Histamine H₁ receptor-mediated CREB phosphorylation via G_q protein signaling and arrestin modulation

RyosukeOgami1

ShotaroMichinaga1✉

YosukeIiboshi1

YasuhiroOgawa1

ShigeruHishinuma1

ShoratoMichinaga

Ph.D.

1Phone(+81)-42-495-8428Emailmichisho@my-pharm.ac.jp

Department of PharmacodynamicsMeiji Pharmaceutical University2-522-1 Noshio, Kiyose204-8588Kiyose, TokyoJapan

Ryosuke Ogami, Shotaro Michinaga^*, Yosuke Iiboshi, Yasuhiro Ogawa, and Shigeru Hishinuma

Department of Pharmacodynamics, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204–8588, Japan

^*Corresponding Author:

Shorato Michinaga, Ph.D.,

Department of Pharmacodynamics, Meiji Pharmaceutical University,

2-522-1 Noshio, Kiyose, Tokyo 204–8588, Japan

TEL: (+ 81)-42-495-8428

E-mail: michisho@my-pharm.ac.jp

ORCID: https://orcid.org/0000-0002-4052-7507

Acknowledgements

We would like to thank Editage (www.editage.com) for English language editing.

Abstract

Histamine H₁ receptors mediate multiple physiological and pathophysiological processes, including inflammation and allergy, by regulating downstream gene expression via transcription factors. Agonist binding activates G_q proteins, increasing intracellular Ca²⁺ and activating protein kinase C (PKC) and stimulating G protein-coupled receptor kinases (ERK) and c-Jun N-terminal kinase (JNK) to promote cytokine production and H₁ receptor upregulation. G protein-coupled receptor kinase (GRK)-mediated phosphorylation and arrestin-dependent internalization enable alternative signaling. cAMP response element-binding protein (CREB) is a major transcription factor whose phosphorylation is regulated by multiple signaling pathways. Therefore, we investigated the roles of G_q proteins and arrestins in H₁ receptor-mediated CREB phosphorylation in Chinese hamster ovary (CHO) cells expressing wild-type (WT) human H₁ receptors and C-terminal mutants, namely S487TR (G_q protein-biased) and S487A (arrestin-biased), in which the Ser487 residue was truncated and substituted with alanine, respectively. Histamine promoted CREB phosphorylation in CHO cells expressing WT or S487TR receptors, but not in cells expressing S487A, suggesting that H₁ receptor-mediated CREB phosphorylation occurs via G_q protein-dependent but arrestin-independent mechanisms. Inhibitors of PKC, ERK, or JNK, and intracellular Ca²⁺ chelator suppressed histamine-induced CREB phosphorylation in CHO cells expressing WT or S487TR receptors. Basal CREB phosphorylation levels increased after β-arrestin1 or β-arrestin2 overexpression and decreased after their siRNA-mediated knockdown, modulating histamine-stimulated CREB phosphorylation in WT CHO cells. Collectively, H₁ receptor-mediated CREB phosphorylation is induced through G_q protein/Ca²⁺/PKC-dependent ERK and JNK activation; arrestins can modulate this process by regulating basal CREB phosphorylation.

Keywords:

Arrestin

CREB

ERK

G_q protein

Histamine H₁ receptor

JNK

PKC

Abbreviations

CHO, Chinese hamster ovary

CREB, cAMP response element binding protein

ERK, extracellular signal-regulated kinase

GPCR, G protein-coupled receptor

GRK, G protein-coupled receptor kinase

JNK, c-Jun N-terminal kinase

MAPK, mitogen-activated protein kinase

PKC, protein kinase C

YFP, yellow fluorescent protein

WT, wild-type

1. Introduction

G protein-coupled receptors (GPCRs) possess seven transmembrane domains and orchestrate various cellular responses in health and disease [1–3]. Upon agonist binding, GPCRs mediate various intracellular signaling pathways via both G proteins and arrestins [4–8]. In receptors such as β-adrenergic, angiotensin II AT₁, and opioid receptors, these proteins mediate distinct signaling pathways that underlie both therapeutic and adverse effects of drugs [9–14]. Accordingly, separate analyses of G protein- and arrestin-dependent signaling pathways are essential to clarify their differential roles in pathological mechanisms and guide the development of novel therapeutic drugs.

The histamine H₁ receptor is a major GPCR expressed throughout the body, including the central nervous system (CNS) and peripheral tissues. It regulates multiple physiological and pathophysiological processes, including allergy, inflammation, arousal, and memory [15–21]. Once this receptor binds to its agonist, it activates G_q proteins, which mediate the increase in intracellular Ca²⁺ concentration and activate protein kinase C (PKC) via phospholipase C-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate. This H₁ receptor-mediated activation of PKC, followed by the activation of mitogen-activated protein kinases, including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), may contribute to allergic and inflammatory reactions. Such reactions are mediated by the production of inflammatory cytokines and growth factors, along with the upregulation of H₁ receptor gene expression [22–38]. Simultaneously, upon agonist binding, H₁ receptors are phosphorylated by G protein-coupled receptor kinases (GRKs) and internalized via the arrestin/clathrin/dynamin pathway [39, 40], enabling arrestin-mediated signal transduction.

We have previously constructed two mutants of human H₁ receptors, S487TR and S487A, in which the Ser487 residue at the end of the intracellular C-terminal was truncated and substituted with alanine, respectively [40]. The S487TR receptor appeared to be G_q protein-biased, mediating histamine-induced inositol phosphate production but not internalization of H₁ receptors. In contrast, the S487A receptor was arrestin-biased, mediating histamine-induced internalization but not inositol phosphate production [40]. Using Chinese hamster ovary (CHO) cells expressing these H₁ receptor mutants, we have demonstrated that S487TR receptors mediate ERK and JNK phosphorylation via G_q protein/Ca²⁺/PKC-dependent signaling pathways. Conversely, S487A receptors mediate ERK phosphorylation, but not JNK phosphorylation, via GRK/arrestin/clathrin/Raf/MEK-dependent signaling pathways [41, 42]. The G_q protein/Ca²⁺/PKC-mediated pathway induces prompt and transient ERK phosphorylation, whereas the GRK/arrestin/clathrin-mediated pathway triggers delayed and sustained ERK phosphorylation via activation of H₁ receptors [41].

cAMP response element binding protein (CREB) is a general transcription factor involved in homeostatic cellular processes, including proliferation, survival, and differentiation [43]. CREB is phosphorylated via multiple signaling pathways and binds to specific DNA sequences known as cAMP response elements, thereby regulating the transcription of target genes. It plays critical roles in inflammatory responses by controlling the production of proinflammatory cytokines and key immune functions in T cells, B cells, and macrophages [43, 44]. Furthermore, CREB is closely involved in neuronal functions in the CNS. For example, it acts as a positive regulator of memory formation and long-term potentiation [45]. Notably, histamine promotes CREB phosphorylation via H₁ receptor-mediated PKC-dependent ERK activation [31]. However, it remains unclear whether H₁ receptor-mediated CREB phosphorylation is regulated by differential activation of ERK via G_q protein/Ca²⁺/PKC-dependent and GRK/arrestin/clathrin/Raf/MEK-dependent pathways. Therefore, in the present study, we aimed to explore the roles of G_q proteins and arrestins in regulating H₁ receptor-mediated CREB phosphorylation in CHO cells expressing wild-type (WT) human H₁, G_q protein-biased S487TR, or arrestin-biased S487A receptors.

2. Materials and Methods

2.1. Preparation of CHO Cells Expressing WT or Mutant Human Histamine H₁ Receptors

The genetic modification studies were approved by the Institutional Safety Committee for Recombinant DNA Experiments of Meiji Pharmaceutical University (Approval No. 1209). CHO-K1 cells (RCB0285, RRID: CVCL_0214) were obtained from RIKEN Bioresource Center (Ibaraki, Japan). CHO cells stably expressing human WT histamine H₁ receptors and two types of C-terminal mutants, S487TR and S487A, in which the Ser487 residue in the C-terminal was truncated and substituted with alanine [40–42], were previously established in our laboratory. These CHO cells were incubated in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) containing G418 sulfate (Enzo Life Sciences, Inc., Farmingdale, NY, USA) and 10% (v/v) fetal bovine serum (Biowest, Nuaillé, France) cultured in 150 cm² culture flasks (BM Bio, Tokyo, Japan) at 37°C in a 5% CO₂ incubator. Upon reaching confluence, CHO cells were dissociated with trypsin/EDTA (Sigma-Aldrich, St. Louis, MO, USA) and reseeded in six-well culture plates (Corning Inc., Corning, NY, USA) for subsequent experiments.

2.2. Drug Treatments

Forty-eight hours before drug treatments, CHO cells were incubated in a serum-free medium in six-well culture plates at 37°C in a 5% CO₂ incubator. First, the concentration- and time-dependent effects of histamine on CREB phosphorylation were investigated in CHO cells treated with varying concentrations (0.1–1000 µM) of histamine (Sigma-Aldrich) for 30 min or with a fixed concentration of histamine (100 µM) for varying time periods (10–360 min). Subsequently, the effects of various inhibitors on histamine-induced CREB phosphorylation were assessed. CHO cells were treated with 100 µM histamine for 10 min in the presence of the following inhibitors: H₁ receptor antagonist (ketotifen; Sigma-Aldrich), G_q protein inhibitor (YM-254890; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), intracellular Ca²⁺ chelator (BAPTA-AM; Abcam, Cambridge, UK), PKC inhibitor (GF109203X; Sigma-Aldrich), ERK inhibitor (SCH772984; Selleckchem, Houston, TX, USA), JNK inhibitor (SP600125; FUJIFILM Wako Pure Chemical Corporation), GRK2/3 inhibitor (cmpd101; Hello Bio, Bristol, UK), and dynamin inhibitor (dynasore; Sigma-Aldrich). Histamine treatment was also performed under hypertonic conditions (0.32 M sucrose; FUJIFILM Wako Pure Chemical Corporation) to inhibit the formation of clathrin-coated pits.

2.3. Immunoblotting Analysis

CHO cells were collected and homogenized in 150 µL of radioimmunoprecipitation assay buffer (Nacalai Tesque, Kyoto, Japan) containing protease cocktails (Nacalai Tesque) and phosphatase inhibitor cocktails (Nacalai Tesque) to extract proteins. The collected cell lysates were centrifuged at 20,000 × g for 10 min (Model 3700; KUBOTA, Tokyo, Japan). The supernatants were collected as protein samples for immunoblotting analyses, and the protein content in the supernatant was determined using the Pierce™ bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The protein samples were subjected to electrophoresis in a 7.5% polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (MilliporeSigma, Burlington, MA, USA). The membranes were then incubated with primary antibodies against CREB (1:4000; #4820, Cell Signaling Technology, Danvers, MA, USA), phosphorylated CREB (1:4000; #9198, Cell Signaling Technology), β-arrestin1 (1:4000; ab32096, Abcam), and β-arrestin2 (1:4000; ab54790, Abcam) and then reacted with peroxidase-conjugated secondary antibody (1:4000; #12–348 or #12–349, MilliporeSigma). After detecting CREB or arrestins, antibodies were stripped, washed, and then incubated with primary antibodies against β-actin (1:4000; ab317794, Abcam), followed by incubation with a peroxidase-conjugated secondary antibody (1:4000; AP180P, MilliporeSigma). Proteins were detected using a chemiluminescence kit (Chemi-Lumi One® L; Nacalai Tesque). The intensity of the protein bands was determined using ImageJ software (version 1.53c; National Institutes of Health, Bethesda, MD, USA). The molecular weights of proteins were assessed using Precision Plus Protein™ Kaleidoscope™ Prestained Protein Standards (Bio-Rad Laboratories, Hercules, CA, USA). In the corresponding figures, representative immunoblot images of phosphorylated CREB (phospho-CREB; 43 kDa), CREB (total CREB; 43 kDa), β-arrestin1 (50 kDa), β-arrestin2 (50 kDa), and β-actin (40 kDa) are presented together with bar graphs. The arrowheads indicate phosphorylated CREB in the immunoblot images. The lower bands of phosphorylated CREB represent phosphorylated ATF-1, a CREB-related protein, according to an antibody datasheet (https://www.cellsignal.jp/products/primary-antibodies/phospho-creb-ser133-87g3-rabbit-mab/9198). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the control in the bar graphs.

2.4. Overexpression of β-arrestin1/2

Overexpression of yellow fluorescent protein (YFP)-conjugated β-arrestin1 or 2 (Addgene plasmid 36916 and 36917, gifted from Robert Lefkowit) was performed via electroporation (Poring pulse: voltage 175 V, pulse width 10 ms, pulse interval 50 ms; Draining pulse: voltage 20 V, pulse width 50 ms, pulse interval 50 ms) [46]. Plasmids (Addgene 36916 or 36917) were then introduced into CHO cells expressing WT H₁ receptors. β-arrestin protein overexpression was confirmed via immunoblotting.

2.5. Knockdown of β-arrestin1/2 Through Small Interfering RNA (siRNA)

Knockdown of β-arrestin1 or β-arrestin2 was achieved via transient transfection of siRNAs. β-arrestin1 siRNA, β-arrestin2 siRNA, and control siRNA were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). These siRNAs were transfected into CHO cells (70–80% confluence) in six-well plates using siRNA transfection reagent in siRNA transfection medium (Santa Cruz Biotechnology), according to the manufacturer’s protocol and our previous study [41]. CHO cells were collected 48 h after siRNA treatment for the immunoblotting assay.

2.6. Statistical Analysis

At least four independent experiments were performed in this study. Normality tests were conducted using normal probability plots in Ekuseru-Toukei (BellCurve for Excel, Social Survey Research Information Co., Ltd., Tokyo, Japan), confirming normally distributed data. Data are presented as mean ± standard error (SE). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by post-hoc tests (Dunnett’s test or Tukey’s test). Statistical significance was set at P < 0.05.

3. Results

3.1. Concentration- and Time-Dependency of Histamine-Induced CREB Phosphorylation

We initially examined the concentration-dependent effects of histamine on CREB phosphorylation in CHO cells expressing WT receptors by treating them with different concentrations of histamine (0.1–1000 µM) for 30 min. The ratio of phosphorylated CREB to total CREB was significantly increased at histamine concentrations above 1 µM and peaked at 100 µM, whereas the ratio of total CREB to β-actin remained unchanged (Fig. 1A and B). In CHO cells expressing G_q protein-biased S487TR receptors, treatment with histamine significantly increased the ratio of phosphorylated CREB to total CREB (Fig. 1C), whereas no significant effect was observed in CHO cells expressing arrestin-biased S487A receptors (Fig. 1D). In CHO-K1 cells without genetic manipulation of H₁ receptor expression, the ratio of phosphorylated CREB to total CREB was unaffected by treatment with 100 µM histamine for 30 min (Fig. 2).

Fig. 1

Concentration-dependent effects of histamine on CREB phosphorylation. (A) Representative uncropped immunoblot images of phosphorylated CREB (phospho-CREB) (left), total CREB (middle), and β-actin (right) in CHO cells expressing WT H₁ receptors. Arrowhead indicates phospho-CREB (43 kDa). Molecular weights are presented on the left side of the images. CHO cells expressing WT (B), S487TR (C), and S487A (D) mutant H₁ receptors were stimulated with or without (control) the indicated concentrations of histamine for 30 min, and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are presented in the upper panels. Arrowheads indicate phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the control in the lower graphs. Values represent the mean ± SE of 4–6 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control (one-way ANOVA followed by Dunnett’s test)

Fig. 2

Effects of histamine on CREB phosphorylation in original CHO-K1 cells. CHO-K1 cells were stimulated with or without (control) 100 µM histamine for 30 min, and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are presented in the upper panels. The arrowhead indicates phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the control in the lower graphs. Values represent the mean ± SE of four independent experiments.

We then assessed the time-dependent effects of histamine (100 µM) on CREB phosphorylation. In CHO cells expressing WT receptors and G_q protein-biased S487TR, histamine-induced CREB phosphorylation occurred within 10 min and persisted for 60–180 min (Fig. 3A and B). In contrast, histamine-induced CREB phosphorylation did not occur in CHO cells expressing arrestin-biased S487A receptors (Fig. 3C).

Fig. 3

Time-dependent effects of histamine on CREB phosphorylation. CHO cells expressing WT (A), S487TR (B), and S487A (C) mutant H₁ receptors were stimulated with or without (control) 100 µM histamine for 10–360 min, and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are depicted in the upper panels (A–C). Arrowheads indicate phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the control in the lower graphs. Values represent the mean ± SE of 4–6 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control (one-way ANOVA followed by Dunnett’s test)

To further evaluate whether arrestin-dependent endocytic pathways could be involved in histamine-induced CREB phosphorylation, we treated CHO cells expressing WT receptors with or without histamine (100 µM, 30 min) in the presence or absence (vehicle) of inhibitors of GRK2/3 (cmpd101; 30 µM), clathrin (0.32 M sucrose), and dynamin (dynasore; 100 µM) (Fig. 4). Notably, none of these inhibitors significantly inhibited CREB phosphorylation, indicating that histamine-induced CREB phosphorylation occurs via G_q protein-dependent rather than arrestin-dependent signaling. Therefore, we conducted subsequent experiments to explore the signaling pathways underlying CREB phosphorylation induced by treatment with 100 µM histamine for 10 min in CHO cells expressing WT and G_q protein-biased S487TR receptors.

Fig. 4

Effects of GRK, clathrin, and dynamin inhibitors on CREB phosphorylation. CHO cells expressing WT H₁ receptors were stimulated with or without 100 µM histamine for 10 min in the presence or absence (vehicle) of inhibitors against GRK2/3 (cmpd101; 30 µM), clathrin (a high concentration of sucrose; 0.32 M), and dynamin (dynasore; 100 µM), and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are depicted in the upper panels. An arrowhead indicates phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the vehicle control without histamine in the lower graphs. Values represent the mean ± SE of four independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. vehicle without histamine treatment (one-way ANOVA followed by Tukey’s test)

3.2. Involvement of G_q Protein/Ca²⁺/PKC-Dependent Pathways in H₁ Receptor-Mediated CREB Phosphorylation

To confirm whether histamine-induced CREB phosphorylation occurred via H₁ receptors, we assessed the effects of an antihistamine, ketotifen (1000 µM), on histamine-induced CREB phosphorylation. Our results indicated that histamine-induced CREB phosphorylation was completely inhibited by ketotifen in CHO cells expressing WT (Fig. 5A) and S487TR receptors (Fig. 5B). We then investigated the involvement of G_q proteins in histamine-induced CREB phosphorylation. Treatment with the G_q protein inhibitor, YM-254890 (20 µM), significantly inhibited histamine-induced CREB phosphorylation in CHO cells expressing WT (Fig. 6A) and S487TR receptors (Fig. 6B). These results suggest that H₁ receptor-mediated CREB phosphorylation occurred via G_q protein-dependent signaling pathways.

Figure 5. Effects of histamine H₁ receptor antagonist on CREB phosphorylation. CHO cells expressing WT (A) and S487TR (B) mutant H₁ receptors were stimulated with or without 100 µM histamine for 10 min in the presence or absence (vehicle) of histamine H₁ receptor antagonist ketotifen (1000 µM), and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are illustrated in the upper panels (A and B). Arrowheads indicate phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the vehicle control without histamine in the lower graphs (A and B). Values represent the mean ± SE of four independent experiments. ^**P < 0.01, ^***P < 0.001 vs. vehicle without histamine, ^##P < 0.01, ^###P < 0.001 vs. vehicle with histamine (one-way ANOVA followed by Tukey’s test)

As G_q proteins mediate Ca²⁺/PKC-dependent signaling pathways, we investigated the effects of the intracellular Ca²⁺ chelator, BAPTA-AM (50 µM), and a PKC inhibitor, GF109203X (10 µM), on histamine-induced CREB phosphorylation. Both BAPTA-AM and GF109203X significantly inhibited histamine-induced CREB phosphorylation in CHO cells expressing WT (Fig. 6C) or S487TR receptors (Fig. 6D). These results suggest that H₁ receptor-mediated CREB phosphorylation occurred via G_q protein/Ca²⁺/PKC-dependent signaling pathways.

Fig. 6

Effects of G_q protein inhibitor, intracellular Ca²⁺ chelator, and PKC inhibitor on CREB phosphorylation. CHO cells expressing WT (A, C) and S487TR mutant (B, D) H₁ receptors were stimulated with or without 100 µM histamine for 10 min in the presence or absence (vehicle) of the G_q protein inhibitor YM-254890 (20 µM), the intracellular Ca²⁺ chelator BAPTA-AM (50 µM), or the PKC inhibitor GF109203X (10 µM). Protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are depicted in the upper panels (A–D). Arrowheads indicate phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the vehicle control without histamine in the lower graphs (A–D). Values represent the mean ± SE of four independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.01 vs. vehicle without histamine, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. vehicle with histamine (one-way ANOVA followed by Tukey’s test)

3.3. Involvement of ERK and JNK in H₁ Receptor-Mediated CREB Phosphorylation

As we previously found that H₁ receptor-mediated activation of the G_q protein/Ca²⁺/PKC pathway induced phosphorylation of ERK and JNK [41, 42], we assessed the potential involvement of ERK and JNK in histamine-induced CREB phosphorylation. Histamine-induced CREB phosphorylation was significantly inhibited by the ERK inhibitor SCH772984 (20 µM) and the JNK inhibitor SP600125 (20 µM) in CHO cells expressing WT (Fig. 7A and C) and S487TR receptors (Fig. 7B and D). These findings suggest that H₁ receptor-mediated CREB phosphorylation occurs via G_q protein/Ca²⁺/PKC-dependent activation of ERK and JNK.

Fig. 7

Effects of ERK and JNK inhibitors on CREB phosphorylation. CHO cells expressing WT (A, C) and S487TR mutant (B, D) H₁ receptors were stimulated with or without 100 µM histamine for 10 min in the presence or absence (vehicle) of the ERK inhibitor SCH772984 (20 µM) or the JNK inhibitor SP600125 (20 µM), and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are depicted in the upper panels (A–D). Arrowheads indicate phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the vehicle control without histamine in the lower graphs (A–D). Values represent the mean ± SE of four independent experiments. ^**P < 0.01, ^***P < 0.001 vs. vehicle without histamine. ^##P < 0.01, ^###P < 0.001 vs. vehicle with histamine (one-way ANOVA followed by Tukey’s test)

3.4. Possible Involvement of Arrestins in Regulating the Basal Level of CREB Phosphorylation and H₁ Receptor-Mediated CREB Phosphorylation

To investigate the possible involvement of arrestins in CREB phosphorylation, we overexpressed β-arrestin1 or β-arrestin2 in CHO cells expressing WT receptors. Overexpression of either β-arrestin1 or β-arrestin2 significantly increased CREB phosphorylation in CHO cells both with and without histamine treatment for 10-min (Fig. 8A), suggesting an increase in basal CREB phosphorylation levels. To further assess the impact of arrestin overexpression on histamine-induced CREB phosphorylation, we treated CHO cells overexpressing β-arrestin1 or β-arrestin2 with histamine for 30–180 min (Fig. 8B, C). Histamine-induced CREB phosphorylation was enhanced in the presence of overexpressed β-arrestin1 (Fig. 8B) or β-arrestin2 (Fig. 8C), suggesting modulation of basal CREB phosphorylation levels. Conversely, siRNA-mediated knockdown of β-arrestin1 or β-arrestin2 in CHO cells expressing WT receptors tended to reduce the basal level of CREB phosphorylation in the absence of histamine (Fig. 9A). The knockdown of either protein did not markedly affect CREB phosphorylation following 10 min of treatment with histamine (Fig. 9A). However, during prolonged stimulation (30–180 min), β-arrestin1 (Fig. 9B) or β-arrestin2 (Fig. 9C) knockdown decreased histamine-induced CREB phosphorylation. Collectively, these results suggest that arrestins regulate the basal level of CREB phosphorylation and thereby modulate H₁ receptor-mediated, G_q protein/Ca²⁺/PKC-dependent phosphorylation of CREB.

Fig. 8

Effects of arrestin overexpression on CREB phosphorylation. (A) β-arrestin1 or β-arrestin2 was overexpressed in CHO cells expressing WT H₁ receptors. CHO cells were stimulated with or without 100 µM histamine for 10 min, and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, YFP-arrestin1/2, and β-actin are shown in the left panels. An arrowhead indicates phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the control without histamine in the right graphs. Values represent the mean ± SE of four independent experiments. ^**P < 0.01, ^***P < 0.001 vs. control; ^##P < 0.01 vs. vehicle with histamine (one-way ANOVA followed by Tukey’s test). (B, C) Time-dependent effects of histamine-induced CREB phosphorylation in cells with β-arrestin1 (B) or β-arrestin2 (C) overexpression. Cells with normal expression (NE) or overexpression (OE) of β-arrestin1/2 were stimulated with histamine for the indicated times, and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are depicted in the upper panels. Arrowheads indicate phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the control (β-arrestin1/2 normal expression) without histamine in the lower graphs. Values represent the mean ± SE of four independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. control (β-arrestin1/2 normal expression) without histamine. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. β-arrestin1/2 normal expression with histamine (one-way ANOVA followed by Tukey’s test).

Fig. 9

Effects of arrestin knockdown on CREB phosphorylation. (A) Knockdown of β-arrestin1 or β-arrestin2 was performed via siRNA treatment in CHO cells expressing WT H₁ receptors. Forty-eight hours after siRNA treatment, CHO cells were stimulated with or without 100 µM histamine for 10 min, and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, β-arrestin1, β-arrestin2, and β-actin are depicted in the left panels. An arrowhead indicates phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the control siRNA without histamine in the upper graphs. The expression levels of β-arrestin1 or β-arrestin2 are indicated in the lower graphs. Values represent the mean ± SE of four independent experiments. ^*P < 0.05, ^***P < 0.001 vs. vehicle without histamine (one-way ANOVA followed by Tukey’s test). (B, C) Time-dependent effects of histamine-induced CREB phosphorylation after β-arrestin1 (B) or β-arrestin2 (C) knockdown. Forty-eight hours after β-arrestin1 or β-arrestin2 siRNA treatment, CHO cells were stimulated with histamine for the indicated times, and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are depicted in the upper panels. Arrowheads show phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the control siRNA without histamine in the lower graphs. Values represent the mean ± SE of four independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. control siRNA without histamine ^#P < 0.05, ^##P < 0.01 vs. control siRNA treatment with histamine (one-way ANOVA followed by Tukey’s test)

4. Discussion

To the best of our knowledge, the present study is the first to reveal the differential roles of G_q proteins and arrestins in H₁ receptor-mediated CREB phosphorylation. We have recently demonstrated that H₁ receptor-mediated ERK phosphorylation is dually regulated by the G_q protein/Ca²⁺/PKC and GRK/arrestin/clathrin/Raf/MEK pathways [41]. Moreover, H₁ receptor-mediated JNK phosphorylation is regulated by G_q protein/Ca²⁺/PKC-dependent but GRK/arrestin/clathrin-independent pathways [42]. Building upon these findings, in the present study, we explored signal transduction pathways underlying H₁ receptor-mediated CREB phosphorylation in CHO cells expressing WT, G_q protein-biased mutant S487TR, and arrestin-biased mutant S487A,

4.1. Histamine H₁ Receptor-Mediated CREB Phosphorylation is Predominantly Induced via G_q Protein/Ca²⁺/PKC-Mediated Activation of ERK and JNK

Our present results indicate that histamine promoted CREB phosphorylation in CHO cells expressing WT and G_q protein-biased S487TR. Moreover, histamine-induced CREB phosphorylation was inhibited by antihistamine, G_q protein inhibitor, intracellular Ca²⁺ chelator, and inhibitors of PKC, ERK, and JNK. These results suggest that H₁ receptor-mediated CREB phosphorylation is regulated via G_q protein/Ca²⁺/PKC-dependent activation of ERK and JNK. Notably, these results are consistent with several reports indicating that CREB phosphorylation is mediated by ERK and JNK [47, 48] and that histamine-induced CREB phosphorylation is mediated via PKC-dependent activation in human aortic endothelial cells [31].

In the present study, histamine failed to increase CREB phosphorylation in CHO cells expressing arrestin-biased S487A. In addition, histamine-induced CREB phosphorylation in CHO cells expressing WT was not affected by inhibitors of GRKs, clathrin, and dynamin. These results suggest that arrestins are not affected by histamine-induced CREB phosphorylation in CHO cells expressing WT receptors, although they have been reported to mediate H₁ receptor-dependent ERK phosphorylation [41]. Differences in the spatial patterns of G protein- and arrestin-mediated ERK activation may lead to differential activation of CREB [49].

4.2. Arrestins Regulate the Basal Level of CREB Phosphorylation and Modulate H₁ Receptor-Mediated CREB Phosphorylation

In the present study, overexpression of β-arrestin1 or β-arrestin2 resulted in increased basal CREB phosphorylation, whereas their knockdown decreased it, thereby modulating H₁ receptor-mediated CREB phosphorylation. These results are consistent with a previous report demonstrating that elevated β-arrestin2 expression leads to increased CREB phosphorylation in cystic fibrosis cells [50]. Thus, basal CREB phosphorylation is likely regulated by arrestin expression within the cell. As our previous studies indicated that basal phosphorylation levels of ERK and JNK are not affected by arrestin2 knockdown [41, 42], arrestin-mediated increases in basal CREB phosphorylation may result from a direct interaction between arrestins and CREB [51, 52].

4.3. Physiological and Pathophysiological Roles of H₁ Receptor-Mediated CREB Regulation

H₁ receptor-mediated signaling pathways induce inflammation by increasing the production of proinflammatory cytokines [27–29, 35]. CREB regulates the production of proinflammatory cytokines and various immune functions [43, 44]. As previously described, H₁ receptors play crucial roles in maintaining wakefulness and memory formation in the CNS [53]. Notably, CREB is reportedly a positive regulator of memory formation and long-term potentiation [45] and is required in excitatory neurons of the forebrain to sustain wakefulness [54]. Therefore, CREB phosphorylation via G_q proteins, and potentially arrestins, may underlie the physiological and pathophysiological regulation of peripheral and CNS function by H₁ receptors.

However, this study has some limitations. First, we did not elucidate the detailed mechanism by which arrestins are involved in CREB phosphorylation. Second, as we only used CHO cells overexpressing H₁ receptors in this study, confirmation using cells that naturally express H₁ receptors is also necessary. Therefore, further studies are required to strengthen the conclusions of this study.

5. Conclusions

We demonstrated that histamine H₁ receptor-mediated CREB phosphorylation is induced via G_q protein/Ca²⁺/PKC-dependent activation of ERK and JNK. In contrast, arrestins appeared to regulate the basal level of CREB phosphorylation, thereby augmenting or maintaining H₁ receptor-mediated CREB phosphorylation. However, further investigations including in vivo studies using experimental animals and in vitro studies using cells that naturally express H₁ receptors are required to provide novel insights into the role of CREB in H₁ receptor-mediated physiological and pathophysiological responses.

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Statements & Declarations

The authors declare no conflict of interest

Funding

The authors received no funding for this study.

Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author Contribution

Ryosuke Ogami : Investigation, Formal analysis, Shotaro Michinaga : Writing – original draft, Investigation, Methodology, Formal analysis, Visualization. Yosuke Iiboshi : Investigation, Formal analysis, Yasuhiro Ogawa : Investigation, Methodology. Shigeru Hishinuma : Project administration, Supervision, Writing – review & editing, Conceptualization.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics Approval

The genetic modification studies were approved by the Institutional Safety Committee for Recombinant DNA Experiments of Meiji Pharmaceutical University (Approval No. 1209).

Consent to Participate

Not applicable.

Consent to Publish

Not applicable.

Yes