Research ArticleHistamine acting on H1 receptor promotes inhibition of proliferation via PLC, RAC, and JNK-dependent pathways
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.
Section snippets
Reagents and antibodies
[3H]-Mepyramine, [3H]-thymidine, and myo-[3H]-inositol were purchased from PerkinElmer Life Sciences (Boston, MA). Histamine dihydrochloride, 2,3-trifluormetilfenilhistamine (H1 agonist), ATP, myo-inositol, bovine serum albumin, G418, Dulbecco's modified Eagle's medium (DMEM), and the inhibitors U73122 and U73343 were purchased from Sigma Chemical Company (St. Louis, MO). Mepyramine maleate and the inhibitors PD98059, U0126, and SP600125 were from Tocris Cookson Inc. (Ballwin, MO). The Rac
Characterization of the CHO-H1R stable cell line
For the generation of CHO cells stably expressing human H1R (CHO-H1R), cells were transfected with pCEFL-humanH1R. Expression of H1R was confirmed using saturation analysis and Scatchard plotting of intact transfected cells which showed specific binding site for [3H]-mepyramine with a Kd within the range value previously reported (2.6 ± 0.2 nM) (n = 3) (Fig. 1A). The number of sites estimated from the Bmax was 35,700 ± 900 sites/cell (n = 3). Binding of [3H]-mepyramine in CHO-naive cells was not
Discussion
In the present study, we established the pattern of Rho-GTPases activation mediated by the H1R and its role in the regulation of cell proliferation. Using CHO cells stably transfected with the human H1R, we showed that histamine couples to the Gq–PLC pathway induced RhoA and Rac activation and inhibits cell growth mediated by Rac and JNK via H1R.
Histamine H1R activation stimulates PLC-β isoform via pertussis toxin-insensitive Gq/G11-proteins in a variety of cell types (for review, see Hill et
Acknowledgments
We are sincerely grateful to Dr. L. Bianciotti for critical reading of the manuscript. This study was supported by grants from the Universidad de Buenos Aires (grant UBACyT B0-50), Consejo Nacional de Investigaciones Científicas y Tecnológica (PIP 6110), ANPCYT (PICT 38318) and National Institutes of Health (grant R01-CA74197).
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