Amino acid deprivation regulates the stress-inducible gene p8 via the GCN2/ATF4 pathway.

In mammals, the GCN2/ATF4 pathway has been described as the main pathway involved in the regulation of gene expression upon amino acid limitation. This regulation is notably conferred by the presence of a cis-element called Amino Acid Response Element (AARE) in the promoter of specific genes. In vivo, the notion of amino acid limitation is not limited to nutritional context, indeed several pathological situations are associated with alteration of endogenous amino acid availability. This is notably true in the context of tumour in which the alteration of the microenvironment can lead to a perturbation in nutrient availability. P8 is a small weakly folded multifunctional protein that is overexpressed in several kinds of cancers and whose expression is induced by different stresses. In this study we have demonstrated that amino acid starvation was also able to induce p8 expression. Moreover, we brought the evidence, in vitro and in vivo, that the GCN2/ATF4 pathway is involved in this regulation through the presence of an AARE in p8 promoter.

Specificity of amino acid regulated gene expression: analysis of genes subjected to either complete or single amino acid deprivation.

Amino acid deprivation activates the amino acid response (AAR) pathway that enhances transcription of genes containing an amino acid response element (AARE). The present data reveal a quantitative difference in the response to deprivation of individual amino acids. The AAR leads to increased eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation and ATF4 translation. When HepG2 cells were deprived of an individual essential amino acid, p-eIF2alpha and activating transcription factor 4 were increased, but the correlation was relatively weak. Complete amino acid starvation in either Earle's balanced salt solution or Krebs-Ringer bicarbonate buffer (KRB) resulted in activation of transcription driven by a SNAT2 genomic fragment that contained an AARE. However, for the KRB, a proportion of the transcription was AARE-independent suggesting that amino acid-independent mechanisms were responsible. Therefore, activation of AARE-driven transcription is triggered by a deficiency in any one of the essential amino acids, but the response is not uniform. Furthermore, caution must be exercised when using a medium completely devoid of amino acids.

Inhibition of CHOP translation by a peptide encoded by an open reading frame localized in the chop 5'UTR.

Chop is a ubiquitously expressed mammalian gene encoding a small nuclear protein related to the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. CHOP protein plays an important role in various cellular processes such as growth, differentiation and programmed cell death. CHOP expression is strongly increased in response to a large variety of stresses including perturbation of the endoplasmic reticulum function, DNA damage and nutrient deprivation. Multiple mechanisms including transcriptional and post-transcriptional controls are involved in the regulation of CHOP expression. We show here that the 5'UTR of the Chop transcript plays an important role in controlling the synthesis of CHOP protein. In particular, the 5'UTR contains a conserved uORF which encodes a 31 amino acid peptide that inhibits the expression of the downstream ORF. Mutational analysis of the 5' leader region and peptide coding sequences suggests that the peptide itself inhibits expression of the downstream ORF. Such results suggest a role for uORF in limiting ribosomal access to downstream initiation sites. With respect to the importance of CHOP protein in the regulation of cellular functions, the mechanisms that regulate its basal level are of considerable interest.

Recent advances on molecular mechanisms involved in amino acid control of gene expression.

In mammals, the impact of nutrients on gene expression has become an important area of research. Because amino acids have multiple and important functions, their homeostasis has to be finely maintained. However, amino acidaemia can be affected by certain nutritional conditions or various forms of aggression. It follows that mammals have to adjust several of their physiological functions involved in the adaptation to amino acid availability by regulating the expression of numerous genes. It has been shown that amino acids by themselves can modify the expression of target genes. However, the current understanding of amino acid-dependent control of gene expression has just started to emerge. This review focuses on the recent advances on mechanisms involved in the amino acids control of gene expression. Several examples discussed in this paper demonstrate that amino acids regulate gene expression at the level of transcription, messenger RNA stability and translation.

Amino acid regulation of gene expression.

The impact of nutrients on gene expression in mammals has become an important area of research. Nevertheless, the current understanding of the amino acid-dependent control of gene expression is limited. Because amino acids have multiple and important functions, their homoeostasis has to be finely maintained. However, amino-acidaemia can be affected by certain nutritional conditions or various forms of stress. It follows that mammals have to adjust several of their physiological functions involved in the adaptation to amino acid availability by regulating the expression of numerous genes. The aim of the present review is to examine the role of amino acids in regulating mammalian gene expression and protein turnover. It has been reported that some genes involved in the control of growth or amino acid metabolism are regulated by amino acid availability. For instance, limitation of several amino acids greatly increases the expression of the genes encoding insulin-like growth factor binding protein-1, CHOP (C/EBP homologous protein, where C/EBP is CCAAT/enhancer binding protein) and asparagine synthetase. Elevated mRNA levels result from both an increase in the rate of transcription and an increase in mRNA stability. Several observations suggest that the amino acid regulation of gene expression observed in mammalian cells and the general control process described in yeast share common features. Moreover, amino acid response elements have been characterized in the promoters of the CHOP and asparagine synthetase genes. Taken together, the results discussed in the present review demonstrate that amino acids, by themselves, can, in concert with hormones, play an important role in the control of gene expression.

Amino acids control mammalian gene transcription: activating transcription factor 2 is essential for the amino acid responsiveness of the CHOP promoter.

In mammals, plasma concentration of amino acids is affected by nutritional or pathological conditions. It has been well established that nutrients, and particularly amino acids, are involved in the control of gene expression. Here we examined the molecular mechanisms involved in the regulation of CHOP (a CCAAT/enhancer-binding protein [C/EBP]-related gene) expression upon amino acid limitation. We have previously shown that regulation of CHOP mRNA expression by amino acid concentration has both transcriptional and posttranscriptional components. We report the analysis of cis- and trans-acting elements involved in the transcriptional activation of the human CHOP gene by leucine starvation. Using a transient expression assay, we show that a cis-positive element is essential for amino acid regulation of the CHOP promoter. This sequence is the first described that can regulate a basal promoter in response to starvation for several individual amino acids and therefore can be called an amino acid response element (AARE). In addition, we show that the CHOP AARE is related to C/EBP and ATF/CRE binding sites and binds in vitro the activating transcription factor 2 (ATF-2) in starved and unstarved conditions. Using ATF-2-deficient mouse embryonic fibroblasts and an ATF-2-dominant negative mutant, we demonstrate that expression of this transcription factor is essential for the transcriptional activation of CHOP by leucine starvation. Altogether, these results suggest that ATF-2 may be a member of a cascade of molecular events by which the cellular concentration of amino acids can regulate mammalian gene expression.

Evidence for multiple signaling pathways in the regulation of gene expression by amino acids in human cell lines.

In mammals, plasma concentrations of amino acids (AA) are affected by nutritional or pathologic conditions. Alterations in AA profiles have been reported as a result of a deficiency of any one of the essential AA, a dietary imbalance of AA or an insufficient intake of protein. In recent years, evidence has accumulated that AA availability regulates the expression of several genes involved in the regulation of a number of cellular functions or AA metabolism. Nevertheless, the molecular mechanisms involved in the AA regulation of mammalian gene expression are limited, particularly the signaling pathways mediating the AA response. This work provides a better understanding of the signaling pathways involved in the AA control of gene expression. We studied the expression of C/EBP homologous protein (CHOP) and asparagine synthetase (AS) in response to deprivation of a single AA and investigated the possible link between protein synthesis inhibition due to amino acid limitation and gene expression. We have shown the following: 1) several mechanisms are involved in the AA control of gene expression. When omitted from the culture medium, each AA can activate one (or several) specific signaling pathways leading to the regulation of one specific pattern of genes. 2) AA limitation by itself can induce gene expression independently of a cellular stress due to protein synthesis inhibition. Together, these results suggest that AA control of gene expression involves several specific mechanisms by which one AA (or one group of AA) can activate one signaling pathway and thus alter one specific pattern of gene expression.

Amino acid limitation regulates gene expression.

In mammals, the plasma concentration of amino acids is affected by nutritional or pathological conditions. For example, an alteration in the amino acid profile has been reported when there is a deficiency of any one or more of the essential amino acids, a dietary imbalance of amino acids, or an insufficient intake of protein. We examined the role of amino acid limitation in regulating mammalian gene expression. Depletion of arginine, cystine and all essential amino acids leads to induction of insulin-like growth factor-binding protein-1 (IGFBP-1) mRNA and protein expression in a dose-dependent manner. Moreover, exposure of HepG2 cells to amino acids at a concentration reproducing the amino acid concentration found in portal blood of rats fed on a low-protein diet leads to a significantly higher (P < 0.0002) expression of IGFBP-1. Using CCAAT/enhancer-binding protein homologous protein (CHOP) induction by leucine deprivation as a model, we have characterized the molecular mechanisms involved in the regulation of gene expression by amino acids. We have shown that leucine limitation leads to induction of CHOP mRNA and protein. Elevated mRNA levels result from both an increase in the rate of CHOP transcription and an increase in mRNA stability. We have characterized two elements of the CHOP gene that are essential to the transcriptional activation produced by an amino acid limitation. These findings demonstrate that an amino acid limitation, as occurs during dietary protein deficiency, can induce gene expression. Thus, amino acids by themselves can play, in concert with hormones, an important role in the control of gene expression.

Amino acid regulation of gene expression.

In mammals, the plasma concentration of amino acids is affected by nutritional or pathological conditions. For example, an amino acid profile alteration has been reported as a result of a deficiency of any one of the essential amino acids, a dietary imbalance of amino acids or an insufficient intake of protein. Amino acid availability regulates the expression of several genes involved in the regulation of growth, cellular function or amino acid metabolism. A limitation of several amino acids strongly increases the expression of insulin-like growth factor binding protein CHOP and asparagine synthetase genes. Elevated messenger RNA levels result from both an increase in the rate of transcription and an increase in messenger RNA stability. DNA amino acid response elements have been characterized in the promoter of CHOP and asparagine synthetase genes. The underlying mechanisms of gene regulation by amino acid limitation are not yet completely understood. The results discussed in this review demonstrate that amino acids by themselves can play, in concert with hormones, an important role in the control of gene expression.

Amino acid limitation regulates CHOP expression through a specific pathway independent of the unfolded protein response.

The gene encoding CHOP (C/EBP-homologous protein) is transcriptionally activated by many stimuli and by amino acid deprivation. CHOP induction was considered to be due to an accumulation of unfolded protein into the ER (unfolded protein response (UPR)). We investigate the role of the UPR in the induction of CHOP by amino acid deprivation and show that this induction is not correlated with BiP expression (an UPR marker). Moreover, amino acid deprivation and UPR inducers regulate the CHOP promoter activity using distinct cis elements. We conclude that amino acid deprivation does not activate the UPR and regulates CHOP expression through a pathway that is independent of the UPR.

Physiological concentration of amino acids regulates insulin-like-growth-factor-binding protein 1 expression.

Protein undernutrition is characterized by growth failure in young growing animals. Current evidence suggests that biosynthesis of insulin-like growth factor (IGF)-I and IGF-binding protein 1 (IGFBP-1) are key control points for nutritional regulation of growth. Here we examined the role of amino acid limitation in regulating the IGFBP-1 expression in the hepatic cell line. Our data show that leucine limitation strongly induces IGFBP-1 without affecting IGF-I and IGF-II expression in human HepG2 cells and in isolated rat hepatocytes. Depletion of arginine, cystine and all essential amino acids leads to induction of IGFBP-1 mRNA and protein expression in a dose-dependent manner. IGFBP-1 expression is significantly induced by leucine concentration in the range of that observed in the blood of rats fed a low-protein diet or in humans affected by kwashiorkor. Moreover, treatment of HepG2 cells with amino acids at a concentration reproducing the amino acid concentration found in portal blood of rats fed a low-protein diet leads to a significantly higher expression of IGFBP-1. These data represent the first demonstration that an amino acid limitation, as occurs during dietary protein deficiency, induces IGFBP-1 expression in hepatic cells. Therefore, amino acids by themselves can play, in concert with hormones, an important role in the control of gene expression.

Amino acid limitation induces expression of CHOP, a CCAAT/enhancer binding protein-related gene, at both transcriptional and post-transcriptional levels.

In mammals, plasma concentrations of amino acids are affected by nutritional or pathological conditions. Here we examined the role of amino acid limitation in regulating the expression of CHOP, a CCAAT/enhancer binding protein (C/EBP)-related gene. CHOP protein is capable of interacting with other C/EBPs to modify their DNA binding activities and may function as a negative regulator of these transcription factors. Our data show that leucine limitation in human cell lines leads to induction of CHOP mRNA and protein in a dose-dependent manner. CHOP mRNA induction is rapidly reversed by leucine replenishment. Elevated mRNA levels result from both an increase in the rate of CHOP transcription and an increase in the CHOP mRNA stability. Using a transient expression assay, we show that a promoter fragment, when linked to a reporter gene, is sufficient to mediate the regulation of CHOP expression by leucine starvation in HeLa cells. In addition, we found that decreasing amino acid concentration by itself can induce CHOP expression independently of a cellular stress due to protein synthesis inhibition. Moreover, CHOP expression is induced at leucine concentrations in the range of those observed in blood of protein-restricted animals suggesting that amino acids can participate, in concert with hormones, in the regulation of gene expression.

Growth factor activation and "H(+)-sensing" of the Na+/H+ exchanger isoform 1 (NHE1). Evidence for an additional mechanism not requiring direct phosphorylation.

Growth factors stimulate the Na+/H+ exchange activity (NHE1 human isoform) and at the same time increase the phosphorylation state of the exchanger at serine residues. To determine the role of NHE1 phosphorylation, a set of deletion and point mutants has been generated. Functional characterization of deletion mutants expressed in fibroblastic cells revealed that the cytoplasmic region between amino acids 567 and 635 is required for both growth factor-induced cytoplasmic alkalinization and maintenance of high intracellular pH (pHi) sensitivity. In contrast to the loss of growth factor activation and pHi sensitivity caused by the deletion of amino acids 567-635, the same deletion had no apparent effect on the pattern of growth factor-induced phosphorylation. In addition, individual replacement of any of the serine residues between amino acids 567 and 635 with alanine also had no effect on growth factor activation of the exchange activity. Comparison of phosphopeptide maps for the wild type with those for the internal deletion mutant exchangers and the expressed cytoplasmic domain revealed that all major in vivo phosphorylation sites including growth factor-sensitive ones map to the cytoplasmic tail (amino acids 636-815). Deletion of these sites preserves high pHi sensitivity and reduces by only 50% growth factor-induced cytoplasmic alkalinization. Taken together, these data support the existence of a mechanism not requiring direct phosphorylation of NHE1, by which growth factor signals transmit to the "H(+)-sensor" and control the set point value of the exchanger. We propose that a regulatory factor(s) controls NHE1, presumably through its interaction with the critical cytoplasmic region prior to amino acid 635.

Evidence that Na+/H+ exchanger isoforms NHE1 and NHE3 exist as stable dimers in membranes with a high degree of specificity for homodimers.

In this study, we have investigated whether members of the Na+/H+ exchanger (NHE) family are oligomers and whether such oligomeric structure is required for function. Fibroblasts overexpressing NHE1 were treated briefly at 0 degrees C with the cross-linker disuccinimidyl suberate, then membranes were prepared and proteins analyzed by SDS-polyacrylamide gel electrophoresis. Disuccinimidyl suberate treatment converted quantitatively the immunoreactive monomeric form of NHE1 (110 kDa) to a putative dimeric form (210 kDa). Utilization of NHE1 mutant deleted of the cytoplasmic domain (delta 515TH) demonstrates that the transmembrane domain of the antiporter is sufficient for dimerization. Moreover, coimmunoprecipitation of NHE1 and delta 515TH, coexpressed in the same cell, formally proved the existence of dimers. This dimerization was also shown to take place with the epithelial and apically expressed NHE3 isoform, suggesting that oligomerization is a common feature of these transporters. However, coexpression of NHE1 and NHE3 in the same cells did not lead to the formation of heterodimers demonstrating an isoform specificity for the subunit interaction. The domain(s) involved in the isoform-specific dimerization is (are) likely to be confined within the transmembrane segments, as deletion of the 300 amino acids of the cytoplasmic domain did not disrupt dimerization. Exploiting the dimeric properties of the receptor tyrosine kinases and the fact that dimerization triggers kinase activity, we constructed a NHE1/insulin receptor chimera to probe NHE1 dimerization in vivo. When transfected into hamster fibroblasts, this chimera containing the N-terminal transmembrane domain of NHE1 and the cytoplasmic beta-subunit domain of the insulin receptor generates a functional transporter that is autophosphorylated on tyrosine and that presents properties of a constitutively active insulin receptor. These findings support the notion that NHE1 exists in an oligomeric state in intact cells. Finally, to test whether individual subunits of NHE1 are the minimum functional unit for Na+/H+ exchange, we coexpressed a truncated form of NHE1 (delta 515) together with an inactive mutant of NHE1 (E262I). In spite of good expression of the inactive transporter and its capacity to dimerize with active NHE1, no dominant negative effect was observed on amiloride-sensitive 22Na+ flux. This observation would suggest that individual subunits of NHE1 function independently within the oligomeric state.

The Na+/H+ antiporter cytoplasmic domain mediates growth factor signals and controls "H(+)-sensing".

The amiloride-sensitive Na+/H+ exchanger (NHE1 human isoform) is activated in response to diverse mitogenic and oncogenic signals presumably through phosphorylation. To get insight into the activating mechanism, a set of deletion mutants within the C-terminal cytoplasmic domain of NHE1 has been generated. These mutant forms expressed in antiporter-deficient fibroblasts revealed that deletion of the complete cytoplasmic domain (i) preserves amiloride-sensitive Na+/H+ exchange and activation by intracellular H+, (ii) reduces the affinity of the internal "H(+)-modifier site" in a manner mimicked by cellular ATP depletion, and (iii) abolishes growth factor-induced cytoplasmic alkalinization. We conclude that NHE1 can be separated into two distinct functional domains. One is an N-terminal transporter domain (T) that has all the features required to catalyze amiloride-sensitive Na+/H+ exchange with a built-in H(+)-modifier site. The other is a C-terminal cytoplasmic regulatory domain (R) that (i) determines the set point value of the exchanger and (ii) mediates growth factor signals by interacting with the "H(+)-sensor" in a phosphorylation-dependent manner.

Functional expression of the human growth factor activatable Na+/H+ antiporter (NHE-1) in baculovirus-infected cells.

We constructed a recombinant baculovirus, based on Autographa californica nuclear polyhedrosis virus, containing the human Na+/H+ antiporter cDNA under control of the polyhedrin promoter. When infected with this recombinant baculovirus, the Sf9 cell line, derived from Spodoptera frugiperda, expresses a fully functional Na+/H+ antiporter as measured by the generation of an amiloride-sensitive Na+ influx in response to an acid load. The Na+/H(+)-exchange activity, not detectable in Sf9 cells, emerges 18 h after infection and continues to increase over the next two days to reach a maximal value about 20-fold higher than in cultured mammalian fibroblasts. Parallel to this activity, infected cells express a single immunoreactive polypeptide of 85 kDa that represents a non-glycosylated form of the 110-kDa mature human antiporter. We estimated that only 10% of the expressed protein is in a functional state. Not only is the antiporter expressed in insect cells phosphorylated, but also, like in mammalian cells, phosphorylation is increased in response to phorbol esters and okadaic acid. Moreover, tumor promoters apparently modify the same antiporter site in both insect and mammalian cells. We conclude that, with this high level of functional expression and apparently conserved signaling machinery, the present system opens the way to the biochemistry of the transporter including identification of the growth factor stimulated phosphorylation sites.

Alpha-thrombin, epidermal growth factor, and okadaic acid activate the Na+/H+ exchanger, NHE-1, by phosphorylating a set of common sites.

The ubiquitous and amiloride-sensitive Na+/H+ exchanger (NHE-1), a plasma membrane phosphoglycoprotein that regulates intracellular pH, is rapidly activated by growth factors. We showed previously that epidermal growth factor (EGF), alpha-thrombin, or serum stimulates Na+/H+ exchange activity in growth-arrested Chinese hamster lung fibroblasts (ER22 cells) in a time-dependent manner which correlates with increased phosphorylation of NHE-1 at serine residues (Sardet, C., Counillon, L., Franchi, A., and Pouysségur, J. (1990) Science 247, 723-726). Here we show that the tumor promoter, okadaic acid, a potent in vivo inhibitor of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A), stimulates Na+/H+ exchange in G0-arrested ER22 cells and in exchanger-deficient fibroblasts transfected with the human NHE-1 cDNA. Okadaic acid effects are maximal at 1 microM (EC50 = 500 nM), detected in 2 min, complete within 15-20 min, and are additives when combined with EGF or alpha-thrombin. Parallel to the pHi-induced rise, okadaic acid alone or together with growth factors stimulated the phosphorylation of NHE-1. More importantly tryptic phosphopeptide maps of NHE-1, immunoprecipitated from cells treated with EGF, alpha-thrombin, or okadaic acid, show a common pattern of phosphorylation. This pattern consists of five major 32P-labeled peptides (P1-P5) present in lower amounts in resting cells. One of them, P5, barely detectable in resting cells is increased up to 15-fold in mitogen-stimulated cells. Taken together these results reinforce the notion that phosphorylation of NHE-1 controls the set point value of the exchanger and suggest that: (i) the proximate step in Na+/H+ exchange activation is mediated by as yet unidentified growth factor-activatable serine "NHE-1 kinase(s)" and (ii) this NHE-1 kinase(s), partly active in resting cells, integrate signals from receptor tyrosine kinases and G protein-coupled receptors.

Fluxes and membrane transport of amino acids in rat liver under different protein diets.

The aim of the present work was to evaluate in vivo the role of the transport step in hepatic amino acid metabolism. To vary hepatic utilization of amino acids, rats were adapted to diets containing various concentrations of casein (5, 15, and 60%). In rats fed 5 or 15% casein diets, Gln and Glu were released by the liver, and there was a significant uptake of Ala. Hepatic fluxes of amino acids increased considerably after adaptation to high-casein diet (up to 1.55 mumol.min-1.g liver-1 for Ala), because of the rise in afferent concentrations as well as enhanced uptake percentage (peaking at 60-75% for most glucogenic amino acids). Adaptation to a high-protein diet led to induction of not only system A but also of most of the other transport systems (Gly, anionic, T, y+, and to a lesser extent system N); only systems ASC and L were unchanged. The study of amino acid repartition between liver and plasma with different diets indicates that transport could modulate utilization of Ala, Ser, Thr, Gly, Gln, and Asp. For Arg and Asn, present in very low concentrations in liver under any condition, the transport step should be the major locus of control of their metabolism. For amino acids chiefly transported by nonconcentrative systems, such as aromatic amino acids, cellular metabolism could also be limited by the transport process. In conclusion, during adaptation to a high-protein diet, there is apparently a coordinated adaptation of amino acid transport and of their intracellular metabolism. For some amino acids, induction of catabolic enzymes seems greater than that of transport, so that the transport step may play an important role in control of metabolic fluxes. For example, concentration of amino acids such as Thr may be markedly depressed in rats adapted to a high-protein diet.

Solubilization and reconstitution characteristics of hepatic system A-mediated amino acid transport.

In the liver, System A-mediated uptake of neutral amino acids may play a key role in metabolic control. Knowing the properties of the solubilized and reconstituted System A activity is important for future studies on the purification of the carrier protein. Solubilization of System A activity by the combination of 2.5% cholate and 4 M urea resulted in greater than 85% extraction of the activity. Previous removal of easily extracted plasma membrane proteins with 1% cholate alone followed by solubilization of the transporter with cholate/urea resulted in a 2-fold enrichment in transport activity. Based on the observation that the carrier protein aggregates in the presence of low detergent concentrations, a selective polyethylene glycol precipitation procedure was developed resulting in recovery of more than 70% of the initial transport activity and less than 10% of the total protein. A concomitant 10-fold enrichment in carrier activity was achieved. The precipitated carrier could be resuspended in buffer containing Triton X-100, asolectin, and glycerol. Transporter activity in this buffer was stable for up to 5 days when maintained at -20 degrees C or for 2 days at 4 degrees C. The general applicability of the devised reconstitution is illustrated by the presence of Systems N and Gly in the reconstituted proteoliposomes at specific activities greater than those in the native vesicles.