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*
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL 32610-0245; and the
Department of Pathology, School of Veterinary Medicine, University of Georgia, Athens, GA, 30602
2To whom correspondence should be addressed. E-mail: mkilberg{at}ufl.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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4 h before an increase in AS mRNA occurred, whereas the elevation of ATA2 mRNA was readily detectable at 2–4 h. Consistent with these observations, de novo protein synthesis was absolutely required for the activation of the AS gene, but the increase in ATA2 mRNA was largely independent of protein synthesis. Furthermore, in contrast to AS, transcription from the ATA2 gene was not increased by glucose deprivation. Given this lack of ATA2 transcriptional activation by glucose starvation and that the induction of the AS gene by glucose or amino acid starvation is mediated by common genomic elements, it is likely that the ATA2 gene does not contain the same genomic amino acid-responsive cis-elements as the AS gene.
KEY WORDS: • metabolite control • transcription • nutrient control • gene expression
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,2
) and transcription of selected genes (3
,4
) occur after amino acid deprivation of mammalian cells.
Asparagine synthetase (AS)3
enzymatic activity is increased in response to asparagine starvation (5
). The molecular basis for this response was revealed by documenting that the AS mRNA content increased after amino acid limitation (6
), and then further research illustrated induction of human AS gene transcription (6
–9
). Consistent with these observations, Guerrini et al. (9
) obtained evidence for an amino acid response element by deletion analysis and mutagenesis of the human AS proximal promoter region. Subsequently, Barbosa-Tessmann et al. (10
–12
) demonstrated transcriptional activation of the AS gene after glucose deprivation as well. Glucose starvation of mammalian cells results in enhanced transcription of numerous genes (13
,14
) that are also up-regulated by a variety of factors that cause accumulation of misfolded proteins within the endoplasmic reticulum (ER) or otherwise disrupt ER function. This activation pathway is referred to as the unfolded protein response in yeast (15
,16
), also called the ER stress response (ERSR) in mammalian cells (17
,18
). In vivo foot-printing analysis and mutagenesis of the human AS promoter region revealed that both amino acid limitation and the ERSR pathway activated the AS gene through a set of genomic elements that were different from those previously reported for genes that respond to one or the other of these pathways (12
). These novel AS sequences are referred to as nutrient sensing response elements (19
).
Another important cellular activity that is induced in response to amino acid deprivation is the neutral amino acid transporter system A (20
–22
). For three decades it has been recognized that system A transport activity (SysA) is enhanced after amino acid deprivation of mammalian cells (23
,24
), but little is known about the molecular mechanisms by which this increase takes place because, until recently, no molecular probes were available. Now cDNA sequences for several isoforms have been cloned and are referred to as ATA1–3 (25
–29
). In particular, human ATA2 mRNA, also known as SAT2 (26
) or SA1 (30
), undergoes adaptive up-regulation in response to amino acid starvation of human fibroblasts, as shown by a direct relationship between ATA2 mRNA expression and SysA transport activity (31
). Furthermore, both the stimulated SysA transport activity and the ATA2 mRNA level decreased after amino acid refeeding of previously starved cells.
The purpose of these experiments was to define the mechanism for ATA2 regulation and to compare the nutritional adaptation processes for AS and ATA2 in response to deprivation of HepG2 human hepatoma cells for a single amino acid, such as histidine, total amino acids or glucose.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cells were maintained in 75-cm2 tissue culture flasks (Costar, Cambridge, MA) at 37°C in a humidified atmosphere of 5% CO2/95% air. The medium used was Modified Eagle Minimal Essential Medium (MEM; Mediatech, Herndon, VA), supplemented with 10% fetal bovine serum (FBS). For transport experiments, the cells were transferred to 24-well dishes and grown to 70–80% confluence.
Nutrient deprivation of cultured cells.
To treat the cells with nutrient-deficient medium, the complete MEM was removed by aspiration and replaced with complete MEM, MEM lacking histidine, MEM lacking glucose, or with Krebs-Ringer bicarbonate (KRB) medium (25 mmol/L sodium bicarbonate, pH 7.4, 119 mmol/L NaCl, 5.6 mmol/L glucose, 5.9 mmol/L KCl, 1.2 mmol/L magnesium sulfate, 1 g/L penicillin G, 100 mg/L streptomycin, 7.5 mg/L phenol red, 2.3 mg/L n-butyl-p-hydroxybenzoate, 2.5 mmol/L calcium chloride, and 10 g/L bovine serum albumin), each supplemented with 10% dialyzed FBS. The length of treatment was indicated in each figure legend.
Amino acid transport assay.
The Na+-dependent system A amino acid transport activity was assayed at 37°C with 50 µmol/L 3H-labeled 2-aminoisobutyrate (3H-AIB), as described previously (32
). Sodium-dependent uptake data, expressed as picomoles of amino acid uptake per milligram of protein per 1 min, were calculated as the difference between the rates measured in the presence and absence of sodium. Protein was measured by a modified Lowry procedure, as reported previously (32
).
Transient transfection and luciferase assays.
HepG2 cells were transfected with a reporter plasmid comprised of the Firefly luciferase gene (pGL3 vector; Promega, Madison, WI) driven by a fragment (nt -512/+770) of the human ATA2 genomic sequence. The cells were transfected at 50–70% confluence in 24-well plates using Superfect reagent, as described previously (10
). After transfection and a subsequent 16-h incubation in MEM plus 10% FBS, cells were incubated for 8 h in either complete MEM, histidine-free MEM, glucose-free MEM, or amino acid-free KRB, each supplemented with 10% dialyzed FBS (11
). A cell extract was prepared and the luciferase activity was assayed using a kit supplied from Promega.
Northern analysis.
Cells were grown to 60% confluence in 60-mm tissue culture dishes (Costar, Cambridge, MA), subjected to the nutrient deprivation treatment as described in each figure legend, and then RNA was collected using the RNeasy Mini Kit (Qiagen, Valencia, CA), according to the manufacturer’s protocol. RNA was subjected to Northern analysis as described by Barbosa-Tessmann et al. (11
). To generate a human ATA2 cDNA probe, primers were designed corresponding to nucleotides 600–619 (5'-TGAGTTTGAGTTTGAGTGGTGCC-3') and 1875–1897 (5'-TGAGTTTGAGTTTGAGTGGTGCC-3') of the coding region of the published sequence (Accession Number AF298897) and were synthesized by Gibco BRL (Gaithersburg, MD). The cDNA probe was generated by RT-PCR using these primers and HepG2 RNA. The resulting product was sequenced by the Center for Mammalian Genetics Sequencing Core at the University of Florida. The cDNA probes for AS, glutamate dehydrogenase (GDH), and ribosomal protein L7a were described previously (11
). Radio-labeled cDNA probes were prepared with (32P)-dATP (Amersham Pharmacia, Piscataway, NJ) using a Strip-EZ DNA probe synthesis kit (Ambion, Austin, TX) according to the manufacturer’s protocol and then purified with a ProbeQuant G-50 column (Amersham Pharmacia). Autoradiographs were made using Kodak Biomax MS film and Biomax MR intensifier screens (Kodak, Rochester, NY). The relative intensity of each experimental band was determined by densitometry and then normalized to the signal intensity of a control mRNA (GDH or L7a) for which expression did not change under the experimental conditions. Replicate blots were analyzed using independently prepared batches to confirm reproducibility.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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To document the induction of SysA transport activity, HepG2 human hepatoma cells were incubated in complete MEM, amino acid-free KRB, KRB + 0.1 mmol/L cycloheximide (CHX), or KRB + 5 µmol/L actinomycin D (ActD) (Fig. 1
). During the initial 3 h, a relatively small increase in transport occurred. A larger enhancement of SysA activity was observed at 6 and 9 h. The inclusion of inhibitors of either RNA or protein synthesis during the incubation largely blocked the increase in SysA transport activity.
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Given the inhibition of the SysA adaptive response by ActD, the accumulation of ATA2 mRNA after amino acid limitation is most likely due to either increased de novo synthesis or mRNA stabilization. To distinguish between these two possibilities, HepG2 cells were incubated in histidine-free MEM without or with ActD (Fig. 2
). Histidine limitation increased ATA2 mRNA to nearly five times the control, and inhibition of RNA synthesis completely prevented the increase. Furthermore, the presence of ActD resulted in a decline in mRNA content to a level even below that in control (MEM) cells, indicating a relatively short half-life for the ATA2 mRNA in the fed state.
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To establish an estimate of the half-life for the ATA2 and AS mRNA species during the decay process that occurs after refeeding, HepG2 cells were incubated for 18 h in amino acid-free KRB. After transfer of those starved cells to amino acid-complete MEM, RNA was collected at specific time-points and analyzed for ATA2 and AS mRNA content (Fig. 3A
). The human AS mRNA exhibited a relatively long half-life, consistent with the estimated half-life of 9 h for the rat AS mRNA (unpublished data, Hutson and Kilberg). In contrast, the ATA2 mRNA had a short half-life of 1 h (Fig. 3
A). The results are in agreement with the half-life of 1.5 h for SysA transport activity after refeeding of amino acid-starved cells (33
).
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To evaluate mRNA stability as a possible mechanism for the increase in ATA2 mRNA, HepG2 cells were incubated in histidine-free medium to elevate ATA2 mRNA content and then transferred to either fresh histidine-free medium or amino acid-complete MEM, both containing 5 µmol/L ActD (Fig. 3
B). The rate of ATA2 mRNA decay was much less in the presence of the RNA synthesis inhibitor than in its absence (compare Fig. 3
, A and B), but when the results in the presence of ActD were analyzed graphically, it was clear that the rate of mRNA turnover was not different in the presence or absence of histidine (Fig. 3
B). Consistent with the blockade of increased ATA2 mRNA by ActD suggesting transcriptional induction (Fig. 2)
, these results indicated that amino acid deprivation does not elevate the ATA2 mRNA content by increasing mRNA stability.
Induction of ATA2 mRNA is due to increased transcription.
To directly test the hypothesis that the starvation-dependent increase in ATA2 mRNA is the result of enhanced transcription, transient reporter gene expression driven by a genomic fragment of the human ATA2 promoter was studied. HepG2 cells were transfected with a firefly luciferase reporter gene driven by nt -512/+770 of the human ATA2 gene (Fig. 4
). Subjecting the cells to histidine or complete amino acid limitation resulted in a 4- or 2.5-fold increase in transcription relative to the cells maintained in complete MEM. In contrast, depriving the cells for glucose did not increase ATA2-driven transcription (Fig. 4)
.
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To evaluate the temporal response of the ATA2 and AS genes to amino acid starvation, HepG2 cells were incubated in MEM or KRB for 0–18 h and RNA collected at specific times (Fig. 5
). An increase in ATA2 mRNA was detected within 2 h and the mRNA level was elevated by >2-fold after 6 h. In contrast, the AS mRNA content was only slightly increased during the first 6 h of amino acid limitation, but there was a substantial, time-dependent increase after 12 and 18 h.
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To determine whether a difference in the adaptive response occurs when cells are deprived of all amino acids compared with a single essential amino acid, ATA2 and AS mRNA content was analyzed in cells incubated for 12 h in either complete MEM, MEM lacking histidine only, or amino acid-free KRB (Fig. 6
). The induction of ATA2 was greater when the cells were deprived of total amino acids than when they were starved for histidine alone, whereas the AS mRNA induction after histidine limitation only was substantially greater than that resulting from total amino acid starvation (Fig. 6)
. Although either condition consistently enhanced mRNA content for both genes, the quantitative difference in sensitivity between ATA2 and AS with respect to single amino acid depletion vs. complete starvation was routinely observed.
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Transient transfection of the ATA2 promoter indicated that glucose deprivation did not increase ATA2 transcription (Fig. 4)
. To determine whether ATA2 mRNA content or SysA transport activity was regulated by glucose availability, HepG2 cells were incubated in complete MEM, MEM lacking glucose, or amino acid-free KRB for either 6 or 18 h. SysA transport activity was then assayed by measuring the Na+-dependent uptake of 50 µmol/L 3H-AIB for 1 min (Fig. 7A
). In contrast to amino acid limitation, depriving the cells of the 5 mmol/L glucose typically present in MEM did not affect SysA activity. Northern blots of RNA from HepG2 cells incubated for 6 h in MEM lacking either histidine or glucose were probed with ATA2 and AS cDNA to monitor the level of these mRNA species (Fig. 7
B). AS mRNA content increased significantly after deprivation of either nutrient. In contrast, ATA2 mRNA expression was elevated after incubation in the absence of histidine as expected, but glucose starvation of the cells caused little or no increase in ATA2 mRNA (Fig. 7
B).
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HepG2 hepatoma cells were incubated in complete MEM or in amino acid-free KRB without or with 0.1 mmol/L cycloheximide (CHX) for 9 h and then RNA was prepared for Northern analysis (Fig. 8A
). Although both AS and ATA2 mRNA levels were increased under amino acid-starved conditions, the induction of AS was blocked completely by the protein synthesis inhibitor, whereas the induction of ATA2 mRNA was only weakly suppressed. To provide a quantitative assessment of this difference, multiple plates (n = 3) of cells were incubated in KRB with or without CHX for 6 h (Fig. 8
B). Relative to control (MEM) cells, the ATA2 mRNA levels were increased significantly after amino acid depletion (KRB) regardless of whether CHX was (3.5-fold) or was not (4-fold) present. In contrast, the AS mRNA content in cells incubated in KRB was increased by 100%, but the increase was totally blocked by CHX (Fig. 8
B). These observations indicate that the enhancement of AS mRNA is completely dependent on de novo protein synthesis, whereas most, if not all, of the induction for ATA2 mRNA is protein synthesis independent.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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4 h before an increase in AS mRNA occurred, whereas the elevation of ATA2 mRNA was readily detectable at 2–4 h. 2) Enhancement of AS and ATA2 mRNA levels was differentially sensitive to either complete amino acid deprivation or limitation of a single essential amino acid. AS mRNA was increased to a greater extent by depriving cells of only a single amino acid rather than all twenty, whereas ATA2 mRNA induction was not as large when only one amino acid was limiting. 3) De novo protein synthesis was absolutely required for the activation of the AS gene, but the increase in ATA2 mRNA was largely independent of protein synthesis. 4) In contrast to the AS gene, the ATA2 gene was not induced by glucose starvation. Given that transcriptional control of the AS gene in response to glucose or amino acid starvation was mediated by the same genomic elements (12
) and that there was no ATA2 transcriptional activation by glucose starvation, it is likely that the ATA2 gene does not contain the same genomic amino acid responsive regulatory elements as the AS gene.
Before the availability of molecular probes, the presumption that at least a component of the starvation-dependant increase in system A activity was transcriptional was supported by the observation that the adaptive response was blocked by inhibitors of RNA polymerase II (actinomycin D) (34
) and poly-A polymerase (cordycepin) (35
). The present data show that a genomic fragment of the human ATA2 gene responds to amino acid limitation with an increase in transcription rate. However, in addition to the adaptive transcription it is also possible that post-translational mechanisms exist for regulating the amount of active system A transport. For example, consistent with the results in this report, Gazzola et al. (31
) found an increase in ATA2 mRNA in human fibroblasts deprived of all amino acids, but Ling et al. (36
) proposed that the initial increase in transport during the 1st h or so after amino acid deprivation resulted from transporter recruitment from an intracellular storage compartment to the plasma membrane.
Franchi-Gazzola et al. (37
) proposed that induction of system A by total amino acid starvation may be a consequence of a change in cell volume, and it is recognized that the transport activity is enhanced in response to hypertonic treatment (38
). However, for the experiments reported here two points should be noted. First, although there was a quantitative difference in the degree of induction, no obvious mechanistic differences were observed between starvation for a single amino acid (histidine) and for all 20 amino acids. Second, the removal of only 30.6 µmol/L histidine from MEM, which induced ATA2 mRNA, resulted in a change of only 0.1% in total osmolarity of the medium. It is unlikely that this difference was sufficient to initiate an osmotic effect. Therefore, it is quite possible that there are two independent pathways for ATA2 induction: one that senses a change in cell volume/osmolarity and another that detects amino acid availability more directly.
The mechanisms underlying metabolite control in mammalian cells are not yet well defined. For amino acid limitation, a number of laboratories are investigating the initial steps in the signal transduction pathway that appear to involve recognition by proteins associated with the translational machinery (39
,40
). The focus of this research is to define the mechanisms that are functional at the target genes and then use those observations to design studies aimed at progressing backwards up the signal pathway. The data reported here document that both the ATA2 and the AS genes are targets for regulation by amino acid limitation but that there are either multiple independent pathways or a pathway that initially has common steps and then branches mechanistically before control of transcription. Future investigation should provide the answer to this interesting question.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: ActD, actinomycin D; AS, asparagine synthetase; ATA2, amino acid transporter A-2; CHX, cycloheximide; ER, endoplasmic reticulum; ERSR, ER stress response; FBS, fetal bovine serum; GDH, glutamate dehydrogenase; KRB, Krebs-Ringer bicarbonate buffer; L7a, large subunit ribosomal protein 7a; MEM, Modified Eagle Minimal Essential Medium; SysA, System A transport activity.
Manuscript received 4 June 2002. Initial review completed 25 June 2002. Revision accepted 15 July 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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 A. Blais, J.-F. Huneau, L. J. Magrum, T. J. Koehnle, J. W. Sharp, D. Tome, and D. W. Gietzen Threonine Deprivation Rapidly Activates the System A Amino Acid Transporter in Primary Cultures of Rat Neurons from the Essential Amino Acid Sensor in the Anterior Piriform Cortex J. Nutr., July 1, 2003; 133(7): 2156 - 2164. [Abstract] [Full Text] [PDF]
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M. Lopez-Fontanals, S. Rodriguez-Mulero, F. J. Casado, B. Derijard, and M. Pastor-Anglada The Osmoregulatory and the Amino Acid-regulated Responses of System A Are Mediated by Different Signal Transduction Pathways J. Gen. Physiol., June 30, 2003; 122(1): 5 - 16. [Abstract] [Full Text] [PDF]
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J. Averous, A. Bruhat, S. Mordier, and P. Fafournoux Recent Advances in the Understanding of Amino Acid Regulation of Gene Expression J. Nutr., June 1, 2003; 133(6): 2040S - 2045. [Abstract] [Full Text] [PDF]
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