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Purpose Transient receptor potential vanilloid 4 (TRPV4) has been reported to be involved in the progression of several human tumors. Nevertheless, clinical significance and molecular mechanism of TRPV4 in gastric cancer (GC) remain poorly defined. Patients and methods Immunohistochemistry assays were used to investigate the correlation between the expression of TRPV4 and epithelial-mesenchymal transition (EMT) markers in human GC tissues. The correlations between TRPV4 expression and clinicopathological features and between TRPV4 expression and survival rates were also examined. TRPV4 knockdown was performed by using small interfering RNAs. In vitro, Cell Counting Kit-8 (CCK-8) assay, colony formation assay, and transwell assay were employed to further explore the biological functions of TRPV4, and Western blotting was used to evaluate the changes in the expression of TRPV4 protein and EMT-related proteins in HGC-27 and MGC-803 human GC cell lines. Results TRPV4 expression was upregulated in GC tissues and cell lines. TRPV4 overexpression was associated with greater depth of tumor invasion, lymph node metastasis, higher TNM stage, poor overall survival, and worse disease-free survival. TRPV4 expression was inversely correlated with E-cadherin expression and positively correlated with vimentin expression. In vitro, knockdown of TRPV4 inhibited GC cell proliferation, colony formation, and invasion. Furthermore, the knockdown of TRPV4 modulated EMT by upregulating E-cadherin expression and downregulating the expression of N-cadherin and vimentin. In addition, the EMT-related transcription factor Snail was downregulated, whereas the expression levels of other transcription factors such as Slug and Twist did not change. Conclusion TRPV4 was upregulated in human GC and the overexpression of TRPV4 could promote GC progression, partially through Snail-mediated EMT.

Bo Shen,1 Aihua Qian,1 Wenji Lao,1 Weiguang Li,1 Xi Chen,1 Benyan Zhang,2 Huafeng Wang,2 Fei Yuan,2 Yunwei Sun11Department of Gastroenterology of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, People’s Republic of China; 2Department of Pathology of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, People’s Republic of ChinaBackground: Many studies have shown that programmed cell death protein 1 (PD-1) and its ligand, PD-L1, are expressed in advanced gastric cancer. Furthermore, detection of these proteins is associated with infiltrating CD8+ T-cells, indicating that an adaptive immune resistance mechanism occurs in advanced gastric cancer. However, PD-L1 and PD-1 expression in gastric intraepithelial neoplasia and early-stage gastric cancer (EGC) has yet to be elucidated.Patients and methods: Fifty-four resections of low-grade intraepithelial neoplasia (LGIN), high-grade intraepithelial neoplasia (HGIN), and EGC were stained by immunohistochemistry for PD-1, PD-L1, and CD8. CD8+ T-cell densities both within tumors and in the tumor-stromal interface were analyzed. Flow cytometry (FACS) was used to analyze the PD-1 expression in tumor tissues and peripheral blood mononuclear cells. Furthermore, the relationship between Helicobacter pylori (Hp) infection and PD-1 and PD-L1 was also evaluated.Results: We demonstrated that PD-L1 expression was significantly increased in HGIN and EGC compared with LGIN, and both PD-1 and PD-L1 showed similar expression patterns, being mainly detected in infiltrating immune cells. FACS also showed that PD-1 was expressed on both CD4+ and CD8+ T-cells. However, no difference was found in CD8+ T-cell infiltration between LGIN and HGIN+EGC, and this was not not found to be associated with PD-L1 or PD-1 expression. However, Hp infection was significantly associated with expression of PD-L1 and PD-1.Conclusions: The PD-1/PD-L1 checkpoint is involved in intraepithelial neoplasia and EGC, but an adaptive immune resistance mechanism does not occur. Expression of PD-1/PD-L1 is also associated with Hp infection, and so Hp infection may be an important initiating factor.Clinical Trial Registration information: This study was approved by the Institutional Review Board of Ruijin Hospital and written informed consent was obtained from all patients.Keywords: PD-L1, PD-1, Helicobacter pylori, LGIN, HGIN

Background Nicotine, an active ingredient in tobacco, can promote epithelial-to-mesenchymal transition (EMT) processes that enhance the aggressiveness of a number of human cancers. In the present study, we investigated whether cigarette smoke/nicotine drives EMT in pancreatic ductal adenocarcinoma (PDAC). Methods Quantitative real-time PCR, western blot, immunohistochemistry, and immunofluorescence assays were used to evaluate Yes-associated protein 1 (YAP1) expression associated with cigarette smoking in human PDAC tissue samples and with nicotine exposure in PDAC cell lines. Bioinformatics, loss- and gain- of- function experiments, luciferase reporter assays, chromatin immunoprecipitation (ChIP), and murine tumor xenograft models were performed to examine the function of YAP1 in PDAC and to identify potential mechanisms of action. Results Exposure to smoking or nicotine promoted EMT and tumor growth in PDAC cells and in xenograft tumors. Functional studies revealed that YAP1 might drive nicotine-stimulated EMT and oncogenic activity in vitro and in vivo. In human PDAC tissues, upregulation of YAP1 was associated with “ever smoking” status and poor overall survival. In term of mechanism, hypoxia inducible factor (HIF)1A promoted YAP1 nuclear localization and YAP1 transactivation by directly binding to the hypoxia responsive elements of the YAP1 promoter upon nicotine treatment. Nicotine stimulated HIF1A and YAP1 expression by activating cholinergic receptor nicotinic alpha7 (CHRNA7). In addition, YAP1 increased and sustained the protein stability of HIF1A. Conclusions These data demonstrate that YAP1 enhances nicotine-stimulated EMT and tumor progression of PDAC through a HIF1A/YAP1 positive feedback loop. Developing inhibitors that specifically target YAP1 may provide a novel therapeutic approach to suppress PDAC growth, especially in PDAC patients who have a history of smoking.

Zinc is a transition metal that has a long history of use as an anti-inflammatory agent. It also soothes pain sensations in a number of animal models. However, the effects and mechanisms of zinc on chemotherapy-induced peripheral neuropathy remain unknown. Here we show that locally injected zinc markedly reduces neuropathic pain in male and female mice induced by paclitaxel, a chemotherapy drug, in a TRPV1-dependent manner. Extracellularly applied zinc also inhibits the function of TRPV1 expressed in HEK293 cells and mouse DRG neurons, which requires the presence of zinc-permeable TRPA1 to mediate entry of zinc into the cytoplasm. Moreover, TRPA1 is required for zinc-induced inhibition of TRPV1-mediated acute nociception. Unexpectedly, zinc transporters, but not TRPA1, are required for zinc-induced inhibition of TRPV1-dependent chronic neuropathic pain produced by paclitaxel. Together, our study demonstrates a novel mechanism underlying the analgesic effect of zinc on paclitaxel-induced neuropathic pain that relies on the function of TRPV1.SIGNIFICANCE STATEMENTThe chemotherapy-induced peripheral neuropathy is a major limiting factor affecting the chemotherapy patients. There is no effective treatment available currently. We demonstrate that zinc prevents paclitaxel-induced mechanical hypersensitivity via inhibiting the TRPV1 channel, which is involved in the sensitization of peripheral nociceptors in chemotherapy. Zinc transporters in DRG neurons are required for the entry of zinc into the intracellular side, where it inhibits TRPV1. Our study provides insight into the mechanism underlying the pain-soothing effect of zinc and suggests that zinc could be developed to therapeutics for the treatment of chemotherapy-induced peripheral neuropathy.

Background Chronic itch is a highly debilitating symptom that underlies many medical disorders with no universally effective treatments. Although unique neuronal signaling cascades in the sensory ganglia and spinal cord have been shown to critically promote the pathogenesis of chronic itch, the role of skin-associated cells remains poorly understood. Objective We sought to examine the cutaneous mechanisms underlying transient receptor potential vanilloid 4 (TRPV4)–mediated allergic and nonallergic chronic itch. Methods Expression of TRPV4 in chronic itch and healthy control skin preparations was examined by using real-time RT-PCR. Trpv4 eGFP mice were used to study the expression and function of TRPV4 in the skin by means of immunofluorescence staining, flow cytometry, calcium imaging, and patch-clamp recordings. Genetic and pharmacologic approaches were used to examine the role and underlying mechanisms of TRPV4 in mouse models of dry skin–associated chronic itch and spontaneous scratching associated with squaric acid dibutylester–induced allergic contact dermatitis. Results TRPV4 is selectively expressed by dermal macrophages and epidermal keratinocytes in mice. Lineage-specific deletion of TRPV4 in macrophages and keratinocytes reduces allergic and nonallergic chronic itch in mice, respectively. Importantly, TRPV4 expression is significantly increased in skin biopsy specimens from patients with chronic idiopathic pruritus in comparison with skin from healthy control subjects. Moreover, TRPV4-dependent chronic itch requires 5-hydroxytryptamine (5-HT) signaling secondary to activation of distinct 5-HT receptors in mice with allergic and those with nonallergic chronic itch conditions. Conclusion Our study reveals previously unrecognized mechanisms by which TRPV4-expressing epithelial and immune cells in the skin critically and dynamically mediate chronic itch and unravels novel targets for therapeutics in the setting of chronic itch.

Background Ca2+/calmodulin-dependent protein kinases (CaMKs) are major downstream mediators of neuronal calcium signaling that regulate multiple neuronal functions. CaMKII, one of the key CaMKs, plays a significant role in mediating cellular responses to external signaling molecules. Although calcium signaling plays an essential role in the enteric nervous system (ENS), the role of CaMKII in neurogenic intestinal function has not been determined. In this study, we investigated the function and expression pattern of CaMKII in the ENS across several mammalian species. Methodology/Principal Findings CaMKII expression was characterized by immunofluorescence analyses and Western Blot. CaMKII function was examined by intracellular recordings and by assays of colonic contractile activity. Immunoreactivity for CaMKII was detected in the ENS of guinea pig, mouse, rat and human preparations. In guinea pig ENS, CaMKII immunoreactivity was enriched in both nitric oxide synthase (NOS)- and calretinin-containing myenteric plexus neurons and non-cholinergic secretomotor/vasodilator neurons in the submucosal plexus. CaMKII immunoreactivity was also expressed in both cholinergic and non-cholinergic neurons in the ENS of mouse, rat and human. The selective CaMKII inhibitor, KN-62, suppressed stimulus-evoked purinergic slow EPSPs and ATP-induced slow EPSP-like response in guinea pig submucosal plexus, suggesting that CaMKII activity is required for some metabotropic synaptic transmissions in the ENS. More importantly, KN-62 significantly suppressed tetrodotoxin-induced contractile response in mouse colon, which suggests that CaMKII activity is a major determinant of the tonic neurogenic inhibition of this tissue. Conclusion ENS neurons across multiple mammalian species express CaMKII. CaMKII signaling constitutes an important molecular mechanism for controlling intestinal motility and secretion by regulating the excitability of musculomotor and secretomotor neurons. These findings revealed a fundamental role of CaMKII in the ENS and provide clues for the treatment of intestinal dysfunctions.

Backgrounds and aims: This study aimed to compare the esophageal motility between gastroesophageal reflux disease (GERD) patients with typical symptoms but without globus sensation and GERD patients only with globus symptoms. Methods: A total of 57 consecutive GERD patients diagnosed by endoscopy or by 24-hour pH monitoring between May 2013 and September 2015 were included retrospectively into the study. The patients were grouped based on the presence or absence of globus. Thirty patients presented with typical reflux symptoms but without globus were assigned to the typical GERD group and 27 patients only with globus symptom were assigned to the globus GERD group. All patients underwent esophageal high resolution manometry (HRM) and the differences in esophageal motility between the two groups were analyzed. Results: The globus GERD group showed a significantly greater lower esophageal sphincter (LES) length, LES basal pressure and upper esophageal sphincter (UES) residual pressure than that of the typical GERD group (3.47 ± 0.76 vs. 2.65 ± 0.62 cm, 21.71 ± 9.68 vs. 16.04 ± 8.49 mmHg, 7.30 ± 4.42 vs. 4.12 ± 2.92 mmHg, all p < 0.05). There was no significant difference between the two groups in terms of the distal wave amplitude, mean wave duration, distal contractile integral (DCI), contractile front velocity (CFV), distal latency (DL), integrated relaxation pressure (IRP) and UES basal pressure. The incidence of esophageal dysmotility in the globus GERD group (33.3%) was higher than in the typical GERD group (23.3%). There was no significant difference in esophageal acid exposure of the non-erosive gastroesophageal reflux disease (NERD) patients between the two groups. Conclusions: Globus GERD patients have a higher UES residual pressure, longer LES length, higher LES basal pressure and greater esophageal dysmotility than typical GERD patients. HRM is useful in evaluating esophageal motility of GERD patients.