G-protein-coupled receptors constitute the largest and most diverse family of integral membrane proteins and are involved in a wide variety of physiological functions. Some of these functions include mediating responses to hormones, neurotransmitters, odorants and light, and regulation of the immune system and inflammation (Yousefi et al. 2001; Lattin et al. 2008; Weis and Kobilka 2008; Gloriam et al. 2009). GPR84 belongs to the rhodopsin subfamily of G-protein-coupled receptors and was first discovered during a comprehensive search of available expressed sequence tags (EST; Wittenberger et al. 2001). Since its discovery, GPR84 has been found in many human tissues, in mouse spleen, liver, spinal cord and sciatic nerve, in embryonic Xenopus laevis tissues, and zebrafish intestine, heart, and liver. This receptor was considered an orphan receptor until late 2006, when it was discovered that GPR84 functions as a receptor for medium-chain free fatty acids (FFAs, Wang et al. 2006) containing 9–14 carbons.
Structure and Activation of GPR84
The arrangement of the transmembrane domains creates a ligand-binding pocket or receptor that is important for signaling events. Activation of this receptor occurs when the ligand binds, resulting in an interaction of the cytoplasmic part of the receptor with the heterotrimeric G protein (Weis and Kobilka 2008). The cellular response to the extracellular signal is mediated through second messenger cascades and this attenuation of GPCR signaling can be achieved by phosphorylation of the receptor. This results either in a conformational change affecting G protein binding or by reduced cell surface expression.
Identification of the ligands that activate GPCRs has been ongoing and many receptors still remain classified as orphan receptors. Initially, a library of biochemical intermediates was used to identify the ligands capable of activating GPR84 (Wang et al. 2006). The study revealed that GPR84 is not activated by short-chain FFAs or by long-chain saturated and unsaturated FFAs but instead by medium-chain free fatty acids (FFAs) with carbon lengths of 9–14. These agonists included nonanoic acid (C9), capric acid (C10), undecanoic acid (C11), lauric acid (C12), tridecanoic acid (C13), and myristic acid (C14). The most potent endogenous agonists identified from this study included capric acid, undecanoic acid, and lauric acid; however, none of these activated GPR84 with greater potency than the small molecule diindolylmethane (Takeda et al. 2003; Wang et al. 2006). The lack of highly potent and selective ligands for the study of GPR84 physiological functions initiated several small molecule screening projects. These efforts resulted in the discovery of a few robust small molecule agonists, including 6-n-octylaminouracil (6-OAU; Suzuki et al. 2013), 2-(hexylthio)pyrimidine-4,6-diol (ZQ-16; Zhang et al. 2016), and the most recently discovered 6-Nonylpyridine-2,-4-diol (Liu et al. 2016). To date, 6-Nonylpyridine-2,-4-diol is the most potent agonist of GPR84.
Role in Immune Response
Several studies have emerged that have given some insight into the role of GPR84 during immune response in humans and mice. During a screen of human peripheral blood neutrophils, the EX33 gene (later named GPR84) was found and identified as a novel G-protein-coupled receptor (Yousefi et al. 2001). Since movement of neutrophils into inflamed tissues is mediated by chemokines that regulate signaling through GPCRs (Adams and Lloyd 1997), it was of particular interest to determine whether EX33 was involved in this type of signaling mechanism. Additionally, identification of chemoattractants and their receptors has implications in the treatment of inflammatory diseases. Experiments were conducted to test EX33 expression and revealed its presence in a variety of human cells and tissues (bone marrow, lung, colon, placenta). Abundant expression was noted in neutrophils and eosinophils in comparison with significantly lower levels in other cell types. This study was of particular interest because it presented the first evidence that EX33 was regulated by agonist-dependent mechanisms. In experiments where neutrophils were stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF; an inflammatory cytokine), lower levels of EX33 proteins were detected, as compared to unstimulated neutrophils. In addition to its accepted involvement in neutrophil activation, the data suggest that GM-CSF might also be involved in the hindrance of neutrophils at the site of inflammation by downregulation of cell surface receptors (Yousefi et al. 2001).
Another study to examine the biological function of GPR84 in mice utilized GPR84 knockout animals (Venkataraman and Kuo 2005). The proliferation rates of T and B cells were found to be similar in wild-type animals versus GPR84 knockout mice and though GPR84 is not necessary to modulate T and B cell proliferation, the absence of GPR84 in CD4+ T cells results in the increased production of IL-4 by anti-CD3-activated cells. Additionally, when T cells were cultured in the appropriate stimuli, differentiation into T helper 1 (Th1) or T helper 2 (Th2) effector populations occurred. Those Th2 effector cells cultured from GPR84-deficient mice consistently secreted higher levels of IL-4, IL-5, and IL-13 in comparison to wild-type animals. Early cytokine signaling mechanisms are poorly understood; yet, it can be suggested that hyper-Th2 cytokine production in GPR84-deficient mice can be correlated with their ability to synthesize more IL-4 protein during initial stimulation under Th2 differentiation conditions.
The inflammatory response of GPR84 was also noted in microglia (macrophage population of the brain) of mice analyzed after lipopolysaccharide (LPS)-induced endotoxemia (Bouchard et al. 2007). GPR84 is strongly expressed here and also in peripheral monocytic cells of the spleen. Additional evidence reveals that GPR84 is also expressed in clusters near blood vessels in microglia with induced experimental autoimmune encephalomyelitis (EAE) and the abundance of labeled clusters increases with the severity of the disease. Endogenous or exogenous inflammatory stimuli are capable of inducing GPR84 gene transcription in the microglia. Soluble mediators such as TNF and IL-1 mediate the effect of LPS on GPR84 expression in the brain (Bouchard et al. 2007). The higher levels in microglia and populations of peripheral macrophages suggest that GPR84 may play a predominant role in populations of macrophages. Additional research has revealed that GPR84 is restricted to bone-marrow macrophage (BMM) and microglia in an unstimulated state (Lattin et al. 2008). However, upon stimulation with LPS, GPR84 is significantly upregulated in BMM and thioglycollate-elicited peritoneal macrophages (TEPMs), consistent with the previously mentioned study. These studies reveal a role for GPR84 in neuroinflammation and may present a useful tool in tracking macrophage populations.
Previous investigations have also shown that chronic inflammatory pathway activation is involved in insulin resistance. In particular, macrophages migrate into adipose tissue and initiate the proinflammatory pathways in macrophages, resulting in the secretion of cytokines. The result is increased inflammation and decreased insulin sensitivity in surrounding insulin-targeted cells. Nagasaki et al. (2012) investigated the role of GPR84 in adipocytes with induced inflammatory situations. Their study found that GPR84 mRNA expression was undetectable in 3T3-L1 cells (preadipocytes) but upregulated in induced 3T3-L1 adipocytes. Obesity-induced expression of GPR84 mRNA was also observed in the epididymal fat pad of lab mice. Macrophage cells (RAW254) cocultured with fully differentiated 3T3-L1 adipocytes enhanced the expression of GPR84 mRNA, suggesting that adipocytes were capable of synthesizing GPR84 in the presence of a bioactive substance secreted by the macrophage cells. This study also determined that the addition of TNFα or LPS markedly increased the levels of GPR84 mRNA expression, leading to the conclusion that GPR84 could serve as a marker for obesity-induced inflammation in adipose tissue (Nagasaki et al. 2012). These results in combination with TNFα-NF-κB pathway inhibition, which suppressed TNFα-induced up-regulation of GPR84 mRNA, suggest that GPR84 is regulated by the TNFα-NF-κB pathway.
Role in Stem Cell Maintenance and Cancer
Stem cell self-renewal and differentiation requires a delicate balance in order to prevent the aberrant overgrowth of certain tissues. The alteration of cell signaling pathways can have disastrous effects and can lead to the transformation of healthy cells into cancerous ones. An example of this is the dysregulation of the Wnt/β-catenin signaling pathway; activation of Wnt/β-catenin can change normally healthy hematopoietic stem cells into leukemic stem cells (Wang et al. 2010; Yeung et al. 2010). More importantly, in these leukemic stem cells GPR84 was identified as a novel regulator of β-catenin signaling (Dietrich et al. 2014). Functional studies revealed that overexpression of GPR84 led to the activation of β-catenin transcription factors (Tcf 712, cFos) and genes involved in Wnt signaling (Sato et al. 2004). Further, depletion of GPR84 impaired leukemic stem cells and inhibited the development of an aggressive β-catenin drug-resistant subtype of acute myeloid leukemia (Dietrich et al. 2014). Rescue of this β-catenin deficient phenotype can be accomplished by the addition of β-catenin (Sato et al. 2004). Overall, these studies present compelling evidence in support of the development of novel therapeutic strategies designed to inhibit GPR/β-catenin signaling in order to target drug-resistant malignant stem cells associated with cancer (Lynch and Wang 2016).
Role in Fatty Acid Metabolism
In the presence of G protein Gqi9, both human and murine GPR84 are activated by medium-chain FFAs, suggesting that signaling occurs through the pertussis-sensitive Gi/o pathway. GPR84 does not signal through the Gs-mediated pathway and is unlikely to signal through the Gq pathway.
Medium-chain FFAs were also shown to amplify LPS-stimulated production of IL-12 p40 directly through GPR84 (Wang et al. 2006). In the presence of naturally occurring medium-chain FFAs (capric acid, undecanoic acid, lauric acid) or in the presence of the small molecule agonist, diindolylmethane, an increase was observed in the secretion of IL-12 p40 subunit from LPS-stimulated RAW264.7 cells. IL-12 p40 is a proinflammatory cytokine responsible for the eradication of pathogens. This cytokine induces and maintains Th1 responses and inhibits Th2 responses (Scott 1993; Hsieh et al. 1993; Kopf et al. 1994). Consistent with these results is the increased production of Th2 cytokines in GPR84-deficient T cells (Venkataraman and Kuo 2005). Combined, these studies emphasize a role for GPR84 as a leukocyte-specific receptor for medium-chain FFAs.
Role in Development
Recent studies have focused on GPR84s role in immune response and so far little is known about its role during development. One study showed that GPR84 expression is important for proper morphogenesis of the eye in the frog, Xenopus laevis (Perry et al. 2010). RT-PCR analysis of various embryonic stages of Xenopus shows that GPR84 mRNA is present beginning at gastrulation through larval stages and more specifically in the retina, lens, and larval cornea epithelium. Previous mouse and human studies have not noted expression in the eye; however, these specific tissues do not appear to have been examined (Yousefi et al. 2001; Venkataraman and Kuo 2005; Wang et al. 2006; Bouchard et al. 2007; Lattin et al. 2008).
GPR84 MO-mediated knockdown experiments revealed a role for GPR84 specifically in lens and retina morphogenesis (Perry et al. 2010). Significantly smaller lenses were formed that did not display secondary fiber cells and had defects in the lens epithelium. The retina was also significantly impacted, showing signs of improper differentiation of various neural retinal layers. Cell proliferation and apoptosis assays also show that GPR84 knockdown tissues not only display a higher number of proliferating cells but also a higher number of apoptotic cells. Previous studies (Ohnuma et al. 2002; Casarosa et al. 2003) have demonstrated that the proper development of cells depends on the delicate balance of cell proliferation and fate determination. Impairing a cell’s ability to exit the cell cycle can have significant implications in terms of the ultimate cell fate. It is possible that GPR84 could be a negative regulator of cell proliferation in the retina and lens, where suppressing proliferation at specific time points may be important for differentiation of the neural retina, lens, and cornea.
GPR84 is a transmembrane receptor originally identified through an EST search. Various medium-chain FFAs act as ligands to activate and induce a conformational change in the receptor. Interaction between the intracellular portion of the receptor and heterotrimeric G proteins results in activation of second messenger cascades and effector proteins. Mediation of cell signaling occurs through phosphorylation of GPR84 and results in lower binding efficiencies or reduced cell surface expression. Further analysis to determine the conformation of the active and inactive receptor will yield a better understanding of this signaling mechanism. GPR84 expression has been noted to be higher in neutrophils and eosinophils. Inflammatory stimuli are able to induce GPR84 transcription in microglia and in clusters near blood vessels of microglia and can be mediated by TNF and IL-1. Chronic inflammatory pathway activation also leads to induced GPR84 expression in adipocytes. GPR84-deficient mice result in CD4+ T cells that produce higher levels of IL-4 and culturing those T cells produces Th2 effector cells that secrete higher levels of IL-4, IL-5, and IL-13. Increased secretion of IL-12 p40 occurs in LPS-stimulated RAW264.7 cells in the presence of medium-chain FFAs or diindolylmethane. GPR84-mediated dysregulation of Wnt/β-catenin in healthy hematopoietic stem cells leads to leukemic stem cells. Xenopus eye development is also dependent on proper GPR84 expression. Knockdown of GPR84 expression results in defective lens formation, an improperly differentiated neural retina, higher proliferation rates, and an increase in cells undergoing apoptosis. Overall, GPR84 plays an important role in development, free fatty acid metabolism, immune system regulation, and stem cell maintenance. Further understanding of the GPR84 signaling cascade may lead to promising therapies/drugs for the treatment of metabolic and autoimmune diseases, as well as cancer.
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