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. 2026 Jan 28;19(2):227. doi: 10.3390/ph19020227

Histamine H1 Receptor-Mediated CREB Phosphorylation via Gq Protein Signaling and Arrestin Modulation

Ryosuke Ogami 1, Shotaro Michinaga 1,*, Yosuke Iiboshi 1, Yasuhiro Ogawa 1, Shigeru Hishinuma 1
Editors: Krzysztof Walczyński1, Marek Staszewski1
PMCID: PMC12943045  PMID: 41754768

Abstract

Background/Objectives: Histamine H1 receptors mediate multiple physiological and pathophysiological processes, including inflammation and allergy, by regulating downstream gene expression via transcription factors. cAMP response element-binding protein (CREB) is a major transcription factor whose phosphorylation is regulated by multiple signaling pathways. Although CREB is closely involved in multiple physiological and pathophysiological processes, the detailed intracellular signaling pathway of H1 receptor-mediated CREB phosphorylation remains to be elucidated. We investigated the roles of Gq proteins and arrestins in H1 receptor-mediated CREB phosphorylation. Methods: We constructed Chinese hamster ovary (CHO) expressing human wild-type (WT) H1 receptors and two types of C-terminal mutants. One mutant was constructed by truncating the serine 487 residue only at the C-terminus (S487Trunc), and the other was constructed by substituting the serine 487 residue at the C-terminus with alanine (S487A). S487Trunc is a Gq protein-biased while S487A is an arrestin-biased receptor. The expressions of CREB and its phosphorylated form were assessed by immunoblotting. Results: Histamine promoted CREB phosphorylation in CHO cells expressing WT or S487Trunc receptors, but not in cells expressing S487A. Inhibitors of protein kinase C (PKC), extracellular signal-regulated kinase (ERK), or c-Jun N-terminal kinase (JNK), and Ca2+ chelator suppressed histamine-induced CREB phosphorylation in CHO cells expressing WT or S487Trunc receptors. Basal CREB phosphorylation levels increased following β-arrestin overexpression and decreased after their siRNA-mediated knockdown, thus modulating histamine-stimulated CREB phosphorylation in WT CHO cells. Conclusions: H1 receptor-mediated CREB phosphorylation is induced through Gq protein/Ca2+/PKC-dependent ERK and JNK activation; arrestins can modulate this process by regulating basal CREB phosphorylation.

Keywords: Arrestin, CREB, ERK, Gq protein, Histamine H1 receptor, JNK, PKC

1. Introduction

G protein-coupled receptors (GPCRs) possess seven transmembrane domains and orchestrate various cellular responses in both health and disease states [1,2,3]. Upon agonist binding, GPCRs mediate various intracellular signaling pathways through both G proteins and arrestins [4,5,6,7,8]. In receptors such as β-adrenergic, angiotensin II AT1, and opioid receptors, these proteins mediate distinct signaling pathways that contribute to both therapeutic and adverse effects of drugs [9,10,11,12,13,14]. Therefore, separate analyses of G protein- and arrestin-dependent signaling pathways are essential to elucidate their differential roles in pathological mechanisms and inform the development of novel therapeutic drugs.

The histamine H1 receptor is a major GPCR expressed throughout the human body, including the central nervous system (CNS) and peripheral tissues. It regulates multiple physiological and pathophysiological processes, including allergy, inflammation, arousal, and memory [15,16,17,18,19,20,21]. Upon binding to its agonist, the H1 receptor activates Gq proteins, leading to an increase in intracellular Ca2+ concentration and activation of protein kinase C (PKC) through phospholipase C-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate. This H1 receptor-mediated activation of PKC stimulates mitogen-activated protein kinases, including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), contributing to allergic and inflammatory reactions. These reactions are mediated by the production of inflammatory cytokines and growth factors, along with the upregulation of H1 receptor gene expression [22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38]. Simultaneously, 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, S487Trunc and S487A, in which the Ser487 residue at the end of the intracellular C-terminus was truncated or substituted with alanine, respectively [40]. The S487Trunc receptor appeared to be Gq 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 demonstrated that S487Trunc receptors mediate ERK and JNK phosphorylation via Gq protein/Ca2+/PKC-dependent signaling pathways. Conversely, we found that S487A receptors mediate ERK phosphorylation, but not JNK phosphorylation, via GRK/arrestin/clathrin/Raf/MEK-dependent signaling pathways [41,42]. The Gq 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 undergoes phosphorylation via multiple signaling pathways and then binds to specific DNA sequences known as cAMP response elements to regulate 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]. CREB is essential for neuronal function 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 Gq protein/Ca2+/PKC-dependent and GRK/arrestin/clathrin/Raf/MEK-dependent pathways. Therefore, in the present study, we aimed to explore the roles of Gq proteins and arrestins in regulating H1 receptor-mediated CREB phosphorylation in CHO cells expressing wild-type (WT) human H1, Gq protein-biased S487Trunc, or arrestin-biased S487A receptors.

2. Results

2.1. 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 in response to exposure to histamine concentrations above 1 µM and peaked at 100 μM, whereas the ratio of total CREB to β-actin remained unchanged (Figure 1A). In CHO cells expressing Gq protein-biased S487Trunc receptors, treatment with histamine significantly increased the ratio of phosphorylated CREB to total CREB (Figure 1B), whereas no significant effect was observed in CHO cells expressing arrestin-biased S487A receptors (Figure 1C). 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 (Figure 2).

Figure 1.

Figure 1

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Concentration-dependent effects of histamine on CREB phosphorylation. CHO cells expressing WT (A), S487Trunc (B), and S487A (C) 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 four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (one-way ANOVA followed by Dunnett’s test). CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type.

Figure 2.

Figure 2

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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. CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type.

We then assessed the time-dependent effects of histamine (100 µM) on CREB phosphorylation. In CHO cells expressing WT receptors and Gq protein-biased S487Trunc, histamine-induced CREB phosphorylation occurred within 10 min and persisted for 60–180 min (Figure 3A,B). In contrast, histamine-induced CREB phosphorylation did not occur in CHO cells expressing arrestin-biased S487A receptors not only for the 30 min histamine treatment shown in Figure 1C but also for other histamine treatment times (Figure 3C).

Figure 3.

Figure 3

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Time-dependent effects of histamine on CREB phosphorylation. CHO cells expressing WT (A), S487Trunc (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 four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (one-way ANOVA followed by Dunnett’s test). CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type.

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, 10 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) (Figure 4). None of these inhibitors significantly inhibited CREB phosphorylation, indicating that histamine-induced CREB phosphorylation occurs via Gq protein-dependent rather than arrestin-dependent signaling. Therefore, we conducted subsequent experiments to explore the signaling pathways underlying the induction of CREB phosphorylation by treatment with 100 µM histamine for 10 min in CHO cells expressing WT and Gq protein-biased S487Trunc receptors.

Figure 4.

Figure 4

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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). CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type; GRK, G protein-coupled receptor kinase.

2.2. Involvement of Gq 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. Histamine-induced CREB phosphorylation was completely inhibited by ketotifen in CHO cells expressing WT (Figure 5A) and S487Trunc receptors (Figure 5B). We then investigated the involvement of Gq proteins in histamine-induced CREB phosphorylation. Treatment with the Gq protein inhibitor, YM-254890 (20 µM), significantly inhibited histamine-induced CREB phosphorylation in CHO cells expressing WT (Figure 6A) and S487Trunc receptors (Figure 6B). These results suggest that H1 receptor-mediated CREB phosphorylation occurred via Gq protein-dependent signaling pathways.

Figure 5.

Figure 5

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Effects of histamine H1 receptor antagonist on CREB phosphorylation. CHO cells expressing WT (A) and S487Trunc (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,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,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). CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type.

Figure 6.

Figure 6

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Effects of a Gq protein inhibitor, intracellular Ca2+ chelator, and PKC inhibitor on CREB phosphorylation. CHO cells expressing WT (A,C) and S487Trunc mutant (B,D) H1 receptors were stimulated with or without 100 µM histamine for 10 min in the presence or absence (vehicle) of the Gq protein inhibitor YM-254890 (20 μM), intracellular Ca2+ chelator BAPTA-AM (50 μM), or 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). CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type; PKC, Protein kinase C.

As Gq 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 (Figure 6C) or S487Trunc receptors (Figure 6D). These results suggest that H1 receptor-mediated CREB phosphorylation occurred via Gq protein/Ca2+/PKC-dependent signaling pathways.

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

In our previous study, we found that H1 receptor-mediated activation of the Gq protein/Ca2+/PKC pathway induces the phosphorylation of ERK and JNK [41,42]. Therefore, in this study, we assessed the potential involvement of ERK and JNK in histamine-induced CREB phosphorylation. Our results indicated that histamine-induced CREB phosphorylation was significantly inhibited by the ERK inhibitor SCH772984 (20 µM) or the JNK inhibitor SP600125 (20 µM) in CHO cells expressing WT (Figure 7A,C) and S487Trunc receptors (Figure 7B,D) although SCH772984 or SP600125 treatment partially suppressed the phosphorylation of basal CREB. These findings suggest that H1 receptor-mediated CREB phosphorylation occurs via Gq protein/Ca2+/PKC-dependent activation of ERK and JNK.

Figure 7.

Figure 7

Figure 7

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Effects of ERK and JNK inhibitors on CREB phosphorylation. CHO cells expressing WT (A,C) and S487Trunc 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). CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type.

2.4. Possible Involvement of Arrestins in Regulating the Basal Levels 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 in the presence and absence of histamine for 10 min (Figure 8A), suggesting increased 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 (Figure 8B,C). Histamine-induced CREB phosphorylation was enhanced in the presence of overexpressed β-arrestin1 (Figure 8B) or β-arrestin2 (Figure 8C). This result suggests modulation of basal CREB phosphorylation levels. Conversely, siRNA-mediated knockdown of β-arrestin1 or β-arrestin2 in CHO cells expressing WT receptors tended to lower the basal level of CREB phosphorylation in the absence of histamine (Figure 9A). The knockdown of either protein did not markedly affect CREB phosphorylation following 10 min of treatment with histamine (Figure 9A). However, during prolonged stimulation (30–180 min), the knockdown of β-arrestin1 (Figure 9B) or β-arrestin2 (Figure 9C) decreased histamine-induced CREB phosphorylation. Collectively, these results suggest that arrestins regulate the basal level of CREB phosphorylation and thereby modulate H1 receptor-mediated, Gq protein/Ca2+/PKC-dependent phosphorylation of CREB.

Figure 8.

Figure 8

Figure 8

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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 presented 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 bottom 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). CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type.

Figure 9.

Figure 9

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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). CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type.

3. Discussion

To the best of our knowledge, the present study is the first to reveal the differential roles of Gq proteins and arrestins in H1 receptor-mediated CREB phosphorylation. We have recently demonstrated that H1 receptor-mediated ERK phosphorylation is regulated through two distinct pathways: the Gq protein/Ca2+/PKC and GRK/arrestin/clathrin/Raf/MEK pathways [41]. We have also revealed that H1 receptor-mediated JNK phosphorylation is regulated by Gq protein/Ca2+/PKC-dependent but GRK/arrestin/clathrin-independent pathways [42]. Building upon these findings, in the present study, we explored the signaling pathways underlying H1 receptor-mediated CREB phosphorylation in CHO cells expressing WT, Gq protein-biased mutant S487Trunc, and arrestin-biased mutant S487A.

3.1. Histamine H1 Receptor-Mediated CREB Phosphorylation Is Predominantly Induced via Gq Protein/Ca2+/PKC-Mediated Activation of ERK and JNK

Our present results revealed that histamine promoted CREB phosphorylation in CHO cells expressing WT and Gq protein-biased S487Trunc. Moreover, histamine-induced CREB phosphorylation was inhibited by antihistamine, Gq protein inhibitor, intracellular Ca2+ chelator, and inhibitors of PKC, ERK, and JNK. These results suggest that H1 receptor-mediated CREB phosphorylation is regulated via Gq 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 [46,47], while histamine-induced CREB phosphorylation is mediated via PKC-dependent activation in human aortic endothelial cells [31]. Furthermore, not only histamine-induced CREB phosphorylation but also basal CREB phosphorylation was partially inhibited by the inhibitors of dynamin, Gq protein, PKC, ERK, and JNK, suggesting that these pathways may also contribute to basal CREB phosphorylation.

We focused only on Gq protein but not G11 protein in this study. Although Gq protein and G11 protein largely initiate similar signaling cascades, they may have distinct roles in specific physiological and pathophysiological situations. For this consideration, observation of CREB phosphorylation by knockdown Gq and G11 with their siRNA should be performed to determine whether G11 participated in the H1 mediated CREB phosphorylation through Ca2+/PKC signaling in the future.

In the present study, histamine did not 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]. These results suggest that differences in the spatial patterns of G protein- and arrestin-mediated ERK activation may lead to differential activation of CREB [48].

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

In the present study, overexpression of β-arrestin1 or β-arrestin2 increased basal CREB phosphorylation levels, whereas their knockdown decreased them, 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 [49]. Collectively, these results suggest that basal CREB phosphorylation is likely regulated by arrestin expression within the cell.

As a mechanism of CREB regulation via arrestins, Manson et al. have suggested that CREB regulation is mediated directly through β-arrestin2 expression via the ERK pathway [50]. Furthermore, β-arrestin can activate ERK signaling independently of G proteins [51]. We previously found that Gq protein-mediated ERK activation was prompt and transient, whereas arrestin-mediated ERK activation was delayed and persistent [41]. These findings suggest that arrestin-mediated ERK activation may be involved in sustaining and enhancing CREB phosphorylation. However, ERK-independent mechanisms of CREB activation may also exist. As our previous studies indicated that basal phosphorylation level of ERK is not affected by arrestin2 knockdown [41], arrestin-mediated increases in basal CREB phosphorylation may also result from a direct interaction between arrestins and CREB [50,52].

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

H1 receptor-mediated signaling pathways induce inflammation by increasing the production of proinflammatory cytokines [27,28,29,35]. 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 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 Gq proteins, and potentially arrestins, may contribute to the physiological and pathophysiological regulation of peripheral and CNS function by H1 receptors.

However, this study has some limitations. First, we did not reveal 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. Third, we have not performed the in vivo experiments to analyze the roles of Gq protein and arrestins on CREB phosphorylation. Although the mutation at serine 487 residue in the C-terminus of the H1 receptor has not been reported in humans and the associated disease phenotype has not been elucidated, analyzing the phenotype through the in vivo knock-in experiments of H1 receptor mutants used in this study may elucidate how Gq protein and arrestins are involved in CREB phosphorylation under physiological and pathophysiological conditions. Therefore, further studies are required to strengthen the conclusions of this study.

4. Materials and Methods

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

All 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. One mutant was constructed by truncating the serine 487 residue only at the C-terminus of the H1 receptor (S487Trunc), and the other was constructed by substituting the serine 487 residue at the C-terminus of the H1 receptor with alanine (S487A), which was previously established in our laboratory [40,41,42]. These CHO cells were incubated in Dulbecco’s modified Eagle’s medium (catalog number: 08460-95, 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.

4.2. Drug Treatments

Forty-eight hours before drug treatments, CHO cells were incubated in a serum-free medium in 6-well 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 durations (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) [41], Gq protein inhibitor (YM-254890; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) [55], intracellular Ca2+ chelator (BAPTA-AM; Abcam, Cambridge, UK) [56], PKC inhibitor (GF109203X; Sigma-Aldrich) [57], ERK inhibitor (SCH772984; Selleckchem, Houston, TX, USA) [58], JNK inhibitor (SP600125; FUJIFILM Wako Pure Chemical Corporation) [59], GRK2/3 inhibitor (cmpd101; Hello Bio, Bristol, UK) [60], and dynamin inhibitor (dynasore; Sigma-Aldrich) [61]. Histamine treatment was performed under hypertonic conditions (0.32 M sucrose; FUJIFILM Wako Pure Chemical Corporation) to inhibit the formation of clathrin-coated pits [62].

4.3. 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). 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). Proteins were subjected to electrophoresis in a 7.5% polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (MilliporeSigma, Burlington, MA, USA). The membranes were placed in the SNAP.i.d.® 2.0 (MilliporeSigma) and were incubated with Blocking One (Nacalai Tesque) for 30 min before the antibody reaction. The antibodies were diluted in Blocking One and were prepared fresh each time. 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) for 1 h and then reacted with peroxidase-conjugated secondary antibody (1:4000; #12-348 or #12-349, MilliporeSigma) for 30 min. After detecting CREB or arrestins, antibodies were stripped by incubating the membranes with WB Stripping Solution (Nacalai Tesque) for 5 min, and the membranes were washed with Tris-buffered saline with Tween 20 (Nacalai Tesque). The membranes were incubated with primary antibodies against β-actin (1:4000; ab317794, Abcam), and then incubated 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 as of 27 January 2026 (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.

4.4. Overexpression of β-arrestin1/2

Overexpression of yellow fluorescent protein (YFP)-conjugated β-arrestin1 or 2 (gifted from Robert Lefkowitz; Addgene plasmids 36916 and 36917, Addgene, Watertown, MA, USA) was performed via electroporation (Poring pulse: voltage 175) [63]. β-arrestin protein overexpression was confirmed via immunoblotting.

4.5. 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 (catalog number: sc-37007) 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.

4.6. Statistical Analysis

Statistical analysis and Normality tests were conducted using Ekuseru-Toukei (BellCurve for Excel, Social Survey Research Information Co., Ltd., Tokyo, Japan). The four independent experiments were performed in this study. Assessment of the normality of the data and testing for outlier was performed using the Shapiro–Wilk test and Smirnov-Grubbs’test, respectively. The Statistical analysis was applied to the normalized data sets. Data are presented as mean ± standard error (SE). Statistical analysis was performed using one-way analysis of variance followed by post hoc tests (Dunnett’s test or Tukey’s test). Statistical significance was set at p < 0.05.

5. Conclusions

We demonstrated that histamine H1 receptor-mediated CREB phosphorylation is induced through Gq protein/Ca2+/PKC-dependent activation of ERK and JNK. In contrast, arrestins regulate the basal level of CREB phosphorylation, thereby enhancing 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.

Abbreviations

The following abbreviations are used in this manuscript:

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
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Author Contributions

Conceptualization: S.H.; Methodology: S.M. and Y.O.; Formal analysis: R.O., S.M. and Y.I.; Investigation: R.O., S.M., Y.I. and Y.O.; Writing—original draft: S.M.; Writing—review & editing: S.H.; Visualization: S.M.; Project administration: S.H.; Supervision: S.H. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not Applicable

Informed Consent Statement

Not Applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research received no external funding.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

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