1A1. They take a genomic approach by utilizing Mouse OneArray Microarrays, and ultimately, their work provides a better understanding of how AhR signaling is modulated during health and disease. Work on AhR is critical, given its importance as an intermediary between the environment, the intestinal microbiota, and inflammation.
At Phalanx Biotech, we are here to help take your research efforts to the next level. Please contact us if you are looking for an experienced contract research organization specializing in genomic techniques. We offer microarray services on either Agilent or our own OneArray platforms. We are also an experienced provider of NGS services, bioinformatics services, and qPCR services. No matter what you need help with, contact us, and we will be sure to elevate your research efforts.
Reference
Manzella C et al. (2018). Serotonin is an endogenous regulator of intestinal CYP1A1 via AhR. Scientific Reports 8(1): 6103.