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

Abstract

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

Introduction

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

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

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

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

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