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Open Access

Peer-reviewed

Research Article

Distinct Signalling Pathways of Murine Histamine H1- and H4-Receptors Expressed at Comparable Levels in HEK293 Cells

Abstract

Histamine (HA) is recognized by its target cells via four G-protein-coupled receptors, referred to as histamine H1-receptor (H1R), H2R, H3R, and H4R. Both H1R and H4R exert pro-inflammatory functions. However, their signal transduction pathways have never been analyzed in a directly comparable manner side by side. Moreover, the analysis of pharmacological properties of the murine orthologs, representing the main targets of pre-clinical research, is very important. Therefore, we engineered recombinant HEK293 cells expressing either mouse (m)H1R or mH4R at similar levels and analyzed HA-induced signalling in these cells. HA induced intracellular calcium mobilization via both mH1R and mH4R, with the mH1R being much more effective. Whereas cAMP accumulation was potentiated via the mH1R, it was reduced via the mH4R. The regulation of both second messengers via the H4R, but not the H1R, was sensitive to pertussis toxin (PTX). The mitogen-activated protein kinases (MAPKs) ERK 1/2 were massively activated downstream of both receptors and demonstrated a functional involvement in HA-induced EGR-1 gene expression. The p38 MAPK was moderately activated via both receptors as well, but was functionally involved in HA-induced EGR-1 gene expression only in H4R-expressing cells. Surprisingly, in this system p38 MAPK activity reduced the HA-induced gene expression. In summary, using this system which allows a direct comparison of mH1R- and mH4R-induced signalling, qualitative and quantitative differences on the levels of second messenger generation and also in terms of p38 MAPK function became evident.

Citation: Beermann S, Vauth M, Hein R, Seifert R, Neumann D (2014) Distinct Signalling Pathways of Murine Histamine H1- and H4-Receptors Expressed at Comparable Levels in HEK293 Cells. PLoS ONE 9(9): e107481. https://doi.org/10.1371/journal.pone.0107481

Editor: Bruno Lourenco Diaz, Universidade Federal do Rio de Janeiro, Brazil

Received: May 8, 2014; Accepted: August 11, 2014; Published: September 22, 2014

Copyright: © 2014 Beermann et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.

Funding: This work was supported by a grant (NE 647/8-1) from the Deutsche Forschungsgemeinschaft (www.dfg.de) to DN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

HA, a biogenic amine, is an important mediator of many physiological and pathological processes such as gastric acid secretion, neurotransmission, cell differentiation, immunomodulation, allergic reactions, peptic ulcer, and tumor progression [1]. Two groups of cells are able to produce HA. The first group comprises mast cells, basophils, histaminergic neurones and ECL-cells of the gastric wall, which are able to store HA in intracellular granules. Specific stimuli lead to degranulation of these cells and to a massive release of HA [2], [3]. The second group of HA-producing cells consists of neutrophils, lymphocytes, macrophages and others. These cells secrete HA immediately following its production, which is regulated by specific stimuli via expression of the histamine-generating enzyme L-histidine decarboxylase [4], [5].

Cellular effects of HA are mediated via four G-protein coupled receptors (GPCRs), H1R - H4R, which are expressed on varying cell types. HxRs differ from each other in their preferred coupling G-protein and, thus, activate different signal transduction pathways. Accordingly, HXRs carry out different functions in the body [6]–[9].

The H1R is expressed ubiquitously, including several immune cells such as B- and T-cells, monocytes, macrophages and dendritic cells [10], [11]. Upon ligand binding the H1R couples to Gq-proteins and activates phospholipase C (PLC) [12], followed by the generation of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) of which IP3 induces an increase in intracellular Ca2+ concentrations ([Ca2+]i) [10], [13]–[15]. Moreover, the activated H1R potentiates forskolin-induced production of the second messenger cAMP [16]. This increase is either mediated via activation of specific adenylylcyclase (AC) isoforms by Ca2+-activated calmodulin [17] or via a Ca2+-independent direct activation of ACs [18]. Further downstream, H1R mediated signalling involves also MAPKs [19], [20].

The H4R is mainly expressed on immune cells [21]–[25], thus can be detected in virtually all organs [26]. The H4R plays a role in inflammatory- and immunoreactions e.g. by inducing chemotaxis in mast cells, T-cells, eosinophils, macrophages, and dendritic cells [22]–[24], [27], [28]. Upon ligand binding the H4R activates pertussis toxin-sensitive Gi-proteins [29], [30] activating PLC which catalyses the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) to IP3 which causes an increase in [Ca2+]i [23], [31]. Furthermore, the activated Gi-protein directly inhibits ACs and, thereby, decreases e.g. forskolin-induced cAMP concentrations [32], [33]. Moreover, similar to the H1R stimulation, H4R activates MAPK signalling pathways [30], [34].

H4R antagonists such as JNJ 7777120 [21], [35] have already been successfully used pre-clinically in animal models. Application of H4R antagonists as well as genetic deletion of the H4R in a mouse asthma model improved asthmatic symptoms. Our own previous studies in murine asthma demonstrated that the H1R antagonist mepyramine affects the ameliorating effect of the H4R antagonist JNJ7777120 [35]. Thus, understanding the signalling pathways activated by the murine (m)H1R and mH4R is of high interest. However, so far HXR-signalling was mainly analysed for the human orthologs while comprehensive information on the murine receptors is missing. In contrast, HXR ligands are analysed pre-clinically mainly in mouse models. Therefore, it is important to compare biochemical and pharmacological parameters in both systems, human and mouse, to estimate if effects that are observed in mice can be translated to humans. In order to analyse mH1R and mH4R signalling, we generated HEK293 cells, which are devoid of endogenous histamine receptors, stably expressing comparable levels of either recombinant mH1R or mH4R. These cells were incubated with HA, the H1R- and H4R-specific antagonists mepyramine and JNJ7777120, and specific inhibitors of MAPKs. The resulting signalling activities were analysed with respect to mobilization of intracellular caclcium, intracellular cAMP accumulation, activation of MAPKs and EGR-1 gene expression.

Materials and Methods

Materials

If not stated otherwise, all chemicals were obtained from Sigma-Aldrich (Taufkirchen,, Germany). PCR primers were purchased from Eurofins MWG Operon (Ebersberg, Germany). The MAPK inhibitors SB 203580 and PD 98059 were purchased from Tocris (Bristol, United Kingdom), and pertussis-toxin from List Biological Laboratories (Campbell, CA, USA). The H4R-selective antagonist JNJ7777120 (1-[(5-chloro-1H-indol-2-yl) carbonyl]-4-methylperazine) was kindly provided by Dr. Armin Buschauer (University of Regensburg, Germany).

HEK293 cells and generation of stably HXR-expressing clones

HEK293 cells (LGC Standards (ATCC), Wesel, Germany) were maintained in DMEM medium (PAA Laboratories, Pasching, Austria) supplemented with 10% [v/v] FCS (Lonza, Basel, Switzerland), penicillin (100 units/ml)/streptomycin (100 µg/ml) and 2 mM L-glutamine at 37°C in a 7% [v/v] CO2 environment. Confluent cell layers were split twice a week.

Cells at 60–80% confluency were transfected with pcDNA3 plasmids containing the coding sequences for either myc-tagged mH1R or Flag-tagged mH4R, or the empty vectors using Fugene HD (Roche, Mannheim, Germany) according to the manufacturer's protocol. Cells with stable integration of vectors or plasmids were selected with G418 (empty vector, mH1R) or Zeocin (empty vector, mH4R) (both Invivogen, San Diego, CA, USA). Clones were generated by limiting dilution technique and receptor expression was checked by flow cytometry (Miltenyi Biotech, Bergisch Gladbach, Germany) and Western blot using anti-Flag and anti-myc (Roche,Penzberg, Germany) reagents.

Detection of intracellular Ca2+ concentration

HEK293 mHXR cells were incubated at a density of 1×107 cells/ml in Krebs-Hepes buffer (120 mM NaCl, 20 mM Hepes, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.25 mM CaCl2, 10 mM glucose, pH 7,4) containing 10 µM Fura-2-AM (Tocris, Bristol, United Kingdom) and 0.2% [m/v] pluronic F127 for 40 min under rotation at room temperature in the dark. Then, cells were diluted 1∶10 with Krebs-Hepes buffer and incubated for an additional 20 min at the same conditions. Cells were sedimented by centrifugation and resuspended at 2×107 cells/ml in fresh Krebs-Hepes buffer. Labelled cells were seeded in a black 96 well plate at 50 µl/well and incubated with increasing concentrations of mepyramine or JNJ7777120, or with Krebs-Hepes buffer for a few minutes. Fluorescence measurement was performed in a Synergy 4 reader (Biotek, Bad Friedrichshall, Germany) with high frequently alternating excitation wavelengths of 340 and 380 nm and an emission wavelength of 508 nm. After 2 min of detection of the baseline, HA at the concentration indicated was added. In antagonists studies, the HA solutions were supplemented with the respective antagonists in order to keep antagonists concentration constant. The effect of HA stimulation on [Ca2+]i was measured over a period of 3 min. Then, Triton X-100 at a final concentration of 0.5% (w/v) was added and the maximal signal (Fmax) was detected over a period of 2 min. Finally, EGTA was added at a final concentration of 12 mM and the minimal signal (Fmin) was detected for additional 2 min. The increase in [Ca2+]i was calculated from these data using the following equitation:

Quantification of intracellular cAMP accumulation using HPLC/MS/MS

HEK293 mHXR cells were seeded in 6-well plates at 1×106 cells/well and cultured for 24 h. Cells were stimulated for 10 min at 37°C with 100 µM forskolin and 100 µM HA in the presence or absence of mepyramine and/or JNJ7777120 at the concentrations indicated. The medium was removed and 300 µl of extraction reagent (AcN/MeOH/H2O (2∶2∶1)) containing 25 ng/ml tenofovir (internal standard) was added to the wells. The extracts were incubated at 98°C for 20 min and aggregated protein was collected by centrifugation for 10 min at 17000 g. Supernatant fluids were transferred into a new tube and evaporated at 40°C under nitrogen flow until complete dryness and residual material was solved in 150 µl H2O. Samples were diluted 1∶100 in H2O containing 50 ng/ml tenofovir and analyzed on an API 5500 (AB SCIEX, Framingham, MA, USA) mass spectrometer after HPLC-separation using a Zorbax Eclipse column XDB-C18 1.8 µ 50×4,6 (Agilent Technologies, Santa Clara, CA). cAMP concentrations of samples were calculated according to standards containing defined cAMP concentrations. The protein pellets were dried at RT and solved in 800 µl 0.1 M NaOH at 95°C for 30–60 min. Protein concentrations were quantified using BCA-assay (Thermo Fischer Scientific, Waltham, MA, USA). cAMP concentrations were calculated in relation to the protein concentration (pmol cAMP/mg protein).

MAPK array

HEK293 mHXR cells were seeded in 6-well plates at 1×106 cells/well and cultured for 24 h. After incubation with or without 10 µM HA for 5 min, cells were analyzed using the MAPK array according to the manufacturer's protocol (Human Phospho-MAPK-Array; R&D System, Minneapolis, MN, USA [36]). Protein concentrations of cellular lysates were determined by Bradford protein assay (BioRad Laboratories) and 300 µg protein were subjected to each membrane. For quantitative analysis, the pixel density of every individual spot was determined and intensities of the spots without HA-stimulation were subtracted from the intensities of the corresponding spots with HA-stimulation.

RNA extraction and real time PCR

HEK293 mHXR cells were seeded in 6-well plates at 1×106 cells/well and cultured for 24 h. After pre-incubation with or without MAPK inhibitors followed by stimulation with or without 10 µM HA (as indicated) for 4 h, the medium was removed and the cells were washed twice with PBS. Total RNA was extracted using the NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's protocol. Two µg RNA were reverse transcribed into cDNA using oligo dT primers (Fermentas, Rockford, IL, USA) and RevertAid Reverse Transcriptase (Fermentas).

Real time PCR was performed by the TaqMan method. Buffers and TaqMan probes were purchased from Applied Biosystems (Darmstadt, Germany) and the assay was performed according to the manufacture's protocol. For standardisation the house-keeping genes β-actin and GUS-β were amplified and gene expression was quantified using the ΔΔCt method [37].

Statistical analysis

If not stated otherwise, statistical analyses were performed by calculating means ± SD of at least three independent determinations. Analysis of significance was performed using Student's t-test or one-way ANOVA with Bonferroni post-test for linear parameters (GraphPad Prism 5). p-Values of ≤0.05 (*), ≤0.01 (**) and ≤0.005 (***) were considered significant.

Results

Histamine increases intracellular Ca2+ concentrations via mH1R and H4R with different potencies and efficacies

Clones of HEK293 cells stably expressing epitop-tagged fusion proteins of either mH1R or mH4R were generated. A myc-tag was added N-terminally at the mH1R, and the mH4R was flanked by a Flag-tag. The presence of the receptor proteins encoded by the exogenous genes was analyzed by Western blot (Figure 1A) and flow cytometry (Figure 1B). The appearance of several species with differing apparent molecular weights (Figure 1A) is due to differing glycosylation of the receptor proteins [38], [39]. For functional analyses, individual clones of both cell lines demonstrating comparable quantities of receptor expression were chosen. This quantification relayed on flow cytometry since radioligand-binding analysis is not feasible for the mH4R due to the lack of a suitable radio-labelled ligand [8]. The chosen clones are referred to as HEK293 mH1R and HEK 293 mH4R.

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Figure 1. Successful generation of HEK293 cells stably expressing mH1R and mH4R.

HEK293 cells were transfected with plasmids encoding the myc-tagged mH1R, the Flag-tagged mH4R, or the empty vectors (e.v.) as indicated and clones thereof were generated by limiting dilution technique. Stable expression of the transgenes was anlayzed by Western blot (A) and flow cytometry (B) using myc- and Flag-specific reagents as indicated. Presented are data from a single clone of each cell line, which has been chosen for further analyses. Arrows in (A) indicate the recombinantly expressed proteins.

https://doi.org/10.1371/journal.pone.0107481.g001

In HEK293 mH1R and HEK293 mH4R cells HA increased [Ca2+]i concentration-dependently (Figure 2A,B). While in HEK293 mH1R a maximal Δ[Ca2+]i of about 400 nM (Figure 2A) was reached, the maximum Δ[Ca2+]i of HEK293 mH4R cells was only 200 nM (Figure 2B). Moreover, HEK 293 mH1R cells displayed a substantially higher potency for HA than HEK293 H4R cells, as reflected by a pEC50 of 8.2 and of 6.9, respectively. In contrast, in empty vector-transfected HEK293 cells HA did not increase [Ca2+]i (Figure 2C).

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Figure 2. Mobilization of [Ca2+]i by histamine is mediated by H1R and H4R.

HEK293 mH1R cells (A+D), HEK293 mH4R cells (B+E), and empty vector-transfected HEK293 cells (C) were labelled with Fura 2-AM and stimulated with different concentrations of histamine (HA) (A–C) or with a constant HA concentration in combination with DMSO or varying concentrations of mepyramine (MEP) or JNJ7777120 (JNJ) (D+E). Changes in [Ca2+]i were determined by fluorescence measurements at an emission wavelength of 508 nm and excitation wavelengths of 340 nm and 380 nm. Data shown are means ± SD (n = 2–3, each consisting of 2 replicates).

https://doi.org/10.1371/journal.pone.0107481.g002

The selective antagonists mepyramine (H1R) and JNJ7777120 (H4R) were used to document the receptor specificity of HA-induced increased [Ca2+]i. In HEK293 mH1R cells the elevated [Ca2+]i was reduced concentration-dependently by mepyramine with a pIC50 of 7.4 and a pKB of 8.6, while JNJ7777120 was without effect (Figure 2D). JNJ7777120, but not mepyramine, inhibited the HA-induced increase in [Ca2+]i in HEK293 mH4R cells displaying a pEC50 of 6.9 and a pKB of 8.8 (Figure 2E). Thus, the HA-induced increase of [Ca2+]i in the transfected HEK 293 cells is mediated indeed by the respective recombinant receptors and not by an unspecific activity of HA.

Intracellular cAMP concentrations are enhanced via mH1R and decreased via mH4R

cAMP is generated by AC, which can be activated directly by forskolin [9], [40]. In comparison to untreated controls, stimulation with forskolin elevated the cAMP-concentration in HEK 293 mH1R cells, HEK 293 mH4R cells, and empty vector-transfected cells, which were defined as 100 % for relative quantification (Figure 3A,B). Additional stimulation with 100 µM HA enhanced cAMP accumulation in HEK293 mH1R cells to about 3500 %. This enhancement was reduced by mepyramine in a concentration dependent manner (Figure 3A). In HEK293 mH4R cells, HA reduced the forskolin-induced cAMP-concentration to basal levels. This effect was concentration-dependently diminished by JNJ7777120 (Figure 3B). The antagonists mepyramine and JNJ7777120 both at concentrations up to 10 µM were selective for their respective receptors since the HA effects were neither affected by mepyramine in HEK293 mH4R cells nor by JNJ777120 in HEK293 mH1R cells (data not shown). Finally, in empty vector-transfected cells, HA did not affect forskolin-induced cAMP generation (Figure 3C) and stimulation with HA alone enhanced cAMP accumulation in HEK293 mH1R cells while it had no effect in HEK293 mH4R cells and empty vector –transfected cells (data not shown).

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Figure 3. Intracellular cAMP concentrations are enhanced by histamine via the H1R and reduced by HA via the H4R.

HEK293 mH1R cells (A), HEK293 mH4R cells (B), and empty vector-transfected HEK293 cells (C) cells were stimulated with forskolin (Forsk) and histamine (HA) in the absence or presence of mepyramine (MEP) or JNJ7777120 (JNJ) for 10 min. Intracellular cAMP was extracted from the cells and the concentration was determined using HPLC/MS/MS and calculated in relation to the protein concentration. The cAMP-concentration after stimulation with forskolin (H1R: 186±57 pmol/mg protein; H4R: 309±111 pmol/mg protein) was set to 100 % and other values were calculated on this base. Data shown are means ± SD (n = 2–5, each consisting of 3 replicates; *: p≤0.05; **: p≤0.01; ***: p≤0.005; ns: not significant).

https://doi.org/10.1371/journal.pone.0107481.g003

Pertussis toxin inhibits mH4R- but not mH1R-mediated effects

Experiments with pertussis toxin (PTX), which selectively inhibits Gi-protein activation by GPCRs [41], [42], were performed. HEK293 mH1R and HEK293 mH4R cells were incubated with or without PTX for 18 h and then the [Ca2+]i and the forskolin-induced cAMP concentrations were evaluated after stimulation with HA.

PTX did not affect the HA-induced increase in [Ca2+]i (∼280 nM) in HEK293 mH1R cells (Figure 4A). In contrast, stimulation of untreated HEK293 mH4R cells with HA enhanced [Ca2+]i to about 100 nM, while such increase was absent in PTX pre-treated HEK293 mH4R cells (Figure 4B). The remaining Δ[Ca2+]i of about 10 nM was consistent with the concentration in unstimulated cells. PTX treatment did not generally inhibit the ability of HEK293 mH4R cells to mobilize Ca2+ ions, since stimulation with ATP increased [Ca2+]i independently of pre-incubation with PTX (Figure 4B).

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Figure 4. Pertussis toxin selectively blocks signaling of the mH4R.

HEK293 mH1R cells (A+C) and HEK293 mH4R cells (B+D) were incubated in the presence or absence of 50 ng/ml pertussistoxin (PTX) for 18 h. Changes in [Ca2+]i were determined as described in Figure 2 after stimulation with 100 µM histamine (HA) or 10 µM ATP as indicated on the x-axis (A+B). Intracellular cAMP concentrations were analyzed as described in Figure 3 after stimulation for 10 min with 100 µM forskolin (Forsk) and histamine (HA) (C+D). Forskolin-induced cAMP concentrations were 2385±160 pmol/mg protein in HEK293 H1R and 2164±436 pmol/mg protein in HEK 293 H4R cells, respectively. Data shown are means ± SD (n = 2, each consisting of 2–3 replicates; *: p≤0.05; **: p≤0.01; ***: p≤0.005; ns: not significant).

https://doi.org/10.1371/journal.pone.0107481.g004

In HEK293 mH1R and HEK293 mH4R cells forskolin induced a cAMP increase independently of PTX treatment (Figure 4C,D). In HEK293 mH1R cells HA enhanced forskolin-induced cAMP concentrations up to 400 % in the absence as well as in the presence of PTX treatment (Figure 4C). HEK293 mH4R cells showed an 80 % reduction of forskolin-induced cAMP concentrations after stimulation with HA which was absent upon pre-incubation with PTX (Figure 4D). Thus, intracellular Ca2+ mobilization and cAMP generation is affected by HA via both mH1R and mH4R, but with differing quantities (Ca2+) and qualities (cAMP). Moreover, only the mH4R, but not the mH1R acts in a PTX-sensitive manner.

HA-induced phosphorylation of MAPKs differs between mH1R- and mH4R-expressing HEK293 cells

In order to analyse signalling events that occur more distally than the enhancement of intracellular Ca2+ and cAMP, the phosphorylation of several MAPKs in lysates of 5 min HA-stimulated HEK293 mH1R and HEK293 mH4R cells was assessed.

In HEK293 mH1R as well as in HEK293 mH4R cells a similar strong HA-induced phosphorylation of ERK 1 and ERK 2 was detected. p38α and CREB were only moderately phosphorylated via activation of mH1R and mH4R, the H1R demonstrating a stronger activity-inducing potential on p38α and CREB phosphorylation as compared to the H4R (Figure 5). Thus, signalling emerging at the mH1R and the mH4R differs basically at the activation of p38 MAPK and the transcription factor CREB.

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Figure 5. Histamine induces phosphorylation of MAP-kinases via H1R and H4R.

HEK293 empty vector cells, HEK293 mH1R cells, and HEK293 mH4R cells were incubated with or without 10 µM HA for 5 min and lysed afterwards. Phosphorylation of different serine/threonine kinases in these lysates were detected by MAPK array. Phosphorylation intensity was quantified by analysis of the pixel density of every single spot (A). Shown are the differences of the densities of corresponding spots obtained using lysates of cells with and without HA stimulation. Data shown are means ± SD (n = 2, each measured in 2 replicates; *: p≤0.05; **: p≤0.01; ***: p≤0.005). In B, exemplarily the spots of ERK1 and ERK2 after histamine induction in the cells as indicated on the left, and quantification control spots (ctr.) are demonstrated.

https://doi.org/10.1371/journal.pone.0107481.g005

HA induced EGR-1 gene expression in mH1R- and mH4R-transfected cells

As an example for MAPK-regulated genes, the expression of the EGR-1 gene was quantified in HEK293 mH1R and HEK293 mH4R cells after 4 h stimulation with HA. Specific kinases involved were analysed by use of inhibitors selective for the MAPKs p38 and ERK, and for CREB.

EGR-1 expression was enhanced 200-fold in HEK293 mH1R cells by stimulation with HA. This HA-induced EGR-1 expression was unaffected by the p38 inhibitor SB 203580 [43] and the CREB inhibitor KG-501 [44]. In contrast, the MEK inhibitor PD 98059, used to assess the involvement of the ERK pathway [43], caused a significant reduction nearly to the level of unstimulated cells (Figure 6A). In HEK293 mH4R cells, EGR-1 expression was roughly doubled by stimulation with HA. This statistically not significant effect was unaffected by KG-501 but reduced to the basal level using PD 98059. Interestingly, a significant 4-fold increase of EGR-1 expression was observed upon incubation of HEK293 mH4R with HA plus SB 203585 (Figure 6B). The treatment of both HEK293 cell lines with the MAPK inhibitors without HA stimulation did not affect EGR-1 expression, with the exception of HEK293 H4R cells incubated with SB 203585, which slightly enhanced EGR-1 expression (data not shown). Thus, HA-induced expression of EGR-1 mediated via mH1R and mH4R involves mainly ERK signalling. In addition, the p38 MAPK pathway seems to reduce mH4R-mediated induction of EGR-1 expression.

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Figure 6. Histamine regulates EGR-1 mRNA expression.

HEK293 mH1R cells (A) and HEK293 mH4R cells (B) were incubated without any inhibitor (Ø) or with 50 µM PD 980598 (PD) for 1 h, with 2 µM SB 203580 (SB) for 30 min, or 25 µM KG-501 (KG) for 20 min and then incubated with or without histamine for 4 h. Total RNA was extracted from the cells and reverse transcribed into cDNA. Expression of EGR-1 was determined by real time PCR using TagMan probes. Data shown are means ± SD of the relative EGR-1 expression in relation to unstimulated cells (basal) (n = 3, each consisting of 2 replicates; **: p≤0.01; ***: p≤0.005; ns: not significant).

https://doi.org/10.1371/journal.pone.0107481.g006

Discussion

In the present study we analyzed the signalling pathways of mH1R and mH4R. A model of stably transfected HEK293 cells expressing either mH1R or mH4R at comparable levels as assessed by FACS analysis was generated and validated. To the best of our knowledge, signal transduction of the mH1R and mH4R has never been studied in parallel in a well-controlled expression system before. However, similar expression levels and identical cellular backgrounds are crucial for a direct comparison because the stoichiometry between receptors, G-proteins, and downstream effectors may have a large impact on the signals measured [45]. An advantage of HEK293 cells is their unresponsiveness to HA stimulation because they do not endogenously express functionally active HxRs. In HEK293 cells stably expressing exogenous HxRs, HA-induced signalling was analysed with respect to [Ca2+]i, intracellular cAMP concentration, activation of MAPKs and EGR-1 gene expression.

Many HxR agonists and antagonists are used in different disease models in the mouse. Therefore, it is important to compare pharmacological parameters like affinity and effectivity in both systems, human and mouse, to estimate if effects that are observed in mouse models can be translated to humans. The mouse and human histamine receptor orthologs have a rather low homology (i.e. 67 % for H4R [46]), and JNJ7777120, which is a partial inverse agonist at the recombinant human H4R expressed in an insect cell system, shows partial agonistic effects at the insect cell-expressed recombinant mH4R [47], [48]. However, so far no further evidence of an agonistic function of JNJ7777120 in other experimental cellular systems has been observed, specifically in eosinophils, the so far only human cell type with confirmed functional expression of the H4R [49]. The concept of functional selectivity, according to which the potency and efficacy of a given ligand depends on the specific signalling pathway studied, may provide a mechanistic explanations for our data [8], [9].

Regulation of intracellular Ca2+ and cAMP concentrations via mH1R and mH4R

Proximal signalling of mH1R and mH4R was analyzed by measurements of [Ca2+]i and intracellular cAMP concentration. The human H1R has been demonstrated to mediate the HA signal to increase [Ca2+]i and intracellular cAMP concentration [13], [18], [50]. Stimulation of the human and the murine H4R increased [Ca2+]i as well [23], [30], [33], while it decreased intracellular cAMP concentrations [23], [32], [51]. Our results on [Ca2+]i and the intracellular cAMP concentration regulated by the recombinant mH1R and mH4R fit well to the above-mentioned literature data. Moreover, we also provide evidence that the mH4R expressed in HEK293 cells couples to Gi-proteins as does the native H4R in murine mast cells [23], and that the mH4R-generated signal towards regulation of both [Ca2+]i and cAMP accumulation is mediated via Gi-proteins. Coupling of the recombinant mH1R to Gq-proteins is highly likely due to the PTX-insensitivity of rises in [Ca2+]i, excluding the possibility of Gi-coupling. Because mH1R and mH4R were expressed at comparable levels, we can also hypothesize that activation of Ca2+ signalling via Gq-proteins is more efficient than via Gi-proteins. Thus, the mH1R may induce a more robust cellular response than the mH4R which may act in a more subtle way. In support of this concept, the hH4R induces only a small pro-inflammatory response in human eosinophils [52].

Activation of MAPK signalling pathways via mH1R and mH4R

Naor et al. [53] reported that different G-proteins can interact in distinct manners with MAPK signalling pathways and, therefore, activate different MAPK cascades. For some GPCRs including the H1R- and the H4R it has been shown that, in addition to G-protein activation, they activate MAPK signalling pathways independently of G-proteins by direct coupling to β-arrestins [54]–[56]. However, while G-protein dependent phosphorylation appears fast, within minutes after stimulation, β-arrestin-mediated MAPK activation is much slower [57]. Since we analyzed MAPK phosphorylation after 5 min of stimulation, it is very unlikely that β-arrestin signalling is responsible for our observations, thus the effects on MAPK phosphorylation described in this study are rather G-protein mediated. A formal experimental proof, however, is still to be provided.

We found the MAPKs ERK 1/2, and p38, and the transcription factor CREB to be activated in response to HA in both mH1R- and mH4R-expressing HEK293 cells. The H1R has already been described to activate ERK 1/2 and p38 most probably via PLC, [Ca2+]i, protein kinase C and Ras [58]. CREB, the ‘cAMP responsive element binding protein’, is activated by enhanced intracellular cAMP concentration followed by PKA activation [59], [60]. Since in mH1R-transfected HEK293 cells HA induced a strong increase in cAMP, CREB phosphorylation can be regarded a direct consequence of this. In HEK293 mH4R cells in contrast, histamine stimulation does not enhance cAMP accumulation, but nevertheless, albeit at a very weak level, it activates CREB. This weak cAMP-independent CREB activation may be induced by e.g. pathways emerging from enhanced [Ca2+]i mobilization [61], [62].

Phosphorylation of ERK 1/2 has also been detected already in human H4R-transfected HEK293 cells [30] and in murine bone marrow-derived mast cells [34]. However, activation of p38 via the H4R has not been described before and is, thus, a novel finding of this study. In mH4R-transfected cells, HA does not induce, but rather reduces cAMP generation. Thus, to detect the phosphorylation of CREB was unexpeceted as well. However, CREB has been found to be activated also by other signalling molecules, such as MAPKAPK-2, a substrate of p38 [58], [63], [64], MSK-1, a substrate of p38 and ERK 1/2 [65], and CaM kinases, which are activated by increased [Ca2+]i [62]. Since we found an activation of p38 and ERK 1/2 and enhanced [Ca2+]i in mH4R-transfected cells upon HA-stimulation, in our system CREB is phosphorylated probably via one of these alternative pathways.

MAPK-mediated gene expression induced by mH1R and mH4R

The activation of MAPK-signalling pathways finally results in regulation of gene expression. The transcription factor EGR-1 plays a decisive role in inflammatory reactions by the induction of the expression of e.g. IL-6, IL-8, CCL-2, TNF-α and MCP-1 [66]–[68]. In mH1R-transfected HEK293 cells, HA induces a strong increase in EGR-1 expression, which is dependent on ERK signalling. This result fits to the data of Guha et al. [69], [70] and Whitmarsh et al. [69], [70] and confirms the evidence of Thiel et al. [71] who demonstrated that activation of the ERK signalling pathway is mainly involved in EGR-1 expression. In contrast, Rolli et al. [72] described a p38 MAPK-dependent induction of EGR-1 expression which was not detected in our HEK293 H1R cells.

In mH4R-transfected cells, histamine induces only a moderate increase in EGR-1 expression which is also dependent on ERK signalling. So far it is not clear why stimulation with histamine leads to a much lower EGR-1 expression in mH4R-transfected cells compared to mH1R-transfected cells although in both cell lines ERK is similarly phosphorylated upon HA stimulation. Surprisingly, in mH4R-transfected cells, p38 MAPK activity inhibited EGR-1 expression. This was unexpected, since it was not detected in HEK293 H1R cells, although in these cells HA induces p38 phosphorylation as well, even stronger as compared to HEK293 mH4R cells. In HEK293 mH1R cells the p38 MAPK-mediated inhibition of EGR-1 expression may be abolished by the high concentration of cAMP, which is not found in HEK293 mH1R cells. Moreover, our finding is also in contrast to the observation by Rolli et al. [72], who demonstrated the induction of EGR-1 expression by p38 MAPK, however using a different stimulus. Consequently, it can be speculated that upon HA-stimulation a mH4R-specific pathway is triggered which, via the activity of p38 MAPK, inhibits EGR-1 expression. Thus, in HEK293 mH1R cells, which lack this pathway, the p38 MAPK activity lacks a cognate target to reduce ERK1/2 MAPK-induced EGR-1 expression. However, the nature of this mH4R-specific pathway has still to be elucidated.

In conclusion, we have shown that HEK293 cells stably expressing comparable quantities of murine histamine receptors represent a valuable model to analyse and directly and reliably compare mH1R and mH4R mediated signal transduction. To our best knowledge, this is the first published side by side approach analyzing signalling of the pro-inflammatory mH1R and mH4R using an identical cellular background and comparable expression levels. Using this system, we provide evidence that both the mH1R and the mH4R mediate the HA-induced [Ca2+]i mobilization, the H1R being more potent and more efficient. Since both receptors are expressed at comparable quantities, this is either an intrinsic property of the H1R, probably indicating that HA-induced coupling of Gq proteins is more effective as compared to that of Gi-proteins, or an intrinsic property of HEK293 cells, probably indicating that in these cells Gq proteins are predominant in comparison to Gi proteins. In contrast to the mH1R, the mH4R indeed, as anticipated from the human ortholog, diminishes AC activity, leading to a reduction of cAMP production. Moreover, we demonstrate that the signalling pathways of these receptors are basically homologous, but specific differences, such as at the functional activity of p38 MAPK, which we have newly defined to be involved in mH4R signalling, were detected. It will be important to study signal transduction of hH1R and hH4R according to the same principles as shown here. Comparing signal transduction between human and murine receptor orthologs is essential for proper interpretation of studies in mouse disease models and their translation to human pathology.

Acknowledgments

We thank Renate Schottmann for excellent technical help and the research core unit ‘Mass spectrometry Metabolomics’ of the Hannover Medical School for cAMP quantification.

Author Contributions

Conceived and designed the experiments: SB RS DN. Performed the experiments: SB MV RH. Analyzed the data: SB MV RS DN. Wrote the paper: SB RS DN.

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