Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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ABSTRACT |
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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 × 106 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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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)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 (1012 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 (1012 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
104 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-PKC
or
anti-PKC
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 (105
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(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 0.3%
-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
[
-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-
Gal
vector.
-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 (1010 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.
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RESULTS |
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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 × 10To 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 × 107 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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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 ( and
I), in
HEK-H2 cells. As shown in Fig.
2, the level of PKC
and
PKC
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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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 PKC and
PKC
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
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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
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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 × 106 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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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
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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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
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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
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DISCUSSION |
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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
Gs,
Gq
,
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
-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.
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ACKNOWLEDGEMENTS |
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We thank Shane Davis and Patricia Richards for typing this manuscript.
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FOOTNOTES |
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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.
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