Unidirectional cross-activation of GRPR by MOR1D uncouples itch and analgesia induced by opioids.

Spinal opioid-induced itch, a prevalent side effect of pain management, has been proposed to result from pain inhibition. We now report that the µ-opioid receptor (MOR) isoform MOR1D is essential for morphine-induced scratching (MIS), whereas the isoform MOR1 is required only for morphine-induced analgesia (MIA). MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, relaying itch information. We show that morphine triggers internalization of both GRPR and MOR1D, whereas GRP specifically triggers GRPR internalization and morphine-independent scratching. Providing potential insight into opioid-induced itch prevention, we demonstrate that molecular and pharmacologic inhibition of PLCß3 and IP3R3, downstream effectors of GRPR, specifically block MIS but not MIA. In addition, blocking MOR1D-GRPR association attenuates MIS but not MIA. Together, these data suggest that opioid-induced itch is an active process concomitant with but independent of opioid analgesia, occurring via the unidirectional cross-activation of GRPR signaling by MOR1D heterodimerization.

Single-cell transcriptomics of suprachiasmatic nuclei reveal a Prokineticin-driven circadian network.

Circadian rhythms in mammals are governed by the hypothalamic suprachiasmatic nucleus (SCN), in which 20,000 clock cells are connected together into a powerful time-keeping network. In the absence of network-level cellular interactions, the SCN fails as a clock. The topology and specific roles of its distinct cell populations (nodes) that direct network functions are, however, not understood. To characterise its component cells and network structure, we conducted single-cell sequencing of SCN organotypic slices and identified eleven distinct neuronal sub-populations across circadian day and night. We defined neuropeptidergic signalling axes between these nodes, and built neuropeptide-specific network topologies. This revealed their temporal plasticity, being up-regulated in circadian day. Through intersectional genetics and real-time imaging, we interrogated the contribution of the Prok2-ProkR2 neuropeptidergic axis to network-wide time-keeping. We showed that Prok2-ProkR2 signalling acts as a key regulator of SCN period and rhythmicity and contributes to defining the network-level properties that underpin robust circadian co-ordination. These results highlight the diverse and distinct contributions of neuropeptide-modulated communication of temporal information across the SCN.

Mas-Related G Protein-Coupled Receptors and the Biology of Itch Sensation.

Chronic, persistent itch is a devastating symptom that causes much suffering. In recent years, there has been great progress made in understanding the molecules, cells, and circuits underlying itch sensation. Once thought to be carried by pain-sensing neurons, itch is now believed to be capable of being transmitted by dedicated sensory labeled lines. Members of the Mas-related G protein-coupled receptor (Mrgpr) family demarcate an itch-specific labeled line in the peripheral nervous system. In the spinal cord, the expression of other proteins identifies additional populations of itch-dedicated sensory neurons. However, as evidence for labeled-line coding has mounted, studies promoting alternative itch-coding strategies have emerged, complicating our understanding of the neural basis of itch. In this review, we cover the molecules, cells, and circuits related to understanding the neural basis of itch, with a focus on the role of Mrgprs in mediating itch sensation.

Descending dopaminergic pathway facilitates itch signal processing via activating spinal GRPR+ neurons.

A11 dopaminergic neurons regulate somatosensory transduction by projecting from the diencephalon to the spinal cord, but the function of this descending projection in itch remained elusive. Here, we report that dopaminergic projection neurons from the A11 nucleus to the spinal dorsal horn (dopaminergicA11-SDH ) are activated by pruritogens. Inhibition of these neurons alleviates itch-induced scratching behaviors. Furthermore, chemogenetic inhibition of spinal dopamine receptor D1-expressing (DRD1+ ) neurons decreases acute or chronic itch-induced scratching. Mechanistically, spinal DRD1+ neurons are excitatory and mostly co-localize with gastrin-releasing peptide (GRP), an endogenous neuropeptide for itch. In addition, DRD1+ neurons form synapses with GRP receptor-expressing (GRPR+ ) neurons and activate these neurons via AMPA receptor (AMPAR). Finally, spontaneous itch and enhanced acute itch induced by activating spinal DRD1+ neurons are relieved by antagonists against AMPAR and GRPR. Thus, the descending dopaminergic pathway facilitates spinal itch transmission via activating DRD1+ neurons and releasing glutamate and GRP, which directly augments GRPR signaling. Interruption of this descending pathway may be used to treat chronic itch.

Bombesin receptor-activated protein homolog deficiency altered the pattern of pathological changes of psoriasis - like skin lesion in mice.

This study investigated the potential role of the mouse homolog of bombesin receptor-activated protein (BRAP) in imiquimod (IMQ) induced psoriasis - like skin inflammation. The expression of both human BRAP, encoded by C6orf89, and its mouse homolog, encoded by BC004004, has been found to be expressed abundantly in the keratinocytes. BC004004 knockout mice (BC004004-/-) were topically treated with IMQ daily for 7 days to test whether they were more vulnerable to psoriasis - like inflammation. We found that those mice exhibited an altered pattern of inflammation process compared to isogenic wild type control mice (BC004004+/+). BC004004-/- mice developed skin lesions with earlier and more acute onset, as well as a quicker remission. The cytokines related to pathogenesis of psoriasis also exhibited different expression patterns in IMQ treated BC004004-/- mice. On day 4 of IMQ treatment, BC004004-/- mice exhibited a higher expression level of IL-17A compared to BC004004+/+ mice, suggesting a more robust activation of Th17 cells in the knockout mice. The serum level of thymic stromal lymphopoietin (TSLP), one of the keratinocyte derived cytokines, was also increased in BC004004-/- mice and reached its peak on day 4. Knockdown of BRAP in cultured human keratinocyte-derived HaCaT cells by siRNA silencing led to increased release of TSLP. Our data suggest that the elevated of level of TSLP released from keratinocytes due to BRAP deficiency might mediate the crosstalk between the epidermal cells and immune cells and thereby contributing to the altered pathological changes observed in psoriasis - like skin lesion in knockout mice.

Lack of bombesin receptor-activated protein homologous protein impairs hippocampal synaptic plasticity and promotes chronic unpredictable mild stress induced behavioral changes in mice.

Bombesin receptor-activated protein (BRAP) and its homologous protein in mice, which is encoded by bc004004 gene, were expressed abundantly in brain tissues with unknown functions. We treated bc004004-/- mice with chronic unpredictable mild stress (CUMS) to test whether those mice were more vulnerable to stress-related disorders. The results of forced swimming test, sucrose preference test, and open field test showed that after being treated with CUMS for 28 days or 35 days both bc004004-/- and bc004004+/+ mice exhibited behavioural changes and there was no significant difference between bc004004+/+ and bc004004-/-. However, behavioural changes were observed only in bc004004-/- mice after being exposed to CUMS for 21 days, but not in bc004004+/+ after 21-day CUMS exposure, indicating that lack of BRAP homologous protein may cause vulnerability to stress-related disorders in mice. In addition, bc004004-/- mice showed a reduction in recognition memory as revealed by novel object recognition test. Since memory changes and stress related behavioural changes are all closely related to the hippocampus function we further analyzed the changes of dendrites and synapses of hippocampal neurons as well as expression levels of some proteins closely related to synaptic function. bc004004-/- mice exhibited decreased dendritic lengths and increased amount of immature spines, as well as altered expression pattern of synaptic related proteins including GluN2A, synaptophysin and BDNF in the hippocampus. Those findings suggest that BRAP homologous protein may have a protective effect on the behavioural response to stress via regulating dendritic spine formation and synaptic plasticity in the hippocampus.

Disruption of neuromedin B receptor improves mitochondrial oxidative phosphorylation capacity in gastrocnemius muscle of female mice.

Neuromedin B (NB), a bombesin-like peptide, exerts its specific actions by binding to the neuromedin B receptor (NBR), a G protein-coupled receptor. Female NBR-knockout (NBR-KO) mice exhibit resistance to diet-induced obesity, without hyperphagia, suggesting possible increase in energy expenditure. Skeletal muscle (SM) is crucial for whole body energy homeostasis, however, the presence of NB-NBR signaling and its effects in SM are unknown. Here, we show that male and female wild type express Nmbr and Nmb mRNA in SM, with higher levels in females. Female NBR-KO gastrocnemius showed increased Myh7 mRNA level, which characterizes type I fibers (oxidative profile). Their permeabilized gastrocnemius fibers, studied by high-resolution respirometry, exhibited higher consumption of O2 coupled to ATP synthesis and unaltered uncoupled respiration. NBR-KO gastrocnemius had higher protein levels of ATP-synthase and Nduf9 mRNA, corresponding to mitochondrial complex I subunit. NBR-KO gastrocnemius exhibited slight increase in mitochondria number, increased thickness of Z line at electron microscopy, and unaltered mitochondrial dynamics markers. Therefore, in the females' gastrocnemius, a predominantly glycolytic SM, the NBR absence promotes changes that favor mitochondrial oxidative phosphorylation capacity. In addition, in L6 myocytes, NB treatment (5 µg/mL/16 h) promoted lower O2 consumption coupled to ATP synthesis, suggesting direct action at SM cells. Altogether, the study reinforces the hypothesis that inhibition of NB-NBR signaling enhances the capacity for oxidative phosphorylation of white SM, encouraging future studies to elucidate their contribution on other types of SM and whole body energy expenditure, which may lead to a new target to drug development for obesity treatment.NEW & NOTEWORTHY This study describes neuromedin B (NB) and NB receptor as new regulators of skeletal muscle mitochondrial function. The white skeletal muscle mitochondrial oxidative phosphorylation capacity was increased by NB receptor genetic disruption in female mice. These findings may contribute to the resistance to diet-induced obesity, previously found in these mice, which requires future studies. Thus, investigations are necessary to clarify if blockade of NB receptor may be an approach to develop drugs to combat obesity.

BR-bombesin: a novel bombesin-related peptide from the skin secretion of the Chaco tree frog (Boana raniceps) with physiological gastric effects.

Bombesin mediates several biological activities in the gastrointestinal (GI) tract and central nervous system in mammals, including smooth muscle contraction, secretion of GI hormones and regulation of homeostatic mechanisms. Here, we report a novel bombesin-like peptide isolated from Boana raniceps. Its amino acid sequence, GGNQWAIGHFM-NH2, was identified and structurally confirmed by HPLC, MS/MS and 454-pyrosequencing; the peptide was named BR-bombesin. The effect of BR-bombesin on smooth muscle contraction was assessed in ileum and esophagus, and its anti-secretory activity was investigated in the stomach. BR-bombesin exerted significant contractile activity with a concentration-response curve similar to that of commercially available bombesin in ileum strips of Wistar rats. In esophageal strips, BR-bombesin acted as an agonist, as many other bombesin-related peptides act, although with different behavior compared to the muscarinic agonist carbachol. Moreover, BR-bombesin inhibited stomach secretion by approximately 50% compared to the untreated control group. This novel peptide has 80% and 70% similarity with the 10-residue C-terminal domain of human neuromedin B (NMB) and human gastrin releasing peptide (GRP10), respectively. Molecular docking analysis revealed that the GRP receptor had a binding energy equal to - 7.3 kcal.mol-1 and - 8.5 kcal.mol-1 when interacting with bombesin and BR-bombesin, respectively. Taken together, our data open an avenue to investigate BR-bombesin in disorders that involve gastrointestinal tract motility and acid gastric secretion.

Constitutively active BRS3 is a genuinely orphan GPCR in placental mammals.

G protein-coupled receptors (GPCRs) play an important role in physiology and disease and represent the most productive drug targets. Orphan GPCRs, with their endogenous ligands unknown, were considered a source of drug targets and consequently attract great interest to identify their endogenous cognate ligands for deorphanization. However, a contrary view to the ubiquitous existence of endogenous ligands for every GPCR is that there might be a significant overlooked fraction of orphan GPCRs that function constitutively in a ligand-independent manner only. Here, we investigated the evolution of the bombesin receptor-ligand family in vertebrates in which one member-bombesin receptor subtype-3 (BRS3)-is a potential orphan GPCR. With analysis of 17 vertebrate BRS3 structures and 10 vertebrate BRS3 functional data, our results demonstrated that nonplacental vertebrate BRS3 still connects to the original ligands-neuromedin B (NMB) and gastrin-releasing peptide (GRP)-because of adaptive evolution, with significantly changed protein structure, especially in three altered key residues (Q127R, P205S, and R294H) originally involved in ligand binding/activation, whereas the placental mammalian BRS3 lost the binding affinity to NMB/GRP and constitutively activates Gs/Gq/G12 signaling in a ligand-independent manner. Moreover, the N terminus of placental mammalian BRS3 underwent positive selection, exhibiting significant structural differences compared to nonplacental vertebrate BRS3, and this domain plays an important role in constitutive activity of placental mammalian BRS3. In conclusion, constitutively active BRS3 is a genuinely orphan GPCR in placental mammals, including human. To our knowledge, this study identified the first example that might represent a new group of genuinely orphan GPCRs that will never be deorphanized by the discovery of a natural ligand and provided new perspectives in addition to the current ligand-driven GPCR deorphanization.

The Neurokinin-1 Receptor is Expressed with Gastrin-Releasing Peptide Receptor in Spinal Interneurons and Modulates Itch.

The neurokinin-1 receptor (NK1R; encoded by Tacr1) is expressed in spinal dorsal horn neurons and has been suggested to mediate itch in rodents. However, previous studies relied heavily on neurotoxic ablation of NK1R spinal neurons, which limited further dissection of their function in spinal itch circuitry. To address this limitation, we leveraged a newly developed Tacr1CreER mouse line to characterize the role of NK1R spinal neurons in itch. We show that pharmacological activation of spinal NK1R and chemogenetic activation of Tacr1CreER spinal neurons increases itch behavior in male and female mice, whereas pharmacological inhibition of spinal NK1R suppresses itch behavior. We use fluorescence in situ hybridization (FISH) to characterize the endogenous expression of Tacr1 throughout the superficial and deeper dorsal horn (DDH), as well as the lateral spinal nucleus (LSN), of mouse and human spinal cord. Retrograde labeling studies in mice from the parabrachial nucleus (PBN) show that less than 20% of superficial Tacr1CreER dorsal horn neurons are spinal projection neurons, and thus the majority of Tacr1CreER are local interneurons. We then use a combination of in situ hybridization and ex vivo two-photon Ca2+ imaging of the mouse spinal cord to establish that NK1R and the gastrin-releasing peptide receptor (GRPR) are coexpressed within a subpopulation of excitatory superficial dorsal horn (SDH) neurons. These findings are the first to suggest a role for NK1R interneurons in itch and extend our understanding of the complexities of spinal itch circuitry.SIGNIFICANCE STATEMENT The spinal cord is a critical hub for processing somatosensory input, yet which spinal neurons process itch input and how itch signals are encoded within the spinal cord is not fully understood. We demonstrate neurokinin-1 receptor (NK1R) spinal neurons mediate itch behavior in mice and that the majority of NK1R spinal neurons are local interneurons. These NK1R neurons comprise a subset of gastrin-releasing peptide receptor (GRPR) interneurons and are thus positioned at the center of spinal itch transmission. We show NK1R mRNA expression in human spinal cord, underscoring the translational relevance of our findings in mice. This work is the first to suggest a role for NK1R interneurons in itch and extends our understanding of the complexities of spinal itch circuitry.

Sensitization of spinal itch transmission neurons in a mouse model of chronic itch requires an astrocytic factor.

BACKGROUND: Chronic itch is a highly debilitating symptom among patients with inflammatory skin diseases. Recent studies have revealed that gastrin-releasing peptide (GRP) and its receptor (gastrin-releasing peptide receptor [GRPR]) in the spinal dorsal horn (SDH) play a central role in itch transmission. OBJECTIVE: We aimed to investigate whether GRP-GRPR signaling is altered in SDH neurons in a mouse model of chronic itch and to determine the potential mechanisms underlying these alterations. METHODS: Patch-clamp recordings from enhanced green fluorescent protein (EGFP)-expressing (GRPR+) SDH neurons were used to examine GRP-GRPR signaling in spinal cord slices obtained from Grpr-EGFP mice. Immunohistochemical, genetic (gene expression and editing through adeno-associated virus vectors), and behavioral approaches were also used for in vivo experiments. RESULTS: We observed potentiation of GRP-evoked excitation in the GRPR+ SDH neurons of mice with contact dermatitis, without concomitant changes in GRPR expression. Interestingly, increases in excitation were attenuated by suppressing the reactive state of SDH astrocytes, which are known to be reactive in patients with chronic itch conditions. Furthermore, CRISPR-Cas9-mediated astrocyte-selective in vivo editing of a gene encoding lipocalin-2 (LCN2), an astrocytic factor implicated in chronic itch, suppressed increases in GRP-induced excitation of GRPR+ neurons, repetitive scratching, and skin damage in mice with contact dermatitis. Moreover, LCN2 potentiated GRP-induced excitation of GRPR+ neurons in normal mice. CONCLUSION: Our findings indicate that, under chronic itch conditions, the GRP-induced excitability of GRPR+ SDH neurons is enhanced through a non-cell-autonomous mechanism involving LCN2 derived from reactive astrocytes.

Impact of natural neuromedin-B receptor variants on iron metabolism.

Iron overload heritability remains partly unexplained. By performing whole exome sequencing in three patients with a clinical phenotype of hemochromatosis not accounted by known genetic risk factors, we identified in all patients rare variants predicted to alter activity of Neuromedin-B receptor (NMBR). Coding NMBR mutations were enriched in 129 patients with hereditary hemochromatosis or iron overload phenotype, as compared to ethnically matched controls, including 100 local healthy blood donors and 1000Genomes project participants (15.5% vs 5%, P = .0038 at burden test), and were associated with higher transferrin saturation in regular blood donors (P = .04). Consistently, in 191 patients with nonalcoholic fatty liver, the most common low-frequency p.L390 M variant was independently associated with higher ferritin (P = .03). In 58 individuals, who underwent oral iron challenge, carriage of the p.L390 M variant was associated with higher transferrin saturation and lower hepcidin release. Furthermore, the circulating concentration of the natural NMBR ligand, Neuromedin-B, was reduced in response to iron challenge. It was also decreased in individuals carrying the p.L390 M variant and with hemochromatosis in parallel with increased transferrin saturation. In mice, Nmbr was induced by chronic dietary iron overload in the liver, gut, pancreas, spleen, and skeletal muscle, while Nmb was downregulated in gut, pancreas and spleen. Finally, Nmb amplified holo-transferrin dependent induction of hepcidin in primary mouse hepatocytes, which was associated with Jak2 induction and abolished by the NMBR antagonist PD168368. In conclusion, NMBR natural variants were enriched in patients with iron overload, and associated with facilitated iron absorption, possibly related to a defect of iron-induced hepcidin release.

Preclinical Evaluation of the Copper-64 Labeled GRPR-Antagonist RM26 in Comparison with the Cobalt-55 Labeled Counterpart for PET-Imaging of Prostate Cancer.

Gastrin-releasing peptide receptor (GRPR) is overexpressed in the majority of prostate cancers. This study aimed to investigate the potential of 64Cu (radionuclide for late time-point PET-imaging) for imaging of GRPR expression using NOTA-PEG2-RM26 and NODAGA-PEG2-RM26. Methods: NOTA/NODAGA-PEG2-RM26 were labeled with 64Cu and evaluated in GRPR-expressing PC-3 cells. Biodistribution of [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was studied in PC-3 xenografted mice and compared to the biodistribution of [57Co]Co-NOTA/NODAGA-PEG2-RM26 at 3 and 24 h p.i. Preclinical PET/CT imaging was performed in tumor-bearing mice. NOTA/NODAGA-PEG2-RM26 were stably labeled with 64Cu with quantitative yields. In vitro, binding of [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was rapid and GRPR-specific with slow internalization. In vivo, [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 bound specifically to GRPR-expressing tumors with fast clearance from blood and normal organs and displayed generally comparable biodistribution profiles to [57Co]Co-NOTA/NODAGA-PEG2-RM26; tumor uptake exceeded normal tissue uptake 3 h p.i.. Tumor-to-organ ratios did not increase significantly with time. [64Cu]Cu-NOTA-PEG2-RM26 had a significantly higher liver and pancreas uptake compared to other agents. 57Co-labeled radioconjugates showed overall higher tumor-to-non-tumor ratios, compared to the 64Cu-labeled counterparts. [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was able to visualize GRPR-expression in a murine PC model using PET. However, [55/57Co]Co-NOTA/NODAGA-PEG2-RM26 provided better in vivo stability and overall higher tumor-to-non-tumor ratios compared with the 64Cu-labeled conjugates.

Gastrin-releasing peptide receptor gene silencing inhibits the development of the epithelial-mesenchymal transition and formation of a calcium oxalate crystal in renal tubular epithelial cells in mice with kidney stones via the PI3K/Akt signaling pathway.

Between 1% and 15% of people are globally affected by kidney stones, and this disease has become more common since the 1970s. Therefore, this study aims to investigate the effects of gastrin-releasing peptide receptor (GRPR) gene silencing via the PI3K/Akt signaling pathway on the development of the epithelial-mesenchymal transition (EMT) and formation of a calcium oxalate crystal in renal tubular epithelial cells (TECs) of kidney stones. A total of 70 clean and healthy C57BL/6J mice were assigned into the normal ( n = 10) and kidney stones groups ( n = 60). The underlying regulatory mechanisms of GRPR were analyzed in concert with the treatment of shGRPR-1, LY294002, and shGRPR-1 + LY294002 in TECs isolated from mice with kidney stones. A series of experiments were conducted for the measurement of urinary oxalate and urinary calcium, the renal calcium salt deposition, the positive rate of GRPR, the expressions of renal TECs related genes and calcium oxalate regulation related genes, and the growth of calcium crystals induced by cells. After treatment of shGRPR-1 and shGRPR-1 + LY294002, levels of urinary oxalate and urinary calcium in the serum, as well as positive rate of GRPR, became relatively low, levels of E-cadherin enhanced, whereas levels of Akt, PI3K, GRPR, extents of PI3K and Akt phosphorylation, α-SMA, Vimentin and FSP-1, OPN, MCP-1, and CD44 decreased and a number of crystals reduced. Taken together, we conclude that GRPR gene silencing suppresses the development of the EMT and formation of the calcium oxalate crystal in renal TECs of kidney stones through the inactivation of the PI3K/Akt signaling pathway.

Development and Characterization of a Novel, High-Affinity, Specific, Radiolabeled Ligand for BRS-3 Receptors.

Bombesin (Bn) receptor subtype 3(BRS-3) is an orphan G-protein-coupled receptor of the Bn family, which does not bind any natural Bn peptide with high affinity. Receptor knockout studies show that the animals develop diabetes, obesity, altered temperature control, and other central nervous system (CNS)/endocrine/gastrointestinal changes. It is present in CNS, peripheral tissues, and tumors; however, its role in normal physiology/pathophysiology, as well as its receptor localization/pharmacology is largely unknown, in part due to the lack of a convenient, specific, direct radiolabeled ligand. This study was designed to address this problem and to develop and characterize a specific radiolabeled ligand for BRS-3. The peptide antagonist Bantag-1 had >10,000-fold selectivity for human BRS-3 (hBRS-3) over other mammalian Bn receptors (BnRs) [i.e., gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR)]. Using iodogen and basic conditions, it was radiolabeled to high specific activity (2200 Ci/mmol) and found to bind with high affinity/specificity to hBRS-3. Binding was saturable, rapid, and reversible. The ligand only interacted with known BRS-3 ligands, and not with other specific GRPR/NMBR ligands or ligands for unrelated receptors. The magnitude of 125I-Bantag-1 binding correlated with BRS-3 mRNA expression and the magnitude of activation of phospholipase C in lung cancer cells, as well as readily identifying BRS-3 in lung cancer cells and normal tissues, allowing the direct assessment of BRS-3 receptor pharmacology/numbers on cells containing BRS-3 with other BnRs, which is usually the case. This circumvents the need for subtraction assays, which are now frequently used to assess BRS-3 indirectly using radiolabeled pan-ligands, which interact with all BnRs.

Receptor-specific crosstalk between prostanoid E receptor 3 and bombesin receptor subtype 3.

Bombesin receptor subtype 3 (BRS-3) is a GPCR that is expressed in the CNS, peripheral tissues, and tumors. Our understanding of BRS-3's role in physiology and pathophysiology is limited because its natural ligand is unknown. In an attempt to identify this ligand, we screened toad skin ( Bufo bufo gargarizans Cantor) extracts and identified prostaglandins as putative ligands. In BRS-3-transfected human embryonic kidney (HEK) cells, we found that prostaglandins, with prostaglandin E2 (PGE2) being the most potent, fulfill the pharmacologic criteria of affinity, selectivity, and specificity to be considered as agonists to the BRS-3 receptor. However, PGE2 is unable to activate BRS-3 in different cellular environments. We speculated that EP receptors might be the cause of this cellular selectivity, and we found that EP3 is the receptor primarily responsible for the differential PGE2 effect. Consequently, we reconstituted the HEK environment in Chinese hamster ovary (CHO) cells and found that BRS-3 and EP3 interact to potentiate PGE2 signaling. This potentiating effect is receptor specific, and it occurs only when BRS-3 is paired to EP3. Our study represents an example of functional crosstalk between two distantly related GPCRs and may be of clinical importance for BRS-3-targeted therapies.-Zhang, Y., Liu, Y., Wu, L., Fan, C., Wang, Z., Zhang, X., Alachkar, A., Liang, X., Civelli, O. Receptor-specific crosstalk between prostanoid E receptor 3 and bombesin receptor subtype 3.

Neuromedin B Induces Acute Itch in Mice via the Activation of Peripheral Sensory Neurons.

Neuromedin B is expressed in nociceptive and itch-sensitive dorsal root ganglia neurons, but its peripheral pruritogenic potential is not well described. The potential of neuromedin B as a pruritogen and pro-inflammatory peptide in the skin was tested in vivo in an acute model in mice and monkeys as well as an allergic dermatitis model in mice. To identify the underlying mechanisms in vitro real time PCR analysis for neuromedin B and its receptor expression in murine mast cells and dorsal root ganglia as well as functional calcium imaging in the ganglia was applied. Neuromedin B induces itch when injected intradermally, and the peripheral signal is likely transmitted through the activation of dorsal root ganglia. Thus, neuromedin B could be an interesting new therapeutic target for peripheral processing of itch at the level of sensory neurons.

Augmented Responses to Ozone in Obese Mice Require IL-17A and Gastrin-Releasing Peptide.

Ozone and obesity both increase IL-17A in the lungs. In mice, obesity augments the airway hyperresponsiveness and neutrophil recruitment induced by acute ozone exposure. Therefore, we examined the role of IL-17A in obesity-related increases in the response to ozone observed in obese mice. Lean wild-type and obese db/db mice were pretreated with IL-17A-blocking or isotype antibodies, exposed to air or ozone (2 ppm for 3 h), and evaluated 24 hours later. Microarray analysis of lung tissue gene expression was used to examine the mechanistic basis for effects of anti-IL-17A. Compared with lean mice, ozone-exposed obese mice had greater concentrations of BAL IL-17A and greater numbers of pulmonary IL-17A+ cells. Ozone-induced increases in BAL IL-23 and CCL20, cytokines important for IL-17A+ cell recruitment and activation, were also greater in obese mice. Anti-IL-17A treatment reduced ozone-induced airway hyperresponsiveness toward levels observed in lean mice. Anti-IL-17A treatment also reduced BAL neutrophils in both lean and obese mice, possibly because of reductions in CXCL1. Microarray analysis identified gastrin-releasing peptide (GRP) receptor (Grpr) among those genes that were both elevated in the lungs of obese mice after ozone exposure and reduced after anti-IL-17A treatment. Furthermore, ozone exposure increased BAL GRP to a greater extent in obese than in lean mice, and GRP-neutralizing antibody treatment reduced obesity-related increases in ozone-induced airway hyperresponsiveness and neutrophil recruitment. Our data indicate that IL-17A contributes to augmented responses to ozone in db/db mice. Furthermore, IL-17A appears to act at least in part by inducing expression of Grpr.

Bombesin/oligoarginine fusion peptides for gastrin releasing peptide receptor (GRPR) targeted gene delivery.

The development of non-viral gene delivery systems, with the capacity to overcome most of the biological barriers facing gene delivery, is challenging. We have developed peptide-based, multicomponent, non-viral delivery systems, incorporating: a bombesin peptide ligand (BBN(6-14)), to selectively target the gastrin releasing peptide receptor (GRPR); oligoarginine peptides (hexa- (R6) and nona-arginine (R9)), for plasmid DNA (pDNA) condensation; and GALA, to facilitate endosome escape. The uptake and endosome escape efficiency of bombesin/oligoarginine and bombesin/oligoarginine/GALA fusion peptides for oligonucleotide delivery was evaluated in terms of their complex size, cellular uptake, endosome escape, and cellular toxicity. Complex size and cell uptake studies demonstrated that the nona-arginine/bombesin delivery system was more efficient at condensing and delivering pDNA into PC-3 prostate cancer cells compared to the hexa-arginine/bombesin delivery system. Further, competition with free bombesin peptide, and comparative uptake studies in Caco-2 cells, which express GRPR at a lower level, suggested that GRPR contributes to the targeted uptake of this system. The addition of GALA into the nona-arginine/bombesin-based system further increased the pDNA cellular uptake at all tested N/P ratios; facilitated endosomal pDNA release; and had limited effects on cell viability. In conclusion, the delivery system combining BBN(6-14) with nona-arginine and GALA had optimal characteristics for the delivery of pDNA into the GRPR overexpressing cell line PC-3.

Neuromedin B and its receptor silencing suppresses osteoclast generation by modulating precursor proliferation via M-CSF/c-Fms/D-type cyclins.

Neuromedin B (NMB), a mammalian bombesin-like peptide, regulates diverse physiological processes, such as energy metabolism, memory and fear behavior, and cellular growth, through its cognate receptor, NMBR. In this study, we report that NMB expression was upregulated during osteoclast development and that silencing NMB or NMBR attenuated osteoclast generation mediated by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). We found that knockdown of NMB or NMBR using a small hairpin RNA suppressed M-CSF-induced proliferation of osteoclast precursor cells without altering osteoclast differentiation. Interestingly, NMB or NMBR knockdown reduced the expression of the M-CSF receptor, c-Fms, which is an important modulator of osteoclast development. Consequently, NMB or NMBR silencing inhibited M-CSF/c-Fms-mediated downstream signaling pathways like activation of ERK and Akt and induction of D-type cyclins, cyclin D1 and D2. Moreover, knockdown of NMB or NMBR accelerated apoptosis in osteoclast lineage cells by inducing caspase-3, caspase-9, and Bim expression. In summary, our study demonstrates that the NMB/NMBR axis plays a pivotal role in osteoclast generation by modulating the proliferation and survival of osteoclast lineage cells.