Liver-enriched Transcriptional Activator Protein Research Articles

CCAAT/Enhancer-Binding Protein δ Is a Critical Mediator of Lipopolysaccharide-Induced Acute Lung Injury

Although inflammation plays a central role in the pathogenesis of acute lung injury, the molecular mechanisms underlying inflammatory responses in acute lung injury are poorly understood, and therapeutic options remain limited. CCAAT/enhancer-binding proteins, C/EBPβ and C/EBPδ, are expressed in the lung and have been implicated in the regulation of inflammatory mediators. However, their functions in lung pathobiological characteristics are not well characterized. Herein, we show that C/EBPβ and C/EBPδ are activated in mouse lung after intrapulmonary deposition of lipopolysaccharide (LPS). Mice carrying a targeted deletion of the C/EBPδ gene displayed significant attenuation of the lung permeability index (lung vascular leak of albumin), lung neutrophil accumulation (myeloperoxidase activity), and neutrophils in bronchial alveolar lavage fluids compared with wild-type mice. These phenotypes were consistent with morphological evaluation of lung, which showed reduced inflammatory cell influx and minimal intra-alveolar hemorrhage. Moreover, mutant mice expressed considerably less tumor necrosis factor-α, IL-6, and macrophage inflammatory protein-2 in bronchial alveolar lavage fluids in LPS-injured lung compared with wild-type mice. In contrast, C/EBPβ deficiency had no effect on LPS-induced lung injury. By using small-interfering RNA-mediated knockdown for C/EBPδ, we demonstrate, for the first time to our knowledge, that C/EBPδ plays a critical role for the tumor necrosis factor-α, IL-6, and macrophage inflammatory protein-2 production in LPS-stimulated alveolar macrophages. These findings demonstrate that C/EBPδ, but not C/EBPβ, plays an important role in LPS-induced lung inflammatory responses and injury.

Differentiation-Dependent Changes in Levels of C/EBPβ Repressors and Activators Regulate Human Papillomavirus Type 31 Late Gene Expression

The liver-enriched transcriptional activator protein (LAP) isoform of CCAAT/enhancer binding protein β (C/EBPβ) is shown to be a major activator of differentiation-dependent human papillomavirus (HPV) late gene expression, while the liver-enriched inhibitory protein (LIP) isoform negatively regulates late expression. In undifferentiated cells, LIPs act as dominant-negative repressors of late expression, and upon differentiation, LIP levels are significantly reduced, allowing LAP-mediated activation of the late promoter. Importantly, knockdown of C/EBPβ isoforms blocks activation of late gene expression from complete viral genomes upon differentiation.

Gap‐Junctional Communication Is Required for Mitotic Clonal Expansion during Adipogenesis

Gap-junctional communication (GJC) plays critical roles in cell growth and differentiation. Several studies have demonstrated the involvement of GJC in myogenesis and osteogenesis; however, the role of GJC in adipogenesis has not been fully studied. Thus, we investigated the role of GJC in adipogenesis. 3T3-L1 preadipocytes were differentiated in the presence of gap junction inhibitor, 18-alpha-glycyrrhetinic acid (AGA), and accumulation of cytoplasmic triglycerides was measured. 3T3-L1 cells were transfected with 100 nM small interfering RNA duplexes targeting connexin (Cx) 43. The mRNA levels of CCAAT/enhancer-binding protein (C/EBP) alpha, peroxisome proliferator-activated receptor gamma, glucose transporter 4, C/EBPbeta, and Cx43 were measured by real-time polymerase chain reaction. The protein levels of C/EBPbeta were quantitated by Western blotting. The cell proliferation was measured by counting cell numbers, and DNA synthesis was measured by bromodeoxyuridine incorporation. AGA inhibited adipocyte differentiation dose-dependently. The lipid accumulation and the mRNA levels of C/EBPalpha, peroxisome proliferator-activated receptor gamma, and glucose transporter 4 were markedly reduced in AGA-treated adipocytes. The mRNA levels of C/EBPbeta did not decrease; however, C/EBPbeta [liver-enriched transcriptional activator protein (LAP)] expression and the C/EBPbeta (LAP)-to-C/EBP [liver-enriched transcriptional inhibitory protein (LIP)] ratio were reduced by AGA treatment. The increase in both cell number and DNA synthesis, which occurs during mitotic clonal expansion, was reduced by AGA in a dose-dependent fashion. The major component of gap junctions in 3T3-L1 cells was Cx43. Down-regulation of Cx43 using small interfering RNA reduced the expression of C/EBPbeta (LAP) and inhibited adipogenesis. Our data suggest that GJC plays some important roles in adipogenesis through inhibiting mitotic clonal expansion and modulating C/EBPbeta (LAP) expression.

Conditional expression of liver‐enriched transcriptional activator protein augments Acholeplasma laidlawii‐induced granulysin gene expression in a human monocytic cell line, THP‐1

The antimicrobial protein granulysin is considered to play an important role in the defence mechanism against bacterial infection. We previously reported that Acholeplasma laidlawii-induced transactivation of the granulysin promoter in a human monocytic cell line, THP-1, is regulated by activator protein-1 and CCAAT/enhancer binding protein-beta (C/EBPbeta), but not by nuclear factor-kappaB. Moreover, liver-enriched transcriptional inhibitory protein (LIP), a C/EBPbeta isoform, was strongly induced in A. laidlawii-stimulated THP-1 cells. However, the level of liver-enriched transcriptional activator protein (LAP), another C/EBPbeta isoform, was essentially constant. Accordingly, we speculated that LIP would down-regulate A. laidlawii-induced granulysin gene expression in THP-1 cells. In the present study, we examined whether LAP augments A. laidlawii-induced granulysin gene expression using conditional LAP-expressing THP-1 cells in a tetracycline-controlled expression system. Our results indicated that conditional expression of LAP augmented A. laidlawii-induced expression of granulysin mRNA. In addition, the granulysin protein was observed in A. laidlawii-stimulated, LAP-expressing THP-1 cells. Our results suggest that the expression of LAP plays a critical role in the expression of the granulysin gene in macrophages.

Opposing roles of activator protein-1 and CCAAT/enhancer binding protein beta in the regulation of inducible granulysin gene expression in a human monocytic cell line, THP-1.

We previously reported that inducible granulysin gene expression in a human monocytic cell line, THP-1 is dominantly dependent on transcription factor activator protein-1 (AP-1). Here, we further examined the precise regulatory mechanisms underlying granulysin gene expression using THP-1 cells treated with Acholeplasma laidlawii. Transfection of reporter gene constructs into THP-1 cells indicated that the presence of a positive regulatory element(s) is located from -329 to -85 base pairs, containing two distinct AP-1 binding sites and one nuclear factor-kappaB (NF-kappaB) binding site. Deletion or mutation of the NF-kappaB binding site failed to affect inducible promoter activity, whereas deletion or mutation of both the AP-1 binding sites abrogated the promoter activity. Interestingly, deletion of the putative CCAAT/enhancer binding protein beta (C/EBPbeta) binding site upstream of the positive regulatory element induced the augmentation of granulysin promoter activity. Electrophoretic mobility shift assays demonstrated that nuclear extract prepared from A. laidlawii-treated THP-1 cells generated a specific binding to oligonucleotides, including AP-1, C/EBPbeta, and NF-kappaB element. Furthermore, over-expression of liver-enriched transcriptional activator protein, a subunit of C/EBPbeta, augmented A. laidlawii-induced granulysin promoter activity, whereas over-expression of liver-enriched transcriptional inhibitory protein inhibited the promoter activity. NF-kappaB p50 homodimer had no transactivation property, although it bound to the NF-kappaB site. These results indicate that AP-1 and C/EBPbeta, but not NF-kappaB participate in the regulation of inducible granulysin gene expression in THP-1 cells. Moreover, the Toll-like receptor 2-dependent signalling pathway may be involved in A. laidlawii-induced transactivation of the granulysin promoter. Thus, these results suggest that the gene expression of granulysin in macrophages would be exquisitely regulated by positive and negative transcription factors when microbial invasion occurs.

Insulin Inhibits Hepatocellular Glucose Production by Utilizing Liver-enriched Transcriptional Inhibitory Protein to Disrupt the Association of CREB-binding Protein and RNA Polymerase II with the Phosphoenolpyruvate Carboxykinase Gene Promoter

Hormones regulate glucose homeostasis, in part, by controlling the expression of gluconeogenic enzymes, such as phosphoenolpyruvate carboxykinase (PEPCK). Insulin and glucocorticoids reciprocally regulate PEPCK expression primarily at the level of gene transcription. We demonstrate here that glucocorticoids promote, whereas insulin disrupts, the association of CREB-binding protein (CBP) and RNA polymerase II with the hepatic PEPCK gene promoter in vivo. We also show that accessory factors, such as CCAAT/enhancer-binding protein beta (C/EBP beta), can recruit CBP to drive transcription. Insulin increases protein levels of liver-enriched transcriptional inhibitory protein (LIP), an inhibitory form of C/EBP beta, in a phosphatidylinositol 3-kinase-dependent manner. LIP concomitantly replaces liver-enriched transcriptional activator protein on the PEPCK gene promoter, which can abrogate the recruitment of CBP and polymerase II, culminating in the repression of PEPCK expression and the attenuation of hepatocellular glucose production.

Transcriptional regulation of human papillomavirus type 16 LCR by different C/EBP? isoforms

During genital human papillomavirus (HPV) infection several cytokines are released, such as interleukin-1 (IL-1), tumor necrosis factoralpha (TNFalpha), IL-6, and IL-8. These cytokines may play a role in the immune surveillance against viral infection. Two of these cytokines, IL-1 and TNFalpha, suppress the transcription of the HPV16 early genes. CAATT/ enhancer binding protein, (C/EBPbeta), which is activated by IL-1 and TNFalpha, has been suggested to act as a mediator of this transcriptional downregulation. C/EBPbeta contains three different translation initiation sites that can lead probably by leaky ribosome scanning to the generation of three isoforms of C/EBPbeta, namely full-length C/EBPbeta, liver enriched transcriptional activator protein (LAP), and liver enriched inhibitory protein (LIP). When transiently expressed in C33A and HeLa cells, the first two C/EBPbeta isoforms activate the HPV16 long control region (LCR). LIP, which acts as an antagonist of C/EBPbeta, represses the HPV16 LCR activity. Our observation that treatment of HeLa cells with IL-1 leads to induction of LIP supports the hypothesis that the LCR downregulation by IL-1 is mediated by LIP.

C/EBPalpha regulates generation of C/EBPbeta isoforms through activation of specific proteolytic cleavage.

C/EBPalpha and C/EBPbeta are intronless genes that can produce several N-terminally truncated isoforms through the process of alternative translation initiation at downstream AUG codons. C/EBPbeta has been reported to produce four isoforms: full-length 38-kDa C/EBPbeta, 35-kDa LAP (liver-enriched transcriptional activator protein), 21-kDa LIP (liver-enriched transcriptional inhibitory protein), and a 14-kDa isoform. In this report, we investigated the mechanisms by which C/EBPbeta isoforms are generated in the liver and in cultured cells. Using an in vitro translation system, we found that LIP can be generated by two mechanisms: alternative translation and a novel mechanism-specific proteolytic cleavage of full-length C/EBPbeta. Studies of mice in which the C/EBPalpha gene had been deleted (C/EBPalpha-/-) showed that the regulation of C/EBPbeta proteolysis is dependent on C/EBPalpha. The induction of C/EBPalpha in cultured cells leads to induced cleavage of C/EBPbeta to generate the LIP isoform. We characterized the cleavage activity in mouse liver extracts and found that the proteolytic cleavage activity is specific to prenatal and newborn livers, is sensitive to chymostatin, and is completely abolished in C/EBPalpha-/- animals. The lack of cleavage activity in the livers of C/EBPalpha-/- mice correlates with the decreased levels of LIP in the livers of these animals. Analysis of LIP production during liver regeneration showed that, in this system, the transient induction of LIP is dependent on the third AUG codon and most likely involves translational control. We propose that there are two mechanisms by which C/EBPbeta isoforms might be generated in the liver and in cultured cells: one that is determined by translation and a second that involves C/EBPalpha-dependent, specific proteolytic cleavage of full-length C/EBPbeta. The latter mechanism implicates C/EBPalpha in the regulation of posttranslational generation of the dominant negative C/EBPbeta isoform, LIP.

Vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, and noradrenaline induce the transcription factors CCAAT/enhancer binding protein (C/EBP)-beta and C/EBP delta in mouse cortical astrocytes: involvement in cAMP-regulated glycogen metabolism

We have described previously a transcription-dependent induction of glycogen resynthesis by the vasoactive intestinal peptide (VIP) or noradrenaline (NA) in astrocytes, which is mediated by cAMP. Because it has been postulated that the cAMP-mediated regulation of energy balance in hepatocytes and adipocytes is channeled at least in part through the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, we tested the hypothesis that C/EBP isoforms could be expressed in mouse cortical astrocytes and that their level of expression could be regulated by VIP, by the VIP-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP), or by NA. We report in this study that in these cells, C/EBP beta and C/EBP delta are induced by VIP, PACAP, or NA via the cAMP second-messenger pathway. Induction of C/EBP beta and -delta mRNA by VIP occurs in the presence of a protein synthesis inhibitor. Thus, c/ebp beta and c/ebp delta behave as cAMP-inducible immediate-early genes in astrocytes. Moreover, transfection of astrocytes with expression vectors selectively producing the transcriptionally active form of C/EBP beta, termed liver-enriched transcriptional activator protein, or C/EBP delta enhance the glycogen resynthesis elicited by NA, whereas an expression vector producing the transcriptionally inactive form of C/EBP beta, termed liver-enriched transcriptional inhibitory protein, reduces this resynthesis. These results support the idea that C/EBP beta and -delta regulate gene expression of energy metabolism-related enzymes in astrocytes.

Transactivation of LAP/NF-IL6 Is Mediated by an Acidic Domain in the N-terminal Part of the Protein

LAP/NF-IL6 is a member of the C/EBP family of transcriptional activators and has been shown to be involved in the regulation of the acute-phase response. We have previously shown that phosphorylation of the liver-enriched transcriptional activator protein (LAP) Ser-105 enhances the activation of LAP-dependent genes. To identify the region which is important for gene activation, a series of LAP mutants were constructed, and domain swapping experiments with the DNA-binding domain of GAL4 were performed. These experiments point to an acidic region located between amino acids 21 and 105 of LAP/NF-IL6 which activates genes independent of the DNA-binding domain and the leucine zipper of LAP/NF-IL6. Computer-assisted predictions reveal two regions, a helical and a hydrophobic region in the transactivation domain, which could be important in mediating the direct interaction with the basal machinery. Site-directed mutagenesis of acidic residues in both regions demonstrates that the hydrophobic region located between amino acids 85 and 95 is the likely motif for the interaction with the basal machinery. Our results demonstrate that a hydrophobic region in the acidic transactivation domain of LAP/NF-IL6 seems to be relevant in mediating gene activation of LAP-dependent genes.

Regulated expression of alpha 2,6-sialyltransferase by the liver-enriched transcription factors HNF-1, DBP, and LAP.

Tissue-specific carbohydrate structures are thought to result from the selective expression of specific terminal glycosyltransferases responsible for their synthesis. However, little is known about the regulation of the expression of these enzymes. Previous analysis of the distribution of one such enzyme, beta-galactoside alpha 2,6-sialyltransferase, revealed that its expression is tissue restricted, with highest levels being found in the liver. Examination of the gene suggested that its expression is regulated at the level of transcription by multiple promoters, one of which is strongly active in the liver. In this present work, an analysis of the liver-restricted promoter was undertaken to identify the promoter elements necessary for liver-restricted expression. Footprinting studies, 5' deletion analysis, and site-directed mutagenesis identified two cis-elements which were potentially important in the tissue-specific expression of this promoter. One of these elements contains a consensus binding site for the liver-enriched transcription factor hepatocyte nuclear factor-1 alpha, while the other is a consensus binding site for the liver-specific factors D-binding protein and liver-enriched transcriptional activator protein. Expression vectors containing cDNAs of these factors are capable of trans-activating transcription of the alpha 2,6ST promoter, demonstrating their ability to regulate transcription of this promoter. Together, these results suggest that tissue-specific glycosylation can be regulated at the level of transcription by the same factors involved in the expression of a number of other tissue-specific genes.

The role of the transcriptional activator protein DBP in circadian liver gene expression.

DBP, a liver-enriched transcriptional activator protein of the leucine zipper protein family, accumulates according to a very strong circadian rhythm (amplitude approx. 1000-fold). In rat parenchymal hepatocytes, the protein is barely detectable during the morning hours. At about 2 p.m., DBP levels begin to rise, reach maximal levels at 8 p.m. and decline sharply during the night. This rhythm is free-running: it persists with regard to both its amplitude and phase in the absence of external time cues, such as daily dark/light switches. Also, fasting of rats for several days influences neither the amplitude nor the phase of circadian DBP expression. Since the levels of DBP mRNA and nascent transcripts also oscillate with a strong amplitude, circadian DBP expression is transcriptionally controlled. While DBP mRNA fluctuates with a similar phase and amplitude in most tissues examined, DBP protein accumulates to high concentrations only in liver nuclei. Hence, at least in nonhepatic tissues, cyclic DBP transcription is unlikely to be controlled by a positive and/or negative feedback mechanism involving DBP itself. More likely, the circadian DBP expression is governed by hormones whose peripheral concentrations also oscillate during the day. Several lines of evidence suggest a pivotal role of glucocorticoid hormones in establishing the DBP cycle. Two genes whose mRNAs and protein products accumulate according to a strong circadian rhythm with a phase compatible with regulation by DBP encode enzymes with key functions in cholesterol metabolism: HMG-coA reductase is the rate-limiting enzyme in cholesterol synthesis; cholesterol 7-alpha hydroxylase performs the rate-limiting step in the conversion of cholesterol to bile acid.(ABSTRACT TRUNCATED AT 250 WORDS)

A liver-enriched transcriptional activator protein, LAP, and a transcriptional inhibitory protein, LIP, are translated from the sam mRNA

LAP, a transcriptional activator, and LIP, a transcriptional repressor, are translated from a single mRNA species by using two AUGs within the same reading frame. These two proteins share the 145 C-terminal amino acids that contain the basic DNA-binding domain and the leucine zipper dimerization helix. Probably owing to its higher affinity for its DNA cognate sequences, LIP can attenuate the transcriptional stimulation by LAP in substoichiometric amounts. As revealed by transient transfection experiments, a moderate increase in the LAP LIP ratio results in a significantly higher transcriptional activation of an appropriate target gene. The LAP LIP ratio increases about 5-fold during terminal rat liver differentiation and is thus likely to modulate the activity of LAP in the intact animal.

Expression of the liver-enriched transcriptional activator protein DBP follows a stringent circadian rhythm

The liver-enriched transcriptional activator protein DBP accumulates in hepatocytes of adult rats according to a strictly controlled circadian rhythm. DBP is not detectable in liver nuclei during the morning hours. Its level raises sharply during the afternoon and reaches a maximum at about 8 p.m. During the night the cellular DBP concentration decreases below detectability. This oscillation is “free running,” transcriptionally regulated, and may be under the negative control of glucocorticoid hormones. In keeping with the rhythmicity of DBP accumulation, the albumin gene, a putative target of DBP, is transcribed more efficiently in the evening than in the morning.

LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein.

A gene, encoding a liver-enriched transcriptional activator protein (LAP) has been isolated. LAP is a 32-kD protein that stimulates the transcription of chimeric genes containing albumin D-promoter elements both in vivo and in vitro. LAP shares extensive sequence homology (71%) in its DNA-binding and leucine zipper domains with C/EBP. As a consequence, these two proteins show an indistinguishable DNA-binding specificity and readily heterodimerize. In addition, both genes, lap and cebp, are devoid of intervening sequences. Although correctly initiated transcripts from the LAP gene accumulate in the six examined tissues--liver, lung, spleen, kidney, brain, and testis--LAP protein is highly enriched in liver nuclei. Thus, the preferential accumulation of LAP protein in liver appears to be regulated post-transcriptionally.