Biomedical subjects

David A Brenner

Publications and source records attributed to David A Brenner.

At least 37 records · Page 2Linked to original sources

PubMed2005

NF-kappaB activation in Kupffer cells after partial hepatectomy.

The transcription factor nuclear factor-kappaB (NF-kappaB) is activated during liver regeneration after partial hepatectomy. However, the physiological role and cellular localization of NF-kappaB activation are unresolved. In this study, we used an adenoviral vector expressing a mutated form of IkappaBalpha to inhibit NF-kappaB activity during liver regeneration. After partial hepatectomy in mice, introduction of Ad5IkappaB, but not a control virus (Ad5GFP), resulted in increased liver injury and decreased hepatocyte proliferation. Hepatocyte apoptosis was not observed. To investigate the kinetics and cellular localization of NF-kappaB-induced transcription during liver regeneration, we generated a transgenic mouse expressing enhanced green fluorescent protein (EGFP) under the transcriptional control of NF-kappaB cis elements (cis-NF-kappaB-EGFP). During liver regeneration, EGFP expression was detected within 12 h and was primarily located in Kupffer cells. Our data demonstrate that activation of NF-kappaB initially occurs in Kupffer cells after partial hepatectomy in mice.

Adenoviridae↗

PubMed2005

Free cholesterol-loaded macrophages are an abundant source of tumor necrosis factor-alpha and interleukin-6: model of NF-kappaB- and map kinase-dependent inflammation in advanced atherosclerosis.

Two key features of atherosclerotic plaques that precipitate acute atherothrombotic vascular occlusion ("vulnerable plaques") are abundant inflammatory mediators and macrophages with excess unesterified, or "free," cholesterol (FC). Herein we show that FC accumulation in macrophages leads to the induction and secretion of two inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). The increases in TNF-alpha and IL-6 mRNA and protein were mediated by FC-induced activation of the IkappaB kinase/NF-kappaB pathway as well as activation of MKK3/p38, Erk1/2, and JNK1/2 mitogen-activated protein kinases (MAPK). Activation of IkappaB kinase and JNK1/2 was needed for the induction of both cytokines. However, MKK3/p38 signaling was specifically involved in TNF-alpha induction, and Erk1/2 signaling was required for IL-6. Most interestingly, activation of all of the signaling pathways and induction of both cytokines required cholesterol trafficking to the endoplasmic reticulum (ER). The CHOP branch of the unfolded protein response, an ER stress pathway, was required for Erk1/2 activation and IL-6 induction. In contrast, one or more other ER-related pathways were responsible for activation of p38, JNK1/2, and IkappaB kinase/NF-kappaB and for the induction of TNF-alpha. These data suggest a novel scenario in which cytokines are induced in macrophages by endogenous cellular events triggered by excess ER cholesterol rather than by exogenous immune cell mediators. Moreover, this model may help explain the relationship between FC accumulation and inflammation in vulnerable plaques.

Animals↗

PubMed2005

JNK mediates hepatic ischemia reperfusion injury.

BACKGROUND/AIMS: Hepatic ischemia followed by reperfusion (I/R) is a major clinical problem during transplantation, liver resection for tumor, and circulatory shock, producing apoptosis and necrosis. Although several intracellular signal molecules are induced following I/R including NF-kappaB and c-Jun N terminal kinase (JNK), their roles in I/R injury are largely unknown. The aim of this study is to assess the role of JNK during warm I/R injury using novel selective JNK inhibitors. METHODS: Male Wistar rats (200+/-25 g) are pretreated with vehicle or with one of three compounds (CC0209766, CC0223105, and CC-401), which are reversible, highly selective, ATP-competitive inhibitors of JNK. In the first study, rats are assessed for survival using a model of ischemia to 70% of the liver for 90 min followed by 30% hepatectomy of the non-ischemic lobes and then reperfusion. In the second study, rats are assessed for liver injury resulting from 60 or 90 min of ischemia followed by reperfusion with analysis over time of hepatic histology, serum ALT, hepatic caspase-3 activation, cytochrome c release, and lipid peroxidation. RESULTS: In the I/R survival model, vehicle-treated rats have a 7-day survival of 20-40%, while rats treated with the three different JNK inhibitors have survival rates of 60-100% (P<0.05). The decrease in mortality correlates with improved hepatic histology and serum ALT levels. Vehicle treated rats have pericentral necrosis, neutrophil infiltration, and some apoptosis in both hepatocytes and sinusoidal endothelial cells, while JNK inhibitors significantly decrease both types of cell death. JNK inhibitors decrease caspase-3 activation, cytochrome c release from mitochondria, and lipid peroxidation. JNK inhibition transiently blocks phosphorylation of c-Jun at an early time point after reperfusion, and AP-1 activation is also substantially blocked. JNK inhibition blocks the upregulation of the pro-apoptotic Bak protein and the degradation of Bid. CONCLUSIONS: Thus, JNK inhibitors decrease both necrosis and apoptosis, suggesting that JNK activity induces cell death by both pathways.

Animals↗

PubMed2005

Deletion of IKK2 in hepatocytes does not sensitize these cells to TNF-induced apoptosis but protects from ischemia/reperfusion injury.

The inhibitor of NF-kappaB (I-kappaB) kinase (IKK) complex consists of 3 subunits, IKK1, IKK2, and NF-kappaB essential modulator (NEMO), and is involved in the activation of NF-kappaB by various stimuli. IKK2 or NEMO constitutive knockout mice die during embryogenesis as a result of massive hepatic apoptosis. Therefore, we examined the role of IKK2 in TNF-induced apoptosis and ischemia/reperfusion (I/R) injury in the liver by using conditional knockout mice. Hepatocyte-specific ablation of IKK2 did not lead to impaired activation of NF-kappaB or increased apoptosis after TNF-alpha stimulation whereas conditional NEMO knockout resulted in complete block of NF-kappaB activation and massive hepatocyte apoptosis. In a model of partial hepatic I/R injury, mice lacking IKK2 in hepatocytes displayed significantly reduced liver necrosis and inflammation than wild-type mice. AS602868, a novel chemical inhibitor of IKK2, protected mice from liver injury due to I/R without sensitizing them toward TNF-induced apoptosis and could therefore emerge as a new pharmacological therapy for liver resection, hemorrhagic shock, or transplantation surgery.

Animals↗

PubMed2005

The role of p70S6K in hepatic stellate cell collagen gene expression and cell proliferation.

During fibrosis the hepatic stellate cell (HSC) undergoes a complex activation process characterized by increased proliferation and extracellular matrix deposition. The 70-kDa ribosomal S6 kinase (p70S6K) is activated by mitogens, growth factors, and hormones in a phosphatidylinositol 3-kinase-dependent manner. p70S6K regulates protein synthesis, proliferation, and cell cycle control. Because these processes are involved in HSC activation, we investigated the role of p70S6K in HSC proliferation, cell cycle control, and type I collagen expression. Platelet-derived growth factor (PDGF) stimulated p70S6K phosphorylation, which was blocked by LY294002, an inhibitor of phosphatidylinositol 3-kinase. Rapamycin blocked phosphorylation of p70S6K but had no affect on PDGF-induced Akt phosphorylation, positioning p70S6K downstream of Akt. Transforming growth factor-beta, which inhibits HSC proliferation, did not affect PDGF-induced p70S6K phosphorylation. Rapamycin treatment did not affect alpha1(I) collagen mRNA but reduced type I collagen protein secretion. Expression of smooth muscle alpha-actin was not affected by rapamycin treatment, indicating that HSC activation was not altered. Rapamycin inhibited serum-induced DNA synthesis approximately 2-fold. Moreover, rapamycin decreased expression of cyclins D1, D3, and E but not cyclin D2, Rb-Ser780, and Rb-Ser795. Together, p70S6K plays a crucial role in HSC proliferation, collagen expression, and cell cycle control, thus representing a potential therapeutic target for liver fibrosis.

Animals↗

PubMed2005

Systemic infusion of angiotensin II exacerbates liver fibrosis in bile duct-ligated rats.

Recent evidence indicates that the renin-angiotensin system (RAS) plays a major role in liver fibrosis. Here, we investigate whether the circulatory RAS, which is frequently activated in patients with chronic liver disease, contributes to fibrosis progression. To test this hypothesis, we increased circulatory angiotensin II (Ang II) levels in rats undergoing biliary fibrosis. Saline or Ang II (25 ng/kg/h) were infused into bile duct-ligated rats for 2 weeks through a subcutaneous pump. Ang II infusion increased serum levels of Ang II and augmented bile duct ligation-induced liver injury, as assessed by elevated liver serum enzymes. Moreover, it increased the hepatic concentration of inflammatory proteins (tumor necrosis factor alpha and interleukin 1beta) and the infiltration of CD43-positive inflammatory cells. Ang II infusion also favored the development of vascular thrombosis and increased the procoagulant activity of tissue factor in the liver. Livers from bile duct-ligated rats infused with Ang II showed increased transforming growth factor beta1 content, collagen deposition, accumulation of smooth muscle alpha-actin-positive cells, and lipid peroxidation products. Moreover, Ang II infusion stimulated phosphorylation of c-Jun and p42/44 mitogen-activated protein kinase and increased proliferation of bile duct cells. In cultured rat hepatic stellate cells (HSCs), Ang II (10(-8) mol/L) increased intracellular calcium and stimulated reactive oxygen species formation, cellular proliferation and secretion of proinflammatory cytokines. Moreover, Ang II stimulated the procoagulant activity of HSCs, a newly described biological function for these cells. In conclusion, increased systemic Ang II augments hepatic fibrosis and promotes inflammation, oxidative stress, and thrombogenic events.

Angiotensin II↗

PubMed2005

Anandamide induces necrosis in primary hepatic stellate cells.

The endogenous cannabinoid anandamide (AEA) is a lipid mediator that blocks proliferation and induces apoptosis in many cell types. Although AEA levels are elevated in liver fibrosis, its role in fibrogenesis remains unclear. This study investigated effects of AEA in primary hepatic stellate cells (HSCs). Anandamide blocked HSC proliferation at concentrations of 1 to 10 micromol/L but did not affect HSC proliferation or activation at nanomolar concentrations. At higher concentrations (25-100 micromol/L), AEA rapidly and dose-dependently induced cell death in primary culture-activated and in vivo-activated HSCs, with over 70% cell death after 4 hours at 25 micromol/L. In contrast to treatment with Fas ligand or gliotoxin, AEA-mediated death was caspase independent and showed typical features of necrosis such as rapid adenosine triphosphate depletion and propidium iodide uptake. Anandamide-induced reactive oxygen species (ROS) formation, and an increase in intracellular Ca(2+). Pretreatment with the antioxidant glutathione or Ca(2+)-chelation attenuated AEA-induced cell death. Although the putative endocannabinoid receptors CB1, CB2, and VR1 were expressed in HSCs, specific receptor blockade failed to block cell death. Depletion of membrane cholesterol by methyl-beta-cyclodextrin inhibited AEA binding, blocked ROS formation and intracellular Ca(2+)-increase, and prevented cell death. In primary hepatocytes, AEA showed significantly lower binding and failed to induce cell death even after prolonged treatment. In conclusion, AEA efficiently induces necrosis in activated HSCs, an effect that depends on membrane cholesterol and a subsequent increase in intracellular Ca(2+) and ROS. The anti-proliferative effects and the selective killing of HSCs, but not hepatocytes, indicate that AEA may be used as a potential anti-fibrogenic tool.

Animals↗

PubMed2005

Roles of AKT and sphingosine kinase in the antiapoptotic effects of bile duct ligation in mouse liver.

Tumor necrosis factor (TNF) receptor- and Fas-mediated apoptosis are major death processes of hepatocytes in liver disease. Although antiapoptotic effects in the injured liver promote chronic hepatitis and carcinogenesis, scant information is known about these mechanisms. To explore this issue, we compared acute liver injury after TNF-alpha or anti-Fas antibody (Jo2) between livers from sham-operated mice and chronic injured liver via bile duct ligation (BDL). BDL inhibited hepatocyte apoptosis induced by TNF-alpha but not by Jo2. On the other hand, BDL inhibited the massive hemorrhage seen in livers treated with either TNF-alpha or Jo2. Inactivation of AKT blocked the antiapoptotic effect of BDL. Sphingosine kinase knockout mice also lost the antihemorrhagic effect of BDL and attenuated the antiapoptotic effects of BDL. In bile duct-ligated livers, hepatic stellate cells (HSCs) were activated and produced tissue inhibitor of metalloproteinase 1 in a sphingosine kinase (SphK)-1-dependent mechanism. In conclusion, BDL exerts antiapoptotic effects that appear to require activation of AKT in hepatocytes and SphK in HSCs.

Animals↗

PubMed2005

Attenuated hepatic inflammation and fibrosis in angiotensin type 1a receptor deficient mice.

BACKGROUND/AIMS: Pharmacological blockade of the renin-angiotensin system (RAS) attenuates liver fibrogenesis in rats. Here, we provide genetic evidence implicating angiotensin type 1 (AT1) receptors in liver fibrogenesis. METHODS: Wild type (WT) and AT1a knockout [AT1a (-/-)] mice were subjected to either sham operation or bile-duct ligation. Fibrosis was assessed by Sirius Red staining and hydroxyproline hepatic content. Fibrogenic and inflammatory cytokines were measured by ELISA. RESULTS: Bile duct ligation-induced elevation of serum liver enzymes was similar in WT and AT1a (-/-) mice. Bile duct ligated WT mice showed inflammatory changes and severe septal fibrosis. In contrast, AT1a (-/-) mice showed minor fibrotic lesions. Collagen accumulation was lower in AT1a (-/-) mice compared to WT mice. The increase in hepatic concentration of TGFbeta1 and pro-inflammatory cytokines was attenuated in AT1a (-/-) mice compared to WT mice. Immunohistochemistry analysis revealed decreased infiltration by inflammatory cells, lipid peroxidation products as well as decreased phosphorylation of c-Jun and p42/44 MAPK in AT1a (-/-) mice compared to AT1 (+/+) mice. CONCLUSIONS: AT1 receptors play an important role in the development of fibrosis. Pharmacological blockade of AT1 receptors appears to be a promising approach to treat liver fibrosis.

Animals↗

PubMed2005

Molecular pathogenesis of alcohol-induced hepatic fibrosis.

Alcohol abuse is a main cause of liver fibrosis and cirrhosis in the western world. Although the major mechanisms of fibrogenesis are independent of the origin of liver injury, alcoholic liver fibrosis features distinctive characteristics, including the pronounced inflammatory response of immune cells due to elevated gut-derived endotoxin plasma levels, increased formation of reactive oxygen species (ROS), ethanol-induced pericentral hepatic hypoxia or formation of cell-toxic and pro-fibrogenic ethanol metabolites (e.g., acetaldehyde or lipid oxidation products). These factors are together responsible for increased hepatocellular cell death and activation of hepatic stellate cells (HSCs), the key cell type of liver fibrogenesis. To date, removing the causative agent is the most effective intervention to prevent the manifestation of liver cirrhosis. A novel experimental approach in fibrosis therapy is the selective induction of cell death in HSCs. Substances such as gliotoxin, anandamide or antibody against tissue inhibitor of metalloproteinase (TIMP)-1 can selectively induce cell death in activated HSCs. These new results in basic science are encouraging for the search of new antifibrotic treatment.

Animals↗

PubMed2005

Information assimilation and distribution challenges and goals for real and virtual journals.

The distribution of biomedical information was transfigured over three centuries ago with the introduction of scientific journals. This enabled the widespread dissemination of data to global audiences and greatly facilitated not only the advance of science but amplified the interaction between investigators despite their different locations. This process continued to expand in a linear fashion prior to the emergence of the Internet. The latter system has prompted a phenomenal augmentation of information accessibility, and its ever-expanding use has resulted in an exponential increase in the demand for digital technology and online resources. This technology has achieved unprecedented acceptance in the scientific domain and enabled publishers to expeditiously produce and distribute journal contents online. Such unparalleled access to information has sparked incendiary debate within the scientific community and among journal publishers in regard to numerous issues. It is thus much debated as to who has the right to "own" or control intellectual property, whether information should be made freely available to the online global community, how to gauge the legitimacy and authenticity of published research, and the need to reexamine the feasibility and profitability of paper journals in consideration of the digital, online formats that continue to gain popularity. To assess the current status of the situation, a meeting of journal editors, research scientists, and publishing executives was held in Constance, Germany, on June 26, 2004, to discuss these issues and formulate strategies and recommendations for the future of biomedical publishing. Herewith we provide a summation (manifesto) of the meeting's proceedings and provide a consensus opinion with the aim of illuminating the subject and also proposing some putative solutions for the major challenges that currently confront the scientific and publishing community.

Forecasting↗

PubMed2005

Clinical syndromes of alcoholic liver disease.

BACKGROUND AND AIMS: Alcoholic liver disease continues to be a major health problem with respect to both morbidity and mortality. To understand the clinical syndromes of alcoholic liver disease, this review highlights the papers on both clinical and basic research of alcoholic liver disease, especially on steatosis, alcoholic hepatitis and fibrosis. METHODS: The various forms of alcoholic liver disease are described, and knowledge about the clinical and pathophysiological features of different stages of alcoholic liver disease are summarized. RESULTS: Clinical studies combined with basic research have established a spectrum of alcoholic liver disease from steatosis to steatohepatitis, fibrosis, and cirrhosis. New insights into the pathogenesis of alcoholic liver disease include the key roles of the excess production of cytokines, reactive oxygen species, and the shortage of protective mediators, including adiponectin. CONCLUSION: These new insights will lead to new specific therapies for the treatment of alcoholic hepatitis and alcoholic liver fibrosis.

Alcoholism↗

PubMed2005

Molecular mechanisms of alcohol-induced hepatic fibrosis.

Alcohol abuse is a major cause of liver fibrosis and cirrhosis in developed countries. Before alcoholic liver fibrosis becomes evident, the liver undergoes several stages of alcoholic liver disease including steatosis and steatohepatitis. Although the main mechanisms of fibrogenesis are independent of the etiology of liver injury, alcoholic liver fibrosis is distinctively characterized by a pronounced inflammatory response due to elevated gut-derived endotoxin plasma levels, an augmented generation of oxidative stress with pericentral hepatic hypoxia and the formation of cell-toxic and profibrogenic ethanol metabolites (e.g. acetaldehyde or lipid oxidation products). These factors, based on a complex network of cytokine actions, together result in increased hepatocellular damage and activation of hepatic stellate cells, the key cell type of liver fibrogenesis. Although to date removal of the causative agent, i.e. alcohol, still represents the most effective intervention to prevent the manifestation of alcoholic liver disease, sophisticated molecular approaches are underway, aiming to specifically blunt profibrogenic signaling pathways in liver cells or specifically induce cell death in activated hepatic stellate cells to decrease the scarring of the liver.

Cytochrome P-450 CYP2E1↗

PubMed2005

Liver fibrosis.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension and often requires liver transplantation. Our knowledge of the cellular and molecular mechanisms of liver fibrosis has greatly advanced. Activated hepatic stellate cells, portal fibroblasts, and myofibroblasts of bone marrow origin have been identified as major collagen-producing cells in the injured liver. These cells are activated by fibrogenic cytokines such as TGF-beta1, angiotensin II, and leptin. Reversibility of advanced liver fibrosis in patients has been recently documented, which has stimulated researchers to develop antifibrotic drugs. Emerging antifibrotic therapies are aimed at inhibiting the accumulation of fibrogenic cells and/or preventing the deposition of extracellular matrix proteins. Although many therapeutic interventions are effective in experimental models of liver fibrosis, their efficacy and safety in humans is unknown. This review summarizes recent progress in the study of the pathogenesis and diagnosis of liver fibrosis and discusses current antifibrotic strategies.

Angiotensin II↗

PubMed2005

Direct hepatotoxic effect of KC chemokine in the liver without infiltration of neutrophils.

KC is a mouse homolog of human chemokine gro-alpha (CXCL1), expression of which is increased in liver diseases. We show that activated, but not quiescent, hepatic stellate cells (HSCs) express KC. Hepatic stellate cells constitutively express the KC receptor, CXCR2. Addition of recombinant KC to HSCs undergoing activation in culture increases secretion and processing of Type I collagen. Overexpression of endogenous KC in the mouse liver could be achieved by an intraperitoneal injection of CCl(4), followed after 24 hrs by an injection of recombinant KC into circulation. This protocol resulted in about a 14-fold increase in concentration of KC protein in the liver. Overexpression of KC was associated with upregulation of the mRNA for CXCR2 and MIP-2 and with necrosis and increased synthesis of Type I collagen. This suggests that KC has a direct hepatotoxic effect, which led to a massive liver necrosis after 48 hrs. No accumulation of neutrophils was seen in the livers as judged by histology and reverse transcriptase-polymerase chain reaction analysis of myeloperoxidase mRNA. Autostimulation of KC and CXCR2 expression by recombinant KC protein in the mice with preexisting liver injury indicates a positive feedback regulation. Such regulation and direct hepatotoxicity of KC with increased collagen synthesis represent novel findings about the role of KC/ gro-alpha in liver pathology.

Animals↗

PubMed2004

c-Jun N-terminal kinase mediates hepatic injury after rat liver transplantation.

BACKGROUND: Orthotopic liver transplantation (OLT) requires cold ischemic storage followed by warm reperfusion. Although c-Jun N-terminal kinase (JNK) is rapidly activated after OLT, the functional consequences of JNK activation are unknown. The aim of this study was to address the role of JNK after OLT using the selective JNK inhibitor CC-401. METHODS: Donors, recipients, or stored liver explants were treated with vehicle or JNK inhibitor before OLT by an arterialized two-cuff method with 40 hours of cold storage. Recipients were assessed for 30-day survival, and graft injury was assessed over time by hepatic histology, serum transaminases, caspase 3 activation, cytosolic cytochrome c, and lipid peroxidation. RESULTS: Survival after OLT increased after donor plus storage and storage only treatment with JNK inhibitor (P<0.05). Treatment of recipient only did not improve survival. Increased survival correlated with improved hepatic histology and serum aspartate aminotransferase levels. JNK inhibition significantly decreased nonparenchymal cell killing at 60 minutes after reperfusion (P<0.05) and pericentral necrosis at 8 hours after reperfusion (P<0.01). JNK inhibition decreased cytochrome c release, caspase 3 activation (P<0.05), and lipid peroxidation (P<0.05). JNK inhibition also transiently blocked phosphorylation of c-Jun at 60 minutes after reperfusion (P<0.05) without affecting other MAPK signaling, including p-38 and Erk activation. CONCLUSIONS: JNK inhibition decreases hepatic necrosis and apoptosis after OLT, suggesting that JNK activation promotes cell death by both pathways. Inhibition of JNK may be a new therapeutic strategy to prevent liver injury after transplantation.

Animals↗

PubMed2004

In vivo pattern of lipopolysaccharide and anti-CD3-induced NF-kappa B activation using a novel gene-targeted enhanced GFP reporter gene mouse.

NF-kappa B is a family of transcription factors involved in regulating cell death/survival, differentiation, and inflammation. Although the transactivation ability of NF-kappa B has been extensively studied in vitro, limited information is available on the spatial and temporal transactivation pattern in vivo. To investigate the kinetics and cellular localization of NF-kappa B-induced transcription, we created a transgenic mouse expressing the enhanced GFP (EGFP) under the transcriptional control of NF-kappa B cis elements (cis-NF-kappa B(EGFP)). A gene-targeting approach was used to insert a single copy of a NF-kappa B-dependent EGFP reporter gene 5' of the X-linked hypoxanthine phosphoribosyltransferase locus in mouse embryonic stem cells. Embryonic fibroblasts, hepatic stellate cells, splenocytes, and dendritic cells isolated from cis-NF-kappa B(EGFP) mice demonstrated a strong induction of EGFP in response to LPS, anti-CD3, or TNF-alpha that was blocked by the NF-kappa B inhibitors BAY 11-7082 and NEMO-binding peptide. Chromatin immunoprecipitation analysis demonstrated RelA binding to the cis-NF-kappa B(EGFP) promoter. Adenoviral delivery of NF-kappa B-inducing kinase strongly induced EGFP expression in the liver of cis-NF-kappa B(EGFP) mice. Similarly, mice injected with anti-CD3 or LPS showed increased EGFP expression in mononuclear cells, lymph node, spleen, and liver as measured by flow cytometry and/or fluorescence microscopy. Using whole organ imaging, LPS selectively induced EGFP expression in the duodenum and proximal jejunum, but not in the ileum and colon. Confocal analysis indicated EGFP expression was primarily found in lamina propria mononuclear cells. In summary, the cis-NF-kappa B(EGFP) mouse will serve as a valuable tool to address multiple questions regarding the cell-specific and real-time activation of NF-kappa B during normal and diseased states.

Animals↗

PubMed2004

Regulation of alpha1(I) collagen messenger RNA decay by interactions with alphaCP at the 3'-untranslated region.

Liver fibrosis is characterized by an increased deposition of extracellular matrix proteins, including collagen type I, by activated hepatic stellate cells (HSCs). Previous studies have shown that this increase is mediated primarily by a post-transcriptional mechanism. In particular, the RNA-binding protein alphaCP binds to the alpha1(I) collagen 3'-untranslated region (UTR) and stabilizes this RNA in activated, but not quiescent, HSCs. This study examines the role of alphaCP in the decay of transcripts containing the collagen 3'-UTR in extracts obtained from NIH fibroblasts and quiescent and activated HSCs. Using an in vitro decay system, alphaCP binding activity was competed out with the addition of wild type oligonucleotides, but not with mutant oligonucleotides. Competition of alphaCP binding activity increased the rate of decay of wild type transcripts containing the alphaCP 3'-UTR binding site, but not of transcripts containing a mutated binding site. Quiescent HSC extracts contain no alphaCP binding activity and have no difference in the rate of decay of transcripts with wild type and mutant binding sites for alphaCP. The addition of recombinant alphaCP was sufficient to increase the half-life of the wild type transcript, whereas that of the mutant transcript was minimally changed. In vitro decay assays performed with activated HSC extracts that contain alphaCP binding activity demonstrate a markedly reduced decay rate of wild type compared with mutant transcripts. In vivo small interfering RNA experiments targeting alphaCP showed a reduction of the binding activity of alphaCP and a concomitant reduction in intracellular levels of alpha1(I) collagen messenger RNA. In conclusion, this study demonstrates the direct role of alphaCP in the stabilization of alpha1(I) collagen messenger RNA by blocking RNA degradation in activated HSCs.

3' Untranslated Regions↗