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CD98 light chain
BBRC Biochemical and Biophysical Research Communications 318 (2004) 529–534 www.elsevier.com/locate/ybbrc
Transcriptional regulation of the LAT-1/CD98 light chain James F. Padbury,a Sri K. Diah,b Bethany McGonnigal,a Carla Miller,b Celine Fugere,b Magdalena Kuzniar,a and Nancy L. Thompsonb,* b
a Department of Pediatrics, Women and Infants’ Hospital, Providence, RI 02903, USA Division of Medical Oncology, Brown Medical School, Rhode Island Hospital, Providence, RI 02903, USA
Received 1 March 2004
Abstract LAT-1/CD98 amino acid transporter expression and activity are induced in hepatic cells deprived of arginine. The promoter dependency of this regulation was investigated. LAT-1 expression, in contrast to that of CD98 heavy chain 4F2, was actinomycin D sensitive in cells cultured without arginine. Transient transfection analysis with promoter reporter constructs including the 2 kbp LAT-1 promoter or a sub-sequence containing multiple potential amino acid response elements failed to show significant amino acid sensitivity in various cell types. Chromatin-dependency did not appear to account for this result as hepatic cell clones stably transfected with the promoter constructs showed little or no arginine or leucine responsive promoter activity. These studies suggest that while amino acid sensitivity of LAT-1 expression is transcriptionally regulated, cis elements within the proximal promoter do not directly mediate this regulation. Understanding mechanisms by which this gene responds to amino acid availability will contribute to our knowledge of how eukaryotic cells sense and respond to their environment. Ó 2004 Elsevier Inc. All rights reserved. Keywords: LAT-1 promoter; Amino acid; Transcriptional regulation; CD98; Hepatic cells; Arginine
Amino acids are translocated across the plasma membrane of mammalian cells by several distinct transport systems originally defined by activity and more recently assigned to specific genes [1,2]. System L (leucine favoring) is widespread and involves Naþ independent uptake of large branched chain, aromatic, and neutral amino acids via heterodimers of a catalytic light chain subunit (LAT-1 or LAT-2) covalently associated with the 4F2 heavy chain glycoprotein [3–5]. The heterodimer is also known as CD98 [6,7]. Adaptive regulation of amino acid transport activity in response to nutrient conditions has been observed in mammalian cells with starvation-dependent enhancement of transport in system A as one of the best characterized examples [2]. While bacteria and yeast have the ability to compensate for the effects of amino acid starvation by inducing enzymes that promote de novo amino acid biosynthesis, mammalian cells do not. They rely on signal(s) that initiate a molecular response to amino acid * Corresponding author. Fax: 1-401-444-8141. E-mail address: [email protected] (N.L. Thompson).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.04.062
starvation and selectively increase gene expression [8–10]. We and others have cloned and characterized TA1/ LAT-1 (referred to here as LAT-1) as an oncofetal gene in rat liver, undetectable in normal adult liver, but highly expressed during liver development and injury and in hepatic tumor cells [11–14]. Moreover, we have shown that expression of LAT-1 in normal hepatic cells is significantly up regulated at the RNA and protein levels by culturing in media deficient in arginine [12,15,16]. Importantly, this mode of regulation is lost in transformed cells which express LAT-1 constitutively [15]. Such loss of amino acid regulation may constitute one of the early mechanisms of oncogenesis. LAT-1 may thus serve as a model for how the cell coordinates metabolic needs with cell cycle during normal growth and how this coordination is altered in neoplasia. We previously cloned approximately 2 kbp of the rat LAT-1 proximal promoter (GenBank Accession No. AF329652), defined the transcription start site by inverse race and primer extension, and demonstrated transcriptional activity in rodent hepatic cell lines [17].
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In the present study we sought to identify the mechanism for amino acid regulation of expression of the LAT-1 gene, assessing its sensitivity to actinomycin D inhibition and using rat LAT-1 promoter-reporter constructs to evaluate whether discrete cis elements, including potential amino acid response elements (AARE), mediate such regulation.
Materials and methods Cell lines and culture conditions. WB cells are a normal diploid hepatic cell line derived from adult male Fischer 344 rats and obtained from the laboratory of William Coleman, University of North Carolina, Chapel Hill. AML12, a normal mouse hepatic epithelial cell line, was obtained from the laboratory of Nelson Fausto, University of Washington, Seattle. Both rodent lines were previously demonstrated to exhibit arginine-sensitive LAT-1 expression [15,16]. Human HEK293 fibroblasts were obtained from American Type Culture Collection. Cells were routinely passaged in DMEM/F12 with 10% FBS and supplements. Evaluation of hepatic amino acid-sensitive gene expression was conducted using a custom formulation of Chee’s essential medium that allows maintenance of differentiated hepatic cell function. The media formulation limits manipulation of amino acids to arginine and leucine. In experiments assessing transcriptional inhibition on ariginine sensitivity of LAT-1 expression, actinomycin D (Sigma) was added as a stock solution at the time of media change to achieve a final concentration of 5 lg/ml. Cells were cultured 4 or 8 h. Control cultures received an identical volume of 100% ethanol vehicle. Northern blot. Total RNA was prepared from cell cultures using Totally RNA extraction kit (Ambion, Texas) and quantified by absorbance at 260 nm. Northern blots were prepared and hybridized as previously described to 32 P-labeled rat LAT-1 cDNA probe [15,16]. Autoradiographs were developed and the 3.3 kb LAT-1 transcript was quantified by densitometry relative to 18S RNA. Promoter reporter constructs, transient transfection, and activity assays. The approximately 2 kbp of the rat LAT-1 50 flanking sequence was subcloned upstream of a b-galactosidase (lacZ) reporter construct (pBlueTOPO) as previously reported [17]. A construct incorporating potential amino acid response elements (AARE) within the LAT-1 50 flanking sequence was generated by amplifying a 379 bp PCR fragment from rat LAT-1 sequences )1238 to )789 and subcloning this into the pGL3P (Promega) luciferase reporter vector. These constructs and empty vector controls were used in transient transfection analysis to examine nutrient regulation in transiently transfected human HEK293 fibroblasts and mouse AML12 hepatic epithelial cells. Co-transfection of luciferase and lacZ vectors was used to monitor transfection efficiency. For each experiment, 6 106 cells per dish were plated on day 1. After 24 h, vector construct and/or vehicle were transfected with pGL3C (7:1) into the cells using Lipofectamine Plus. Cells were allowed to express for 24 h. To test amino acid responsiveness, the medium was replaced with Chee’s essential medium with or without arginine (+/)Arg). Cells were incubated for 4–24 h, harvested in b-galactosidase assay buffer, and assayed for activity with a commercial kit (Promega). Luciferase activity was measured in the same extracts (Pharmingen). Assays were normalized to protein concentration in the whole cell extracts. All results represent the results of triplicate or quadruplicate determinations that were carried out in at least three separate experiments. Generation and confirmation of hepatic clones stably incorporating lat-1 proximal promoter. The full-length LAT-1 proximal promoter construct in pBlueTOPO was linearized, complexed with Liposome Plus (Gibco), and added to a 100 mm plate with 4–5 106 AML-12
cells in the absence of serum. Serum containing media were added after expression. Cells were passaged and plated at very low densities in separate plates. On day 5 after transfection, G418 (geneticin) was added to the media to select for stable transfectants. The cells were observed for colony formation and the media was changed regularly (2–4 weeks). Individual clones (n ¼ 32) were moved from 100 mm dishes to single wells of a 96-well plate, maintained in selection media (G418), and expanded. To confirm incorporation of the construct, genomic DNA was isolated from individual clones and subjected to PCR amplifications using primers designed to span the LAT-1 promoter and neomycin resistance genes. Sufficiently expanded cells from stable transfectants were then plated as described above, exposed to (+/)Arg), (Leu) or both in Chee’s essential medium, and assessed for reporter activity.
Results LAT1/CD98 light chain arginine-sensitive expression is transcriptionally dependent We first assessed amino acid-sensitive regulation of LAT-1 RNA levels by analyzing steady state message levels in normal hepatic cells cultured in media with and without Arginine (Arg), with and without the RNA synthesis inhibitor actinomycin D. As previously observed, arginine depletion for 4 and 8 h resulted in an approximately 10-fold elevation in steady state levels of LAT-1 RNA (Fig. 1, lane 1 vs. 3). Culture in the presence of actinomycin D (lanes 2, 4, 6, and 8) prevented this upregulation. There was no effect of actinomycin D in the presence of arginine at 4 h (lane 4 vs. 3). This suggests that the increase in LAT-1 expression under
Fig. 1. The effects of altered arginine and actinomycin D on LAT-1 and 4F2 mRNA levels. Autoradiograph of Northern blot hybridization of 10 mg total RNA/lane with 32 P-labeled cDNAs for LAT-1/ CD98 light chain (top) and 4F2/CD98 heavy chain (middle). Ethidium bromide stained blot shown below for evaluation of RNA loading and quality. Lanes 1–4, WB normal hepatic cells 4 h treatment; lanes 5–8, WB cells 8 h treatment; lanes 2, 4, 6, and 8, with Act D treatment; lanes 1, 2 and 5, 6, without Arginine; lane 9, normal adult rat liver positive control for 4F2; and lane 10, 1683 rat transplantable hepatoma positive control for LAT-1-1.
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conditions of amino acid depletion requires new gene transcription. By contrast, CD98 heavy chain/4F2 message level was significantly increased in the presence of Act D (Fig. 1, lanes 2 and 4), suggesting transcriptional repression of gene expression under basal conditions. This is consistent with work in lymphoid cells characterizing a block to mRNA elongation in the exon1/intron-1 boundary of the 4F2 gene that was removed following mitogen stimulation [18]. Clearly, the two CD98 subunits are independently regulated in normal hepatic cells.
Fig. 2. (A) b-Galactosidase activity of the full-length (around 2 kb pairs) promoter in HEK293 cells in the presence (+Arg) and absence ()Arg) of arginine. There was a 1.6-fold increase in activity, p < 0:07. (B) Transcription activity of 379 bp fragment encompassing AAREs from the rat LAT-1 promoter as shown in Table 1. Activities expressed as luciferase activity normalized to protein concentration. Results are shown for the AARE construct in pGL3P in the presence (+Arg) or the absence ()Arg) of arginine-containing media.
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Neither the LAT-1 proximal promoter nor 50 AARE-like sequence elements alone are sufficient for arginine-sensitive transient expression in fibroblasts or hepatic cells To determine if the arginine-responsive transcriptional effect involves elements in the LAT-1 proximal promoter, we used LAT-1 promoter/reporter constructs in transient transfection experiments and studied their expression in different amino acid conditions. WB cells are consistently difficult to transfect. Therefore, in order to utilize the lacZ reporter constructs to examine nutrient regulation in transiently transfected cells, we used human HEK293 fibroblasts and mouse AML12 normal hepatic epithelial cells. In data not shown, we demonstrated that the optimal time for harvesting of cells and amino acid responsiveness was between 4 and 8 h. We first carried out experiments using the originally reported full-length, approximately 2 kbp promoter [17]. Data for the reporter constructs were compared to the level of expression of the empty pBlueTOPOTm expression vector. Fig. 2A shows the results of pooled data from four experiments in HEK293 cells. The full-length promoter gave strong levels of expression (15–40-fold increase in activity, p < 0:05) compared to the empty reporter vector. Removal of arginine from the incubation media for 4 h resulted in an average 1.6-fold increase in transcription activity which was just at the level of significance, p < 0:07. There was some variation in this response. In five identical experiments, the fold increase in activity varied from 0- to over 2-fold. Very similar results were seen in AML12 cells (data not shown). Computer analysis of the LAT-1 promoter was carried out using several transcription factor matrices [19]. Sequence analysis identified putative amino acid regulatory elements (AARE) in a region between 1200 and 800 base pairs upstream from the transcription start site, Table 1. In order to eliminate the possibility that a repressor element, or other silencing mechanism in the 2 kbp promoter, was inhibiting amino acid responsiveness and to increase the sensitivity of detection of
Table 1 Nucleotide sequence of the 50 flanking region of the rat LAT-1 gene (GenBank Accession No. AF329652)
Putative cis-acting elements predicted by database analysis (MatInspector and TRANSFAC) are underlined [19]. AARE, amino acid response element consensus sequence 50 -(A/G)TT(G/T)CATCA-30 . The primer sites for the PCR amplification of the fragment containing the AAREs are shown in ARIAL in italics.
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nutrient responsiveness, we cloned a fragment incorporating these putative AARE sequences into the pGL3P (Promega) luciferase reporter vector. This heterologous expression vector contains a core promoter element from SV40 which expresses strongly in most eukaryotic systems, a luciferase reporter gene, selectable markers and was developed specifically to analyze activity of discrete, putative cis elements. A representative experiment is shown in Fig. 2B. The basal promoter activity was comparable to the empty pGL3P vector. Though activity was somewhat higher in )Arg media, the increase was not statistically significant. Chromatin context of LAT-1 proximal promoter in stable hepatic cell clones is not sufficient for amino acid-sensitive expression The experiments shown in Fig. 2 suggested but did not conclusively demonstrate increased expression in nutrient-depleted conditions ()Arg). This could be because these were transient transfection experiments and genomic integration within a chromatin context is required for regulation and induction. Several promoter elements that lack activity in transient transfection assays have been demonstrated to be transcriptionally active after stable incorporation into genomic DNA [20]. To explore this possibility, we created stable transfectants of the full-length promoter in AML-12 cells. This hepatic cell line was chosen as it had been shown in prior work to demonstrate nutrient regulation of LAT-1 and the transcription induction seen in Fig. 1. Sufficiently expanded cells from stable transfectants confirmed by PCR to contain the 50 flanking sequence within their genomic DNA were plated as described above and exposed to + or )Arg in Chee’s essential medium. In this series of experiments we also examined
Fig. 3. Transcription activity of stable transfectants of the rat LAT-1 promoter in AML12 cells. Activities expressed as b-galactosidase activity normalized to protein concentration of cell extracts. Results are shown for AML 12 cells and three different stable transfectants, 2B2, 3A1, and 3A5. Transcription activity is shown in four different media amino acid conditions as indicated.
the effect of removal of the amino acid leucine (Leu) alone or in combination with modulation of Arg. We screened several different stably transfected clones in order to control for variations in expression due to the site of incorporation. Cell lines with low, medium, and high basal levels of b-galactosidase activity were expanded for additional studies. We first demonstrated in preliminary experiments the optimal timing of harvest for maximal induction in b-gal activity. The results with three representative clones relative to the parent line are shown in Fig. 3. As can be seen, the results were similar to the transient transfection data. Although a modest increase in b-galactosidase activity in ()Arg) or ()Leu) media was observed in several of the 8–10 stably transformed clones we screened, the effect did not reach statistical significance.
Discussion Several mammalian genes involved in the control of growth or amino acid metabolism are now known to be regulated in response to changes in amino acid availability [8,10]. Amino acid deprivation in Chinese hamster ovary cells results in increased mRNA for c-jun, c-myc, and ornithine decarboxylase [9]. Induction of the C/EBP homologous protein (CHOP) by amino acid deprivation [21] and the induction of asparagine synthetase [22] are other recent examples of alterations in gene expression in response to amino acid restriction (AAR). Several mechanisms may underlie changes in gene expression upon amino acid depletion. Activation of transcription factors which are constitutively bound to DNA but require phosphorylation for their activation (e.g., immediate early genes like fos, jun, myc, and CREB) has been observed [9,10,23]. Activation is produced by nuclear translocation of a stimulus-dependent kinase such as the ERK kinases or their effector kinases. A second mechanism may involve activation of transcription factors in the cytoplasm via the same or other kinase pathways [9,23]. Upon activation, these factors translocate to the nucleus and affect transcription via their respective regulatory elements, e.g., STATs, NFkB. Transactivation also requires chromatin remodeling and alteration in the accessibility of cognate DNA elements to activated transcription factors [24]. Thus, transcription factor activation and/or chromatin remodeling are likely to be affected by signal transduction cascades which themselves are activated in response to alterations in the nutrient environment. Additional mechanisms for gene induction following amino acid depletion have been examined for several important genes including asparagine synthetase [25,26], C/EBP homologous protein or CHOP [21,25], the system A amino acid transporter ATA2 [27], and the Cat-1 amino acid transporter [28–30]. These actions
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occur by a number of complex transcriptional and posttranscriptional mechanisms of regulation. Well studied are the mechanisms of regulation of AS, CHOP, and cat-1. Cis elements which confer transcriptional regulation have been identified in both the AS and the CHOP gene through functional assays, footprinting, gel shift, and site-directed mutagenesis [21,25,27]. In the case of the ATA2, amino acid transporter, the increase in expression is due to the increase in transcriptional activation, not differences in amino acid stability [27]. Unlike the AS gene, the ATA2 gene is not activated by glucose starvation. The nutrient-sensing response unit (NSRU) in the AS promoter contains two cis elements termed NSRE1 and NSRE2, both of which are required for gene activation [27]. In the case of the Cat-1 gene, a high affinity transporter for amino acids, the increase in Cat-1 protein expression during amino acid deprivation is due to increased mRNA stability mediated by sequences within the 30 untranslated region and to enhanced translation of the messenger RNA [29]. The increase in translation efficiency is due to the presence of an internal ribosomal entry sequence (IRES) within the 50 -untranslated region of the Cat-1 mRNA which allows an increase in translation under conditions of amino acid starvation [28]. Regulation of Cat-1 expression during nutrient depletion is also dependent on an increased rate of transcription via activation of GCN2 kinase which phosphorylates eIF2a. This phosphorylation induces an increase in transcription of the Cat-1 gene promoter and is dependent on an amino acid response element located in the first exon [30]. Rapid modulation of the high affinity L-type amino acid transporter, LAT-1, expression and function in response to fluctuations in amino acid content and composition in the tissue environment is likely to be an important adaptive homeostatic mechanism in liver development and injury-response as well as starvation. These are all conditions in which LAT-1 expression is transiently increased in vivo [12,13,31]. The mechanism by which this occurs is unknown. Such regulation is lost in hepatic tumor cells where levels of LAT-1 RNA are constitutively high. Given our prior data demonstrating an increase in steady state LAT-1 mRNA, increased LAT-1 protein expression, and increased amino acid transport following incubation in media lacking Arg and/or Leu, we anticipated the effects might be due to AAREs within the LAT-1 proximal promoter. We failed, however, to demonstrate promoter dependency of increased LAT-1 expression within the cloned sequence using the reporter constructs and expression systems described above. This may be because (1) the cis elements necessary for this effect were not contained within our 2 kb promoter sequence; (2) the cell system(s) chosen did not express key transcription factors for this effect; (3) the effect involves a large change in RNA stability; or (4) a combination of these and other ex-
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planations. Further elucidation of the mechanisms by which cells specifically alter gene expression in response to amino acids remains of interest in order to understand how eukaryotic cells coordinate cellular processes with their tissue microenvironment under normal conditions as well as in disease states.
Acknowledgments This work was supported by American Institute for Cancer Research 02A127, NIH 1RO1 CA73611 and NCRR P20 RR17695 and P20 RR018728.
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Transcriptional regulation of the LAT-1/CD98 light chain James F. Padbury,a Sri K. Diah,b Bethany McGonnigal,a Carla Miller,b Celine Fugere,b Magdalena Kuzniar,a and Nancy L. Thompsonb,* b
a Department of Pediatrics, Women and Infants’ Hospital, Providence, RI 02903, USA Division of Medical Oncology, Brown Medical School, Rhode Island Hospital, Providence, RI 02903, USA
Received 1 March 2004
Abstract LAT-1/CD98 amino acid transporter expression and activity are induced in hepatic cells deprived of arginine. The promoter dependency of this regulation was investigated. LAT-1 expression, in contrast to that of CD98 heavy chain 4F2, was actinomycin D sensitive in cells cultured without arginine. Transient transfection analysis with promoter reporter constructs including the 2 kbp LAT-1 promoter or a sub-sequence containing multiple potential amino acid response elements failed to show significant amino acid sensitivity in various cell types. Chromatin-dependency did not appear to account for this result as hepatic cell clones stably transfected with the promoter constructs showed little or no arginine or leucine responsive promoter activity. These studies suggest that while amino acid sensitivity of LAT-1 expression is transcriptionally regulated, cis elements within the proximal promoter do not directly mediate this regulation. Understanding mechanisms by which this gene responds to amino acid availability will contribute to our knowledge of how eukaryotic cells sense and respond to their environment. Ó 2004 Elsevier Inc. All rights reserved. Keywords: LAT-1 promoter; Amino acid; Transcriptional regulation; CD98; Hepatic cells; Arginine
Amino acids are translocated across the plasma membrane of mammalian cells by several distinct transport systems originally defined by activity and more recently assigned to specific genes [1,2]. System L (leucine favoring) is widespread and involves Naþ independent uptake of large branched chain, aromatic, and neutral amino acids via heterodimers of a catalytic light chain subunit (LAT-1 or LAT-2) covalently associated with the 4F2 heavy chain glycoprotein [3–5]. The heterodimer is also known as CD98 [6,7]. Adaptive regulation of amino acid transport activity in response to nutrient conditions has been observed in mammalian cells with starvation-dependent enhancement of transport in system A as one of the best characterized examples [2]. While bacteria and yeast have the ability to compensate for the effects of amino acid starvation by inducing enzymes that promote de novo amino acid biosynthesis, mammalian cells do not. They rely on signal(s) that initiate a molecular response to amino acid * Corresponding author. Fax: 1-401-444-8141. E-mail address: [email protected] (N.L. Thompson).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.04.062
starvation and selectively increase gene expression [8–10]. We and others have cloned and characterized TA1/ LAT-1 (referred to here as LAT-1) as an oncofetal gene in rat liver, undetectable in normal adult liver, but highly expressed during liver development and injury and in hepatic tumor cells [11–14]. Moreover, we have shown that expression of LAT-1 in normal hepatic cells is significantly up regulated at the RNA and protein levels by culturing in media deficient in arginine [12,15,16]. Importantly, this mode of regulation is lost in transformed cells which express LAT-1 constitutively [15]. Such loss of amino acid regulation may constitute one of the early mechanisms of oncogenesis. LAT-1 may thus serve as a model for how the cell coordinates metabolic needs with cell cycle during normal growth and how this coordination is altered in neoplasia. We previously cloned approximately 2 kbp of the rat LAT-1 proximal promoter (GenBank Accession No. AF329652), defined the transcription start site by inverse race and primer extension, and demonstrated transcriptional activity in rodent hepatic cell lines [17].
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In the present study we sought to identify the mechanism for amino acid regulation of expression of the LAT-1 gene, assessing its sensitivity to actinomycin D inhibition and using rat LAT-1 promoter-reporter constructs to evaluate whether discrete cis elements, including potential amino acid response elements (AARE), mediate such regulation.
Materials and methods Cell lines and culture conditions. WB cells are a normal diploid hepatic cell line derived from adult male Fischer 344 rats and obtained from the laboratory of William Coleman, University of North Carolina, Chapel Hill. AML12, a normal mouse hepatic epithelial cell line, was obtained from the laboratory of Nelson Fausto, University of Washington, Seattle. Both rodent lines were previously demonstrated to exhibit arginine-sensitive LAT-1 expression [15,16]. Human HEK293 fibroblasts were obtained from American Type Culture Collection. Cells were routinely passaged in DMEM/F12 with 10% FBS and supplements. Evaluation of hepatic amino acid-sensitive gene expression was conducted using a custom formulation of Chee’s essential medium that allows maintenance of differentiated hepatic cell function. The media formulation limits manipulation of amino acids to arginine and leucine. In experiments assessing transcriptional inhibition on ariginine sensitivity of LAT-1 expression, actinomycin D (Sigma) was added as a stock solution at the time of media change to achieve a final concentration of 5 lg/ml. Cells were cultured 4 or 8 h. Control cultures received an identical volume of 100% ethanol vehicle. Northern blot. Total RNA was prepared from cell cultures using Totally RNA extraction kit (Ambion, Texas) and quantified by absorbance at 260 nm. Northern blots were prepared and hybridized as previously described to 32 P-labeled rat LAT-1 cDNA probe [15,16]. Autoradiographs were developed and the 3.3 kb LAT-1 transcript was quantified by densitometry relative to 18S RNA. Promoter reporter constructs, transient transfection, and activity assays. The approximately 2 kbp of the rat LAT-1 50 flanking sequence was subcloned upstream of a b-galactosidase (lacZ) reporter construct (pBlueTOPO) as previously reported [17]. A construct incorporating potential amino acid response elements (AARE) within the LAT-1 50 flanking sequence was generated by amplifying a 379 bp PCR fragment from rat LAT-1 sequences )1238 to )789 and subcloning this into the pGL3P (Promega) luciferase reporter vector. These constructs and empty vector controls were used in transient transfection analysis to examine nutrient regulation in transiently transfected human HEK293 fibroblasts and mouse AML12 hepatic epithelial cells. Co-transfection of luciferase and lacZ vectors was used to monitor transfection efficiency. For each experiment, 6 106 cells per dish were plated on day 1. After 24 h, vector construct and/or vehicle were transfected with pGL3C (7:1) into the cells using Lipofectamine Plus. Cells were allowed to express for 24 h. To test amino acid responsiveness, the medium was replaced with Chee’s essential medium with or without arginine (+/)Arg). Cells were incubated for 4–24 h, harvested in b-galactosidase assay buffer, and assayed for activity with a commercial kit (Promega). Luciferase activity was measured in the same extracts (Pharmingen). Assays were normalized to protein concentration in the whole cell extracts. All results represent the results of triplicate or quadruplicate determinations that were carried out in at least three separate experiments. Generation and confirmation of hepatic clones stably incorporating lat-1 proximal promoter. The full-length LAT-1 proximal promoter construct in pBlueTOPO was linearized, complexed with Liposome Plus (Gibco), and added to a 100 mm plate with 4–5 106 AML-12
cells in the absence of serum. Serum containing media were added after expression. Cells were passaged and plated at very low densities in separate plates. On day 5 after transfection, G418 (geneticin) was added to the media to select for stable transfectants. The cells were observed for colony formation and the media was changed regularly (2–4 weeks). Individual clones (n ¼ 32) were moved from 100 mm dishes to single wells of a 96-well plate, maintained in selection media (G418), and expanded. To confirm incorporation of the construct, genomic DNA was isolated from individual clones and subjected to PCR amplifications using primers designed to span the LAT-1 promoter and neomycin resistance genes. Sufficiently expanded cells from stable transfectants were then plated as described above, exposed to (+/)Arg), (Leu) or both in Chee’s essential medium, and assessed for reporter activity.
Results LAT1/CD98 light chain arginine-sensitive expression is transcriptionally dependent We first assessed amino acid-sensitive regulation of LAT-1 RNA levels by analyzing steady state message levels in normal hepatic cells cultured in media with and without Arginine (Arg), with and without the RNA synthesis inhibitor actinomycin D. As previously observed, arginine depletion for 4 and 8 h resulted in an approximately 10-fold elevation in steady state levels of LAT-1 RNA (Fig. 1, lane 1 vs. 3). Culture in the presence of actinomycin D (lanes 2, 4, 6, and 8) prevented this upregulation. There was no effect of actinomycin D in the presence of arginine at 4 h (lane 4 vs. 3). This suggests that the increase in LAT-1 expression under
Fig. 1. The effects of altered arginine and actinomycin D on LAT-1 and 4F2 mRNA levels. Autoradiograph of Northern blot hybridization of 10 mg total RNA/lane with 32 P-labeled cDNAs for LAT-1/ CD98 light chain (top) and 4F2/CD98 heavy chain (middle). Ethidium bromide stained blot shown below for evaluation of RNA loading and quality. Lanes 1–4, WB normal hepatic cells 4 h treatment; lanes 5–8, WB cells 8 h treatment; lanes 2, 4, 6, and 8, with Act D treatment; lanes 1, 2 and 5, 6, without Arginine; lane 9, normal adult rat liver positive control for 4F2; and lane 10, 1683 rat transplantable hepatoma positive control for LAT-1-1.
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conditions of amino acid depletion requires new gene transcription. By contrast, CD98 heavy chain/4F2 message level was significantly increased in the presence of Act D (Fig. 1, lanes 2 and 4), suggesting transcriptional repression of gene expression under basal conditions. This is consistent with work in lymphoid cells characterizing a block to mRNA elongation in the exon1/intron-1 boundary of the 4F2 gene that was removed following mitogen stimulation [18]. Clearly, the two CD98 subunits are independently regulated in normal hepatic cells.
Fig. 2. (A) b-Galactosidase activity of the full-length (around 2 kb pairs) promoter in HEK293 cells in the presence (+Arg) and absence ()Arg) of arginine. There was a 1.6-fold increase in activity, p < 0:07. (B) Transcription activity of 379 bp fragment encompassing AAREs from the rat LAT-1 promoter as shown in Table 1. Activities expressed as luciferase activity normalized to protein concentration. Results are shown for the AARE construct in pGL3P in the presence (+Arg) or the absence ()Arg) of arginine-containing media.
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Neither the LAT-1 proximal promoter nor 50 AARE-like sequence elements alone are sufficient for arginine-sensitive transient expression in fibroblasts or hepatic cells To determine if the arginine-responsive transcriptional effect involves elements in the LAT-1 proximal promoter, we used LAT-1 promoter/reporter constructs in transient transfection experiments and studied their expression in different amino acid conditions. WB cells are consistently difficult to transfect. Therefore, in order to utilize the lacZ reporter constructs to examine nutrient regulation in transiently transfected cells, we used human HEK293 fibroblasts and mouse AML12 normal hepatic epithelial cells. In data not shown, we demonstrated that the optimal time for harvesting of cells and amino acid responsiveness was between 4 and 8 h. We first carried out experiments using the originally reported full-length, approximately 2 kbp promoter [17]. Data for the reporter constructs were compared to the level of expression of the empty pBlueTOPOTm expression vector. Fig. 2A shows the results of pooled data from four experiments in HEK293 cells. The full-length promoter gave strong levels of expression (15–40-fold increase in activity, p < 0:05) compared to the empty reporter vector. Removal of arginine from the incubation media for 4 h resulted in an average 1.6-fold increase in transcription activity which was just at the level of significance, p < 0:07. There was some variation in this response. In five identical experiments, the fold increase in activity varied from 0- to over 2-fold. Very similar results were seen in AML12 cells (data not shown). Computer analysis of the LAT-1 promoter was carried out using several transcription factor matrices [19]. Sequence analysis identified putative amino acid regulatory elements (AARE) in a region between 1200 and 800 base pairs upstream from the transcription start site, Table 1. In order to eliminate the possibility that a repressor element, or other silencing mechanism in the 2 kbp promoter, was inhibiting amino acid responsiveness and to increase the sensitivity of detection of
Table 1 Nucleotide sequence of the 50 flanking region of the rat LAT-1 gene (GenBank Accession No. AF329652)
Putative cis-acting elements predicted by database analysis (MatInspector and TRANSFAC) are underlined [19]. AARE, amino acid response element consensus sequence 50 -(A/G)TT(G/T)CATCA-30 . The primer sites for the PCR amplification of the fragment containing the AAREs are shown in ARIAL in italics.
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nutrient responsiveness, we cloned a fragment incorporating these putative AARE sequences into the pGL3P (Promega) luciferase reporter vector. This heterologous expression vector contains a core promoter element from SV40 which expresses strongly in most eukaryotic systems, a luciferase reporter gene, selectable markers and was developed specifically to analyze activity of discrete, putative cis elements. A representative experiment is shown in Fig. 2B. The basal promoter activity was comparable to the empty pGL3P vector. Though activity was somewhat higher in )Arg media, the increase was not statistically significant. Chromatin context of LAT-1 proximal promoter in stable hepatic cell clones is not sufficient for amino acid-sensitive expression The experiments shown in Fig. 2 suggested but did not conclusively demonstrate increased expression in nutrient-depleted conditions ()Arg). This could be because these were transient transfection experiments and genomic integration within a chromatin context is required for regulation and induction. Several promoter elements that lack activity in transient transfection assays have been demonstrated to be transcriptionally active after stable incorporation into genomic DNA [20]. To explore this possibility, we created stable transfectants of the full-length promoter in AML-12 cells. This hepatic cell line was chosen as it had been shown in prior work to demonstrate nutrient regulation of LAT-1 and the transcription induction seen in Fig. 1. Sufficiently expanded cells from stable transfectants confirmed by PCR to contain the 50 flanking sequence within their genomic DNA were plated as described above and exposed to + or )Arg in Chee’s essential medium. In this series of experiments we also examined
Fig. 3. Transcription activity of stable transfectants of the rat LAT-1 promoter in AML12 cells. Activities expressed as b-galactosidase activity normalized to protein concentration of cell extracts. Results are shown for AML 12 cells and three different stable transfectants, 2B2, 3A1, and 3A5. Transcription activity is shown in four different media amino acid conditions as indicated.
the effect of removal of the amino acid leucine (Leu) alone or in combination with modulation of Arg. We screened several different stably transfected clones in order to control for variations in expression due to the site of incorporation. Cell lines with low, medium, and high basal levels of b-galactosidase activity were expanded for additional studies. We first demonstrated in preliminary experiments the optimal timing of harvest for maximal induction in b-gal activity. The results with three representative clones relative to the parent line are shown in Fig. 3. As can be seen, the results were similar to the transient transfection data. Although a modest increase in b-galactosidase activity in ()Arg) or ()Leu) media was observed in several of the 8–10 stably transformed clones we screened, the effect did not reach statistical significance.
Discussion Several mammalian genes involved in the control of growth or amino acid metabolism are now known to be regulated in response to changes in amino acid availability [8,10]. Amino acid deprivation in Chinese hamster ovary cells results in increased mRNA for c-jun, c-myc, and ornithine decarboxylase [9]. Induction of the C/EBP homologous protein (CHOP) by amino acid deprivation [21] and the induction of asparagine synthetase [22] are other recent examples of alterations in gene expression in response to amino acid restriction (AAR). Several mechanisms may underlie changes in gene expression upon amino acid depletion. Activation of transcription factors which are constitutively bound to DNA but require phosphorylation for their activation (e.g., immediate early genes like fos, jun, myc, and CREB) has been observed [9,10,23]. Activation is produced by nuclear translocation of a stimulus-dependent kinase such as the ERK kinases or their effector kinases. A second mechanism may involve activation of transcription factors in the cytoplasm via the same or other kinase pathways [9,23]. Upon activation, these factors translocate to the nucleus and affect transcription via their respective regulatory elements, e.g., STATs, NFkB. Transactivation also requires chromatin remodeling and alteration in the accessibility of cognate DNA elements to activated transcription factors [24]. Thus, transcription factor activation and/or chromatin remodeling are likely to be affected by signal transduction cascades which themselves are activated in response to alterations in the nutrient environment. Additional mechanisms for gene induction following amino acid depletion have been examined for several important genes including asparagine synthetase [25,26], C/EBP homologous protein or CHOP [21,25], the system A amino acid transporter ATA2 [27], and the Cat-1 amino acid transporter [28–30]. These actions
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occur by a number of complex transcriptional and posttranscriptional mechanisms of regulation. Well studied are the mechanisms of regulation of AS, CHOP, and cat-1. Cis elements which confer transcriptional regulation have been identified in both the AS and the CHOP gene through functional assays, footprinting, gel shift, and site-directed mutagenesis [21,25,27]. In the case of the ATA2, amino acid transporter, the increase in expression is due to the increase in transcriptional activation, not differences in amino acid stability [27]. Unlike the AS gene, the ATA2 gene is not activated by glucose starvation. The nutrient-sensing response unit (NSRU) in the AS promoter contains two cis elements termed NSRE1 and NSRE2, both of which are required for gene activation [27]. In the case of the Cat-1 gene, a high affinity transporter for amino acids, the increase in Cat-1 protein expression during amino acid deprivation is due to increased mRNA stability mediated by sequences within the 30 untranslated region and to enhanced translation of the messenger RNA [29]. The increase in translation efficiency is due to the presence of an internal ribosomal entry sequence (IRES) within the 50 -untranslated region of the Cat-1 mRNA which allows an increase in translation under conditions of amino acid starvation [28]. Regulation of Cat-1 expression during nutrient depletion is also dependent on an increased rate of transcription via activation of GCN2 kinase which phosphorylates eIF2a. This phosphorylation induces an increase in transcription of the Cat-1 gene promoter and is dependent on an amino acid response element located in the first exon [30]. Rapid modulation of the high affinity L-type amino acid transporter, LAT-1, expression and function in response to fluctuations in amino acid content and composition in the tissue environment is likely to be an important adaptive homeostatic mechanism in liver development and injury-response as well as starvation. These are all conditions in which LAT-1 expression is transiently increased in vivo [12,13,31]. The mechanism by which this occurs is unknown. Such regulation is lost in hepatic tumor cells where levels of LAT-1 RNA are constitutively high. Given our prior data demonstrating an increase in steady state LAT-1 mRNA, increased LAT-1 protein expression, and increased amino acid transport following incubation in media lacking Arg and/or Leu, we anticipated the effects might be due to AAREs within the LAT-1 proximal promoter. We failed, however, to demonstrate promoter dependency of increased LAT-1 expression within the cloned sequence using the reporter constructs and expression systems described above. This may be because (1) the cis elements necessary for this effect were not contained within our 2 kb promoter sequence; (2) the cell system(s) chosen did not express key transcription factors for this effect; (3) the effect involves a large change in RNA stability; or (4) a combination of these and other ex-
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planations. Further elucidation of the mechanisms by which cells specifically alter gene expression in response to amino acids remains of interest in order to understand how eukaryotic cells coordinate cellular processes with their tissue microenvironment under normal conditions as well as in disease states.
Acknowledgments This work was supported by American Institute for Cancer Research 02A127, NIH 1RO1 CA73611 and NCRR P20 RR17695 and P20 RR018728.
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