Structures of human gastrin-releasing peptide receptors bound to antagonist and agonist for cancer and itch therapy.

Gastrin releasing peptide receptor (GRPR), a member of the bombesin (BBN) G protein-coupled receptors, is aberrantly overexpressed in several malignant tumors, including those of the breast, prostate, pancreas, lung, and central nervous system. Additionally, it also mediates non-histaminergic itch and pathological itch conditions in mice. Thus, GRPR could be an attractive target for cancer and itch therapy. Here, we report the inactive state crystal structure of human GRPR in complex with the non-peptide antagonist PD176252, as well as two active state cryo-electron microscopy (cryo-EM) structures of GRPR bound to the endogenous peptide agonist gastrin-releasing peptide and the synthetic BBN analog [D-Phe6, ß-Ala11, Phe13, Nle14] Bn (6-14), in complex with Gq heterotrimers. These structures revealed the molecular mechanisms for the ligand binding, receptor activation, and Gq proteins signaling of GRPR, which are expected to accelerate the structure-based design of GRPR antagonists and agonists for the treatments of cancer and pruritus.

A non-canonical retina-ipRGCs-SCN-PVT visual pathway for mediating contagious itch behavior.

Contagious itch behavior informs conspecifics of adverse environment and is crucial for the survival of social animals. Gastrin-releasing peptide (GRP) and its receptor (GRPR) in the suprachiasmatic nucleus (SCN) of the hypothalamus mediates contagious itch behavior in mice. Here, we show that intrinsically photosensitive retina ganglion cells (ipRGCs) convey visual itch information, independently of melanopsin, from the retina to GRP neurons via PACAP-PAC1R signaling. Moreover, GRPR neurons relay itch information to the paraventricular nucleus of the thalamus (PVT). Surprisingly, neither the visual cortex nor superior colliculus is involved in contagious itch. In vivo calcium imaging and extracellular recordings reveal contagious itch-specific neural dynamics of GRPR neurons. Thus, we propose that the retina-ipRGC-SCN-PVT pathway constitutes a previously unknown visual pathway that probably evolved for motion vision that encodes salient environmental cues and enables animals to imitate behaviors of conspecifics as an anticipatory mechanism to cope with adverse conditions.

A spinal neural circuitry for converting touch to itch sensation.

Touch and itch sensations are crucial for evoking defensive and emotional responses, and light tactile touch may induce unpleasant itch sensations (mechanical itch or alloknesis). The neural substrate for touch-to-itch conversion in the spinal cord remains elusive. We report that spinal interneurons expressing Tachykinin 2-Cre (Tac2Cre) receive direct Aß low threshold mechanoreceptor (LTMR) input and form monosynaptic connections with GRPR neurons. Ablation or inhibition markedly reduces mechanical but not acute chemical itch nor noxious touch information. Chemogenetic inhibition of Tac2Cre neurons also displays pronounced deficit in chronic dry skin itch, a type of chemical itch in mice. Consistently, ablation of gastrin-releasing peptide receptor (GRPR) neurons, which are essential for transmitting chemical itch, also abolishes mechanical itch. Together, these results suggest that innocuous touch and chemical itch information converge on GRPR neurons and thus map an exquisite spinal circuitry hard-wired for converting innocuous touch to irritating itch.

PAR2 Mediates Itch via TRPV3 Signaling in Keratinocytes.

Animal studies have suggested that transient receptor potential ion channels and G-protein coupled receptors play important roles in itch transmission. TRPV3 gain-of-function mutations have been identified in patients with Olmsted syndrome, which is associated with severe pruritus. However, the mechanisms causing itch remain poorly understood. Here, we show that keratinocytes lacking TRPV3 impair the function of protease-activated receptor 2 (PAR2), resulting in reduced neuronal activation and scratching behavior in response to PAR2 agonists. Moreover, we show that TRPV3 and PAR2 were upregulated in skin biopsies from patients and mice with atopic dermatitis, whereas their inhibition attenuated scratching and inflammatory responses in mouse atopic dermatitis models. These results reveal a previously unrecognized link between TRPV3 and PAR2 in keratinocytes to convey itch information and suggest that a blockade of PAR2 or TRPV3 individually or both may serve as a potential approach for antipruritic therapy in atopic dermatitis.

mir-1 inhibits migration of gastric cancer cells.

Gastric cancer is the fourth most common malignancy world-wide that bears a high mortality by invasiveness and metastases. To this end, we examined the role of miR-1 in mobility and migration of gastric cancer cells. miR-1 was down-regulated and Sorcin, which supports invasion, was highly expressed in gastric cancer cell lines as compared to the control. The overexpression of miR-1 significantly inhibited the mobility and migration of gastric cancer cells, while, its knockdown exerted an oppoiste effect. In addition, while overexpression of miR-1 suppressed the expression of Sorcin, the siRNA knockdown of Sorcin significantly counteracted the effect of miR-1 inhibitor on cell invasion and migration of gastric cancer cells. A miR-1 mimic decreased while its inhibitor increased the MMP-7 and VEGF required for invasion. Taken together, the findings support the view that miR-1 controls the mobility and migration of gastric cancer cells and might be a therapeutic target for blocking gastric cancer invasion.

Sedated gastroscopy improves detection of gastric polyps.

The detection rate of gastric polyps (GPs) is low, improving the detection rate would be good. The present study aimed to evaluate the role of sedated gastroscopy in GP detection. The data of patients who underwent gastroscopic examination from January 2014 to December 2016 at the First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) were retrospectively reviewed. Endoscopic records of 6,195 patients diagnosed with GPs were analyzed. The GP detection rate was 3.12 and 5.11% in the unsedated and sedated gastroscopy group, respectively (P<0.05). Also after stratification by sex, the GP detection rate was significantly higher in the sedated gastroscopy group (P<0.05). In addition, patients aged ≥20 years in the sedated gastroscopy group had a higher GP detection rate than those in the unsedated gastroscopy group (P<0.05). The incidence of cardiac, gastric fundus, gastric body and multiple-site GPs was significantly different between the two groups (P<0.05). GPs ≤0.5 and >0.5 cm were more common in the sedated gastroscopy group than in the unsedated gastroscopy group (P<0.05). The common pathologic types of GPs were gastric fundus gland (52.27%) and hyperplastic polyps (34.74%). In conclusion, the GP detection rate may be improved by inhibition of gastric muscle cramping with sedation.

The combined effect of non-alcoholic fatty liver disease and metabolic syndrome on colorectal carcinoma mortality: a retrospective in Chinese females.

BACKGROUND: This research aimed to investigate whether metabolic syndrome (MetS) and non-alcoholic fatty liver disease (NAFLD) had both individual and synergistic effects on the prognosis for female colorectal carcinoma (CRC) patients. METHODS: The relationship between CRC prognosis and NAFLD as well as MetS was evaluated in 764 female participants. Based on the NAFLD level, patients were divided into significant NAFLD (SNAFLD), "moderate" and "severe" level, and non-SNAFLD, "non" and "mild" level. All the patients were categorized into four subgroups according to the status of SNAFLD and MetS and then a comparison of CRC prognosis among those four groups was performed. RESULTS: NAFLD, SNAFLD, and MetS were independent factors for CRC-specific mortality with the adjustment of age and other confounders. The hazard ratio (HR) of CRC-specific mortality in MetS (+) SNAFLD (+) group was significantly higher than that in other three groups. Relative excess risk of interaction (RERI) was 2.203 with 95% CI ranged from 0.197 to 4.210, attributable proportion (AP) was 0.444 with range from 0.222 to 0.667, and synergy index (SI) of 2.256 with 95% CI from 1.252 to 4.065, indicating SNAFLD and MetS had a significant synergic effect on CRC-specific mortality. CONCLUSIONS: SNAFLD and MetS are independent risk factors for CRC-specific mortality in females. Moreover, those two diseases have a synergistic effect on promoting CRC-specific mortality.

REG3A promotes the proliferation, migration, and invasion of gastric cancer cells.

The mechanism underlying the metastasis of gastric cancer (GC) cells remains elusive. REG3A is considered an oncogene in various cancers, but in GC its role is unclear. Here, we report that the expression of REG3A was significantly increased in the tumor tissues of patients with GC compared with the matched normal tissues. Knockdown of REG3A induced by specific small interfering RNA (siRNA) significantly repressed the proliferation of GC cells for 24 h or 48 h. Moreover, knockdown of REG3A significantly suppressed the migration, invasion, and adhesion of GC cells in vitro. Furthermore, knockdown of REG3A reduced the phosphorylation of JAK2 and STAT3, and altered the messenger RNA (mRNA) and protein expression levels of E-cadherin, Snail, RhoC, MTA1, MMP-2, and MMP-9. Taken together, REG3A is overexpressed in GC and promotes the proliferation, migration, invasion, and adhesion of GC cells by regulating the JAK2/STAT3 signal pathway. REG3A may be a potential therapeutic target for GC.

Chronic itch development in sensory neurons requires BRAF signaling pathways.

Chronic itch, or pruritus, is associated with a wide range of skin abnormalities. The mechanisms responsible for chronic itch induction and persistence remain unclear. We developed a mouse model in which a constitutively active form of the serine/threonine kinase BRAF was expressed in neurons gated by the sodium channel Nav1.8 (BRAF(Nav1.8) mice). We found that constitutive BRAF pathway activation in BRAF(Nav1.8) mice results in ectopic and enhanced expression of a cohort of itch-sensing genes, including gastrin-releasing peptide (GRP) and MAS-related GPCR member A3 (MRGPRA3), in nociceptors expressing transient receptor potential vanilloid 1 (TRPV1). BRAF(Nav1.8) mice showed de novo neuronal responsiveness to pruritogens, enhanced pruriceptor excitability, and heightened evoked and spontaneous scratching behavior. GRP receptor expression was increased in the spinal cord, indicating augmented coding capacity for itch subsequent to amplified pruriceptive inputs. Enhanced GRP expression and sustained ERK phosphorylation were observed in sensory neurons of mice with allergic contact dermatitis­ or dry skin­elicited itch; however, spinal ERK activation was not required for maintaining central sensitization of itch. Inhibition of either BRAF or GRP signaling attenuated itch sensation in chronic itch mouse models. These data uncover RAF/MEK/ERK signaling as a key regulator that confers a subset of nociceptors with pruriceptive properties to initiate and maintain long-lasting itch sensation.

Itch signaling in the nervous system.

Itch is a major somatic sensation, along with pain, temperature, and touch, detected and relayed by the somatosensory system. Itch can be an acute sensation, associated with mosquito bite, or a chronic condition, like atopic dermatitis (29, 59). The origins of the stimulus can be localized in the periphery or systemic, and associated with organ failure or cancer. Itch is also a perception originating in the brain. Itch is broadly characterized as either histamine-dependent (histaminergic) or histamine-independent (nonhistaminergic), both of which are relayed by subsets of C fibers and by the second-order neurons expressing gastrin-releasing peptide receptor (GRPR) and spinothalamic track (STT) neurons in the spinal cord of rodents. Historically, itch research has been primarily limited to clinical and psychophysical studies and to histamine-mediated mechanisms. In contrast, little is known about the signaling mechanisms underlying nonhistaminergic itch, despite the fact that the majority of chronic itch are mediated by nonhistaminergic mechanisms. During the past few years, important progress has been made in understanding the molecular signaling of itch, largely due to the introduction of mouse genetics. In this review, we examine some of the molecular mechanisms underlying itch sensation with an emphasis on recent studies in rodents.

The c-kit signaling pathway is involved in the development of persistent pain.

Protein kinase signal transduction pathways play critical roles in regulating nociception. Here we show that c-kit, a tyrosine kinase receptor, is expressed in lamina I and II layer of the dorsal horn. Moreover, the superficial c-kit(+) fibers originate from the dorsal root ganglion, and c-kit in lamina II inner layer comes from intrinsic expression of the spinal cord. Kit(W-v) mice, which contain a hypomorphic mutation, exhibited normal acute pain in most pain behavior tests. In the formalin test, the first phase was not affected, whereas the second phase pain response of Kit(W-v) mice was significantly reduced relative to wild-type littermates. Kit(W-v) mice also showed abnormal neuropathic pain, notably in the contralateral side of nerve injury. The expression and release of CGRP and substance P were not altered by the c-kit mutation. Together, these results implicate c-kit-mediated signal transduction in the development of persistent pain.

Lmx1b is essential for Fgf8 and Wnt1 expression in the isthmic organizer during tectum and cerebellum development in mice.

Secreted factors FGF8 and WNT1 are essential either for the inductive activity of the isthmus organizer or for the regionalization of the midbrain-hindbrain boundary (MHB). However, transcriptional regulation of these secreted factors during development remains to be elucidated. Here we show that the LIM homeobox gene Lmx1b is expressed in the anterior embryo as early as E7.5 and its expression becomes progressively restricted to the isthmus at E9.0. Analysis of gene expression in the MHB of the mutant embryos showed that many genes were lost by E9.5. In the MHB of Lmx1b-/- embryos, the expression of Fgf8, which normally occurs at the 4-somite stage, was completely absent, whereas Wnt1 was downregulated before the 4-somite stage. Moreover, transcription factors En1 and Pax2 were also downregulated prior to the 4-somite stage, whereas Gbx2 downregulation occurred at the 4-somite stage. By contrast, Otx2 and Pax6 expression was not affected in Lmx1b-/- embryos. The requirement of specific Lmx1b expression in the MHB was further confirmed by Wnt1-Cre-mediated region-specific conditional knockout of Lmx1b. As a result of these molecular defects, the development of the tectum and cerebellum was severely impaired in Lmx1b-/- mice. Taken together, our results indicate that Lmx1b plays an essential role in the development of the tectum and cerebellum by regulating expression of Fgf8, Wnt1 and several isthmus-related transcription factors in the MHB, and is a crucial component of a cross-regulatory network required for the induction activity of the isthmic organizer in the MHB.

Lmx1b is required for maintenance of central serotonergic neurons and mice lacking central serotonergic system exhibit normal locomotor activity.

Central serotonergic neurons have been implicated in numerous animal behaviors and psychiatric disorders, but the molecular mechanisms underlying their development are not well understood. Here we generated Lmx1b (LIM homeobox transcription factor 1 beta) conditional knock-out mice (Lmx1b(f/f/p)) in which Lmx1b was only deleted in Pet1 (pheochromocytoma 12 ETS factor-1)-expressing 5-HT neurons. In Lmx1b(f/f/p) mice, the initial generation of central 5-HT neurons appeared normal. However, the expression of both 5-HT-specific and non-5-HT-specific markers was lost in these neurons at later stages of development. The loss of gene expression is concomitant with downregulation of Lmx1b expression, with the exception of serotonin transporter Sert and tryptophan hydroxylase TPH2, whose expression appears to be most sensitive to Lmx1b. Interestingly, the expression of Pet1 is tightly coupled with expression of Lmx1b during later stages of embryonic development, indicating that Lmx1b maintains Pet1 expression. In Lmx1b(f/f/p) mice, almost all central 5-HT neurons failed to survive. Surprisingly, Lmx1b(f/f/p) mice survived to adulthood and exhibited normal locomotor activity. These data reveal a critical role of Lmx1b in maintaining the differentiated status of 5-HT neurons. Lmx1b(f/f/p) mice with normal locomotor function should provide a unique animal model for examining the roles of central 5-HT in a variety of animal behaviors.

Ventral migration of early-born neurons requires Dcc and is essential for the projections of primary afferents in the spinal cord.

Neuronal migration and lamina-specific primary afferent projections are crucial for establishing spinal cord circuits, but the underlying mechanisms are poorly understood. Here, we report that in mice lacking Dcc (deleted in colorectal cancer), some early-born neurons could not migrate ventrally in the spinal cord. Conversely, forced expression of Dcc caused ventral migration and prevented dorsolateral migration of late-born spinal neurons. In the superficial layer of the spinal cord of Dcc-/- mutants, mislocalized neurons are followed by proprioceptive afferents, while their presence repels nociceptive afferents through Sema3a. Thus, our study has shown that Dcc is a key molecule required for ventral migration of early-born neurons, and that appropriate neuronal migration is a prerequisite for, and coupled to, normal projections of primary afferents in the developing spinal cord.

Focal adhesion kinase in netrin-1 signaling.

Netrins are a family of secreted molecules that are important for axonal outgrowth and guidance in the developing nervous system. However, the signaling mechanisms that lie immediately downstream of netrin receptors remain poorly understood. Here we report that the netrin receptor DCC (deleted in colorectal cancer) interacts with the focal adhesion kinase (FAK), a kinase implicated in regulating cell adhesion and migration. FAK was expressed in developing brains and was localized with DCC in cultured neurons. Netrin-1 induced FAK and DCC tyrosine phosphorylation. Disruption of FAK signaling abolished netrin-1-induced neurite outgrowth and attractive growth cone turning. Taken together, these results indicate a new signaling mechanism for DCC, in which FAK is activated upon netrin-1 stimulation and mediates netrin-1 function; they also identify a critical role for FAK in axon navigation.

Lmx1b controls the differentiation and migration of the superficial dorsal horn neurons of the spinal cord.

The differentiation and migration of superficial dorsal horn neurons and subsequent ingrowth of cutaneous afferents are crucial events in the formation of somatosensory circuitry in the dorsal spinal cord. We report that the differentiation and migration of the superficial dorsal horn neurons are regulated by the LIM homeobox gene Lmx1b, and its downstream targets Rnx and Drg11, two transcription factors implicated in the development of dorsal horn circuitry. An analysis of Lmx1b mutants shows that Lmx1b normally acts to maintain the expression of the Ebf genes and to repress the Zic genes. Lmx1b mutants also exhibit the disruption of the cutaneous afferent ingrowth, suggesting that the dorsal horn cells might provide important cues guiding sensory axons into the dorsal spinal cord. Our results thus indicate that Lmx1b has a pivotal role in genetic cascades that control the assembly of circuitry in the superficial dorsal horn.

Genetic elimination of behavioral sensitization in mice lacking calmodulin-stimulated adenylyl cyclases.

Adenylyl cyclase types 1 (AC1) and 8 (AC8), the two major calmodulin-stimulated adenylyl cyclases in the brain, couple NMDA receptor activation to cAMP signaling pathways. Cyclic AMP signaling pathways are important for many brain functions, such as learning and memory, drug addiction, and development. Here we show that wild-type, AC1, AC8, or AC1&8 double knockout (DKO) mice were indistinguishable in tests of acute pain, whereas behavioral responses to peripheral injection of two inflammatory stimuli, formalin and complete Freund's adjuvant, were reduced or abolished in AC1&8 DKO mice. AC1 and AC8 are highly expressed in the anterior cingulate cortex (ACC), and contribute to inflammation-induced activation of CREB. Intra-ACC administration of forskolin rescued behavioral allodynia defective in the AC1&8 DKO mice. Our studies suggest that AC1 and AC8 in the ACC selectively contribute to behavioral allodynia.