Epithelia-Sensory Neuron Cross Talk Underlies Cholestatic Itch Induced by Lysophosphatidylcholine.

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.

Hes1, an important gene for activation of hepatic stellate cells, is regulated by Notch1 and TGF-ß/BMP signaling.

AIM: To determine the role of Notch1 and Hes1 in regulating the activation of hepatic stellate cells (HSCs) and whether Hes1 is regulated by transforming growth factor (TGF)/bone morphogenetic protein (BMP) signaling. METHODS: Immunofluorescence staining was used to detect the expression of desmin, glial fibrillary acidic protein and the myofibroblastic marker α-smooth muscle actin (α-SMA) after freshly isolated, normal rat HSCs had been activated in culture for different numbers of days (0, 1, 3, 7 and 10 d). The expression of α-SMA, collagen1α2 (COL1α2), Notch receptors (Notch1-4), and the Notch target genes Hes1 and Hey1 were analyzed by reverse transcriptase-polymerase chain reaction. Luciferase reporter assays and Western blot were used to study the regulation of α-SMA, COL1α1, COL1α2 and Hes1 by NICD1, Hes1, CA-ALK3, and CA-ALK5 in HSC-T6 cells. Moreover, the effects of inhibiting Hes1 function in HSC-T6 cells using a Hes1 decoy were also investigated. RESULTS: The expression of Notch1 and Hes1 mRNAs was significantly down-regulated during the culture of freshly isolated HSCs. In HSC-T6 cells, Notch1 inhibited the promoter activities of α-SMA, COL1α1 and COL1α2. On the other hand, Hes1 enhanced the promoter activities of α-SMA and COL1α2, and this effect could be blocked by inhibiting Hes1 function with a Hes1 decoy. Furthermore, co-transfection of pcDNA3-CA-ALK3 (BMP signaling activin receptor-like kinase 3) and pcDNA3.1-NICD1 further increased the expression of Hes1 compared with transfection of either vector alone in HSC-T6 cells, while pcDNA3-CA-ALK5 (TGF-ß signaling activin receptor-like kinase 5) reduced the effect of NICD1 on Hes1 expression. CONCLUSION: Selective interruption of Hes1 or maintenance of Hes1 at a reasonable level decreases the promoter activities of α-SMA and COL1α2, and these conditions may provide an anti-fibrotic strategy against hepatic fibrosis.