Role of G protein-coupled receptors-microRNA interactions in gastrointestinal pathophysiology

Abstract

G protein-coupled receptors (GPCRs) make up the largest transmembrane receptor superfamily in the human genome and are expressed in nearly all gastrointestinal cell types. Coupling of GPCRs and their respective ligands activates various phosphotransferases in the cytoplasm, and, thus, activation of GPCR signaling in intestine regulates many cellular and physiological processes. Studies in microRNAs (miRNAs) demonstrate that they represent critical epigenetic regulators of different pathophysiological responses in different organs and cell types in humans and animals. Here, we reviewed recent research on GPCR-miRNA interactions related to gastrointestinal pathophysiology, such as inflammatory bowel diseases, irritable bowel syndrome, and gastrointestinal cancers. Given that the presence of different types of cells in the gastrointestinal tract suggests the importance of cell-cell interactions in maintaining gastrointestinal homeostasis, we also discuss how GPCR-miRNA interactions regulate gene expression at the cellular level and subsequently modulate gastrointestinal pathophysiology through molecular regulatory circuits and cell-cell interactions. These studies helped identify novel molecular pathways leading to the discovery of potential biomarkers for gastrointestinal diseases.

receptor activation and its subsequent signaling represent an important part of cellular responses to a variety of external stimuli from the changing environment. Among different families of receptors, G protein-coupled receptors (GPCRs), which are also known as seven-transmembrane domain receptors, make up the largest transmembrane receptor superfamily in the human, mouse, Caenorhabditis elegans, and Drosophila genomes (96). Based on sequence homology analysis, the International Union of Basic and Clinical Pharmacology (IUPHAR)/The British Pharmacological Society Guide to Pharmacology has included a classification scheme that divides GPCRs as follows: class A (rhodopsin like), class B (secretin receptor family), class C (metabotropic glutamate), class D (fungal mating pheromone receptors), class E (cAMP receptors), and class F (frizzled/smoothened) (1), in which only classes A, B, C, and F are found in vertebrates. Alternatively, GPCRs are also grouped in five classes in the “GRAFS” classification system in which the five classes overlap with classes A–F in the IUPHAR classification scheme (108). In this article, the names or abbreviations of the GPCRs are used in agreement with official IUPHAR nomenclature, and the human gene symbols are written in italics.

GPCR Signal Transduction

GPCRs mediate a broad range of cellular responses to various extracellular stimuli, often in response to their ligands. GPCRs are essential to normal gastrointestinal functions, and their ligands include different types of molecules, such as peptides (e.g., neuropeptides), lipids (e.g., endocannabinoids), and proteins (e.g., chemokines) (32, 104). Structurally, a GPCR consists of an NH₂-terminal extracellular end, a core made of seven transmembrane α-helices, and a COOH-terminal cytoplasmic tail. In the unbound (inactivate) state, GPCRs are associated with the membrane-bound heterotrimeric guanine nucleotide-binding proteins (G proteins) at the COOH-terminus. GPCR-associated G proteins are comprised of α-, β-, and γ-subunits in which the α-subunit binds to a GDP molecule, and the β- and γ-subunits form stable Gβγ dimers. According to the classical view of GPCR signaling activation, coupling of a ligand at the NH₂-terminus mediates conformational changes in a GPCR and activates Gα subunit by exchanging the GDP for GTP, leading to the dissociation of the Gα subunit from the COOH-terminus and the Gβγ complex. The dissociated G protein subunits then interact with adenylyl cyclase or phospholipases, which are signal transducers located at the cell membrane, and generate secondary messengers, such as cAMP or phosphatidylinositol. On the other hand, activated GPCRs also transactivate various membrane-associated receptors, including epidermal growth factor receptor (EGFR), insulin-like growth factor receptor (IGFR), and transforming growth factor-α through highly regulated cross talk with receptor tyrosine kinases, matrix metalloproteinases, and integrins. Subsequently, the secondary messengers generated by the activation of GPCRs and the transactivation of other membrane-associated receptors initiate various signaling cascades by activating various downstream phosphokinases. These include mitogen-activated protein kinases (MAPKs), c-Jun NH₂-terminal kinases (JNKs), phosphatidylinositol 3-kinase (PI3K), and inhibitor of κB kinases (IκB kinases) and mediate appropriate cellular responses to external stimuli (reviewed in Refs. 19 and 104).

To prevent the detrimental effect from sustained G protein-dependent signaling activation induced by extracellular ligand binding, stimulated cells achieve cellular desensitization by internalization of the ligand-bound GPCRs. In β-arrestin (β-arrs)-dependent GPCR internalization, the dissociation of G proteins from a ligand-bound GPCR is followed by phosphorylation of the intracellular region and/or COOH-terminal cytoplasmic tail of the ligand-bound GPCR by G protein-coupled receptor kinases. β-Arrs are then recruited to the COOH-terminus of GPCR and, together with adaptor protein 2 (AP2) complex, dynamin, and clathrin, initiate the process of internalization with invagination of the cell membrane. Still bound to β-arrs, the ligand-bound GPCR is believed to be inactivated and transported in a clathrin-coated vesicle to the early endosomes, where β-arrs will be dissociated from the GPCR. The GPCR will then be either transported to lysosomal vesicles for degeneration or recycled to the cell membrane for ligand binding. This β-arrs-dependent clathrin-mediated internalization mechanism appears to be common among most of the GPCRs during the desensitization process, although there are exceptions where β-arrs are not involved (reviewed in Ref. 94). However, the functional relevance of β-arrs in GPCR signal transduction is not restricted to its role in GPCR desensitization. Accumulated evidence showed that β-arrs act as functional adaptors to signaling molecules, such as c-Src, IκB-α, and other key players in MAPK-, JNK-, and PI3K-protein kinase B (AKT) signaling pathways by bringing these signaling molecules to proximity to the ligand-bound GPCRs in the intracellular vesicles. This second wave of signal transduction is dependent on β-arrs as GPCR signal transducers in early endosomes, but independent of G protein signaling activation that occurred on the cell membrane. To add to the complexity of spatial and temporal aspects of GPCR signaling, a recent study by Irannejad et al. showed that the endosome-localized β₂-adrenoceptor is able to activate Gα subunits and generate cAMP after receptor internalization, in contrast to the classic G protein-dependent GPCR signaling activation model (51). Taken together, GPCR signal transduction exhibits complex spatial and temporal patterns and, thus, can mediate highly regulated and appropriate cellular responses to a broad range of external stimuli.

Activation of GPCR signaling in the intestine regulates many cellular and physiological processes, such as cell growth and proliferation (44, 63, 71), inflammation (131, 134), angiogenesis (3, 4, 50), absorption and secretion (45, 86), wound healing (44, 64), leukocyte trafficking (40), fibrogenesis (62), tumorigenesis (2, 14, 103), and intestinal motility (reviewed in Ref. 45). Importantly, GPCR signaling pathways are also targets of several existing gastrointestinal agents, such as drugs against diarrhea (opioid receptors), functional bowel disorder agents (receptors for acetylcholine, glutamate, and serotonin), gastrointestinal stimulants (dopamine and serotonin receptors), and H2 antagonists. Conventionally, studies on GPCRs and their associated protein effector signaling pathways provided significant information toward our understanding in gastrointestinal pathophysiology. However, epigenetic regulatory mechanisms of GPCR signaling evolved in recent years and represent a new interesting area to help us understand mechanisms regulating gastrointestinal pathophysiology. In this review, the interaction between GPCRs and a novel class of epigenetic regulators, microRNAs (miRNAs), will be discussed with emphasis on the gastrointestinal tract.

miRNA, an Epigenetic Regulator in GPCR Signaling

miRNAs are endogenously expressed short single-stranded RNA molecules (∼22 nucleotides) acting mainly as negative regulators in gene expression. Similar to GPCRs, miRNAs are found in C. elegans, Drosophila, mice, and humans. At the time of writing of this review, 2,588 mature human miRNAs registered in the miRbase (http://mirbase.org, Release 21, accessed February 2017) have been identified (66). Analysis from bioinformatics and proteomics estimated that 30–60% of gene expression in humans is regulated by miRNAs (33, 74, 109).

Biogenesis of miRNAs begins with the transcription of primary miRNAs (pri-miRNAs) by RNA polymerase II (11, 73) at the nucleus. pri-miRNAs are double-stranded RNA molecules in stem-loop structures, with sizes ranging from several hundred nucleotides to several kilobases. Inside the nucleus, pri-miRNAs are cleaved by a type III RNase, Drosha, to generate smaller-hairpin RNAs of ∼70 nucleotides called pre-miRNA (72). The shorter pre-miRNAs in stem-loop structures are then exported from the nucleus to the cytoplasm by exportin 5 (82, 126). This is followed by a second cleavage process in the cytoplasm. pre-miRNAs are cleaved by Dicer, another type III RNase, which releases double-stranded miRNAs from the loop regions (48). This RNA duplex immediately associates with members from the Argonaute protein family (AGO1–5 in humans) to form RNA-induced silencing complexes (RISC). The mature strand of miRNA is retained in the complex, whereas the other strand is degraded. RISC then binds to mRNA transcripts through imperfect base pairing. This results in mRNA decay by promoting deadenylation, decapping, and exonucleolytic degradation, in addition to translational repression (9). A more detailed description of miRNA biogenesis and processing is described elsewhere (8, 9, 41, 59).

Similar to GPCRs, miRNAs regulate a large number of signaling pathways, thereby modulating the pathophysiology of several gastrointestinal disease states, such as colon cancer and inflammatory bowel disease (IBD) (52, 56, 129). The ability of miRNAs to regulate gene expression at the epigenetic level by binding to their corresponding complementary sequences in the 3′-untranslated regions (UTRs) of different target genes allows GPCR signaling pathways to regulate different targets simultaneously upon activation. GPCR expression, in some instances, is also modulated by expression of miRNAs induced by extracellular stimuli. Thus, the potential GPCR-miRNA interactions can be summarized as follows. An external stimuli or GPCR-ligand coupling activates or attenuates the transcription of miRNA in the nucleus. Once in the cytoplasm, mature miRNAs bind to their complementary mRNA sequences and reduce target protein expression either by destabilizing mRNA transcripts and/or by attenuating the translation process. Target proteins can be downstream effectors of GPCR signaling pathways, proteins that facilitate intracellular trafficking of GPCRs, or a GPCR itself. It should be noted that multiple miRNAs are transcribed upon signal activation; therefore, GPCR-miRNA interactions orchestrate a cascade of modifications in protein expression or signaling activation at the cellular level (Fig. 1). In this review, examples of different GPCR-miRNA interactions relevant to various gastrointestinal physiological conditions and their significance will be discussed.

Fig. 1.

A representative diagram of possible G protein-coupled receptor (GPCR)-microRNA (miRNA) interactions at the cellular level. miRNA transcription is initiated by either external stimuli (a) or GPCR activation (b). Primary miRNAs (pri-miRNAs) are transcribed in nucleus (c) and exported to cytoplasm as smaller-hairpin RNAs of ∼70 nucleotides (pre-miRNAs) (d). miRNAs, together with Argonaute protein family (AGO) proteins, then bind to their corresponding complementary sequence in the 3′-untranslated region (UTR) of their target mRNA transcript, in which the target mRNA transcripts will be destabilized or their translation will be inhibited. Subsequently, the expression of target proteins is reduced. The above process affects the expression of proteins that are downstream of GPCR signaling activation (e), components that facilitate GPCR signaling activation (f), or GPCR itself (g).

Inflammatory Bowel Diseases

The intestinal wall is divided into different layers comprised of different tissues and cell types. The presence of different types of cells in specialized tissues in the gastrointestinal tract strongly suggests the importance of cell-cell interactions in maintaining gastrointestinal homeostasis. Furthermore, GPCRs are expressed in nearly all gastrointestinal cell types while their associated signaling pathways are crucial in maintaining various physiological and pathophysiological functions (3, 4, 40, 44, 45, 50, 63, 86). Taken together, GPCR-miRNA interactions may affect gastrointestinal homeostasis through cell-cell communication. Studies related to NK₁ receptor (TACR1), neurotensin-1 receptor (NTSR1), and their cellular and physiological roles in IBD are discussed below as examples.

IBD is a group of chronic inflammatory gastrointestinal disorders that includes Crohn’s disease and ulcerative colitis (UC), characterized by periods of persistent inflammation followed by remission. Studies have shown that miRNAs are differentially expressed in pediatric (65) and adult (98, 124, 125) colonic tissues, suggesting that miRNA expression is differentially regulated during colonic inflammation. NK₁ receptor is a GPCR from the class A (rhodopsin-like) family and a high-affinity receptor for the neuropeptide substance P (SP). NK₁ receptors are expressed in various gastrointestinal cell types, including colonic epithelial cells (36, 132), immune cells (116), fibroblasts (17, 62), mesenteric fat depots (37, 115), and the enteric nervous system (101). Thus, SP/NK₁ receptor signaling is involved in a wide range of physiological responses, including motility, pain sensation, neurotransmission, and inflammation in the gastrointestinal tract (reviewed in Ref. 119). In cellular responses associated with inflammation, binding of SP to NK₁ receptor activates the Rho family small GTPases and protein kinase C, leading to activation of kinases in MAPK, NF-κB, AKT, and JAK-STAT signaling pathways. Activation of these signaling pathways, in turn, is linked to increased interleukin-8 (IL-8) and prostaglandin E₂ synthesis via a cyclooxygenase-2-dependent mechanism (37, 132). In addition, SP/NK₁ receptor coupling also transactivates EGFR in murine colonic fibroblasts (17). Interestingly, part of the SP/NK₁ receptor-miRNA interactions represents a regulatory circuit on NK₁ receptor expression. In human astrocytoma cells, for example, prolonged exposure to SP leads to a reduction in NK₁ receptor expression that coincides with an elevated expression of miR-449b and miR-500 (105). Further bioinformatics analysis suggested NK₁ receptor as a direct downstream target of both miRNAs, and overexpression of miR-449b and miR-500 in human astrocytoma cells reduced NK1 receptor expression and activity (105). This study demonstrates the possibility that GPCR-miRNA interactions can directly regulate GPCR expression in a negative feedback manner. At present, the miRNAs that regulate NK₁ receptor expression in human colonic epithelial cells or other gastrointestinal cell types have not been identified.

The importance of NK₁ receptor signaling in IBD pathophysiology was demonstrated by the ability of NK₁ receptor antagonist to attenuate mucosal healing in an experimental colitis mouse model (64). NK₁ receptor expression is also increased in colonic tissues from experimental colitis models and in the colon of IBD patients (29, 36, 99). In addition, a recent study also showed that SP/NK₁ receptor signaling activation induced differential expression of 29 miRNAs in human colonic epithelial cells, including miR-221–5p (29). miR-221–5p is an oncogenic miRNA associated with prostate, breast, and colorectal cancer (34, 127, 135). In UC patients, miR-221–5p expression is increased in colonic tissues, alongside with expression of SP and NK₁ receptor (29). SP/NK₁ receptor-induced miR-221–5p expression is NF-κB- and JNK- dependent in human colonic epithelial cells and directly targets on expression of IL-6 receptor (IL6R), which is also downregulated in the tissues from colonic biopsies taken from UC patients, supporting the notion that IL6R is a direct target of SP/NK₁ receptor-miR-221–5p (29). Thus, expression levels of SP, NK₁ receptor, miR-221–5p, and IL6R can be used as markers of disease or disease progression in UC patients. Recent development of intracolonic delivery of oligonucleotides in vivo is shown to be mainly targeted at the gene expression level in colonic epithelial cells (47). With the use of this mode of delivery in experimental colitis mouse models, intracolonic administration of locked nucleic acid (LNA)-antisense-miR-221–5p exacerbated colitis development, as shown by increased proinflammatory cytokine expression and higher histology scores in colonic tissues (29). Together, these results suggest that miR-221–5p is involved in the anti-inflammatory network in colonic tissues comprised of the SP/NK₁ receptor-miR-221–5p-IL6R axis.

Several studies have shown that the 13-amino peptide/hormone neurotensin (NTS) is closely associated with IBD pathophysiology. NTS is found in the ileum (97) and colon (18) while its high-affinity receptor, NTSR1, is also a GPCR member of the class A (rhodopsin-like) family (121) expressed in different colonic epithelial cell lines, colonic adenocarcinoma tissues, and the enteric nervous system (2, 16, 38). In human colonic epithelial cells, NTS/NTS₁ receptor coupling activates NK-κB, MAPK signaling (131, 133, 134), and AKT (2, 130), at least in part through Rho GTPases (133) and/or transactivation of EGFR1 (134) and IGFR1 (130). Activation of these signaling pathways regulates chloride secretion (102), angiogenesis (3, 4), colonic inflammation (14, 18), wound healing (13), and tumorigenesis (2, 14). Importantly, NTS and NTS₁ receptor expression is increased in inflamed colonic tissues of experimental colitis models and UC patients (13, 14, 18, 61).

Recently, miRNA-associated epigenetic regulation induced by NTS/NTS₁ receptor signaling was studied in human colonic epithelial cells in vitro (2). The results demonstrated that NTS/NTS₁ receptor signaling activation led to a simultaneous dysregulation of 38 miRNAs in human colonic epithelial cells, including miR-210 and miR-133α (2). miR-210 has been previously associated with hypoxia, a process that plays a crucial role in regulating mucosal homeostasis during colitis development (110), through hypoxia-inducible factor-1α (HIF-1α) (20, 25, 46, 67). On the other hand, miR-133α expression is also closely related to the development of colorectal cancer (26, 55, 87, 122), of which IBD patients have a higher risk of developing. Similar to NTS and NTS₁ receptor, miR-210 and miR-133α levels are increased in colonic biopsies from UC patients (3, 69), suggesting the possibility that they can be used as biomarkers for UC. Along these lines, in vitro studies in human colonic epithelial cells show that gene silencing of miR-210 and miR-133α expression attenuates NTS-induced proinflammatory cytokine production (3, 51). Importantly, in vivo intracolonic knockdown of miR-210 and miR-133α in colon tissues alleviated colitis development and reduced proinflammatory cytokine production in colon tissues taken from experimental colitis mouse models (3, 69). In summary, blocking miR-210 and miR-133α overexpression in colonic epithelial cells ameliorates NTS/NTS₁ receptor-associated colonic inflammation in vivo.

Further investigation on the functional roles of individual miRNAs revealed the cellular and molecular pathways linking the GPCR-miRNA interactions observed at the cellular level to their effects in IBD pathophysiology. Similar to other GPCRs expressed on the cell membrane, membrane-bound NTS₁ receptor is internalized to the cytoplasm upon NTS binding. This leads to desensitization of the NTS₁ receptor to extracellular NTS (71, 92, 93) and prevents the cells from being overstimulated. On the other hand, the cells regain sensitivity toward NTS after the internalized NTS₁ receptor is recycled to the cell surface and ready for ligand binding (71). Taken together, continuous NTS/NTS₁ receptor signaling transduction in colonic epithelial cells largely depends on the regulation of NTS₁ receptor internalization and recycling to the cell surface. In human colonic epithelial cells, NTS/NTS₁ receptor-induced miR-133α expression promotes the recycling of the internalized NTS₁ receptor to the cell membrane of human colonic epithelial cells (70) through its direct downstream target, aftiphilin (AFTPH) (69), a protein localized in early endosomes and the trans-golgi network (43, 70). AFTPH is originally associated with proteins involved in intracellular trafficking, such as clathrin, AP1, and AP2 complexes (15, 43, 81), and its expression is reduced upon NTS exposure and miR-133α in human colonic epithelial cells in vitro (63). Further studies suggested that reduced AFTPH expression promotes NTS₁ receptor recycling and resensitization of human colonic epithelial cells to NTS stimulation (70). The above observations suggest that NTS/NTS₁ receptor-miR-133α-AFTPH interactions in colonic epithelial cells regulate NTS/NTS₁ receptor signaling by promoting resensitization of the cells toward NTS stimulation, which, in turn, directly affect other NTS/NTS₁ receptor effector pathways, such as NTS/NTS₁ receptor-miR-210 interactions.

In inflamed colonic tissues of experimental colitis mouse models and IBD patients, metabolism shifts toward hypoxia, which promotes inflammation and pathogenic angiogenesis (22). A recent study shows that NTS/NTS₁ receptor signaling activation promotes angiogenesis by stabilizing HIF-1α protein and increases vascular endothelial growth factor A expression in human colonic epithelial cells in vitro and in colonic tissues from experimental colitis mouse models (4). Because H1F-1α is a primary transcription regulator of miR-210 expression (25, 46, 67), NTS/NTS₁ receptor signaling activation also increases miR-210 expression in human colonic epithelial cells in vitro and in inflamed colonic tissues from experimental colitis mouse models (3, 4). Moreover, NTS/NTS₁ receptor-induced miR-210 expression lowers the expression of ephrin A3 (EFNA3), a direct miR-210 downstream target and an inhibitor of angiogenesis, both in vitro and in vivo (3, 30). Last, in vivo miR-210 silencing by intracolonic administration of LNA-antisense-miR-210 reduced colitis and colitis-associated angiogenesis (3). Taken together, NTS/NTS₁ receptor signaling activation in colonic epithelial cells promotes angiogenesis in vitro and in colonic mucosa during colitis development, at least partially through NTS/NTS₁ receptor-miR-210-EFNA3 interactions (3) (Fig. 2). Most importantly, however, these studies suggest that miR-210 represents a novel target for treatment of colonic inflammation and IBD, whereas measurements of the expression levels of miR-210 and its downstream target EFNA3 may represent potential biomarkers for UC diagnosis.

Fig. 2.

Representative diagram showing the physiological role of NK₁ and neurotensin-1 (NTS₁) receptor interactions with miRNAs on cellular and tissue levels. In colonic epithelial cells, substance P (SP)/NK₁ receptor coupling increases miR-221–5p expression through NF-κB and c-Jun NH₂-terminal kinase (JNK) activation, leading to a reduction in the expression of interleukin-6 receptor (IL6R), the downstream target of miR-221–5p. On the other hand, NTS/NTS₁ receptor coupling activates hypoxia-inducible factor-1α (HIF-1α)-regulated miR-210 transcription and reduces the expression of ephrin A3 (EFNA3), a direct target of miR-210, whereas NTS/NTS₁ receptor activation alleviates inhibition of miR-133α expression by zinc finger E-box-binding homeobox 1 and therefore reduces aftiphilin (AFTPH) levels. Our studies show that NTS/NTS₁ receptor-associated EFNA3 reduction contributes to increased angiogenesis, whereas NTS/NTS₁ receptor-associated AFTPH reduction promotes NTS₁ receptor recycling in vitro. Importantly, intracolonic administration of antisense miR-221–5p exacerbates experimental colitis development, whereas intracolonic administration of antisense miR-133α and antisense miR-210 ameliorates 2,4,6-trinitrobenzenesulfonic acid-induced colonic inflammation by reducing proinflammatory cytokine production and attenuating development of colitic phenotype.

In summary, the above studies of colonic inflammation in vitro and of mouse colitis models suggest that interactions between neuropeptides, their respective GPCRs, and their downstream miRNA targets modulate cellular signaling pathways that can be self-regulatory or involved in cell-cell interactions. Thus, GPCR-miRNA interactions regulate important components of gastrointestinal mucosal homeostasis.

Irritable Bowel Syndrome

Another intestinal disorder closely related to GPCR signaling activation is irritable bowel syndrome (IBS). IBS is characterized by chronic abdominal pain or discomfort associated with changes in bowel habits, including diarrhea, constipation, or both. Thus, IBS can be further divided into IBS-D, IBS-C, and IBS-M. Although the disease mechanisms of IBS are not well understood, studies have identified a list of related risk factors, including acute psychological stress and experience of stressful life events (reviewed in Ref. 28).

Corticotropin-releasing hormone (CRH), through its receptors, CRF₁ and CRF₂ receptors, is an important mediator in stress-related physiological responses from the GPCR class B (secretin receptor) family. Besides CRH, other ligands of the CRF receptors include urocortin (UCN), UCN2, and UCN3. Originally identified in the central nervous system, CRF₁ receptor can also be localized in myenteric and submucosal nervous plexus of the distal gut, whereas CRF₂ receptors are localized in the luminal surface of crypts and myenteric neurons (reviewed in Ref. 120). Previous studies showed that chronic stress activates CRF₁ receptor signaling and mediates the visceral hyperalgesia and inflammation in mesenteric adipose tissue in rats (58, 68). Although little is known about the effect of chronic stress on miRNA expression in intestine and colon, chronic water avoidance stress in rats induces visceral hyperalgesia and increases miR-17–5p expression in the dorsal horn of the spinal cord, which subsequently is found to regulate IL-6 expression and STAT3 activity (12). More importantly, reducing miR-17–5p levels in the spinal cord by intrathecal administration exacerbates stress-induced visceral hyperalgesia, supporting the notion that miR-17–5p has a potential modulatory role in stress-associated glial activity and neuroimmunomodulation (12). On the other hand, acute stress induces glucocorticoid feedback and reduces CRF₁ receptor expression by increasing miR-449a levels in the rat pituitary (89). In addition, UCN2 increases miR-325–3p expression in rat pituitary cells in vitro and suppresses luteinizing hormone in rat pituitary during acute stress in vivo (88). In summary, signaling activation and expression of CRF receptors are associated with dysregulated miRNA expression in the central nervous system during acute and chronic stress and possibly mediates downstream effects in the gastrointestinal tract. Therefore, it is likely that stress regulates miRNA expression in the enteric nervous system along the gastrointestinal tract and mediates localized effects.

Opioid receptors belong to the GPCR class A (rhodopsin-like) family and are drug targets to relieve symptoms in IBS patients. Opioid receptors are expressed in the central and the enteric nervous system and classified as μ (OPRM1)-, κ (OPRK1)-, and δ (OPRD1)-receptors, with μ receptor representing a major opioid receptor in the small intestine and colon. Ligands of μ-receptors include the alkaloid morphine, which is extracted and purified from opium, and the endogenously produced ligands β-endorphin (β-endorphin, POMC), methionine-enkephalin, and leucine-encephalin ([met]- or [leu]-enkephalin, respectively, gene symbol: PENK). Along the gastrointestinal tract, μ-receptor signaling activation regulates motility, secretion, and peristalsis (reviewed in Ref. 45). Recently, studies on opioid tolerance in human neuroblastoma cells in vitro and in mouse models show that morphine treatment in vitro and in vivo increases expression of miRNAs of the let-7 family (42) and miR-103/-107 (80) and reduces μ-receptor expression by directly targeting sequences on the 3′-UTR of μ-receptor, which leads to opioid tolerance in vitro and in vivo. On the other hand, miR-134 also directly regulates μ-receptor expression in human neuroblastoma cells in vitro, whereas in a chronic inflammatory pain model miR-134 expression is reduced in the dorsal root ganglia, resulting in μ-receptor overexpression in mice (90). The above studies suggest that μ-receptor-miRNA interactions in neurons are regulated by μ-receptor signaling activation and peripheral inflammation. The role of miRNAs in the endogenous μ-opioid system expressed in the central nervous system was recently reviewed by Barbierato et al. (6). The enteric nervous system is an important component in gastrointestinal tract associated with inflammation and intestinal motility. Therefore, further studies on GPCR-miRNA interactions in the enteric nervous system will contribute to our understanding on their role in maintaining gastrointestinal homeostasis and functions.

miRNAs and Gastrointestinal Cancers

According to World Health Organization statistics, there were 774,000 and 754,000 deaths caused by colorectal cancer and gastric cancer, respectively, in 2015 (http://www.who.int/mediacentre/factsheets/fs297/en/, February 9, 2017), making these two gastrointestinal cancers the third and the fourth most common causes of cancer deaths in the world. Table 1 presents a list of GPCRs associated with gastric and colorectal cancer pathophysiology and the miRNAs involved in regulating their expression or downstream signaling pathways. These GPCRs include C-C motif chemokine receptor type 7 (CCR7) (123), C-X-C motif chemokine receptor 4 (CXCR4) (5, 27, 76, 78, 79), CCK₂ receptor (cholecystokinin B receptor, CCKBR) (118), calcium-sensing receptor (CASR) (31, 117), NTS₁ receptor, EP₂ receptor (prostaglandin E₂ receptor, PTGER2) (113), and EP₄ receptor (PTGER4) (21, 113). Among these GPCRs, studies in patients and tissues from biopsy material suggest that chemokine receptors, such as CXCR4, are one of the GPCRs initiating important signaling pathways in gastrointestinal cancer pathophysiology (reviewed in Ref. 85).

Table 1.

Summary of studies related to GPCR-associated miRNAs and gastrointestinal cancers

Pathophysiology	microRNA	Association with GPCR	Ref. No.
Gastric cancer	let-7a	Negatively correlates with CCR7 gene expression	123
	miR-139	Regulate CXCR4 levels	5
	miR-16, miR-21	EP2- and EP4- receptors signaling regulates miR-16 and miR-21 levels	113
Colon cancer	miR-21, miR-155	These miRNAs are differentially regulated by NTS1 receptor signaling	2
	miR-135b, miR-146b	Regulates CaS receptor	31
	miR-145, miR-21, miR-135a, miR135b	These miRNAs are differentially regulated in CaS receptor-null cells	117
	miR-101	Regulates EP4 receptor expression	21
	miR-126	Regulates CXCR4 expression	76, 78, 79
	miR-133b	Regulates CXCR4 expression	27
	miR-148b	Regulates CCK2 receptor expression	118

miRNA, microRNA; GPCR, G protein-coupled receptor; CCR7, C-C motif chemokine receptor type 7; CXCR4, C-X-C motif chemokine receptor 4; EP₂, prostaglandin E receptor 2; EP₄, prostaglandin E receptor 4; NTS1, neurotensin 1 receptor; CaS, calcium-sensing receptor; CCK2, cholecystokinin B receptor. Bold type indicates GPCRs.

In gastric cancer, expression of erb-b2 receptor tyrosine kinase 2 (HER2) is associated with an increased rate of metastases in gastric cancers (23, 35). Previous studies showed that HER2/CD44 signaling activation downregulates CXCR4 expression by increasing histone deacetylation in the miR-139 promoter region (5). Importantly, two additional studies also provided evidence that miR-139 is downregulated in HER2-positive gastric carcinomas (57) and primary gastric cancer tissues from patients (39). Furthermore, similar observations were made in patients with laryngeal squamous cell carcinoma in which miR-139 also targets CXCR4 and inhibits the proliferation and metastasis of laryngeal squamous carcinoma cells (83). Results from the above studies suggest that miR-139 is a miRNA with tumor-suppressing properties; however, the functional role of miR-139 in gastric cancer in vivo needs further investigation. On the other hand, studies in colon cancer have also identified miRNAs acting as tumor suppressors by directly regulating the expression of CXCR4, such as miR-126 (76, 78, 79) and miR-133b (27). Previous studies showed that miR-126 directly targets the 3′-UTR sequence of CXCR4 in colon cancer cells (76, 78). Low expression of miR-126 is associated with poor prognosis (79) and increased migration and invasion of colon cancer cells (76). Furthermore, increased miR-126 levels in human colon cancer cells in vitro reduce cell migration and invasion by reducing CXCR4 expression (76, 78), possibly by suppressing AKT and ERK1/2 activation (78). On the other hand, expression of miR-133b and CXCR4 is inversely correlated in human colorectal cancer tissues compared with nonneoplastic adjacent tissues (27). In addition, miR-133b also directly regulates CXCR4-associated cell invasion and migration in vitro (27). These studies suggested that increased CXCR4 is associated with increased metastasis and poor prognosis in human colon cancer, possibly by promoting cell migration resulting from the downregulation of tumor suppressor miR-126 and miR-133b. In the above examples, miRNA expression induced by upstream signaling events or endogenously expressed miRNAs directly regulate CXCR4 expression in gastrointestinal cancer cells and thus affect CXCR4-mediated cellular functions along the gastrointestinal tract.

As noted in the examples of NK₁ receptor (105) and CXCR4 (27, 76, 78, 79), expression of these GPCRs can be simultaneously targeted by two miRNAs in the same cell type. The recent development of high-throughput gene expression analysis and the subsequent bioinformatics analysis have enabled researchers to study the complex pattern and dynamic nature of gene expression in many tissue and cell types. Results from these studies revealed that miRNA-mRNA interactions often participate in molecular regulatory circuits. NTS₁ receptor signaling pathways in human colon cancer cells are discussed below as an example.

NTS₁ receptor activation in human colonic epithelial cells induced dysregulation of the expression of 38 miRNAs simultaneously (2). Among these miRNAs, NTS-induced miR-21 and miR-155 expression suppresses the expression of phosphatase and tensin homolog (PTEN) and suppressor of cytokine signaling, which represent downstream targets of miR-21 and miR-155, respectively (49, 54), in human colorectal carcinoma HCT-116 and adenocarcinoma DLD-1 cells (2). Whereas NTS/NTS₁ receptor signaling has been shown to activate AKT in human colonic epithelial cells (130), PTEN is also a well-established negative regulator of AKT activity. Therefore, this study provides evidence that miR-21 is involved in NTS-induced AKT activation through suppressing PTEN expression. More importantly, this study also revealed a synergistic effect of AKT activation by NTS-induced miR-21 and miR-155 overexpression. Bioinformatics analysis suggested that miR-155 directly inhibited the expression of protein phosphatase 2 catalytic subunit-α (PPP2CA), which is another well-known negative regulator of AKT activity (100). Subsequent examination showed that NTS/NTS₁ receptor-miR-21-miR-155 interactions regulate cell proliferation, migration, and invasion in vitro and in mouse cancer xenograft models (2). Last, this study also identified a positive feedback loop regulating NTS₁ receptor-associated NF-κB and AKT activities by showing increased AKT activity suppresses NT-induced NF-κB activation. Importantly, this positive feedback loop is regulated by NTS-induced miR-21 and miR-155. Taken together, NTS/NTS₁ receptor-miR-21-miR-155 interactions alleviate suppression on PTEN and PPP2CA expression and thus activate AKT and initiate a positive feedback loop of NTS/NTS₁ receptor signaling in human colon cancer cells.

Stem Cells

Accumulated evidence from studies on various GPCRs indicate that GPCR-miRNA interactions help maintain important aspects of gastrointestinal homeostasis, including cell proliferation (84, 111), immune tolerance (24), and angiogenesis (60, 106, 136) (summarized in Table 2). Recently, intestinal stem cells became an important component of mucosal healing. Moreover, primary intestinal stem cells taken from different mouse models can now be routinely differentiated into organoids and used as tools to study intestinal epithelial cell biology. Leucine-rich repeat-containing GPCR 5 (LGR5) is an important marker of small intestinal and colonic stem cells for crypt regeneration and in vitro expansion (7, 107, 128). It is a class A (rhodopsin-like) GPCR and a part of the Wnt signaling pathway expressed in intestinal and colonic stem cells (7) and colonic adenomas (10). Previous studies showed that miR-142–3p and miR-100 directly target the 3′-UTR of LGR5 gene in colon cancer cells (111, 136) and therefore have been proposed as tumor suppressors. Importantly, both miRs are downregulated in colon cancer tissues from the patients (111, 136). Furthermore, miR-142-LGR5 interaction in colon cancer cells is associated with drug-induced apoptosis in vitro (111). However, these particular interactions have yet to be confirmed in normal intestinal and colonic stem cells in vitro and in vivo. Understanding the interactions between miRNAs and LGR5 may provide insights to the epigenetic regulation in intestinal and colonic stem cells during normal crypt regeneration and healing processes.

Table 2.

Smmary of studies related to GPCRs, their associated miRNAs, and their proposed physiological roles in gastrointestinal studies

Function	miRNA	Association with GPCR	Ref. No.
Proliferation	miR-142–3p	Regulate CD133, ABCG2, and LGR5	111
	miR-34a	Regulate PAR2-induced CyclinD1	84
Monocytes/immune tolerance	miR-525–5p	Negatively regulates VPAC1 receptor expression	24
Angiogenesis	miR-802	Regulates AT1 receptor expression	106
	miR-27b, miR-218a	Regulates A2B receptor expression	60
	miR-100	Inhibit LGR5 expression	136

LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; PAR2, protein-activated receptor 2; VPAC1, vasoactive intestinal peptide receptor 1; AT₁, angiotensin II receptor type 1; A_2B, adenosine A_2b receptor. Bold type indicates GPCRs.

Clinical Implication of GPCR-miRNA Interactions.

As discussed in this review, studies with miRNAs identified novel GPCR signaling pathways as participating in several important gastrointestinal responses. Given the importance of GPCR signaling activation in gastrointestinal pathophysiology, components of GPCR signaling pathways, including miRNAs, can be potentially developed into therapeutic targets or biomarkers of drug efficacy and gastrointestinal disease modulation. From the clinical perspective, because GPCR signaling pathways in gastrointestinal tissues are highly regulated and organized in molecular regulatory circuits, measuring biomarkers developed from a combination of miRNAs and other GPCR signaling components may represent a more comprehensive view for gastrointestinal disease prognosis. In the case of human colorectal cancer, the study on NTS₁ receptor-associated miRNA expression in human colon cancer cells provided evidence showing that the NTS-induced signaling pathway is activated in a molecular regulatory circuit involving increased expression of miR-155 and miR-21 and AKT activity in vitro (2). To associate these gene expression changes to human colon cancer development, expression of NTS₁ receptor, miR-155, and miR-21 was examined and found upregulated significantly in human colon tumors compared with normal colon tissues. Furthermore, the expression of these two miRNAs is positively correlated to NTS₁ receptor expression in human colon tumors, whereas the expression of all these genes is also correlated with tumor stages (2). Interestingly, miR-21 expression has also been shown to be increased in 5-fluorouracil-resistant human colon cancer cells and negatively correlated to patient survival (91). Taken together, combined use of NTS₁ receptor, miR-155, and miR-21 expression in human colon tumors as biomarkers may serve as a useful indicator to the disease state and possibly response to treatment.

Alternatively, miRNAs could by themselves serve as drug targets, since therapies directed against the downstream mediators of GPCR signaling may provide more selective targeting of aberrant signaling cascades implicated in gastrointestinal pathophysiology. Although, in theory, delivery of antisense oligonucleotides to the appropriate tissue targets can neutralize the effects of the corresponding miRNAs, unmodified antisense oligonucleotides are prone to degradation by serum exonucleases and cellular endonucleases and have relatively poor binding affinity to tissues. Thus, antisense oligonucleotides with various chemical modifications, such as 2′-O-methyl modification of nucleotides, formation of phosphorothioate bonds by replacing nonbridging oxygen atoms in the phosphate backbone with sulfur atoms, and LNA modifications that lock the sugar structure of the nucleotide into a 3′-endo conformation, are devised to optimize the nuclease resistance of antisense oligonucleotides and their binding affinity to tissues (reviewed in Ref. 77). These modified antisense miRNAs have shown great promise in bringing therapies directed against miRNAs to clinical use. In a phase 2a study, a LNA-modified DNA phosphorothioate antisense oligonucleotide directed against miR-122 was developed to sequester mature miR-122 and inhibit its function in the context of hepatitis C virus (HCV) infection (53). The results suggested that the chemically optimized antisense miR-122 elicited a durable dose-dependent reduction in HCV RNA levels without evidence of viral resistance (53), opening up the possibilities for therapeutic use of antisense miRNA approaches in patients. Last, because various GPCRs, such as the ones mentioned in this review, are overexpressed in gastrointestinal cell types in different disease states, the recent development of surface receptor-targeted delivery using nanovectors may provide increased cellular specificity to the antisense oligonucleotide delivery. Although, at present, there are no known studies of miRNA delivery using nanovectors targeting GPCRs, results from two recent studies suggested the potential of target delivery of miRNAs using GPCRs as targets. In one study, Li et al. showed that administration of nanovectors labeled with antibodies against surface markers of pancreatic cancer cells encapsulating antisense miR-21 showed increased cellular uptake of antisense miR-21 and inhibited tumor growth in mice (75). In another study, nanovectors conjugated with octreotide, a synthetic ligand of somatostatin receptors, have successfully targeted somatostatin receptor-overexpressing tumor cells (95). Taken together, development of nanovectors conjugated with GPCR ligands or labeled with antibodies against GPCRs for delivering chemically optimized miRNAs may be one of the potential therapeutic strategies for GPCR-associated pathophysiology.

In summary, studies on GPCR-miRNA interactions may also lead to novel biomarkers or pharmaceutical interventions targeting gastrointestinal disorders. These studies suggest the possibility of using miRNAs in clinical settings, such as biomarkers or drugs that intervene with GPCR-associated miRNA functioning in disease pathology.

Conclusions

GPCR signaling plays a major role in gastrointestinal pathophysiology and is targeted by various pharmacological agents. miRNAs are a new class of negative regulators regulating gene expression. In this review, studies on miRNAs regulating GPCR expression and miRNA expression induced by GPCR signaling activation in cell types residing in gastrointestinal tracts were discussed. Although miRNAs are non-protein-coding nucleotides, results demonstrating that GPCR signaling activation induces expression of multiple miRNAs and the ability of a miRNA to target on 3′-UTRs of different genes make miRNA an important amplifier of the GPCR signaling cascade. Furthermore, GPCR-miRNA interactions occurring in a single cell type in the gastrointestinal tract can potentially regulate physiology at both cellular and tissue levels because of the close proximity of different cell types and their frequent interactions within the gastrointestinal tract. This complex network of gene regulation may explain the complexity of interactions modulating gastrointestinal homeostasis.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-60729, DK-47373, CURE:DDRC P30-DK-41301(C. Pothoulakis), DK-110003 (D. Iliopoulos), T32-AM-41301 (D. Padua), fellowships from the Crohn’s and Colitis Foundation of America (I. K. M. Law), the Blinder Research Foundation for Crohn’s Disease (C. Pothoulakis), and the Eli and Edythe Broad Chair (C. Pothoulakis).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

I.K.L. prepared figures; I.K.L. drafted manuscript; I.K.L., D.M.P., D.I., and C.P. edited and revised manuscript; C.P. approved final version of manuscript.