Research paperERK activated by Histamine H1 receptor is anti-proliferative through spatial restriction in the cytosol
Graphical abstract
Introduction
Histamine coupled G-protein coupled receptors (GPCRs) are involved in the regulation of allergy, inflammation, acid secretion, neuromodulation amongst many other physiological processes. Histamine regulated signaling responses are orchestrated by canonical as well as non-canonical activation of the downstream regulators present inside a cell. MAPK pathway activation is an example of one of the prominent non − canonical signaling pathway cross-activated by histamine stimulation. Over the years, three major pathways have been identified through which MAP kinases can be activated by GPCRs. First, GPCR can activate matrix metallo-proteinases which release membrane bound growth factors, which in turn initiates typical MAP kinase pathway/s (Prenzel et al., 1999). Second, activated GPCRs recruit β-arrestins, which can directly activate scaffolding proteins like KSR, MEK and phosphorylate ERK (Luttrell, 2002). And lastly, downstream effectors of G protein signaling cascade such as protein kinase A (Daaka et al., 1997) or protein kinase C (Tsai et al., 1997), can interact with the downstream proteins of MAP kinase pathways such as MEK and activate ERK. Though the cascades underlying GPCR mediated cross-activation of MAPK signaling pathways are characterized in detail, the physiological responses generated through this non-canonical activation are not conserved and tend to differ significantly.
Physiologically, GPCR mediated activation of the MAP kinase pathway has been associated with many physiological disorders including multiple types of cancers such as prostate cancers, intraperitoneal cancer (Westermann et al., 1998, Xu et al., 1995) and cardiac hypertrophy (Luttrell, 2005). However, the physiological effects of pERK generated through GPCRs are often inconclusive, as they are not typical of canonical RTK cascades. For example, it has been observed that when histamine coupled GPCRs are activated, generation of pERK is recorded, as with other GPCRs (Luttrell, 2002, Pierce et al., 2001), but it may not be linked to pro-proliferative responses (Notcovich et al., 2010) except in the cases of chronic allergy (Francis et al., 2013). This could possibly be because of the differences in the dynamics of spatial or temporal activation of pERK between various cascades, which is compromised when activation of ERK is monitored using only the phosphorylation status of the conserved TEY motif of ERK. Temporally analysis has revealed that while direct RTK transactivation and the effector mediated MAP kinase activations are fast, β-arrestins cause slow and sustained activation of MAPK (Luttrell, 2002). Further, a number of studies have shown that subcellular localization of the activated ERK can discriminately prime a cell either towards proliferation or differentiation. Based on this premise, the current study was aimed to identify the molecular regulators involved in non-canonical activation of ERK by histamine stimulation, alongwith monitoring the spatio-temporal dynamics of the activated ERK. In a previous study, it has been shown that histamine stimulation generates pERK which is responsible for induction of histamine H1 receptor expression (Mizuguchi et al., 2011). This induction in the H1 receptor expression is projected as the reason of enhanced sensitivity to allergic rhinitis (Shirasaki et al., 2012). However, no account of the typical physiological responses associated with MAPK activation was shown.
In the present study, we address the reason behind the absence of pro-proliferative changes in response to pERK generated after histamine stimulation using live cell imaging approach, which can reveal the spatio − temporal dynamics of ERK activation and phosphorylation. The differences in pERK activity under histamine or EGF stimulated conditions were monitored using GFP-tagged ERK2 as a reporter, which provided details of ERK and pERK translocation and shuttling movement. Along with this, analysis of activation of downstream targets revealed that histamine induced pERK is spatially restricted to the cytosol and does not translocate to the nucleus, thereby delinking the RTK mediated and the GPCR mediated activation of MAPK (ERK). Physiologically, this cytosolic restriction of pERK was associated with enhancement of oxidative stress in the cells thereby revealing a novel signal transduction cascade associated with histamine mediated inflammatory responses.
Section snippets
Materials
DMEM, Histamine dihydrochloride (PubChem CID: 5818), Cimetidine (PubChem CID: 2756), Doxorubicin hydrochloride (PubChem CID: 31703) were purchased from Sigma-Aldrich, USA). Fetal bovine serum (FBS), EGF, Live cell imaging solution; trypsin-EDTA (0.05%), Turbofect™ transfection reagent, restriction enzymes and T4 DNA ligase were from Thermo Fischer Scientific, USA. Pharmacological inhibitors such as Gefitinib (PubChem CID: 123631), PD 181461 (PubChem CID: 9937619), U-73122 (PubChem CID: 5631),
Histamine H1R receptor stimulation causes ERK2 phosphorylation
Our preliminary experiments were aimed to identify if stimulation of endogenous histamine G-protein coupled receptor/s (GPCR) leads to phosphorylation of ERK protein in various cell lines. In our experiments, robust ERK phosphorylation was detected in HeLa cells stimulated with 100 μM histamine for 5 min, by western blot analysis using anti-pERK antibody targeting phospho-TEY motif in the ERK1/2 proteins (Fig. 1A; Supplementary Fig. 1A), as well as in A549 cells stimulated with 50 μM of histamine
Discussion
GPCR mediated cross-activation of MAP kinase is an area of intense research, however, the process still lacks detailed mechanistic insights and the observed physiological outcomes still don’t have clear molecular correlates. Histamine receptor mediated cross activation of MAPK pathways presents one such example wherein histamine stimulation has been reported to promote cellular proliferation in some cell types (Francis et al., 2012), while inhibits the proliferation in other cells (Notcovich et
Conclusions
In conclusion, we have reported that histamine mediated cross-activation of ERK is regulated by H1R activation, which tethers the activated ERK in the cytoplasm. Overall our study proposes an alternate GPCR- MAPK signaling cascade where the spatio − temporal dynamics of pERK generated in response to GPCR stimulation regulates the final outcome of MAP kinase signaling and hence opens up another layer of regulation of MAP kinase cascades by GPCR signaling.
Funding
This work was supported by Department of Science and Technology, India (Grant No. EMR/2014/000997) to DKS. The work is also support in part from Department of Science and Technology-Funds for Improvement of Science & Technology (DST-FIST), University Grants Commission, and Department of Biotechnology funding to Department of MRDG, IISc. RJ also acknowledges University Grants Commission for a Research Fellowship.
Author contributions
RJ, designed the study, performed the experiments, analyzed the data and wrote the manuscript; UW, performed the experiments and DKS, conceived the idea, analyzed the data and wrote the manuscript.
Conflict of interest
Authors have no conflict of interest to declare.
Acknowledgements
Technical help of Neha Somalwar for imaging experiments and Krishna Kumar Singh for MMP2 inhibitor analysis is acknowledged.
References (36)
- et al.
Differential kinetic and spatial patterns of Î2-Arrestin and G protein-mediated ERK activation by the angiotensin II receptor
J. Biol. Chem.
(2004) - et al.
Live cell imaging of ERK and MEK: simple binding equilibrium explains the regulated nucleocytoplasmic distribution of ERK
J. Biol. Chem.
(2005) - et al.
Regulation of CXCR4 signaling
Biochim. Biophys. Acta
(2007) - et al.
Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP(3)/Ca(2+) and cAMP-dependent signaling mechanisms
Lab. Investig. J. Tech. Methods Pathol.
(2012) - et al.
Distinct beta-arrestin- and G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2 activation
J. Biol. Chem.
(2006) - et al.
Activation of mitogen-activated protein kinase by HO: role in cell survival following oxidant injury
J. Biol. Chem.
(1996) - et al.
Examining the mechanism of Erk nuclear translocation using green fluorescent protein
Exp. Cell Res.
(2003) - et al.
Critical role of NADPH oxidase-derived reactive oxygen species in generating Ca2+ oscillations in human aortic endothelial cells stimulated by histamine
J. Biol. Chem.
(2002) - et al.
Beta-arrestin2 enhances beta2-adrenergic receptor-mediated nuclear translocation of ERK
Cell. Signal.
(2005) - et al.
Involvement of protein kinase Cdelta/extracellular signal-regulated kinase/poly(ADP-ribose) polymerase-1 (PARP-1) signaling pathway in histamine-induced up-regulation of histamine H1 receptor gene expression in HeLa cells
J. Biol. Chem.
(2011)