Activation of the human histamine H2 receptor is linked to cell proliferation and c-fos gene transcription

L.-D. Wang, M. Hoeltzel, K. Butler, B. Hare, A. Todisco, M. Wang, and J. Del Valle

Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We examined whether histamine could regulate cell proliferation and expression of the early response gene c-fos in HEK-293 cells stably transfected with the human H2 receptor (HEK-H2). Histamine stimulated [3H]thymidine incorporation [50% effective concentration (EC50) = 3.6 × 10-6 M] in HEK-H2 cells in a cimetidine-sensitive manner and increased c-fos mRNA in a time-dependent fashion, reaching maximal induction after 30 min. Histamine induced luciferase activity in HEK-H2 cells transiently transfected with a construct containing the luciferase reporter gene (Luc) coupled to the serum response element (SRE) of the c-fos gene promoter (EC50 = 1.5 × 10-6 M) or a plasmid containing the SRE core fragment (bases -320 to -298). The protein kinase C (PKC) inhibitor staurosporine and long-term pretreatment of HEK cells with phorbol ester inhibited the effect of histamine on PKC activation, SRE-Luc activity, and [3H]thymidine incorporation. We have demonstrated that activation of the human H2 receptor can lead to induction of c-fos gene transcription and cell proliferation through a PKC-dependent mechanism.

signal transduction; biogenic amine receptor

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ACTIVATION OF THE HISTAMINE H2 receptor is associated with a wide range of physiological actions extending from stimulation of gastric acid secretion to induction of human promyelocyte differentiation (23). Additionally, several studies (1, 5, 36) have suggested that histamine can regulate cell proliferation via the H2 receptor. Notably, Tonnesen and co-workers (33) made the interesting observation that the H2 receptor antagonist cimetidine improved survival in patients with gastric cancer. Contrary to this, others have failed to observe a significant proliferative effect of histamine in several experimental models tested (8).

The mechanism through which H2 receptor activation may lead to regulation of cell proliferation in vivo is unknown. Some have hypothesized that histamine acts through an H2 receptor located on suppressor T cells, which when activated leads to a decrease in tumor cell kill (3, 28). Others have demonstrated a direct proliferative effect of histamine via an H2 receptor found on several tumor cell lines derived from gastric and colonic neoplasms (5, 36). The observation that the histamine H2 receptor is expressed on multiple malignant cell types, coupled with the finding that it can activate the phosphoinositide signaling pathway, lends further support to the hypothesis that this receptor may be involved in cell growth. We and others have shown that histamine can stimulate both adenosine 3',5'-cyclic monophosphate (cAMP) and inositol trisphosphate (IP3)/intracellular Ca2+ concentration ([Ca2+]i) signaling cascades (12, 15, 23, 27). Moreover, we have recently demonstrated that the dual stimulatory action of histamine occurs via separate GTP-dependent mechanisms (34). Although H2 receptor-mediated stimulation of cAMP is important for histamine-induced gastric secretion (26), the physiological role for histamine-mediated activation of the phosphoinositide pathway is unknown. The established role of the phosphoinositide pathway in cell growth and proliferation, coupled with the large body of intriguing literature suggesting a role for H2 receptor activation in tissue growth, led us to examine whether the cloned human histamine H2 receptor could regulate proliferation in HEK-293 cells. In addition, we explored whether the immediate early response protooncogene c-fos, which is known to play an important role in cell proliferation (21, 24), is regulated by histamine.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chemicals

Bovine serum albumin (BSA), trichloroacetic acid (TCA), Triton X-100, forskolin, histamine, cimetidine, epinephrine, dithiothreitol (DTT), and EDTA were purchased from Sigma Chemical (St. Louis, MO). [methyl-3H]tiotidine (87 Ci/mmol) and myo-[2-3H]inositol (15.8 Ci/mmol) were products of DuPont NEN (Boston, MA). Staurosporine, calphostin C, and 12-O-tetradecanoylphorbol-13-acetate (TPA; phorbol 12-myristate 13-acetate) were purchased from Calbiochem (La Jolla, CA). Fura 2-acetoxymethyl ester (AM) was purchased from Molecular Probes (Eugene, OR). Anti-protein kinase C (PKC) alpha  and anti-PKCbeta I were purchased from Santa Cruz Biotech (Santa Cruz, CA). [3H]thymidine (15.1 Ci/mmol) and cAMP assay kits were purchased from Amersham (Arlington Heights, IL). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from GIBCO (Grand Island, NY). Formaldehyde and phenol were from Fisher Scientific (Pittsburgh, PA). Plasmids of serum response element (SRE)-luciferase reporter gene (Luc), SRE core-Luc, and thymidine kinase (TK)-Luc were obtained from J. Pessin (Univ. of Iowa College of Medicine, Iowa City, IA) (37).

Methods

H2 receptor expression. The full-length coding region of the human H2 receptor gene was subcloned into a pBK CMV expression vector as previously described (19). HEK-293 cells were transfected using the technique of lipofectamine, and permanently transfected cells were selected by resistance to the neomycine analog G418 (600 mg/l). Single clones of transfected cells were selected and screened for expression of the human H2 histamine receptor by Northern blot analysis and receptor binding studies using [methyl-3H]tiotidine as previously described (15).

Intracellular cAMP measurement. Transfected HEK-293 cells (0.5 × 106 cells/ml) were cultured on 12-well plates as described previously (15). After 24 h of culture, cells were incubated for 60 min in Earle's balanced salt solution (EBSS) containing 0.1% BSA with or without histamine (10-12 to 10-4 M). Cells were then extracted with 1 ml of 100% ethanol, supernatants were evaporated to dryness at 55°C under a stream of nitrogen, and cAMP levels were measured using the cAMP competitive binding protein assay kit (Amersham).

Measurement of IP3. Transfected HEK-293 cells were grown to confluence in 12-well plates and prelabeled for 10 min with myo-[2-3H]inositol at 37°C for 18 h. LiCl (10 mM) was added 10 min before completion of the preincubation period. Ligands were added at varying concentrations (10-12 to 10-4 M), incubations were finalized by addition of 1.5 ml of chloroform-methanol (1:2), and the water-soluble products were separated by ion-exchange chromatography on Dowex-1 resin columns (formate form) as previously described (15). The fraction containing IP3 represents a mixture of the 1,4,5 and 1,3,4 isomers.

Measurement of [Ca2+]i. [Ca2+]i was measured using previously described methods (15, 38). HEK-293 cells stably transfected with the histamine H2 receptor were seeded onto 22-mm-diameter glass coverslips and cultured overnight in DMEM (high glucose) with 10% FBS to 30-50% confluency. Cells were then incubated for 30 min at 37°C and 5% CO2, with 1 mM of fura 2-AM added to the existing media. Each coverslip was transferred to a closed chamber, mounted on the stage of a Zeiss Axiovert inverted microscope, and continuously superfused (1 ml/min, 37°C) with EBSS containing 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 13 mM NaHCO3, and 0.1% BSA. Superfusion was accomplished by means of a gravity-driven, temperature-regulated, eight-chamber superfusion reservoir equipped with a valve allowing rapid changes between control and experimental solutions. The Attofluor digital imaging system (Rockville, MD) used to measure [Ca2+]i of individual cells utilized a computer-selectable filter and shutter system for the excitation of fura 2-AM-loaded cells at 334 nm and 380 nm (10-nm band pass). Subsequent emission at 520 nm was detected by an intensified charge-coupled device camera and digitized. Calibration of [Ca2+]i was determined as previously described (15, 38).

PKC translocation. PKC translocation in cell membranes was measured using previously described methods (35). Transfected HEK-293 cells were serum starved for 18 h and stimulated with 10-4 M histamine or 10-8 M TPA for 5 min at 37°C. Cells were suspended in 62.5 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 7.4), 2 mM EDTA, and 2 mM DTT and sonicated three times for 5 s. Sonicates were centrifuged at 500 g for 5 min at 4°C. Supernatants were harvested and centrifuged at 120,000 g for 24 min at 4°C, and the pellets were resuspended in 10 mM Tris · HCl (pH 7.4) containing 1 mM DTT. Membrane samples (50-100 µg) were electrophoresed on discontinuous 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose. Nitrocellulose blots were incubated in Tris-buffered saline-Tween 20 [TBST; 10 mM Tris (pH 7.5), 100 mM NaCl, and 0.3% Tween 20] containing 5.0% nonfat dry milk for 1 h at room temperature to block nonspecific binding sites. Blots were washed in TBST for 5 min, followed by incubation with the primary antibody (anti-PKCalpha or anti-PKCbeta I) at a final dilution of 1:2,000 in TBST for 2 h at room temperature. Blots were washed repeatedly with TBST containing 0.25% dry milk, followed by incubation with peroxidase-linked secondary antibody (Zymed rabbit anti-mouse horseradish peroxidase, 1:1,250) for 60 min. Immunoreactive bands were visualized using the standardized enhanced chemiluminescence-like immunoblotting detection system (Amersham). The nitrocellulose membranes were then stripped of bound antibody by incubation with 100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate, and 62.5 mM Tris · HCl (pH 6.7) for 30 min at 50°C, rinsed three times for 5 min with 200 ml TBST, and reprobed with a different antibody.

Measurement of PKC enzyme activity. PKC enzyme activity was measured in HEK-H2 cells as previously described (35) using a standardized assay kit (Amersham protein kinase enzyme assay) that is based on the ability of PKC to specifically phosphorylate a synthetic peptide substrate. After 18 h of serum starvation, HEK-H2 cells were treated with histamine (10-5 M), epidermal growth factor (EGF; 10-8 M), or TPA (10-6 M) for 5 min at 37°C. The incubation was stopped by the addition of 1 ml ice-cold phosphate-buffered saline, and the mixture was immediately centrifuged. For membrane preparation, cells were suspended in 1 ml of sonicate buffer [50 mM Tris · HCl (pH 7.5), 5 mM EDTA, 10 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 0.3% beta -mercaptoethanol, 10 mM benzamidine, and 25 mg/ml phenylmethylsulfonyl fluoride] on ice and sonicated twice for 15 s. Sonicates were centrifuged at 1,000 g for 5 min at 4°C. Supernatants were harvested and centrifuged at 120,000 g for 25 min at 4°C. The membrane-containing pellets were resuspended in 0.5 ml sonicate buffer and stored at -70°C for later assay. Total PKC activity in membranes (30-60 µg protein) was measured in a final reaction volume of 100 µl that contained 50 mM Tris · HCl (pH 7.5), 0.05% sodium azide, 900 µM peptide, 300 mM DTT, 150 µM ATP, 45 mM magnesium acetate, and 1 × 106 counts/min of [gamma -32P]ATP (Amersham; sp. act., 1.66 mCi/ml). After incubation for 15 min at 25°C, the reaction was terminated by adding 100 µl stop solution and 125 µl of the terminated reaction mixture were pipetted onto binding paper. After the paper was washed with 5% acetic acid twice, the extent of phosphorylation was detected by scintillation counting.

Luciferase assays. Transfected HEK-293 cells were grown at 37°C (5% CO2-95% O2) in 12-well plates in DMEM supplemented with 10% FBS. Subconfluent cells were transfected with 5 µg of the Luc plasmids and, where indicated, with 5 µg of the expression vectors. The SRE-Luc construct contains a dimer of the rat c-fos genomic sequence from nucleotides -357 to -276 relative to the transcription start site. This construct contains the SRE, which includes ternary complex factor (TCF) and serum response factor (SRF) binding sites, the sis-inducible element (SIE), and E box inserted into TK-Luc. To assess the specificity of the response observed, we utilized the SRE core construct, which contains the rat c-fos genomic sequence from -320 to -298 and includes all of the binding sites outlined above except the SIE. The TK minimal promoter was also utilized as a control construct. Transfections were carried out using lipofectamine (Life Technologies) as described previously (32). On the day after transfection the medium was removed, and the cells were fed with serum-free medium (DMEM) for 24 h and then incubated for 6 h with or without histamine. At the end of the incubation period, the cells were washed and lysed and luciferase assays were performed as previously described (32). Luciferase activity was expressed as relative light units and then normalized for protein content in the cell lysate to correct for differences in cell numbers and transfection efficiency among the different treatment groups. In some experiments the cells were cotransfected with the pCMV-beta Gal vector. beta -galactosidase activity was measured by the luminescent light derived from 10 µl of Lumi-Gal 530 (Lumigen, Southfield, MI) and used to normalize the luciferase assay data for transfection efficiency. Protein concentrations were measured by the Bradford method. Similar results were obtained with both methods of normalization.

Northern blot analysis. After different time intervals of incubation with ligands, the transfected HEK-293 cells were lysed with TRIzol (Life Technologies), according to the manufacturer's instructions. Northern blot hybridization assays were performed as described previously (31). Equal amounts of each RNA sample, with ethidium bromide (10 mg/ml) in a final volume of 20 µl, were electrophoresed on a 1.25% agarose gel containing formaldehyde, and the RNA was transferred from the gel to nitrocellulose filters. The ethidium-stained ribosomal RNA bands in the gel were photographed before and after transfer to ensure that equivalent amounts of RNA were loaded onto each lane and that no residual RNA was left on the gel. The probes used for hybridization analysis included c-fos cDNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA obtained from the American Type Culture Collection (Rockville, MD). The c-fos (and GAPDH) cDNA was labeled with [32P]dCTP by the random priming procedure, and the nitrocellulose filters were hybridized with the 32P-labeled cDNA probes as described previously (31).

Measurement of [3H]thymidine incorporation. Transfected HEK-293 cells grown in DMEM with 10% FBS were plated in 12-well plates, allowed to attach overnight, and then cultured for 24 h in serum-free medium. After the cells were washed with serum-free medium, histamine was added at different concentrations (10-10 to 10-4 M). DNA synthesis was estimated by measurement of [3H]thymidine incorporation into the TCA-precipitable material. The [3H]thymidine (0.1 mCi/ml) was added during the last 1 h of the 18-h treatment period. Cells were then washed with serum-free medium to remove unincorporated [3H]thymidine. DNA was precipitated with 5% TCA at 4°C for 15 min. Precipitates were washed twice with 95% ethanol and dissolved in 1 ml of 0.1 N NaOH, and radioactivity was measured in a liquid scintillation counter.

Data analysis. Data are presented as means ± SE; n is the number of separate transfections performed with the HEK cells. Statistical analysis was performed using Student's t-test; P < 0.05 was considered significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of Histamine on cAMP and IP3 Generation, [Ca2+]i, and PKC Activation in HEK-293 Cells Transfected With the H2 Receptor

We examined binding of the H2 receptor antagonist [3H]tiotidine to transfected HEK-293 (HEK-H2) cells, using cimetidine as the nonradioactive ligand. [3H]tiotidine bound to transfected HEK-293 cells in a saturable manner (maximum binding capacity, 250 ± 8.9 fmol/mg protein). Cimetidine displaced tiotidine binding dose dependently with a 50% inhibitory concentration (IC50) of 1.5 × 10-7 M (data not shown).

To examine whether the H2 receptor stably expressed in HEK-293 cells was functional, we measured the effect of histamine on intracellular cAMP and IP3 generation. Histamine dose dependently stimulated cAMP generation with a 50% effective concentration (EC50) of 2.5 × 10-7 M (n = 8; data not shown). The maximal stimulation of cAMP (10-4 M histamine) was 698.5 ± 67.5% above control. Histamine stimulated IP3 formation in a dose-dependent manner, with an EC50 of 5.5 × 10-7 M (n = 6; data not shown). Histamine (10-5 M) stimulated an increase in [Ca2+]i in >90% of the transfected HEK cells (Fig. 1A). Activation of the H2 receptor led to a biphasic rise in [Ca2+]i, with a plateau phase that was dependent on the presence of extracellular Ca2+ (Fig. 1B).


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Fig. 1.   Effect of histamine (His) on intracellular Ca2+ concentration ([Ca2+]i) in transfected HEK-293 cells. A: histamine (10-5 M) stimulated a biphasic increase in [Ca2+]i in HEK cells transfected with human histamine H2 receptor. B: removal of extracellular Ca2+ led to a decrease in histamine-stimulated sustained phase of response. Although not shown here, in Ca2+-free conditions, histamine led to a rapid and transient rise in [Ca2+]i without an associated plateau. Tracings shown here represent average of a minimum of 20 cells tested. Similar results were obtained during 3-4 separate experimental days.

Consistent with coupling of the H2 receptor to IP3/[Ca2+]i signaling, we observed that histamine could stimulate membrane translocation of two isoforms of PKC (alpha  and beta I), in HEK-H2 cells. As shown in Fig. 2, the level of PKCalpha and PKCbeta found in HEK-H2 cell membranes was significantly increased after treatment with histamine (10-5 M) for 5 min. We also examined whether the agents of interest would activate PKC enzyme activity in HEK-H2 cells. As illustrated in Fig. 3A, histamine (10-5 M), EGF (10-8 M), and the phorbol ester TPA (10-8 M) led to a significant increase in PKC-mediated substrate phosphorylation in a staurosporine (10-7 M)-sensitive manner. Prolonged treatment (18 h) of HEK-H2 cells with TPA (10-6 M) led to complete inhibition of ligand-stimulated PKC activity (10-5 M His, 10-8 M EGF, 10-8 M TPA; Fig. 3B), supporting the specificity of this assay system.


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Fig. 2.   Histamine mediated translocation of protein kinase C (PKC) alpha  and PKCbeta into HEK-H2 cell membranes. Histamine (10-4 M; lane 2) and 12-O-tetradecanoylphorbol-13-acetate (TPA; 10-7 M; lane 3) significantly increased translocation of PKCalpha and PKCbeta into membranes of HEK-H2 cells. Lane 1, membranes obtained from unstimulated cells. Similar results were obtained in 3 additional experiments.


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Fig. 3.   Histamine-, epidermal growth factor (EGF)-, and TPA-mediated regulation of PKC enzyme activity. A: histamine (10-5 M), EGF (10-8 M), and TPA (10-8 M) stimulated PKC activity in a staurosporine (Stau; 10-7 M)-sensitive manner. # P < 0.05 compared with control (Cont); * P < 0.05 compared with stimulant alone. B: pretreatment of HEK-H2 cells with TPA (10-6 M) for 18 h abolished the stimulatory effect of all ligands tested on PKC activity. H, histamine; E, EGF; T, TPA; C, control. Data are expressed as means ± SE of 3 experiments.

Histamine did not increase cAMP, IP3, [Ca2+]i, PKC translocation, or PKC enzyme activity in nontransfected cells. These data are consistent with our previously published studies demonstrating that the histamine H2 receptor can couple in a stimulatory manner to both the adenylate cyclase and IP3/[Ca2+]i signaling systems (15, 34) in HEK-293 cells. We have also demonstrated that H2 receptor activation leads to translocation of PKCalpha and PKCbeta I with a concomitant increase in PKC enzyme activity in this cell model.

Effect of Histamine on [3H]thymidine Incorporation in HEK-293 Cells

As shown in Fig. 4A, histamine increased [3H]thymidine incorporation into transfected HEK-293 cells in a dose-dependent manner with an EC50 of 3.6 × 10-6 M (n = 8). Maximal stimulatory effect of histamine was 107.5 ± 10.6% above control and was achieved with 10-4 M histamine. Histamine did not affect nontransfected HEK-293 cells. The effect of histamine on [3H]thymidine incorporation was dose dependently inhibited by cimetidine, with an IC50 of 2.2 × 10-7 M (Fig. 4B).


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Fig. 4.   Histamine mediated stimulation of [3H]thymidine into transfected HEK cells in a dose-dependent manner, with a 50% effective concentration (EC50) of 3.6 × 10-6 M. A: nontransfected cells did not respond to histamine. B: H2 receptor antagonist cimetidine dose dependently inhibited the stimulatory action of histamine, with a 50% inhibitory concentration (IC50) of 2.2 × 10-7 M. Basal [3H]thymidine incorporation was 1,761.6 ± 174 counts/min (cpm), and maximal level achieved with histamine (10-4 M) was 3,196 ± 475.4 cpm. Data are expressed as means ± SE of experiments performed on different days.

Effect of Histamine on c-fos Regulation

In an effort to explore the potential mechanisms through which histamine stimulates cell proliferation, we examined the effect of H2 receptor activation on regulation of the protooncogene c-fos. As shown in Fig. 5, histamine (10-5 M) and EGF (10-8 M) increased c-fos-specific mRNA in a time-dependent fashion. Maximal stimulatory effect of histamine and EGF occurred after 30 min.


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Fig. 5.   Effect of histamine on c-fos mRNA levels in transfected HEK cells. A: histamine (10-5 M) and EGF (10-8 M) treatment led to a time-dependent increase in c-fos mRNA, with a peak effect noted between 30 and 60 min. Increase achieved with histamine was similar to that observed in response to EGF. B: histamine and EGF did not alter levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Data shown are representative of 3 additional experiments.

Transcriptional regulation of c-fos involves several elements within its promoter (25). SRE was initially found to mediate c-fos induction by serum. Other growth factors, such as EGF and platelet-derived growth factor, and cytokines like interleukin-2, in addition to phorbol esters (TPA) and antioxidants, also induce c-fos transcription through this element. More recently, it has been shown that G protein-coupled receptors such as the one for lysophosphatidic acid also activate SRE, although it is felt that the mechanism through which this occurs is different from tyrosine kinase or classic growth-factor-mediated activation (22). We examined whether the effect of histamine on c-fos expression was through SRE-mediated transcriptional regulation of the c-fos gene. As shown in Fig. 6A, histamine dose dependently stimulated SRE-Luc activity (EC50 = 1.5 × 10-6 M; n = 8) in HEK-H2 cells. The maximal effect of histamine on SRE-Luc (677.6 ± 12.3% above control) was achieved at 10-4 M. As shown in Fig. 6B, cimetidine dose dependently inhibited histamine-stimulated SRE-Luc activity (IC50 = 5.6 × 10-7 M; n = 6). Histamine did not stimulate SRE-Luc activity in wild-type HEK cells.


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Fig. 6.   Effect of histamine on c-fos transcription. A: histamine stimulated serum response element (SRE)-luciferase reporter gene (Luc) activity in transfected HEK cells, with an EC50 of 1.5 × 10-6 M. Histamine did not stimulate SRE-Luc activity in nontransfected (H2 receptor) HEK cells. B: cimetidine dose dependently inhibited the stimulatory effect of histamine, with an IC50 of 5.6 × 10-7 M. Data are expressed as means ± SE of experiments performed on different days.

The SRE-Luc construct contains two key regulatory regions, including the SIE and the SRE, which consists of TCF and SRF binding sites (25). SIE and SRE have different regulatory effects on c-fos gene transcription (16). To distinguish which element is mediating histamine-induced c-fos gene transcription, we employed two additional constructs, SRE core-Luc, which corresponds to positions -320 to -298 of the c-fos gene, and the TK-Luc reporter (37). As shown in Fig. 7, histamine (10-4 M) significantly stimulated SRE-Luc and SRE core-Luc activity, with 5.7 ± 0.49- and 5.5 ± 0.8-fold induction (P < 0.001; n = 6) in c-fos transcriptional activity, respectively. Histamine had no effect on TK-Luc-mediated activity. EGF (10-7 M) induced an increase in c-fos transcriptional activity to a level similar to that achieved with histamine. Epinephrine failed to significantly increase c-fos transcriptional activity in HEK cells stably transfected with the human beta 2-adrenergic receptor. Forskolin, a known activator of adenylate cyclase, did not stimulate c-fos activation in HEK cells. Notably, both epinephrine and forskolin stimulated cAMP formation to 514.5 ± 30.5 and 853.6 ± 45.3% above control in HEK cells, respectively. These findings suggest that the ability of histamine to stimulate c-fos transcription through SRE is independent of the effect of this biogenic amine on cAMP formation.


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Fig. 7.   Ligand-mediated regulation of SRE-, SRE core-, and thymidine kinase (TK)-Luc. Both histamine (10-4 M) and EGF (10-7 M) stimulated SRE- and SRE core-Luc activity but did not significantly alter TK-Luc activity. Neither epinephrine nor forskolin stimulated SRE-, SRE core-, or TK-Luc activity. Data are expressed as means ± SE of 4 experiments. * P < 0.05 compared with control luciferase activity. SIE, sis-inducible element; TCF, ternary complex factor; SRF, serum response factor.

Role of PKC in Histamine-Stimulated SRE-Luc Activity and c-fos mRNA

We hypothesized that part of the action of histamine on cell proliferation and c-fos regulation is through activation of the phosphoinositide signaling pathway. One of the key effectors through which the phosphoinositide cascade regulates nuclear events is stimulation of PKC. We examined whether inhibition of PKC altered the action of histamine on c-fos transcription. As shown in Fig. 8, the PKC inhibitor staurosporine (11) dose dependently inhibited histamine-stimulated SRE-Luc activity. Similarly, calphostin C (10-6 M) (6), another PKC inhibitor, abolished the effect of histamine on c-fos transcription (data not shown). To further assess the specificity of our findings with staurosporine and calphostin C, we explored whether downregulation of PKC with prolonged incubation of HEK-H2 cells with the phorbol ester TPA also altered the stimulatory action of histamine. As demonstrated in Fig. 9, pretreatment of HEK cells with TPA for 18 h essentially abolished the stimulatory effect of histamine on SRE-Luc activation. Figure 10 illustrates the effect of staurosporine (10-7 M) on histamine (10-5 M)-stimulated c-fos mRNA. Consistent with our SRE-Luc studies, the PKC inhibitor also inhibited the ability of histamine to increase c-fos message. In summary, this series of experiments suggests that the effect of histamine on c-fos transcription involves the PKC signaling pathway.


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Fig. 8.   Effect of staurosporine on histamine-stimulated SRE-Luc activity. PKC inhibitor staurosporine did not affect basal SRE-Luc activity but inhibited histamine-stimulated c-fos transcription in a dose-dependent manner. Data are expressed as means ± SE of 4 experiments. * P < 0.05 compared with histamine-stimulated activity.


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Fig. 9.   Effect of TPA pretreatment on histamine (Hist)-stimulated SRE-Luc activity. Pretreatment of HEK-H2 cells with TPA (10-8 M) for 18 h significantly decreased the stimulatory effect of histamine (10-4 M) and TPA on SRE-Luc activity. Data are expressed as means ± SE of 5 experiments. * P < 0.05 compared with histamine- and TPA-stimulated activity without pretreatment with TPA.


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Fig. 10.   Effect of staurosporine (Staur; 10-7 M) on histamine (10-5 M)-stimulated c-fos mRNA. Staurosporine treatment led to a significant decrease in histamine-stimulated c-fos mRNA. Histamine and staurosporine did not alter GAPDH mRNA. Similar results were obtained in 3 additional experiments.

Role of PKC in Histamine-Stimulated [3H]thymidine Incorporation

We examined whether PKC was involved in H2 receptor-mediated cell proliferation by examining the effect of staurosporine and TPA preincubation on histamine-stimulated [3H]thymidine incorporation. Staurosporine (10-7 M) significantly inhibited histamine (10-4 M)-stimulated [3H]thymidine incorporation into HEK-H2 cells (Fig. 11A). As shown in Fig. 11B, pretreatment of HEK-H2 cells with TPA (10-8 M) for 18 h nearly abolished the stimulatory effect of histamine on cell proliferation.


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Fig. 11.   Role of PKC in histamine-stimulated [3H]thymidine incorporation. A: staurosporine (Stau; 10-7 M) significantly inhibited histamine (10-4 M)-stimulated [3H]thymidine incorporation into HEK-H2 cells. B: maximal concentrations of histamine (10-4 M) failed to stimulate [3H]thymidine incorporation into HEK-H2 cells pretreated with TPA (10-8 M) for 18 h. Data are expressed as means ± SE of 4 experiments. * P < 0.05 compared with histamine-stimulated activity.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have demonstrated that activation of the human histamine H2 receptor directly leads to stimulation of cell proliferation in transfected HEK cells. In addition, our studies indicate that histamine induces transcriptional activation of the protooncogene c-fos through a pathway linked to stimulation of the SRE of the corresponding promoter.

The role of histamine in regulating gastric acid secretion and inflammation is well established, but the evidence linking histamine H2 receptor activation to cell proliferation has been controversial. Earlier studies (7, 10) documenting increased histamine content in proliferating or tumor tissues led to the hypothesis that this biogenic amine may be involved in the regulation of cell growth and proliferation. Gespach and co-workers (20) hypothesized that the H2 receptor found on the human monocyte-like cell line U-937 was involved in the regulation of cell proliferation and differentiation. The beneficial effect of the H2 receptor antagonist cimetidine on the survival of gastric cancer patients (33) was another intriguing observation suggesting a potential role for the H2 receptor in the proliferation of malignant cells. Subsequent studies in patients with carcinomas of different types, including adenocarcinoma of the colon (2, 9, 29), have also suggested that H2 receptor antagonists may be beneficial for patient outcome. Contrary to these observations, others have not demonstrated that histamine exerts a trophic action on gastric tissues (4, 14).

Our studies indicate that human H2 receptor activation can lead to stimulation of cell proliferation. It is difficult to assess whether our findings are due to the particular cell model or receptor species we chose to examine. To explore this further, we determined whether stimulation of the canine H2 receptor expressed in either the human colon cancer cell line colo DM or in the rat hepatoma cell (HEPA) could lead to cell proliferation. As in the experiments shown in this study, histamine stimulated proliferation in both cell lines in a cimetidine-sensitive manner (data not shown). These findings suggest that the proliferative response we observed in our studies is not limited to the HEK-293 cells nor to the human H2 receptor subtype.

The mechanism through which histamine leads to cell proliferation is unknown. A large body of recent literature has demonstrated that heptahelical G protein-linked receptors can promote malignant transformation and stimulation of cell proliferation (30). Because of the importance of c-fos in cell proliferation, we chose to examine whether this protooncogene was regulated by histamine. The c-fos protooncogene belongs to a family of cellular response genes that are rapidly and transiently induced on stimulation of cells with growth factors. Moreover, treatment of cells with c-fos antisense RNA can lead to inhibition of cell growth (24). In our studies, we demonstrated that histamine treatment induced c-fos mRNA in a time-dependent manner. The level of c-fos induction with histamine was similar to that achieved with the potent growth factor EGF. We speculate that the increase in c-fos transcription is important in mediating the proliferative effect of histamine. Direct proof of this would require blocking the action of c-fos with a specific antibody or by using antisense probes. The observation that the EC50 values for histamine-stimulated cell proliferation and c-fos transcription are identical is indirect evidence supporting the hypothesis that these two events are linked.

The postreceptor events coupling H2 receptor activation to c-fos transcription are unknown. Three elements within the c-fos promoter have been identified as important sites for factors that stimulate transcription of this protooncogene (25). These sites include the SIE, the cAMP response element (CRE), and the SRE. Although the H2 receptor has been traditionally considered to stimulate the cAMP pathway, we have recently demonstrated that this receptor can also couple to the phosphoinositide cascade through a separate GTP-dependent mechanism (34). The importance of histamine-stimulated adenylate cyclase in mediating acid secretion has been established, but the role of H2 receptor-mediated increases in IP3/[Ca2+]i is unknown. In view of the established importance of the phosphoinositide pathway in cell growth and proliferation, we hypothesized that activation of this signaling system by histamine may be responsible for the proliferative response we observed. Our studies illustrating that inhibition and downregulation of PKC decrease the effect of histamine on c-fos transcription and cell proliferation support our hypothesis.

H2 receptor activation of the adenylate cyclase/cAMP pathway may also contribute to the proliferative responses observed. There are examples of G protein-linked receptors that stimulate cell proliferation and differentiation through a cAMP-dependent mechanism (17). In addition, stimulation of cAMP may lead to inhibition of the proliferative or mitogenic response induced by certain G protein-linked receptors (13). The cAMP-mediated effects on cellular proliferation vary depending on the specific cell type examined. In our studies, we did not observe stimulation of [3H]thymidine incorporation in response to agents that increase cAMP. Therefore, it appears that this pathway of mediating proliferation is not prominent in HEK cells. This does not exclude, however, a modulatory effect of cAMP in histamine-mediated cell proliferation.

The cAMP/protein kinase A signaling pathway is also important in regulating c-fos transcription (25). As mentioned earlier, the c-fos promoter region contains a CRE that serves as a binding site for the transcription factor CRE binding protein or CREB. Although this pathway may be in part responsible for the increase in c-fos mRNA observed after treatment with histamine, our data support a direct effect of H2 receptor activation on c-fos transcription through the SRE. As noted in our studies, stimulation of cAMP by epinephrine or forskolin did not increase SRE-Luc activity, supporting the specificity of our observations.

An interesting aspect regarding the H2 receptor is that its activation leads to stimulation of both the phosphoinositide and adenylate cyclase pathways. As noted above, each of these signaling systems has been associated with regulation of cell proliferation to varying degrees. It is unclear from our studies what each individual signaling pathway contributes to the proliferative response observed. It may be that these pathways act synergistically to alter cell growth. Frodin and colleagues (18) have demonstrated that activation of the cAMP cascade acted synergistically with phospholipase C in the stimulation of the mitogen-activated protein kinase (MAPK) cascade. These investigators also observed that the peptide pituitary adenylate cyclase-activating polypeptide 38, which activates both the adenylate cyclase and phosphoinositide pathway, led to efficient stimulation of the MAPK signaling cascade.

The signaling events leading to H2 receptor-mediated activation of c-fos transcription are unknown. Recent literature has illustrated that G protein-coupled receptors can stimulate cell proliferation and differentiation (30). Mitogenic responses to Gsalpha , Gqalpha , Gi/Go, and, more recently, G12/G13 have been demonstrated. Multiple signaling pathways have been associated with heptahelical receptor-mediated regulation of cell growth (30). The possibilities that account for histamine-mediated activation of c-fos transcriptional activation and cell proliferation are multiple and may include stimulation of the low-molecular-weight guanosine triphosphatase Ras with subsequent stimulation of the Raf-1/MAPK pathway, PKC-mediated stimulation of Raf-1/MAPK cascade, activation of the c-Jun NH2-terminal protein kinase, or stimulation of a tyrosine kinase-signaling pathway as in the case of bombesin. The mediation of a proliferative response through beta gamma -subunits, as in the case of receptors that stimulate Gi/Go, seems less likely in view of our previous studies demonstrating that H2 receptor coupling to cAMP and IP3 is pertussis toxin insensitive (15, 34).

In summary, we have demonstrated that the human histamine H2 receptor can stimulate cell proliferation in transfected HEK cells. Moreover, the proliferative effect of the H2 receptor may involve activation of the early response gene c-fos through a PKC-dependent mechanism.

    ACKNOWLEDGEMENTS

We thank Shane Davis and Patricia Richards for typing this manuscript.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant R01-DK-47424 and funds from the University of Michigan Gastrointestinal Peptide Research Center (NIDDK Grant P30-DK-34933). A. Todisco is the recipient of NIDDK Clinical Investigator Award K08-DK-02336.

Address for reprint requests: J. Del Valle, Div. of Gastroenterology, University of Michigan, 6520 MSRB-I, Box 0682, Ann Arbor, MI 48109.

Received 7 April 1997; accepted in final form 22 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Adams, W. J., J. A. Lawson, and D. L. Morris. Cimetidine inhibits in vivo growth of human colon cancer and reverses histamine stimulated in vitro and in vivo growth. Gut 35: 1632-1636, 1994[Abstract].

2.   Adams, W. J., and D. L. Morris. Short course cimetidine and survival with colorectal cancer. Lancet 344: 1768-1769, 1994[Medline].

3.   Altomare, D. F., L. Lupo, O. C. Pannarale, M. G. DiCorcia, and V. Memeo. Reduction of post-operative immunosuppression with ranitidine in patients with cancer of the stomach or large bowel. Eur. J. Surg. 161: 109-113, 1995[Medline].

4.   Anderson, K., R. Hakanson, H. Mattsson, B. Ryberg, and F. Sundler. Hyperplasia of histamine-depleted enterochromaffine-like cells in rat stomach using omeprazole and alpha -fluoromethylhistidine. Gastroenterology 103: 897-904, 1992[Medline].

5.   Arima, N., Y. Yamashita, H. Nokata, A. Nakamura, Y. Kinoshita, and T. Chiba. Presence of histamine H2 receptors on human gastric carcinoma cell line MKN-45 and their increase by retinoic acid treatment. Biochem. Biophys. Res. Commun. 176: 1027-1032, 1991[Medline].

6.   Barg, J., M. M. Belcheva, and C. J. Coscia. Evidence for the implication of phosphoinositol signal transduction in mu-opioid inhibition of DNA synthesis. J. Neurochem. 59: 1145-1152, 1992[Medline].

7.   Bartholeyns, J., and M. Bouclier. Involvement of histamine in growth of mouse and rat tumors: antitumoral properties of monfluoromethylhistidine, an enzyme activated irreversible inhibitor of histidine decarboxylase. Cancer Res. 44: 639-645, 1984[Abstract].

8.   Brenna, E., H. G. P. Swarts, C. H. W. Klaassen, J. J. H. H. M. de Pont, and H. L. Waldum. Evaluation of the trophic effect of long-term treatment with the histamine H2 receptor antagonist loxtidine on rat oxyntic mucosa by differential counting of dispersed cells. Gut 35: 1547-1550, 1994[Abstract].

9.   Burtin, C., C. Noirot, P. Scheinmann, L. Galoppin, D. Sabolovic, and P. Bernard. Clinical improvement in advanced cancer disease after treatment combining histamine and H2-antihistaminics (ranitidine and cimetidine). Eur. J. Cancer Clin. Oncol. 24: 161-167, 1988[Medline].

10.   Burtin, C., P. Scheinmann, J. C. Salomon, G. Lespinats, C. Frayssinet, B. Lebel, and P. Canu. Increased tissue histamine in tumor-bearing mice and rats. Br. J. Cancer 43: 684-688, 1981[Medline].

11.   Casnellie, J. E. Protein kinase inhibitors: probes for the functions of protein phosphorylation. Adv. Pharmacol. 22: 167-203, 1994.

12.   Chew, C. S. Cholecystokinin, carbachol, gastrin, histamine, and forskolin increase [Ca2+]i in gastric glands. Am. J. Physiol. 238 (Gastrointest. Liver Physiol. 1): G814-G823, 1986.

13.   Cook, S. J., and F. McCormick. Inhibition of cAMP of Ras-dependent activation of Raf. Science 262: 1069-1072, 1993[Medline].

14.   Crean, G. P., M. W. Marshall, and R. D. E. Rumsey. Parietal cell hyperplasia induced by the administration of pentagastrin (ICI 50,123) to rats. Gastroenterology 57: 147-156, 1969[Medline].

15.   Del Valle, J., L. D. Wang, I. Gantz, and T. Yamada. Characterization of H2 histamine receptor: linkage to both adenylate cyclase and [Ca2+]i signaling systems. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G967-G972, 1992[Abstract/Free Full Text].

16.   DeMouzon, S. H., P. Csermely, G. Zoppini, and C. R. Kahn. Quantitative dissociation between EGF effects on c-myc and c-fos gene expression, DNA synthesis, and epidermal growth factor receptor tyrosine kinase activity. J. Cell. Physiol. 150: 180-187, 1992[Medline].

17.   Dumont, J. E., J. C. Jauniaux, and P. P. Roger. The cyclic AMP-mediated stimulation of cell proliferation. Trends Biochem. Sci. 14: 67-71, 1989[Medline].

18.   Frodin, M., P. Peraldi, and E. Van Obberghen. Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells. J. Biol. Chem. 269: 6207-6214, 1994[Abstract/Free Full Text].

19.   Gantz, I., M. Schaffer, J. Del Valle, C. Logsdon, V. Campbell, M. Uhler, and T. Yamada. Molecular cloning of a gene encoding the histamine H2 receptor. Proc. Natl. Acad. Sci. USA 88: 429-433, 1991[Abstract].

20.   Gespach, C., H. Cost, and J.-P. Abita. Histamine H2 receptor activity during the differentiation of the human monocytic-like cell line U-937. FEBS Lett. 184: 207-213, 1985[Medline].

21.   Herschman, H. R. Primary response genes induced by growth factors and tumor promoters. Annu. Rev. Biochem. 60: 281-319, 1991[Medline].

22.   Hill, C. S., and R. Treisman. Differential activation of c-fos promoter elements by serum, lysophosphatidic acid, G proteins and polypeptide growth factors. EMBO J. 14: 5037-5047, 1995[Abstract].

23.   Hill, S. J. Distribution, properties, and functional characteristics of three classes of histamine receptor. Pharmacol. Rev. 42: 45-83, 1990[Abstract].

24.   Holt, J. T., T. V. Gopal, D. Moulton, and A. W. Nienhuis. Inducible production of c-fos anti-sense RNA inhibits 3T3 cell proliferation. Proc. Natl. Acad. Sci. USA 83: 4794-4798, 1986[Abstract].

25.   Janknecht, R., M. A. Cahill, and A. Nordheim. Signal integration at the c-fos promoter. Carcinogenesis 16: 443-450, 1995[Medline].

26.   Ljungstrom, M., and C. S. Chew. Calcium oscillation and morphological transformations in single cultured gastric parietal cells. Am. J. Physiol. 260 (Gastrointest. Liver Physiol. 23): G67-G78, 1991.

27.   Mitsuhashi, M., T. Mitsuhashi, and D. G. Payan. Multiple signaling pathways of histamine H2 receptor. J. Biol. Chem. 264: 18356-18362, 1989[Abstract/Free Full Text].

28.   Nielsen, H. J., J. H. Hammer, F. Moesgaard, and H. Kehlet. Possible role of histamine-2 receptor antagonists for adjuvant treatment in colorectal cancer. Clinical review. Eur. J. Surg. 157: 437-441, 1990.

29.   Nielsen, H. J., J. H. Hammer, and S. Gronvall. The effect of ranitidine on immune function, tumor response and survival in patients with liver metastases from colorectal cancer. Gastrointest. Cancer 1: 183-190, 1996.

30.   Post, G. R., and J. H. Brown. G protein-coupled receptors and signaling pathways regulating growth responses. FASEB J. 10: 741-749, 1996[Abstract/Free Full Text].

31.   Todisco, A., V. Campbell, C. J. Dickinson, J. Del Valle, and T. Yamada. Molecular basis for somatostatin action: inhibition of c-fos expression and AP-1 binding. Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G245-G253, 1994[Abstract/Free Full Text].

32.   Todisco, A., Y. Takeuchi, C. Seva, C. J. Dickinson, and T. Yamada. Gastrin and glycine-extended progastrin processing intermediates induce different programs of early gene activation. J. Biol. Chem. 270: 28337-28341, 1995[Abstract/Free Full Text].

33.   Tonnesen, H., S. Bulow, K. Fischerman, A. Hjortrup, V. M. Pedersen, L. B. Svendsen, U. Knigge, P. Damm, P. Hesselfeld, I. K. Pedersen, O. J. Siemssen, and P. M. Christiansen. Effect of cimetidine on survival after gastric cancer. Lancet 2: 990-992, 1988[Medline].

34.   Wang, L., I. Gantz, and J. Del Valle. Histamine H2 receptor activates adenylate cyclase and PLC via separate GTP-dependent pathways. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G613-G620, 1996[Abstract/Free Full Text].

35.   Wang, L., E. J. Wilson, J. Osburn, and J. Del Valle. Epidermal growth factor inhibits carbachol-stimulated canine parietal cell function via protein kinase C. Gastroenterology 110: 469-477, 1996[Medline].

36.   Watson, S. A., L. J. Wilkinson, J. F. R. Robertson, and J. D. Hardcastle. Effect of histamine on the growth of human gastrointestinal tumours: reversal by cimetidine. Gut 34: 1091-1096, 1993[Abstract].

37.   Yamauchi, K., K. Holt, and J. E. Pessin. Phosphatidyinositol 3-kinase functions upstream of ras and raf in mediating insulin stimulation of c-fos transcription. J. Biol. Chem. 268: 14597-14600, 1993[Abstract/Free Full Text].

38.   Yule, D. I., D. Wu, T. E. Essington, J. A. Shayman, and J. A. Williams. Sphingosine metabolism induces Ca2+ oscillations in rat pancreatic acinar cells. J. Biol. Chem. 268: 12353-12358, 1993[Abstract/Free Full Text].


AJP Cell Physiol 273(6):C2037-C2045
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