Modified Proteinase-Activated Receptor-1 and -2 Derived Peptides Inhibit Proteinase-Activated Receptor-2 Activation by Trypsin

BAHJAT AL-ANI, MAHMOUD SAIFEDDINE, SURANGA J. WIJESURIYA and MORLEY D. HOLLENBERG

ABSTRACT
Trypsin activates proteinase-activated receptor-2 (PAR2) by a mechanism that involves the release of a tethered receptor activating sequence. We have identified two peptides, FSLLRY-NH2 (FSY-NH2) and LSIGRL-NH2 (LS-NH2) that block the ability of trypsin to activate PAR2 either in PAR2-expressing Kirsten virus-transformed kidney (KNRK) cell lines or in a rat aorta ring preparation. The reverse PAR2 peptide, LRGILS-NH2(LRG-NH2) did not do so and FSY-NH2 failed to block thrombin activation of PAR1 in the aorta ring or in PAR1-expressing human embryonic kidney cells. Half-maximal inhibition (IC50) by FSY-NH2 and LS-NH2 of the activation of PAR2 by trypsin in a PAR2 KNRK calcium-signaling assay was observed at about 50 and 200 M, respectively. In contrast, the activation of PAR2 by the PAR2-activating peptide, SLIGRL-NH2 (SL-NH2) was not inhibited by FSY-NH2, LS-NH2, or LRG-NH2. In a casein proteolysis assay, neither FSY-NH2 nor LS-NH2 inhibited the proteolytic action of trypsin on its substrate. In addition, FSY-NH2
and LS-NH2 were unable to prevent trypsin from hydrolyzing a 20-amino acid peptide, GPNSKGR/SLIGRLDTPYGGC representing the trypsin cleavage/activation site of rat PAR2. Similarly, FSY-NH2 and LS-NH2 failed to block the ability of trypsin to release the PAR2 N-terminal epitope that is cleaved from the receptor upon proteolytic activation of receptor-expressing KNRK cells. We conclude that the peptides FSY-NH2 and LSNH2 block the ability of trypsin to activate PAR2 by a mechanism that does not involve a simple inhibition of trypsin proteolytic activity, but possibly by interacting with a tethered ligand receptor-docking site.

Presenter: Gi Baek Lee

IL-31 levels correlate with pruritus in patients with cholestatic and metabolic liver diseases and is farnesoid X receptor responsive in NASH

Jun Xu , Ya Wang , Mina Khoshdeli , Matt Peach , Jen-Chieh Chuang , Julie Lin , Wen-Wei Tsai , Sangeetha Mahadevan , Wesley Minto , Lauri Diehl , Ruchi Gupta , Michael Trauner , Keyur Patel , Mazen Noureddin , Kris V Kowdley , Aliya Gulamhusein , Christopher L Bowlus , Ryan S Huss , Robert P Myers , Chuhan Chung , Andrew N Billin 

Abstract

Background and aims: Pruritus is associated with multiple liver diseases, particularly those with cholestasis, but the mechanism remains incompletely understood. Our aim was to evaluate serum IL-31 as a putative biomarker of pruritus in clinical trials of an farnesoid X receptor (FXR) agonist, cilofexor, in patients with NASH, primary sclerosing cholangitis (PSC), and primary biliary cholangitis (PBC).

Approach and results: Serum IL-31 was measured in clinical studies of cilofexor in NASH, PSC, and PBC. In patients with PSC or PBC, baseline IL-31 was elevated compared to patients with NASH and healthy volunteers (HVs). IL-31 correlated with serum bile acids among patients with NASH, PBC, and PSC. Baseline IL-31 levels in PSC and PBC were positively correlated with Visual Analog Scale for pruritus and 5-D itch scores. In patients with NASH, cilofexor dose-dependently increased IL-31 from Week (W)1 to W24. In patients with NASH receiving cilofexor 100 mg, IL-31 was higher in those with Grade 2-3 pruritus adverse events (AEs) than those with Grade 0-1 pruritus AEs. IL-31 weakly correlated with C4 at baseline in patients with NASH, and among those receiving cilofexor 100 mg, changes in IL-31 and C4 from baseline to W24 were negatively correlated. IL-31 messenger RNA (mRNA) was elevated in hepatocytes from patients with PSC and NASH compared to HVs. In a humanized liver murine model, obeticholic acid increased IL-31 mRNA expression in human hepatocytes and serum levels of human IL-31.

Conclusions: IL-31 levels correlate with pruritus in patients with cholestatic disease and NASH, with FXR agonist therapy resulting in higher serum levels in the latter group. IL-31 appears to derive in part from increased hepatocyte expression. These findings have therapeutic implications for patients with liver disease and pruritus.

GPR15L is an epithelial inflammation-derived pruritogen

Pang-Yen Tseng and Mark A. Hoon*

Molecular Genetics Section, National Institute of Dental and Craniofacial Research/ NIH, 35 Convent Drive, Bethesda, MD 20892, USA.
*Corresponding author. Email: mark.hoon@nih.gov

Itch is an unpleasant sensation that often accompanies chronic dermatological conditions. Although many of the itch receptors and the neural pathways underlying this sensation are known, the identity of endogenous ligands is still not fully appreciated. Using an unbiased bioinformatic approach, we identified GPR15L as a candidate pruritogen whose expression is robustly up-regulated in psoriasis and atopic dermatitis. Although GPR15L was previously shown to be a cognate ligand of the receptor GPR15, expressed in dermal T cells, here we show that it also contributes to pruritogenesis by activating Mas-related G protein–coupled receptors (MRGPRs). GPR15L can selectively stimulate mouse dorsal root ganglion neurons that express Mrgpra3 and evokes intense itch re- sponses. GPR15L causes mast cell degranulation through stimulation of MRGPRX2 and Mrgprb2. Genetic dis- ruption of GPR15L expression attenuates scratch responses in a mouse model of psoriasis. Our study reveals unrecognized features of GRP15L, showing that it is a potent itch-inducing agent.

Sphingomyelin Deacylase, the Enzyme Involved in the Pathogenesis of Atopic Dermatitis, Is Identical to the β-Subunit of Acid Ceramidase

Yasuhiro Teranishi 1,†,‡, Hiroshi Kuwahara 1,†,§, Masaru Ueda 1, Tadashi Takemura 1, Masanori Kusumoto 1,§, Keiji Nakamura 1, Jun Sakai 1, Toru Kimura 1, Yasuji Furutani 1, Makoto Kawashima 2, Genji Imokawa 3,* and Mari Nogami-Itoh 4,*

Abstract: A ceramide deficiency in the stratum corneum (SC) is an essential etiologic factor for the
dry and barrier-disrupted skin of patients with atopic dermatitis (AD). Previously, we reported that
sphingomyelin (SM) deacylase, which hydrolyzes SM and glucosylceramide at the acyl site to yield
their lysoforms sphingosylphosphorylcholine (SPC) and glucosylsphingosine, respectively, instead of
ceramide and/or acylceramide, is over-expressed in AD skin and results in a ceramide deficiency.
Although the enzymatic properties of SM deacylase have been clarified, the enzyme itself remains
unidentified. In this study, we purified and characterized SM deacylase from rat skin. The activities
of SM deacylase and acid ceramidase (aCDase) were measured using SM and ceramide as substrates
by tandem mass spectrometry by monitoring the production of SPC and sphingosine, respectively.
Levels of SM deacylase activity from various rat organs were higher in the order of skin > lung >
heart. By successive chromatography using Phenyl-5PW, Rotofor, SP-Sepharose, Superdex 200 and
Shodex RP18-415, SM deacylase was purified to homogeneity with a single band of an apparent
molecular mass of 43 kDa with an enrichment of > 14,000-fold. Analysis by MALDI-TOF MS/MS
using a protein spot with SM deacylase activity separated by 2D-SDS-PAGE allowed its amino acid
sequence to be determined and identified as the β-subunit of aCDase, which consists of α- and
β-subunits linked by amino bonds and a single S-S bond. Western blotting of samples treated with
2-mercaptoethanol revealed that, whereas recombinant human aCDase was recognized by antibodies
to the α-subunit at ~56 kDa and ~13 kDa and the β-subunit at ~43 kDa, the purified SM deacylase was
detectable only by the antibody to the β-subunit at ~43 kDa. Breaking the S-S bond of recombinant
human aCDase with dithiothreitol elicited the activity of SM deacylase with ~40 kDa upon gel chromatography. These results provide new insights into the essential role of SM deacylase expressed as an aCDase-degrading β-subunit that evokes the ceramide deficiency in AD skin.

Keywords: atopic dermatitis; ceramide; ceramide deficiency; barrier function; water reservoir faction;
stratum corneum; sphingomyelin deacylase; sphingosylphosphorylcholine; acid ceramidase

Functional Interaction between Transient Receptor Potential V4 Channel and Neuronal Calcium Sensor 1 and the Effects of Paclitaxel

Julio C. Sanchez and Barbara E. Ehrlich

ABSTRACT
Neuronal calcium sensor 1 (NCS1), a calcium-binding protein, and transient receptor potential V4 (TRPV4), a plasma membrane calcium channel, are fundamental in the regulation of calcium homeostasis. The interactions of these proteins and their regulation by paclitaxel (PTX) were investigated using biochemical, pharmacological, and electrophysiological approaches in both a breast cancer epithelial cell model and a neuronal model. TRPV4 and NCS1 reciprocally immunoprecipitated each other, suggesting that they make up a signaling complex. The functional consequence of this physical association was that TRPV4 currents increased with increased NCS1 expression. Calcium fluxes through TRPV4 correlated with the magnitude of TRPV4 currents, and these calcium fluxes depended on NCS1
expression levels. Exposure to PTX amplified the acute effects of TRPV4 expression, currents, and calcium fluxes but decreased the expression of NCS1. These findings augment the understanding of the properties of TRPV4, the role of NCS1 in the regulation of TRPV4, and the cellular mechanisms of PTX-induced neuropathy.

Presenter: Ki Baek Lee

TRPV4 inhibition prevents paclitaxel-induced neurotoxicity in preclinicalmodels

Wolfgang Boehmerlea,b,c,⁎,1, Petra Huehnchena,b,c,1, Sabrina Lin Lin Leeb,d, Christoph Harmsa,b,c,d,1, Matthias Endresa,b,c,d,e,f,1

Paclitaxel is a cytotoxic drug which frequently causes sensory peripheral neuropathy in patients. Increasing
evidence suggests that altered intracellular calcium (Ca2+) signals play an important role in the pathogenesis of
this condition. In the present study, we examined the interplay between Ca2+ release channels in the endoplasmic reticulum (ER) and Ca2+ permeable channels in the plasma membrane in the context of paclitaxel
mediated neurotoxicity. We observed that in small to medium size dorsal root ganglia neurons (DRGN) the
inositol-trisphosphate receptor (InsP3R) type 1 was often concentrated in the periphery of cells, which is in
contrast to homogenous ER distribution. G protein-coupled designer receptors were used to further elucidate
phosphoinositide mediated Ca2+ signaling: This approach showed strong InsP3 mediated Ca2+ signals close to
the plasma membrane, which can be amplified by Ca2+ entry through TRPV4 channels. In addition, our results
support a physical interaction and partial colocalization of InsP3R1 and TRPV4 channels. In the context of
paclitaxel-induced neurotoxicity, blocking Ca2+ influx through TRPV4 channels reduced cell death in cultured
DRGN. Pretreatment of mice with the pharmacological TRPV4 inhibitor HC067047 prior to paclitaxel injections
prevented electrophysiological and behavioral changes associated with paclitaxel-induced neuropathy.
In summary, these results underline the relevance of TRPV4 signaling for the pathogenesis of paclitaxelinduced neuropathy and suggest novel preventive strategies

Keywords: Paclitaxel, Neuropathy, Calcium, TRPV4, Inositol-trisphosphate receptor

Presenter: Hye In Kim

A re‐innervated in vitro skin model of non‐histaminergic itch and skin neurogenic inflammation: PAR2‐, TRPV1‐ and TRPA1‐agonist induced functionality

N. Lebonvallet, J. W. Fluhr, C. Le Gall‐Ianotto, R. Leschiera, M. Talagas, J.‐L. Carré, A. Reux, L. Misery, A. Bataille, C. Brun3, T. Oddos3, J.‐P. Pennec4

• Published in:- Skin Health and Disease

Abstract

Background: Skin, and epidermis, is innervated by sensory nerve fibres. Interactions between them and signal transduction are only partially eluci- dated in physiological/pathological conditions, especially in pruritus.

Objectives: To study the mechanisms involved in pruritus in vitro, we developed a skin explant model re‐innervated by sensory neurons.

Methods: This model is based on the co‐culture of human skin explants and sensory neurons from dorsal root ganglia of rats. Innervation and the expression of protease activated receptor 2 (PAR2), transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin one (TRPA1) was analysed by immunostaining. The response of the model to TRPV1, PAR2 and TRPA1 agonists was analysed by patch‐clamp, qPCR and enzyme‐linked immunosorbent assay.

Results: After 5 days of re‐innervating nerve fibres was evidenced in the epidermis. Re‐innervation was correlated with decrease of epidermal thickness and the number of apoptotic cells in the tissue. The major actors of non‐histaminergic itch (PAR‐2, thymic stromal lymphopoietin [TSLP], TSLP‐R, TRPA1 and TRPV1) were expressed in neurons and/or epidermal cells of skin explants. After topical exposure of TRPV1‐(Capsaicin), TRPA1‐ (Polygodial) and PAR2‐agonist (SLIGKV‐NH2) activation of reinnervating neurons could be shown in patch‐clamp analysis. The release of TSLP was increased with capsaicin or SLIGKV but decreased with polygodial. Release of CGRP was increased by capsaicin and polygodial but decreased with SLIGKV. Activation by SLIGKV showed a decrease of VEGF; polygodial induced an increase of TSLP, Tumour necrosis factor (TNF) and nerve growth factor and capsaicin lead to a decrease of sema3 and TNF expression.

Conclusion: The present model is suitable for studying itch and neurogenic inflammation pathways in vitro. We observed that activation of TRPV1, TRPA1 and PAR‐2 leads to different response profiles in re‐innervated skin explants.

The role of Na _v 1.7 and methylglyoxal-mediated activation of TRPA1 in itch and hypoalgesia in a murine model of type 1 diabetes

Ruo-Xiao Cheng, Yu Feng, Di Liu, Zhi-Hong Wang, Jiang-Tao Zhang, Li-Hua Chen, Cun-Jin Su, Bing Wang, Ya Huang, Ru-Rong Ji, Ji Hu, Tong Liu

Abstract

Methylglyoxal (MGO), an endogenous reactive carbonyl compound, plays a key role in the pathogenesis of diabetic neuropathy. The aim of this study is to investigate the role of MGO in diabetic itch and hypoalgesia, two common symptoms associated with diabetic neuropathy. 

Methods: Scratching behavior, mechanical itch (alloknesis), and thermal hypoalgesia were quantified after intradermal (i.d.) injection of MGO in naïve mice or in diabetic mice induced by intraperitoneal (i.p.) injection of streptozotocin (STZ). Behavioral testing, patch-clamp recording, transgenic mice, and gene expression analysis were used to investigate the mechanisms underlying diabetic itch and hypoalgesia in mice. 

Results: I.d. injection of MGO evoked dose-dependent scratching in normal mice. Addition of MGO directly activated transient receptor potential ankyrin 1 (TRPA1) to induce inward currents and calcium influx in dorsal root ganglia (DRG) neurons or in TRPA1-expressing HEK293 cells. Mechanical itch, but not spontaneous itch was developed in STZ-induced diabetic mice. Genetic ablation of Trpa1 (Trpa1^-/- ), pharmacological blockade of TRPA1 and Na_v1.7, antioxidants, and mitogen-activated protein kinase kinase enzyme (MEK) inhibitor U0126 abrogated itch induced by MGO or in STZ-induced diabetic mice. Thermal hypoalgesia was induced by intrathecal (i.t.) injection of MGO or in STZ-induced diabetic mice, which was abolished by MGO scavengers, intrathecal injection of TRPA1 blockers, and in Trpa1^-/- mice. 

Conclusion: This study revealed that Na_v1.7 and MGO-mediated activation of TRPA1 play key roles in itch and hypoalgesia in a murine model of type 1 diabetes. Thereby, we provide a novel potential therapeutic strategy for the treatment of itch and hypoalgesia induced by diabetic neuropathy.

Keywords: Diabetes; Hypoalgesia; Itch; Methylglyoxal; TRPA1.

The Role of Transient Receptor Potential A1 and G Protein-Coupled Receptor 39 in Zinc-Mediated Acute and Chronic Itch in Mice

Yue Hu^1†, Qing-Yue Fu^1†, Dan-Ni Fu^1†, Xue-Long Wang^2†, Zhi-Hong Wang¹, Jiang-Tao Zhang³, Wen-Jing Xu³, Guo-Kun Zhou³, Li-Hua Chen^4* and Tong Liu^3,5,6*

• ¹Jiangsu Key Laboratory of Neuropsychiatric Diseases and Institute of Neuroscience, Soochow University, Suzhou, China
• ²Department of Thoracic Surgery, Capital Medical University Electric Power Teaching Hospital Beijing, Beijing, China
• ³Institute of Pain Medicine and Special Environmental Medicine, Nantong University, Nantong, China
• ⁴Department of Nutrition and Food Hygiene, School of Public Health, Nantong University, Nantong, China
• ⁵College of Life Sciences, Yanan University, Yan’an, China
• ⁶Suzhou Key Laboratory of Intelligent Medicine and Equipment, Soochow University, Suzhou, China

Itching is a common symptom of many skin or systemic diseases and has a negative impact on the quality of life. Zinc, one of the most important trace elements in an organism, plays an important role in the regulation of pain. Whether and how zinc regulates itching is largely unclear. Herein, we explored the role of Zn²⁺ in the regulation of acute and chronic itch in mice. It is found that intradermal injection (i.d.) of Zn²⁺ dose-dependently induced acute itch and transient receptor potential A1 (TRPA1) participated in Zn²⁺-induced acute itch in mice. Moreover, the pharmacological analysis showed the involvement of histamine, mast cells, opioid receptors, and capsaicin-sensitive C-fibers in Zn²⁺-induced acute itch in mice. Systemic administration of Zn²⁺ chelators, such as N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), pyrithione, and clioquinol were able to attenuate both acute itch and dry skin-induced chronic itch in mice. Quantitative polymerase chain reaction (Q-PCR) analysis showed that the messenger RNA (mRNA) expression levels of zinc transporters (ZIPs and ZnTs) significantly changed in the dorsal root ganglia (DRG) under dry skin-induced chronic itch condition in mice. Activation of extracellular signal-regulated kinase (ERK) pathway was induced in the DRG and skin by the administration of zinc or under dry skin condition, which was inhibited by systemic administration of Zn²⁺ chelators. Finally, we found that the expression of GPR39 (a zinc-sensing GPCR) was significantly upregulated in the dry skin mice model and involved in the pathogenesis of chronic itch. Together, these results indicated that the TRPA1/GPR39/ERK axis mediated the zinc-induced itch and, thus, targeting zinc signaling may be a promising strategy for anti-itch therapy.

Keywords: GPR39; Itch; TRPA1; Zn2+; p-ERK.

Sensory neuron expressed TRPC3 mediates acute and chronic itch.

Yan Liu , Yutong Liu , Nathachit Limjunyawong , Claire Narang , Hanna Jamaldeen, Shimeng Yu , Shivanie Patiram , Hong Nie , Michael J. Caterina , Xinzhong Dong , Lintao Qu

Abstract

Chronic pruritus is a prominent symptom of allergic contact dermatitis(ACD) and represent a huge unmet health problem. However, its underlying cellular and molecular mechanisms remain largely unexplored. TRPC3 is highly expressed in primary sensory neurons and has been implicated in peripheral sensitization induced by proinflammatory mediators. Yet, the role of TRPC3 in acute and chronic itch is still not well defined. Here, we show that, among mouse trigeminal ganglion (TG) neurons, Trpc3 mRNA is predominantly expressed in nonpeptidergic small diameter TG neurons of mice. Moreover, Trpc3 mRNA signal was present in the majority of presumptively itch sensing neurons. TRPC3 agonism induced TG neuronal activation and acute nonhistaminergic itch- and pain-like behaviors in naïve mice. In addition, genetic deletion of Trpc3 attenuated acute itch evoked by certain common nonhistaminergic pruritogens, including endothelin-1 and SLIGRL-NH2. In a murine model of contact hypersensitivity (CHS), Trpc3 mRNA expression level and function were upregulated in the TG following CHS. Pharmacological inhibition and global knockout of Trpc3 significantly alleviated spontaneous scratching behaviors without affecting concurrent cutaneous inflammation in the CHS model. Furthermore, conditional deletion of Trpc3 in primary sensory neurons but not in keratinocytes produced similar antipruritic effects in this model. These findings suggest that TRPC3 expressed in primary sensory neurons may contribute to acute and chronic itch via a histamine independent mechanism and that targeting neuronal TRPC3 might benefit the treatment of chronic itch associated with ACD and other inflammatory skin disorders.

Keywords: TRPC3, itch, pain, primary sensory neurons, dermatitis