Article Figures & Data
Figures
- Fig. 1.
Chemical structure of natural compound forsythoside B.
- Fig. 2.
Identification of natural forsythoside B as an inhibitor of hTRPV3 channels expressed in HEK-293 cells. (A) Dose-dependent inhibition of intracellular Ca2+ increase mediated through activation of TRPV3 channels in response to 200 μM 2-APB by different concentrations of forsythoside B ranging from 50 to 200 μM in a FlexStation 3 calcium fluorescent assay. The right panel shows a summary of the inhibition of the intracellular fluorescent Ca2+ increase shown in the left panel. The blank control is 0.2% DMSO. Asterisks denote statistical significance compared with the ruthenium red control (n = 6; *P < 0.05, by one-way analysis of variance). (B) Whole-cell patch-clamp recordings of TRPV3 currents in response to 2-APB alone (50 μM, red bar) or coapplication of 50 μM forsythoside B (blue bar). The right panel shows current-voltage curves of hTRPV3 in response to voltage ramps from −100 to +100 mV before (1) and after 50 μM 2-APB (2), after the coaddition of 50 μM 2-APB and 50 μM forsythoside B (3), and after washout (4). (C) The left panel shows the representative hTRPV3 currents in response to 50 μM 2-APB before and after its coaddition with increasing concentrations of forsythoside B (from 1 to 100 μM). The right panel shows curve fitting of dose-dependent inhibition of 2-APB–mediated hTRPV3 activation by forsythoside B, with an IC50 of 6.7 ± 0.7 μM (n = 5–10). Data are presented as means ± S.E.M. FB, forsythoside B; RFU, relative fluorescence unit; RR, ruthenium red.
- Fig. 3.
Selective inhibition of TRPV3 current by forsythoside B over TRPV1, TRPV4, and TRPA1 channels. (A) Representative hTRPV1 current activated by 1 μM capsaicin (red bar) before and after its coapplication with 50 μM forsythoside B (blue bar) and washout. The right panel shows current-voltage curves of hTRPV1 in response to voltage ramps from −100 to +100 mV shown in the left panel before (1) and after 1 μM capsaicin (2), after the coaddition of 50 μM forsythoside B and 1 μM capsaicin (3), and after washout (4). (B) Representative hTRPV4 current activated by 50 nM GSK1016790A (red bar) or in the absence or presence of 50 μM forsythoside B (blue bar) before inhibition by 20 μM ruthenium red (pink bar). The right panel shows current-voltage curves of hTRPV4 in response to voltage ramps from −100 to +100 mV shown in the left panel before (1) and after 50 nM GSK101 (2), after coaddition of 50 μM forsythoside B and 50 nM GSK101 (3), and after addition of 20 μM ruthenium red (4). (C) Representative hTRPA1 current activated by 300 μM AITC (red bar) or in the absence or presence of 50 μM forsythoside B (blue bar) before complete inhibition by 20 μM ruthenium red (pink bar). The right panel shows the current-voltage curves of hTRPA1 in response to voltage ramps from −100 to +100 mV shown in the left panel before (1) and after 300 μM AITC (2), after coaddition of 50 μM forsythoside B and 300 μM AITC (3), and after addition of 20 μM ruthenium red (4). (D) Summary of the percent inhibition of hTRPV3, hTRPV1, hTRPV4, and hTRPA1 channel activation by 50 μM forsythoside B. Data are presented as means ± S.E.M. Asterisks denote statistical significance compared with the hTRPV3 control (n = 5 to 6; ***P < 0.001, by unpaired t test). AITC, allyl isothiocyanate; FB, forsythoside B; GSK101, GSK1016790A; RR, ruthenium red.
- Fig. 4.
Forsythoside B reduces scratching behavior induced by carvacrol and histamine or AEW in mice. (A) The left panel shows the time course of neck-scratching activity observed in C57BL/6 mice after intradermal injection of 50 μl carvacrol (0.1%) into the mouse neck, with pretreatment with vehicle (0.3% DMSO) and different concentrations (3, 30, or 300 μM) of 50 μl forsythoside B in the same site. Time for scratching bouts was plotted for each 5-minute bin over 30 minutes. The right panel shows quantification of scratching bouts during the 30 minutes shown in the left panel (n = 7–9; no significance; *P < 0.05; **P < 0.01; ***P < 0.001, by one-way ANOVA). (B) The left panel shows the representative time course of neck-scratching activity observed after intradermal injection of 50 μl histamine (100 μM) into the mouse neck, with pretreatment with vehicle (0.03% DMSO) and different concentrations (0.3, 3, or 30 μM) of 50 μl forsythoside B into the same site. Time for scratching bouts was plotted for each 5-minute bin over 30 minutes. The right panel shows the quantification of scratching bouts during the 30 minutes shown in the left panel (n = 5 to 6; no significance; **P < 0.01; ***P < 0.001; ****P < 0.0001 by one-way ANOVA). (C) Dose-dependent inhibition of spontaneous scratching bouts in AEW-treated mice by pretreatment with topical application of forsythoside B (n = 5–9; no significance; *P < 0.05; **P < 0.01, by one-way ANOVA). (D) Locomotor activity assessment (total travel distance, in centimeters) after intradermal injection with forsythoside B (0.3, 3, 30, or 300 μM; 50 μl/site) into the nape of the mouse neck (n = 5; no significance, by one-way ANOVA). ANOVA, analysis of variance; Car, carvacrol; FB, forsythoside B; N.S., no significance.
- Fig. 5.
Forsythoside B reduces cell death of HEK 293 cells and HaCaT cells expressing the gain-of-function TRPV3 G573S mutant. (A) Forsythoside B reduced the death of HEK293 cells expressing the TRPV3 G573S mutant in a dose-dependent manner. Representative Hoechst staining of cell nuclei (top images) and PI staining for dead cells (bottom images). Scale bar, 100 μm. (B) Statistical analysis for normalized cell death from (A). Five independent experiments were performed. Data are presented as means ± S.E.M. (C) Forsythoside B reduced the death of HaCaT cells expressing the TRPV3 G573S mutant in a dose-dependent manner. (D) Summary of the bar graph for (C). Four independent experiments were performed. Data are presented as means ± S.E.M. *P < 0.05; **P < 0.01; ****P < 0.0001. FB, forsythoside B; RR, ruthenium red
- Fig. 6.
Forsythoside B attenuates cell death in HEK293 cells expressing TRPV3 channels or HaCaT cells activated by TRPV3 agonist carvacrol. (A) The left panel shows the representative hTRPV3 current in response to 300 μM carvacrol (red bar) before and after its coaddition with ruthenium red (pink bar). The right panel shows the representative hTRPV3 current activated by 300 μM carvacrol (red bar) in the absence or presence of forsythoside B from 1 to 100 μM (blue bar) and washout. (B) A summary of TRPV3 current in response to different compounds in (A) (n = 6; by one-way analysis of variance). (C) Cell death assay after transient transfection with WT TRPV3 and after treatment with different compounds for 12 hours in HEK293 cells. (D) Summary of the cell death ratio after treatment with different compounds (n = 4). (E) Cell death assay after treatment with different compounds for 12 hours in HaCaT cells. (F) Summary of the cell death ratio after treatment with different compounds (n = 4). Data are presented as means ± S.E.M. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Car, carvacrol; FB, forsythoside B; N.S., no significance; RR, ruthenium red. Scale bar, 100 μm in (C) and (E).
- Fig. 7.
Putative binding pocket for forsythoside B bound to the central cavity of mouse TRPV3. (A) Representative view of a coronal section for forsythoside B bound to the structure of TRPV3 subunits labeled in different colors of green, pink, purple, and cyan for each subunit. Four forsythoside B molecules bound to the binding pocket are also labeled in different colors of red, blue, purple, and hot pink. (B) Top view of four forsythoside B molecules bound to the TRPV3 structure. (C) Expanded view of putative binding sites of forsythoside B in TRPV3. Different subunits of TRPV3 are shown in different colors, and forsythoside B is shown in yellow mesh. The surrounding residues T636 from the pore helix and F666 from S6 of one subunit in green and G638 and L639 from the selectivity filter of the neighboring subunit in yellow are shown as blue sticks and indicated by white arrows. (D) Schematic diagram illustrating inhibition of TRPV3 by forsythoside B based on the model proposed by Singh et al. (2018). (Left) Putative lipids (blue) are located between the S1–S4 (orange) and S5 to S6 (blue) domains in the closed state. (Middle) Open TRPV3 upon binding of agonist 2-APB (in green triangles) to the top of S1–S4 domains, bringing the S1–S4 and pore domains close together. (Right) Forsythoside B (in red) binds to the putative pocket formed by residues from S6 and pore helices of TRPV3, thus blocking the region right below the selectivity filter and the central cavity.
- Fig. 8.
Proposed mechanism for the causative role of overactive TRPV3 in itch signaling and cytotoxicity of keratinocytes. For TRPV3-mediated itch signaling, activation of TRPV3 by channel agonist carvacrol or gain-of-function mutation results in an elevation of intracellular calcium that leads to the activation of calcineurin. Calcineurin dephosphorylates NFAT and induces the translocation of NFAT from the cytosol to the nucleus. Translocation of NFAT induces TSLP transcription and thus promotes TSLP secretion in the keratinocytes. TSLP, histamine, and cytokines released by TRPV3 activation stimulate the afferent nerve endings that transmit the itch signal through the dorsal horn of the spinal cord to the CNS to evoke scratching behavior. For overactive TRPV3-mediated cytotoxicity, the excessive influx of calcium induces keratinocyte cell death via activation of an unidentified Ca2+-dependent kinase. ARD, ankyrin repeat domain; CNS, central nervous system; NFAT, nuclear factor of activated T cells; TSLP, thymic stromal lymphopoietin.
In this issue
Novel TRPV3 Inhibitor Forsythoside B Reduces Pruritus and Cytotoxicity
