Regulation by glucagon of the rat histidase gene promoter in cultured rat hepatocytes and human hepatoblastoma cells
Gabriela Alemán,1,4
Victor Ortíz,1
Elizabeth Langley,2,3
Armando R. Tovar,1 and
Nimbe Torres1
1Departamento de Fisiología de la Nutrición, 2Unidad de Biología Molecular y Medicina Genómica, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán; and 3Departamento Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, and 4Faculdad de Medicina, Universidad Nacional Autonomica de México, Mexico City, Mexico
Submitted 10 December 2004
; accepted in final form 24 February 2005
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ABSTRACT
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Histidase (Hal), the amino acid-degrading enzyme of histidine, is regulated by the protein content of the diet and by hormones such as glucocorticoids and glucagon. However, glucagon can activate the following two possible transduction pathways: protein kinase A (PKA) and protein kinase C (PKC). The aim of this study was to isolate the 5'-flanking region of rat Hal gene to locate possible cAMP- and glucocorticoid-responsive elements and to identify whether the activation of the Hal promoter by glucagon occurs via PKA or PKC. The results showed that glucagon was able to induce Hal expression 1.5-fold in primary hepatocytes. The addition of phorbol 12-myristate,13-acetate (PMA) and forskolin to hepatocytes increased Hal mRNA concentration by 100 and 40%, respectively. To identify the Hal gene regulatory region, a 1248-bp fragment of the 5'-region was obtained. The transcription initiation site was located at 404 bp from ATG. The sequence did not show consensus TATA-like or CAAT-like boxes in the first 100 bp upstream from the transcription start site. The promoter contained six GC rich boxes, seven putative AP1 binding sites, and four glucocorticoid-responsive elements. The putative Hal promoter region was cloned into the pGL3basic vector and transfected into HepG2 cells. Luciferase expression was significantly stimulated by glucagon (0.9-fold), forskolin (0.9-fold), PMA (2.0-fold), and dexamethasone (2.9-fold). This evidence supports that the Hal gene is turned on by glucocorticoids and by glucagon either via PKC or PKA, but prefers the PKA pathway.
protein kinase A; protein kinase C; gene regulation; amino acid catabolism
AMINO ACID CATABOLISM IS ESSENTIAL for maintaining normal amino acid concentrations in tissues and body fluids, since there is no storage for the excess amino acids from the diet. Defects in the catabolic pathways for each amino acid may lead to metabolic abnormalities that in some instances can be life-threatening (11). There are rate-limiting enzymes in the degradative pathways for each amino acid; however, there is scarce information on the mechanisms of gene regulation for the amino acid-degrading enzymes. An integrative knowledge of gene expression control of these enzymes can help to elucidate the basic mechanism by which the body is able to control its nitrogen content.
Catabolism of most amino acids occurs in the liver, with the exception of the branched-chain amino acids. Histidine is a typical example of an amino acid degraded in the liver. The histidine-degrading rate-limiting enzyme is the histidine-ammonia lyase (E.C. 4.3.1.3
[EC]
), also known as histidase (Hal; see Ref. 26). In the rat, and in the mouse and human, the Hal gene is found as a single copy, and the cDNA from these species has high homology (18, 19). In the rat, the Hal gene codes for an mRNA of 1.97 kb that is translated into a protein of 657 amino acids with a relative molecular mass of
72.1 kDa (20). The activity and gene expression of this enzyme is regulated mainly by the protein content in the diet, the greater the concentration, the higher the Hal expression (23). Hal expression rises rapidly after the protein requirement is met to eliminate excess histidine.
The increase in Hal mRNA concentration also occurs under catabolic stress (2) or through the ingestion of imbalanced histidine diets (21). Thus the administration of glucocorticoids, hormones involved in stress responses, also increase the expression of Hal mRNA (1). Conversely, severe undernutrition decreases the expression of Hal (25). These changes in Hal expression are associated with specific hormonal changes (22). Dietary protein content is associated with glucagon serum levels in the rat, and glucagon levels in turn are associated with hepatic Hal activity and mRNA concentration (24). In fact, when rats are injected with glucagon, Hal activity and mRNA concentration increase rapidly after 3 h of glucagon administration (1). It is now clear that liver cells possess a single type of glucagon receptor. Upon glucagon binding, the receptor is able to couple to multiple G proteins, thereby activating two distinct signaling pathways, one coupled to phospholipase C, generating diacylglycerol and inositol 1,4,5-trisphosphate, and the other coupled to adenylate cyclase, increasing cAMP levels. However, it is not known which of these signaling pathways activate Hal gene expression.
At present, there is no information about the regulation of the Hal gene promoter. Suchi et al. (17) reported the sequence of the Hal human gene and a short segment of its promoter; however, its functionality was not assessed. Additionally, the regulatory region of the Hal gene in the rat has not been established. Therefore, the purpose of the present work was to determine the promoter region of the rat Hal gene, to identify the potential cis-acting elements, and to assess its functionality. Furthermore, we studied which of the signaling pathways activated by glucagon was involved in Hal gene expression. Our results showed that Hal gene expression in cultured hepatocytes is activated by either protein kinase A (PKA) or protein kinase C (PKC), and that the promoter of the gene contains the responsive elements that are activated by both signaling pathways. Furthermore, Hal promoter is activated by dexamethasone, indicating the presence of active glucocorticoid-responsive elements (GRE).
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MATERIALS AND METHODS
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Animals.
Male Wistar rats, obtained from the Experimental Research Department and Animal Care Facilities at the National Institute of Medical Sciences and Nutrition, were housed individually in wire, stainless steel cages at 22°C with a 12:12-h light-dark cycle and with free access to diet.
Diets.
Diets were administered in dry form and contain (in g/kg diet) 60, 180, or 350 vitamin-free casein, 50 corn oil, 50 mineral mix, and 10 vitamin mix. Cornstarch and sucrose, in 1:1 proportion, were added to complete 1 kg diet. The composition of the diets was described previously (23). Vitamin-free casein and the rest of the ingredients were obtained from Teklad (Madison, WI).
Dietary treatments.
Twelve rats, weighing 7590 g, had free access to the appropriate diet for 10 days and were randomly divided into three groups of four rats each: 1) 6% casein, 2) 18% casein, and 3) 35% casein. At the end of the 10-day period, rats were anesthetized with ether for hepatic perfusion to obtain hepatocytes. The protocols used in these experiments were approved by the Animal Care Committee of the National Institute of Medical Sciences and Nutrition.
Preparation and culture of primary rat hepatocytes.
Rat hepatocytes were isolated by the collagenase perfusion technique and separated from nonparenchymal liver cells and debris by centrifugation. Cell viability was assessed by the Trypan blue exclusion test and was always higher than 90%. Cells (65,000/cm2) were plated on treated culture dishes (100 mm diameter) and maintained in DMEM (GIBCO-BRL) supplemented with glucose, L-glutamine, pyridoxine hydrochloride, and sodium pyruvate. After 2 h, cells were washed, and the culture was continued in DMEM containing 10% heat-inactivated fetal bovine serum and 100 mg/ml streptomycin (4).
Hal activity.
Isolated hepatocytes (1 x 107 cells) were washed with ice-cold saline and lysed with 5 mmol/l NaOH in 14 mmol/l KCl. The lysed cells were centrifuged for 60 min at 105,000 g, and the clear supernatant was stored at 80°C before measuring Hal activity. The activity was assayed as described previously (23). The method is based on the spectrophotometric measurement of the appearance of urocanic acid at 277 nm. The reaction was linear for 10 min at 25°C in 0.1 mol/l pyrophosphate buffer, pH 9.2. An enzyme unit was defined as the formation of 1 nmol urocanic acid/min. The protein concentration was measured by Lowry assay with BSA standards.
Northern blot analysis.
Total cellular RNA was isolated from cultured hepatocytes with guanidine thiocyanate according to Chomczynski and Sachi (5). For Northern analysis, 15 µg RNA was electrophoresed in a 8 g/l agarose gel containing 2.2 mol/l formaldehyde, transferred to a nylon membrane filter (Hybond-N+), and cross-linked with an ultraviolet cross-linker (Amersham). RNA integrity and location of the 28S and 18S ribosomal RNA bands were determined under ultraviolet light. The cDNA probe was a 1005-bp PCR product amplified from rat liver Hal cDNA. The forward and reverse primers used for the PCR reaction were 5'-GCATCACCACGGGTTTT-3' and 5'-GGGCTATCATGAATCCAGAAT-3', respectively. The PCR product was purified with the high pure PCR product purification kit (Roche) and labeled with Redivue [
-32P]deoxycytidine triphosphate (110 TBq/mmol) by using the Rediprime DNA labeling kit. Membranes were prehybridized with rapid-hyb buffer at 65°C for 1 h and then hybridized with the cDNA probe (53.3 MBq/l) for 2.5 h at 65°C. Membranes were washed one time with 2x citrate saline solution (SSC; 1x SSC = 0.15 mol/l sodium chloride and 15 mol/l sodium citrate) and 0.1% SDS (wt/vol) at room temperature for 20 min and then two times for 15 min with 0.1x SSC/0.1% SDS (wt/vol) at 65°C. Digitized images and quantification of radioactivity (dpm) of the bands were carried out using the Instant Imager (Packard Instrument, Meriden, CT). Membranes were also exposed to Ecktascan film (Kodak de México, Guadalajara, México) at 70°C with an intensifying screen.
Growth and culture of HepG2 cells.
Human HepG2 hepatoblastoma cells were grown to confluence in tissue-culture bottles (75 cm2) in 12 ml RPMI medium containing 100 g/l FCS. For subcultivation, trypsin-treated cells were diluted in the same medium and plated again on 12-well plates at a density of 3 x 105 cells/well. The next day, cells were transfected with lipofectamine 2000 in serum-free medium, and, 4 h later, fresh serum-containing medium was applied. After transfection (24 h), experiments were started by applying fresh medium containing hormones or drugs at the concentrations indicated in Figs. 17.

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Fig. 1. Schematic representation of the rat histidase (Hal) promoter and its deletions. The arrow indicates the transcription start site. Numbering refers to the position of the transcription start site, which was set as +1. GRE, glucocorticoid-responsive element.
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Fig. 7. Transient transfection analysis of induction by hormones and drugs. HepG2 cells were transfected with Hal-pGL3b plasmid as described in MATERIALS AND METHODS. The cells were treated with medium alone (control), 10 µM dexamethasone (Dex), 0.1 µM glucagon, 10 µM forskolin, and 1 µM PMA. For dexamethasone assays, cells were cotransfected with 50 ng glucocorticoid receptor. Luciferase activity was normalized to -gal activity. Data for the 5 groups were taken from 3 representative experiments and are given as the mean ± range from triplicate transfections.
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Isolation of rat Hal 5'-flanking region.
Isolation of the promoter region of rat Hal gene was carried out using nested PCR reactions with specific antisense primers designed based on the Hal cDNA sequence and sense adaptor primers to amplify rat genomic DNA fragments (Genome Walker Kit). The external and nested gene specific reverse primers were 5'-TTG TTC TTC ATG TAG CGC CGC ACA G-3' and 5'-AAC ACT GAG CTT CCC GTC CTG GCA G-3' corresponding to nucleotides +116 to +91 and +72 to +48 of rat Hal cDNA, respectively. The external and nested sense adaptor primers were provided by the manufacturer. The amplified product was digested with the restriction enzyme Rsa I, subcloned in the EcoR V site of p-Bluescript KS(+), and sequenced with the Thermosequenase radiolabeled terminator cycle sequencing kit. The reaction products were separated in a polyacrylamide gel (80 g/l acrylamide-4 mol/l urea) that was dried before autoradiography at 70°C.
Primer extension analysis.
To find the transcription initiation site, primer extension analysis was performed using the primer extension system-AMV RT kit described by the manufacturer (Promega). To determine the size of the products, sequencing reactions were performed and run on the same gel.
Reporter gene assays.
To assess the functionality of the Hal promoter, different promoter fragments were inserted in the pGL3b-vector containing a luciferase reporter gene. The first PCR construct was generated using oligonucleotide primers [upper 5'-cttactcgagACTATAGGGCACGCGTGGT-3' (815 to 797 bp) and lower 5'-gcgaagcttATCAGGGTTCCTAGTTCTC-3'(+39 to +22)]. The product was ligated via pGEM-T overnight at room temperature. The ligated product was digested with Hind III/Xho I and subcloned into pGL3b, which had been digested with Hind III and Xho I. The rest of the inserts were 5'-deleted derivatives of the first insert. The first and second deletions (Hal-420 and Hal-302) were amplified using the following upper primers: 5'-gcgctcgagAAGACACAACTTTTCGTTG-3', and 5'-gcgctcgagATTAGGATTGCTACACTCC-3', respectively. A summary of all Hal-pGL3b constructs made is shown in Fig. 1.
Transfection protocol.
Transient transfection using lipofectamine 2000 (Invitrogen) was carried out according to the manufacturer's instructions. For assays with a luciferase reporter gene, plasmid pSV
containing
-galactosidase gene (25 ng/well) was cotransfected to normalize the transfection efficiency differences among samples. The total amount of DNA for assays not containing the glucocorticoid receptor plasmid was held constant by adding empty pCMV5 vector.
Luciferase and
-galactosidase assays.
After the last hormone treatment (3 h), cells were processed for luciferase and
-galactosidase activity according to Promega's protocol for the luciferase assay system. For each well, 100 µl lysis buffer were added. Luciferase activity was measured as relative light units with a BD Monolight 3010C luminometer (BD Biosciences Pharmingen, San Diego, CA). The
-galactosidase activity was measured as described previously (3). The luciferase activity from different samples was normalized by
-galactosidase activity from the same sample. Each treatment was done by triplicate, and all experiments were repeated at least three times. Within each experiment, the promoterless vector (pGL3 basic) was also transfected into HepG2 cells as a negative control. The results are presented as degree of induction.
Reagents and chemicals.
Nylon membrane filters (Hybond-N+), the Rediprime DNA labeling system, the Thermosequenase radiolabeled terminator cycle sequencing kit, deoxycytidine 5'-[
-32P]triphosphate (110 TBq/mmol), Redivue adenosine 5'-[
-33P]triphosphate (110 TBq/mmol), and Redivue 5'-[
-33P]dideoxyribonucleotides (55.5 TBq/mmol) were purchased from Amersham (Buckinghamshire, UK). The vitamin-free casein and the remaining ingredients of the diets were obtained from Teklad. Cell culture mediums and components and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA). The luciferase assay system,
-galactosidase system, and primer extension system-AMV RT were obtained from Promega (Madison, WI). Signaling pathway reagents were obtained from Sigma (Indianapolis, IN). Genome walker was obtained from Clontech (Palo Alto, CA). The high Pure PCR product purification kit was obtained from Roche (Mannheim, Germany). The endofree plasmid purification kit was purchased from Qiagen (Chatsworth, CA).
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RESULTS
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Structure of the 5'-flanking region of the Hal gene.
Isolation of the 5'-flanking region of the Hal gene was performed by DNA walking using specific nested primers located in the first 120 bp after the initiation codon in the cDNA sequence (17, 20). This sequence in the human cDNA is located in exon 2 of the genomic sequence of the Hal gene (18). The PCR amplification product using genomic rat DNA cut with EcoR V restriction enzyme produced a single fragment of 1265 bp. The amplified product was digested with Rsa I and then cloned in pBS II KS(+). The cloned fragment was sequenced, and the first 430 bp coincided with the untranslated region sequence of the rat Hal gene previously reported by Sano et al. (16). The remaining 818 bp had not been reported previously. The initiation of transcription site of the Hal gene was determined by primer extension analysis using a specific antisense primer flanking the 5'-end of the cDNA sequence. The amplified product was located 403 bp upstream from the initiation codon (Fig. 2). Computational analysis using MatInspector version 2.2 software (16) coincided with our primer extension assays in indicating that the nucleotide located 403 bp from the initiation of translation was the putative transcription initiation site.

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Fig. 2. Primer extension analysis of the hepatic Hal gene to determine the transcription initiation site.
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Sequence analysis of the 5'-flanking region of the Hal gene.
Blast sequence analysis showed that the rat Hal promoter region obtained in the present study showed homology with a small sequence of the mouse genome located close to the Hal gene in chromosome 10, whereas there was no homology with any sequence of the human genome. Interestingly, the rat Hal cDNA sequence has an 87% identity for mouse and human over the coding region. At the amino acid level, rat Hal is 93% conserved with both mouse and human Hal. The 818-bp DNA sequence of the 5'-flanking region of the Hal gene is shown in Fig. 3. Computational analysis to determine putative transcriptional binding sites was also performed with the MatInspector version 2.2 program at the TRANSFAC 7.2-Public 4.0 database (http://transfac.gbf.de/TRANSFAC/). The sequence did not show consensus TATA-like or CAAT-like boxes in the first 100 bp upstream from the transcription start site. Instead, there are two putative TATA-like boxes at 152 and 263 bp and two putative CAAT-like boxes at 190 and 283 bp. There are also six GC-rich boxes at 78, 130, 211, 312, 418, and +2 nt that are potential binding sites for the transcription factor Sp1 (8). Additionally, there are seven putative AP1, four GATA, and one Oct-1 binding site. Sequences homologous to several binding sites for liver-specific transcription factors including C/EBP, NF-IL-6, and HNF4 were found. There are also four GRE at 158, 390, 446, and 528 and single estrogen receptor and progesterone receptor sites at 218 and 697, respectively (Fig. 3).

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Fig. 3. Nucleotide sequence of the rat hepatic Hal 5'-flanking region. The filled circle (assigned +1) represents the putative major transcription initiation site. Underlined are identified cis-element sequences homologous to binding sites for various transcription factors. Italic type shows previously reported nontranslated region. Bold type indicates transcription and translation initiation sites. Box areas indicate putative TATA and CAAT boxes.
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Effect of dietary protein on Hal expression in rat hepatocytes.
To examine the effect of different concentrations of dietary protein on Hal activity and mRNA concentration in rat isolated hepatocytes, rats were fed for 10 days with diets containing 6% (low-protein diet), 18% (adequate-protein diet), or 35% (high-protein diet) casein. Hal activity increased by
11-fold in rats fed 18% casein diet compared with rats fed 6% casein diet, whereas rats fed 35% casein diet showed 30% increased Hal activity with respect to rats fed 18% casein diet. Increments in Hal activity were accompanied with corresponding increments in Hal mRNA, as shown in Fig. 4. These results indicate that the consumption of graded dietary protein concentrations increased Hal expression in rat isolated hepatocytes.

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Fig. 4. mRNA concentration and Hal activity in hepatocytes of rats fed 6, 18, or 35% casein diets. A: Northern blot analysis of Hal mRNA in hepatocytes. B: ethidium bromide-stained gels. C: Hal activity expressed as U/mg protein.
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Effect of glucagon on Hal expression in rat cultured hepatocytes.
Because glucagon is a hormone that increases when the protein content of the diet increases, we studied the effect of this hormone on Hal mRNA concentration in primary cultured rat hepatocytes. The effect of physiological concentration of glucagon (107 mol/l) on the time course response of Hal mRNA concentration showed an increase of 46% 1 h after the addition of glucagon. Hal mRNA concentration returned to basal levels after 2 h of exposure to the hormone (Fig. 5A). Furthermore, we assessed the effect of graded concentrations of glucagon (106 to 1010 mol/l) on Hal mRNA concentration in cultured isolated rat hepatocytes. The highest stimulation of Hal mRNA was obtained after 1 h of incubation when cultured hepatocytes were incubated with 1010 mol/l glucagon (Fig. 5B). We carried out a time course experiment at this hormone concentration to see the maximal response of Hal mRNA abundance to glucagon. This experiment showed that the maximal induction of 1.5-fold was obtained after 4575 min of incubation (Fig. 5C). These data clearly show that glucagon induces Hal gene expression in cultured rat hepatocytes.

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Fig. 5. Northern blot analysis of Hal mRNA in hepatocytes with different concentrations of glucagon. A: time course of glucagon (107 M)-treated hepatocytes on Hal mRNA. B: hepatocytes treated with different concentrations of glucagon. C: time course of glucagon (1010 M)-treated hepatocytes on Hal mRNA. AC show Northern blot analysis of 15 µg total RNA from hepatocytes under different conditions (ethidium bromide-stained gels and quantification of Hal mRNA concentrations by electronic autoradiography).
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Effect of PMA, forskolin, and ANG II on Hal gene expression in rat hepatocytes.
There are two possible different signal transduction pathways that can be activated by glucagon. The first one is coupled to phospholipase C, causing breakdown of phosphatidylinositol 4,5-bisphosphate, and the second one is coupled to adenylate cyclase, generating cAMP (10). To investigate which of these two signaling pathways was preferentially used to induce Hal transcription, we used phorbol 12-myristate,13-acetate (PMA), forskolin in the presence of 3-isobutyl-1-metylxanthine, or ANG II as activators of PKC, PKA, and calcium-calmodulin kinase, respectively. The results showed that addition of 100 nmol/l PMA doubled Hal mRNA concentration after 4 h of incubation in cultured rat hepatocytes (Fig. 6A). Cultured hepatocytes incubated for 12 h with 10 µmol/l forskolin also increased Hal mRNA concentration by
40% (Fig. 6B). ANG II did not change Hal mRNA levels. (Fig. 6C). These data indicated that Hal gene expression can be activated via either PKA or PKC.

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Fig. 6. Northern blot analysis of Hal mRNA in hepatocytes treated with different drugs. A: time course of the effect of 100 nM phorbol 12-myristate,13-acetate (PMA) on Hal mRNA. B: time course of the effect of 10 µM forskolin on Hal mRNA. C: time course of the effect of 100 nM ANG II on Hal mRNA. AC show Northern blot analysis of 15 µg total RNA from hepatocytes under different conditions (ethidium bromide-stained gels and quantification of Hal mRNA concentrations by electronic autoradiography).
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Functional analysis of the rat liver Hal promoter.
To analyze the function of the Hal promoter and to study whether the Hal gene was turned on via PKA or PKC, we cloned the Hal gene promoter in the pGL3 basic vector. The complete promoter region and two different 5'-deletion fragments of the rat Hal gene promoter, upstream of the transcription initiation site, were generated by PCR and ligated into the luciferase reporter vector pGL3-basic. These reporter constructs were transfected into HepG2 cells. The common downstream boundary of all these constructs was at the nucleotide position +22 from the transcription start site. In HepG2 cells, luciferase expression, under the control of the rat liver full-length Hal promoter, was significantly stimulated by dexamethasone (2.9-fold), glucagon (0.9-fold), forskolin (0.9-fold), and PMA (2.0-fold) over basal nonstimulated expression (Fig. 7). This evidence supported our findings using cultured hepatocytes that the Hal gene is turned on via PKC or PKA and that activation is higher with PMA than forskolin. As we have demonstrated previously, dexamethasone in vivo increases hepatic Hal mRNA concentration (1). Our present data support a direct role for dexamethasone on the activation of the Hal promoter. Deletion of nucleotides 815 to 340 from the rat Hal promoter, which removes the region containing three AP1 sites, a TAX/CREB site, and three glucocorticoid response elements, abolished the stimulation induced by PMA, forskolin, and glucagon but still showed stimulation by dexamethasone, although to a lower extent compared with the full segment. Deletion of the segment containing nucleotides 815 to 202 was insensitive to treatment with PMA, forskolin, or glucagon. However, despite of the deletion of 613 nucleotides, dexamethasone was able to induce luciferase activity by 1.6-fold compared with pGL3 promoterless vector. These results suggest that the GRE sites in this region may be more active when a potential repressive site found between 340 and 202 is deleted (Fig. 7).
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DISCUSSION
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Several studies in mammals have shown a lack of protein storage in the body after the consumption of a high-protein diet. Furthermore, high concentrations of many of the individual indispensable amino acids in body fluids lead to adverse effects and even toxicity. This implies the presence of specific mechanisms to control the excess of dietary amino acids to prevent deleterious effects. Conservation of body nitrogen and elimination of excess amino acids is mainly controlled by the amino acid-degrading enzymes. Understanding the mechanisms of regulation of expression of these enzymes will give insight into the fine tuning of amino acid catabolism.
In the present study, we observed that Hal mRNA concentration from cultured hepatocytes obtained from rats fed a high-protein diet was higher than those fed an adequate- or low-protein diet, indicating the induction of this amino acid-degrading enzyme to eliminate the excess histidine. Previous studies have demonstrated that induction of several amino acid-degrading enzymes, including Hal, is mediated by an increase in glucagon concentration, which enhances amino acid catabolism (5). Also, it was demonstrated that ingestion of a high-histidine diet did not increase Hal expression, indicating that Hal gene is regulated by hormones but not by its substrate (22). In this study, we demonstrate that addition of glucagon to cultured hepatocytes increases Hal mRNA concentration, and the stimulation is dose and time dependent. These data are in agreement with previous studies that showed that other hepatic amino acid-degrading enzymes are regulated by the protein content of the diet, by the injection of glucagon to rats, or by the addition of this hormone to cultured hepatocytes (7, 9, 1214, 27). In these studies, it has been demonstrated that glucagon stimulates amino acid-degrading enzyme activities and induces their mRNA. However, it has not been clearly established what signal transduction pathway(s) are involved in the activation of the expression of these enzymes by glucagon. In addition, with the exception of serine dehydratase (SDH), whose promoter contains functional cAMP-response element (CRE) sites for its activation by cAMP (9), other amino acid-degrading enzyme promoters have not been studied to determine whether they contain putative cis-acting elements associated with changes in gene expression due to the presence of metabolic hormones such as glucagon or glucocorticoids.
Glucagon exerts its action through a receptor coupled to a G protein; however, it was demonstrated that this hormone increases cAMP concentration and intracellular calcium concentration via inositol 1,4,5-trisphosphate (10). This indicates that glucagon signaling occurs via PKA or PKC. To assess if Hal induction by glucagon was mediated via PKA or PKC, isolated hepatocytes were incubated with forskolin, an activator of adenylate cyclase that causes an increase in cAMP levels and activates PKA, or PMA, a structural analog of diacylglycerol that activates PKC. Our results show that Hal mRNA is induced by both compounds, indicating that the expression of this enzyme occurs via either PKA or PKC. Interestingly, ANG II, which increases the release of intracellular calcium and has no effect on cellular cAMP levels, did not change Hal gene expression. Thus the increase of Hal expression is not mediated by calcium-calmodulin kinases.
To understand whether the activation of the Hal gene occurs as a result of an increase in Hal transcription mediated by signaling pathways involving PKA or PKC, it was necessary to isolate and sequence the promoter region of the rat Hal gene to identify potential cis-acting elements activated by cAMP or PMA. The analysis of the promoter region of the rat Hal gene revealed the lack of a typical TATA box but was GC-rich, resulting in the presence of several Sp1 and AP1 motifs near the transcription initiation site (Fig. 2). Conversely, a segment of 610 nucleotides from the human Hal promoter has been previously reported and shows a consensus TATA box (18). Additionally, the adenine repeat in the rat promoter sequence is not present in the human gene. Nonetheless, in the promoters of both organisms, there are a similar number of potential GRE, GATA, C/EBP, and NF-IL-6 sites. The rat Hal promoter shows four AP1 sites compared with the human promoter that only contains two sites, and this difference may alter the capacity of both promoters to respond to cAMP and/or PMA. On the other hand, the regulatory region of the rat SDH gene promoter has been studied (13, 15). The SDH promoter region, similar to the rat Hal promoter, lacks typical TATA and CAAT boxes and has several GC boxes that may be binding sites for transcription factor Sp1. In addition, the SDH promoter contains a 22-bp adenine repeat between nucleotides 157 to 178, which resembles the 17-bp adenine repeat present in the rat Hal promoter between nucleotides 25 to 44. Furthermore, rat SDH promoter contains at least two GRE and two CRE sites, providing it with the capacity to respond to glucocorticoids and cAMP in a similar fashion as the rat Hal promoter.
The functional analysis of the rat Hal promoter showed that the region between nucleotides 340 to 815 is important for the response to glucagon and to forskolin and PMA. This region contains a TAX/CREB site at 629 bp and three AP1 sites at 404, 452, and 691 bp that are the potential regulator sites for glucagon response. On the other hand, there are at least six GRE sites in the complete fragment of 815 bp and all seem to be functional, although GRE sites at positions 455, 628, and 780 bp are apparently stronger sites for glucocorticoid stimulation. A more detailed study is required for the analysis of cis-acting elements in the region between 340 and 815 bp.
In summary, this analysis showed that the rat Hal gene promoter contains active response elements for the metabolic hormones, glucagon and glucocorticoids, that are able to upregulate rat Hal gene expression. In addition, glucagon stimulation of the Hal promoter occurs preferentially via PKA, although it is also stimulated via PKC. Thus, when rats consume a high-protein diet, there is a proportional increase in serum glucagon levels that in turn activate PKC and PKA pathways and increase Hal transcription, leading to an increase in the amount of this enzyme to degrade excess histidine. Contrariwise, under stress conditions, glucocorticoids are also able to induce Hal expression to oxidize histidine to meet energy requirements. It seems that most of the amino acid-degrading enzymes respond to both hormones in a similar fashion; thus, it seems that the mechanism that controls Hal gene expression is a concerted mechanism through which all of the amino acid-degrading enzymes control body nitrogen balance. Further studies are required to prove this hypothesis.
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GRANTS
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G. Alemán was supported by fellowships from the program of Doctorado en Ciencias Biomédicas, UNAM (Direccion General de Estudios Profesionales), and by Consejo Nacional de Ciencia y Tecnologia (CONACYT). This work was supported by Grant 26591-M from CONACYT (to N. Torres).
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FOOTNOTES
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Address for reprint requests and other correspondence: N. Torres, Instituto Nacional de Ciencias Médicas y Nutrición, Depto. Fisiología de la Nutrición, Vasco de Quiroga No 15, Tlalpan, México, D.F., 14000, México (e-mail: nimbet{at}quetzal.innsz.mx)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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