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