ABSTRACT
Cholestatic itch is a severe and debilitating symptom in liver diseases with limited treatment options. The class A G protein-coupled receptor (GPCR) Mas-related GPCR subtype X4 (MRGPRX4) has been identified as a receptor for bile acids, which are potential cholestatic pruritogens. An increasing number of GPCRs have been shown to interact with receptor activity-modifying proteins (RAMPs), which can modulate different aspects of GPCR biology. Using a combination of multiplexed immunoassay and proximity ligation assay, we show that MRGPRX4 interacts with RAMPs. The interaction of MRGPRX4 with RAMP2, but not RAMP1 or 3, causes attenuation of basal and agonist-dependent signaling, which correlates with a decrease of MRGPRX4 cell surface expression as measured using a quantitative NanoBRET pulse-chase assay. Finally, we use AlphaFold Multimer to predict the structure of the MRGPRX4-RAMP2 complex. The discovery that RAMP2 regulates MRGPRX4 may have direct implications for future drug development for cholestatic itch.
Subject(s)
Pruritus , Receptor Activity-Modifying Proteins , Receptors, G-Protein-Coupled , Cell Membrane/metabolism , Receptor Activity-Modifying Protein 1/metabolism , Receptor Activity-Modifying Protein 2/metabolism , Receptor Activity-Modifying Protein 3/metabolism , Receptor Activity-Modifying Proteins/chemistry , Receptor Activity-Modifying Proteins/metabolism , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Pruritus/metabolism , Protein Binding , HumansABSTRACT
BACKGROUND & AIMS: Limited understanding of pruritus mechanisms in cholestatic liver diseases hinders development of antipruritic treatments. Previous studies implicated lysophosphatidic acid (LPA) as a potential mediator of cholestatic pruritus. METHODS: Pruritogenicity of lysophosphatidylcholine (LPC), LPA's precursor, was examined in naïve mice, cholestatic mice, and nonhuman primates. LPC's pruritogenicity involving keratinocyte TRPV4 was studied using genetic and pharmacologic approaches, cultured keratinocytes, ion channel physiology, and structural computational modeling. Activation of pruriceptor sensory neurons by microRNA-146a (miR-146a), secreted from keratinocytes, was identified by in vitro and ex vivo Ca2+ imaging assays. Sera from patients with primary biliary cholangitis were used for measuring the levels of LPC and miR-146a. RESULTS: LPC was robustly pruritic in mice. TRPV4 in skin keratinocytes was essential for LPC-induced itch and itch in mice with cholestasis. Three-dimensional structural modeling, site-directed mutagenesis, and channel function analysis suggested a TRPV4 C-terminal motif for LPC binding and channel activation. In keratinocytes, TRPV4 activation by LPC induced extracellular release of miR-146a, which activated TRPV1+ sensory neurons to cause itch. LPC and miR-146a levels were both elevated in sera of patients with primary biliary cholangitis with itch and correlated with itch intensity. Moreover, LPC and miR-146a were also increased in sera of cholestatic mice and elicited itch in nonhuman primates. CONCLUSIONS: We identified LPC as a novel cholestatic pruritogen that induces itch through epithelia-sensory neuron cross talk, whereby it directly activates skin keratinocyte TRPV4, which rapidly releases miR-146a to activate skin-innervating TRPV1+ pruriceptor sensory neurons. Our findings support the new concept of the skin, as a sensory organ, playing a critical role in cholestatic itch, beyond liver, peripheral sensory neurons, and central neural pathways supporting pruriception.
Subject(s)
Cholestasis/complications , Keratinocytes/metabolism , Lysophosphatidylcholines , Pruritus/metabolism , Sensory Receptor Cells/metabolism , Skin/innervation , TRPV Cation Channels/metabolism , Adult , Aged , Animals , Behavior, Animal , Cells, Cultured , Cholestasis/genetics , Cholestasis/metabolism , Cholestasis/physiopathology , Disease Models, Animal , Female , Humans , Macaca mulatta , Male , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Pruritus/chemically induced , Pruritus/genetics , Pruritus/physiopathology , Signal Transduction , TRPV Cation Channels/geneticsABSTRACT
The spinal origin of cholestatic itch in experimental obstructive jaundice mouse model remains poorly understood. In this study, the jaundice model was established by bile duct ligation (BDL) in mice, and differential gene expression patterns were analyzed in the lower thoracic spinal cord involved in cholestatic pruritus after BDL operation using high-throughput RNA sequencing. At 21st day after BDL, the expression levels of ENSRNOG00000060523, ENSRNOG00000058405 and ENSRNOG00000055193 mRNA were significantly up-regulated, and those of ENSRNOG00000042197, ENSRNOG00000008478, ENSRNOGOOOOOO19607, ENSRNOG00000020647, ENSRNOG00000046289, Gemin8, Serpina3n and Trim63 mRNA were significantly down-regulated in BDL group. The RNAseq data of selected mRNAs were validated by RT-qPCR. The expression levels of ENSRNOG00000042197, ENSRNOG00000008478, ENSRNOGOOOOOO 19607, ENSRNOG00000020647, ENSRNOG00000046289 and Serpina3n mRNA were significantly down-regulated in BDL group. This study suggested that cholestatic pruritus in experimental obstructive jaundice mouse model is related with in the changes of gene expression profiles in spinal cord.
Subject(s)
Cholestasis/complications , Pruritus/genetics , Spinal Cord/metabolism , Transcriptome , Animals , Gene Expression Profiling , Male , Mice , Mice, Inbred C57BL , Pruritus/etiology , Pruritus/metabolismABSTRACT
The spinal origin of cholestatic itch in experimental obstructive jaundice mouse model remains poorly understood.In this study,the jaundice model was established by bile duct ligation (BDL) in mice,and differential gene expression patterns were analyzed in the lower thoracic spinal cord involved in cholestatic pruritus after BDL operation using high-throughput RNA sequencing.At 21st day after BDL,the expression levels of ENSRNOG00000060523,ENSRNOG00000058405 and ENSRNOG00000055193 mRNA were significantly up-regulated,and those of ENSRNOG00000042197,ENSRNOG00000008478,ENSRNOG00000019607,ENSRNOG00000020647,ENSRNOG00000046289,Gemin8,Serpina3n and Trim63 mRNA were significantly down-regulated in BDL group.The RNAseq data of selected mRNAs were validated by RT-qPCR.The expression levels of ENSRNOG00000042197,ENSRNOG00000008478,ENSRNOG00000019607,ENSRNOG00000020647,ENSRNOG00000046289 and Serpina3n mRNA were significantly down-regulated in BDL group.This study suggested that cholestatic pruritus in experimental obstructive jaundice mouse model is related with in the changes of gene expression profiles in spinal cord.
ABSTRACT
The spinal origin of cholestatic itch in experimental obstructive jaundice mouse model remains poorly understood.In this study,the jaundice model was established by bile duct ligation (BDL) in mice,and differential gene expression patterns were analyzed in the lower thoracic spinal cord involved in cholestatic pruritus after BDL operation using high-throughput RNA sequencing.At 21st day after BDL,the expression levels of ENSRNOG00000060523,ENSRNOG00000058405 and ENSRNOG00000055193 mRNA were significantly up-regulated,and those of ENSRNOG00000042197,ENSRNOG00000008478,ENSRNOG00000019607,ENSRNOG00000020647,ENSRNOG00000046289,Gemin8,Serpina3n and Trim63 mRNA were significantly down-regulated in BDL group.The RNAseq data of selected mRNAs were validated by RT-qPCR.The expression levels of ENSRNOG00000042197,ENSRNOG00000008478,ENSRNOG00000019607,ENSRNOG00000020647,ENSRNOG00000046289 and Serpina3n mRNA were significantly down-regulated in BDL group.This study suggested that cholestatic pruritus in experimental obstructive jaundice mouse model is related with in the changes of gene expression profiles in spinal cord.