Fig 1.
Characterization of TRPV4 channels in A375 melanoma cells.
A) Upper panel: Exemplary whole-cell recordings showing activation of TRPV4 channels by GSK1016790A (200 nM) and inhibition of currents by HC067047 (1 μM). The arrow indicates a positive reversal potential of GSK1016790A-activated currents. Baseline currents were not considerable inhibited by HC067047. Lower panel: Co-activation of KCa-currents. The right arrow indicates a negative reversal potential (Erev) of ca. -35 mV of the mixed TRPV4 and KCa current and the left arrow indicates an Erev of ca. -75 mV of the isolated KCa-current after inhibition of TRPV4 currents by HC067047. The KCa-current was fully blocked by the negative-gating modulator of KCa3.1 channels, 13b (1 μM). B) Upper panel: Mean normalized currents at clamp potentials of -80 and +80 mV before and after addition GSK1016790A (n = 8, experiments) and after addition of HC067047 (n = 8). Lower panel: Mean mixed TRPV4/KCa3.1 currents at a clamp potential of 0 mV after addition of GSK1016790A (n = 5) and inhibition of TRPV4 currents by HC067047 (n = 4) and of KCa3.1 currents by 13b (n = 5). C) Quantitative RT-PCR analysis of TRPV4 and KCa3.1 gene expression as percentage of GAPDH expression (replicates, n = 3). Data points are means ± SEM.
Fig 2.
GSK1016790A-induced TRPV4-currents and inhibition by HC067047 in the melanoma lines, MKTBR and SK-MEL-28, and the human non-cancer keratinocyte line, HaCaT.
Data points are means ± SEM (cells, n = 4–6 each). Panel on right: Quantitative RT-PCR analysis of TRPV4 and KCa3.1 gene expression in HaCaT as percentage of GAPDH expression (replicates, n = 3). Data points are means ± SEM.
Fig 3.
A) Changes of intracellular calcium ([Ca2+i]) in response to TRPV4 activation in A375 cells. Exemplary images of GSK1016790A(1 μM)-induced increase of [Ca2+i] (upper left panel), its time course (upper panel in middle), and (on right) summary data of % change of fluorescence to GSK1016790A (n = 10 independent experiments) and in combination with HC067047 (n = 5 independent experiments). HC067047 (10 μM) partially reversed the response. Note that artifacts caused by compound addition were removed. No change of fluorescence was recorded in the presence of vehicle (DMSO 0.1%, n = 3). B) Lower panel on left: Exemplary images of changes of intracellular calcium ([Ca2+]i) to TRPV4 activation in HaCaT cells. Note that artefacts caused by compound addition to the bath were removed. In middle: Time course of changes in [Ca2+]i in response to GSK1016790A (n = 4 independent experiments) alone and in combination with HC067047 (n = 4 independent experiments). The vehicle (DMSO 0.1%, n = 3) produced virtually no response. On right: Summary of data. Data are means ± SEM; *P<0.001, Student’s T test.
Fig 4.
Alterations of cell morphology, cell detachment and cell death induced by GSK1016790A.
A) Upper two panels: Exemplary light microscopic images illustrating time course of cell retraction, membrane blebbing (indicated by arrows), and cell detachment during the first hour of exposure to GSK1016790A (1 μM). HC067047 (1 μM) prevented visibly GSK1016790A-induced changes. HC067047 or vehicle (DMSO) had no visible effect. Lower panel: Giemsa-stained A375 cells after 1 h exposure to GSK1016790A, in combination with HC067047, or DMSO. Note the densification of nuclei (dark-grey dots indicated by arrows) in GSK1016790A-treated cells. B) Counts of non-viable, “death” cells in supernatant. Data points are means ± SEM (number of independent experiments, n = 3). *P<0.05 vs. DMSO, #P <0.05 vs. GSK1016790A; Student’s T test.
Fig 5.
A) Representative flow cytometry dot plots with double Annexin V-FITC/PI staining for control cells (DMSO 0,2%), cells exposed to GSK1016790A (10 nM), and cells exposed to GSK1016790A and HC067047 (1 μM) at 1 h, 24 h, and 72 h. B) Summary data. C) Induction of apoptosis in HaCaT cells and protective effects of HC067047. D) Summary data. *P<0.05 vs. Control, #P<0.05 vs. GSK1016790A, ANOVA, n = 3). Data are means ± SEM (number of independent experiments, n = 3).
Fig 6.
Pharmacological TRPV4 activation did not impair the cell cycle in A375 cells (A) and HaCaT keratinocytes (B).
Fig 7.
Impact of GSK1016790A on cell proliferation/survival.
A) Upper panel on left: Concentration-dependent reduction of cell proliferation/survival of A375 as measured by Janus Green-Assay. Upper panel on right: Note that half-maximal inhibition was achieved at ca. 1 nM GSK1016790A for all time intervals, except day-1. Lower panel on left: HC067047 antagonized the response to GSK1016790A. Lower panel on right: The negative gating-modulator of KCa3.1, the 13b derivate, RA-2 (10 μM) reduced cell proliferation/survival and potentiated the response to GSK1016790A. The positive-gating modulator of KCa3.1, SKA-121, had no effects. Data points are means ± SEM (n = 6–36 from n = 2–6 independent experiments). B) GSK1016790A impaired proliferation/survival of HaCaT cells. HC067047 partially antagonized the response. The negative and positive KCa3.1-gating modulators, RA-2 and SKA-121, respectively, did not modulate the response. Data points are means ± SEM (n = 18; number of independent experiments, n = 3). *P<0.05 vs. DMSO, #P<0.05 vs. GSK1016790A; Student’s T test.