Abstract

Previously, we demonstrated that activation of the human H2 receptor (hH2R) leads to an increase in c-fos transcription and cell proliferation. The purpose of these studies was to examine whether hH2R regulates c-jun expression and, if so, explore the mechanisms by which it does so. Histamine induced an increase in c-jun mRNA in human embryonic kidney cells stably transfected with the hH2R (maximal effect: 554.6 ± 86.8% of control). The protein kinase C (PKC) inhibitors staurosporine (10−6 M) and GF-109203X (10−6 M) significantly inhibited histamine-stimulated c-fos mRNA while not altering c-jun expression. The protein kinase A (PKA) pathway inhibitors Rp-cAMP and protein kinase inhibitor did not affect the action of histamine on c-jun or c-fos mRNA. Histamine (10−4M) stimulated extracellularly regulated kinase 2 tyrosine phosphorylation. The specific inhibitor of the mitogen-activated protein (MAP) kinase pathway, PD-98059 (5 × 10−5 M), significantly inhibited histamine-induced c-fos and c-jun mRNA. Of interest, the p70 S6 kinase inhibitor rapamycin (10−6 M) but not wortmannin decreased histamine-stimulated c-jun mRNA by 58.5 ± 12% (mean ± SE, n = 4) while not significantly altering c-fos message. Histamine (10−4 M) also led to an ∼4.5-fold increase in Jun NH2-terminal kinase activity in a PKC-, PKA-, and MAP kinase-independent but rapamycin-sensitive manner. Our findings suggest that histamine stimulates both c-fos and c-jun mRNA in a differential manner. PKC is involved in histamine-mediated c-fos activation, whereas p70 S6 kinase is important for linkage of this receptor to c-jun.

  • signal transduction
  • mitogen-activated protein kinase

histamine is a biogenic amine that is widely distributed throughout the body (32). The observation that this chemical mediator is synthesized and made available in an unstored diffusable form in tissues undergoing rapid growth and repair suggests that histamine may have a role beyond responding to inflammation (20,24). Several reports have suggested that activation of the H2 receptor can lead to cell proliferation (5, 34, 38). More recently, we have shown that this G protein-linked receptor can also activate transcription of the gene encoding c-fos in a protein kinase C (PKC)-dependent manner (40). Cell proliferation often requires a series of signaling steps that act in a coordinated manner to regulate nuclear events responsible for controlling cell division. Transcriptional regulation of target genes regulated by c-fosoften involves parallel activation of c-jun with dimerization of c-fos and c-jun forming the transcription factor AP-1 (26, 36). AP-1 in turn binds to specific DNA elements important in driving transcription of target genes. The purpose of the following study was to examine whether activation of the human H2receptor (hH2R) leads to regulation of c-jun mRNA. We have also explored in greater detail the postreceptor events coupling the H2 receptor to regulation of c-fos and c-jun.

MATERIALS AND METHODS

Chemicals.

Fetal bovine serum (FBS) and DMEM were purchased from GIBCO (Grand Island, NY). Formaldehyde and phenol were from Fisher Scientific (Pittsburgh, PA). Myelin basic protein (MBP), TCA, Triton X-100, histamine, cimetidine, BSA, dithiothreitol, and EDTA were purchased from Sigma Chemical (St. Louis, MO). Earle's balanced salts was purchased from Irvine Scientific (Santa Ana, CA). [3H]tiotidine (87 Ci/mmol) was obtained from Amersham (Arlington Heights, IL). The c-fos and c-jun40 base single-stranded synthetic oligonucleotide probes were purchased from Calbiochem (San Diego, CA). Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA control probe was purchased from Clontech Laboratories (Palo Alto, CA). The c-junNH2-terminal kinase 1 (JNK1), extracellularly regulated kinase 2 (ERK2), and p70 S6 kinase antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The phospho-p70 S6 kinase and the phospho-cAMP response element binding protein (CREB; Ser-133) antibody were obtained from New England Biolabs (Beverly, MA).

Cell culture and transfection.

HEK-293 cells were maintained in DMEM supplemented with 10% FBS at 37°C in a humidified 5% CO2 atmosphere. Transfections were performed on 50% confluent monolayers in 100-mm dishes. For stable transfection, cells were incubated at 37°C in 5-ml serum-free DMEM containing 2 μg of the full-length coding region of the human H2 DNA subcloned into a pBK cytomegalovirus expression vector as previously described (14, 40). After 5 h of exposure to the transfection media containing Lipofectamine (25 μl), monolayers were placed in DMEM containing 10% FBS and incubated overnight. Permanently transfected cells were selected by resistance to the neomycin analog G418 (500 mg/l). Single clones of transfected cells were selected and screened for expression of the human H2histamine receptor by Northern blot analysis and receptor-binding studies using [methyl-3H]tiotidine as the radioligand. Stably transfected human embryonic kidney (HEK) cells will be referred to as HEK-H2 cells. HEK-H2cells express 87,392 ± 1,823 (mean ± SE; n = 4) receptors per cell.

Characterization of ERK and JNK activity.

Assays for ERK2 and JNK activity were performed as previously described (11, 29). Briefly, transfected HEK-293 cells were serum starved for 24 h and then treated with ligands for 30 min at 37°C. Cells were harvested in lysis buffer [10 mM KPO4, pH 7.4, 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 50 mM glycerophosphate, 1 mM sodium orthovanadate (Na3VO4), 2 mM dithiothreitol, 40 μg/ml phenylmethylsulfonyl fluoride, 0.8 μg/ml leupeptin, 10 mg/ml p-nitrophenylphosphate, and 10 μg/ml aprotinin]. Endogenous ERK2 and JNK were immunoprecipitated with 2 μg of the corresponding antibodies for 18 h at 4°C followed by an additional 1 h incubation with protein A-agarose beads. The immunocomplexes were washed two times in lysis buffer and kinase buffer. The activity of the immune complex was assayed at 30°C for 30 min in 20 μl of kinase buffer (1 M HEPES, pH 7.4, 0.5 M magnesium acetate, 1 mM ATP) in the presence of 2 μCi of [γ-32P]ATP (10 Ci/mmol) with the appropriate substrates [MBP or glutathione S-transferase-jun (GST-c-Jun)]. The reactions were terminated by boiling in Laemmli sample buffer. The proteins were resolved by SDS-13% PAGE followed by Coomassie blue staining and autoradiography. The phosphorylated proteins were quantitated by phosphoimaging.

Measurement of CREB phosphorylation.

HEK-H2 cells were grown in six-well plates containing DMEM supplemented with 10% FBS for 24 h and then placed in serum-free conditions for 18 h. Cells were pretreated with the protein kinase A (PKA) inhibitors Rp-cAMP and protein kinase inhibitor (PKI) for 15 min followed by incubation with either histamine (10−4 M) or forskolin (10−5 M) for 30 min. Cells were then washed, lysed, and Western blot was performed as described above. Blots were incubated with a phospho-CREB antibody for 4 h, and immunoreactive bands were visualized using the standard immunoblotting detection system. Blots were then stripped and reprobed with a CREB-specific antibody.

Measurement of 12-O-tetradecanoylphorbol-13-acetate response element luciferase activity.

HEK-H2 cells were grown at 37°C in 12-well plates containing DMEM supplemented with 10% FBS. Subconfluent cells were transfected with 5 μg of the 12-O-tetradecanoylphorbol-13-acetate response element (TRE, perfect AP-1) luciferase expression vector (TRE-Luc) using Lipofectamine as previously described (14, 40). The plasmid containing the herpes simplex virus thymidine kinase minimal promoter was used as a control construct. Media were removed 24 h after transfection, serum-free DMEM was added for 24 h, and cells were treated with histamine for 6 h. Cells were then washed, lysed, and luciferase assays performed as previously described (40). Luciferase activity was expressed as relative light units, then normalized for protein content (measured by Bradford method) in the cell lysate to correct for differences in cell number and transfection efficiency. In several experiments, cells were cotransfected with the pCMV-Gal vector. Galactosidase activity was measured by luminescence derived from 10 μl of Lumin-Gal 530 (Lumigen, Southfield, MI) and used to normalize the luciferase assay data for transfection efficiency. Similar results were obtained when data were normalized for either protein concentration or luciferase activity.

Northern blot analysis.

HEK-293 cells stably transfected with the H2 receptor were treated with ligands and lysed with TRIzol (Life Technologies) according to manufacturer's instructions. Northern blot hybridization assays were performed as described previously (40). 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 then transferred from the gel to nitrocellulose filters. Ethidium-stained ribosomal RNA bands in the gel were photographed before and after 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 were human c-fos and c-jun 40 base single-stranded synthetic oligonucleotides labeled with [γ-32P]ATP by 5′-end labeling procedure. The human GAPDH control cDNA probe was labeled with [α-32P]dCTP by using the random priming procedure. The same nitrocellulose filters were hybridized to three different 32P-labeled (c-fosand c-jun GAPDH) probes as described previously (40).

Detection of p70 S6 kinase by immunoblotting.

Transfected HEK-293 cells were serum starved for 24 h and treated with ligands for 15 min. Cells were pretreated with antagonists for 15 min in selected experiments. Treated cells were lysed in 500 μl lysis buffer [50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 1.5 mM MgCl2, 1 mM Na3VO4, 10 mM NaF, 10 mM Na4P2O7 ⋅ 10 H2O, 1 mM 4-(2-aminoethyl)benzenesulfonylfluoride hydrochlorine, 1 μg/ml leupeptin, and 1 μg/ml aprotinin] and lysates transferred to microcentrifuge tubes and spun at 16,000g for 10 min. Equal amounts of sample proteins (80 μg) were loaded on a discontinuous 10% SDS polyacrylamide gel and run at 25 V for 18 h. Gels were transferred onto Immobilon-P transfer membrane (Millipore, Bedford, MA) in 25 mM Tris, 150 mM glycine, and 20% methanol and then incubated in Tris-buffered saline (TBS; 20 mM Tris and 0.15 NaCl containing 0.33% Tween) and 0.5% nonfat dry milk for 1 h at room temperature to block nonspecific binding. For experiments measuring p70 S6 kinase phosphorylation, membranes were incubated with the phospho-p70 S6 kinase antibody (1:1,000) in TBS for 4 h. Membranes were washed repeatedly with TBS buffer containing 0.25% dry milk. Membranes incubated with the phospho-p70 S6 kinase antibody were then incubated with the peroxidase-linked secondary antibody (Zymed horseradish peroxidase-goat anti-rabbit antibody, 1:1,250) for 60 min. Immunoreactive bands were visualized using the standardized enhanced chemiluminescence immunoblotting detection system (Amersham). The same Immobilon-P transfer membranes were then stripped of bound antibody by incubation with 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris ⋅ HCl (pH 6.7) for 30 min at 50°C, rinsed three times for 5 min with 200 ml TBS, and reprobed with p70 S6 kinase antibody (1:1,000).

Measurement of [3H]thymidine incorporation.

Histamine-stimulated cell proliferation was measured by using [3H]thymidine incorporation as previously described (40). 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 media. Cells were washed with serum-free media and treated with ligands for 18 h. 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 hour 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.

Statistical analysis.

Data are presented as means ± SE, where n is equal to the number of cell preparations. Statistical analysis was performed using either Student's t-test or ANOVA if multiple comparisons were performed. P < 0.05 was considered significant.

RESULTS

Histamine-mediated regulation of c-fos and c-junmRNA.

We first examined the effect of histamine on c-fos and c-jun mRNA. As previously reported (40), histamine (10−4 M) significantly increased c-fosmessage in a staurosporine and bisindolylmaleimide (GF-109203)-sensitive manner (Fig.1 A). Histamine also stimulated c-jun mRNA (Fig. 1 B), but of note, neither staurosporine nor GF blocked this effect, suggesting to us a difference in the mechanism by which the H2 receptor regulates these two protooncogenes. Our previous studies demonstrating that staurosporine at the concentrations shown here inhibited histamine-stimulated PKC activation (40) support the efficacy of this antagonist in our system.

Fig. 1.

Effect of protein kinase C (PKC) inhibitors on histamine stimulated c-fos and c-jun gene expression in HEK-293 cells stably transfected with the human H2 receptor (HEK-H2cells). In these and subsequent experiments, HEK-H2 cells were starved for 24 h. After pretreatment with the PKC inhibitors staurosporine (Stau)/GF-109203 (GF) or vehicle for 15 min, the cells were exposed to histamine (His) for 30 min at 37°C. Total RNA was extracted and Northern blot analysis performed. A: the maximal level of c-fos mRNA was 554.6 ± 86.8% of control (mean ± SE, n = 4) after 30 min of treatment with histamine (10−4 M). The PKC inhibitor Stau (10−6 M) and GF (10−6 M) nearly abolished histamine-stimulated c-fos mRNA level. B: the maximal stimulatory effect of His (10−4 M) on c-jun mRNA was 581.4 ± 52.5% of control (mean ± SE,n = 4). Both PKC inhibitors Stau and GF did not alter the stimulatory effect of histamine on c-jun gene transcription. Cont, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

In view of the known stimulatory effect of the H2receptor on adenylate cyclase, we examined whether this signaling system was involved in histamine-mediated stimulation of c-fos and c-jun mRNA. As shown in Fig.2, the inhibitors of the adenylate cyclase signaling pathway, Rp-cAMP and PKI, did not affect histamine's stimulatory action on c-jun and c-fos message. We also examined the effect of a known stimulant of cAMP, forskolin, on c-jun mRNA. As illustrated in Fig.3, forskolin did not stimulate an increase in the c-jun message.

Fig. 2.

Effect of protein kinase A (PKA) inhibitors on histamine stimulated c-fos and c-jun gene expression in HEK-H2. HEK-H2 cells were pretreated with the myristoylated protein kinase A inhibitor amide (PKI) or Rp-cAMP (or vehicle) for 15 min, then exposed to histamine for 30 min at 37°C. Total RNA was extracted and Northern blot analysis performed. Histamine (10−4 M) significantly stimulated c-fos and c-jun gene transcription. The PKA inhibitor (10−6 M) or Rp-cAMP (10−5M) did not alter the stimulatory effect of histamine on c-fos(A) and c-jun (B) gene activation.

Fig. 3.

Effect of forskolin and PKI on histamine stimulated c-junexpression. HEK-H2 were pretreated with PKI (10−5 M; or vehicle) for 15 min, then exposed to histamine (10−4 M) or forskolin (Fors, 10−5 M) for 30 min at 37°C. Total RNA was extracted and Northern blot analysis was performed. Histamine significantly stimulated c-jun mRNA to 539.4 ± 38.9% of control in a PKI-insensitive manner. Forskolin did not alter c-jun message. Study shown is representative of 3 experiments.

In an effort to document the efficacy of Rp-cAMP and PKI to inhibit the adenylate cyclase signaling pathway, we examined their effect on histamine and forskolin-stimulated phosphorylation of the transcription factor CREB that binds the cAMP response element (CRE) (17, 37). As shown in Fig. 4, both histamine (10−4 M) and forskolin (10−5 M) significantly increased phosphorylation of CREB. Both PKI (10−6 M) and Rp-cAMP (10−5 M) inhibited histamine and forskolin-stimulated CREB phosphorylation while not altering CREB levels. These findings support the efficacy of these two antagonists in blocking the adenylate cyclase pathway. Moreover, these data support that histamine is regulating c-fos and c-jun mRNA via a cAMP-independent mechanism.

Fig. 4.

Histamine and forskolin mediated phosphorylation of cAMP response element binding protein (CREB). HEK-H2 cells were pretreated with PKI (10−6 M) or Rp-cAMP (10−5 M; or vehicle) for 15 min, followed by treatment with histamine (10−4 M) or forskolin (10−5 M) for 30 min. Cell lysates were prepared and Western blotting performed as described in materials and methods. Blots were incubated with an anti-phospho-CREB (Ser-133) antibody for 4 h. Blots were stripped then reprobed with an anti-CREB antibody. A: histamine and forskolin stimulated phosphorylation of CREB to 263.3 ± 34.5% and 398.5 ± 56.9% control, respectively (mean ± SE, n = 3; P < 0.05).B: both PKI and Rp-cAMP nearly abolished the stimulatory effect of both secretogogues on CREB phosphorylation.

Histamine-mediated regulation of ERK2 and JNK.

Our observation that the ability of histamine to increase c-junmRNA independent of PKC and PKA led us to examine additional signaling systems to which the H2 receptor may be coupling. A growing body of literature has documented the ability of G protein-coupled receptors to activate the mitogen-activated protein (MAP) kinase signaling cascades (19). We first examined whether the H2 receptor was linked to the MAP kinase signaling pathways previously shown to be involved in regulating c-fos and c-jun. As illustrated in Fig.5, histamine stimulated both ERK2 and JNK activity in a time-dependent manner. Histamine's stimulatory effect on ERK2 was rapid in onset (5 min) and sustained in duration. In contrast, the effect of histamine on JNK activation was transient, with a peak effect observed after 30 min of treatment.

Fig. 5.

Histamine mediated regulation of extracellularly regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK) in HEK-H2. HEK-H2 cells were treated with histamine (10−4 M) for different time intervals at 37°C and lysed directly in 400 μl of ice-cold lysis buffer. The stimulatory effect of histamine on ERK and JNK activation was examined by using a specific immune complex assay. A: histamine significantly stimulated ERK and JNK activity in a time-dependent fashion. B: histamine (10−4 M) increased ERK activity to 4.1 ± 0.5-fold within 5 min, with a sustained effect noted for up to 3 h stably. The maximal stimulation of histamine (10−4 M) on JNK activity was 4.8 ± 0.4-fold induction at 30 min, returning to basal levels after 3 h. MBP, myelin basic protein; GST-c-Jun, glutathioneS-transferase-c-Jun.

Effect of histamine on p70 S6 kinase.

The ribosomal p70 S6 kinase is a serine/threonine kinase involved in cell proliferation through regulation of the cell cycle. Recent studies have demonstrated that this kinase can be regulated by a host of different mitogenic factors. As shown in Fig.6, activation of the H2receptor led to phosphorylation of p70 S6 kinase in a rapamycin-sensitive manner. As noted in panel 6B, the amount of p70 S6 kinase protein did not change with treatment, whereas phosphorylation levels of this enzyme were clearly regulated by histamine and rapamycin (Fig. 6, A and C).

Fig. 6.

Histamine mediated phosphorylation of p70 S6 kinase in HEK-H2 cells. HEK-H2 cells were pretreated with the p70 S6 kinase inhibitor rapamycin (Rapa) for 15 min at 37°C, exposed to histamine (10−4 M) for 15 min at 37°C, and lysed directly in 500 μl of ice-cold lysis buffer. A: phospho-p70 S6 kinase antibody was used as a probe to perform the Western blot. Histamine potently stimulated p70 S6 kinase phosphorylation in a rapamycin-sensitive manner. B: the same membrane was stripped and probed with a specific nonphosphorylated p70 S6 kinase antibody. The data shown confirm that the amount of p70 S6 kinase protein was stable. C: histamine (10−4M) stimulated p70 S6 kinase phosphorylation from 250.5 ± 101.1 to 1,575.6 ± 150.5 densitometric units (mean ± SE, P < 0.05). The p70 S6 kinase inhibitor rapamycin (10−7 M) significantly inhibited histamine-stimulated p70 S6 kinase phosphorylation by 55.2 ± 12.5% (mean ± SE, P < 0.05).

Role of MAP kinase and p70 S6 kinase in histamine-mediated regulation of c-fos and c-jun mRNA.

In view of the stimulatory effect of histamine on ERK activity, we explored whether this pathway was involved in H2receptor-mediated regulation of c-fos and c-jun. As shown in Fig. 7, the specific MAP or ERK kinase (MEK) inhibitor PD-98059 (2) significantly decreased histamine-mediated induction of both c-fos and c-junmRNA. We explored in greater detail the inhibitory effect of PD-98059 on c-fos and c-jun mRNA. As shown in Fig.8, PD-98059 dose dependently inhibited both c-fos and c-jun message with virtually identical IC50 (Fig. 8), suggesting MEK is involved in the regulation of both these early response genes in a similar manner.

Fig. 7.

Effect of the PD-98059 on histamine stimulated c-fos and c-jun gene expression in HEK-H2 cells. HEK-H2 cells were pretreated with PD-98059 (PD) for 15 min and exposed to histamine for 30 min at 37°C. Total RNA was extracted and Northern blot analysis performed. A: the maximal level of c-fos mRNA was 595.8 ± 76.9% of control (mean ± SE, n = 4) after 30 min of treatment with histamine (10−4 M). PD-98059 (5 × 10−5 M) nearly abolished histamine-stimulated c-fos mRNA level (79.2 ± 6.55% inhibition; mean ± SE,n = 4). B: the maximal stimulatory effect of histamine (10−4 M) on c-jun mRNA was 553.4 + 45.5% control (mean ± SE, n = 6). The ERK inhibitor PD-98059 (5 × 10−5 M) also abolished histamine-stimulated c-fos mRNA level (64.2 ± 15.3% inhibition; n = 5,P < 0.05).

Fig. 8.

PD-98059 mediated inhibition of c-fos and c-jun mRNA. HEK-H2 cells were pretreated with various concentrations of PD (or vehicle) for 30 min (maximal inhibitory effect for both c-fos and c-jun). Cells were then treated with histamine (10−4 M) for 30 min. Northern blotting was performed as described above. A: PD inhibited histamine-stimulated c-fos and c-jun mRNA in a dose-dependent fashion. B: the IC50 for PD was virtually identical for both c-fos (7.5 × 10−7 M) and c-jun (7.0 × 10−7 M).

We next examined whether the p70 S6 kinase pathway was linked to H2 receptor-mediated regulation of c-fos and c-jun (Fig. 9). Of interest, rapamycin inhibited the action of histamine on c-jun while leaving unaltered the action of this biogenic amine on the c-fosmessage. Phosphatidylinositol 3-kinase (PI 3-kinase) is an important upstream regulator of p70 S6 kinase. We examined the role of this kinase in histamine-mediated c-jun activation. Of note, the PI 3-kinase inhibitor wortmannin failed to inhibit histamine's stimulatory effect on c-jun (Fig. 9 B).

Fig. 9.

Effect of the p70 S6 kinase and phosphatidylinositol 3-kinase inhibitors on histamine stimulated c-fos and c-jungene transcription in HEK-H2 cells. HEK-H2 cells were pretreated with rapamycin or wortmannin (Wort) for 15 min and exposed to histamine for 30 min at 37°C. Total RNA was extracted and Northern blot analysis performed.A: the maximal level of c-fos mRNA was 595.8 ± 76.9% of control (mean ± SE, n = 4) after 30 min of treatment with histamine (10−4 M). Rapamycin (10−7 M) and wortmannin (10−6 M) did not significantly alter histamine-stimulated c-fos mRNA level. B: the maximal stimulatory effect of histamine (10−4 M) on c-jun mRNA was 553.4 ± 45.5% of control (mean ± SE, n = 6). The p70 S6 kinase inhibitor rapamycin (10−7 M) decreased histamine-stimulated c-jun mRNA level (58.5 ± 12.6% inhibition; mean ± SE,n = 4). In contrast, wortmannin did not alter histamine's stimulatory effect.

In an effort to examine the specificity of the antagonists utilized, we investigated their effect on histamine-mediated kinase regulation. As anticipated (Fig. 10), PD-98059 significantly inhibited histamine-stimulated ERK activity, whereas the PKC inhibitor GF and the p70 S6 kinase inhibitor rapamycin did not affect this parameter. The role of p70 S6 kinase on JNK activity was also assessed. Rapamycin significantly inhibited histamine-stimulated JNK activity (Fig. 11). The inhibitors of PKC and MEK did not alter the stimulatory effect of histamine on JNK. Finally, we examined the effect of GF and 2′-amino-3′-methoxyflavone (PD) on histamine-mediated phosphorylation of p70 S6 kinase, and neither had an effect on this pathway (Fig. 12).

Fig. 10.

Effect of different inhibitors on ERK activation in HEK-H2cells. HEK-H2 cells were pretreated with PD-98059, the PKC inhibitor GF-109203, or the p70 S6 kinase inhibitor rapamycin (or vehicle) for 15 min, exposed to histamine for 30 min at 37°C, and lysed directly in 400 μl of ice-cold lysis buffer. A: histamine significantly stimulated ERK activity in a PD-sensitive manner. B: histamine (10−4 M) increased ERK activity 4.5 ± 0.6-fold (mean ± SE, n = 3, P< 0.05). GF-109203 and the p70 S6 kinase inhibitor rapamycin did not significantly affect histamine's action.

Fig. 11.

Effect of rapamycin on histamine stimulated JNK activation in HEK-H2 cells. HEK-H2 cells were pretreated with PD-98059, GF-109203, or rapamycin (or vehicle) for 15 min, exposed to histamine for 30 min at 37°C, and lysed directly in 400 μl of ice-cold lysis buffer. A: histamine significantly stimulated JNK activity in a rapamycin-sensitive manner. B: histamine (10−4 M) increased JNK activity 5.5 ± 1.0-fold (mean ± SE, n = 3, P < 0.05). Rapamycin (10−7 M) inhibited histamine's action by 75.4 ± 5.9% (mean ± SE, n = 4). GF-109203 and PD-98059 did not affect histamine's action.

Fig. 12.

Effect of GF-109203 and PD-98059 on histamine stimulated phosphorylation of p70 S6 kinase. HEK-H2 cells were pretreated with GF or PD (or vehicle) for 15 min, then incubated with histamine for 30 min. Phosphorylation of p70 S6 kinase was determined by Western blotting (A) as described before. B: histamine (10−4 M) stimulated phosphorylation of p70 S6 kinase to 517.5 ± 29.3% control (mean ± SE, n = 4). GF (10−4 M) and PD (10−5 M) failed to alter histamine's effect on p70 S6 kinase.

Role of MAP kinase and p70 S6 kinase in histamine-stimulated cell proliferation.

We examined the role of MAP kinase and p70 S6 kinase on histamine-mediated cell proliferation. As shown in Fig.13, both PD-98059 and rapamycin significantly inhibited histamine-mediated cell proliferation. Combination of these two inhibitors abolished histamine's proliferative effect.

Fig. 13.

Effect of PD-98059 and rapamycin on histamine stimulated [3H]thymidine incorporation in transfected HEK-H2 cells. HEK-H2 cells were pretreated with PD, rapamycin, or vehicle for 15 min and exposed to histamine for 18 h at 37°C. Histamine (10−4 M) stimulated [3H]thymidine incorporation to 214.5 ± 69.4% of control (n = 8, P < 0.05). PD-98059 (5 × 10−5 M) decreased histamine-stimulated [3H]thymidine incorporation by 60.5 ± 7.8% (n = 4, P < 0.05). Rapamycin (10−7M) inhibited histamine-stimulated [3H]thymidine incorporation by 50.8 ± 8.9% (n = 4, P < 0.05). Combination of the antagonists abolished histamine's action.

DISCUSSION

We have demonstrated that the hH2R can induce both c-fos and c-jun mRNA in HEK-293 cells through both common and divergent signaling pathways. It appears that ERK is involved in the regulation of both c-fos and c-jun by histamine. In contrast, PKC appears to be most important for c-fos activation, whereas p70 S6 kinase is primarily involved in c-jun regulation via a JNK-dependent mechanism.

The ability of a receptor to activate downstream effectors is in part dictated by the cell type in which it is found. In an effort to assess the potential cell specificity of histamine-mediated MAP kinase regulation, we have begun to examine the effect of this amine on these pathways in isolated parietal cells (39). In preliminary studies, we have demonstrated that activation of the H2 receptor leads to stimulation of ERK2 and JNK, as well as p38 in isolated canine parietal cells. The mechanism and the potential physiological significance in parietal cell activation is being pursued. These findings demonstrate that histamine-mediated regulation of the MAP kinase signaling system is not limited to transfected HEK-293 cells. Receptor-mediated signaling is also dictated in part by the level of receptor expression. Specifically, events that ordinarily do not occur under physiological circumstances may be promoted if an excessive number of receptors are expressed. Our observation that histamine could stimulate the MAP kinase cascade in isolated canine parietal cells, which express 45,302 ± 457 receptors per cell, supports that our findings in HEK-H2 are not due to receptor overexpression in a nonphysiological system.

In contrast to histamine's action on c-fos, the effect of this ligand on c-jun was not dependent on the coupling of this receptor to PKC. In view of the known stimulatory action of the H2 receptor on adenylate cyclase/cAMP, we examined whether histamine's effect on c-jun was via this later signaling pathway. Of interest, despite a robust increase in cAMP in response to histamine (data not shown) and phosphorylation of CREB, there did not appear to be a linkage between this pathway and either c-fos or c-jun regulation in these cells. This observation is different from that made with the dopamine D1 receptor that was shown to activate the JNK signaling pathway via a PKA-dependent mechanism (45).

Recent studies have documented the importance of MAP kinases in regulating the transcription factor AP-1 (42). Moreover, it has been established that G protein-coupled receptors can regulate MAP kinase pathways in multiple cell systems (19). Although both ERK and JNK activity were stimulated by histamine in a time-dependent manner, the profile of activation was different. The stimulatory effect of histamine on JNK was transient, returning to baseline levels by 2 h, whereas ERK activity was sustained throughout the entire period examined. It has been suggested that a sustained increase in ERK may point toward a role of this kinase in regulating cell differentiation, whereas a transient rise is most consistent with a role in cell proliferation (10, 28). It is not clear at this time which of these two events is primarily linked to H2 receptor signaling; however, our studies showing that PD-98059 inhibits histamine-stimulated cell proliferation suggests that ERK may be more important for cell growth in this particular model.

As in the case of other G protein-coupled receptors (GPCRs) (7), we have demonstrated that the H2receptor can activate JNK. It appears that H2-related signaling diverges at this point. The PKC pathway is not involved in histamine-mediated JNK or c-jun-mediated activation, whereas the p70 S6 kinase system in part mediates the action of the H2 receptor on c-jun. The inability of wortmannin to inhibit histamine-stimulated c-jun mRNA suggests that the H2 receptor is coupling to p70 S6 kinase in a PI 3-kinase-independent manner. Moreover, the negative results obtained with PD and GF suggest that PKC does not mediate p70 S6 kinase activation in response to histamine. Although the PI 3-kinase pathway has been traditionally coupled to p70 S6 kinase (23, 31), more recently it has been demonstrated that activation of this pathway can also occur via a PI 3-kinase-independent mechanism (1, 25, 35). Our studies are the first to demonstrate that a G protein-linked receptor can stimulate c-jun expression via a p70 S6 kinase pathway that is PI 3-kinase independent. As in the case of studies with the fibroblast growth factor receptor-1 (25), we have demonstrated that activation of p70 S6 kinase by the H2 receptor is independent of PI 3-kinase and PKC. We have not, however, established how the H2 receptor regulates JNK. Possible links include small GTP-binding proteins such as Rac1 and Cdc42 (8, 9). Other possibilities include free βγ dimers (9) or a different type of α-subunit such as Gα12 (30). Others have demonstrated that rapamycin can inhibit JNK activity in lymphocytes (21), but the actual mechanism by which these two pathways interact has not been established.

The endothelin-1 (ET-1) receptor expressed in Rat1 cells has also been shown to activate both ERK and JNK through what appear to be parallel pathways (41). In contrast to the H2 receptor, the action of ET-1 on JNK was inhibited by activation of PKC. It was also noted that inhibition of PKC potentiated the effect of ET-1 on JNK. Of interest, the effect of ET-1 on ERK was transient, as opposed to our studies with the H2 receptor, which reveal a sustained increase in kinase activity in response to histamine. Moreover, it appeared that the action of ET-1 on both ERK and JNK was in part mediated through a Gi or Go-regulated pathway and that chelation of intracellular and extracellular Ca2+potentiated the effect of ET-1 on JNK activity. The results reported with ET-1 receptor (41) are somewhat different from those shown here and data published previously (6). One similarity seen between ET-1 and H2 receptor signaling is the relative independence of these on PKC for ERK activation. Therefore, although both the ET-1 and histamine H2 receptors can regulate the ERK and JNK signaling pathways, the cellular mechanisms linking a single receptor to these kinase systems appear to be quite different.

We observed with interest that the MEK inhibitor significantly decreased the stimulatory effect of histamine on both c-fos and c-jun. This observation suggests a point of convergence for histamine-mediated regulation of these two early response genes. The convergence is downstream from PKC, JNK, and p70 S6 kinase because these pathways appear to be regulated by the H2 receptor via independent mechanisms. The point of convergence is most likely at the nuclear transcription factor level. Previous studies have demonstrated the ability of ERK and JNK to share several potential substrates. Specifically, both have been shown to phosphorylate the transcription factor Elk-1 (15, 22, 27). Moreover, the transcription factor AP-1, initially described as one that mediated gene regulation in response to phorbol esters (4), is modulated by multiple stimuli including growth factors and cell stress (4, 12, 26). We examined this possibility in our system by measuring the effect of histamine on TRE activation. As shown in Fig. 14, histamine dose dependently increased TRE-Luc activity. These data suggest that the H2 receptor is activating gene transcription of c-jun through a true AP-1 site.

Fig. 14.

Histamine mediated regulation of 12-O-tetradecanoylphorbol-13-acetate luciferase expression vector (TRE-Luc) activity. HEK-H2 cells were transiently transfected with either the TRE-Luc or thymidine kinase minimal promoter (TK-Luc) construct and treated with various concentrations of histamine for 5 h. Cells were lysed and luciferase activity measured. Histamine dose dependently stimulated TRE-Luc activity with an EC50 = 5.0 × 10−6 M, whereas not altering TK-Luc activity (mean ± SE, n = 6).

Transcriptional regulation of c-jun primarily involves two TREs, Jun 1 and Jun 2 (2). These promoter elements are regulated by binding heterodimers of c-Jun and ATF-2. JNK phosphorylates both c-jun and ATF-2, leading to their activation. Our work demonstrates that histamine-mediated regulation of c-jun may occur via a JNK-dependent mechanism. Of interest, we also observed that the ERK pathway may be involved in c-jun regulation. The mechanism by which the ERK pathway impacts c-jun expression is unclear.

Regulation of the gene encoding c-fos involves three important elements: the CRE, the serum response element (SRE), and thecis inducible element. In previous studies, we have shown that the H2 receptor activates c-fosthrough the SRE in a PKC-dependent mechanism. Although it is not shown here, we have also demonstrated that the MEK inhibitor PD-98059 can inhibit histamine-stimulated SRE activity. This observation suggests that part of the mechanism by which histamine regulates c-fosexpression is via the SRE through an ERK-dependent pathway. This is consistent with prior studies demonstrating that ERK can phosphorylate and activate members of the ternary complex factor (TCF) proteins, which in conjunction with serum response factor regulates SRE (42). Specifically, the TCFs Elk-1 and SAP-1 can be phosphorylated by ERK. In addition, Elk-1 can be phosphorylated by JNK, demonstrating a level of cross talk between these two systems in c-fos regulation.

In summary, our studies demonstrate for the first time that the hH2R activates both c-fos and c-junthrough both parallel and divergent pathways (Fig.15). Parallel stimulation of these two protooncogenes involves the ERK signaling system. In addition, PKC appears to be important for H2-mediated c-fosregulation, whereas the p70 S6 kinase system is involved in activation of c-jun, possibly via a JNK-dependent mechanism.

Fig. 15.

Model summarizing H2 receptor-mediated regulation of c-fos and c-jun.

Acknowledgments

We are grateful to Pamela Glazer for typing this manuscript.

Footnotes

  • Address for reprint requests and other correspondence: J. Del Valle, 6520 MSRBI, Box 0682, The Univ. of Michigan Medical School, Ann Arbor, MI 48109 (E-mail: jdelvall{at}umich.edu).

  • This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-34306 and funds from the Michigan Gastrointestinal Peptide Research Center (Grant P30-DK-34933).

  • 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. §1734 solely to indicate this fact.

REFERENCES

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