Journal of Advanced Research 23 (2020) 25–35

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Journal of Advanced Research

journal homepage: www.elsevier.com/locate/jare

TMEM16A Ca2+-activated Cl
channel inhibition ameliorates acute

pancreatitis via the IP3R/Ca2+/NFjB/IL-6 signaling pathway
Qinghua Wang a,b,1, Lichuan Bai a,1, Shuya Luo a, Tianyu Wang a, Fan Yang a, Jialin Xia a, Hui Wang a, Ke Ma a,
Mei Liu a, Shuwei Wu a, Huijie Wang a, Shibin Guo c, Xiaohong Sun d, Qinghuan Xiao a,⇑
a
Department of Ion Channel Pharmacology, School of Pharmacy, China Medical University, Shenyang 110122, China
b
Department of Experimental Center, The Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang 110032, China
c
Department of Gastroenterological Endoscopy, the First Affiliated Hospital of Dalian Medical University, Dalian 116011, China
d
Department of Neurology, The Fourth Affiliated Hospital of China Medical University, Shenyang 110032, China

g r a p h i c a l a b s t r a c t
A positive activation loop between TMEM16A and the IP3R/Ca2+/NFjB/IL-6 pathway is important for Ca2+ elevation, NFjB activation and IL-6 release, and
thus cooperatively promotes the pathogenesis of AP.

Abbreviations: AP, acute pancreatitis; T16Ainh-A01, TMEM16A inhibitor-A01; CaCCinh-A01, Ca2+-activated Cl
channel inhibitor-A01; PACs, pancreatic acinar cells; CCK,
cholesystokinin; IP3R, inositol 1,4,5-trisphosphate receptor; ER, endoplasmic reticulum; IL-6, interleukin 6; IL-6R, interleukin 6 receptor; STAT3, signal transducers and
activators of transcription 3; NFjB, nuclear factor-jB; shRNAs, short hairpin RNAs; FBS, fetal bovine serum; EGFP, green fluorescent protein; RIPA, radio immunoprecipitation
assay; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; Tris, tris(hydroxymethyl)aminomethane; NP-40,
Nonidet P-40; HEPES, N-2-hydroxyethil-piperazine-N’-2-ethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis(2-aminoethyl ether)-N,N,N’,
N’-tetraacetic acid; NMDG, N-methyl-D-glucamine; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid-acetyloxymethyl ester; WT, wild type; EGF,
epidermal growth factor; EGFR, epidermal growth factor receptor; CFBE, cystic fibrosis bronchial epithelial.
Peer review under responsibility of Cairo University.
⇑ Corresponding author at: Department of Ion Channel Pharmacology, School of Pharmacy, China Medical University, No. 77 Puhe Road, Shenyang North New Area,
Shenyang 110122, China.
E-mail address: qinghuanxiao12345@163.com (Q. Xiao).
1
Q.W. and L.B. contributed equally to this work.

https://doi.org/10.1016/j.jare.2020.01.006
2090-1232/Ó 2020 The Authors. Published by Elsevier B.V. on behalf of Cairo University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
26 Q. Wang et al. / Journal of Advanced Research 23 (2020) 25–35

a r t i c l e i n f o a b s t r a c t

Article history: TMEM16A Ca2+-activated Cl
channels are expressed in pancreatic acinar cells and participate in
Received 24 November 2019 inflammation-associated diseases. Whether TMEM16A contributes to the pathogenesis of acute pancre-
Revised 14 January 2020 atitis (AP) remains unknown. Here, we found that increased TMEM16A expression in the pancreatic tis-
Accepted 18 January 2020
sue was correlated with the interleukin-6 (IL-6) level in the pancreatic tissue and in the serum of a
Available online 21 January 2020
cerulein-induced AP mouse model. IL-6 treatment promoted TMEM16A expression in AR42J pancreatic
acinar cells via the IL-6 receptor (IL-6R)/signal transducers and activators of transcription 3 (STAT3) sig-
Keywords:
naling pathway. In addition, TMEM16A was co-immunoprecipitated with the inositol 1,4,5-trisphosphate
TMEM16A
Inositol 1,4,5-trisphosphate receptor
receptor (IP3R) and was activated by IP3R-mediated Ca2+ release. TMEM16A inhibition reduced the
Acute pancreatitis IP3R-mediated Ca2+ release induced by cerulein. Furthermore, TMEM16A overexpression activated
NFjB nuclear factor-jB (NFjB) and increased IL-6 release by increasing intracellular Ca2+. TMEM16A knock-
Interleukin-6 down by shRNAs reduced the cerulein-induced NFjB activation by Ca2+. TMEM16A inhibitors inhibited
NFjB activation by decreasing channel activity and reducing TMEM16A protein levels in AR42J cells,
and it ameliorated pancreatic damage in cerulein-induced AP mice. This study identifies a novel mecha-
nism underlying the pathogenesis of AP by which IL-6 promotes TMEM16A expression via IL-6R/STAT3
signaling activation, and TMEM16A overexpression increases IL-6 secretion via IP3R/Ca2+/NFjB signaling
activation in pancreatic acinar cells. TMEM16A inhibition may be a new potential strategy for treating AP.
Ó 2020 The Authors. Published by Elsevier B.V. on behalf of Cairo University. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction 1,4,5-trisphosphate receptor (IP3R) Ca2+ channel in the endoplas-

mic reticulum (ER) [20]. TMEM16A binds directly to IP3R and is
Acute pancreatitis (AP) is an inflammatory disease with a sever- activated by IP3R-mediated Ca2+ release in dorsal root ganglia cells
ity ranging from mild edema in the pancreas to severe inflamma- and HeLa cells (the human epithelial cancer cells of the cervix that
tion with systemic involvement [1]. The incidence of AP is have been maintained in culture since 1951 and are often used in
approximately 13–45 cases/100,000 people and is increasing research) [21,22], though this direct interaction between
worldwide [2]. No therapeutic agents that inhibit the progression TMEM16A and IP3R is not found in cerebral artery smooth muscles
of AP are currently available [3]. Therefore, it is urgent to develop [23]. In addition, several lines of evidence have shown that
a novel agent that can ameliorate AP. TMEM16A overexpression increases intracellular Ca2+ concentra-
TMEM16A (anoctamin 1) is a Ca2+-activated Cl
channel that tions [17,22]. However, it is unknown whether TMEM16A is
participates in various physiological functions ranging from the involved in the pathogenesis of AP by increasing IP3R-mediated
secretion of epithelial fluid and the contraction of skeletal and
Ca2+ elevation.
smooth muscles to blood pressure control [4–6]. Recent studies
Here, we studied the mechanism of TMEM16A channels in AP
have revealed that TMEM16A expression is upregulated in many
induced by cerulein, a CCK analog that is experimentally used for
diseases including cancer, hypertension, and cystic fibrosis [6–8].
creating AP models. Both animal and cellular studies showed that
In addition, TMEM16A overexpression is found in many
TMEM16A expression was upregulated in PACs. TMEM16A upreg-
inflammation-associated diseases such as asthma, cystic fibrosis,
ulation was caused by interleukin 6 (IL-6)/IL-6 receptor (IL-6R)/
and chronic rhinosinusitis [8–11]. TMEM16A inhibition by its inhi-
signal transducers and activators of transcription 3 (STAT3) signal-
bitors such as T16Ainh-A01 (TMEM16A inhibitor-A01) and
CaCCinh-A01 (Ca2+-activated Cl
channel inhibitor-A01) (the struc- ing activation in AR42J cells. TMEM16A overexpression activated
ture of the inhibitors is shown in Fig. S1) blocks the development of nuclear factor-jB (NFjB) and increased IL-6 secretion. TMEM16A
cancer and inflammation [8,9]. Therefore, TMEM16A inhibitors inhibition by short hairpin RNAs (shRNAs) to silence the targeted
may be promising for treating TMEM16A overexpression- gene or by inhibitors to block channel currents reduced the
associated inflammatory diseases. TMEM16A currents, inhibited IP3R-mediated Ca2+ release, and
TMEM16A has been immunohistochemically detected in the blocked NFjB activation and IL-6 secretion in AR42J cells following
pancreatic tissues [12], including in acinar cells [13,14], islets cerulein treatment. TMEM16A inhibitors ameliorated pancreatic
[15], and duct cells [16], as well as in pancreatic cancer cells damage in cerulein-induced AP mice. Our findings suggest that
[17]. TMEM16A mediates Ca2+-activated Cl
and HCO–3 transport TMEM16A promotes the pathogenesis of AP via activation of the
in pancreatic acinar cells (PACs) [18,19]. TMEM16A-mediated Cl
IP3R/Ca2+/NFjB/IL-6 pathway, and TMEM16A inhibition may be a
secretion may function as a main driving force for secreting fluids promising strategy for the treatment of AP.
in PACs [14]. TMEM16A-mediated HCO–3 transport in PACs is
important for luminal pH regulation, and TMME16A inhibition
increases luminal acidosis in AP induced by supramaximal Materials and methods
cholesystokinin (CCK) treatment [19]. However, although
TMEM16A has been implicated in luminal pH regulation in AP, Animals
no studies have investigated whether TMEM16A contributes to
the pathogenesis of AP. The Animal Ethics Committee of Liaoning University of Tradi-
Sustained intracellular Ca2+ elevation in PACs activates tional Chinese Medicine approved the experimental protocol for
trypsinogen, causes mitochondrial dysfunction, induces NFjB acti- animal use (No. 2019YS (DW)-024-01). All animal experiments
vation, and has been recognized as a critical mechanism underly- were conducted in accordance with the National Institutes of
ing the pathogenesis of AP [3,20]. Many known stimuli that Health Guide for the Care and Use of Laboratory Animals.
induce AP, such as alcohol, CCK hyperstimulation, and bile acids, C57BL/6N mice (male, 8–10 weeks old, weighing 20–24 g) were
elicit sustained Ca2+ elevation in PACs by activating the inositol housed at 25 °C with 3.3. water and food ad libitum.
 Q. Wang et al. / Journal of Advanced Research 23 (2020) 25–35 27

Mouse model of cerulein-induced AP IL-6 ELISA kit (AMEKO, Shanghai, China) according to the manufac-
turer’s protocols and were detected using a microplate reader
The mouse model of AP was created by a series of 7 intraperi- (Bio-Rad, USA).
toneal injections of supramaximal cerulein (50 lg/kg; Sigma-
Aldrich, USA) spaced one hour apart. To investigate the time course Co-immunoprecipitation
of TMEM16A expression in AP mice, 24 mice were randomly
assigned to 4 groups (n = 6 per group) based on sacrifice time at AR42J cells were homogenized for 30 min in ice-cold RIPA
0, 6, 12, and 24 h after the last cerulein injection. To study the lysis buffer containing 50 mM Tris(hydroxymethyl)aminome
in vivo effect of T16Ainh-A01, 18 mice were randomly assigned thane-HCl (Tris-HCl; pH 7.4), 150 mM NaCl, 1% Nonidet P-40
to 3 groups (n = 6 per group): the control group, the AP group, (NP-40), 0.25% sodium deoxycholate, sodium orthovanadate,
and the AP + T16Ainh-A01 group. For the AP + T16Ainh-A01
ethylenediaminetetraacetic acid (EDTA) and aprotinin, a protease
group, AP mice received intraperitoneal injection of T16Ainh-A01
inhibitor that inhibits proteolysis (Absin Biotechnology, China).
(1 mg/kg) 30 min prior to the first cerulein injection. Equal
After centrifugation, the supernatant was incubated with anti-
volumes of normal saline were injected in control mice. The mice
TMEM16A antibodies or anti-IP3R antibodies overnight at 4 °C,
were sacrificed 12 h after the last cerulein injection. Blood and
followed by incubation with pre-cleaned protein A/G agarose
pancreatic tissues were collected for further analysis.
beads (20 ll) for 2 h at 4 °C. The beads were then centrifuged
at 3,000 rpm for 3 min at 4 °C, washed with lysis buffer, and
Histological examination of the pancreas
resuspended in the sample buffer. The samples were then ana-
lyzed by Western blot.
Pancreatic tissue sections (5 lm thick) were stained with hema-
toxylin and eosin. The severity of pancreatitis was assessed using a
scoring system that evaluated four pathological parameters: Measurement of intracellular Ca2+
edema (0–4), acinar cell necrosis (0–4), inflammation (0–4), and
intrapancreatic hemorrhage (0–4) [24]. The final pathological score AR42J cells were loaded with the cell-permeable fluorescent
(range, 0–16) was determined by the summation of the score for Ca2+ dye fluo-4- acetyloxymethyl ester (Fluo-4-AM) (2 lM,
each parameter. Invitrogen, USA) and 0.1% F127 (Invitrogen, USA) for 50 min
at 37 °C in Hank’s solution, which provided physiological pH,
Cell culture and transfection osmotic balance and essential inorganic ions. The cells
were plated on a coverslip in Hank’s solution without Ca2+
Pancreatic acinar AR42J cells (ATCC, Manassas, USA) were cul- (containing 5 mM Ca2+ chelator ethylene glycol-bis(2-
tured in RPMI 1640 medium (HyClone) supplemented with 20% aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA)). The
fetal bovine serum (FBS) and 1% penicillin and streptomycin to pre- intracellular Ca2+ concentration in response to cerulein
vent bacterial contamination in a humid incubator (37 °C, 5% CO2). (10 nM) was measured using a confocal microscope (Nikon C2
The cell model of AP was created by treating cells with cerulein plus, Japan) (excitation wavelength, 485 nm; emission wave-
(10 nM) for 24 h. length, 515 nm). Fluo-4 fluorescence signal normalized to the
AR42J cells were transfected with TMEM16A-expressing plas- resting level (F/F0) was used for analysis.
mids in the pEGFP-N1 vector and the control empty vector [12]
or with TMEM16A-shRNAs and scrambled control shRNAs in the
Patch clamp recordings
pGPU6-EGFP vector (constructed by Shanghai GenePharma, China)
using Lipofectamine 2000 (Invitrogen) according to the manufac-
The patch clamp technique was used to record Cl
currents in a
ture’s protocol. The pEGFP-N1 vector and the pGPU6-EGFP vector
whole-cell configuration. A P97 puller (Sutter Instruments, CA) was
encode enhanced green fluorescent protein (EGFP), which exhibits
used to make electrodes with resistances of ~2–4 mX when filled
green fluorescence under a fluorescence microscope and can be
with pipette solution. The data were recorded using Clampex 10
used as a reporter to detect the transfected cells.
software on a computer connected to an Axopatch 200B amplifier
via a Digidata system (Molecular Devices, CA, USA). AR42J cells
Western blot
were voltage clamped at a holding potential of 0 mV. Voltage
ramps from
100 to +100 mV were applied at an interval of 10 s.
For TMEM16A expression, pancreatic tissues or AR42J cells
Pipette solutions contained (in mM): 146 CsCl, 2 MgCl2, 0.5 EGTA
were homogenized in radio immunoprecipitation assay (RIPA) buf-
and 8 HEPES (N-2-hydroxyethil-piperazine-N’-2-ethanesulfonic
fer (Beyotime Biotechnology, China). For NFjB/p65 (65 kD) nuclear
acid), pH 7.3, adjusted with NMDG (N-methyl-D-glucamine).
translocation, nuclear and cytoplasmic pools were generated using
the nuclear and cytoplasmic protein extraction kit (KeyGEN, External solutions contained (in mM): 144 NaCl, 4 KCl, 5 EGTA, 1
China). After protein separation by sodium dodecyl sulfate poly- MgCl2, 10 glucose, and 10 HEPES (pH 7.3). Cerulein was applied
acrylamide gel electrophoresis (SDS-PAGE) and electroblot trans- to the external solution to record Ca2+-activated Cl
currents acti-
fer, the membranes were incubated with primary antibodies vated by IP3R.
against TMEM16A (1:2,000), STAT3 (1:1,000), phosphorylated
STAT3 (p-STAT3; 1:1,000), NFjB/p65 (1:1,000) or IP3R (1:1,000) Statistical analysis
overnight at 4 °C, followed by secondary antibodies (1:10,000) at
room temperature for 1 h. Bands were visualized using chemilumi- Origin9 software was used for graphical presentations. Clamp-
nescence detection agents. All primary and secondary antibodies fit10 software was used for analyzing current traces. SPSS 13.0
were from Abcam Biotechnology, UK. software was used for statistical analyses. Student’s t-test or one-
way analysis of variance (ANOVA) was used to compare the differ-
Enzyme-linked immunosorbent assay (ELISA) ences between groups. Spearman correlation analysis was used to
evaluate the association of TMEM16A expression with the IL-6
The IL-6 levels in the AR42J cell culture medium and in the levels in AP mice. P < 0.05 was considered to be statistically
mouse serum and pancreatic tissues were determined using an significant.
28 Q. Wang et al. / Journal of Advanced Research 23 (2020) 25–35

Results AP, TMEM16A expression was upregulated in AR42J cells following

cerulein treatment for 6–24 h (Fig. 1D).
TMEM16A expression is upregulated in cerulein-induced AP
TMEM16A expression correlates with IL-6 levels in cerulein-induced
We examined TMEM16A expression in cerulein-induced AP AP mice
mice. Histological examination showed that cerulein treatment
induced pancreatic damage in the mice (Fig. 1A, B). TMEM16A It is known that IL-6 is increased in AP and contributes to the
expression was upregulated in the pancreatic tissue of AP mice pathogenesis of AP [25,26]. We examined the IL-6 levels in the
(Fig. 1C). Consistent with the animal model of cerulein-induced pancreatic tissue and the serum of AP mice using ELISA. The IL-6

Fig. 1. TMEM16A expression was increased in cerulein-induced AP and was correlated with IL-6 expression. A. Representative H&E stains of pancreatic tissues of control mice
and AP mice. Mice were sacrificed at 6, 12, and 24 h after the last injection of cerulein. Scale bar: 100 lm. B. The pathologic scores of pancreatic tissues in control mice and AP
mice at 6, 12, and 24 h after cerulein treatment. n = 6 mice. *p < 0.05 vs control. C. Western blot analysis of TMEM16A expression in the pancreatic tissues of control mice and
AP mice at 6, 12, and 24 h after the last injection of cerulein. D. Western blot analysis of TMEM16A expression in control AR42J cells and cells treated with 10 nM cerulein for
6–24 h. n = 3.*p < 0.05 vs control. E, F. ELISA results of the IL-6 levels in the pancreatic tissues (E) and in the serum (F) of control mice and AP mice at 6, 12 and 24 h after
cerulein injection. n = 6. *p < 0.05 vs control. G, H. Correlation of TMEM16A with the IL-6 levels in the pancreatic tissues (G) and in the serum (H) of control mice and AP mice
at 6, 12 and 24 h after cerulein injection. The association was analyzed using Spearman correlation analysis.
 Q. Wang et al. / Journal of Advanced Research 23 (2020) 25–35 29

Fig. 2. IL-6 increased TMEM16A expression via the IL-6R/STAT3 signaling pathway in AR42J cells. A. ELISA results of the IL-6 levels in the culture medium in control AR42J
cells and in cerulein-treated cells. n = 6.*p < 0.05 vs control. B. Western blot analysis of TMEM16A expression in AR42J cells treated with different concentrations of IL-6 (0–
0.25 lg/ml) for 24 h. C. Western blot analysis of the expression of p-STAT3, STAT3, and TMEM16A in AR42J cells treated with 0.25 lg/ml IL-6 for 24 h. D. E. Quantification
results of pSTAT3 (D) and TMEM16A (E) expression in C. n = 3.*p < 0.05 vs control; #p < 0.05 vs IgG. F. Western blot results of TMEM16A expression in control AR42J cells and
cells treated with 0.25 lg/ml IL-6 for 24 in the presence or absence of the STAT3 inhibitor JSI-124 (1 lM). n = 3.*p < 0.05 vs control; #p < 0.05 vs IL-6 alone. G. H. Western blot
results of TMEM16A expression in control AR42J cells and cerulein-treated cells in the presence or absence of antibodies against IL-6R (1 lg/ml) (G) or JSI-124 (1 lM) (H). IgG
was used as control for IL-6R antibodies. n = 3.*p < 0.05 vs control; #p < 0.05 vs IgG (G) or cerulein alone (H).

levels in the pancreatic tissues and in the serum were increased in IL-6 promotes TMEM16A expression via the IL-6R/STAT3 pathway in
AP mice 6–24 h after cerulein injection (Fig. 1E, F). The amount of cerulein-induced AP
TMEM16A protein was significantly correlated with IL-6 levels in
the pancreatic tissues (r = 0.99, p = 0.011; Fig. 1G) and in the serum The ELISA results confirmed that the IL-6 concentration in the
(r = 0.99, p = 0.0057; Fig. 1F), suggesting that IL-6 may promote culture medium was significantly increased after cerulein treat-
TMEM16A expression in AP. ment for 24 h (Fig. 2A), suggesting that IL-6 secretion was
30 Q. Wang et al. / Journal of Advanced Research 23 (2020) 25–35

Fig. 3. TMEM16A and IP3R activated each other in AR42J cells. A. Immunoprecipitation of IP3R by antibodies against TMEM16A (anti-T16A, left) and of TMEM16A by
antibodies against IP3R (right) from lysates of AR42J cells. IgG was used as control. B. The time course of activation of Cl
currents by cerulein in AR42J cells transfected with
scrambled shRNAs or TMEM16A-shRNAs. Cells were recorded with 750-ms voltage ramps from
100 to + 100 mV at an interval of 10 s. The external solution contained no
Ca2+. Currents at –100 mV and + 100 mV are shown. Bottom trace: the representative currents recorded before (a) and after (b) cerulein application and at the peak current
(c). C. The representative currents at the time points a, b, c in B. D. The representative current at c recorded with a step voltage pulse from a holding potential of 0 mV to
potentials between –100 mV and + 100 mV in 20 mV increments for 750 ms. E. Mean peak current densities at + 100 mV for cells treated with scrambled shRNA or TMEM16A-
shRNA, as recorded in (B). n = 3–4. *p < 0.05 vs scrambled shRNA. F. The time course of cerulein-induced Cl
currents inhibited by T16Ainh-A01 (20 lM) in AR42J cells. G.
Mean Fluo-4 intensity changes (F/F0) in AR42J cells in the presence or absence of T16Ainh-A01 (20 lM). Cerulein (10 nM) was applied to induce Ca2+ release from IP3R. Cells
were treated with T16Ainh-A01 30 min before cerulein application. H. Quantification of the amplitude of the curve of Fluo-4 intensity changes (F/F0). n = 10–12 cells. *p < 0.05
vs control.

increased in the AR42J cell model of AP. IL-6 (0.25 lg/ml) treatment AR42J cells. The co-immunoprecipitation results showed that
increased TMEM16A expression in AR42J cells (Fig. 2B). Antibodies TMEM16A directly interacted with IP3R in AR42J cells (Fig. 3A).
against IL-6 receptor (anti-IL-6R) reduced IL-6-induced STAT3 acti- We then investigated whether IP3R-mediated Ca2+ release acti-
vation and TMEM16A upregulation in AR42J cells (Fig. 2C-E). The vated TMEM16A Cl
currents in AR42J cells by acute application
STAT3 inhibitor JSI-124 inhibited IL-6-induced TMEM16A upregula- of cerulein. Under the condition of ‘‘000 Ca2+ extracellular solution
tion in AR42J cells (Fig. 2F). IL-6R antibodies and JSI-124 significantly to exclude the possible effect of Ca2+ influx, whole-cell Cl
currents
inhibited cerulein-induced TMEM16A upregulation in AR42J cells in AR42J cells were gradually activated after the application of cer-
(Fig. 2G, H). These findings suggested that increased IL-6 secretion ulein. The currents activated by cerulein exhibited outward rectifi-
from acinar cells after cerulein treatment promoted TMEM16A cation at the beginning of cerulein application and showed liner
expression via IL-6R/STAT3 signaling activation. voltage-current relationships when currents were maximally acti-
vated, exhibiting the characteristic feature of TMEM16A currents
TMEM16A channels and IP3R activate each other in AR42J cells (Fig. 3B-D). Cerulein-induced Cl
currents were significantly
reduced by TMEM16A-shRNA treatment (Fig. 3B-E). The TMEM16A
We performed co-immunoprecipitation experiments to investi- inhibitor T16Ainh-A01 inhibited cerulein-induced Cl
currents in
gate whether TMEM16A and IP3R directly bind to each other in AR42J cells (Fig. 3F). Furthermore, T16Ainh-A01 inhibited the
 Q. Wang et al. / Journal of Advanced Research 23 (2020) 25–35 31

Fig. 4. TMEM16A overexpression activated NFjB signaling in AR42J cells. A. Western blot results of p65 expression in the nucleus and cytosol in control AR42J cells and cells
transfected with empty vector or TMEM16A-overexpressing plasmids. Quantification results of the ratio of nuclear/cytosolic p65 expression in the bottom. n = 3 *p < 0.05 vs
vector. B. ELISA results of the IL-6 levels in the culture medium in control AR42J cells and cells transfected with empty vector or TMEM16A-overexpressing plasmids. n = 6.
*p < 0.05 vs vector. C. Representative Western blot results of p65 expression in the nucleus and cytosol in control AR42J cells and cells transfected with empty vector or
TMEM16A-overexpressing plasmids in the presence or absence of BAPTA-AM (13 lM). DMSO was used as a vehicle control for BAPTA-AM. D. Quantification results of the
ratio of nuclear/cytosolic p65 expression in C. n = 3. *p < 0.05 vs vector; #p < 0.05 vs DMSO.

IP3R-mediated Ca2+ release induced by cerulein application TMEM16A inhibitors ameliorate cerulein-induced AP via inhibition of
(Fig. 3G, H). Taken together, these results suggested that TMEM16A channel activities and protein expression
and IP3R directly interacted with and activated each other in AR42J
cells. We investigated the effect of TMEM16A inhibitors on NFjB acti-
vation in cerulein-induced AP. T16Ainh-A01 treatment inhibited
TMEM16A overexpression activates NFjB signaling via Ca2+ in AR42J cerulein-induced NFjB activation (Fig. 6A, B), suggesting that inhi-
cells bition of TMEM16A channel function reduced the cerulein-induced
NFjB activation in AR42J cells. To further confirm whether
Since NFjB activation by Ca2+ in PACs contributes to the patho- TMEM16A channel activities were essential for NFjB activation,
genesis of AP [25,27], we further investigated whether TMEM16A we transfected AR42J cells with D444EEEEEAVKD452 TMEM16A
activates NFjB signaling in AR42J cells. TMEM16A overexpression mutants with reduced channel activities [28]. Compared with
increased the nuclear expression of NFjB/p65 and decreased the wild-type (WT) TMEM16A, overexpression of D444EEEEEAVKD452
cytosolic expression of NFjB/p65 (Fig. 4A). TMEM16A overexpres- mutants resulted in less NFjB activation (Fig. 6C), suggesting that
sion also increased the IL-6 secretion from AR42J cells (Fig. 4B). TMEM16A channel activity was essential for NFjB activation.
These results suggested that TMEM16A promoted NFjB activation We then examined whether T16Ainh-A01 inhibited TMEM16A
in AR42J cells. Furthermore, the Ca2+ chelator 1,2-bis(2-aminophe expression in cerulein-treated AR42J cells. The cerulein-induced
noxy)ethane-N,N,N0 ,N0 -tetraacetic acid-acetyloxymethyl ester increase in TMEM16A expression was inhibited by T16Ainh-A01
(BAPTA-AM) (Fig. S1C) inhibited TMEM16A overexpression- treatment (Fig. 6D). This effect was also found in the in vivo mouse
induced NFjB activation (Fig. 4C, D). These findings suggested that model of cerulein-induced AP (Fig. 6E). Furthermore, histological
TMEM16A activated NFjB signaling by increasing intracellular examination showed that T16Ainh-A01 reduced the pancreatic
Ca2+. damage in AP mice (Fig. 6F, G). T16Ainh-A01 treatment inhibited
the IL-6 level in the pancreatic tissues and the serum of AP mice
(Fig. 6H, I). These findings indicated that TMEM16A inhibition
TMEM16A knockdown blocks cerulein-induced NFjB activation
ameliorated AP.
We next investigated the effect of TMEM16A inhibition on
NFjB activation in the AR42J cell model of cerulein-induced AP. Discussion
BAPTA-AM treatment inhibited cerulein-induced (24 h) NFjB acti-
vation (Fig. 5A), suggesting that Ca2+ signaling was critical for NFjB Our study demonstrated that TMEM16A expression was upreg-
activation in cerulein-induced AP. Cerulein-induced NFjB activa- ulated in PACs via the IL-6/IL-6R/STAT3 signaling pathway in
tion and IL-6 secretion were inhibited by TMEM16A-shRNA treat- cerulein-induced AP, and increased TMEM16A expression acti-
ment (Fig. 5B, C), suggesting that TMEM16A mediated NFjB vated NFjB signaling and promoted IL-6 secretion by increasing
activation and IL-6 secretion in cerulein-induced AP. IP3R-mediated Ca2+ release in acinar cells (Fig. 7). Thus, these
32 Q. Wang et al. / Journal of Advanced Research 23 (2020) 25–35

Fig. 5. TMEM16A inhibition blocked cerulein-induced NFjB activation in AR42J cells. A. Representative Western blot results (Top) of p65 expression in the nucleus and
cytosol in control AR42J cells and cerulein-treated cells in the presence or absence of BAPTA-AM (13 lM). DMSO was used as vehicle control. Bottom: quantification results of
the ratio of nuclear/cytosolic p65 expression. n = 3. *p < 0.05 vs control; #p < 0.05 vs DMSO. B. Western blot results (Top) of p65 expression in the nucleus and cytosol in
control AR42J cells and cerulein-treated cells with or without transfection of scrambled shRNAs (Scr) or TMEM16A-shRNAs. Bottom: quantification results of the ratio of
nuclear/cytosolic p65 expression. n = 3. *p < 0.05 vs control; #p < 0.05 vs scrambled shRNAs. C. ELISA results of the IL-6 levels in the culture medium in control AR42J cells and
cerulein-treated cells with or without transfection of scrambled shRNAs or TMEM16A-shRNAs. n = 6. *p < 0.05 vs control; #p < 0.05 vs scrambled shRNAs.

results reveal a positive activation loop between TMEM16A and IL- persistent high TMEM16A expression and the constitutive secre-
6 in PACs. Since Ca2+, NFjB, and IL-6 are known contributors to the tion of IL-6 in PACs during AP. Since IL-6 is a pro-inflammatory
pathogenesis of AP [20,27], our findings suggest that TMEM16A cytokine that contributes to the development of AP [25], our find-
may aggravate AP by promoting Ca2+ elevation, NFjB activation ings also suggest that TMEM16A upregulation in PACs may pro-
and IL-6 release. Furthermore, TMEM16A inhibition by mote AP by increasing IL-6 secretion.
TMEM16A-shRNA or T16Ainh-A01 was able to block NFjB activa- Sustained Ca2+ elevation is an early cellular event in PACs dur-
tion and IL-6 secretion in the cellular model of AP, and T16inh-A01 ing AP [3,20,27]. NFjB activation and the subsequent release of
treatment ameliorated pancreatic damage and reduced the IL-6 many pro-inflammatory cytokines including IL-6 play key roles
levels in the AP animal model. Our findings suggest that TMEM16A in the pathogenesis of AP [25,27]. Several studies have demon-
inhibition may be a new strategy for treating AP. strated that Ca2+ is required for NFjB activation in PACs treated
TMEM16A expression is upregulated in inflammation [10], and with cerulein or bile acids [27,32]. Consistent with these studies,
TMEM16A overexpression contributes to inflammation-associated we found that the Ca2+ chelator BAPTA-AM reduced cerulein-
respiratory diseases such as cystic fibrosis, chronic rhinosinusitis, induced NFjB activation in PACs. In addition, TMEM16A inhibition
and asthma [8,9,11]. Inflammatory cytokines such as IL-4 and IL- by T16Ainh-A01 reduced the IP3R-mediated Ca2+ release induced
13 increase TMEM16A expression by activating STAT6 in airway by cerulein, and BAPTA-AM treatment inhibited the TMEM16A
epithelial cells and biliary epithelial cells [10,29,30]. We previously overexpression-induced NFjB activation in PACs. Thus, TMEM16A
found that TMEM16A expression is upregulated by epidermal overexpression activates NFjB by increasing Ca2+ levels. Further-
growth factor (EGF)/EGF receptor (EGFR)/STAT3 signaling activa- more, TMEM16A inhibition by shRNAs or T16Ainh-A01 reduced
tion in breast cancer cells [31]. The current study showed that the cerulein-induced NFjB activation. Therefore, our findings sug-
TMEM16A expression was correlated with the IL-6 levels in the gest that TMEM16A may promote AP by increasing intracellular
pancreatic tissues and in the serum of AP mice, and IL-6 promoted Ca2+ concentrations and subsequently activating NFjB in PACs.
TMEM16A expression in PACs by activating the IL-6R/STAT3 sig- TMEM16A is expressed in the ER–plasma membrane contact
naling pathway. Furthermore, TMEM16A overexpression resulted sites [21,22], where ion channels such as IP3R are expressed and
in an increase in IL-6 secretion in PACs, and TMEM16A knockdown participate in the regulation of Ca2+ signaling [33]. TMEM16A
reduced IL-6 secretion in cerulein-treated cells. These findings sug- directly binds to IP3R and is activated by IP3R-mediated Ca2+
gest that TMEM16A expression and IL-6 secretion mutually acti- release in the dorsal root ganglia and HeLa cells [21,22]. Further-
vate each other in PACs, and the positive activation loop between more, TMEM16A knockdown inhibits ATP-induced Ca2+ release in
TMEM16A and IL-6 may be important for the maintenance of HeLa cells and human cystic fibrosis bronchial epithelial (CFBE)
 Q. Wang et al. / Journal of Advanced Research 23 (2020) 25–35 33

Fig. 6. T16Ainh-A01 inhibited cerulein-induced AP. A. Representative Western blot results of p65 expression in the nucleus and cytosol in control AR42J cells and cerulein-
treated cells in the presence or absence of T16Ainh-A01 treatment applied 30 min before cerulein treatment. B. Quantification results of the ratio of nuclear/cytosolic p65
expression in (A). n = 3. *p < 0.05 vs control; #p < 0.05 vs cerulein alone. C. Western blot results of p65 expression in the nucleus and cytosol in AR42J cells transfected with
empty vector, WT TMEM16A, or D444EEEEAVKD452 TMEM16A mutants. D. E. Western blot results (Top) of TMEM16A expression in control AR42J cells, cerulein-treated cells
(D), control mice and mice with cerulein treatment (E) in the presence or absence of T16Ainh-A01. Bottom: quantification results of TMEM16A expression. n = 3. *p < 0.05 vs
control; #p < 0.05 vs cerulein alone. F. G. Representative H&E stains (F) and pathological scores (G) of pancreatic tissues in control mice and mice with cerulein treatment in
the presence or absence of T16Ainh-A01. H. I. The IL-6 levels in the pancreatic tissues (H) and in the serum (I) of control mice and mice with cerulein treatment in the
presence or absence of T16Ainh-A01. n = 6. *p < 0.05 vs control; #p < 0.05 vs cerulein alone.

cells [22,34]. However, the binding of TMEM16A to IP3R is cell cerulein. In turn, TMME16A inhibition by T16Ainh-A01 reduced
type-dependent, since TMEM16A does not co-immunoprecipitate IP3R-mediated Ca2+ release. These results demonstrate that
with IP3R in cerebral artery smooth muscles [23]. In addition, IP3R-mediated Ca2+ release can activate TMEM16A channels, and
TMEM16A has been found to control EGF-induced Ca2+ signaling TMEM16A channels can promote IP3R-mediated Ca2+ release from
in pancreatic cancer cells [17]. Here, we found that TMEM16A the ER. Thus, our findings suggest that TMEM16A and IP3R directly
and IP3R were co-immunoprecipitated in PACs. TMEM16A interact with and activate each other, thus forming a positive
channels were activated by Ca2+ release from IP3R activation by activation loop between TMEM16A and IP3R.
34 Q. Wang et al. / Journal of Advanced Research 23 (2020) 25–35

Conclusions

Our study identified a novel mechanism by which TMEM16A

channels are upregulated in PACs via IL-6/IL-6R/STAT3 signaling
activation, and TMEM16A overexpression activates IP3R/Ca2+/
NFjB/IL-6 signaling in PACs (Fig. 7). The mutual activation
between TMEM16A and IL-6 may be essential for persistent high
TMEM16A expression, sustained Ca2+ elevation, and constitutive
NFjB activation and IL-6 secretion in PACs, all of which are known
to contribute to the pathogenesis of AP. Furthermore, TMEM16A
inhibition by T16Ainh-A01 reduced the serum IL-6 levels and ame-
liorated pancreatic damage in AP mice. Therefore, our findings sug-
gest that targeting TMEM16A may represent a novel therapy for
AP. The identification of novel TMEM16A inhibitors may be
promising for the treatment of AP. Consistent with this idea, some
Fig. 7. The mechanisms by which TMEM16A promotes the pathogenesis of AP by agents such as sikonin that inhibit TMEM16A [38] can ameliorate
activating the IP3R/Ca2+/NFjB signaling pathways. TMEM16A is upregulated by IL-6 AP in mice [39].
via the IL-6R/STAT3 signaling pathway. Increased TMEM16A expression promotes
intracellular Ca2+ release from the ER via direct interaction with IP3R. Intracellular
Ca2+ elevation subsequently activates NFjB signaling, resulting in an increase in IL- Compliance with ethics requirements
6 secretion from acinar cells. IL-6 further promotes TMEM16A expression via the IL-
6R/STAT3 signaling pathway. Therefore, a positive activation loop between
All Institutional and National Guidelines for the care and use of
TMEM16A and the IP3R/ Ca2+/NFjB/IL-6 pathway is important for Ca2+ elevation,
NFjB activation and IL-6 release, and thus cooperatively promotes the pathogenesis animals (fisheries) were followed.
of AP.
Declaration of Competing Interest

IP3R-mediated Ca2+ elevation in PACs is important for the All authors declare no conflicts of interest.
pathogenesis of AP [20], since IP3R inhibition by IP3R knockout
and by the pharmacological inhibitor caffeine protects against AP Acknowledgements
induced by cerulein, taurolithocholate, or ethanol and palmitoleic
acid [35]. However, caffeine is not a specific IP3R inhibitor, and This work was supported by grants from the National Natural
intoxication doses (>25 mg/kg) are required to ameliorate AP in Science Foundation of China (No. 81572613 and No. 31371145 to
mice [35]. This study found that TMEM16A inhibition by Qinghuan Xiao; No. 81702611 to Hui Wang), the Liaoning Pandeng
T16Ainh-A01 inhibited the IP3R-mediated Ca2+ elevation and Scholar (to Qinghuan Xiao), the Natural Science Foundation of
cerulein-induced NFjB activation in the AR42J cell model of AP, Liaoning Province (No. 2019-MS-222 to Qinghua Wang), and the
and it reduced the serum IL-6 levels and ameliorated pancreatic Natural Science Foundation of Liaoning Province for Guidance Pro-
damage in the animal model of AP. Our findings indicate that gram (No. 20180551125 to Lichuan Bai).
TMEM16A may be a potential new target for treating AP.
We found that TMEM16A expression was upregulated in PACs, Appendix A. Supplementary material
and reduced TMEM16A expression via transfection of TMEM16A-
shRNAs inhibited the cerulein-induced NFjB activation, suggesting Supplementary data to this article can be found online at
that reduced TMEM16A channel function via decreasing protein https://doi.org/10.1016/j.jare.2020.01.006.
levels is important for NFjB activation in PACs. However, knock-
down of TMEM16A protein could not determine whether
References
TMEM16A channel activity contributes to NFjB activation. Our
findings show that overexpression of D444EEEEEAVKD452 mutants [1] Goodchild G, Chouhan M, Johnson GJ. Practical guide to the management of
with decreased channel activities resulted in less NFjB activation, acute pancreatitis. Frontline Gastroenterol 2019;10:292–9. doi: https://doi.
suggesting that TMEM16A channel activity is important for NFjB org/10.1136/flgastro-2018-101102.
[2] Roberts SE, Akbari A, Thorne K, Atkinson M, Evans PA. The incidence of acute
activation in AR42J cells. Furthermore, T16Ainh-A01 not only pancreatitis: impact of social deprivation, alcohol consumption, seasonal and
inhibited the TMEM16A current activated by cerulein, but it also demographic factors. Aliment Pharmacol Ther 2013;38:539–48. doi: https://
reduced TMEM16A expression in cell and mouse models of doi.org/10.1111/apt.12408.
[3] Habtezion A, Gukovskaya AS, Pandol SJ. Acute pancreatitis: a multifaceted set
cerulein-induced AP. Reduced TMEM16A protein expression is of organelle and cellular interactions. Gastroenterology 2019;156:1941–50.
found for CaCCinh-A01 (but not for T16Ainh-A01) in head and neck doi: https://doi.org/10.1053/j.gastro.2018.11.082.
squamous cell carcinoma Te11 cells and in pharyngeal squamous [4] Oh U, Jung J. Cellular functions of TMEM16/anoctamin. Pflugers Arch
2016;468:443–53. doi: https://doi.org/10.1007/s00424-016-1790-0.
cancer FaDu cells due to decreased protein stability or increased [5] Dayal A, Ng SFJ, Grabner M. Ca(2+)-activated Cl(-) channel TMEM16A/ANO1
internalization via an ER-associated, proteasome-dependent mech- identified in zebrafish skeletal muscle is crucial for action potential
anism [36]. However, T16Ainh-A01 has been found to reduce acceleration. Nat Commun 2019;10:115. doi: https://doi.org/10.1038/
s41467-018-07918-z.
TMEM16 expression in SKBR3 breast cancer cells [37], similar to [6] Ma MM, Gao M, Guo KM, Wang M, Li XY, Zeng XL, et al. TMEM16A contributes
our findings. This cell-specific effect of T16Ainh-A01 on TMEM16A to endothelial dysfunction by facilitating Nox2 NADPH oxidase-derived
expression suggests that the reduction of TMEM16A expression by reactive oxygen species generation in hypertension. Hypertension
2017;69:892–901. doi: https://doi.org/10.1161/HYPERTENSIONAHA.
T16Ainh-A01 may depend on a specific cellular environment. In
116.08874.
this study, T16Ainh-A01 inhibited NFjB activation by cerulein in [7] Wang H, Zou L, Ma K, Yu J, Wu H, Wei M, et al. Cell-specific mechanisms of
AR42J cells, and reduced the IL-6 levels in the pancreatic tissues TMEM16A Ca(2+)-activated chloride channel in cancer. Mol Cancer
and serum. Since IL-6 promoted TMEM16A expression (Fig. 7), 2017;16:152. doi: https://doi.org/10.1186/s12943-017-0720-x.
[8] Kunzelmann K, Ousingsawat J, Cabrita I, Dousova T, Bahr A, Janda M, et al.
the reduction of TMEM16A expression by T16Ainh-A01 may be TMEM16A in cystic fibrosis: activating or inhibiting?. Front Pharmacol
due to reduced IL-6 levels via NFjB inhibition in PACs. 2019;10:3. doi: https://doi.org/10.3389/fphar.2019.00003.
 Q. Wang et al. / Journal of Advanced Research 23 (2020) 25–35 35

[9] Wang P, Zhao W, Sun J, Tao T, Chen X, Zheng YY, et al. Inflammatory mediators vasoconstriction in cerebral arteries. Am J Physiol Cell Physiol 2016;310:
mediate airway smooth muscle contraction through a G protein-coupled C1001–9. doi: https://doi.org/10.1152/ajpcell.00092.2016.
receptor-transmembrane protein 16A-voltage-dependent Ca(2+) channel axis [24] Wang Y, Kayoumu A, Lu G, Xu P, Qiu X, Chen L, et al. Experimental models in
and contribute to bronchial hyperresponsiveness in asthma e1211. J Allergy syrian golden hamster replicate human acute pancreatitis. Sci Rep
Clin Immunol 2018;141:1259–68. doi: https://doi.org/10.1016/ 2016;6:28014. doi: https://doi.org/10.1038/srep28014.
j.jaci.2017.05.053. [25] Manohar M, Verma AK, Venkateshaiah SU, Sanders NL, Mishra A. Pathogenic
[10] Caputo A, Caci E, Ferrera L, Pedemonte N, Barsanti C, Sondo E, et al. TMEM16A, mechanisms of pancreatitis. World J Gastrointest Pharmacol Ther
a membrane protein associated with calcium-dependent chloride channel 2017;8:10–25. doi: https://doi.org/10.4292/wjgpt.v8.i1.10.
activity. Science 2008;322:590–4. doi: https://doi.org/10.1126/ [26] Suzuki S, Miyasaka K, Jimi A, Funakoshi A. Induction of acute pancreatitis by
science.1163518. cerulein in human IL-6 gene transgenic mice. Pancreas 2000;21:86–92.
[11] Zhang Y, Wang X, Wang H, Jiao J, Li Y, Fan E, et al. TMEM16A-mediated mucin [27] Jakkampudi A, Jangala R, Reddy BR, Mitnala S, Nageshwar Reddy D, Talukdar R.
secretion in IL-13-induced nasal epithelial cells from chronic rhinosinusitis NF-kappaB in acute pancreatitis: Mechanisms and therapeutic potential.
patients. Allergy Asthma Immunol Res 2015;7:367–75. doi: https://doi.org/ Pancreatology 2016;16:477–88. doi: https://doi.org/10.1016/j.pan.2016.
10.4168/aair.2015.7.4.367. 05.001.
[12] Yang YD, Cho H, Koo JY, Tak MH, Cho Y, Shim WS, et al. TMEM16A confers [28] Xiao Q, Cui Y. Acidic amino acids in the first intracellular loop contribute to
receptor-activated calcium-dependent chloride conductance. Nature voltage- and calcium- dependent gating of anoctamin1/TMEM16A. PLoS ONE
2008;455:1210–5. doi: https://doi.org/10.1038/nature07313. 2014;9:e99376. doi: https://doi.org/10.1371/journal.pone.0099376.
[13] Yokoyama T, Takemoto M, Hirakawa M, Saino T. Different [29] Qin Y, Jiang Y, Sheikh AS, Shen S, Liu J, Jiang D. Interleukin-13 stimulates
immunohistochemical localization for TMEM16A and CFTR in acinar and MUC5AC expression via a STAT6-TMEM16A-ERK1/2 pathway in human airway
ductal cells of rat major salivary glands and exocrine pancreas. Acta Histochem epithelial cells. Int Immunopharmacol 2016;40:106–14. doi: https://doi.org/
2019;121:50–5. doi: https://doi.org/10.1016/j.acthis.2018.10.013. 10.1016/j.intimp.2016.08.033.
[14] Huang F, Rock JR, Harfe BD, Cheng T, Huang X, Jan YN, et al. Studies on [30] Dutta AK, Khimji AK, Kresge C, Bugde A, Dougherty M, Esser V, et al.
expression and function of the TMEM16A calcium-activated chloride channel. Identification and functional characterization of TMEM16A, a Ca2+-activated
Proc Natl Acad Sci USA 2009;106:21413–8. doi: https://doi.org/10.1073/ Cl- channel activated by extracellular nucleotides, in biliary epithelium. J Biol
pnas.0911935106. Chem 2011;286:766–76. doi: https://doi.org/10.1074/jbc.M110.164970.
[15] Crutzen R, Virreira M, Markadieu N, Shlyonsky V, Sener A, Malaisse WJ, et al. [31] Wang H, Yao F, Luo S, Ma K, Liu M, Bai L, et al. A mutual activation loop
Anoctamin 1 (Ano1) is required for glucose-induced membrane potential between the Ca(2+)-activated chloride channel TMEM16A and EGFR/STAT3
oscillations and insulin secretion by murine beta-cells. Pflugers Arch signaling promotes breast cancer tumorigenesis. Cancer Lett 2019;455:48–59.
2016;468:573–91. doi: https://doi.org/10.1007/s00424-015-1758-5. doi: https://doi.org/10.1016/j.canlet.2019.04.027.
[16] Wang J, Haanes KA, Novak I. Purinergic regulation of CFTR and Ca(2+)- [32] Muili KA, Jin S, Orabi AI, Eisses JF, Javed TA, Le T, et al. Pancreatic acinar cell
activated Cl(-) channels and K(+) channels in human pancreatic duct nuclear factor kappaB activation because of bile acid exposure is dependent on
epithelium. Am J Physiol Cell Physiol 2013;304:C673–84. doi: https://doi. calcineurin. J Biol Chem 2013;288:21065–73. doi: https://doi.org/10.1074/jbc.
org/10.1152/ajpcell.00196.2012. M113.471425.
[17] Crottes D, Lin YT, Peters CJ, Gilchrist JM, Wiita AP, Jan YN, et al. TMEM16A [33] Chung WY, Jha A, Ahuja M, Muallem S. Ca(2+) influx at the ER/PM junctions.
controls EGF-induced calcium signaling implicated in pancreatic cancer Cell Calcium 2017;63:29–32. doi: https://doi.org/10.1016/j.ceca.2017.02.009.
prognosis. Proc Natl Acad Sci USA 2019;116:13026–35. doi: https://doi.org/ [34] Benedetto R, Ousingsawat J, Wanitchakool P, Zhang Y, Holtzman MJ, Amaral M,
10.1073/pnas.1900703116. et al. Epithelial chloride transport by CFTR requires TMEM16A. Sci Rep
[18] Ousingsawat J, Martins JR, Schreiber R, Rock JR, Harfe BD, Kunzelmann K. Loss 2017;7:12397. doi: https://doi.org/10.1038/s41598-017-10910-0.
of TMEM16A causes a defect in epithelial Ca2+-dependent chloride transport. J [35] Huang W, Cane MC, Mukherjee R, Szatmary P, Zhang X, Elliott V, et al. Caffeine
Biol Chem 2009;284:28698–703. doi: https://doi.org/10.1074/jbc. protects against experimental acute pancreatitis by inhibition of inositol 1,4,5-
M109.012120. trisphosphate receptor-mediated Ca2+ release. Gut 2017;66:301–13. doi:
[19] Han Y, Shewan AM, Thorn P. HCO3-transport through https://doi.org/10.1136/gutjnl-2015-309363.
anoctamin/transmembrane protein ANO1/TMEM16A in pancreatic acinar [36] Bill A, Hall ML, Borawski J, Hodgson C, Jenkins J, Piechon P, et al. Small
cells regulates luminal pH. J Biol Chem 2016;291:20345–52. doi: https://doi. molecule-facilitated degradation of ANO1 protein: a new targeting approach
org/10.1074/jbc.M116.750224. for anticancer therapeutics. J Biol Chem 2014;289:11029–41. doi: https://doi.
[20] Gerasimenko JV, Peng S, Tsugorka T, Gerasimenko OV. Ca(2+) signalling org/10.1074/jbc.M114.549188.
underlying pancreatitis. Cell Calcium 2018;70:95–101. doi: https://doi.org/ [37] Kulkarni S, Bill A, Godse NR, Khan NI, Kass JI, Steehler K, et al. TMEM16A/ANO1
10.1016/j.ceca.2017.05.010. suppression improves response to antibody-mediated targeted therapy of
[21] Jin X, Shah S, Liu Y, Zhang H, Lees M, Fu Z, et al. Activation of the Cl- channel EGFR and HER2/ERBB2. Genes Chromosom Cancer 2017;56:460–71. doi:
ANO1 by localized calcium signals in nociceptive sensory neurons requires https://doi.org/10.1002/gcc.22450.
coupling with the IP3 receptor. Sci Signal 2013;6:ra73. doi: https://doi.org/ [38] Jiang Y, Yu B, Yang H, Ma T. Shikonin inhibits intestinal calcium-activated
10.1126/scisignal.2004184. chloride channels and prevents rotaviral diarrhea. Front Pharmacol
[22] Cabrita I, Benedetto R, Fonseca A, Wanitchakool P, Sirianant L, Skryabin BV, 2016;7:270. doi: https://doi.org/10.3389/fphar.2016.00270.
et al. Differential effects of anoctamins on intracellular calcium signals. FASEB J [39] Xiong J, Ni J, Hu G, Shen J, Zhao Y, Yang L, et al. Shikonin ameliorates cerulein-
2017;31:2123–34. doi: https://doi.org/10.1096/fj.201600797RR. induced acute pancreatitis in mice. J Ethnopharmacol 2013;145:573–80. doi:
[23] Wang Q, Leo MD, Narayanan D, Kuruvilla KP, Jaggar JH. Local coupling of https://doi.org/10.1016/j.jep.2012.11.032.
TRPC6 to ANO1/TMEM16A channels in smooth muscle cells amplifies