Techniques to measure nucleic acid-protein binding and specificity. Nuclear extract preparations, DNase I footprinting, and mobility shift assays.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
D A Brenner
Publications and source records attributed to D A Brenner.
Long-term alcohol exposure changes sensitivity of rat Kupffer cells to lipopolysaccharide.
BACKGROUND: Chronic ethanol treatment enhances Kupffer cell sensitivity to lipopolysaccharide (LPS). In this model, CD14 in Kupffer cells was increased significantly 4 weeks after ethanol. Moreover, it was shown that prostaglandin E2 produced by activated Kupffer cells participated in the mechanism of ethanol-induced fatty liver. This study was designed to elucidate the temporal effect of chronic ethanol exposure on Kupffer cell sensitization to LPS. METHODS: Rats were given ethanol every 24 hr intragastrically for up to 12 weeks, and Kupffer cells were isolated 24 hr after the final ethanol administration and cultured in RPMI 1640 with 10% fetal bovine serum. After addition of LPS to Kupffer cells, intracellular calcium ([Ca2+]i) was measured. RESULTS: CD14 in Kupffer cells was increased approximately 2-fold, and then it decreased and returned to control levels. The LPS-induced increases in [Ca2+]i and tumor necrosis factor-alpha by Kupffer cells were also increased approximately 3-fold over control values, but they also returned to control levels. Triglyceride content increased with the duration of chronic ethanol treatment. At 8 weeks, prostaglandin E2 produced by Kupffer cells increased approximately 3-fold over control values and triglycerides by approximately 4-fold before gradually decreasing to basal levels. After 12 weeks of ethanol exposure, LPS-induced increases in [Ca2+]i and tumor necrosis factor-alpha production were only approximately 50% as high as peak levels at 4 weeks. Liver triglyceride content at 12 weeks was reduced significantly compared with values at 8 weeks. CONCLUSIONS: Kupffer cells at the early stage of chronic ethanol exposure exhibited sensitization to LPS, but this sensitivity was blunted later. This correlated with triglyceride accumulation in the liver. These data indicate that long-term alcohol exposure changes the sensitivity of rat Kupffer cells to LPS but that the magnitude of the effect is time dependent.
Animals↗
Characterization of the interaction between alphaCP(2) and the 3'-untranslated region of collagen alpha1(I) mRNA.
Activated hepatic stellate cells produce increased type I collagen in hepatic fibrosis. The increase in type I collagen protein results from an increase in mRNA levels that is mainly mediated by increased mRNA stability. Protein-RNA interactions in the 3'-UTR of the collagen alpha1(I) mRNA correlate with stabilization of the mRNA during hepatic stellate cell activation. A component of the binding complex is alphaCP(2). Recombinant alphaCP(2) is sufficient for binding to the 3'-UTR of collagen alpha1(I). To characterize the binding affinity of and specificity for alphaCP(2), we performed electrophoretic mobility shift assays using the poly(C)-rich sequence in the 3'-UTR of collagen alpha1(I) as probe. The binding affinity of alphaCP(2) for the 3'-UTR sequence is approximately 2 nM in vitro and the wild-type 3' sequence binds with high specificity. Furthermore, we demonstrate a system for detecting protein-nucleotide interactions that is suitable for high throughput assays using molecular beacons. Molecular beacons, developed for DNA-DNA hybridization, are oligonucleotides with a fluorophore and quencher brought together by a hairpin sequence. Fluorescence increases when the hairpin is disrupted by binding to an antisense sequence or interaction with a protein. Molecular beacons displayed a similar high affinity for binding to recombinant alphaCP(2) to the wild-type 3' sequence, although the kinetics of binding were slower.
3' Untranslated Regions↗
The focal adhesion kinase suppresses transformation-associated, anchorage-independent apoptosis in human breast cancer cells. Involvement of death receptor-related signaling pathways.
The focal adhesion kinase (FAK) is a mediator of cell-extracellular matrix signaling events and is overexpressed in tumor cells. In order to rapidly down-regulate FAK function in normal and transformed mammary cells, we have used adenoviral gene transduction of the carboxyl-terminal domain of FAK (FAK-CD). Transduction of adenovirus containing FAK-CD in breast cancer cells caused loss of adhesion, degradation of p125(FAK), and induced apoptosis. Furthermore, breast tumor cells that were viable without matrix attachment also underwent apoptosis upon interruption of FAK function, demonstrating that FAK is a survival signal in breast tumor cells even in the absence of matrix signaling. In addition, both anchorage-dependent and anchorage-independent apoptotic signaling required Fas-associated death domain and caspase-8, suggesting that a death receptor-mediated apoptotic pathway is involved. Finally, FAK-CD had no effect on adhesion or viability in normal mammary cells, despite the loss of tyrosine phosphorylation of p125(FAK). These results indicate that FAK-mediated signaling is required for both cell adhesion and anchorage-independent survival and the disruption of FAK function involves the Fas-associated death domain and caspase-8 apoptotic pathway.
Apoptosis↗
Analysis of ferrochelatase expression during hematopoietic development of embryonic stem cells.
Ferrochelatase, the last enzyme in the heme pathway, chelates protoporphyrin IX and iron to form heme and is mutated in protoporphyria. The ferrochelatase gene is expressed in all tissues at low levels to provide heme for essential heme-containing proteins and is up-regulated during erythropoiesis for the synthesis of hemoglobin. The human ferrochelatase promoter contains 2 Sp1 cis-elements and GATA and NF-E2 sites, all of which bind their cognate trans-acting factors in vitro. To investigate the role of these elements during erythropoiesis, we introduced expression of the green fluorescent protein (EGFP) transgenes driven by various ferrochelatase promoter fragments into a single locus in mouse embryonic stem cells. EGFP expression was monitored during hematopoietic differentiation in vitro using flow cytometry. We show that a promoter fragment containing the Sp1 sites, the NF-E2 and GATA elements, was sufficient to confer developmental-specific expression of the EGFP transgene, with an expression profile identical to that of the endogenous gene. In this system the -0.275 kb NF-E2 cis-element is required for erythroid-enhanced expression, the GATA cis-element functions as a stage-specific repressor and enhancer, and elements located between -0.375kb and -1.1kb are necessary for optimal levels of expression. Ferrochelatase mRNA increased before the primitive erythroid-cell stage without a concomitant increase in ferrochelatase protein, suggesting the presence of a translational control mechanism. Because of the sensitivity of this system, we were able to assess the effect of an A-to-G polymorphism identified in the promoters of patients with protoporphyria. There was no effect of the G haplotype on transcriptional activity of the -1.1 kb transgene.
Animals↗
The mitochondrial permeability transition augments Fas-induced apoptosis in mouse hepatocytes.
Tumor necrosis factor-alpha receptor 1 and Fas recruit overlapping signaling pathways. To clarify the differences between tumor necrosis factor alpha (TNFalpha) and Fas pathways in hepatocyte apoptosis, primary mouse hepatocytes were treated with TNFalpha or an agonist anti-Fas antibody after infection with an adenovirus expressing an IkappaB superrepressor (Ad5IkappaB). Treatment with TNFalpha induced apoptosis in Ad5IkappaB-infected mouse hepatocytes, as we previously reported for rat hepatocytes. Ad5IkappaB plus anti-Fas antibody or actinomycin D plus anti-Fas antibody rapidly induced apoptosis, whereas anti-Fas antibody alone produced little cytotoxicity. The proteasome inhibitor (MG-132) and a dominant-negative mutant of nuclear factor-kappaB-inducing kinase also promoted TNFalpha- and Fas-mediated apoptosis. Expression of either crmA or a dominant-negative mutant of the Fas-associated death domain protein prevented TNFalpha- and Fas-mediated apoptosis. In addition, the caspase inhibitors, DEVD-cho and IETD-fmk, inhibited TNFalpha- and Fas-mediated apoptosis. In Ad5IkappaB-infected hepatocytes, caspases-3 and -8 were activated within 2 h after treatment with anti-Fas antibody or within 6 h after TNFalpha treatment. Confocal microscopy demonstrated onset of the mitochondrial permeability transition (MPT) and mitochondrial depolarization by 2-3 h after anti-Fas antibody treatment and 8-10 h after TNFalpha treatment, followed by cytochrome c release. The combination of the MPT inhibitors, cyclosporin A, and trifluoperazine, protected Ad5IkappaB-infected hepatocytes from TNFalpha-mediated apoptosis. After anti-Fas antibody, cyclosporin A and trifluoperazine decreased cytochrome c release but did not prevent caspase-3 activation and cell-death. In conclusion, nuclear factor-kappaB activation protects mouse hepatocytes against both TNFalpha- and Fas-mediated apoptosis. TNFalpha and Fas recruit similar but nonidentical, pathways signaling apoptosis. The MPT is obligatory for TNFalpha-induced apoptosis. In Fas-mediated apoptosis, the MPT accelerates the apoptogenic events but is not obligatory for them.
Animals↗
Cellular differentiation causes a selective down-regulation of interleukin (IL)-1beta-mediated NF-kappaB activation and IL-8 gene expression in intestinal epithelial cells.
Interleukin (IL)-1beta signals through various adapter proteins and kinases that lead to activation of numerous downstream targets, including the transcription factors including NF-kappaB. In this study, we analyzed and characterized the effect of the differentiation of intestinal epithelial cells on IL-1beta-mediated NF-kappaB activation and IL-8 gene expression. We report that IL-8 mRNA accumulation and protein secretion were down-regulated in IL-1beta- and lipopolysaccharide-stimulated differentiated HT-29 cells (HT-29/MTX, where MTX is methotrexate) compared with undifferentiated cells (HT-29/p), whereas no differential effects were found following tumor necrosis factor (TNF)-alpha or phorbol myristate acetate stimulation. Cross-linking and affinity binding studies reveal that IL-1beta exclusively binds the type I receptor (IL-1RI) and not IL-1RII in both HT-29/p and HT-29/MTX cells. IL-1beta-mediated IkappaB kinase and c-Jun N-terminal kinase (JNK) activity were both diminished in differentiated HT-29 cells. DNA binding activity in differentiated HT-29 cells relative to HT-29/p cells was strongly reduced following IL-1beta exposure but not after TNF-alpha stimulation. The proximal IL-1 signaling molecule IL-1 receptor-associated kinase was not degraded in IL-1beta-stimulated HT-29 cells, in contrast to Caco-2 cells. kappaB-luciferase reporter gene activity was 16-fold higher following TNF receptor-associated factor-6 transfection after IL-1beta stimulation in HT-29/MTX cells. We conclude that cellular differentiation of HT-29 cells selectively impairs the IL-1beta signaling pathway inhibiting both NF-kappaB and JNK activity in response to IL-1beta. This relative unresponsiveness to IL-1beta may represent an important regulatory mechanism of differentiated intestinal epithelial cells.
Cell Differentiation↗
The 5' stem-loop regulates expression of collagen alpha1(I) mRNA in mouse fibroblasts cultured in a three-dimensional matrix.
The stability of collagen alpha1(I) mRNA is regulated by its 5' stem-loop, which binds a cytoplasmic protein in a cap-dependent manner, and its 3'-untranslated region (UTR), which binds alphaCP. When cultured in a three-dimensional gel composed of type I collagen, mouse fibroblasts had decreased collagen alpha1(I) mRNA steady-state levels, which resulted from a decreased mRNA half-life. In cells cultured in gel, hybrid mouse-human collagen alpha1(I) mRNA with a wild-type 5' stem-loop decayed faster than the same mRNA with a mutated stem-loop. When the 5' stem-loop was placed in a heterologous mRNA, the mRNA accumulated to a lower level in cells grown in gel than in cells grown on plastic. This suggests that the 5' stem-loop down-regulates collagen alpha1(I) mRNA. Protein binding to the 5' stem-loop was reduced in cells grown in gel, which was associated with destabilization of the collagen alpha1(I) mRNA. In addition to the binding of a cytoplasmic protein, there was also a nuclear binding activity directed to the collagen alpha1(I) 5' stem-loop. The nuclear binding was increased in cells grown in gel, suggesting that it may negatively regulate expression of collagen alpha1(I) mRNA. Binding of alphaCP, a protein involved in stabilization of collagen alpha1(I) mRNA, was unchanged by the culture conditions.
3T3 Cells↗
c-Jun does not mediate hepatocyte apoptosis following NFkappaB inhibition and partial hepatectomy.
Inhibition of the transcription factor nuclear factor kappa B (NFkappaB) induces marked hepatocyte apoptosis and liver dysfunction after partial hepatectomy (PH) in rats. Hepatocyte apoptosis may be due to direct inhibition of NFkappaB-induced hepatocyte survival genes or due to indirect increased signaling through the stress-activated protein kinase pathway (SAPK), resulting in increased c-Jun. c-Jun, an AP-1 transcription factor, induces apoptosis in fibroblasts. Our aim was to determine if hepatocyte apoptosis following inhibition of NFkappaB and partial hepatectomy in rats is due to increased c-Jun. Adult male Sprague-Dawley rats (200 g) were injected intraportally with 6 x 10(9) PFU adenoviral vector containing luciferase (Ad5Luc) or superrepressor IkappaB (Ad5IkappaB) transgene that inhibits NFkappaB translocation into the nucleus. Two-thirds PH was performed 24 h after vector administration, and the remnant liver was harvested 30 min or 24 h after PH. Northern and Western blots were performed to examine the presence of IkappaB and c-Jun. A GST c-Jun kinase assay was used to examine Jun-N-terminal kinase (JNK) activity. AP-1 DNA binding activity was assessed by electrophoretic mobility shift assay. TUNEL assay was performed to assess apoptosis. All rats receiving adenoviral vectors expressed the luciferase or superrepressor IkappaB transgenes. c-Jun mRNA, protein levels, and DNA binding activity were not increased in rats treated with Ad5IkappaB at 30 min after PH compared to rats injected with Ad5Luc. Jun kinase activity increased following partial hepatectomy, but activity was similar in Ad5Luc- and Ad5IkappaB-treated animals. AP-1 DNA binding activity was not altered substantially in rats treated with Ad5IkappaB. The percentage of apoptotic hepatocytes was similar between Ad5Luc- and Ad5IkappaB-injected animals at 0 h, but livers from Ad5IkappaB-treated rats had increased apoptosis at 24 h compared to Ad5Luc rats (24% vs. 4%) after PH. Hepatocyte apoptosis after NFkappaB inhibition and PH is not mediated by increased JNK activity or c-Jun.
Adenoviridae↗
Glutamine metabolism stimulates intestinal cell MAPKs by a cAMP-inhibitable, Raf-independent mechanism.
BACKGROUND & AIMS: Infectious diarrhea caused by viruses plus enterotoxigenic bacteria is often more severe than diarrhea induced by either pathogen alone. We postulated that the increased cell adenosine 3',5'-cyclic monophosphate (cAMP) concentration observed during infection by enterotoxigenic organisms retards the intestinal repair process by blocking activation of mitogen-activated protein kinases (MAPKs) in proliferating intestinal cells. METHODS: We evaluated the effects of glutamine on MAPK activity, thymidine incorporation, and cell number in glutamine-starved and -sufficient rat intestinal crypt cells (IEC-6). RESULTS: In glutamine-starved cells, 10 mmol/L glutamine in the absence of serum stimulated [(3)H]thymidine incorporation 8-fold. This effect was inhibited by 60% with 8-(4-chlorophenylthio) (8-CPT)-cAMP (100 micromol/L) + isobutyl methylxanthine (100 micromol/L). In cells not starved of glutamine, glutamine stimulated thymidine incorporation by 3-fold, and 8-CPT-cAMP completely blocked the mitogenic effect. Inhibition of proliferation by cAMP persisted for at least 68 hours after cAMP removal. In vitro kinase assays showed that glutamine signaling requires an intact ERK (extracellular signal-related kinase) pathway in unstarved cells. In starved cells, at least one other pathway (JNK) was activated by glutamine, and the mitogenic inhibition by 8-CPT-cAMP was incomplete. Other intestinal fuels (glucose and acetate) were not mitogenic. CONCLUSIONS: Increased levels of intracellular cAMP inhibit ERKs but only partially reduce glutamine-stimulated proliferation in enterocytes adapted to low glutamine.
Cell Division↗
Nuclear factor kappaB in proliferation, activation, and apoptosis in rat hepatic stellate cells.
BACKGROUND/AIMS: Activation of the transcription factor NFkappaB has been demonstrated in activated hepatic stellate cells (HSCs). We investigated the role of NFkappaB in proliferation, in activation, and in TNFalpha-induced apoptosis of HSCs. METHODS: NFkappaB activation was inhibited using an adenovirus expressing an IkappaB dominant negative protein (Ad5IkappaB) in both quiescent and activated HSCs. Quiescent HSCs were infected with Ad5IkappaB or an adenovirus expressing beta-galactosidase (Ad5LacZ). The cells were cultured for 7 days. HSCs activation was determined by cell morphology, smooth muscle alpha-actin (alpha-sma) expression, and steady-state mRNA levels of alpha1(I) collagen as assessed by Western blot and RNase protection assay, respectively. Proliferation was determined in culture-activated HSCs by 3H-thymidine incorporation and direct cell counting. Apoptosis was analyzed by infecting quiescent or activated HSCs with Ad5IkappaB or Ad5LacZ, and then treating with TNFalpha. Apoptosis was demonstrated by determining cell number, assessing nuclear morphology, TUNEL assay and caspase 3 activity. RESULTS: After 7 days in culture no differences were noted between the Ad5IkappaB- and the Ad5LacZ-infected cells in the morphology, alpha-sma expression or in alpha1(I) collagen mRNA levels. Ad5IkappaB infection did not modify proliferation in activated HSCs. TNFalpha induced apoptosis only in Ad5IkappaB-infected activated, but not quiescent HSCs. Apoptosis was initially demonstrated 12 h after exposure to TNFalpha. Twenty-four h after the TNFalpha treatment, 60% of the activated HSCs were apoptotic. CONCLUSION: NFkappaB activity is not required for proliferation or activation of HSCs; however, NFkappaB protects activated HSCs against TNFalpha-induced apoptosis.
Adenoviridae Infections↗
New aspects of hepatic fibrosis.
Hepatic stellate cells are the major source of extracellular matrix proteins in hepatic fibrosis, including Type I collagen. In response to liver injury, the hepatic stellate cells change from a quiescent to an activated phenotype. This activation process includes a phenotypic change to a myofibroblast-like cell, increased proliferation rate, loss of retinoid stores, increased production of extracellular matrix proteins, chemokines, and cytokines, and contractility. Ongoing studies are characterizing the genes that are differentially expressed in the quiescent and activated hepatic stellate cells. We have also investigated the regulation of Type I collagen expression, the cleavage of collagen propeptides, and the formation of collagen cross-links. Understanding these pathways may provide new insights into the molecular pathogenesis of hepatic fibrosis.
Bone Morphogenetic Proteins↗
Role of Kupffer cells and gut-derived endotoxins in alcoholic liver injury.
The hepatotoxic effects of alcohol have been described in detail, but factors responsible for its hepatotoxicity have only partially been characterized. For example, it is known that chronic ethanol ingestion increases hepatotoxicity and produces fatty liver, hepatitis and cirrhosis. However, acute ethanol consumption reduces endotoxin hepatotoxicity. It now appears that Kupffer cells participate in several aspects of these phenomena. Previously, most studies on the effects of alcohol on liver function have focused chiefly on the hepatocyte. Recently, attention has been directed towards the effect of ethanol ingestion on Kupffer cell function, which is stimulated by gut-derived endotoxins (lipopolysaccharides) via mechanisms dependent on increased gut permeability and the possible relationship between Kupffer cells and alcohol-induced liver injury. Here we will review new evidence for the proposal that Kupffer cells and endotoxins play a pivotal role in hepatotoxicity following alcohol exposure, based on studies using the continuous intragastric enteral feeding model developed by Tsukamoto and French and an acute model developed by us.
Ethanol↗
Moderate alcohol drinking: effects on the heart and liver.
Explore the source record for details and available documents.
Alcohol Drinking↗
Gene delivery of Cu/Zn-superoxide dismutase improves graft function after transplantation of fatty livers in the rat.
Oxygen-derived free radicals play a central role in reperfusion injury after organ transplantation, and fatty livers are particularly susceptible. Endogenous radical scavengers such as superoxide dismutase (SOD) degrade these radicals; however, SOD is destroyed rapidly when given exogenously. Therefore, an adenoviral vector encoding the Cu/Zn-SOD gene (Ad.SOD1) was used here to test the hypothesis that organ injury would be reduced and survival increased in a rat model of transplantation of fatty livers. Donors received chow diet (untreated), high-fat diet, or ethanol-containing high-fat diet. Some of the ethanol-fed donors were infected either with the gene lacZ encoding bacterial beta-galactosidase (Ad.lacZ), or Ad.SOD1. After liver transplantation, SOD activity and protein expression in liver, survival, histopathology, release of transaminases, free radical adducts in bile, and activation of NF-kappaB, IkappaB kinase (IKK), Jun-N-terminal kinase (JNK), and TNFalpha were evaluated. Ad.SOD1 treatment increased survival dramatically, blunted transaminase release, and reduced necrosis and apoptosis significantly. Free radical adducts were increased two-fold in the ethanol group compared with untreated controls. Ad. SOD1 blunted this increase and reduced the activation of NF-kappaB. However, release of TNFalpha was not affected. Ad.SOD1 also blunted JNK activity after transplantation. This study shows that gene therapy with Ad.SOD1 protects marginal livers from failure after transplantation because of decreased oxygen radical production. Genetic modification of fatty livers using viral vectors represents a new approach to protect marginal grafts against primary nonfunction.
Adenoviridae↗
Tumor necrosis factor alpha prevents tumor necrosis factor receptor-mediated mouse hepatocyte apoptosis, but not fas-mediated apoptosis: role of nuclear factor-kappaB.
Tumor necrosis factor alpha (TNF-alpha) binding to the TNF receptor (TNFR) initiates apoptosis and simultaneously activates the transcription factor, nuclear factor-kappaB (NF-kappaB), which suppresses apoptosis by an unknown mechanism. Pretreatment with TNF-alpha or interleukin-1beta (IL-1beta), which activated NF-kappaB in the liver, dramatically prevented TNF-alpha-induced liver-cell apoptosis in D-galactosamine (GalN)-sensitized mice, but not anti-Fas antibody-induced hepatotoxicity. This protective effect of TNF-alpha continued for 5 hours after TNF-alpha administration, a time course similar to that found in NF-kappaB activation after TNF-alpha administration. In mice treated with adenoviruses expressing a mutant form of IkappaB, the antiapoptotic effect of TNF-alpha was inhibited in part. Prior TNF-alpha administration was not found to block the activation of caspase-8, although caspase-3 was inhibited in mice treated with TNF-alpha plus GalN/TNF-alpha compared with mice treated with GalN/TNF-alpha. These results indicate that TNFR and Fas independently regulate murine apoptotic liver failure, and that a rapid defense mechanism induced by the activation of NF-kappaB blocks death-signaling at the initiation stage of hepatic apoptosis mediated by TNFR, probably downstream of caspase-8, but not by Fas.
Animals↗
Impact of burn injury on hepatic TGF-beta1 expression and plasma TGF-beta1 levels.
BACKGROUND: The liver plays a critical regulatory role in the acute inflammatory response to injury, although the mechanisms of this regulation are not well understood. transforming growth factor-beta1 (TGF-beta1) is induced after burn injury and may contribute to an inhibitory or fatal effect on hepatocytes. We investigated the association over time between plasma concentration of TGF-beta1, expression of TGF-beta1 m-RNA in liver tissue, and histologic analysis of liver apoptosis after burn injury. METHODS: Male BALB/c mice were anesthetized and randomized to receive 0% (sham), moderate (approximately 25%) (M), or large (approximately 50%) (L) body surface area full-thickness contact burn, followed by resuscitation and analgesia. Animals were killed over a time course from 15 minutes to 24 hours after burn injury, and liver tissue and peripheral blood were collected. Plasma levels of TGF-beta1 (nanograms per milliliter) were measured by enzyme-linked immunosorbent assay. TGF-beta1 m-RNA was extracted from liver and measured by reverse transcription-polymerase chain reaction. Histology of liver apoptosis was examined after fixation and staining with TdT-mediated dUTP nick-end labeling (TUNEL) method. RESULTS: The plasma concentration of TGF-beta in burn group L was significantly increased at 4 hours after burn when compared with sham and M burn groups. This rise in plasma TGF-beta1 was preceded by an increase in hepatic TGF-beta1 m-RNA expression at 30 minutes, 1, 2, and 4 hours after burn in the L group. Histologic analysis found greater hepatocyte death in the L group than in the M group at 8 hours after burn. CONCLUSION: The levels of induced TGF-beta1 and TGF-beta1 m-RNA after L burn injury are higher and peak earlier than after M burn injury. Elevated TGF-beta1 may be associated with cell death in hepatocytes. The TGF-beta1 rise may be associated with hepatocyte injury and systemic response to massive burn.
Acute Disease↗
Kupffer cell-derived prostaglandin E(2) is involved in alcohol-induced fat accumulation in rat liver.
Destruction of Kupffer cells with gadolinium chloride (GdCl(3)) and intestinal sterilization with antibiotics diminished ethanol-induced steatosis in the enteral ethanol feeding model. However, mechanisms of ethanol-induced fatty liver remain unclear. Accordingly, the role of Kupffer cells in ethanol-induced fat accumulation was studied. Rats were given ethanol (5 g/kg body wt) intragastrically, and tissue triglycerides were measured enzymatically. Kupffer cells were isolated 0-24 h after ethanol, and PGE(2) production was measured by ELISA, whereas inducible cyclooxygenase (COX-2) mRNA was detected by RT-PCR. As expected, ethanol increased liver triglycerides about threefold. This increase was blunted by antibiotics, GdCl(3), the dihydropyridine-type Ca(2+) channel blocker nimodipine, and the COX inhibitor indomethacin. Ethanol also increased PGE(2) production by Kupffer cells about threefold. This increase was also blunted significantly by antibiotics, nimodipine, and indomethacin. Furthermore, tissue triglycerides were increased about threefold by PGE(2) treatment in vivo as well as by a PGE(2) EP(2)/EP(4) receptor agonist, whereas an EP(1)/EP(3) agonist had no effect. Moreover, permeable cAMP analogs also increased triglyceride content in the liver significantly. We conclude that PGE(2) derived from Kupffer cells, which are activated by ethanol, interacts with prostanoid receptors on hepatocytes to increase cAMP, which causes triglyceride accumulation in the liver. This mechanism is one of many involved in fatty liver caused by ethanol.
8-Bromo Cyclic Adenosine Monophosphate↗