Transcriptional activation of the haem oxygenase-1 gene by cGMP via a cAMP response element/activator protein-1 element in primary cultures of rat hepatocytes.
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ABSTRACT:
SUBMITTER: Immenschuh S
PROVIDER: S-EPMC1219672 | biostudies-other | 1998 Aug
REPOSITORIES: biostudies-other
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Project description:Nitro-fatty acid products of oxidative inflammatory reactions mediate anti-inflammatory cell signalling responses. LNO2 (nitrolinoleic acid) induces expression of HO-1 (haem oxygenase-1), an enzyme that catabolizes haem into products exhibiting potent anti-inflammatory properties. In the present manuscript, the molecular mechanisms underlying HO-1 induction by LNO2 were examined in HAEC (human aortic endothelial cells), HEK-293 (human embryonic kidney 293) cells, and in transcription factor-deficient MEF (mouse embryonic fibroblasts). LNO2 induced HO-1 expression in Nrf2 [NF-E2 (nuclear factor-erythroid 2)-related factor 2]-deficient MEF and in HEK-293 cells transfected with Nrf2-specific shRNA (small-hairpin RNA), supporting the fact that LNO2-mediated HO-1 induction can be regulated by Nrf2-independent mechanisms. LNO2 activated expression of a -4.5 kb human HO-1 promoter construct, whereas a -4.0 kb construct with deletion of 500 bp from the 5' region was unresponsive. Site-directed mutagenesis of a CRE (cAMP-response element) or of a downstream NF-E2/AP-1 (activating protein-1) element, individually, within this 500 bp region modestly reduced activation of the HO-1 promoter by LNO2. Mutations of both the CRE and the NF-E2/AP-1 site also attenuated LNO2-mediated HO-1 promoter expression, whereas the addition of a third mutation in the proximal E-box sequence completely abolished LNO2-induced HO-1 expression. Chromatin immunoprecipitation assays confirmed CREB (CRE-binding protein)-1 binding to the CRE (located at -4.0 kb) and E-box regions (located at -44 bp) of the human HO-1 promoter. A 3C (Chromosome Conformation Capture) assay of intact cells showed LNO2-induced interactions between the CRE- and E-box- containing regions. These observations indicate that regulation of human HO-1 expression by LNO2 requires synergy between CRE, AP-1 and E-box sequences and involves the participation of CREB-1.
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Project description:Haem oxygenase 1 (HO-1) is an inducible protein that plays a major protective role in conditions such as ischaemia-reperfusion injury and inflammation. In this study, we have investigated the role of haem arginate (HA) in human male subjects in the modulation of HO-1 expression and its correlation with the GT length polymorphism (GT(n)) in the promoter of the HO-1 gene.In a dose-escalation, randomized, placebo-controlled trial, seven healthy male subjects with a homozygous short (S/S) and eight with a long (L/L) GT(n) genotype received intravenous HA. HO-1 protein expression and mRNA levels in peripheral blood monocytes, bilirubin, haptoglobin, haemopexin and haem levels were analysed over a 48?h observation period.We found that the baseline mRNA levels of HO-1 were higher in L/L subjects, while protein levels were higher in S/S subjects. HA induced a dose-dependent increase in the baseline corrected area under the curve values of HO-1 mRNA and protein over 48?h. The response of HO-1 mRNA was more pronounced in L/L subjects but the protein level was similar across the groups.HA is an effective inducer of HO-1 in humans irrespective of the GT(n) genotype. The potential therapeutic application of HA needs to be evaluated in clinical trials.
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Project description:Cyanide is a well known potent inhibitor of haem proteins, including haem oxygenase (HO). Generally, cyanide coordinates to the ferric haem iron with a linear binding geometry; the Fe-C-N angle ranges from 160 to 180 degrees . The Fe-C-N angle observed in the crystal structure of haem-HO bound to cyanide prepared at alkaline pH was 166 degrees . Here, it is reported that cyanide can bind to the haem iron in HO in a bent mode when the ternary complex is prepared at neutral pH; a crystal structure showed that the Fe-C-N angle was bent by 47 degrees . Unlike the ternary complex prepared at alkaline pH, in which the haem group, including the proximal ligand and the distal helix, was displaced upon cyanide binding, the positions of the haem group and the distal helix in the complex prepared at neutral pH were nearly identical to those in haem-HO. Cyanide that was bound to haem-HO with a bent geometry was readily photodissociated, whereas that bound with a linear geometry was not photodissociated. Thus, alternative cyanide-binding modes with linear and bent geometries exist in the crystalline state of haem-HO.
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Project description:The present studies were undertaken to determine whether the interaction between cAMP-dependent and insulin-dependent pathways in primary cultures of rat hepatocytes affects biological functions and tyrosine phosphorylation. Quiescent hepatocytes were pretreated with dibutyryl cAMP or cAMP-generating agents such as glucagon, and then treated or not with insulin. Preincubation for 6 h with dibutyryl cAMP or glucagon enhanced the effect of insulin on DNA synthesis, but not the effect of insulin on amino acid transport or glycogen and protein synthesis. Tyrosine phosphorylation of intracellular proteins was determined by immunoblot analysis using an anti-phosphotyrosine antibody. Maximum tyrosine phosphorylation of a 195 kDa protein, which may be a substrate of insulin receptor kinase, of 175-180 kDa proteins, including insulin receptor substrate (IRS)-1, and of 90-95 kDa proteins, including the insulin receptor beta-subunit, was reached within 30 s of incubation with insulin. Pretreatment for about 3 h with dibutyryl cAMP or cAMP-generating agents clearly increased insulin-dependent tyrosine phosphorylation of the 195 kDa protein, but not IRS-1, IRS-2 or the insulin receptor beta-subunit. Because dibutyryl cAMP and cAMP-generating agents did not increase insulin receptor number or its kinase activity, the effect of cAMP on this potentiation of tyrosine phosphorylation is assumed to be exerted at a step distal to insulin receptor kinase activation. The potentiation by cAMP pretreatment of insulin-stimulated tyrosine phosphorylation may in part be secondary to inhibition of phosphotyrosine phosphatase activity, because cAMP pretreatment blunted the effect of Na3VO4 on the net tyrosine phosphorylation of the 195 kDa protein as compared with cells pretreated with no additive. In summary, the interactions between cAMP-dependent and insulin-dependent pathways that lead to augmentation of DNA synthesis appear to parallel the changes in tyrosine phosphorylation. Further studies will be required to determine whether there is a causal relationship between these phenomena.
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Project description:Bacterial sepsis is characterized by a rapid increase in the expression of inflammatory mediators to initiate the acute phase response in liver. Inflammatory mediator release is counterbalanced by the coordinated expression of anti-inflammatory molecules such as interleukin 1 receptor antagonist (IL1-Ra) through time. This study determined whether activation of pregnane X receptor (PXR, NR1I2) alters the lipopolysaccharide (LPS)-inducible gene expression program in primary cultures of hepatocytes (PCHs). Preactivation of PXR for 24 hours in PCHs isolated from wild-type mice suppressed the subsequent LPS-inducible expression of the key inflammatory mediators interleukin 1? (IL-1?), interleukin 6 (IL-6), and tumor necrosis factor ? (TNF?) but not in PCHs isolated from Pxr-null (PXR-knockout [KO]) mice. Basal expression of key inflammatory cytokines was elevated in PCHs from PXR-KO mice. Stimulation of PCHs from PXR-KO mice with LPS alone produced enhanced levels of IL-1? when compared with wild-type mice. Experiments performed using PCHs from both humanized-PXR transgenic mice as well as human donors indicate that prolonged activation of PXR produces an increased secretion of IL1-Ra from cells through time. Our data reveal a working model that describes a pivotal role for PXR in both inhibiting as well as in resolving the inflammatory response in hepatocytes. Understanding the molecular details of how PXR is converted from a positive regulator of drug-metabolizing enzymes into a transcriptional suppressor of inflammation in liver will provide new pharmacologic strategies for modulating inflammatory-related diseases in the liver and intestine.
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Project description:The liver is the primary source of a large number of plasma proteins and plays a critical role in multiple biological processes. Inadequate oxygen supply characterizing various clinical settings such as liver transplantation exposes the liver to hypoxic conditions. Studies assessing hypoxia-induced global translational changes in liver are lacking. Here, we employed a recently developed ribosome-profiling technique to assess global translational responses of human primary hepatocytes exposed to acute hypoxic stress (1% O2) for the short term. In parallel, transcriptome profiling was performed to assess mRNA expression changes. We found that translational responses appeared earlier and were predominant over transcriptional responses. A significant decrease in translational efficiency of several ribosome genes indicated translational inhibition of new ribosome protein synthesis in hypoxia. Pathway enrichment analysis highlighted altered translational regulation of MAPK signaling, drug metabolism, oxidative phosphorylation, and nonalcoholic fatty liver disease pathways. Gene Ontology enrichment analysis revealed terms related to translation, metabolism, angiogenesis, apoptosis, and response to stress. Transcriptional induction of genes encoding heat shock proteins was observed within 30 min of hypoxia. Induction of genes encoding stress response mediators, metabolism regulators, and proangiogenic proteins was observed at 240 min. Despite the liver being the primary source of coagulation proteins and the implicated role of hypoxia in thrombosis, limited differences were observed in genes encoding coagulation-associated proteins. Overall, our study demonstrates the predominance of translational regulation over transcription and highlights differentially regulated pathways or biological processes in short-term hypoxic stress responses of human primary hepatocytes. NEW & NOTEWORTHY The novelty of this study lies in applying parallel ribosome- and transcriptome-profiling analyses to human primary hepatocytes in hypoxia. To our knowledge, this is the first study to assess global translational responses using ribosome profiling in hypoxic hepatocytes. Our results demonstrate the predominance of translational responses over transcriptional responses in early hepatic hypoxic stress responses. Furthermore, our study reveals multiple pathways and specific genes showing altered regulation in hypoxic hepatocytes.
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