Spexin Diminishes Atrial Fibrillation Vulnerability by Acting on Galanin Receptor 2.

BACKGROUND: G protein-coupled receptors play a critical role in atrial fibrillation (AF). Spexin is a novel ligand of galanin receptors (GALRs). In this study, we investigated the regulation of spexin and GALRs on AF and the underlying mechanisms. METHODS: Global spexin knockout (SPX-KO) and cardiomyocyte-specific GALRs knockout (GALR-cKO) mice underwent burst pacing electrical stimulation. Optical mapping was used to determine atrial conduction velocity and action potential duration. Atrial myocyte action potential duration and inward rectifying K+ current (IK1) were recorded using whole-cell patch clamps. Isolated cardiomyocytes were stained with Fluo-3/AM dye, and intracellular Ca2+ handling was examined by CCD camera. A mouse model of AF was established by Ang-II (angiotensin II) infusion. RESULTS: Spexin plasma levels in patients with AF were lower than those in subjects without AF, and knockout of spexin increased AF susceptibility in mice. In the atrium of SPX-KO mice, potassium inwardly rectifying channel subfamily J member 2 (KCNJ2) and sarcolipin (SLN) were upregulated; meanwhile, IK1 current was increased and Ca2+ handling was impaired in isolated atrial myocytes of SPX-KO mice. GALR2-cKO mice, but not GALR1-cKO and GALR3-cKO mice, had a higher incidence of AF, which was associated with higher IK1 current and intracellular Ca2+ overload. The phosphorylation level of CREB (cyclic AMP responsive element binding protein 1) was upregulated in atrial tissues of SPX-KO and GALR2-cKO mice. Chromatin immunoprecipitation confirmed the recruitment of p-CREB to the proximal promoter regions of KCNJ2 and SLN. Finally, spexin treatment suppressed CREB signaling, decreased IK1 current and decreased intracellular Ca2+ overload, which thus reduced the inducibility of AF in Ang-II-infused mice. CONCLUSIONS: Spexin reduces atrial fibrillation susceptibility by inhibiting CREB phosphorylation and thus downregulating KCNJ2 and SLN transcription by GALR2 receptor. The spexin/GALR2/CREB signaling pathway represents a novel therapeutic avenue in the development of agents against atrial fibrillation.

Jaw size variation is associated with a novel craniofacial function for galanin receptor 2 in an adaptive radiation of pupfishes.

Understanding the genetic basis of novel adaptations in new species is a fundamental question in biology. Here we demonstrate a new role for galr2 in vertebrate craniofacial development using an adaptive radiation of trophic specialist pupfishes endemic to San Salvador Island, Bahamas. We confirmed the loss of a putative Sry transcription factor binding site upstream of galr2 in scale-eating pupfish and found significant spatial differences in galr2 expression among pupfish species in Meckel's cartilage using in situ hybridization chain reaction (HCR). We then experimentally demonstrated a novel role for Galr2 in craniofacial development by exposing embryos to Garl2-inhibiting drugs. Galr2-inhibition reduced Meckel's cartilage length and increased chondrocyte density in both trophic specialists but not in the generalist genetic background. We propose a mechanism for jaw elongation in scale-eaters based on the reduced expression of galr2 due to the loss of a putative Sry binding site. Fewer Galr2 receptors in the scale-eater Meckel's cartilage may result in their enlarged jaw lengths as adults by limiting opportunities for a circulating Galr2 agonist to bind to these receptors during development. Our findings illustrate the growing utility of linking candidate adaptive SNPs in non-model systems with highly divergent phenotypes to novel vertebrate gene functions.

Galanin 2 Receptor: A Novel Target for a Subset of Pancreatic Ductal Adenocarcinoma.

Galanin is a 30 amino acid peptide that stimulates three subtype receptors (GAL1-3R). M89b is a lanthionine-stabilized, C-terminally truncated galanin analog that specifically stimulates GAL2R. We investigated the potential of M89b as a therapeutic for pancreatic ductal adenocarcinoma (PDAC) and assessed its safety. The anti-tumor activity of subcutaneously injected M89b on the growth of patient-derived xenografts of PDAC (PDAC-PDX) in mice was investigated. In addition, the safety of M89b was assessed in vitro using a multi-target panel to measure the off-target binding and modulation of enzyme activities. In a PDAC-PDX with a high GAL2R expression, M89b completely inhibited the growth of the tumor (p < 0.001), while in two PDAC-PDXs with low GAL2R expression, low or negligeable inhibition of tumor growth was measured, and in the PDX without GAL2R expression no influence on the tumor growth was observed. The M89b treatment of the GAL2R high-PDAC-PDX-bearing mice led to a reduction in the expression of RacGap1 (p < 0.05), PCNA (p < 0.01), and MMP13 (p < 0.05). In vitro studies involving a multi-target panel of pharmacologically relevant targets revealedexcellent safety of M89b. Our data indicated that GAL2R is a safe and valuable target for treating PDACs with high GAL2R expression.

Galanin Receptors (GalR1, GalR2, and GalR3) Expression in Colorectal Cancer Tissue and Correlations to the Overall Survival and Poor Prognosis of CRC Patients.

Colorectal cancer (CRC) is the second most common cause of cancer in women and the third in men. The postoperative pathomorphological evaluation of patients with CRC is extremely important for future therapeutic decisions. Although our previous studies demonstrated high galanin (GAL) presence within tumor tissue and an elevated concentration of GAL in the serum of CRC patients, to date, there is a lack of data regarding GAL receptor (GalR) protein expression in CRC cells. Therefore, the aim of this study was to evaluate the presence of all three types of GalRs (GalR1, GalR2 and GalR3) within epithelial cells of the human colon and CRC tissue with the use of the immunohistochemical method and to correlate the results with the clinical-pathological data. We found stronger immunoreactivity of GalR1 and GalR3 in CRC cells compared to epithelial cells of the unchanged mucosa of the large intestine. No differences in the GalR2 protein immunoreactivity between the studied tissues were noted. We also found that the increased immunoexpression of the GalR3 in CRC tissue correlated with the better prognosis and longer survival (p < 0.0079) of CRC patients (n = 55). The obtained results suggest that GalR3 may play the role of a prognostic factor for CRC patients. Based on data from the TCGA-COAD project deposited in the GDC Data Portal, we also found that GalR mRNA in cancer samples and the adjacent normal tissue did not correlate with immunoexpression of the GalR proteins in CRC cells and epithelial cells of the unchanged mucosa.

Involvement of galanin and galanin receptor 2 in a mouse model of allergic rhinitis.

BACKGROUND: Allergic rhinitis (AR) is caused by allergic reaction to allergens such as pollen. Galanin (GAL), a neuropeptide that regulates inflammatory processes, is widely expressed in the central and peripheral nervous systems. Although neuropeptides are implicated in arthritis and chemically induced ileitis, their roles in AR remain unclear. METHODS: We developed a murine model of AR and generated control, systemic sensitization, mild AR, and severe AR groups. We examined GAL and GAL receptor (GALR) mRNA and protein levels and localization patterns in each group using reverse transcription PCR, western blotting, and immunohistochemical analyses. Additionally, we evaluated the effects of M871, a GALR2 antagonist, on mice with severe AR. RESULTS: Gal and Galr2 are expressed in nasal mucosa and brain (control) samples from control and AR mice. GAL and GALR2 were expressed at similar levels and localized to ciliated epithelial and submucosal gland cells of the nasal mucosa in all four groups. Intranasal M871 administration significantly reduced the incidence of nose rubbing behaviors and sneezing (p < 0.001 in 30 min, respectively) in severe AR mice relative to that in controls. Mechanistically, we postulate that GALR2 is expressed in B cells, and M871 administration reduces IgE production, as well as the number of B cells in tissues. CONCLUSIONS: GAL signaling may not change progressively with increasing nasal sensitization, suggesting that this signaling process exacerbates, rather than directly trigger, AR. GAL-GALR2 signaling likely mediates AR development, suggesting that its inhibition represents a novel therapeutic strategy for AR.

Coordinated Targeting of Galanin Receptors on Cholangiocytes and Hepatic Stellate Cells Ameliorates Liver Fibrosis in Multidrug Resistance Protein 2 Knockout Mice.

Galanin (Gal) is a peptide with a role in neuroendocrine regulation of the liver. In this study, we assessed the role of Gal and its receptors, Gal receptor 1 (GalR1) and Gal receptor 2 (GalR2), in cholangiocyte proliferation and liver fibrosis in multidrug resistance protein 2 knockout (Mdr2KO) mice as a model of chronic hepatic cholestasis. The distribution of Gal, GalR1, and GalR2 in specific liver cell types was assessed by laser-capture microdissection and confocal microscopy. Galanin immunoreactivity was detected in cholangiocytes, hepatic stellate cells (HSCs), and hepatocytes. Cholangiocytes expressed GalR1, whereas HSCs and hepatocytes expressed GalR2. Strategies were used to either stimulate or block GalR1 and GalR2 in FVB/N (wild-type) and Mdr2KO mice and measure biliary hyperplasia and hepatic fibrosis by quantitative PCR and immunostaining of specific markers. Galanin treatment increased cholangiocyte proliferation and fibrogenesis in both FVB/N and Mdr2KO mice. Suppression of GalR1, GalR2, or both receptors in Mdr2KO mice resulted in reduced bile duct mass and hepatic fibrosis. In vitro knockdown of GalR1 in cholangiocytes reduced α-smooth muscle actin expression in LX-2 cells treated with cholangiocyte-conditioned media. A GalR2 antagonist inhibited HSC activation when Gal was administered directly to LX-2 cells, but not via cholangiocyte-conditioned media. These data demonstrate that Gal contributes not only to cholangiocyte proliferation but also to liver fibrogenesis via the coordinate activation of GalR1 in cholangiocytes and GalR2 in HSCs.

Involvement of Neuropeptide Galanin Receptors 2 and 3 in Learning, Memory and Anxiety in Aging Mice.

The neuropeptide galanin (GAL), which is expressed in limbic brain structures, has a strong impact on the regulation of mood and behavior. GAL exerts its effects via three G protein-coupled receptors (GAL1-3-R). Little is known about the effects of aging and loss of GAL-Rs on hippocampal-mediated processes connected to neurogenesis, such as learning, memory recall and anxiety, and cell proliferation and survival in the dorsal dentate gyrus (dDG) in mice. Our results demonstrate that loss of GAL3-R, but not GAL2-R, slowed learning and induced anxiety in older (12-14-month-old) mice. Lack of GAL2-R increased cell survival (BrdU incorporation) in the dDG of young mice. However, normal neurogenesis was observed in vitro using neural stem and precursor cells obtained from GAL2-R and GAL3-R knockouts upon GAL treatment. Interestingly, we found sub-strain differences between C57BL/6J and C57BL/6N mice, the latter showing faster learning, less anxiety and lower cell survival in the dDG. We conclude that GAL-R signaling is involved in cognitive functions and can modulate the survival of cells in the neurogenic niche, which might lead to new therapeutic applications. Furthermore, we observed that the mouse sub-strain had a profound impact on the behavioral parameters analyzed and should therefore be carefully considered in future studies.

A non-functional galanin receptor-2 in a multiple sclerosis patient.

Multiple Sclerosis (MS) is an inflammatory neurodegenerative disease that affects approximately 2.5 million people globally. Even though the etiology of MS remains unknown, it is accepted that it involves a combination of genetic alterations and environmental factors. Here, after performing whole exome sequencing, we found a MS patient harboring a rare and homozygous single nucleotide variant (SNV; rs61745847) of the G-protein coupled receptor (GPCR) galanin-receptor 2 (GALR2) that alters an important amino acid in the TM6 molecular toggle switch region (W249L). Nuclear magnetic resonance imaging showed that the hypothalamus (an area rich in GALR2) of this patient exhibited an important volumetric reduction leading to an enlarged third ventricle. Ex vivo experiments with patient-derived blood cells (AKT phosphorylation), as well as studies in recombinant cell lines expressing the human GALR2 (calcium mobilization and NFAT mediated gene transcription), showed that galanin (GAL) was unable to stimulate cell signaling in cells expressing the variant GALR2 allele. Live cell confocal microscopy showed that the GALR2 mutant receptor was primarily localized to intracellular endosomes. We conclude that the W249L SNV is likely to abrogate GAL-mediated signaling through GALR2 due to the spontaneous internalization of this receptor in this patient. Although this homozygous SNV was rare in our MS cohort (1:262 cases), our findings raise the potential importance of impaired neuroregenerative pathways in the pathogenesis of MS, warrant future studies into the relevance of the GAL/GALR2 axis in MS and further suggest the activation of GALR2 as a potential therapeutic route for this disease.

Central administration of galanin N-terminal fragment 1-15 decreases the voluntary alcohol intake in rats.

Alcohol consumption is considered a major risk factor for disease and mortality worldwide. In the absence of effective treatments in alcohol use disorders, it is important to find new biological targets that could modulate alcohol consumption. We tested the role of the N-terminal galanin fragment (1-15) [GAL(1-15)] in voluntary ethanol consumption in rats using the two-bottle choice paradigm as well as compare the effects of GAL(1-15) with the whole molecule of GAL. We describe for the first time that GAL(1-15), via central mechanisms, induces a strong reduction in preference and ethanol consumption in rats. These effects were significantly different than GAL. GAL receptor (GALR) 2 was involved in these effects, because the specific GALR2 antagonist M871 blocked GAL(1-15) mediated actions in preference and ethanol intake. Importantly, the mechanism of this action involves changes in GALR expression and also in immediate-early gene C-Fos and receptors-internalization-related gene Rab5 in the striatum. The relevance of the striatum as a target for GAL(1-15) was supported by the effect of GAL(1-15) on the locomotor activity of rats after ethanol administration. These results may give the basis for the development of novel therapeutics strategies using GAL(1-15) analogues for the treatment of alcohol use disorders in humans.

Galanin Protects Rat Cortical Astrocyte from Oxidative Stress: Involvement of GalR2 and pERK1/2 Signal Pathway.

The neuropeptide galanin and its receptors have been found to have protective effects on neurons. However, the role of galanin on astrocytes is still unclear. The present study is aimed at investigating the effects of galanin on the viability of cultured rat cortical astrocytes after oxidative stress induced by H2O2 and possible receptor and signaling mechanisms involved. Treatment of galanin had significant protective effects against H2O2-induced toxicity in the cultured cortical astrocytes. H2O2 induced an upregulation of phosphorylated extracellular signal-related kinase1/2 (pERK1/2) in astrocytes, which was suppressed by coapplication of galanin, suggesting an involvement of the pERK1/2 signal pathway in the protective effects of galanin. GalR2 has higher expression levels than GalR1 and GalR3 in the cultured cortical astrocytes, and GalR2 agonist AR-M1896 mimicked galanin effects on the astrocytes, implying that galanin protective effects mainly mediated by GalR2. Meanwhile, galanin had no effect on the A1-type transformation of rat cortical astrocytes. All those results suggest that galanin protects rat cortical astrocytes from oxidative stress by suppressing H2O2-induced upregulation of pERK1/2, mainly through GalR2.

Functional µ-Opioid-Galanin Receptor Heteromers in the Ventral Tegmental Area.

The neuropeptide galanin has been shown to interact with the opioid system. More specifically, galanin counteracts the behavioral effects of the systemic administration of µ-opioid receptor (MOR) agonists. Yet the mechanism responsible for this galanin-opioid interaction has remained elusive. Using biophysical techniques in mammalian transfected cells, we found evidence for selective heteromerization of MOR and the galanin receptor subtype Gal1 (Gal1R). Also in transfected cells, a synthetic peptide selectively disrupted MOR-Gal1R heteromerization as well as specific interactions between MOR and Gal1R ligands: a negative cross talk, by which galanin counteracted MAPK activation induced by the endogenous MOR agonist endomorphin-1, and a cross-antagonism, by which a MOR antagonist counteracted MAPK activation induced by galanin. These specific interactions, which represented biochemical properties of the MOR-Gal1R heteromer, could then be identified in situ in slices of rat ventral tegmental area (VTA) with MAPK activation and two additional cell signaling pathways, AKT and CREB phosphorylation. Furthermore, in vivo microdialysis experiments showed that the disruptive peptide selectively counteracted the ability of galanin to block the dendritic dopamine release in the rat VTA induced by local infusion of endomorphin-1, demonstrating a key role of MOR-Gal1R heteromers localized in the VTA in the direct control of dopamine cell function and their ability to mediate antagonistic interactions between MOR and Gal1R ligands. The results also indicate that MOR-Gal1R heteromers should be viewed as targets for the treatment of opioid use disorders. SIGNIFICANCE STATEMENT: The µ-opioid receptor (MOR) localized in the ventral tegmental area (VTA) plays a key role in the reinforcing and addictive properties of opioids. With parallel in vitro experiments in mammalian transfected cells and in situ and in vivo experiments in rat VTA, we demonstrate that a significant population of these MORs form functional heteromers with the galanin receptor subtype Gal1 (Gal1R), which modulate the activity of the VTA dopaminergic neurons. The MOR-Gal1R heteromer can explain previous results showing antagonistic galanin-opioid interactions and offers a new therapeutic target for the treatment of opioid use disorder.

Activation of galanin receptor 1 inhibits locus coeruleus neurons via GIRK channels.

The noradrenergic neurons of the locus coeruleus (LC) are associated with various brain functions and psychiatric disorders, such as addiction and depression. It has been shown that neuropeptide galanin (GAL) inhibits neuronal excitability in LC, but the mechanisms remain unclear. In the present study, we investigated the ionic and signal transduction mechanisms underlying inhibitory effect of GAL on LC neurons using whole-cell patch clamp recording in rat brain slices. Bath application of GAL decreased the spontaneous firings and induced a dose-dependent hyperpolarization of LC neurons and this effect was attenuated by knockdown of Galr1, but not Galr2, confirming that mainly GALR1 mediates the inhibition effect of GAL. The inhibitory effect of GAL was also blocked by treatments of pertussis toxin (PTX), GTP-γ-s or GDP-ß-s, respectively, indicating that the functions of PTX sensitive Gi/o protein are required for GAL-induced hyperpolarization. Moreover, the blockers of GIRK (tertiapin-Q or SCH2 3390 hydrochloride) attenuated the GAL response while blocker of BK/SK/KATP channels or TASK-1/3 channels did not affect it significantly, suggesting that GIRK channels play an important role in GAL-induced hyperpolarization in LC neurons. Taken together, the inhibitory effect of GAL on LC neurons is mediated by GALR1 via PTX-sensitive Gi/o proteins, which activate GIRK channels.

Effects of galanin receptor 2 and receptor 3 knockout in mouse models of acute seizures.

There exists solid evidence that endogenous galanin and galanin agonists exert anticonvulsive actions mediated both by galanin 1 receptor (GAL1-R) and galanin 2 receptor (GAL2-R). We have now investigated whether depletion of the recently identified third galanin receptor, GAL3-R, and that of GAL2-R, alters the threshold to the systemically applied γ-aminobutyric acid (GABA) antagonist pentylenetetrazole (PTZ) or to intrahippocampally administered kainic acid (KA). In neither model, GAL3-KO mice differed in their latency to the first seizure, mean seizure duration, total number of seizures, or time spent in seizures compared to wild-type controls. In addition, consistent with previous data, the response to PTZ was not altered in GAL2-KO mice. In contrast, intrahippocampal KA resulted in a significantly increased number of seizures and time spent in seizures in GAL2-KO mice, although the latency to the first seizure and the duration of individual seizures was not altered. These results are consistent with the previous data showing that GAL2-R knockdown does not affect the number of perforant path stimulations required for initiating status epilepticus but significantly increases the seizure severity during the ongoing status. In conclusion, our data support a specific role of GAL2-R but not of GAL3-R in mediating the anticonvulsive actions of endogenous galanin.

Site-specific methylation patterns of the GAL and GALR1/2 genes in head and neck cancer: Potential utility as biomarkers for prognosis.

The aim of this study was to evaluate the prognostic value of the promoter methylation status of galanin (GAL) and galanin receptor 1/2 (GALR1/2) by assessing their association with disease-free survival and known prognostic factors in head and neck cancer. We generated methylation profiles of GAL and GALR1/2 in tumor samples obtained from 202 patients with head and neck squamous cell carcinoma (HNSCC); these included 43 hypopharynx, 42 larynx, 59 oral cavity, and 58 oropharynx tumor samples. CpG island hypermethylation status of the three genes was analyzed using quantitative methylation-specific PCR (Q-MSP). In order to determine the prognostic value of the methylation status of these genes, the associations between methylation index and various clinical characteristics, especially tumor site, were assessed for tumors from patients with HNSCC. The methylation index was positively correlated with female gender (P = 0.008) and disease recurrence (P = 0.01) in oral cancer and human papillomavirus (HPV)-positive (P = 0.004) status and disease recurrence (P = 0.005) in oropharyngeal cancer. Among patients with oral and oropharyngeal cancer, promoter hypermethylation of GAL, GALR1, or GALR2 was statistically correlated with a decrease in disease-free survival (log-rank test, P = 0.036 and P = 0.042, respectively). Furthermore, methylation of GAL, GALR1, or GALR2 exhibited the highest association with poor survival (log-rank test, P = 0.018) in patients with HPV-negative oropharyngeal cancers. As such, GAL and GALR1/2 methylation status may serve as an important site-specific biomarker for prediction of clinical outcome in patients with HNSCC. © 2016 Wiley Periodicals, Inc.

The generation of knock-in mice expressing fluorescently tagged galanin receptors 1 and 2.

The neuropeptide galanin has diverse roles in the central and peripheral nervous systems, by activating the G protein-coupled receptors Gal1, Gal2 and the less studied Gal3 (GalR1-3 gene products). There is a wealth of data on expression of Gal1-3 at the mRNA level, but not at the protein level due to the lack of specificity of currently available antibodies. Here we report the generation of knock-in mice expressing Gal1 or Gal2 receptor fluorescently tagged at the C-terminus with, respectively, mCherry or hrGFP (humanized Renilla green fluorescent protein). In dorsal root ganglia (DRG) neurons expressing the highest levels of Gal1-mCherry, localization to the somatic cell membrane was detected by live-cell fluorescence and immunohistochemistry, and that fluorescence decreased upon addition of galanin. In spinal cord, abundant Gal1-mCherry immunoreactive processes were detected in the superficial layers of the dorsal horn, and highly expressing intrinsic neurons of the lamina III/IV border showed both somatic cell membrane localization and outward transport of receptor from the cell body, detected as puncta within cell processes. In brain, high levels of Gal1-mCherry immunofluorescence were detected within thalamus, hypothalamus and amygdala, with a high density of nerve endings in the external zone of the median eminence, and regions with lesser immunoreactivity included the dorsal raphe nucleus. Gal2-hrGFP mRNA was detected in DRG, but live-cell fluorescence was at the limits of detection, drawing attention to both the much lower mRNA expression than to Gal1 in mice and the previously unrecognized potential for translational control by upstream open reading frames (uORFs).

Distinct features of neurotransmitter systems in the human brain with focus on the galanin system in locus coeruleus and dorsal raphe.

Using riboprobe in situ hybridization, we studied the localization of the transcripts for the neuropeptide galanin and its receptors (GalR1-R3), tryptophan hydroxylase 2, tyrosine hydroxylase, and nitric oxide synthase as well as the three vesicular glutamate transporters (VGLUT 1-3) in the locus coeruleus (LC) and the dorsal raphe nucleus (DRN) regions of postmortem human brains. Quantitative real-time PCR (qPCR) was used also. Galanin and GalR3 mRNA were found in many noradrenergic LC neurons, and GalR3 overlapped with serotonin neurons in the DRN. The qPCR analysis at the LC level ranked the transcripts in the following order in the LC: galanin >> GalR3 >> GalR1 > GalR2; in the DRN the ranking was galanin >> GalR3 >> GalR1 = GalR2. In forebrain regions the ranking was GalR1 > galanin > GalR2. VGLUT1 and -2 were strongly expressed in the pontine nuclei but could not be detected in LC or serotonin neurons. VGLUT2 transcripts were found in very small, nonpigmented cells in the LC and in the lateral and dorsal aspects of the periaqueductal central gray. Nitric oxide synthase was not detected in serotonin neurons. These findings show distinct differences between the human brain and rodents, especially rat, in the distribution of the galanin system and some other transmitter systems. For example, GalR3 seems to be the important galanin receptor in both the human LC and DRN versus GalR1 and -2 in the rodent brain. Such knowledge may be important when considering therapeutic principles and drug development.

Novel anti-tumor mechanism of galanin receptor type 2 in head and neck squamous cell carcinoma cells.

Galanin and its receptors, GALR1 and GALR2, are known tumor suppressors and potential therapeutic targets in head and neck squamous cell carcinoma (HNSCC). Previously, we demonstrated that, in GALR1-expressing HNSCC cells, the addition of galanin suppressed tumor proliferation via upregulation of ERK1/2 and cyclin-dependent kinase inhibitors, whereas, in GALR2-expressing cells, the addition of galanin not only suppressed proliferation, but also induced apoptosis. In this study, we first transduced HEp-2 and KB cell lines using a recombinant adeno-associated virus (rAAV)-green fluorescent protein (GFP) vector and confirmed a high GFP expression rate (>90%) in both cell lines at the standard vector dose. Next, we demonstrated that GALR2 expression in the presence of galanin suppressed cell viability to 40-60% after 72 h in both cell lines. Additionally, the annexin V-positive rate and sub-G0/G1 phase population were significantly elevated in HEp-2 cells (mock vs GALR2: 12.3 vs 25.0% (P < 0.01) and 9.1 vs 32.0% (P < 0.05), respectively) after 48 h. These changes were also observed in KB cells, although to a lesser extent. Furthermore, in HEp-2 cells, GALR2-mediated apoptosis was caspase-independent, involving downregulation of ERK1/2, followed by induction of the pro-apoptotic Bcl-2 protein, Bim. These results illustrate that transient GALR2 expression in the presence of galanin induces apoptosis via diverse pathways and serves as a platform for suicide gene therapy against HNSCC.

Tumor suppressor activity and inactivation of galanin receptor type 2 by aberrant promoter methylation in head and neck cancer.

BACKGROUND: There is accumulating evidence that galanin receptors (GALRs) may be tumor suppressors in head and neck squamous cell carcinoma (HNSCC). Promoter methylation status and gene expression were assessed in a large panel of head and neck primary tumors, based on the hypothesis that cytosine-guanine dinucleotide (CpG) hypermethylation might silence the galanin receptor 2 (GALR2) gene. METHODS: GALR2 expression was examined in a panel of cell lines by using quantitative reverse transcription polymerase chain reaction (RT-PCR). The methylation status of the GALR2 promoter was studied using quantitative methylation-specific PCR (Q-MSP). UM-SCC-1 was stably transfected to express GALR2. RESULTS: GALR2 expression was suppressed in UM-SCC cell lines, whereas nonmalignant cell lines exhibited stable expression. GALR2 methylation found in 31 of 100 (31.0%) tumor specimens was significantly correlated with the methylation status of both GALR1 and Galanin. The observed GALR2 promoter hypermethylation was statistically correlated with a decrease in disease-free survival (log-rank test, P=.045). A multivariate logistic-regression analysis revealed a high odds ratio for recurring methylation of GALR2 and the gene pair GALR2 and Galanin, 8.95 (95% confidence interval, 2.29-35.03; P=.024) and 9.05 (95% confidence interval, 1.76-46.50; P=.008), respectively. In addition, exogenous expression of GALR2 suppressed cell proliferation in UM-SCC-1 cells with hypermethylated Galanin and GALR2-proficient cell lines. CONCLUSIONS: Frequent promoter hypermethylation in association with prognosis, and growth suppression after re-expression, supports the hypothesis that GALR2 may act to suppress tumor activity. GALR2 is a potentially significant therapeutic target and prognostic factor for this cancer type.

Preferential activation by galanin 1-15 fragment of the GalR1 protomer of a GalR1-GalR2 heteroreceptor complex.

The three cloned galanin receptors show a higher affinity for galanin than for galanin N-terminal fragments. Galanin fragment (1-15) binding sites were discovered in the rat Central Nervous System, especially in dorsal hippocampus, indicating a relevant role of galanin fragments in central galanin communication. The hypothesis was introduced that these N-terminal galanin fragment preferring sites are formed through the formation of GalR1-GalR2 heteromers which may play a significant role in mediating galanin fragment (1-15) signaling. In HEK293T cells evidence for the existence of GalR1-GalR2 heteroreceptor complexes were obtained with proximity ligation and BRET(2) assays. PLA positive blobs representing GalR1-GalR2 heteroreceptor complexes were also observed in the raphe-hippocampal system. In CRE luciferase reporter gene assays, galanin (1-15) was more potent than galanin (1-29) in inhibiting the forskolin-induced increase of luciferase activity in GalR1-GalR2 transfected cells. The inhibition of CREB by 50nM of galanin (1-15) and of galanin (1-29) was fully counteracted by the non-selective galanin antagonist M35 and the selective GalR2 antagonist M871. These results suggested that the orthosteric agonist binding site of GalR1 protomer may have an increased affinity for the galanin (1-15) vs galanin (1-29) which can lead to its demonstrated increase in potency to inhibit CREB vs galanin (1-29). In contrast, in NFAT reporter gene assays galanin (1-29) shows a higher efficacy than galanin (1-15) in increasing Gq/11 mediated signaling over the GalR2 of these heteroreceptor complexes. This disbalance in the signaling of the GalR1-GalR2 heteroreceptor complexes induced by galanin (1-15) may contribute to depression-like actions since GalR1 agonists produce such effects.