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. Author manuscript; available in PMC: 2026 Mar 14.
Published in final edited form as: Exp Cell Res. 2009 Nov 11;316(3):401–411. doi: 10.1016/j.yexcr.2009.11.002

Histamine acting on H1 receptor promotes inhibition of proliferation via PLC, RAC, and JNK-dependent pathways

Cintia Notcovich a,b, Federico Diez b, Maria Rosario Tubio a,b, Alberto Baldi a,c, Marcelo G Kazanietz d, Carlos Davio b,c, Carina Shayo a,c,*
PMCID: PMC12985397  NIHMSID: NIHMS2137756  PMID: 19913013

Abstract

It is well established that histamine modulates cell proliferation through the activation of the histamine H1 receptor (H1R), a G protein-coupled receptor (GPCR) that is known to couple to phospholipase C (PLC) activation via Gq. In the present study, we aimed to determine whether H1R activation modulates Rho GTPases, well-known effectors of Gq/G11-coupled receptors, and whether such modulation influences cell proliferation. Experiments were carried out in CHO cells stably expressing H1R (CHO-H1R). By using pull-down assays, we found that both histamine and a selective H1R agonist activated Rac and RhoA in a time- and dose-dependent manner without significant changes in the activation of Cdc42. Histamine response was abolished by the H1R antagonist mepyramine, RGS2 and the PLC inhibitor U73122, suggesting that Rac and RhoA activation is mediated by H1R via Gq coupling to PLC stimulation. Histamine caused a marked activation of serum response factor activity via the H1R, as determined with a serum-responsive element (SRE) luciferase reporter, and this response was inhibited by RhoA inactivation with C3 toxin. Histamine also caused a significant activation of JNK which was inhibited by expression of the Rac-GAP β2-chimaerin. On the other hand, H1R-induced ERK1/2 activation was inhibited by U73122 but not affected by C3 or β2-chimaerin, suggesting that ERK1/2 activation was dependent on PLC and independent of RhoA or Rac. [3H]-Thymidine incorporation assays showed that both histamine and the H1R agonist inhibited cell proliferation in a dose-dependent manner and that the effect was independent of RhoA but partially dependent on JNK and Rac. Our results reveal that functional coupling of the H1R to Gq–PLC leads to the activation of RhoA and Rac small GTPases and suggest distinct roles for Rho GTPases in the control of cell proliferation by histamine.

Keywords: Histamine, Histamine H1 receptor, Rho GTPases, Cell proliferation, Cell signaling

Introduction

Histamine is an intercellular signal molecule that exerts its effects through four different G protein-coupled receptors (GPCR) subtypes: H1, H2, H3, and H4 receptors [1]. These receptors are structurally characterized by seven transmembrane α-helices, and functionally by their ability to transmit signals to effector molecules via G proteins [2]. Histamine is involved in a wide spectrum of biological effects. Its role in allergy and inflammatory reactions has been extensively studied [1,3]. Histamine also functions as a neurotransmitter in the central nervous system, stimulates gastric acid secretion [1,3], and plays a role in the maintenance of blood–brain barrier [4]. In the last decade, a great body of evidence supports its participation in the immune response and in cell proliferation [3,5]. The role of histamine in cell proliferation is rather controversial given that histamine has been shown not only to inhibit but also to promote cell growth depending on the cell type. For example, in confluent airway smooth muscle cells [6], human articular chondrocytes [7], and mammary carcinoma cells [8], histamine markedly increases cell proliferation, while it reduces growth in human HuH-6 hepatocellular carcinoma cells. Although this cell line expresses H1R and H2R only the selective H1R antagonist terfenadine abolishes histamine response [9]. In melanoma cells, growth arrest induced by IL-6 is partially mediated by a characteristic pattern of histamine receptor expression and an elevation of locally produced histamine [10].

In most mammalian cells, including smooth muscle, endothelial cells, and neurons, histamine binding to the H1R triggers Gαq protein activation with the subsequent stimulation of phospholipase C (PLC), and this leads to the generation of inositol phosphates (IP3) and diacylglycerol (DAG) [11]. Increasing evidence suggests that Gq-coupled receptors can also activate small GTP-binding proteins of the Rho family [12,13]. Vogt et al. [14] demonstrated that in pertussis toxin-treated Gα12/Gα13-deficient mouse embryonic fibroblasts, in which coupling of receptors is restricted to Gq/G11, endogenous receptor stimulation results in a rapid activation of RhoA. Moreover, microinjection of activated Gαq into fibroblasts promotes actin stress filaments formation via Rho [15].

RhoGTPases belong to the Ras superfamily of small GTPases that cycle between inactive GDP-bound and active GTP-bound conformations. These proteins are normally kept in an inactive, GDP-bound conformation in the cytoplasm through binding RhoGDI before stimulation [16,17]. GTP loading onto Rho GTPases upon membrane receptor activation is mediated by guanine nucleotide-exchange factors (GEFs). The binding of GTP eventually leads to a conformational change that allows for the activation of downstream effectors and the subsequent activation of a number of signalling pathways that modulate cell motility, cell cycle progression, and gene transcription. On the other hand, inactivation of Rho GTPases is mediated by GTPase-activating proteins (GAPs) which accelerate GTP hydrolysis to GDP. RhoA, Cdc42, and Rac1 are the most extensively studied Rho GTPases [18].

In the present study, we sought to establish the role of H1R in the modulation of RhoA, Rac1, and Cdc42. While little is known regarding the coupling of histamine receptors to Rho small GTPases, studies have found that histamine can cause the activation of Rho in rabbit aortic smooth muscle cells and in HEK-293 cells ectopically expressing the H1R and the p63RhoGEF [19,20]. However, the role of histamine as a vascular permeability enhancer through a Rho-dependent pathway is still controversial [21,22].

We found that in CHO cells stably transfected with the human H1R, histamine time and dose-dependently activates Rac1 and RhoA, but not Cdc42, through the Gq–PLC pathway. H1R stimulation also led to the expression of a SRE-luciferase reporter via RhoA, as well as to JNK activation via Rac. Furthermore, histamine dose-dependently inhibited cell proliferation in a JNK-, PLC-, and Rac-dependent manner but independent of RhoA.

Materials and methods

Reagents and antibodies

[3H]-Mepyramine, [3H]-thymidine, and myo-[3H]-inositol were purchased from PerkinElmer Life Sciences (Boston, MA). Histamine dihydrochloride, 2,3-trifluormetilfenilhistamine (H1 agonist), ATP, myo-inositol, bovine serum albumin, G418, Dulbecco’s modified Eagle’s medium (DMEM), and the inhibitors U73122 and U73343 were purchased from Sigma Chemical Company (St. Louis, MO). Mepyramine maleate and the inhibitors PD98059, U0126, and SP600125 were from Tocris Cookson Inc. (Ballwin, MO). The Rac inhibitor NSC-23766 was from Calbiochem (San Diego, CA). Dowex AG-1X8 formate form resin was obtained from Bio-Rad (Hercules, CA), whereas fetal calf serum was from Natocor (Argentina).

Anti-phosho-Akt1/2/3, anti-phospho-Erk1/2, anti-phospho-p38, anti-phospho-JNK, anti-Erk1, anti-Akt1, anti-p38, anti-JNK1, anti-RhoA, and anti-Cdc42 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-Rac antibody was obtained from Upstate Biotechnology Inc. (Lake Placid, NY).

All other chemicals used were of analytical grade and obtained from standard sources.

Cell culture

CHO/dhFr (−) cells (CHO) and clones generated by stable transfection were cultured at 37 °C in a humidified atmosphere with 5% CO2 in DMEM supplemented with 0.1 mM hypoxanthine, 0.016 mM thymidine, 10% fetal calf serum, and 50 μg/ml gentamicin.

Stable transfection

CHO cells were harvested from cultures in exponential growth phase, washed once in phosphate-buffered saline (PBS), and resuspended at 106cells/ml in fresh DMEM on ice. pCEFL-humanH1R (10 μg) linearized with SalI was added to cell suspension (400 μl) and kept on ice for 5 min. Cells and DNA were then subjected to a pulse of 150 V at a capacitance of 250 μF using a Gene Pulser (Bio-Rad, Hercules, CA). Cells were returned to ice for 5 min and further incubated in a nonselective medium overnight. Cells were then plated on a 96-well culture plate in DMEM medium containing 0.8 mg/ml G-418. After 2–3 weeks, [3H]-mepyramine binding studies and phosphoinositide hydrolysis assays were performed in amplified surviving clones to determine if they expressed functional H1R. To avoid promiscuous G-protein coupling, a clone (CHO-H1R) expressing less than 50,000 sites/cells was used for the experiments.

Transient transfection

CHO or CHO-H1R cells were grown to 80–90% confluence. cDNA constructs pCDNA3-RGS2, pCEFL-HA-RacN17, pCDNA3-C3 toxin, pCDNA3-GFP-β2-chimaerin, or pSRE.L-luciferase reporter plasmid and pRL-TK control reporter vector were transfected into cells using LipofectAMINE 2000. The transfection protocol was optimized as recommended by the manufacturer (Invitrogen, Argentina). Assays were performed 48 h after transfection. The expression of the pCDNA3-GFP-β2-chimaerin construct was confirmed by fluorescence microscopy and that of β2-chimaerin and C3 functionality by a RacGTP or RhoGTP pull-down assay. pcDNA3-C3 toxin and pCEFL-HA-RacN17 plasmids were a generous gift from Dr. O. Coso (Universidad de Buenos Aires, Argentina).

Radioligand binding assay

Triplicate binding assays were performed for CHO/dhFr (−) and CHO/dhFr (−) H1R clones. For saturation studies, cells were incubated for 40 min at 4 °C in 50 mM Tris–HCl, pH 7.4 with increasing concentrations of [3H]-mepyramine in the presence or in the absence of 1 μM mepyramine. Specific binding was calculated by subtraction of nonspecific binding from total binding. Incubation was stopped by adding 3 ml of ice-cold 50 mM Tris–HCl, pH 7.4, followed by washes with ice-cold buffer. Experiments were carried out on intact cells at 4 °C to avoid ligand internalization. Kinetic studies showed that the equilibrium was reached after 30 min and persisted for 4 h (data not shown). Kd and Bmax values were calculated using the equation for one binding sites.

[3H]-Inositol phosphate production

Total inositol phosphate production was assessed as previously described [23]. Briefly, cells were seeded in 24-well cluster dishes and cultured for 24 h (70–80% confluence) in DMEM. Cells were then washed, and the medium was replaced by DMEM with calf serum plus the addition of myo-[3H]-inositol (5 μCi/ml) and cultured for 6 h. Thereafter, the medium was aspirated and replaced by DMEM without calf serum containing 10 mM LiCl and incubated for 20 min. Cells were then stimulated for 20 min with histamine, the H1R agonist, or ATP in a final volume of 300 μl, either in the presence or in the absence of the specific H1R antagonist mepyramine. The reaction was stopped by the addition of 2 ml of cold chloroform–methanol (1:2 vol./vol., freshly prepared), and phases were separated by adding 1 ml of water and 620 μl of chloroform. The mixture was then centrifuged at 1500 × g for 10 min, and the total water-soluble inositol phosphate fraction was purified by anion exchange chromatography. Radioactivity in the eluted fractions was measured using a Wallac 1410 liquid scintillation counter. Results were expressed as percentage relative to basal levels.

Pull-down assays

The cellular levels of GTP loaded Rac, Cdc42, and RhoA were determined using a GST fusion protein containing the p21-binding domain (PBD) of p21-activated kinase (Pak) for Rac and Cdc42 [24] and the Rho binding domain of rhotekin (RBD) for RhoA [25]. In brief, subconfluent monolayers of CHO or CHO-H1R cells were starved 16 h. Following the corresponding treatment, they were lysed, and centrifuged for 10 min at 12,000 × g. A supernatant aliquot was separated for total Rac, Cdc42, and RhoA measurement, and the remaining supernatant was incubated for 1 h at 4 °C with PBD or RBD bound to glutathione–Sepharose beads. Beads were washed 3 times, and bound proteins were eluted with a sample buffer and separated by SDS–PAGE. Rac, Cdc42, and RhoA were detected by Western blot using specific antibodies.

Western blot

Cells were lysed in 50 mM Tris–HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 100 mM 2-mercaptoethanol, 10% glycerol, and 0.05% bromophenol blue and sonicated to shear DNA. Total cell lysates were resolved by SDS–PAGE, immunoblotted with the indicated primary antibodies, followed by horseradish peroxidase-conjugated anti-rabbit or anti-mouse (Santa Cruz Biotechnology, Santa Cruz, CA), and developed by enhanced chemiluminescence (ECL) following the manufacturer’s instructions (Amersham Life Science, England). Band intensity was quantified by densitometry using Scion Image (Scion Corporation, Frederick, MD) software.

SRF activation assay

CHO or CHO-H1R cells seeded on 12-well plates were cotransfected with the pSRE.L-luciferase reporter plasmid and the thymidine kinase promoter-Renilla luciferase reporter plasmid (pRL-TK) as a control reporter vector. In some experiments, cells were also cotransfected with C3, β2-chimaerin, or empty vector. After 24 h, cells were deprived from serum for 24 h, and then stimulated with histamine or pretreated with mepyramine for 30 min before the addition of histamine. Luciferase activity was measured 16 h later; cells were washed with PBS and lysed with Passive lysis buffer (Promega Biosciences, Inc., San Luis Obispo, CA, USA). Luciferase activity was determined using the Dual-Luciferase reporter assay system, according to the manufacturer’s instructions (Promega Biosciences Inc. San Luis Obispo, CA, USA). Experimental reporter activity was normalized to control vector activity.

Proliferation assay

Cells were seeded in quadruplicates at 1 × 104 cells per well in 96-well plates and cultured at 37 °C and 5% CO2 in complete DMEM in a 75-μl final volume. After 24 h, 0.2 μCi [3H]-thymidine per well was added in a volume of 25 μl of DMEM, in the presence of the H1R agonist or histamine, and cells were incubated overnight at 37 °C. Pharmacological inhibitors of PLC, Rac, MEK, and JNK were added 30 min before histamine stimulation. Samples were harvested and analyzed in a Wallac 1410 liquid scintillation counter.

Statistical analysis

Binding data and sigmoidal dose–response fittings were performed with GraphPad Prism 3.00 for Windows, GraphPad Software (San Diego, CA). Statistical analysis was performed by wed by the Bonferroni test. A p < 0.05 was considered statistically significant.

Results

Characterization of the CHO-H1R stable cell line

For the generation of CHO cells stably expressing human H1R (CHO-H1R), cells were transfected with pCEFL-humanH1R. Expression of H1R was confirmed using saturation analysis and Scatchard plotting of intact transfected cells which showed specific binding site for [3H]-mepyramine with a Kd within the range value previously reported (2.6 ± 0.2 nM) (n = 3) (Fig. 1A). The number of sites estimated from the Bmax was 35,700 ± 900 sites/cell (n = 3). Binding of [3H]-mepyramine in CHO-naive cells was not detectable. To assess the functionality of transfected H1R in CHO-H1R cells, InsP production was measured. Fig. 1B shows that stimulation with histamine (100 μM) or 2,3-trifluormetilfenilhistamine (H1R agonist, 10 μM) elicited a 3- to 4-fold increase in InsP production, and this effect was prevented by the H1R antagonist mepyramine. As expected, CHO-naive cells failed to increase InsP in response to histamine or the H1R agonist. As a positive control, we used ATP, which promoted a strong elevation of InsP levels.

Fig. 1 –

Fig. 1 –

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Characterization of the CHO-H1R clone.

(A) [3H]-Mepyramine binding assay. Saturation assays for [3H]-mepyramine in CHO-H1R cells. Data were calculated as the mean±SD of assay triplicates. Similar results were obtained in at least three independent experiments. Inset: Scatchard representation of the saturation binding experiment. Kd and Bmax values were calculated using the equation for one binding site. Values are the mean±SEM (n = 3). Kd = 2.6 ± 0.2 nM, Bmax = 35,700 ± 900 sites/cell. (B) Histamine stimulates InsP production through H1 receptor. CHO or CHO-H1R cells were exposed to 100 μM histamine (H), 10 μM H1R agonist (H1), 10 μM ATP, 10 μM mepyramine (Mep), alone or in combination, and InsP production was measured as described under Materials and methods. Data were calculated as the mean±SEM (n = 3). **p < 0.01 with respect to basal levels.

Histamine induces Rac and RhoA activation in CHO-H1R cells

To determine whether histamine promotes the activation of RhoA, Rac, and Cdc42 in CHO-H1R, we used pull-down assays based on the specific GTP-dependent association of Rho with the Rho-binding domain of Rhotekin, and Rac1/Cdc42 with the p21-binding domain of Pak. A time course analysis of RhoA, Rac, and Cdc42 in the presence of histamine shows that histamine induces a rapid and potent activation of RhoA and Rac but not of Cdc42 in H1R-transfected cells (Figs. 2A and B). Activation of RhoA and Rac1 was detectable as early as 30 sec, and the effect was attenuated at 3 min, reaching basal levels at 10 min. Activation of RhoA and Rac by histamine was dose-dependent as shown in Figs. 2C and D, reaching the maximum activation at 10 μM. CHO-naive cells exhibited no activation of the small G proteins in the concentration range of histamine used in the experiments. The histamine response was mimicked by the selective H1R agonist, 2,3-trifluormetilfenilhistamine, and the effects abolished by the antagonist mepyramine. Transfection with RGS2, a Gq alpha-mediated signaling inhibitor, impaired RhoA and Rac activation (Fig. 2F). These findings suggest that the activation of RhoA and Rac by histamine is mediated by Gq-coupled H1R.

Fig. 2 –

Fig. 2 –

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Histamine induces Rac and RhoA activation in CHO-H1R cells. (A) Time-dependent activation of Rac, RhoA but not Cdc42 by histamine. Cells were stimulated with 100 μM histamine at different periods of time and Rac/RhoA/Cdc42-GTP levels were determined using a protein specific “pull-down” assay. (B) Densitometric analysis of time-dependent activation of small GTPases, normalized to their corresponding total levels, obtained with the Scion Image program. Data are presented as the means±SEM (n = 3). (C) Concentration-dependent activation of Rac and RhoA by histamine. Rac-GTP or RhoA-GTP levels were assessed following 1 min of cell stimulation with histamine. (D) Densitometric analysis of concentration-dependent activation of Rac and RhoA in CHO-H1R cells. Data are presented as the means±SEM (n = 3). (E) Specificity of Rac and RhoA activation by H1 receptor. Cells were stimulated for 1 min with different concentrations of a H1R agonist, 100 μM histamine, or 10 μM mepyramine and Rac-GTP or RhoA-GTP levels were determined. Similar results were observed in three independent experiments. (F) Gq-dependent activation of Rac and RhoA by H1 receptor stimulation. CHO-H1R cells transfected with mock or RGS2 were stimulated for 1 min with 100 μM histamine and Rac-GTP or RhoA-GTP levels were determined. Similar results were observed in three independent experiments.

Next, we assessed whether H1R-mediated RhoA and Rac activation is dependent on PLC stimulation. Histamine-evoked RhoA and Rac activation in CHO-H1R cells was inhibited by the PLC inhibitor U73122 but not by the inactive analogue U73343, suggesting that PLC mediates the activation of small G proteins in response to H1R receptor activation (Fig. 3).

Fig. 3 –

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Histamine-induced Rac and RhoA activation is PLC-dependent. (A) CHO-H1R cells were treated either with 10 μM U73122 or 10 μM U73343 for 20 min and then stimulated with 100 μM histamine for 1 min, and Rac-GTP or RhoA-GTP levels were determined. (B) Densitometric analysis obtained with the Scion Image program. Data are presented as the means±SEM (n = 3). **p < 0.01 with respect to histamine.

Histamine induces SRE reporter activation via RhoA in CHO-H1R cells

Since the activation of serum response factor (SRF) by extracellular factors generally requires Rho function, the ability of histamine to induce SRE luciferase reporter activity was tested. We observed that in CHO-H1R cells, histamine dose-dependently increased luciferase activity. The EC50 for the histamine effect was 0.41 ± 0.03 μM (mean ±SEM, n =3). Maximum activation in luciferase activity (∼ 4-fold) was observed at ∼ 10 μM (Fig. 4A). The response was abolished by mepyramine (Fig. 4B). To determine the involvement of RhoA in SRE luciferase activation, we expressed botulinum C3 exoenzyme, which functions as an ADP-ribosyltransferase that inactivates Rho protein. As shown in Fig. 4C, C3 effectively suppressed histamine-induced SRE reporter activation. However, inhibition of Rac by expression of the Rac-GAP β2-chimerin [24,26] did not have any appreciable effect on the activation of SRE luciferase by histamine. Therefore, SRE activation in response to H1R activation is Rho-dependent.

Fig. 4 –

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Effect of histamine on SRE luciferase reporter gene activity in CHO-H1R cells. (A) Concentration-dependent activation of the SRE luciferase reporter gene activity. Cells were stimulated for 16 h with different concentration of histamine before being lysed, and SRE luciferase expression was determined. Data were calculated as the mean±SEM (n = 3). (B) SRE luciferase reporter gene activity is activated by H1 receptor. Cells were pretreated for 30 min with 10 μM mepyramine and stimulated for 16 h with 100 μM histamine before being lysed, and SRE luciferase expression was determined. Data were calculated as the mean±SEM (n = 3). **p < 0.01 with respect to histamine treatment. (C) C3 toxin inhibited SRE luciferase reporter gene activity. CHO-H1R cells were transfected with mock, C3 toxin (C3), or β2-chimaerin (β-chim) and stimulated for 16 h with 100 μM histamine (H) or unstimulated (basal) before being lysed and SRE luciferase expression was determined. Data were calculated as the mean±SEM (n = 3). **p < 0.01 with respect to histamine treatment in mock cells.

Histamine induces JNK activation via Rac in CHO-H1R cells

Studies in human aortic endothelial and vascular smooth muscle cells show that H1R stimulation leads to the activation of JNK [27,28]. As shown in Figs. 5A and B, 100 μM histamine caused a marked activation of JNK in CHO-H1R cells that was detectable within 10 min and persisted for at least 2 h. To investigate the role of Rac in histamine-induced JNK activation, we expressed the Rac-GAP β2-chimaerin in CHO-H1R cells. As shown in Figs. 5C and D, expression of β2-chimerin abolished histamine-induced JNK phosphorylation. On the other hand, the effect is not affected by C3, arguing for the involvement of Rac but not RhoA in histamine-induced JNK activation.

Fig. 5 –

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Effect of histamine on JNK activation in CHO-H1R cells. (A) Time-dependent activation of JNK by histamine. Cells were stimulated with 100 μM histamine and harvested at the indicated times. Equal amounts of protein were subjected to SDS–PAGE and analyzed by Western blot with anti p-JNK and JNK antibodies. (B) Densitometric analysis of time-dependent activation of JNK, normalized to the corresponding total level, obtained by the Scion Image program. Data are presented as the means±SEM (n = 3). (C) β2-chimaerin inhibited JNK activation. CHO-H1R cells transfected with mock, β2-chimaerin (β-chim), or C3 toxin (C3) were stimulated for 30 min with 100 μM histamine. Equal amounts of protein were subjected to SDS–PAGE and analyzed by Western blot with anti p-JNK and JNK antibodies. (D) Densitometric analysis of JNK activation in histamine-treated cells, normalized to the corresponding total level, obtained by the Scion Image program. One hundred percent (100%) corresponds to p-JNK levels in histamine-treated mock cells. Data are presented as the means±SEM (n = 3). **p <0.01 with respect to histamine-stimulated mock levels.

H1R activation inhibits CHO-H1R cell proliferation via PLC, Rac, and JNK

Next, the effect of histamine on CHO-H1R cell proliferation was assessed. Histamine or the selective H1R agonist, 2,3-trifluormetilfenilhistamine, reduced [3H]-thymidine incorporation in CHO-H1R cells in a concentration-dependent manner. The EC50 were 1.24 ± 0.31 and 23.1 ± 3.6 μM (mean ±SEM, n =3), respectively. The antiproliferative effect of histamine was prevented by the H1R antagonist mepyramine. Furthermore, [3H]-thymidine incorporation in parental CHO cells was not affected by histamine (Fig. 6A).

Fig. 6 –

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Effect of histamine and H1 agonist on CHO-H1R cell proliferation. (A) Histamine and H1 agonist inhibit CHO-H1R cell proliferation. CHO and CHO-H1R cells were stimulated with different concentration of histamine, H1 agonist or pretreated for 20 min with 10 μM mepyramine and then stimulated with 30 μM histamine in the presence of [3H]-thymidine and samples treated as described under Materials and methods. Data are the mean±SEM (n = 3) and expressed as a percent of basal, in the absence of ligand. **p < 0.01 with respect to basal levels. (B) Reversion of histamine-induced inhibition on cell proliferation by β2-chimaerin. CHO-H1R cells were transfected with mock, C3 toxin (C3), or β2-chimaerin (β-chim) and after 24 h, stimulated with different concentration of histamine or H1 agonist for 16 h in the presence of [3H]-thymidine. (C) Inhibition of cell proliferation by histamine is abolished by a pharmacological inhibition of Rac and a DN-Rac. Cells preincubated for 30 min with NSC-23766 or transfected with mock or HA-RacN17 were exposed to different concentrations of histamine for 16 h in the presence of [3H]-thymidine. Data are the mean±SEM (n = 3) and expressed as a percent of basal in the absence of histamine. **p < 0.01 with respect to similar treatment in control cells (black bars). (D) Inhibition of proliferation is PLC- and JNK-dependent. CHO-H1R cells were pretreated for 30 min with the different concentration of inhibitors and stimulated with 30 μM histamine for 16 h in the presence of [3H]-thymidine and samples treated as described under Materials and methods. Data are the mean±SEM (n = 3). One hundred percent (100%) corresponds to thymidine uptake after inhibitor treatment in the absence of histamine. **p < 0.01 with respect to histamine.

As Rac and RhoA were activated by H1R stimulation, we sought to establish their potential roles in histamine-induced growth inhibition. CHO-H1R cells transfected with either C3 toxin or β2-chimaerin were treated with histamine or 2,3-trifluormetilfenilhistamine. As shown in Fig. 6B, C3 toxin failed to affect [3H]-thymidine incorporation, whereas β2-chimaerin abolished the histamine and H1 agonist response. To further characterize this response, Rac pharmacological inhibitors and dominant negative Rac (DN-Rac) approach were used. Both, NSC-23766 (Rac inhibitor) and RacN17 abolished the inhibition of cell proliferation induced by histamine similarly to β2-chimaerin (Fig. 6C). In addition, histamine-induced growth inhibition in CHO-H1R cells was prevented by U7312 (PLC inhibitor) but not by its inactive analogue U73343 (Fig. 6D).

To assess the contribution of JNK in the antiproliferative effect of histamine, we used the JNK inhibitor SP600125. Interestingly, SP600125 inhibited histamine response in a dose-dependent manner (Fig. 6D), suggesting a key role for JNK in histamine-mediated inhibition of cell growth.

Interestingly, histamine caused a marked activation of ERK1/2, as revealed by Western blot using a phospho-specific antibody (Fig. 7A). ERK activation was detected within 3 min after histamine stimulation, and it was sustained for at least 30 min. On the other hand, no activation of p38 or Akt could be detected. A densitometric analysis of these signalling readouts normalized to the expression of their corresponding total levels is shown in Fig. 7B. Moreover, the MEK inhibitors PD98059 and U0126 were unable to rescue the inhibitory effect of histamine in CHO-H1R cells (Fig. 7C). Furthermore, ERK1/2 activation by histamine was inhibited by U73122 (PLC inhibitor) but not by β2-chimaerin or C3 toxin (Fig. 7D).

Fig. 7 –

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Effect of histamine on kinase pathway activation in CHO-H1R cells. (A) Time-dependent activation of ERK, Akt, and p38 by histamine. Cells were stimulated with 100 μM histamine and harvested at the indicated times. Equal amounts of protein were subjected to SDS–PAGE and analyzed by Western blot with anti p-ERK, ERK, p-Akt, Akt, p-p38, and p38 antibodies. Data are representative of at least three independent experiments. (B) Densitometric analysis of ERK, Akt, and p38 time-dependent activation, normalized to the corresponding total levels, obtained with the Scion Image program. Data are presented as the means±SEM (n = 3). (C) CHO-H1R cells were pretreated for 30 min with different concentrations of ERK inhibitors and stimulated with 30 μM histamine for 16 h in the presence of [3H]-thymidine and were samples treated as described under Materials and methods. Data are the mean±SEM (n = 3). One hundred percent (100%) corresponds to thymidine uptake after inhibitor treatment in the absence of histamine. (D) ERK activation is PLC-dependent and Rac- and RhoA-independent. CHO-H1R cells pretreated for 20 min with 10 μM U73122 or transfected with mock, C3 toxin (C3), or β2-chimaerin (β2-chim) were stimulated for 30 min with 100 μM histamine. Equal amounts of protein were subjected to SDS–PAGE and analyzed by Western blot with anti p-ERK and ERK antibodies. Data are representative of at least three independent experiments.

Discussion

In the present study, we established the pattern of Rho-GTPases activation mediated by the H1R and its role in the regulation of cell proliferation. Using CHO cells stably transfected with the human H1R, we showed that histamine couples to the Gq–PLC pathway induced RhoA and Rac activation and inhibits cell growth mediated by Rac and JNK via H1R.

Histamine H1R activation stimulates PLC-β isoform via pertussis toxin-insensitive Gq/G11-proteins in a variety of cell types (for review, see Hill et al. [11]). Furthermore, Gαq as well as Gq/G11-coupled receptors also activate Rac1 and RhoA [12,15,29]. In fact, Rho GTPases activation by H1R stimulation has been reported for RhoA, but not for the other members of this family [19]. Such activation has been monitored by Rho-dependent gene transcription (SRE) measurement. However, SRE activation may be also induced by other Rho GTPases like Rac and Cdc42 [30], therefore limiting the specificity of the assay in those studies. In the present study, we demonstrated that histamine and H1R agonist induced Rac and RhoA activation by pull-down assay of Rac/RhoA-GTP, which was further confirmed by read-out experiments. RhoA and Rac but not Cdc42 were strongly stimulated by histamine in a time- and concentration-dependent manner. In addition, activation of small GTPases was linked to SRE and JNK. The specificity of the signaling was assessed by inhibiting RhoA and Rac with C3 toxin and the Rac-GAP β2-chimaerin, respectively.

Evidence shows that Gq/G11 subfamily activation induces PLC/PKC-independent activation of Rho [31]. A recent study suggests that p63RhoGEF, a member of the Dbl GEFs, is one of the specific GEFs involved in the response [19]. Gq-coupled GPCRs were directly linked to RhoA- and RhoA-dependent processes, apparently in competition with the canonical PLCβ/PKC pathway. On the other hand, it was reported that thrombin increases endothelial permeability by inducing RhoA activation through PAR-1. Thrombin binding to PAR-1 can stimulate both Gα12/13 and Gαq. Rho activation in endothelial cells requires the cooperative interaction of both G12/13 and Gαq-PLC-TRPC6–PKCα pathways that converge at p115RhoGEF [32]. These findings are consistent with the notion that signals generated by Gαq/PLC may not be sufficient to induce Rho activation but are required to enhance responses elicited through Rho signaling pathways. In the present study, we provide evidence that in CHO-H1R cells the H1R activates RhoA in a PLC-dependent manner. It is likely that activation of RhoA in these cells may require the cooperation of RhoGEF and PLC downstream effectors. However, it is also possible that the RhoGEF involved in Rho activation might be a PLC downstream effector. Further studies are needed to elucidate this issue.

In CHO-H1R cells, Rac was also activated by histamine through PLC stimulation. In other models, various Gq/G11 GPCRs, including bradykinin receptors, M1 and M3 muscarinic receptors, have been shown to activate Rac [33]. In CHO cells transfected with the M3 muscarinic receptor, RhoA and Rac1 activity increases following receptor activation, but only Rac1 participates in the inhibition of cell growth. Furthermore, Rac1 levels correlate with the ability of muscarinic ligands to induce apoptosis. Here, we showed that only Rac mediates histamine-induced inhibition of cell proliferation although H1R stimulation activates both RhoA and Rac. Consistent with our results, other authors reported that RhoA and Rac1 activity increases in tumor necrosis factor-α-treated U937 cells, but only Rac1 participates in the inhibition of cell growth and the induction of apoptosis [34]. In CHO-H1R cells, the inhibition of cell proliferation induced by histamine and its relationship with apoptosis remains to be determined.

It has been shown that histamine may induce or inhibit cell proliferation. For example, in human astrocytoma U373 MG cells, H1R stimulates mitogenesis through PKC and MAPK activation [5]. However, in melanoma cells, histamine has a dual role in the cell proliferation since its effect depends on its local concentration as well as on the type and density of histamine receptors in the cell [10]. Histamine also affects cell growth in different human hepatocellular carcinoma cells; it reduces cell viability and proliferation in HuH-6 cells, but in HA22T/VGH cells, it induces a weak but significant increase in cell growth. H1R and H2R are expressed in both cell lines, and the histamine response is reverted by a selective H1R antagonist in HuH-6 cells and by a H2R antagonist in HA22T/VGH cells. As CHO cells do not express histamine receptors, the effect of histamine through H1R in our study is strictly dependent on this receptor without the interference of the other receptor subtypes in our study. To our knowledge, this is the first report to show in CHO-H1R cells the antiproliferative response of histamine as previously reported in melanoma and hepatocarcinoma cells [9,10].

The signalling pathways activated by histamine largely depend on the cell context. MAPKs are a group of serine/threonine protein kinases comprising three main subfamilies: the p42/p44 extracellular regulated kinases (ERKs), also known as p42/p44 MAPKs; the c-Jun N-terminal kinases (JNKs) which are also known as stress-activated protein kinases (SAPKs) and the p38 MAPKs [35,36]. Initially, p42/p44 MAPK pathway was associated with tyrosine kinase receptor activation, whereas p38 MAPK and JNK activation was triggered by stimuli such as UV irradiation, osmotic stress, and inflammatory cytokines [37,38]. However, many studies in the past years provided clear evidence that a wide range of GPCRs, including those coupled to Gq, are also involved in the regulation MAPKs (for reviews, see [39,40]). Histamine activates p42/p44 MAPK and p38 MAPK signalling pathways without affecting JNK activation in DDT1MF-2 smooth muscle cells, but it has no effect on cell proliferation [41]. In the present study, we show that histamine activated JNK and p42/p44 MAPK, but only JNK activation was involved in histamine inhibition of cell growth.

The activation of p42/p44 MAPK in CHO-H1R cells has been previously reported [42]; however, neither JNK activation nor cell proliferation inhibition induced by H1R activation was assessed. In the present study, we chose for the experiment a CHO-H1R clone that expressed less than 50,000 sites per cell. Nevertheless, it would be interesting to assess the effects of histamine on clones expressing different densities of H1R since the stoichiometry of the components involved in the signaling might condition the response. The activation of p42/p44 MAPK in our system resulted independent of RhoA and Rac activation, since the specific inhibitors C3 toxin and β2-chimaerin failed to inhibit the response but dependent on PLC. Megson et al. [42] suggested that the H1R receptor activates the p42/p44 MAPK pathway via PKCα following phospholipase Cβ activation. Our present findings show that histamine H1R coupling to Gq–PLC stimulation activates both RhoA and Rac small G proteins in CHO-H1R cells. Moreover, PLC-dependent Rac activation was associated with the inhibition of cell proliferation involving the JNK pathway, supporting that the activation of Rho GTPases is likely a novel step in the H1R signaling pathway leading to the inhibition of cell proliferation.

A challenge for future research would be to establish a potential link between this signaling stimulation with physiopathological conditions that depend on the hyperactivation of the H1R.

Acknowledgments

We are sincerely grateful to Dr. L. Bianciotti for critical reading of the manuscript. This study was supported by grants from the Universidad de Buenos Aires (grant UBACyT B0-50), Consejo Nacional de Investigaciones Científicas y Tecnológica (PIP 6110), ANPCYT (PICT 38318) and National Institutes of Health (grant R01-CA74197).

Abbreviations:

H1R

histamine 1 receptor

GPCR

G protein-coupled receptor

PLC

phospholipase C

CHO-H1R

CHO cells stably expressing H1R

SRE

serum-responsive element

IP3

inositol phosphates

DAG

diacylglycerol

GEFs

nucleotide-exchange factors

GAPs

GTPase-activating proteins

DMEM

Dulbecco’s modified Eagle’s medium

CHO

CHO/dhFr cells

PBS

phosphate-buffered saline

PBD

p21-binding domain

RBD

Rho binding domain of rhotekin

SDS

sodium dodecyl sulfate

ECL

enhanced chemiluminescence

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