Silica nanoparticles inhibit the cation channel TRPV4 in airway epithelial cells
Sanchez et al. Particle and Fibre Toxicology
Silica nanoparticles inhibit the cation channel TRPV4 in airway epithelial cells
Alicia Sanchez 0
Julio L. Alvarez 0
Kateryna Demydenko 0 3
Carole Jung 2
Yeranddy A. Alpizar 0
Julio Alvarez-Collazo 0
Stevan M. Cokic 1
Miguel A. Valverde 2
Peter H. Hoet 4
Karel Talavera 0
0 Department of Cellular and Molecular Medicine, Laboratory of Ion Channel Research, KU Leuven; VIB Center for Brain & Disease Research , Leuven , Belgium
1 KU Leuven BIOMAT, Department of Oral Health Sciences, KU Leuven & Dentistry University Hospitals Leuven , Leuven , Belgium
2 Department of Experimental and Health Sciences, Laboratory of Molecular Physiology and Channelopathies, Universitat Pompeu Fabra , Barcelona , Spain
3 Present address: Department of Cardiovascular Sciences, Laboratory of Experimental Cardiology , Leuven, KU , Belgium
4 Department of Public Health and Primary Care, KU Leuven , Leuven , Belgium
Background: Silica nanoparticles (SiNPs) have numerous beneficial properties and are extensively used in cosmetics and food industries as anti-caking, densifying and hydrophobic agents. However, the increasing exposure levels experienced by the general population and the ability of SiNPs to penetrate cells and tissues have raised concerns about possible toxic effects of this material. Although SiNPs are known to affect the function of the airway epithelium, the molecular targets of these particles remain largely unknown. Given that SiNPs interact with the plasma membrane of epithelial cells we hypothesized that they may affect the function of Transient Receptor Potential Vanilloid 4 (TRPV4), a cation-permeable channel that regulates epithelial barrier function. The main aims of this study were to evaluate the effects of SiNPs on the activation of TRPV4 and to determine whether these alter the positive modulatory action of this channel on the ciliary beat frequency in airway epithelial cells. Results: Using fluorometric measurements of intracellular Ca2+ concentration ([Ca2+]i) we found that SiNPs inhibit activation of TRPV4 by the synthetic agonist GSK1016790A in cultured human airway epithelial cells 16HBE and in primary cultured mouse tracheobronchial epithelial cells. Inhibition of TRPV4 by SiNPs was confirmed in intracellular Ca2+ imaging and whole-cell patch-clamp experiments performed in HEK293T cells over-expressing this channel. In addition to these effects, SiNPs were found to induce a significant increase in basal [Ca2+]i, but in a TRPV4-independent manner. SiNPs enhanced the activation of the capsaicin receptor TRPV1, demonstrating that these particles have a specific inhibitory action on TRPV4 activation. Finally, we found that SiNPs abrogate the increase in ciliary beat frequency induced by TRPV4 activation in mouse airway epithelial cells. Conclusions: Our results show that SiNPs inhibit TRPV4 activation, and that this effect may impair the positive modulatory action of the stimulation of this channel on the ciliary function in airway epithelial cells. These findings unveil the cation channel TRPV4 as a primary molecular target of SiNPs.
silica nanoparticles; TRPV4; GSK1016790A; epithelial cells; ciliary beat frequency
Synthetic amorphous SiNPs are extensively used due to its
interesting physico-chemical properties, low cost and
relatively easy production. This material has many applications
in industrial manufacturing, cosmetics, biotechnology,
medicine, and food, pharmaceutical and chemical industries
]. SiNPs are widely used in consumer products and as
a consequence, human exposure to this nanomaterial has
highly increased. However, there is very little information
available about the risks associated to the exposure to this
It is known that SiNPs can penetrate cells, interacting
with the plasma membrane, intracellular structures and
organelles, thereby posing potential health threats [
The toxicity generated by nanoparticles has been
related to an increased generation of reactive oxygen
species (ROS) [
]. This results in oxidative stress,
mitochondrial perturbation and the generation of
inflammatory mediators leading to cell dysfunction and
2, 3, 11–19
One of the main entry pathways of nanoparticles into
the body is the epithelium of the airways. In addition to
its function in gas exchange, the respiratory epithelium
protects the body against hazardous environmental
substances and pathogens, constituting an active diffusion
barrier, and supporting the mechanisms of mucociliary
clearance and recruitment of inflammatory cells [
Several cellular responses to SiNPs in the airways have
been reported. Rabiolli et al. demonstrated that SiNPs
induce lung inflammation through the stimulation of IL-1β
production by alveolar macrophages [
]. Skuland et al.
showed evidence of pro-inflammatory responses induced
by amorphous SiNPs in lung epithelial cells [
et al. reported that SiNPs pre-exposure in pneumonia
induced by Pseudomonas aeruginosa increases lung
permeability and enhance mortality [
], and Kasper et al.
showed inflammatory and cytotoxic responses such as
DNA damage, hypoxia and ER-stress induced by SiNPs in
an alveolar-capillary co-culture model [
However, little is known about the influence of SiNPs
on specific molecular targets and cell signaling events,
especially at the level of the plasma membrane. In this
study we hypothesized that SiNPs may affect the
function of TRPV4, a Ca2+-permeable cation channel
expressed in airway epithelial cells. This channel plays a
role in the transduction of physical and chemical stimuli
into Ca2+ signals that regulate ciliary beat frequency and
mucociliary transport [
]. Moreover, TRPV4
contributes to the barrier integrity in the lung and to the
regulation of endothelial and epithelial permeability [
], and has been implicated in the modulation of the
respiratory function and proposed as target for the
treatment of respiratory diseases such chronic obstructive
pulmonary disease and asthma [
We used intracellular Ca2+ imaging and patch-clamp
to evaluate the effects of SiNPs on TRPV4 activation.
We found that SiNPs inhibit the activation of native
TRPV4 channels in human and mouse airway
epithelial cells, as well as recombinant TRPV4 in the
heterologous expression system HEK293T. Furthermore,
SiNPs inhibited the TRPV4-mediated increase in
ciliary beat frequency in mouse airway epithelial cells.
TRPV4 emerges therefore as a defined molecular
target of SiNPs, with possible deleterious consequences
for epithelial barrier function.
SM30 Ludox® SiNPs were purchased from Sigma-Aldrich
(Bornem, Belgium) as the commercial source of 30% wt
suspension in H2O. For the biological experiments the
nanoparticle suspension was diluted to the desired
concentrations in Krebs solution containing (in mM):
150 NaCl, 6 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, 10
4(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
and titrated to pH 7.4 with NaOH.
Dynamic Light Scattering (DLS) and Zeta potential
The stock suspension of SiNPs particles was diluted in
water to 30 μg/ml. DLS and Zeta potential measurements
were performed with a Brookhaven 90 Plus/ZetaPlus
instrument (Brookhaven Instruments Ltd, Redditch, UK).
DLS measurements were performed using a NanoParticle
Size Distribution Analyser (scattering angle 90 u,
wavelength 659 nm, power 15 mW). Correlation functions
were analyzed using the Clementine package (maximum
entropy method) for Igor Pro 6.02A (WaveMetrics,
Portland, OR, USA).
Zeta potential measurements were done by applying
electrophoretic light scattering. A primary and reference
beam (659 nm, 35 mW), modulated optics and a dip-in
electrode system were used. The frequency shift of
scattered light (relative to the reference beam) from a charged
particle moving in an electric field is related to the
electrophoretic mobility of the particle. The Smoluchowski limit
was used to calculate the Zeta potential from the
Transmission Electron Microscopy (TEM)
Suspensions (5 μl of stock suspension and 30 μg/ml) of
the SiNPs particles were applied on formvar-coated
cupper mesh grids (drop on grid). After drying overnight
(25 °C in the dark), the particles were characterized by
TEM (JEOL JEM-1200 EX-II, Tokyo, Japan).
We used the Endosafe-PTS LAL assay for FDA-licensed
endotoxin detection. The cartridges contained four
channels to which LAL reagent and a chromogenic substrate
were applied. Two of these channels contained also an
endotoxin spike that served as positive control. The
sensitivity of the assay was 0.05 EU/ml.
Human bronchial epithelial cell line, 16HBE, were grown
in Dulbecco's modified Eagle's medium: nutrient mixture
F-12 (DMEM/F-12) containing 5% (v/v) fetal calf serum
(FCS), 2 mM L-glutamine, 2 U/ml penicillin and 2 mg/
ml streptomycin at 37 °C in a humidity-controlled
incubator with 5% CO2 and were seeded on 18-mm glass
cover slips coated with poly-L-lysine (0.1 mg/ml).
Human embryonic kidney cells, HEK293T, were grown
in Dulbecco's modified Eagle's medium (DMEM)
containing 10% (v/v) fetal calf serum (FCS), 2 mM L-glutamine, 2
U/ml penicillin, 1% non-essential amino acids (Invitrogen,
Erembodegem - Aalst, Belgium) and 2 mg/ml
streptomycin at 37 °C in a humidity-controlled incubator with
10% CO2 and were seeded on 18-mm glass cover slips
coated with poly-L-lysine (0.1 mg/ml). For intracellular
Ca2+ imaging and patch-clamp experiments, HEK293T
cells were transiently transfected with mouse TRPV4 in
the CAGGSM2/Ires/GFP/R1R2 vector, using Mirus
TransIT-293 (Mirus Corporation; Madison, WI, USA). In
all experiments, transfected cells were identified by green
fluorescent protein (GFP) expression.
C57Bl/6J male mice from 8-12 weeks old were used for
the experiments. The animals were maintained under
standard conditions with a maximum of four animals
per cage on a 12-h light/12-h dark cycle and with food
and water ad libitum.
Culture of mouse tracheal epithelial cells
Mouse tracheal epithelial cells (mTEC) were isolated
following the protocol described by Lam et al. [
seeded on 18-mm glass cover slips coated with collagen
solution containing 50 μg/ml collagen (type I solution
from rat tail, Sigma-Aldrich). Cells were grown for 2-3
days in the appropriate proliferation medium and
maintained at 37 °C in a humidity-controlled incubator with
Intracellular Ca2+ imaging experiments
Ca2+-imaging experiments were conducted with the
ratiometric fluorescent indicator Fura-2 acetoxymethyl (AM)
ester. Cells were incubated with 2 μM Fura-2 AM for 30
min at 37 °C. Bath solutions were perfused by gravity via a
multi-barreled pipette tip with a single outlet of 0.8 mm
inner diameter. This system allows full exchange of the
medium bathing the recorded cell in less than 2-4 s. For
recording in control condition cells were rinsed with
Krebs solution. The [Ca2+]i was monitored through the
ratio of fluorescence measured upon alternating
illumination at 340 and 380 nm using an MT-10 illumination
system and the xcellence pro software (Olympus, Planegg,
Germany). All experiments conducted in the native
16HBE and mTEC cells were performed at 35 °C.
Experiments in HEK293T cells were performed at 25 °C because
at 35 °C the TRPV4-transfected cells were heavily
overloaded with Ca2+ in basal condition.
The concentration-dependent effects of SiNPs on basal
[Ca2+]i of 16HBE cells was fit with a Hill function of the
¼ Δ Ca2þ Max ½SiNPs H
where Δ[Ca2+]Max is the maximal amplitude of the
response to SiNPs, [SiNPs] is the concentration of SiNPs,
EC50 is the effective concentration and H is the Hill
The concentration-dependent effects of SiNPs on the
Ca2+ responses to the TRPV4 agonist GSK1016790A
were fit with a Hill function of the form:
½SiNPs H þ IC5H0
Δ Ca2þ ¼
Δ Ca2þ Max−Δ Ca2þ Inf
þ Δ Ca2þ Inf
where Δ[Ca2+]Max is the amplitude of the response in
the absence of SiNPs, Δ[Ca2+]Inf is the amplitude of the
response the presence of saturating concentrations of
SiNPs, [SiNPs] is the concentration of SiNPs, IC50 is the
effective inhibitory concentration and H is the Hill
Whole-cell voltage-clamp recordings were performed at
35 °C with standard patch pipettes (2-3 MΩ resistance)
pulled using a DMZ-Universal puller (Zeitz Instruments,
Augsburg, Germany). The pipette solution contained (in
mM): 2 ATPNa2, 5 EGTA, 10 HEPES, 1 MgCl2, 135
CsCl2 (292 mOsm/kg; pH 7.2, adjusted with CsOH). For
perforated patch experiments, 250 μg/ml of
Amphotericin B was added to the pipette solution and data were
collected after the access resistance reached stable values
of ~15 MΩ.
An Ag-AgCl wire was used as reference electrode. The
cover slips with cells were placed in the stage of an
inverted microscope (Olympus IX70, Tokyo, Japan) and
stabilized for a few minutes in Krebs solution, containing
(in mM): 150 NaCl, 6 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose,
and 10 HEPES and titrated to pH 7.4 with NaOH. The
control bath solution was kept at room temperature and
contained (in mM): 140 NaCl, 1.3 MgCl2, 2.4 CaCl2, 10
HEPES, 10 glucose and (311 mOsm/kg; pH 7.4, adjusted
with NaOH). Bath solutions were perfused by gravity via a
multi-barreled pipette. A bath solution in which all cations
were isotonically substituted by NMDG+
(N-methyl-Dglucamine) was used to monitor the size of the leak
currents during the patch-clamp recordings [
signals were recorded using the patch-clamp technique by
using an EPC-7 (LIST Electronics, Darmstadt, Germany)
amplifier and the Clampex 9.0 software program (Axon
instruments, Sunnyvale, CA, USA). Currents were
acquired at 10 kHz, filtered at 2 kHz, and stored for off-line
analysis on a personal computer. In order to minimize
voltage errors, the series resistance was compensated by
30-50% and the capacitance artifact was reduced using the
amplifier circuitry. Membrane TRPV4 currents were
elicited by a 600 ms long voltage ramp from -100 mV to
+100 mV every 5 s with a holding potential of 0 mV.
Patch-clamp data was analyzed with the WinASCD
software written by Dr. Guy Droogmans and Origin 7.0
(OriginLab Corporation, Northampton, MA, USA). The
concentration dependence of TRPV4 current amplitude
was fit with a Hill function of the form:
½SiNPs H þ IC5H0 þ ΔIInf
where ΔIMax is the current density increase in the
absence of SiNPs, ΔIInf is the current density increase in the
presence of saturating concentrations of SiNPs, [SiNPs] is
the concentration of SiNPs, IC50 is the effective inhibitory
concentration and H is the Hill coefficient.
Ciliary beat frequency (CBF) measurements
CBF was measured in primary cultures ciliated cells using
with a high-speed digital imaging system as previously
]. Briefly, phase-contrast images (512 × 512
pixels) were collected at 120–135 frames per second with
a high speed CCD camera using a frame grabber
(Infaimon, Barcelona, Spain) and recording software from
Video Savant (IO Industries, London, ON, Canada). The
ciliary beat frequency was determined from the frequency
of variation in light intensity of the image as a result of
repetitive motion of cilia.
All chemicals were purchased from Sigma-Aldrich (Bornem,
Data are given as mean ± standard error of the mean.
Comparisons tests are indicated in the text were
appropriate. Statistical significance were taken at P < 0.05
or P < 0.01.
Characterization of the SM30 Ludox® SiNPs
Analysis of the SiNPs by DLS showed a single
population of average size 10.2 nm (P10: 8.1 nm - P90: 11.8).
The particles had a Zeta-potential of 20 ± 3 mV. TEM
analysis of undiluted samples showed large aggregates,
but in the diluted samples only a few aggregates could
be found, and the particles appeared as spherical entities.
No endotoxin contamination was detected in 30 μg/ml
Silica NPs inhibit TRPV4 activation in cultured human airway epithelial cells
To determine whether SiNPs modulate native human
TRPV4 channels we used fluorometric measurements of
[Ca2+]i in cultured human bronchial epithelial 16HBE cells,
which were reported to express this channel [
found that 10 nM GSK1016790A induced intracellular Ca2
+ responses in 100% (n = 333) of these cells, indicating for a
prevalent functional expression of TRPV4 (Fig. 1a).
Extracellular application of SiNPs increased the basal
[Ca2+]iin a concentration-dependent manner (Fig. 1b-d),
which was characterized by an EC50 of 99 ± 13 μg/ml, a
Hill coefficient of 0.71 ± 0.07 and a maximal response of
0.6 ± 0.1 μM (Fig. 1e). To evaluate the effect of SiNPs on
TRPV4 activation we compared the amplitude of the [Ca2
+]i responses measured 2 min after application of 10 nM
GSK1016790A, in the absence and in the presence of
nanoparticles. SiNPs induced a concentration-dependent
inhibition of the responses to GSK1016790A, with an IC50
of 130 ± 40 μg/ml and a Hill coefficient of -1.2 ± 0.4 (Fig.
1f ). Of note, SiNPs failed to completely abolish the
response to GSK1016790A up to a concentration of 3000
μg/ml, leaving ~30% of the response to the channel
SiNPs increase basal [Ca2+]i in a TRPV4-independent
It has been previously suggested that TRPV4 is
implicated in intracellular Ca2+ responses to SiNPs in a cell
subpopulation of the GT1-7 neuron-derived cell line
]. Thus, we tested whether TRPV4 is involved in the
[Ca2+]i increases triggered by SiNPs in 16HBE cells.
First, we determined whether the amplitude of the
responses to SiNPs correlated with the amplitude of the
responses to GSK1016790A, i.e., with the level of
functional expression of TRPV4 in each cell. We found that
this was not the case, with correlation values (R) of
0.074 (Fig. 2a). This value is lower than those we have
previously found for the correlation between the
amplitudes of responses to very low and high concentrations
of GSK1016790A [
]. The average increase in basal
[Ca2+]i elicited by 300 μg/ml SiNPs in 16HBE was not
significantly different in the absence (0.40 ± 0.04 μM)
and in the presence of the specific TRPV4 blocker
] (0.43 ± 0.05 μM; P = 0.64; Fig. 2b).
These data demonstrate that TRPV4 does not mediate
the basal Ca2+ responses triggered by SiNPs.
An increase in basal [Ca2+]i does not inhibit a subsequent
Next, we determined if an increase in basal [Ca2+]i such
as that induced by SiNPs could cause a decrease in
TRPV4 activation. For this we tested the effect of
extracellular application of ATP on a subsequent response of
16HBE cells to 10 nM GSK1016790A. We found that
ATP triggered a robust intracellular Ca2+ signal, and that
this did not reduced, but rather increased the amplitude
of the TRPV4 response measured at 2 min of
GSK1016790A application (0.54 ± 0.05 vs. 0.82 ± 0.08 in
control and after ATP application, respectively, P =
0.005; Fig. 3a, b).
SiNPs inhibit TRPV4 activation in mouse tracheal epithelial (mTEC) cells
In order to determine whether the effect of SiNPs is
conserved for native mouse TRPV4 we used primary
cultured mouse tracheal epithelial cells. Application of
10 nM GSK1016790A triggered intracellular Ca2+
signals in 94.4% (187 out of 198) of these cells (Fig. 4a),
consistent with a previous report on the functional
expression of TRPV4 channels in these cells [
induced a concentration-dependent inhibition of the
responses to GSK1016790A, with an IC50 of 1.2 ± 0.2
μg/ml and a Hill coefficient of -1.1 ± 0.3 (Fig. 4b-d).
As observed in 16HBE cells, application of SiNPs at
high concentrations did not abolish the response to
the TRPV4 agonist, but left ~20% of the maximal
SiNPs inhibit activation of recombinant TRPV4
To test whether SiNPs inhibit activation of TRPV4 in a
heterologous expression system we performed
intracellular Ca2+ imaging experiments in HEK293T cells
transiently transfected with the mouse channel isoform.
These cells showed a wide spectrum of basal [Ca2+]i, a
fact that we ascribe to the variable efficacy of TRPV4
transfection in each cell and the constitutive activity of
this Ca2+-permeable channel. Analysis of the
distribution of these values suggested the presence of two cell
populations, which could be divided using a cutoff
value of 250 nM. Both groups of cells responded
robustly to 10 nM GSK1016790A (Fig. 5a).
Application of SiNPs induced a
concentrationdependent increase of [Ca2+]i in cells with low basal
[Ca2+]i (Fig. 5b, c), an effect reminiscent of that we
observed in 16HBE cells (Fig. 1b-e). In contrast, SiNPs
reduced [Ca2+]i in cells with high basal Ca2+ levels (Fig.
5b, c), which may be an indicative of an inhibitory effect
of the SiNPs on the basal activity of TRPV4. In both
groups SiNPs reduced the response to GSK1016790A
with an IC50 of 1.44 ± 0.06 μg/ml and a Hill coefficient
of -2.0 ± 0.16 (Fig. 5d). Again, we found that application
of SiNPs at high concentrations left ~30% of the
response to the TRPV4 agonist.
Next, we determined whether SiNPs inhibit the
activation of TRPV4 by another synthetic chemical agonist,
4α-phorbol 12,13-didecanoate (4αPDD). We found that
SiNPs (300 μg/ml) strongly inhibited the response to
4αPDD (Fig. 5e, f ), showing that their effect is not
exclusive for channel activation with GSK1016790A.
In all experiments described above we allowed
sufficient time for the SiNPs effects on basal [Ca2+]i to
roughly reach a steady-state (~10 min). However, we
were also interested in estimating the time required for
these particles to reduce TRPV4 activation. Thus, we
performed a series of experiments in which we varied
the time of application of SiNPs before stimulating
TRPV4 with 10 nM GSK1016790A. This time varied
from zero (simultaneous application of SiNPs and
GSK1016790A) to 10 min. We found that TRPV4
responses were significantly smaller in the presence of
SiNPs (P < 0.05) and that the strength of inhibition was
not significantly different when comparing across the
various pre-application times tested (Tukey's Multiple
Comparison Test; Fig. 6). This indicates that the
inhibitory action of these particles on TRPV4 activation was
prior to the full activation of TRPV4 by GSK1016790A
(~2 - 3 min).
To directly test the effects of SiNPs on TRPV4 we
performed whole-cell patch-clamp experiments (Fig. 7a, b).
We recorded currents during the application of repetitive
voltage ramps applied from -100 to +100 mV. Application
of SiNPs at increasing concentrations had a tendency to
augment the amplitude of basal currents, but this was
statistically significant only at 300 μg/ml (Fig. 7c). To
evaluate the effect of SiNPs on TRPV4 we compared the
amplitude of the current responses measured 1 min after
application of 10 nM GSK1016790A, in the absence and
in the presence of nanoparticles (Fig. 7a and b). The
response to GSK1016790A was significantly reduced when
this compound was applied in the presence of SiNPs. This
effect was dependent on the concentration of SiNPs, and
was characterized by an IC50 of 2.4 ± 0.5 μg/ml, a Hill
coefficient of -0.54 ± 0.07 and minimum value of 0.7 ± 0.2
pA/pF, for the currents measured at -75 mV (Fig. 7d).
To test whether the inhibition of TRPV4 activation is
observed also in experimental conditions in which the
intracellular milieu is better preserved we performed
perforated patch-clamp experiments using Amphotericin
B in the patch pipette (Fig. 8a, b). We found that
extracellular application of SiNPs (30 μg/ml) significantly
reduced the response of TRPV4 current to 10 nM
GSK1016790A (Fig. 8c).
Effects of SiNPs on the capsaicin receptor TRPV1
Next, we performed intracellular Ca2+ imaging
experiments to determine the effects of 300 μg/ml SiNPs on
the response of TRPV1 to its specific agonist capsaicin
(1 μM). TRPV1 is the founding member of the vanilloid
subfamily of TRP channels, and its amino acid sequence
has 42.3% identity and 58.9% similarity with that of
TRPV4 (EMBOSS Needle application for Protein
help/index-protein.html). We found that the responses
of HEK293T cells transfected with mouse TRPV1 to
capsaicin were increased by 44% in the presence of SiNPs (P
= 0.0011; Fig. 9).
SiNPs inhibit TRPV4-mediated increase of ciliary beat frequency in airway epithelial cells
The cilia of airway epithelial cells are considered to be
sensory organelles with the ultimate function of
sweeping mucous loaded with pollutants and pathogens out of
the airways. TRPV4 is expressed in the cilia, and has
been proposed to regulate mucociliary transport by
transducing physical and chemical stimuli such as
viscosity or fluid tonicity into a Ca2+ signal that enhances
ciliary beat frequency [
26, 27, 41, 42
]. Thus, to
determine whether the inhibition of TRPV4 by SiNPs has a
correlate at the level of a cellular function, we
determined the effects of these nanoparticles on the response
of cilia to GSK1016790A. Application of 10 nM
GSK1016790A in control condition induced a significant
26 ± 3% increase in CBF (n = 22; P < 10-4; paired t test;
Fig. 10a). This effect was very similar to that reported by
Alenmyr et al. in human nasal epithelial cells .
Application of 300 μg/ml SiNPs modestly reduced the basal
CBF (~10%; n = 34; P < 10-4; paired t test; Fig. 10b) and
fully abrogated the response to GSK1016790A (n = 34;
P = 0.41; paired t test between CBF immediately before
and after 3 min application of the TRPV4 agonist).
Washout of SiNPs in the presence of the TRPV4
agonist led to an increase in CBF (n = 34; P = 0.014; paired
t test). Because SiNPs reduced the basal CBF only
slightly and fully inhibited the response to GSK1016790A
we argue that the latter effect was mainly mediated by
inhibition of TRPV4, and not by an unspecific effect of the
nanoparticles on other mechanisms regulating the CBF.
Despite the current advances in the characterization of
the toxicological properties of SiNPs, little is known
about how this material interacts with specific cellular
components. Under the plausible assumption that SiNPs
interact primarily with the plasma membrane of epithelial
cells, in this study we evaluated the effects on TRPV4, a
cation-permeable channel that is highly enriched in these
In essence, we found that SiNPs inhibit intracellular
Ca2+ signals triggered by activation of native TRPV4
channels in human and mouse airway epithelial cells.
TRPV4 inhibition by SiNPs was confirmed with Ca2+
imaging and direct measurements of TRPV4 currents in
the heterologous expression system HEK293T. In sharp
contrast to these results, we show that SiNPs enhanced
the activation of TRPV1, demonstrating that these
particles have a specific inhibitory action on TRPV4
channels. Finally, we found that SiNPs abrogate the increase
in ciliary beat frequency induced by TRPV4 activation in
mouse airway epithelial cells.
The comparison of the data obtained in mTEC and
HEK293T cells transfected with mouse TRPV4 indicate
that SiNPs have very similar effects on the responses to
GSK1016790A, with IC50 values around 1 μg/ml. In
contrast, SiNPs appeared to be much less effective in
16HBE cells, with a 100-fold higher IC50 value. This may
indicate that human TRPV4 is less sensitive than the
mouse isoform. Nevertheless, the SiNPs concentrations
required to inhibit TRPV4 mediated responses in the
human-derived cells (100 - 3000 μg/ml) are in the same
range or lower than those used in cytotoxicity and
cytokine release in vitro experiments performed in previous
studies (25 - 6000 μg/ml [
18, 23, 24
]. Moreover, we
observed the inhibitory effect on TRPV4 in a matter of
minutes, which represents a time scale 3- to 150-fold
shorter than that of those previous reports. This strongly
suggests TRPV4 as a primary and sensitive target of
As for the mechanism underlying the effects of SiNPs,
it may be speculated that these particles somehow
disrupt the binding site of GSK1016790A. SiNPs are
roughly the same size of the whole channel protein, and
more than twice the size of the length of the channel’s
transmembrane segments. So, it is unlikely that these
nanoparticles interact directly with a relatively small
binding pocket for GSK1016790A, unless this would be
located on the channel’s outer interface. However, to the
best of our knowledge, the binding site for GSK1016790A
is not yet known. On the other hand, we gained some
insight into this issue from the result that SiNPs also
strongly inhibit TRPV4 activation by 4αPDD, a compound
that was reported to interact with an internal pocket of
the channel formed between transmembrane segments 3
and 4 [
]. Notably, SiNPs also altered the response of
TRPV1 to capsaicin, which was reported to bind to an
occluded region of this channel [
]. Thus, according to
our reasoning above, SiNPs seem not to act on TRPV4
and TRPV1 activation mechanisms by competitive
inhibition. At this point we may just speculate that SiNPs
induce mechanical perturbations in the plasma membrane
that may disrupt activation of TRPV4 and enhance
activation of TRPV1.
An interesting observation was that the SiNPs failed to
completely inhibit TRPV4 activation. This could result
from the presence of two channel populations with
distinct sensitivities to SiNPs. This might be the case for
the HEK293T cells, in which an endogenous human
TRPV4 channel population may co-exist with the
transfected mouse TRPV4 channels. However, we observed
the lack of complete inhibition also in native 16HBE and
mTEC, for which there is no evidence for separate
populations of TRPV4. Although further studies are required
to address this point, we may also consider that if SiNPs
inhibit channel activation by inducing mechanical
perturbations in the plasma membrane, these might not be
sufficient to completely silence channel activity.
A concomitant finding in our experiments was that
SiNPs induce an increase in basal [Ca2+]i in the
humanderived cells. However, our data demonstrates that
TRPV4 channels are not involved in this effect (e.g., lack
of inhibitory effect of the TRPV4 blocker HC067047).
This is different from what was previously suggested by
Gilardino et al., who found that the unspecific TRPV
channel blocker ruthenium red inhibited intracellular
Ca2+ responses to SiNPs [
]. A possible cause for this is
that these authors used particles of 50 nm in diameter,
which represents about a 170-fold larger volume than
that of the ones we used here. Considering that TRPV4
can be activated by mechanical stress at the plasma
], it is conceivable that only the larger
particles may induce TRPV4 activation. On the other
hand, Gilardino et al.  did find TRPV4-independent
responses to SiNPs, which could be triggered via
mechanisms similar to those underlying the responses we
found in 16HBE cells and in HEK293T cells displaying
low basal Ca2+ concentration. Of note, for some yet
unclear reasons mTEC did not display Ca2+ responses
upon SiNPs application.
Other features of our results are also qualitatively
comparable to those obtained by Gilardino et al. [
For instance, the intracellular Ca2+ responses to SiNPs
occurred after a significant delay and showed a transient
initial phase (Fig. 2b). The mechanisms underlying these
responses remain fully unknown, but could be related to
Ca2+ release from intracellular stores. However, the fact
that we found SiNPs to increase inward and outward
basal currents in HEK293T cells is more consistent with
enhanced activities of Ca2+-permeable channels in the
plasma membrane (e.g., the ubiquitously expressed
TRPM7 channels) [
]. These mechanisms should be
addressed in future studies because they may bare
relevance for the toxic effects of SiNPs (Ca2+ overload)
in airway epithelial cells.
Our results show that SiNPs inhibit TRPV4 activation,
and that this effect may impair the positive modulatory
action of the stimulation of this channel on the ciliary
function in airway epithelial cells. It has been proposed
that inhibition of TRPV4 could have therapeutic benefits
in several respiratory conditions, such as chronic heart
failure, hypoxia-induced pulmonary hypertension, acute
lung injury, chronic obstructive pulmonary disease and
]. However, TRPV4 activity has been
shown to underlie protective responses in airway
epithelial cells, including the increase in CBF [
addition, TRPV4 function was reported to be important
for essential functions in other cells that are direct
targets of polluting SiNPs. These include the enhancement
of barrier function is skin keratynocytes [
endothelium-dependent vasorelaxation in pulmonary
arteries , and ATP release from oesophageal
]. Thus, inhibition of TRPV4 by SiNPs is
expected to have complex effects on airway
pathophysiology and rather certain detrimental effects on several
epithelial cell functions. Our findings unveil TRPV4
and TRPV1 as defined molecular targets of SiNPs, and
prompt for further exploration of the role of these
channels in the cellular effects of other types of
The authors would like to thank Prof. Bernd Nilius and the members of the
LICR laboratory for helpful discussions and to Melissa Benoit for the
maintenance of the cell cultures.
Availability of data and materials
All data generated or analysed during this study are included in this
Y.A.A. held a Postdoctoral Mandate of the KU Leuven and is currently a
Postdoctoral Fellow of the Fund for Scientific Research Flanders (FWO).
Research was supported by grants from the Research Foundation Flanders
FWO (G076714), the Research Council of the KU Leuven (Grants GOA/14/011
and PF-TRPLe), The Spanish Ministry of Economy and Competitiveness
(SAF2015-69762R and María de Maeztu Programme for Units of Excellence in
R&D MDM-2014-0370), and the FEDER Funds.
AS, KD and YAA performed Ca2+ imaging experiments; AS, JLA and JAC
performed the patch-clamp experiments; CJ performed the CBF measurements;
SMC performed the characterization of the particles; MAV, PHH and KT
supervised the project; AS and KT wrote the manuscript. All authors edited the
manuscript, and have given approval to its final version.
Experiments were performed in accordance with the guidelines established
by the European Communities Council Directive (86/609/ECC) and by the
Ethics Committee of the KU Leuven, Leuven (021/2012).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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