04 Fakultät Energie-, Verfahrens- und Biotechnik
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Item Open Access Role of inflammatory cytokine signaling in the regulation of detoxifying functions in human hepatocytes and liver(2014) Klein, Marcus; Zanger, Ulrich (Prof. Dr.)During inflammation, circulating pro-inflammatory cytokines such as TNFα, IL-1ß, and IL-6, which are produced by, e.g., Kupffer cells, macrophages, or tumor cells, play important roles in hepatocellular signalling pathways and in the regulation of cellular homeostasis. In particular, these cytokines are responsible for the acute phase response (APR) but also for a dramatic reduction of drug detoxification capacity due to impaired expression of numerous genes coding for drug metabolic enzymes and transporters (DMETs). Several pathways are known to be activated by IL-6 such as the JAK/STAT, MAPK/ERK, and PI3K/AKT pathways. Earlier work by others has shown that downregulation of CYP3A4 is independent of the JAK/STAT and MAPK/ERK pathways. However, there is evidence that MAPKs are able to phosphorylate nuclear receptors (NRs) such as RXR-α, which alters their function. Moreover, AKT, downstream of PI3K, may induce nuclear translocation of NF-κB which antagonizes RXR-α and other NRs. RXR-α, which heterodimerizes with subfamily 1 NRs (e.g., CAR and PXR), is an important regulator of detoxifying functions in liver. Inhibition of RXR-α or other NRs could therefore explain the simultaneous downregulation of large gene batteries including many DMET genes. The contributing signaling events and mechanisms remained, however, largely unexplained. Therefore, the major focus of this thesis was the investigation of the impact of the major inflammatory mediator IL-6 on the regulation of detoxifying functions in human liver. For this purpose, a large-scale investigation of DMET gene expression changes in IL-6-stimulated primary human hepatocytes (PHH) was carried out. Many important DMET genes were found to be downregulated in response to IL-6 stimulation of PHH. Most significantly suppressed were genes coding for cytochrome P450s (e.g., CYP1A2, 2C9, 2D6, and 3A4) and ATP-binding cassette (e.g., ABCB1 and ABCC2) and solute carrier (e.g., SLC10A1 and SLCO1B1) drug transporters. The average phase II metabolism gene expression appeared to be only moderately affected by IL-6, showing much stronger variability in gene expression, including genes with a trend towards upregulation (SULTs). Most notably, CYPs appeared to be highly downregulated in a coordinated fashion, demonstrating the broad suppressive potency of IL-6 towards this particular family of drug metabolizing enzymes (DMEs). Moreover, determination of metabolite formation rates in IL-6-treated PHH revealed impaired metabolic functionality of the major CYP isoenzymes CYP1A2, 2B6, 2C8, 2C9, 2C19, and 3A4. Therefore, it was shown that IL-6 signaling extensively interferes with drug detoxification capacity in human hepatocytes. Phosphoprotein analyses revealed activation of the JAK/STAT, MAPK, and PI3K cascades by IL-6. Whereas individual chemical inhibition of the two latter pathways attenuated many IL-6-mediated effects on DMET gene expression, co-inhibition almost completely abolished these effects. Inhibition of JAK/STAT signaling barely affected IL-6-mediated effects. Notably, activation of PI3K and knock-down (KD) of RXR-α demonstrated strikingly similar DMET gene expression patterns compared to IL-6 stimulation. Therefore, these data indicated a MAPK/ERK- and PI3K/AKT-dependent but JAK/STAT-independent downregulation of DMET genes in response to IL-6, possibly via interference with RXR-α. In conclusion, these data suggest that MAPKs and AKT-activated NF-κB antagonize NR signaling, causing a coordinated downregulation of DMET genes. The investigation of sensitive regulatory mechanisms is complicated by the interindividual variability of PHH. The human hepatocellular carcinoma derived HepaRG cell line has been shown to retain many functional characteristics of PHH, including the expression of functional DMETs, but the influence of inflammation has not been investigated so far. Thus, HepaRG cells were tested for their robustness and suitability in studying the inflammation-mediated impact on the drug detoxification capacity in human liver. Indeed, IL-6 stimulation of HepaRG cells led to highly induced expression of acute phase (AP) genes (e.g., CRP) and significantly repressed DMET gene expression in a coordinated fashion. The selectivity and magnitude of these effects were strikingly similar to those observed in IL-6-exposed PHH, with only few exceptions (e.g., CYP2E1 and SULTs). This was further supported by a strong positive correlation of IL-6-mediated expression changes of DMET and critical modifier genes in both cell models. Moreover, decreased protein expression and activity of major P450s could be determined in HepaRG cells, comparable to PHH. Exposure of HepaRG cells to different cytokines resulted in moderately different gene expression patterns, indicating specific responsiveness to particular pro-inflammatory cytokines. These data indicate that HepaRG cells retain the regulatory mechanisms that are responsible for the downregulation of the liver’s drug detoxification capacity during inflammation. This cell line may therefore provide a good alternative model for detailed mechanistic analyses during such conditions. The inflammation-mediated transcriptional changes that have major effects on drug detoxification in the liver have not been analyzed on a transcriptome-wide scale so far. Therefore, the last part of this work focused on the unbiased assessment of genome-wide transcriptional changes in response to inflammatory signaling in the human liver. For this purpose, microarray analysis was carried out in IL-6-stimulated PHH and compared to transcriptome data, previously acquired in samples from a liver cohort, including patients having undergone an APR (elevated CRP). Remarkably, major human-relevant CYPs, 2C8, 3A4, and 2A6 were the most strongly downregulated genes in IL-6-challenged PHH. Their transcription was at least 4-fold repressed. A total of 40 DMET genes were identified as significantly altered, of which 30 were downregulated, including almost all transcripts of major CYPs of importance in humans (e.g., 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 3A4 and 3A5), phase II drug metabolizing enzymes (e.g., GSTAs, SULTs and UGTs), and drug transporters (ABCs and SLCs). In liver samples from patients with elevated CRP, 29 DMET genes were downregulated including important genes coding for phase I/II drug metabolizing enzymes (e.g., ADHs, ALDHs, CYPs, GSTs and UGTs) and drug transporters (e.g., ABCG2 and SLCs). In both studies, gene term enrichment analyses indicated a very strong influence on xenobiotic metabolic and related processes, containing mostly downregulated DMET genes. Moreover, pathway enrichment (KEGG) analyses revealed that drug and xenobiotic metabolic signaling pathways were the most strongly impacted reaction networks, clearly demonstrating that the drug detoxification system in the liver is largely affected during inflammation. Gene annotation analysis also identified enriched processes related to diverse lipid metabolic processes such as fatty-acid and steroid metabolism. Moreover, enriched biological processes and regulatory pathways related to amino acid metabolism were found, particularly in the retrospective study. The data indicated a conservation and allocation of specific amino acids, possibly in favor of acute phase protein (APP) synthesis. Taken together, these findings highlight the scale on which the human liver transcriptome is affected during inflammation. Extensive reorganization related to xenobiotic, lipid, and amino acid metabolism takes place. It appears that the liver devotes its transcriptional machinery to the immune response while other major liver functions are shut down. This may help to pave the way towards a better understanding of how the liver organizes its many responsibilities in different conditions.Item Open Access Regulation of the BH3-only protein NOXA in mantle cell lymphoma : relevance for novel treatment concepts(2014) Dengler, Michael; Scheurich, Peter (Prof. Dr.)Mantle cell lymphoma (MCL) is an aggressive form of non-Hodgkin lymphoma and characterized by only transient responses to chemotherapy and relatively short survival. Extensive research is ongoing to improve current therapy and identify novel targets for treatment of this B cell malignancy. The central role of BH3-only proteins in mediating the response of tumor cells to anticancer drugs has been shown by many studies. The present work aimed at the investigation of the role of the BH3-only protein NOXA in the decision between life and death in MCL. Astonishingly, basal NOXA mRNA and NOXA protein expression levels were found in this study to be extremely discrepant in MCL cell lines as well as primary cells from MCL patients. The malignant B cells express very high constitutive levels of NOXA mRNA. In strong contrast, NOXA protein levels are very low in MCL cell lines and even hardly detectable in primary MCL cells. It could be demonstrated in this study that chronic active B-cell receptor (BCR)-signaling and Cyclin D1 overexpression, the hallmark of MCL, contribute to the maintenance of the high constitutive transcript levels of the pro-apoptotic Bcl-2 family member. Furthermore, the phoshatidyl-inositol-3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway was determined as the major mediator responsible for the high NOXA mRNA expression downstream of the BCR and surprisingly also Cyclin D1. Overexpression of the cell cycle regulatory protein Cyclin D1 contributes to enhanced NOXA transcription by exerting a positive feedback loop on the PI3K/AKT/mTOR signaling. Increased expression of the pro-apoptotic Bcl-2 family member NOXA is generally implicated in induction of cell death. Intriguingly, the high basal transcript levels of the BH3-only protein do not impair viability of MCL cells. The malignant B cells adapt to this permanent pro-apoptotic signal by extensive polyubiquitination and rapid proteasome-mediated degradation of NOXA (T½ < 30 min). Importantly, the resulting phenotype of high NOXA mRNA and low NOXA protein expression appears to constitute a druggable Achilles heel of MCL. Exposure of the cells to the proteasome inhibitor Bortezomib accumulates NOXA protein and efficiently induces apoptosis. In addition to Bortezomib, the neddylation inhibitor MLN4924 and the fatty acid synthase inhibitor Orlistat were identified in the present study as effective inducers of NOXA protein and NOXA-dependent apoptosis in MCL. Such as Bortezomib, both inhibitors target rapid NOXA protein turnover thereby stabilizing the pre-existing pool of the pro-apoptotic protein. Interestingly, in contrast to the proteasome inhibitor, MLN4924 as well as Orlistat interfere with ubiquitination of NOXA and stabilize the BH3-only protein in a proteasome independent manner. Therefore, these substances not only represent alternatives to Bortezomib treatment but may also offer new approaches to treat Bortezomib resistant MCL cells. Indeed, results from the present work demonstrate that Bortezomib resistant MCL cells were still sensitive to Orlistat and MLN4924. Remarkably, active PI3K/AKT/mTOR signaling is needed for effective accumulation of NOXA and induction of cell death upon treatment with Bortezomib, Orlistat or MLN4924 indicating that the high constitutive NOXA mRNA levels are essential for the sensitivity of MCL cells to inhibitors targeting NOXA turnover. Especially fatty acid metabolism appears to represent a very promising target for treatment of MCL. Constitutive Cyclin D1 overexpression in MCL was found to have an inhibitory effect on cellular metabolism thereby rendering MCL cells susceptible to drugs targeting the fatty acid synthase such as Orlistat. Furthermore, inhibition of fatty acid metabolism may not only constitute a new and selective strategy to kill MCL cells but also sensitize the malignant B cells to other anticancer drugs. In summary, the present study highlights a critical role of the BH3-only protein NOXA in MCL and provides evidence that the regulation of the pro-apoptotic protein might represent an Achilles heel of this aggressive tumor. Targeting the phenotype of high NOXA mRNA/low NOXA protein expression by inhibitors inferring with different steps of the ubiquitin-proteasome system may provide a novel therapeutic approach to kill MCL cells and improve clinical outcome of this lymphoid neoplasm.