L-Histidine decarboxylase decreases its own transcription through downregulation of ERK activity

Rocchina Colucci*, John V. Fleming*, Ramnik Xavier, and Timothy C. Wang

Harvard Medical School and Gastrointestinal Unit, Massachusetts General Hospital, Boston, Massachusetts 02114


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A poorly defined negative feedback loop decreases transcription of the L-histidine decarboxylase (HDC) gene. To help understand this regulation, we have studied the effect of HDC protein expression on HDC gene transcription in transfected AGS-B cells. Expression of the rat HDC protein inhibited HDC promoter activity in a dose-dependent fashion. The region of the HDC promoter mediating this inhibitory effect corresponded to a previously defined gastrin and extracellular signal-related kinase (ERK)-1 response element. Overexpression of the HDC protein reduced nuclear factor binding in this region. Experiments employing specific histamine receptor agonists indicated that the inhibitory effect was not dependent on histamine production, and studies with the HDC inhibitor alpha -fluoromethylhistidine revealed that inhibition was unrelated to enzyme activity. Instead, an enzymatically inactive region at the amino terminal of the HDC enzyme (residues 1-271) was shown to mediate inhibition. Fluorescent chimeras containing this domain were not targeted to the nucleus, arguing against specific inhibition of the HDC transcription machinery. Instead, we found that overexpression of HDC protein decreased ERK protein levels and ERK activity and that the inhibitory effect of HDC protein could be overcome by overexpression of ERK1. These data suggest a novel feedback-inhibitory role for amino terminal sequences of the HDC protein.

extracellular signal-related kinase; gastrin; gastrin response element-binding protein


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

EXPRESSION OF THE histamine-producing enzyme L-histidine decarboxylase (HDC; 4.1.1.22) has been detected in a variety of cell and tissue types, reflective of the diverse regulatory role of histamine in numerous physiological processes (5, 30). This includes expression in the gastrointestinal tract, where enterochromaffinlike (ECL) cells of the gastric mucosa appear to be the main source of histamine responsible for the stimulation of gastric acid secretion by the hormone gastrin (32). HDC expression and histamine production have also been linked to a number of physiological processes related to growth and differentiation (18, 19, 24, 25). For example, HDC is expressed at high levels during certain stages of fetal development, suggestive of a role in tissue modeling or organogenesis (24, 35). Increased expression has also been observed in the adult during conditions of growth or regeneration, including the rat liver after partial hepatectomy (21, 23), the tissue of healing skin wounds (23), after ischemia-reperfusion injury to the small intestine (14), and in the gastric tissues surrounding restraint and cold-induced stress ulcerations (7). Given the importance of histamine in normal physiology, it is not surprising that expression of the HDC gene is under tight regulatory control.

Stimulation of ECL cells by the hormone gastrin leads to activation of CCK-B/gastrin receptor signaling and the immediate release of stored histamine from secretory vesicles (9, 16). Levels of stored histamine are then replenished over a 6- to 8-h period, first through increases in HDC protein stability (and, consequently, increases in HDC activity) and then through increased HDC gene transcription (9, 11, 13, 32). There is no evidence for feedback inhibition of either HDC activity or HDC gene transcription immediately after gastrin stimulation, which is when extracellular histamine concentrations are at their highest (9, 32). Instead there is a decrease in HDC activity and in the rate of HDC gene transcription a number of hours after gastrin stimulation (9).

The molecular mechanisms responsible for gastrin stimulation of HDC transcription have been addressed in a number of studies. These include experiments in which HDC promoter constructs have been transfected into the AGS-B cell line, a gastric cell line that stably expresses the human CCK-B/gastrin receptor (16, 18). In these experiments, a 28-nucleotide cis-acting element composed of two overlapping transcription factor binding sites (+1/+19 and +11/+27) could mediate gastrin stimulation of HDC gene transcription [gastrin response element (GAS-RE); Ref. 31]. GAS-RE stimulation by gastrin was shown to occur through a mitogen activated protein kinase (MAPK)-dependent pathway that involved the phosphorylation of tyrosine residues on both extracellular signal-related kinase (ERK)-1 and -2 proteins. This resulted in increased ERK activity, as indicated by increased phosphorylation of myelin basic protein. In addition to promoting the increase in HDC gene transcription, gastrin could also increase Elk-1- and c-myc-dependent transactivation (16, 18, 40). Identical results involving ERK phosphorylation/activation, HDC gene transcription, and Elk-1/c-myc transactivation were obtained when gastrin was replaced with phorbol esters (PMA) (16). Thus these studies have shown that MAPK is crucial to the normal physiological control of HDC transcription and suggest that HDC gene transcription is only one of several downstream targets of the ERK signaling pathway.

Although increases in histamine synthesis and histamine release have been reasonably well studied, considerably less is known about the mechanisms that downregulate this system, particularly the mechanism involved in downregulation of HDC transcription. Therefore, although recent studies indicate that H3 receptor stimulation by histamine can decrease both histamine secretion (3) and, to a lesser extent, HDC activity (20), the decreased rate of HDC gene transcription observed 6-8 h after gastrin stimulation (9) has not been directly studied. Nevertheless, there is some indirect evidence to suggest that histamine promotes feedback inhibition of the HDC promoter (2). The possibility of feedback inhibition through a nonenzymatic mechanism has not been examined.

To investigate feedback inhibition of HDC promoter activity by HDC protein, we carried out cotransfection experiments with two expression constructs; the first overexpresses enzymatically active rat HDC protein from the cytomegalovirus (CMV) promoter, whereas the second contains the luciferase reporter cassette (-luc) under the regulation of the human HDC promoter. A combination of studies with these two constructs revealed that HDC promoter activity could be inhibited independently of both histamine production and HDC enzyme activity and that this inhibition was linked to downregulation of ERK protein expression. The inhibitory domain of HDC was localized to the amino terminal of the protein. Given the close temporal correlation between the accumulation of HDC protein and decreased rate of HDC transcription observed in gastric mucosa, we believe that the regulatory mechanism identified in this study is of importance in the maintenance of gastric homeostasis as well as regulation of ERK-dependent transcription in histamine-producing cells.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials. DMEM, FCS, penicillin-streptomycin, and EDTA-trypsin were obtained from BioWhittaker. Human sulfated gastrin-1 was purchased from Peninsula Laboratories (San Diego, CA). L-[14C]histidine (0.02 mCi/ml, 50 mCi/mmol) was supplied by New England Nuclear (Boston, MA). The following drugs were used: histamine dihydrochloride, R-alpha -methylhistamine dihydrochloride, clobenpropit dihydrobromide, alpha -fluoromethylhistidine hydrochloride (alpha -FMH) (all from RBI), N,N-diethyl-2[4-(phenylmethyl)phenoxy]ethanamine fumarate (DPPE; Tocris, Cookson, MO), propidium iodide (Sigma, St. Louis, MO), anti-ERK1/2 and anti-active MAPK antibodies (Promega, Madison, WI), anti-BFP antibody (Clontech), and ranitidine hydrochloride (Glaxo Pharmaceuticals).

Cell culture and transfections. AGS-B cells were grown in complete medium (DMEM containing 10% FCS, 100 IU/ml penicillin, and 100 µg/ml streptomycin) in a humidified atmosphere (5% CO2-95% air). The AGS-B/HDC cell line was generated by transfection of AGS cells (ATCC CRL 1739) with the expression vectors pEF1alpha -CCKB and pCMVHDC-18 (gift from D. Joseph and hereafter referred to as pCMV-HDC; Ref. 22), which express full-length human CCK-B/gastrin receptor and rat HDC proteins, respectively. Stably transfected cells were initially selected in puromycin (1 mg/ml in complete medium). Cells were subsequently screened for overexpression of HDC by Northern blotting. AGS-B cells were used for luciferase assays, Western blots, enzymatic assays, and fluorescence experiments, whereas both AGS-B and AGS-B/HDC cells were used for electrophoretic mobility shift assays (EMSA) and ERK phosphorylation assays. Transient transfections were carried out using the calcium-phosphate precipitation method (DNA transfection kit, 5'-3'). Cells were plated at a density of 1 × 106 cells/35-mm well and transfected the next day. Gastrin was added at the maximal stimulatory concentration (0.1 µM) 12-24 h after transfection, and cells were harvested and luciferase assays were performed at 48 h (Promega) with a Monolight luminometer (Analytical Luminescence Laboratory) as previously described (18). In experiments in which agonist and antagonist compounds were used, incubation was initiated either 2 h after transfection or 2 h before gastrin stimulation. Transfections were performed in triplicate. Values for luciferase activity, obtained from at least three independent experiments, were expressed as absolute values or as multiples of increase compared with untreated controls. Statistical significance was evaluated by ANOVA followed by Dunnett test.

DNA constructs. A human HDC promoter fragment containing 1.8-kb of 5'-flanking DNA and 126 nucleotides of the noncoding first exon (p1.8HDC-luc) and a series of 5' deletion constructs containing 125 and 59 nucleotides upstream of the start site as well as 126 nucleotides of the noncoding first exon (pHDC-luc125bp and pHDC-luc59bp) are all based on the promoterless luciferase reporter gene vector pXP2, and all have been previously reported (40). The pHDC-luc +1/+27 construct contained the GAS-RE (+1/+27) ligated upstream of the enhancerless herpes simplex thymidine kinase promoter (16).

The pCMV-HDC expression construct has been described and characterized elsewhere as pCMVHDC-18 (22). Sense and antisense expression plasmids were confirmed by asymmetric double-restriction digestion. A carboxy terminal deletion of the rat HDC cDNA that was 2,033 nucleotides in length (pCMV-HDC2033) was obtained by digestion of pCMV-HDC with Pst I and subsequent relegation. Additional deletion constructs were generated by PCR using primers containing EcoR I sites in the sense primers and Sal I sites in the antisense primers and using pCMV-HDC as template. PCR products of sizes 890, 436, and 196 nucleotides were used to generate the inserts for pCMV-HDC890, pCMV-HDC436, and pCMV-HDC196, respectively. These PCR products, which are from the 5' end of the rat HDC cDNA and included the 5' untranslated region (UTR), were digested with EcoR I and Sal I and subcloned into the pCMV5 vector. Chimeric fluorescent constructs were obtained by cloning in frame into the EcoR I and Sal I sites of the blue fluorescence protein (BFP) pEBFP-C1 or green fluorescence protein (GFP) pEGFP-C1 expression vectors. PCR products for these fluorescent constructs did not contain the 5' UTR of HDC and corresponded to nucleotides +75 to +196, +75 to +890, and +196 to + 890 of the rat HDC full-length cDNA (pBF-75/196HDC, pBF-75/890HDC, and pBF or pGF-75/890HDC, respectively). Cells transfected with these expression constructs were viewed by fluorescent microscopy, assayed for luciferase activity, or assayed for peptide expression with Western blots.

The pRSV-luc plasmid carried the luciferase gene driven by the Rous sarcoma virus promoter. The 4.8-kb mCgA-luc plasmid, which contains 4.8 kb of the mouse chromogranin A promoter ligated into the polylinker region of the promoterless luciferase reporter vector pXP1, has previously been described (31). The wild-type ERK1 and ERK2 expression vectors (pCMV-ERK1 and pCMV-ERK2) have also been described (16). The pGAL4-Elk-1 vector expresses the GAL4 DNA binding domain linked to the ELK-1 transactivation domain. p5xGAL-luc contains five consensus GAL4 DNA binding sites (17-mer) subcloned into p20-luc. The pGAL4-c-Myc construct contains the GAL4 DNA binding domain fused to amino acids 1-103 of c-Myc (16).

EMSAs. Nuclear extracts from AGS-B and AGS-B/HDC cells were prepared from unstimulated and gastrin-stimulated cells. Protein content was assessed in the nuclear lysates using the Bradford assay (Bio-Rad). Double-stranded oligonucleotides representing the sequence +1/+19 and +11/+27 of the GAS-RE1 and the GAS-RE2 binding sites, respectively, were radiolabeled with [alpha -32P]dCTP, and EMSAs were performed with 5 µg of nuclear extracts in a final volume of 20 µl of binding buffer containing (in mM) 10 Tris · HCl (pH 7.5), 50 NaCl, 5 MgCl2, 1 dithiothreitol, and 1 EDTA with 1 µg of poly(dA-dT) and 10% glycerol as previously described (31). The binding reactions were performed by adding 10 fmol of double-stranded oligonucleotide probes for 20 min at room temperature. DNA-protein complexes were electrophoresed on a 6% nondenaturing polyacrylamide gel containing 0.25× (Tris-borate-EDTA) at a constant current of 15 mA. Gels were dried and exposed to Kodak X-AR films at room temperature.

Western blotting. After transfection with appropriate HDC expression constructs, AGS-B cells were washed with ice-cold PBS twice and lysed with ice-cold lysis buffer containing 10 mM HEPES (pH 7.4), 30 mM NaCl, 2% glycerol, 0.2% Triton X-100, 0.3 mM MgCl2, 0.2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 mM sodium orthovanadate, and 1 mM sodium fluoride. Fifty micrograms of the lysates were electrophoresed on 10% SDS-polyacrylamide gels. After gel separation, proteins were transferred at room temperature for 1 h at 100 V to nitrocellulose membranes using transfer buffer consisting of 192 mM glycine, 25 mM Tris, and 20% methanol. Nonspecific binding was blocked with 5% nonfat dried milk in PBS plus Tween 20 (PBS-T; in mM: 136 NaCl, 2.7 KCl, 8.1 Na2HPO4, and 1.5 KH2PO4 plus 0.1% Tween 20) for 2 h at room temperature. The membrane was then incubated with the Living Colors antibody (1 µg/ml; Clontech) at a 1:5,000 dilution for 1 h. The membrane was washed three times with PBS-T and then incubated with the anti-rabbit IgG-horseradish peroxidase conjugate (1:5,000 dilution; Amersham) in PBS-T for 1 h at room temperature. After four washes with PBS-T, bands were visualized with the enhanced chemiluminescence system (New England Nuclear).

ERK phosphorylation assay. Cells were treated for 5 min at 37°C with 0.1 µM PMA or left untreated (control), rinsed with ice-cold PBS, and then lysed. Ten micrograms of the lysates were analyzed by SDS-PAGE. After transfer to nitrocellulose membranes, proteins were detected with anti-active MAPK antibody, which recognizes only the active phosphorylated ERKs. The membranes were then stripped with appropriate buffer and reprobed with anti-ERK1/2 antibody to reveal the total ERKs present in the samples. Scanning densitometry of the Western blots was performed using the NIH Image program.

HDC enzymatic assay. Cells were harvested in 2 ml of PBS and assayed for HDC activity as described elsewhere (10, 13). All samples were normalized to total protein content, and the protein concentration was determined using the method of Bradford.

Fluorescence. Cells were grown to a low density on poly-D-lysine-coated microscope slides under standard conditions and transfected with 20 µg of either pEGFP-C1 (pGF) or pGF-75/890HDC expression constructs and Superfect as advised by the manufacturer (Qiagen). The cells were then washed with PBS and viewed with a fluorescent microscope at ×60 magnification.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HDC protein expression decreases HDC promoter activity. In a first series of experiments, AGS-B cells were transiently cotransfected with the 1.8HDC-luc construct and increasing amounts of pCMV-HDC, which expresses the full-length rat HDC protein. Increasing HDC protein levels resulted in a dose-dependent decrease in HDC promoter activity, with greatest inhibition (82 ± 11%; n = 3) observed for cells transfected with 1 µg/well of pCMV-HDC (Fig. 1). Transfection with 1 µg/well of either pCMV-empty vector or pCMV-HDC antisense did not significantly alter promoter activity (Fig. 1), showing that inhibition was dependent on the production of HDC protein. In this series of experiments, the expression of functionally active HDC protein was confirmed by Western blotting and enzymatic assays (data not shown).


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Fig. 1.   Effect of L-histidine decarboxylase (HDC) protein expression on HDC promoter activity. AGS-B cells were cotransfected with 1 µg of p1.8HDC-luc and increasing amounts of the pCMV-HDC expression vector. Control cells were cotransfected with p1.8HDC-luc and either pCMV empty or pCMV-HDC antisense constructs. CMV, cytomegalovirus; -luc, luciferase cassette. Values are means ± SE of 4 independent experiments. *P < 0.01 vs. 0-µg pCMV-HDC control.

To test whether inhibition by HDC protein is limited to the HDC promoter, AGS-B cells were cotransfected with pCMV-HDC and one of the following plasmids: p4.8mCgA-luc, pRSV-luc, or pT81-luc. For these plasmids, the luciferase reporter cassette is under the control of the chromogranin A, Rous sarcoma virus, or thymidine kinase promoters, respectively. The chromogranin A and Rous sarcoma virus promoters, which were both activated by gastrin stimulation, showed 57 ± 8% (n = 3) and 71 ± 9% (n = 3) inhibition, respectively in response to HDC protein expression. In contrast, the activity of the pT81-luc construct, which showed minimal responses to gastrin, was not inhibited by the HDC protein expression (data not shown).

Inhibition of HDC promoter activity is mediated by the GAS-RE. These data suggested that inhibition is not specific to the HDC promoter but may instead be a feature of gastrin-inducible promoters. To identify the DNA sequence that mediates transcriptional inhibition, we used a series of HDC promoter deletion constructs that have previously been described (18, 40). Overexpression of the HDC protein inhibited transcription of p1.8HDC-luc, pHDC-luc125bp, and pHDC-luc59bp by 57 ± 8, 63 ± 6, and 56 ± 7%, respectively (Fig. 2A). Similar inhibitory effects were observed when cotransfections were performed with region +1 to +27 of the HDC promoter (pHDC-luc+1/+27), which has previously been defined as the minimal HDC GAS-RE (40). When cells were transfected with a construct containing this region inserted upstream of the thymidinekinase promoter, a 40 ± 12% inhibition of transcription was observed in response to HDC protein expression (Fig. 2A). Thus the HDC promoter sequence that is targeted for inhibition by the HDC protein is also that minimal region that activates transcription in response to gastrin stimulation, the GAS-RE.


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Fig. 2.   Inhibition of the HDC promoter is mediated by the gastrin response element (GAS-RE). AGS-B cells were cotransfected with pCMV-HDC and with either the full-length promoter (p1.8HDC-luc) or deleted promoter (pHDC-luc125bp, pHDC-luc59bp, or pHDC-luc +1/+27bp) constructs. Luciferase activities were assessed in the absence (A) and presence (B) of 0.1 µM gastrin. Values are means ± SE of 4 independent experiments. *P < 0.01. C: nuclear extracts were prepared from unstimulated and gastrin-stimulated AGS-B or AGS-B/HDC cells. Nuclear extracts were incubated with GAS-RE1 (+1/+19) or GAS-RE2 (+11/+27) probes and then analyzed by electrophoretic mobility shift assay. Bound complexes are indicated with an arrow.

Transfected cells were additionally cultured in the presence of gastrin, and an increase in basal luciferase activity was observed for all four HDC promoter constructs (Fig. 2B). Gastrin stimulation of the HDC promoter was also observed during coexpression of the HDC protein (Fig. 2B); however, stimulation was not as great as that observed in its absence. Thus, although HDC protein expression inhibits gastrin-dependent activation of the HDC promoter, the inhibitory effect is clearly observed on basal promoter activity. Consequently, in all later experiments, HDC promoter inhibition observed in the absence of gastrin was also repeated in its presence.

HDC protein overexpression decreases GAS-REBP1/2 binding to the HDC promoter. Previous studies have shown that two distinct nuclear transcription factor complexes, GAS-REBP1 and GAS-REBP2, bind specifically and in a gastrin-responsive manner to nucleotides +1 to +19 and +11 to +27, respectively, of the human HDC promoter (31). To determine whether the inhibition of HDC transcription by the HDC protein is related to reduced binding of GAS-REBP1 and -2, we generated an AGS-B cell line stably transfected to overexpress HDC. Elevated HDC mRNA levels were confirmed by Northern blot analysis (data not shown). EMSA experiments on nuclear extracts from both the AGS-B and AGS-B/HDC cell lines were performed using radiolabeled probes corresponding to nucleotides +1 to +19 and +11 to +27 of the GAS-RE. In confirmation of previous reports (31), we noted that gastrin stimulation of AGS-B cells resulted in increased binding activities for both GAS-REBP1 and -2 (Fig. 2C). The stable expression of HDC protein in the AGS-B/HDC cell line, on the other hand, resulted in decreased binding of these nuclear factors to both elements of the promoter (Fig. 2C).

Histamine synthesis and histaminic receptors are not involved. Previous studies suggest that feedback inhibition of histamine secretion and HDC activity depends on histamine binding to the histamine H3 receptor (3, 20). One hypothesis to explain our initial results is that HDC protein expression leads to increased histamine production and, consequently, ligand stimulation of a histamine receptor (possibly even the H3 receptor). This in turn might result in decreased binding of GAS-REBP1 and -2 to the HDC promoter, with the net effect of decreased HDC transcription. To test this hypothesis, and to determine whether the inhibition of HDC transcription observed in transfected AGS-B cells is dependent on H3 receptor stimulation, we performed transfection studies using histaminic agonists and antagonists with the aim of mimicking or blocking the inhibitory effects of HDC protein. In cells transfected with only p1.8HDC-luc, and at dose ranges of 0.01-1.0 µM, neither histamine nor the H3 receptor agonist R-alpha -methylhistamine significantly decreased HDC promoter activity (data not shown). This suggested that transcriptional inhibition was not mediated by H3 receptor stimulation.

In a separate series of experiments, cells were cotransfected with both p1.8HDC-luc and pCMV-HDC and incubated in the presence of selective histamine and histamine receptor antagonists. The antagonists used in this study were specific against a broad spectrum of histamine receptors, including the H3 antagonist clobenpropit and the H2 antagonist ranitidine. DPPE, an antagonist for events regulated by intracellular histamine was also used. At doses ranging from 0.01 to 10 µM, none of the receptor antagonists were able to prevent promoter inhibition by HDC protein (Fig. 3A). As final confirmation that feedback regulation of HDC transcription in AGS-B cells was not dependent on either histamine production or histamine receptor stimulation, we performed experiments overexpressing HDC in the presence of alpha -FMH, a specific and irreversible HDC inhibitor. Inhibition of HDC promoter activity by expression of pCMV-HDC was not affected by treatment with alpha -FMH at doses ranging from 1 to 1,000 µM (Fig. 3A). At the dose of 100 µM, complete inhibition of HDC activity by alpha -FMH was confirmed in enzymatic assays (data not shown). Once again, identical patterns of inhibition were observed when transfected cells were cultured in the presence of gastrin (Fig. 3B).


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Fig. 3.   Inhibition of the HDC promoter by HDC protein is not related to the activation of histaminic receptors or to enzymatic activity. AGS-B cells were cotransfected with p1.8HDC-luc and pCMV-HDC expression constructs (open circle ) and exposed to the histaminic antagonists clobenpropit (H3 antagonist; ), ranitidine (H2 antagonist ), N,N-diethyl-2[4-(phenylmethyl)phenoxy]ethanamine fumarate (a histamine intracellular antagonist; black-triangle), or alpha -fluoromethylhistadine (an irreversible inhibitor of HDC enzymatic activity; black-lozenge ). Luciferase activity is shown as a percentage of the unstimulated control (p1.8HDC-luc without cotransfection of pCMV-HDC; ). Cells were cultured in the absence (A) and presence (B) of 0.1 µM gastrin. Values are means ± SE of 5 independent experiments. P < 0.01 for all points vs. control ().

Carboxy terminal deletions of rat HDC cDNA leads to identification of an inhibitory domain in the HDC protein. These results suggested that a region of the HDC protein other than the catalytic site is responsible for transcriptional inhibition. To identify the portion of the HDC protein that codes for the inhibitory domain, we generated a series of deletion constructs that express carboxy terminal-truncated HDC proteins. The deletion constructs, which were all generated from the pCMV-HDC parent vector, are shown schematically in Fig. 4A. Cotransfection of the three larger constructs pCMV-HDC, pCMV-HDC2033, and pCMV-HDC890, which produce HDC proteins of 655, 653, and 271 amino acids, respectively, all significantly inhibited p1.8HDC-luc promoter activity compared with control (i.e., cotransfection of p1.8HDC-luc with pCMV-HDC empty; Fig. 4B). However, only the two larger constructs pCMV-HDC and pCMV-HDC2033 exhibited enzyme activity (data not shown). For pCMV-HDC890, this confirmed earlier results that showed that inhibition was not dependent on HDC enzyme activity. Significant inhibition of HDC promoter activity was not observed when the two smaller constructs (pCMV-HDC432 and pCMV-HDC196), which produce amino terminal HDC proteins of 119 and 41 amino acids, were cotransfected with p1.8HDC-luc (Fig. 4B). Neither of these two smaller proteins were enzymatically active. Although the results shown in Fig. 4B are from cells cultured in the presence of gastrin, an identical pattern of promoter inhibition was observed in its absence (data not shown).


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Fig. 4.   Effect of HDC protein truncation on inhibition of the HDC promoter. A: diagrammatic representation of the inserts of the carboxy-truncated constructs used in B. Numbered base pairs from the rat HDC cDNA sequence are shown at right. B: AGS-B cells were cotransfected with p1.8HDC-luc and constructs expressing carboxy-terminal-truncated HDC proteins (pCMV-HDC2033, -890, -436, and -196). Control cells were cotransfected with p1.8HDC-luc and either pCMV empty or pCMV-HDC antisense vectors. Identical trends were observed for cells cultured in the presence or absence of 0.1 µM gastrin. Results from gastrin-stimulated cells are shown. Values are means ± SE of 4 independent experiments. *P < 0.01 vs. pCMV-empty vector control. C: AGS-B cells were cotransfected with p1.8HDC-luc and blue fluorescence protein (BFP)-HDC chimeras (pBF-75/890HDC, -196/890HDC, and -75/196HDC). Identical trends were observed for cells cultured in the presence or absence of 0.1 µM gastrin. Results from gastrin-stimulated cells are shown. Values are means ± SE of 4 independent experiments. *P < 0.01 vs. pBF-empty control. D: total lysates from the transfected cells in C were analyzed by Western blot, and peptides were detected using an anti-BFP antibody.

These results, which identified a transcriptional-inhibitory domain in the amino terminal of the HDC enzyme, were confirmed by independently generating HDC constructs fused to the carboxy terminal of the BFP. The inserts for two of these constructs, pBF-75/890HDC and pBF-75/196HDC, are identical to those used in Fig. 4B for pCMV-890HDC (which caused inhibition of the promoter) and pCMV-HDC196 (which did not cause inhibition), except that they lacked any 5' UTR sequence from HDC. As shown in Fig. 4C, it was once again noted that the chimera that contained 271 amino acids from the amino terminal of HDC (pBF-75/890HDC) was capable of inhibiting HDC promoter activity. In contrast, the chimera that contained only 41 amino acids from the amino terminal of HDC (pBF-75/196HDC) was unable to inhibit the HDC promoter. A third fusion protein containing amino-truncated HDC sequences (generated from pBF-197/890HDC) also significantly inhibited the HDC promoter. These data independently confirmed results shown in Fig. 4B. Although the results in Fig. 4C are from cells cultured in the presence of gastrin, an identical pattern of promoter inhibition was observed in its absence (data not shown). Chimeric proteins of the correct sizes were confirmed by Western blotting (Fig. 4D).

Intracellular localization of the inhibitory element in the amino terminal of HDC protein. After translation of the primary 74-kDa HDC enzyme, the protein is processed into multiple smaller HDC isoforms (10). Such a pattern of processing could theoretically generate an amino terminal product capable of translocation to the nucleus, where it could interfere with transcription factor binding to the GAS-RE of the HDC promoter. To test this hypothesis, we used fluorescently tagged fusion proteins to examine the cellular location of the inhibitory 271 amino acids from the amino terminal of HDC. A construct identical to the inhibitory pBF-75/890HDC plasmid described was generated, except that BFP was replaced with GFP to aid in visualization. Transfection experiments confirmed that the GFP-75/890HDC protein was also capable of inhibiting HDC promoter activity (61 ± 6% inhibition, n = 2). AGS-B cells were transfected with pGFP-75/890HDC, and fluorescent microscopy was used to monitor intracellular localization. These studies revealed localization of the GFP-75/890HDC protein expression to cellular regions outside of the nucleus (Fig. 5B). This pattern of cellular localization was specific for the GFP-HDC chimera, because expression of GFP alone was distributed throughout the cell, including in the nucleus (Fig. 5A).


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Fig. 5.   Intracellular localization of the inhibitory domain of the HDC protein AGS-B cells were transfected with the construct expressing the green fluorescent protein alone (from pGF-empty; A) or the GFP-75/890HDC fusion protein (from pGF-75/890HDC; B). Patterns of intracellular localization were examined by fluorescent microscopy at ×60 magnification.

HDC overexpression interferes with MAPK pathways. These results clearly demonstrate that the inhibitory function of HDC is exercised outside the nucleus. To determine whether the inhibition of the HDC promoter occurs as a consequence of interactions between HDC and proteins in the signaling pathway upstream of GAS-REB1/2, we transfected cells with the pCMV-HDC construct along with expression constructs for wild-type ERK1 and ERK2 (pCMV-ERK1 and pCMV-ERK2). The inhibitory effect of HDC protein expression on the HDC promoter was overcome by overexpression of ERK1 and to a lesser extent by ERK2 (Fig. 6A).


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Fig. 6.   Evidence for inhibition of mitogen-activated protein kinase (MAPK) pathways by the HDC protein. A: AGS-B cells were cotransfected with 1 µg of p1.8HDC-luc and pCMV-HDC, pCMV-ERK1, or pCMV-ERK2 expression vectors. Values are means ± SE from 4 independent experiments. ERK, extracellular signal-related kinase. *P < 0.01 vs. controls cotransfected with p1.8 HDC-luc and pCMV-HDC. B: AGS-B cells were cotransfected with 1.0 µg each of pGAL4-Elk-1 or pGAL4-c-Myc transactivator plasmids along with 1.0 µg of the p5xGAL4-luciferase reporter plasmid and incubated with or without phorbol esters (PMA; 0.1 µM). Luciferase activity is expressed as multiples of expression relative to controls (means ± SE; n = 4). *P < 0.01 vs. cotransfection of pCMV-HDC. C: AGS-B and AGS-B/HDC cells were treated for 5 min with 0.1 µM PMA (+) or left untreated (-). Cell lysates were obtained from both cell types, and Western blotting was performed using an anti-active MAPK antibody (top). Blots were stripped and reprobed with an anti ERK1/2 antibody (bottom). D: data obtained by scanning densitometry of the Western blots.

Further evidence for an interaction between HDC protein and the MAPK pathway was provided by transfection experiments with pGAL4-c-Myc and pGAL4-Elk-1 fusion genes, used in combination with a p5xGAL-luc reporter construct. The pGAL4 fusion constructs contained the DNA binding domain of the yeast transcription factor GAL4 linked to the carboxy terminal transcriptional activation domain of either Elk-1 or c-Myc. Both Elk-1 and c-Myc are recognized downstream targets of the gastrin- and PMA-stimulated MAPK/MAPK-ERK kinase (MEK) pathways (16). These constructs were cotransfected with a reporter gene containing five GAL4 binding sites upstream of a minimal promoter linked to luciferase (p5xGAL-luc). In the presence of PMA (0.1 µM), so as to achieve maximal promoter transactivation by both Elk-1 and c-Myc, overexpression of the HDC protein inhibited promoter activity by 66 ± 6 and 80 ± 8%, respectively (Fig. 6B).

To further elucidate the inhibitory mechanism exerted by HDC protein, we performed Western blots and ERK phosphorylation assays on cell lysates derived from both AGS-B and AGS-B/HDC stable cell lines. Cells were stimulated with PMA or left untreated. In the absence of stimulation, the level of ERK activity in AGS-B/HDC cells was barely detectable and reduced when compared with the level of phosphorylated ERKs present in AGS-B. After PMA stimulation, both total ERKs and phosphorylated ERK remained decreased in AGS-B/HDC cells compared with the corresponding levels in AGS-B cells (Fig. 6C). Scanning densitometry showed that the total amount of ERKs detected with anti-ERK1/2 antibody was reduced by 89% in the HDC-expressing cells compared with AGS-B cells (Fig. 6D).

Effects of HDC protein expression on AGS-B cell morphology. Previous studies have shown that ERK expression plays a major role in the differentiation state of gastrointestinal epithelial cells (1, 38). Since our results indicated that HDC expression leads to downregulation of the ERKs, we decided to investigate whether HDC expression could also influence gastric epithelial cell morphology. AGS-B and AGS-B/HDC stable cells were grown on microscope slides, fixed in formaldehyde, stained with propidium iodide, and examined using light microscopy. Although the parent AGS-B cells appeared fairly rounded, the HDC-expressing cells showed flattened morphology, with the appearance of extended processes, consistent with a more differentiated phenotype (Fig. 7). These data suggested that HDC expression, possibly modulating ERK activity, could lead to a more differentiated appearance of the gastric cancer cells.


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Fig. 7.   Morphological characteristics of AGS-B and AGS-B/HDC cells AGS-B (A) and AGS-B/HDC (B) cells were grown on microscope slides under standard conditions. Twenty-four hours later, the cells were rinsed with PBS, fixed in 4% paraformaldehyde, and stained with propidium iodide, then viewed by fluorescence microscope at ×40 magnification.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Gastrin regulates the secretion and synthesis of histamine in ECL cells in a biphasic manner. The first phase involves the rapid release of stored histamine from secretory vesicles. This event, which is initiated within minutes of stimulation, depends on intracellular calcium mobilization and is widely believed to be complete within 30 min (9). The second phase in gastrin stimulation of ECL cells relates to the replenishment of intracellular histamine stores. This involves first an increase in HDC activity (due to stabilization of the protein) and then an increase in HDC gene transcription (9, 13, 32). There are no in vivo data to suggest that histamine acts to feedback-inhibit either of these latter two events; indeed HDC activity and HDC gene transcription both increase at a time when extracellular histamine concentrations are at their highest. Therefore, in the context of gastric physiology, it is not only impractical for such an inhibition to exist (given that histamine stores need to be replenished in advance of subsequent feeding) but it would represent a very insensitive mechanism for downregulation (given its dependence on coexpression of histamine receptors as well as hormone-dependent secretion of histamine to activate receptors). Instead, results obtained from in vivo studies demonstrate that a decrease in the rate of HDC gene transcription is not observed until a number of hours after gastrin stimulation (9, 13). Thus downregulation of HDC gene transcription does not occur when extracellular histamine concentrations are highest and when stimulation of extracellular receptors (such as H2 and H3) would likely occur.

In this study, we present evidence for inhibitory feedback control of HDC gene transcription that is mediated by the HDC protein itself. This result is consistent with the data obtained from physiological studies showing that downregulation occurs at a time when HDC protein levels are high and hours after extracellular histamine levels have peaked (9). The inhibitory effect was mapped to the GAS-RE (+1 to +27), a region of the HDC promoter that has previously been shown to contain a MAPK-responsive element and that binds two presumably novel nuclear factors, GAS-RE1 and GAS-RE2 (31). Overexpression of HDC protein inhibited, in a dose-dependent fashion, the activity of the full-length HDC promoter construct as well as the activity of heterologous promoter constructs containing the GAS-RE enhancer. The inhibitory effect could be overcome through transfection of an ERK1 overexpression construct, consistent with the observation that MAPK is downregulated in response to HDC expression. However, in contrast to what was hypothesized in earlier work, the feedback-inhibitory effect was independent of histamine production and histamine receptors.

Histamine is a potent regulator of a number of physiological processes and not just acid secretion in the stomach. It is logical therefore that controls should be in place to downregulate its effects. There are three obvious levels at which this control could be exercised, namely histamine release, HDC enzymatic activity, and HDC gene transcription. In relation to the downregulation of histamine release, studies have demonstrated that secretion can be decreased by H3 receptor stimulation (3). Similarly, it would appear that H3 receptor-dependent mechanisms are also capable of decreasing HDC enzymatic activity. However, this downregulation of activity is maximal at low histamine concentrations, leading to proposals that it acts primarily to protect against leakage from storage vesicles that might occur in the absence of stimulated release (20). There is no evidence that histamine can act at high concentrations to significantly inhibit HDC activity immediately after its release from ECL cells (9).

In relation to downregulation of HDC transcription, it has been suggested that histamine receptor stimulation might also play a role. This has been discussed in the context of experiments in which administration of alpha -FMH to rats increased the mRNA levels for a number of genes, including that of HDC (2). A potential role for histamine receptor stimulation was consequently considered on the basis of the fact that H3 stimulation decreases histamine release and HDC activity. In light of the results presented here, it is now important to obtain a greater understanding of the molecular mechanisms underlying alpha -FMH-mediated inhibition of HDC; in particular, it is necessary to study whether HDC protein processing in vivo is influenced by binding of the suicide inhibitor.

Although we cannot completely exclude any histamine receptor-mediated effect, our results define a mechanism of inhibition that occurs independently of both histamine synthesis and HDC enzymatic activity. In this model there is a close correlation between levels of HDC protein and the onset of transcriptional inhibition as observed in vivo. The effect of HDC protein expression on promoter activity was not mimicked by treatment of cells with histamine or the H3 agonist R-alpha -methylhistamine. Feedback inhibition was not blocked by recognized histamine receptor antagonists, including the H3 receptor antagonist clobenpropit and the H2 receptor antagonist ranitidine (6). DPPE, which inhibits events regulated by intracellular histamine (8), also had no effect. Finally, although the deletion of 492 amino acids from the carboxy terminal eliminated HDC enzymatic activity, it did not lead to a reduction in the transcriptional inhibitory properties. Thus, although we cannot exclude the presence of a histamine inhibitory effect, and there are a number of cases in which the transcription of genes as diverse as insulin, leptin, and p53 (26, 29, 39) is all somehow regulated by the gene product, our study indicates a novel inhibitory pathway mediated by the HDC protein that is independent of its histamine-producing function. In addition, we show that the minimal inhibitory domain could be localized to a 271-amino acid amino terminal fragment that was devoid of enzymatic activity but possessed the full transcriptional inhibitory activity present in the full-length HDC protein.

There are clearly a number of possible mechanisms by which the amino terminal, an enzymatically inactive portion of the HDC protein, could inhibit transcription from the HDC promoter. In theory, the HDC protein could interact directly with the nuclear factors involved in HDC transcription, resulting in either decreased promoter binding or reduced transactivation. Indeed, EMSA studies using nuclear extracts from cells stably transfected with HDC-expressing constructs did show reduced binding of GAS-REBP1 and GAS-REBP2 to their cognate cis-acting elements. However, EMSA studies did not reveal the presence of any new DNA-protein complexes, suggesting an indirect interaction, and studies utilizing GFP-tagged chimeras indicated that the inhibitory domain did not localize to the nucleus. Since we have previously shown that the GAS-REBPs are downstream targets of the MAPK signaling cascade, we investigated the possibility that the inhibitory effect was a more general phenomenon that occurred upstream of the GAS-REBPs. The inhibitory effect on the HDC promoter could be overcome by ERK1 overexpression and, to a lesser extent, by ERK2 overexpression. HDC protein overexpression was found to block both Elk-1 and c-Myc transactivation, both well-characterized downstream targets of the ERK pathway. Finally, Western blot studies of lysates from control and stably transfected cells showed quite clearly that overexpression of HDC led to reduced expression and activity of ERKs.

The ERK family of serine-theronine kinases represents a critical step of signaling control within the eukaryotic cell and influences numerous processes, including transcription, growth, differentiation, and apoptosis. Several studies have shown that ERKs are regulated in part by the activity of MAPK-specific phosphatases (MKP-1 or PAC-1), which remove phosphate residues from key serine and theronine residues, thereby reducing MAPK activity (34). However, although our immunoblot studies do show decreased levels of phosphorylated ERKs in HDC-expressing cells, they also show equivalent reductions in the basal level of ERK expression. Thus the major effect of HDC protein expression is the reduction in total ERK protein expression. Further studies will be needed to address the mechanisms involved in downregulation of ERK expression.

The effect of HDC protein expression on ERKs in gastric cancer cells is of interest given recent studies indicating that MAPK activity has a profound influence on the differentiation of gastrointestinal epithelial cells. In HT-29 cells, treatment with a MEK inhibitor (PD-98059) induces downregulation of MAPK activity and terminal differentiation (38). Similarly, in Caco-2 cells, high levels of p42/p44 MAPK activities stimulate cell proliferation, whereas low levels of MAPK activity are associated with cellular differentiation (1). In our study, overexpression of HDC led to both reduced MAPK activity and altered cellular morphology, which was consistent with a more differentiated phenotype. This finding is of some interest given that HDC is upregulated during late stages of development and during conditions of growth and regeneration, and our data raise the hypothesis that HDC expression in these conditions may be associated with cellular differentiation, possibly through modulation of ERK activity.

Finally, we were able to localize the inhibitory domain to a specific portion of the HDC protein. Initial deletions from the 3' end of cDNA (from 2,033 nucleotides to 890 nucleotides) eliminated HDC enzymatic activity, consistent with previous notions that the catalytic domain is not contained within the carboxy terminal of the protein but is located toward the central portion of the enzyme (13, 36). Thus our findings support the idea that the catalytic domain and the inhibitory domain reside in distinct portions of the protein. The localization of these functions to separate domains needs to be interpreted in light of the fact that the full-length HDC protein undergoes significant processing through a complicated but poorly understood series of cleavages to a number of different isoforms (10, 13, 34, 36). Recent studies suggest that the amino terminal of the HDC protein is processed to sequentially remove a signal sequence as well as an amino terminal proline-glutamic acid-serine-threonine domain enriched in hydrophilic residues that promote intracellular degradation (12, 13, 37). Further studies will be required to determined whether cleavage and/or degradation at the amino terminal modulates the inhibitory effect on HDC promoter activity.


    ACKNOWLEDGEMENTS

R. Colucci was supported by a fellowship issued by the Italian National Council of Research (Committee for Biotechnology and Molecular Biology Grant no. 106089/14/97/03566, TMWAC). T. C. Wang is supported National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-48077.


    FOOTNOTES

* R. Colucci and J. V. Fleming contributed equally to this work.

Current address for R. Colucci: Division of Pharmacology and Chemotherapy, Department of Oncology, University of Pisa, I-56126 Pisa, Italy.

Address for reprint requests and other correspondence: T. C. Wang, Gastroenterology Division, Univ. of Massachusetts Medical Center, Biotech Two Suite 202, 373 Plantation St., Worcester, MA 01605-2377 (E-mail: Timothy.Wang{at}UMassmed.edu).

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.

Received 8 September 2000; accepted in final form 13 June 2001.


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RESULTS
DISCUSSION
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