Characterization of the transcriptional signature of C/EBPbeta isoforms (LAP/LIP) in Hep3B cells: implication of LIP in pro-survival functions.

BACKGROUND & AIMS: C/EBPbeta is an important mediator of several cellular processes, such as differentiation, proliferation, and survival of hepatic cells. However, a complete catalog of the targets of C/EBPbeta or the mechanism by which this transcription factor regulates certain liver-dependent pathways has not been clearly determined. Two major natural isoforms of this transcription factor exist: the liver-enriched activating protein (LAP) and the liver-enriched inhibitory protein (LIP), a functional LAP antagonist. In this study, we used the opposing transcriptional effects driven by LAP and LIP to determine the genuine C/EBPbeta molecular signature in the Hep3B human hepatoma cell line. We subsequently investigated the role of each of the LAP and LIP isoforms in drug-induced Hep3B cell death. METHODS: We engineered Hep3B cells with regulated LAP or LIP expression using the Tet-off expression system. The genes that showed inverse regulation by LAP and LIP were identified by cDNA array analysis. The cohort of direct-C/EBPbeta-targets was distinguished from indirect-targets by ChIP-on-chip analysis. RESULTS: We characterized 676 genes by this approach. Among these genes, 39 are novel direct targets of C/EBPbeta. Eleven of these new direct targets are involved in cell survival, suggesting critical roles for LAP/LIP isoforms in this cellular process. Therefore, we examined the effects of LAP and LIP over-expression on cell survival. We show that LIP promotes survival in staurosporine- or taxol-induced Hep3B cell death. CONCLUSIONS: Our study provides new molecular and cellular insights into the role of C/EBPbeta in cells of hepatic origin.

Gene profiling predicts rheumatoid arthritis responsiveness to IL-1Ra (anakinra).

OBJECTIVES: The overall non-response rate to biologics remains 30-40% for patients with RA resistant to MTX. The objective of this study was to predict responsiveness to the anakinra-MTX combination by peripheral blood mononuclear cell gene profiling in order to optimize treatment choice. METHODS: Thirty-two patients treated with anakinra (100 mg/day s.c.) and MTX were categorized as responders when their 28-joint DAS (DAS-28) had decreased by ≥1.2 at 3 months. Pre-treatment blood samples had been drawn. RESULTS: For seven responders and seven non-responders, 52 microarray-identified mRNAs were expressed as a function of the response to treatment, and unsupervised hierarchical clustering correctly separated responders from non-responders. The levels of seven of these 52 transcripts, as assessed by real-time, quantitative RT-PCR, were able to accurately classify 15 of 18 other patients (8 responders and 10 non-responders), with 87.5% specificity and 77.8% negative-predictive value for responders. Among the 52 genes, 56% were associated with IL-1ß. CONCLUSION: This predictive gene expression profile was obtained with a non-invasive procedure. After further validation in other cohorts of patients, it could be proposed and used on a large scale to select likely RA responders to combined anakinra-MTX. Trial registration. Clinical Trials; NCT00213538 (http://www.clinicaltrials.gov).

Novel serum markers of fibrosis progression for the follow-up of hepatitis C virus-infected patients.

Liver biopsy is considered the gold-standard method for the assessment of liver fibrosis during follow-up of hepatitis C virus-infected patients, but this invasive procedure is not devoid of complications. The aim of the present study was to identify novel non-invasive markers of fibrosis progression. By microarray analysis, we compared transcript levels in two extreme stages of fibrosis from 16 patients. Informative transcripts were validated by real-time PCR and used for the assessment of fibrosis in 23 additional patients. Sixteen transcripts were found to be dysregulated during the fibrogenesis process. Among them, some were of great interest because their corresponding proteins could be serologically measured. Thus, the protein levels of inter-alpha inhibitor H1, serpin peptidase inhibitor clade F member 2, and transthyretin were all significantly different according to the four Metavir stages of fibrosis. In conclusion, we report here that dysregulation, at both the transcriptional and protein levels, exists during the fibrogenesis process. Our description of three novel serum markers and their potential use as serological tests for the non-invasive diagnosis of liver fibrosis open new opportunities for better follow-up of hepatitis C virus-infected patients.

Can rheumatoid arthritis responsiveness to methotrexate and biologics be predicted?

This review briefly recapitulates the existing markers predictive of RA responsiveness to treatment, focusing on MTX alone or combined with a biologic. In addition to the demographic and clinical factors, an update is provided of the predictive biomarkers identified by large-scale gene and protein analyses that generated new insights into the ability of high-throughput analysis of biological systems to select new potential indicators. Among the large-scale analysis tools now available, pharmacogenetics and pharmacogenomics (including transcriptomic and proteomic approaches) have been shown to provide such new putative biomarkers of therapeutic responses.

Down-regulated expression of the TSAP6 protein in liver is associated with a transition from cirrhosis to hepatocellular carcinoma.

AIMS: Hepatocellular carcinoma (HCC) results from cirrhosis and, in Western Europe, hepatitis C virus and alcoholism are the predominant causes of this disease. We recently documented a global transcript repression in hepatocarcinoma nodules. The tumour suppressor activated pathway-6 (TSAP6) transcript codes for a transmembrane molecule that is an inducer of a caspase-3-dependent apoptotic pathway. The down-regulation of TSAP6 transcripts in HCC and perinodular cirrhosis, which contrasts with a sustained transcript level in HCC-free cirrhosis, has suggested that this hepatic protein level may provide a prognostic marker for HCC occurrence. METHODS AND RESULTS: This protein was quantified by semiquantitative assessment of immunohistochemistry on samples from 42 cases HCC-free cirrhosis, 49 cases cirrhosis with HCC, 43 HCC associated with healthy liver and 31 controls. TSAP6 expression was linked to the liver state, healthy or cirrhotic without or with an HCC and to tumour grade. CONCLUSIONS: With biopsies periodically performed for surveillance purposes, the decreased expression of TSAP6 in cirrhotic tissue could reflect a decrease in the apoptotic process and could be interpreted as a warning sign. This evaluation of the TSAP6 level in cirrhotic liver conveys predictive information for the development of HCC.

Tumor necrosis factor (TNF)-alpha persistently activates nuclear factor-kappaB signaling through the type 2 TNF receptor in chromaffin cells: implications for long-term regulation of neuropeptide gene expression in inflammation.

Chromaffin cells of the adrenal medulla elaborate and secrete catecholamines and neuropeptides for hormonal and paracrine signaling in stress and during inflammation. We have recently documented the action of the cytokine TNF-alpha on neuropeptide secretion and biosynthesis in isolated bovine chromaffin cells. Here, we demonstrate that the type 2 TNF-alpha receptor (TNF-R2) mediates TNF-alpha signaling in chromaffin cells via activation of nuclear factor (NF)-kappaB. Microarray and suppression subtractive hybridization have been used to identify TNF-alpha target genes in addition to those encoding the neuropeptides galanin, vasoactive intestinal polypeptide, and secretogranin II in chromaffin cells. TNF-alpha, acting through the TNF-R2, causes an early up-regulation of NF-kappaB, long-lasting induction of the NF-kappaB target gene inhibitor kappaB (IkappaB), and persistent stimulation of other NF-kappaB-associated genes including mitogen-inducible gene-6 (MIG-6), which acts as an IkappaB signaling antagonist, and butyrate-induced transcript 1. Consistent with long-term activation of the NF-kappaB signaling pathway, delayed induction of neuropeptide gene transcription by TNF-alpha in chromaffin cells is blocked by an antagonist of NF-kappaB signaling. TNF-alpha-dependent signaling in neuroendocrine cells thus leads to a unique, persistent mode of NF-kappaB activation that features long-lasting transcription of both IkappaB and MIG-6, which may play a role in the long-lasting effects of TNF-alpha in regulating neuropeptide output from the adrenal, a potentially important feedback station for modulating long-term cytokine effects in inflammation.

Genome-wide differences in hepatitis C- vs alcoholism-associated hepatocellular carcinoma.

AIM: To look at a comprehensive picture of etiology-dependent gene abnormalities in hepatocellular carcinoma in Western Europe. METHODS: With a liver-oriented microarray, transcript levels were compared in nodules and cirrhosis from a training set of patients with hepatocellular carcinoma (alcoholism, 12; hepatitis C, 10) and 5 controls. Loose or tight selection of informative transcripts with an abnormal abundance was statistically valid and the tightly selected transcripts were next quantified by qRTPCR in the nodules from our training set (12 + 10) and a test set (6 + 7). RESULTS: A selection of 475 transcripts pointed to significant gene over-representation on chromosome 8 (alcoholism) or -2 (hepatitis C) and ontology indicated a predominant inflammatory response (alcoholism) or changes in cell cycle regulation, transcription factors and interferon responsiveness (hepatitis C). A stringent selection of 23 transcripts whose differences between etiologies were significant in nodules but not in cirrhotic tissue indicated that the above dysregulations take place in tumor but not in the surrounding cirrhosis. These 23 transcripts separated our test set according to etiologies. The inflammation-associated transcripts pointed to limited alterations of free iron metabolism in alcoholic vs hepatitis C tumors. CONCLUSION: Etiology-specific abnormalities (chromo-some preference; differences in transcriptomes and related functions) have been identified in hepatocellular carcinoma driven by alcoholism or hepatitis C. This may open novel avenues for differential therapies in this disease.

The inflammation-induced down-regulation of plasma Fetuin-A (alpha2HS-Glycoprotein) in liver results from the loss of interaction between long C/EBP isoforms at two neighbouring binding sites.

Fetuin-A is an hepatic protein whose mRNA transiently falls during the inflammatory acute phase via unknown transcriptional mechanisms. Various FETUA promoter/cat constructs transiently transfected in the Hep3B hepatoma cell line allowed us to identify four NF-1 and C/EBP binding sites (N, C) arranged in a 5'-N2-C2-N1-C1-3' order and required for basal promoter activity. Mutant constructs demonstrated that C1 and C2 but not N1 nor N2 are required for the cytokine-driven down-regulation of the promoter. A variable spacing between C2 and N1 showed that the alignment of the (C1+N1) and (C2+N2) areas is critical for the promoter activity in quiescent but not cytokine-stimulated cells. Co-transfection of a plasmid only producing either a long or short C/EBPbeta isoform prevented FETUA regulation by cytokines. Electromobility shift assays with liver nuclear extracts showed that during the acute phase the complexes formed over N1 and N2 are not modified whereas short C/EBPalpha and -beta isoforms replace the long isoforms bound to C1 and C2 in the quiescent liver. Therefore the basal promoter activity requires an interaction between the long C/EBP isoforms bound to C1 and C2 whereas the inflammation-induced down-regulation results from the loss of interaction between the cytokine-induced, short C/EBP isoforms.

Microarray and suppression subtractive hybridization analyses of gene expression in pheochromocytoma cells reveal pleiotropic effects of pituitary adenylate cyclase-activating polypeptide on cell proliferation, survival, and adhesion.

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts trophic effects on several neuronal, neuroendocrine, and endocrine cells. To gain insight into the pattern of the transcriptional modifications induced by PACAP during cell differentiation, we studied the effects of this neuropeptide on rat pheochromocytoma PC12 cells. We first analyzed the transcriptome of PC12 cells in comparison to that of terminally differentiated rat adrenomedullary chromaffin cells, using a high-density microarray, to identify genes associated with the proliferative phenotype that are possible targets of PACAP during differentiation of sympathoadrenal normal and tumoral cells. We then studied global gene expression in PC12 cells after 48 h of exposure to PACAP, using both cDNA microarray and suppression subtractive hybridization technologies. These complementary approaches resulted in the identification of 75 up-regulated and 70 down-regulated genes in PACAP-treated PC12 cells. Among the genes whose expression is modified in differentiated cells, a vast majority are involved in cell proliferation, survival, and adhesion/motility. Expression changes of most of these genes have been associated with progression of several neoplasms. A kinetic study of the effects of PACAP on some of the identified genes showed that the neuropeptide likely exerts early as well as late actions to achieve the gene expression program necessary for cell differentiation. In conclusion, the results of the present study underscore the pleiotropic role of PACAP in cell differentiation and provide important information on novel targets that could mediate the effects of this neuropeptide in normal and tumoral neuroendocrine cells.

An interplay of Sp1, GKLF and CREB-2 controls human Pre-alpha-Inhibitor gene (ITIH3) transcription.

Pre-alpha-Inhibitor is a plasma protease inhibitor and a heterodimeric molecule whose one polypeptide chain is encoded by the ITIH3 gene. In order to understand the expression of this protein that is regulated in health and disease, we have analyzed the 5' flanking region of ITIH3, specifically focussing on its proximal promoter. A combination of methods including wild-type (wt) or mutant promoter linked to a reporter cat gene, co-transfections of cat constructs with expression plasmids for nuclear factors and electrophoretic mobility shift assays revealed that two antagonistic sets of regulatory elements and nuclear proteins are critical for the activity of this promoter. Indeed, several overlapping Sp1/Sp3-binding sites are required for a sustained activity. However, a tripartite complex including CREB-2 and two molecules of the gut-enriched, Krüppel-like factor cooperate to bind to an upstream area whose 3' end overlaps the Sp1-binding sites. The resulting competition between this tripartite complex and Sp1 results in impaired occupancy of Sp1-binding sites by Sp1 and a consequent reduction in ITIH3 transcription. Competition between Sp1 and a Krüppel-like factor for GC-rich sites has been previously reported, but this is the first description of an elaborate tripartite cooperation of two Krüppel-like factors and CREB as a key step in such a competition.

A new tool for rheumatology: large-scale analysis of gene expression.

Large-scale analysis of gene expression with cDNA arrays is spreading over many biological fields, including rheumatology. In this report, we wish to explain the principle and main advantages of this tool in the context of our discipline. Until 1995, analysis of gene expression was conducted for a few genes at a time but DNA chips now allow one to monitor the expression of thousands of genes in a single experiment and analyze the transcriptome, i.e. the whole of the transcripts in a given cell or tissue. Whatever the platform used (macro- or microarrays, oligo-chips), this technology rests upon the hybridization of i) a set of cDNA clones tethered to a solid support (nylon or glass) as probes, and ii) labelled cDNAs that are reverse-transcribed from bulk mRNAs extracted from a cell or tissue sample as a target. The end result is information on the relative abundance of every mRNA between two or more samples. The transcriptome analysis has two main objectives in rheumatology: i) identifying a gene expression profile that is a hallmark of a pathology and using it for a diagnostic or prognostic purpose, and ii) gathering genes with similar changes of expression, which allows one to specify the identity of novel proteins involved in a well-known intracellular cascade of regulation or even to identify new cascades.

Transient and etiology-related transcription regulation in cirrhosis prior to hepatocellular carcinoma occurrence.

AIM: To search for transcription dysregulation that could (1) differentiate hepatocellular carcinoma (HCC)-free from HCC-related cirrhosis (2) differentiate HCC-free cirrhosis related to HCV from that related to alcohol intake. METHODS: Using microarray analysis, we compared transcript levels in HCC-free cirrhosis (alcoholism: 7; hepatitis C: 7), HCC-associated cirrhosis (alcoholism: 10; hepatitis C: 10) and eight control livers. The identified transcripts were validated by qRT-PCR in an independent cohort of 45 samples (20 HCC-free cirrhosis; 15 HCC-associated cirrhosis and 10 control livers). We also confirmed our results by immunohistochemistry. RESULTS: In HCC-free livers, we identified 70 transcripts which differentiated between alcoholic-related cirrhosis, HCV-related cirrhosis and control livers. They mainly corresponded to down-regulation. Dysregulation of Signal Transduction and Activator of Transcription-3 (STAT-3) was found along with related changes in STAT-3 targets which occurred in an etiology-dependent fashion in HCC-free cirrhosis. In contrast, in HCC, such transcription dysregulations were not observed. CONCLUSION: We report that transcriptional dysregulations exist in HCC-free cirrhosis, are transiently observed prior to detectable HCC onset and may be appear like markers from cirrhosis to HCC transition.

Early and long-standing rheumatoid arthritis: distinct molecular signatures identified by gene-expression profiling in synovia.

INTRODUCTION: Rheumatoid arthritis (RA) is a heterogeneous disease and its underlying molecular mechanisms are still poorly understood. Because previous microarray studies have only focused on long-standing (LS) RA compared to osteoarthritis, we aimed to compare the molecular profiles of early and LS RA versus control synovia. METHODS: Synovial biopsies were obtained by arthroscopy from 15 patients (4 early untreated RA, 4 treated LS RA and 7 controls, who had traumatic or mechanical lesions). Extracted mRNAs were used for large-scale gene-expression profiling. The different gene-expression combinations identified by comparison of profiles of early, LS RA and healthy synovia were linked to the biological processes involved in each situation. RESULTS: Three combinations of 719, 116 and 52 transcripts discriminated, respectively, early from LS RA, and early or LS RA from healthy synovia. We identified several gene clusters and distinct molecular signatures specifically expressed during early or LS RA, thereby suggesting the involvement of different pathophysiological mechanisms during the course of RA. CONCLUSIONS: Early and LS RA have distinct molecular signatures with different biological processes participating at different times during the course of the disease. These results suggest that better knowledge of the main biological processes involved at a given RA stage might help to choose the most appropriate treatment.

Gene profiling in white blood cells predicts infliximab responsiveness in rheumatoid arthritis.

As indicators of responsiveness to a tumour necrosis factor (TNF)alpha blocking agent (infliximab) are lacking in rheumatoid arthritis, we have used gene profiling in peripheral blood mononuclear cells to predict a good versus poor response to infliximab. Thirty three patients with very active disease (Disease Activity Score 28 >5.1) that resisted weekly methotrexate therapy were given infliximab at baseline, weeks 2 and 6, and every 8th week thereafter. The patients were categorized as responders if a change of Disease Activity Score 28 = 1.2 was obtained at 3 months. Mononuclear cell RNAs were collected at baseline and at three months from responders and non-responders. The baseline RNAs were hybridised to a microarray of 10,000 non-redundant human cDNAs. In 6 responders and 7 non-responders, 41 mRNAs identified by microarray analysis were expressed as a function of the response to treatment and an unsupervised hierarchical clustering perfectly separated these responders from non-responders. The informativeness of 20 of these 41 transcripts, as measured by qRT-PCR, was re-assessed in 20 other patients. The combined levels of these 20 transcripts properly classified 16 out of 20 patients in a leave-one-out procedure, with a sensitivity of 90% and a specificity of 70%, whereas a set of only 8 transcripts properly classified 18/20 patients. Trends for changes in various transcript levels at three months tightly correlated with treatment responsiveness and a down-regulation of specific transcript levels was observed in non-responders only. Our gene profiling obtained by a non-invasive procedure should now be used to predict the likely responders to an infliximab/methotrexate combination.

Genome-wide response of the human Hep3B hepatoma cell to proinflammatory cytokines, from transcription to translation.

Given the unknown timing of the onset of an acute systemic inflammation in humans, the fine tuning of cascades and pathways involved in the associated hepatocyte response cannot be appraised in vivo. Therefore, the authors used a genome-wide and kinetic analysis in the human Hep3B hepatoma cell line challenged with a conditioned medium from bacterial lipopolysaccharide-stimulated macrophages. A complete coverage of the liver transcriptome disclosed 648 mRNAs whose change in abundance allowed for their clustering in mRNA subsets with an early, intermediate, or late regulation. The contribution of transcription, stability, or translation was appraised with genome-wide studies of the changes in nuclear primary transcripts, mRNA decay, or polysome-associated mRNAs. A predominance of mRNAs with decreased stability and the fact that translation alone controls a significant number of acute phase-associated proteins are prominent findings. Transcription and stability act independently or, more rarely, cooperate or even counteract in a gene-by-gene manner, which results in a unidirectional change in mRNA abundance. Waves of mRNAs for groups of functionally related proteins are up- or downregulated in an ordered fashion. This includes an early regulation of transcription-associated proteins, an intermediate repression of detoxication and metabolism proteins, and finally an enhanced translation and transport of a number of membranous or secreted proteins along with an enhanced protein degradation. In conclusion, this study provides a comprehensive and simultaneous overview of events in the human hepatocyte during the inflammatory acute phase.

Global gene repression in hepatocellular carcinoma and fetal liver, and suppression of dudulin-2 mRNA as a possible marker for the cirrhosis-to-tumor transition.

BACKGROUND/AIMS: Whether the transcriptional reprogramming process induced by hepatocellular carcinoma recapitulates that of the developing liver is at present unclear. METHODS: With a complete coverage of the liver transcriptome by microarray using adult livers as controls, we searched for similarities and differences in mRNA abundances between hepatocellular carcinoma nodules and fetal livers taken before (early) or after (late) the 22-24th week of gestation. Changes in some mRNA levels were studied in further liver samples by quantitative RT-PCR. RESULTS: Altered gene expression in hepatocellular carcinoma mostly results in down-regulated mRNAs which largely overlap with those repressed in the late fetal liver. Different frequencies of transcription factor binding sites in the down-regulated genes vs control genes as well as changes in abundance of mRNAs for relevant transcription factors point to a transcriptional repression. The down-regulated mRNAs code for proteins involved in (i) transcription and translation, (ii) specific functions of the differentiated hepatocyte or (iii) activation of proliferation and apoptosis. CONCLUSIONS: Apoptosis limitation is likely to predominate over active proliferation during liver development and hepatocellular carcinoma. Repression of the apoptosis-associated dudulin-2 mRNA points to a potential marker for the transition from a carcinoma-free to carcinoma-associated cirrhosis.

The liver-specific human alpha(1)-microglobulin/bikunin precursor (AMBP) is capable of self-association.

alpha-1-Microglobulin (A1M) and bikunin are two plasma glycoproteins encoded by an alpha-1-microglobulin/bikunin precursor (AMBP) gene. Despite their lack of any structural or functional relationship, both A1M and bikunin originate from AMBP cleavage by a furin-like protease that releases the two mature molecules. The AMBP gene maintains a tight control over its expression by a unique enhancer, which is controlled by several hepatocyte-enriched nuclear factors; however, the mechanisms of regulation of the intracellular levels of the AMBP protein are currently unknown. We report the ability of the AMBP protein to self-associate and form a dimer in a yeast environment using the yeast two-hybrid system and an in vitro dimerization assay. We also show that the A1M protein binds to its precursor protein, AMBP, whereas bikunin does not. This observation warrants further investigations for a dimerization-dependent intracellular control that AMBP may be involved in. The relevance of AMBP dimerization and its possible biological significance are postulated.

Expression of a functional C5a receptor in regenerating hepatocytes and its involvement in a proliferative signaling pathway in rat.

Activation of the complement system generates the anaphylatoxin C5a whose activities are mediated through its binding to the widely expressed C5aR. C5aR mRNA and protein expressions are known to be induced in rat hepatocytes under inflammatory conditions. However, little is known about the role of the C5a/C5aR complex in liver and its involvement during a proliferative process. We have evaluated the expression of C5aR in regenerating rat hepatocytes following a partial hepatectomy and in hepatocyte cultures. C5aR induction was observed in hepatocytes from regenerating liver, as well as in normal hepatocytes under a culture-induced stress. The effect of a stimulation by a C5a agonist upon the synthesis of a growth factor/receptor pair (hepatocyte growth factor/c-Met) was also evaluated. Our data demonstrated an up-regulated expression of hepatocyte growth factor and c-Met mRNAs, but we failed to observe a direct mitogenic effect of C5a in culture. However, a significantly increased expression of cyclin E and D1mRNA levels, as well as an increased BrdU incorporation, were observed in rats given an i.v. C5a agonist injection following an 80% partial hepatectomy. These studies demonstrate for the first time that: 1) C5aR is up-regulated during liver regeneration, 2) the binding of C5a to C5aR promotes a growth response, and 3) C5aR is involved in a cell cycle signaling pathway. Taken together, these findings point to a novel role for the hepatic C5aR implicating this complement system in the context of normal or abnormal proliferative pathways.

Amino acid control of the human glyceraldehyde 3-phosphate dehydrogenase gene transcription in hepatocyte.

Glutamine (Gln) is the most potent of the amino acids (AAs) that regulate liver anabolism, and its effect is similar to that of insulin in peripheral tissues. However, the influence of AAs on regulation of metabolic enzyme-encoding genes is not known at the molecular level in liver. We now report that Gln and some essential AAs activate the human GAPDH gene that codes for GAPDH, a central enzyme of glycolysis and a target for insulin regulation. In HepG2 cells, Gln upregulated the GAPDH mRNA level, and this effect was additive to that of insulin. Transient transfection of GAPDH promoter/cat constructs demonstrated that a gene-specific and insulin-independent transcriptional step is involved in the Gln responsiveness of GAPDH. Transfected HepG2 cells challenged with various AAs, Gln metabolites or inhibitors of Gln metabolism showed that the Gln-induced effect is similar to that of some essential AAs and that Gln metabolism is a necessary step for GAPDH activation. Deletion mutants and site-directed mutagenesis of the GAPDH promoter indicated that the Gln responsiveness is mediated by a sequence that is distinct from insulin-responsive elements and from positively acting elements previously described in this promoter. This motif located at -126/-118 clearly differs from AA-responsive elements recently identified in other genes. Electromobility shift assay and supershifts showed that the transcription factors bound to the Gln-responsive element in the GAPDH promoter are C/EBPalpha and -delta. This finding is consistent with the role of C/EBP family members in controlling the hepatic expression of genes involved in nutrient metabolism.