Souren Mkrtchian

Signal transduction-mediated activation of the aryl hydrocarbon receptor in rat hepatoma H4IIE cells.

We have investigated mechanisms of omeprazole (OME)-mediated induction of CYP1A1 and CYP3A, using the rat hepatoma H4IIE cell line, in comparison with mechanisms exerted by traditional aryl hydrocarbon receptor (AhR) ligands such as benso(a)pyrene (B(a)P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). OME did not bind specifically to AhR, and it could not activate the AhR complex in rat cytosol to a xenobiotic-responsive element (XRE)-binding formin vitro. Genistein, a tyrosine kinase inhibitor, and daidzein, an inhibitor of casein kinase II, efficiently inhibited OME-mediated but not B(a)P- or TCDD-mediated induction of CYP1A1, as monitored at the transcriptional, mRNA, and protein levels as well as by analysis of activation of XRE-luciferase reporter constructs transfected into H4IIE cells. The protease inhibitorN α-p-tosyl-l-lysine chloromethyl ketone (TLCK) and lavendustin A also had similar OME-specific effects. In addition, insulin pretreatment caused an almost complete inhibition of OME-dependent CYP1A1 induction but only partially affected TCDD and B(a)P-mediated induction of CYP1A1. Staurosporine, an inhibitor of protein kinase C, impaired the induction by both B(a)P and OME. OME caused an approximately 2-fold increase in the level of CYP3A expression, but all inhibitors used were ineffective in preventing this induction. Gel shift analysis with radiolabeled XRE and specific peptide antibodies toward AhR and aryl hydrocarbon receptor nuclear translocator protein (Arnt) revealed an OME-mediated translocation of the AhR·Arnt complex into the nuclei. Genistein inhibited the specific nuclear XRE binding caused by OME, but it potentiated the formation of the TCDD-induced XRE·AhR complex. Although daidzein was able to effectively inhibit the OME-stimulatedCYP1A1 gene transcription, it did not influence the OME-dependent AhR·XRE complex formation. The data are consistent with a mechanism for OME-mediated induction of CYP1A1 that involves activation of the AhR complex via intracellular signal transduction systems and that is distinct from induction mediated by AhR ligands.

Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease

Liver biology and function, drug-induced liver injury (DILI) and liver diseases are difficult to study using current in vitro models such as primary human hepatocyte (PHH) monolayer cultures, as their rapid de-differentiation restricts their usefulness substantially. Thus, we have developed and extensively characterized an easily scalable 3D PHH spheroid system in chemically-defined, serum-free conditions. Using whole proteome analyses, we found that PHH spheroids cultured this way were similar to the liver in vivo and even retained their inter-individual variability. Furthermore, PHH spheroids remained phenotypically stable and retained morphology, viability, and hepatocyte-specific functions for culture periods of at least 5 weeks. We show that under chronic exposure, the sensitivity of the hepatocytes drastically increased and toxicity of a set of hepatotoxins was detected at clinically relevant concentrations. An interesting example was the chronic toxicity of fialuridine for which hepatotoxicity was mimicked after repeated-dosing in the PHH spheroid model, not possible to detect using previous in vitro systems. Additionally, we provide proof-of-principle that PHH spheroids can reflect liver pathologies such as cholestasis, steatosis and viral hepatitis. Combined, our results demonstrate that the PHH spheroid system presented here constitutes a versatile and promising in vitro system to study liver function, liver diseases, drug targets and long-term DILI.

Identification of ERp29, an Endoplasmic Reticulum Lumenal Protein, as a New Member of the Thyroglobulin Folding Complex

Folding and post-translational modification of the thyroid hormone precursor, thyroglobulin (Tg), in the endoplasmic reticulum (ER) of the thyroid epithelial cells is facilitated by several molecular chaperones and folding enzymes, such as BiP, GRP94, calnexin, protein disulfide isomerase, ERp72, and others. They have been shown to associate simultaneously and/or sequentially with Tg in the course of its maturation, thus forming large heterocomplexes in the ER of thyrocytes. Here we present evidence that such complexes include a novel member, an ER-resident lumenal protein, ERp29, which is present in all mammalian tissues with exceptionally high levels of expression in the secretory cells. ERp29 was induced upon treatment of FRTL-5 rat thyrocytes with the thyroid-stimulating hormone, which is essential for the maintenance of thyroid cells and Tg biosynthesis. Chemical cross-linking followed by the cell lysis and immunoprecipitation of ERp29 or Tg revealed association of these proteins and additionally, immunocomplexes that also included major ER chaperones, BiP and GRP94. Sucrose density gradient analysis indicated co-localization of ERp29 with Tg and BiP in the fractions containing large macromolecular complexes. This was supported by immunofluorescent microscopy showing co-localization of ERp29 with Tg in the putative transport vesicular structures. Affinity chromatography using Tg as an affinity ligand demonstrated that ERp29 might be selectively isolated from the FRTL-5 cell lysate or purified lumenal fraction of rat liver microsomes along with the other ER chaperones. Preferential association with the urea-denatured Tg-Sepharose was indicative of either direct or circuitous ERp29/Tg interactions in a chaperone-like manner. Despite the presence of the C-terminal ER-retrieval signal, significant amounts of ERp29 were also recovered from the culture medium of stimulated thyrocytes, indicating ERp29 secretion. Based on these data, we suggest that the function of ERp29 in thyroid cells is connected with folding and/or secretion of Tg.

Thioredoxin Fold as Homodimerization Module in the Putative Chaperone ERp29: NMR Structures of the Domains and Experimental Model of the 51 kDa Dimer

BACKGROUND ERp29 is a ubiquitously expressed rat endoplasmic reticulum (ER) protein conserved in mammalian species. Fold predictions suggest the presence of a thioredoxin-like domain homologous to the a domain of human protein disulfide isomerase (PDI) and a helical domain similar to the C-terminal domain of P5-like PDIs. As ERp29 lacks the double-cysteine motif essential for PDI redox activity, it is suggested to play a role in protein maturation and/or secretion related to the chaperone function of PDI. ERp29 self-associates into 51 kDa dimers and also higher oligomers. RESULTS 3D structures of the N- and C-terminal domains determined by NMR spectroscopy confirmed the thioredoxin fold for the N-terminal domain and yielded a novel all-helical fold for the C-terminal domain. Studies of the full-length protein revealed a short, flexible linker between the two domains, homodimerization by the N-terminal domain, and the presence of interaction sites for the formation of higher molecular weight oligomers. A gadolinium-based relaxation agent is shown to present a sensitive tool for the identification of macromolecular interfaces by NMR. CONCLUSIONS ERp29 is the first eukaryotic PDI-related protein for which the structures of all domains have been determined. Furthermore, an experimental model of the full-length protein and its association states was established. It is the first example of a protein where the thioredoxin fold was found to act as a specific homodimerization module, without covalent linkages or supporting interactions by further domains. A homodimerization module similar as in ERp29 may also be present in homodimeric human PDI.

Expression of CYP2W1 in colon tumors: regulation by gene methylation.

INTRODUCTION CYP2W1 is a novel enzyme shown to be selectively expressed in rat fetal colon and in human colon cancer and has previously been suggested as a potential drug target for cancer therapy. Here, the expression and gene methylation of CYP2W1 were analyzed in human colon carcinoma cell lines, colon tumors and in corresponding normal colon tissue. METHODS CYP2W1 mRNA and protein expression in HepG2 and Caco-2TC7 cells and normal colon and colon tumor tissue samples were analyzed using real-time PCR and Western blotting. CYP2W1 gene methylation status in the same samples was analyzed using the sodium bisulfite sequencing method. RESULTS & DISCUSSION CYP2W1 mRNA was detected in all (n = 39) tumor samples analyzed. Moreover, in 60% (12/20) of the colon tumors, CYP2W1 mRNA levels were substantially higher than in corresponding normal tissues. CYP2W1 protein was detected in most of the colon tumor samples analyzed (n = 16), which appeared to be of two apparent phenotypes: those with five- to ten-fold induced CYP2W1 (approximately 50% of the tumors), and those with low expression, harboring similar or only slightly higher amounts of CYP2W1 as compared with surrounding control tissue. Methylation analysis of the CpG island in the exon 1-intron 1 junction of the CYP2W1 gene from both cell lines, tumors and normal tissues revealed that demethylated CpG dinucleotides appeared as a requirement for high CYP2W1 gene expression. CONCLUSION The expression of CYP2W1 is colon tumor-specific and is associated with methylation status of the CYP2W1 gene, suggesting a potential causal link between the gene hypomethylation and its enhanced expression.

The human carotid body transcriptome with focus on oxygen sensing and inflammation – a comparative analysis

•  The carotid body (CB) is the key oxygen sensor and governs the ventilatory response to hypoxia. •  CB oxygen sensing and signalling gene expression is well described in animals whereas human data are absent. •  Here we have characterized the human CB global gene expression in comparison with functionally related tissues and mouse CB gene expression. •  We show that the human CB expresses oxygen sensing genes in common with mice but also differs on key genes such as certain K+ channels. There is moreover increased expression of inflammatory response genes in human and mouse CBs in comparison with related tissues. •  The study establishes similarities but also important differences between animal and human CB gene expression profiles and provides a platform for future functional studies on human CBs.

Regulation of CYP2C19 Expression by Estrogen Receptor α: Implications for Estrogen-Dependent Inhibition of Drug Metabolism

Cytochrome P4502C19 (CYP2C19) is an important drug-metabolizing enzyme involved in the biotransformation of, for example, proton pump inhibitors and antidepressants. Several in vivo studies have shown that the CYP2C19 activity is inhibited by oral contraceptives, which can cause important drug interactions. The underlying molecular mechanism has been suggested to be competitive inhibition. However, the results presented here indicate that estradiol derivatives down-regulate CYP2C19 expression via estrogen receptor (ER) α, which interacts with the newly identified ER-binding half site [estrogen response element (ERE)] at the position −151/−147 in the CYP2C19 promoter. In gene reporter experiments in Huh-7 hepatoma cells, the activity of the luciferase construct carrying a 1.6-kb long CYP2C19 promoter fragment cotransfected with ERα was down-regulated upon treatment with 17β-estradiol (EE) or 17α-ethinylestradiol (ETE) at half-maximum concentrations of 10−7 and 10−8 M, respectively. Mutations introduced into the ERE half site −151/−147 significantly inhibited these ligand-dependent effects. Electrophoretic mobility shift assays and quantitative chromatin immunoprecipitation experiments revealed that estrogen receptor α binds to this element. A significant suppression of CYP2C19 transcription by female sex steroids was confirmed by reverse transcription polymerase chain reaction after hormonal treatment of human hepatocytes. Inhibition experiments using a stable human embryonic kidney 293 CYP2C19 cell line revealed competitive inhibition at much higher concentrations of EE and ETE compared with those required for transcriptional inhibition. These results indicate that both EE and ETE inhibit CYP2C19 expression via an ERα-dependent regulatory pathway, thus providing a new insight into the molecular mechanism behind the inhibitory effect of oral contraceptives on CYP2C19 activity.

The human carotid body: expression of oxygen sensing and signaling genes of relevance for anesthesia.

Background:Hypoxia is a common cause of adverse events in the postoperative period, where respiratory depression due to residual effects of drugs used in anesthesia is an important underlying factor. General anesthetics and neuromuscular blocking agents reduce the human ventilatory response to hypoxia. Although the carotid body (CB) is the major oxygen sensor in humans, critical oxygen sensing and signaling pathways have been investigated only in animals so far. Thus, the aim of this study was to characterize the expression of key genes and localization of their products involved in the human oxygen sensing and signaling pathways with a focus on receptor systems and ion channels of relevance in anesthesia. Methods:Six CBs were removed unilaterally from patients undergoing radical neck dissection. The gene expression and cell-specific protein localization in the CBs were investigated with DNA microarrays, real-time polymerase chain reaction, and immunohistochemistry. Results:We found gene expression of the oxygen-sensing pathway, heme oxygenase 2, and the K+ channels TASK (TWIK-related acid sensitive K+ channel)-1 and BK (large-conductance potassium channel). In addition, we show the expression of critical receptor subunits such as &ggr;-aminobutyric acid A (&agr;2, &bgr;3, and &ggr;2), nicotinic acetylcholine receptors (&agr;3, &agr;7, and &bgr;2), purinoceptors (A2A and P2X2), and the dopamine D2 receptor. Conclusions:In unique samples of the human CB, we here demonstrate presence of critical proteins in the oxygen-sensing and signaling cascade. Our findings demonstrate similarities to, but also important differences from, established animal models. In addition, our work establishes an essential platform for studying the interaction between anesthetic drugs and human CB chemoreception.

Colorectal cancer-specific cytochrome P450 2W1: intracellular localization, glycosylation, and catalytic activity.

Cytochrome P450 2W1 (CYP2W1) is expressed at high levels in colorectal cancer cells. Moreover, we have shown previously that a higher tumor expression is associated with less survival. In this study, we characterize post-translational modification, inverted endoplasmic reticulum (ER) topology, and catalytic activity of CYP2W1. The analysis of colorectal normal and cancer tissues and CYP2W1 overexpressing human embryonic kidney (HEK) 293 cells showed that a fraction of CYP2W1 is modified by N-glycosylation. Bioinformatic analysis identified Asn177 as the only possible glycosylation site of CYP2W1, which was supported by the inability of an N177A mutant to be glycosylated in HEK 293 cells. Analysis of the membrane topology indicated that unlike other cytochromes P450, CYP2W1 in HEK 293-transfected cells and in nontransfected Caco2TC7 and HepG2 cells is oriented toward the lumen of the ER, a topology making CYP2W1 available to the ER glycosylation machinery. Immunofluorescence microscopy and cell surface biotinylation experiments revealed approximately 8% of the CYP2W1 on the cell surface. Despite the reverse orientation of CYP2W1 in the ER membrane, apparently making functional interactions with NADPH-cytochrome P450 reductase impossible, CYP2W1 in HEK 293 cells was active in the metabolism of indoline substrates and was able to activate aflatoxin B1 into cytotoxic products. The study identifies for the first time a cytochrome P450 enzyme with a luminal ER orientation and still retaining catalytic activity. Together, these results suggest the possibility of using CYP2W1 as a drug target in the treatment of colon cancer using antibodies and/or specific CYP2W1 activated prodrugs.

Massive rearrangements of cellular MicroRNA signatures are key drivers of hepatocyte dedifferentiation

Hepatocytes are dynamic cells that, upon injury, can alternate between nondividing differentiated and dedifferentiated proliferating states in vivo. However, in two‐dimensional cultures, primary human hepatocytes (PHHs) rapidly dedifferentiate, resulting in loss of hepatic functions that significantly limits their usefulness as an in vitro model of liver biology, liver diseases, as well as drug metabolism and toxicity. Thus, understanding the underlying mechanisms and stalling of the dedifferentiation process would be highly beneficial to establish more‐accurate and relevant long‐term in vitro hepatocyte models. Here, we present comprehensive analyses of whole proteome and transcriptome dynamics during the initiation of dedifferentiation during the first 24 hours of culture. We report that early major rearrangements of the noncoding transcriptome, hallmarked by increased expression of small nucleolar RNAs, long noncoding RNAs, microRNAs (miRNAs), and ribosomal genes, precede most changes in coding genes during dedifferentiation of PHHs, and we speculated that these modulations could drive the hepatic dedifferentiation process. To functionally test this hypothesis, we globally inhibited the miRNA machinery using two established chemically distinct compounds, acriflavine and poly‐l‐lysine. These inhibition experiments resulted in a significantly impaired miRNA response and, most important, in a pronounced reduction in the down‐regulation of hepatic genes with importance for liver function. Thus, we provide strong evidence for the importance of noncoding RNAs, in particular, miRNAs, in hepatic dedifferentiation, which can aid the development of more‐efficient differentiation protocols for stem‐cell‐derived hepatocytes and broaden our understanding of the dynamic properties of hepatocytes with respect to liver regeneration. Conclusion: miRNAs are important drivers of hepatic dedifferentiation, and our results provide valuable information regarding the mechanisms behind liver regeneration and possibilities to inhibit dedifferentiation in vitro. (Hepatology 2016;64:1743‐1756)