Biochem. J. (2010) 429, 53–61 (Printed in Great Britain)
53
doi:10.1042/BJ20100223
Roles of COMM-domain-containing 1 in stability and recruitment of the
copper-transporting ATPase in a mouse hepatoma cell line
Takamitsu MIYAYAMA*1 , Daisuke HIRAOKA†1 , Fumika KAWAJI†, Emi NAKAMURA†, Noriyuki SUZUKI† and Yasumitsu OGRA*‡2
*Laboratory of Chemical Toxicology and Environmental Health, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan, †Graduate School of Pharmaceutical Sciences,
Chiba University, Chuo, Chiba 260-8675, Japan, and ‡High Technology Research Center, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan
A novel function of COMMD1 {COMM [copper metabolism
MURR1 (mouse U2af1-rs1 region 1)]-domain-containing 1},
a protein relevant to canine copper toxicosis, was examined
in the mouse hepatoma cell line Hepa 1-6 with multidisciplinary techniques consisting of molecular and cellular
biological techniques, speciation and elemental imaging. To
clarify the function of COMMD1, COMMD1-knockdown was
accomplished by introducing siRNA (small interfering RNA) into
the cells. Although COMMD1-knockdown did not affect copper
incorporation, it inhibited copper excretion, resulting in copper
accumulation, which predominantly existed in the form bound to
MT (metallothionein). It is known that the liver copper transporter
Atp7b (ATP-dependent copper transporter 7β), localizes on the
trans-Golgi network membrane under basal copper conditions
and translocates to cytoplasmic vesicles to excrete copper when
its concentration exceeds a certain threshold, with the vesicles
dispersing in the periphery of the cell. COMMD1-knockdown
reduced the expression of Atp7b, and abolished the relocation
of Atp7b back from the periphery to the trans-Golgi network
membrane when the copper concentration was reduced by
treatment with a Cu(I) chelator. The same phenomena were
observed during COMMD1-knockdown when another Atp7b
substrate, cis-diamminedichloroplatinum, and its sequestrator,
glutathione ethylester, were applied. These results suggest that
COMMD1 maintains the amount of Atp7b and facilitates
recruitment of Atp7b from cytoplasmic vesicles to the trans-Golgi
network membrane, i.e. COMMD1 is required to shuttle Atp7b
when the intracellular copper level returns below the threshold.
INTRODUCTION
(Cu transporter) family proteins are also known Cu transporters
[7]. Ctr1-knockout mice are embryonic lethal due to severe Cu
deficiency, and mice bearing conditional knockout in the intestinal
epithelium presented with systemic Cu deficiency [8]. Thus
Ctr1 mediates Cu influx via high affinity uptake on the plasma
membrane. As embryonic fibroblasts established from Ctr1knockout embryos survived in culture, the cells were suggested to
also have a low affinity uptake of Cu capability, in addition to that
provided by Ctr1 [9]. Ctr2, a homologue of Ctr1, is expressed on
the plasma membrane and acts as a transporter with low affinity
uptake of Cu [10]. It was reported previously that Ctr2 is also
expressed on the surface of late endosomes and lysosomes where
it delivers excess Cu into endosomal and lysosomal Cu pools in
order to maintain cellular Cu homoeostasis [11]. The second group
of Cu-regulating proteins consists of intracellular Cu delivery
proteins, or the so-called ‘Cu chaperones’. Cu transported by Ctr1
associates with one of three Cu chaperones, Atox1 (antioxidant
protein 1), Cox17 (cytochrome c oxidase subunit 17) or Ccs
(copper chaperone for superoxide dismutase), to be escorted
to organelles or cuproenzymes. Atox1 hands over Cu to Atp7a
and Atp7b expressed on the surface of Golgi apparatus [12].
Cox17 is required to donate Cu to RYR1 (ryanodine receptor
1) and/or SCO1 (suppressor of cytochrome oxidase deficiency
1), a recipient protein of Cu on the mitochondrial membrane
Copper (Cu) is an essential metal for living organisms that
utilize oxygen for respiration, and is required as a cofactor of
redox-regulating enzymes, such as SOD1 (superoxide dismutase
1), ceruloplasmin, lysyl oxidase, tyrosinase and dopamine βhydroxylase [1,2]. However, the redox-active property of this
metal may have toxic effects on cells due to the generation
of harmful ROS (reactive oxygen species) [3]. Given these
circumstances, it is necessary that cells have a dependable system
for Cu homoeostasis that efficiently distributes this essential metal
to cuproenzymes, thereby avoiding damage to proteins, DNA,
sugars and lipids. In particular, influx, efflux and intracellular
distribution, with fixation of the oxidation state, of Cu are strictly
regulated.
Several groups of Cu-regulating proteins have been identified
in mammalian cells. The first group consists of Cu transporters
that transport Cu across the membrane. These P-type ATPases
Atp7a (ATP-dependent copper transporter 7α) and Atp7b (ATPdependent copper transporter 7β), are expressed on the Golgi
apparatus and participate in the efflux of Cu from cells [4].
Disorders of ATP7A and ATP7B in humans result in Cu
deficiency (Menkes disease and occipital horn syndrome) and
Cu toxicosis (Wilson disease) respectively [5,6]. In addition, Ctr
Key words: ATP-dependent copper transporter 7β (Atp7b),
COMM-domain-containing 1 (COMMD1), copper sensor 1
(CS1), copper transport, metallothionein (MT), speciation.
Abbreviations used: Atox1, antioxidant protein 1; Atp7a, ATP-dependent copper transporter 7α; Atp7b, ATP-dependent copper transporter 7β; BCS,
bathocuproinedisulfonic acid disodium salt; Ccs, copper chaperone for superoxide dismutase; COMMD1, COMM [copper metabolism MURR1 (mouse
U2af1-rs1 region 1)]-domain-containing 1; Cox17, cytochrome c oxidase subunit 17; CS1, copper sensor 1; Ctr, copper transporter; FAB, fast atom
bombardment; GSH-E; GSH ethyl ester; HEK-293T cells, HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus
40); HRP, horseradish peroxidase; ICP-MS, inductively coupled plasma MS; LEC, Long Evans Cinnamon; MT, metallothionein; qRT-PCR, quantitative
real-time PCR; RT, reverse transcriptase; siRNA, small interfering RNA; SOD1, superoxide dismutase 1; TBS-T, Tris-buffer saline containing Tween 20;
TGN, trans -Golgi network.
1
These authors contributed equally to this work.
2
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2010 Biochemical Society
54
T. Miyayama and others
[13]. Ccs transports Cu to SOD1 in the cytosol by forming a
heterodimer between Ccs and SOD1 [14]. The third group consists
of MTs (metallothioneins), which are also suggested to act as
Cu-regulating proteins in cytoplasm. MT actually binds excess
intracellular Cu via Cu-thiolate clusters to mask the toxicity [15].
It has been also suggested that MT alleviates Cu deficiency by
maintaining the intracellular Cu concentration. Thus MT may
play a dual role in Cu homoeostasis in mammalian cells [16].
The fourth group includes a novel type of Cu-regulating protein:
COMMD1 {COMM [copper metabolism MURR1 (mouse
U2af1-rs1 region 1)]-domain-containing 1} [17]. Although it does
not have any apparent Cu(I)-binding motifs in its molecule, unlike
Cu-regulating proteins in the first three groups, it is reported that
COMMD1 binds Cu(II) [18] and is implicated in the Cu efflux
pathway by co-operating with Atp7b [19]. Indeed, the dysfunction
of this protein causes the Cu toxicosis that resulted from abnormal
Cu accumulation in the liver in Bedlington terriers, similar to in
human Wilson disease patients and LEC (Long Evans Cinnamon)
rats with a cinnamon coat colour, which have dysfunctional
ATP7B and Atp7b respectively [20–22]. However, the role of
COMMD1 in the regulation of Cu homoeostasis remains unclear.
In the present study, multi-disciplinary techniques were used
to elucidate the role of COMMD1 in Cu homoeostasis. In
order to reveal the role of COMMD1 in Cu regulation, siRNA
(small interfering RNA) targeting COMMD1 was transfected
into a mouse hepatoma cell line and the effects of COMMD1knockdown on the expression of other Cu-regulating genes were
evaluated. In addition to the determination of gene expression
levels in COMMD1-knockdown cells, the cellular localization of
proteins known to co-operate with COMMD1, i.e. Atp7b, was
also evaluated. In addition, a previously developed Cu-specific
fluorescent imaging probe, CS1 (copper sensor 1) [23,24], was
utilized. CS1 has some valuable properties for imaging of Cu
distribution in cells. Namely, it is applicable to living cells,
has appropriate water solubility and is a ‘turn-on’ fluorescent
sensor with high selectivity and sensitivity to Cu(I). Moreover, the
speciation of Cu in the soluble fraction of cells was determined by
using an hyphenated (hybrid) technique, HPLC–ICP-(inductively
coupled plasma)-MS; as the amount of samples from cultured
cells was too small to analyse by conventional HPLC–ICP-MS,
microHLPC coupled with the ICP-MS system was adopted. This
technique can directly provide information of the intracellular
behaviour of Cu associated with MT, which may play the most
important role in Cu toxicosis [25]. The aim of the present study
was to reveal the role of COMMD1 in Cu homoeostasis using
COMMD1-knockdown cells with multi-disciplinary techniques.
EXPERIMENTAL
Cell culture
The minimum-deviated mouse hepatoma cell line, Hepa 1-6, was
obtained from the RIKEN Cell Bank (Tsukuba, Japan). Hepa 1-6
cells were grown and maintained in DMEM (Dulbecco’s modified
eagle’s medium; Sigma–Aldrich) supplemented with 10 % (v/v)
fetal bovine serum (Wako), 100 units/ml penicillin (Invitrogen)
and 100 μg/ml streptomycin (Invitrogen) at 37 ◦C under a 5 %
CO2 atmosphere. Cells were passaged after reaching confluence
using 0.025 % trypsin/EDTA in PBS.
GCGAAGAUGAGAGGACUUCUCAA-3 ) was selected following general recommendations (Invitrogen) and was designed
from the mouse COMMD1 gene sequence (GenBank® accession
number NM_144514). Hepa 1-6 cells were seeded on to a six-well
plastic plate at 105 cells/well and were pre-incubated for 24 h.
The pre-incubated cells were transfected with 100 nM control
(StealthTM RNAi, negative universal control of medium GC
content; Invitrogen) or COMMD1-targeting siRNA in the medium
optimized for siRNA transfection (Opti-MEM® I; Invitrogen)
containing 1.0 % LipofectamineTM 2000 (Invitrogen) for 24 h.
Determination of Cu concentration
Cells transfected with COMMD1 or control siRNA were either
exposed or not exposed to 10 μM Cu(CH3 COO)2 (Wako) plus
30 μM GSH (Wako) for 24 h. As Cu is transported into cells by
Ctr1 as a monovalent ion, the complex of Cu(I) and GSH was
used. After exposure to Cu, the cells were washed to remove Cu
in the medium and continuously cultured in medium without
Cu addition for another 20 h. Then, the cells were collected and
wet-ashed with analytical grade nitric acid (Wako) to obtain the
samples for Cu determination. Cu concentration was measured
with ICP-MS (in an Agilent 7500ce ICP-MS system) equipped
with a micro concentric nebulizer (Burgener Ari Mist HP) at m/z
65 using a standard addition method.
Preparation of the Cu+ -fluorescent probe CS1 and Cu imaging
The synthesis of CS1 was performed according to the
same procedure as reported previously by Zeng et al.
[23]. Briefly, 8-chloromethyl-2,6-diethyl-4,4-difluoro-1,3,5,7tetramethyl-4-bora-3a,4a-diaza-s-indacene (1) is obtained via
condensation of 2,4-dimethyl-3-ethylpyrrole (Wako) with
chloroacetyl chloride (Wako) followed by treatment with boron
trifluoride diethyl etherate (BF3 · OEt2 ). The Cu+ chelator
moiety is also delivered in two steps. Ethyl 2-hydroxyethyl
sulfide (Wako) was converted into 3-thiapentan-1-thiol, and
this thiol compound was treated with bis(2-chloroethyl)amine
hydrochloride under basic conditions to afford 3,6,12,15-tetrathia9-monoazaheptadecane (2). Coupling of (1) and (2) in refluxing
acetonitrile under basic conditions affords CS1. The structure was
confirmed by FAB (fast atom bombardment)-MS (AX-550 FAB
mass spectrometer; JEOL, Tokyo) and NMR spectroscopy (JEOL
400α 1 H-NMR spectrometer at 400 MHz).
Hepa 1-6 cells were seeded on to a glass-bottomed culture
dish at 5 × 104 cells/dish and were pre-incubated for 24 h.
The pre-incubated cells were transfected with 100 nM siRNA
targeting COMMD1 or control siRNA for 24 h. Then, the
cells were exposed to 10 μM Cu(CH3 COO)2 plus 30 μM GSH
for 24 h and either treated or not treated with 1.0 mM BCS
(bathocuproinedisulfonic acid disodium salt, a Cu(I) chelator;
Wako) for an additional 24 h. The cells were washed and incubated
for further 20 h in fresh medium. After the incubation, the cells
were treated with 2 μM CS1 dissolved in DMSO for 5 min at
37 ◦C, and fluorescence of the Cu(I)–CS1 complex in the cells
was detected by confocal laser microscopy (FluoView FV500
microscope; Olympus) at excitation and emission wavelengths of
543 and 560 nm respectively.
Determination of Cu speciation
Gene knockdown
dsRNAs (double-stranded RNAs) were used as siRNAs (StealthTM
RNAi; Invitrogen). The targeted sequence of COMMD1 (5 -UG
c The Authors Journal compilation c 2010 Biochemical Society
siRNA-transfected cells at 20 h after Cu treatment were collected
and suspended in 10 mM Tris/HCl, pH 7.2. Then, the suspended
cells were disrupted with an ultrasonic homogenizer (Bioruptor®
UCD-200, Cosmo Bio) on ice at 200 W and 20 kHz for three
Roles of COMMD1 in Atp7b function
times of 30 s with 30 s intervals. The cytosolic fraction for Cu
speciation was obtained by ultracentrifugation of the homogenate
at 52 000 rev./min using a S70AT rotor (Hitachi Koki) for 60 min
at 4 ◦C in a 250 μl ultracentrifuge tube (Hitachi Koki). A 5.0 μl
aliquot of the supernatant was applied to a narrow-bore gelfiltration column (Shodex Protein KW802.5-2E, 2.0 mm internal
diameter × 250 mm; Showa Denko), and the column was eluted
with 100 mM ammonium acetate, pH 7.2, at a flow rate of
40 μl/min on a Prominence HPLC system (Shimadzu). The eluate
was introduced into the microconcentric nebulizer of the ICP-MS.
Cu in the eluate was monitored at m/z 65.
qRT-PCR (quantitative real-time PCR) and semi-quantitative RT
(reverse transcriptase)–PCR
Total cellular RNA was extracted from Hepa 1-6 cells using
an RNAqueous® -Micro kit (Ambion). MT-I and MT-II mRNA
expression was determined by qRT-PCR. cDNA was synthesized
from 0.5 μg of total RNA with a QuantiTect reverse transcription
kit (Qiagen). Amplification reactions were performed with
1× ABsoluteTM SYBR® Green Mix (ABgene House), 70 nM
each of forward and reverse primers for MT-I (5 -CACCAGATCTCGGAATGGAC-3 and 5 -AGGAGCAGCAGCTCTTCTTG-3 ) and MT-II (5 -CGCTCCTAGAACTCTTCAAACC3 and 5 -GAGCAGCAGCTTTTCTTGC-3 ), and 50 ng of cDNA.
Samples were analysed in triplicate in a total volume of 50 μl
using an ABI Prism 7000 sequence detection system (Applied
Biosystems). β-Actin (5 -TTCTTTGCAGCTCCTTCGTT-3 and
5 -GAGTCCTTCTGACCCATTCC-3 ) was used as the internal
control for RNA quantity and the efficiency of reverse transcription. The amount of each gene was expressed as the Ct
(threshold cycle) value. The relative RNA expression of each
MT gene to the control was calculated.
The expression levels of COMMD1, Ctr1, Atox1, CCS,
Cox17 and β-actin (as a control) were quantified by
semi-quantitative RT–PCR. A 0.5 μg portion of total RNA
extracted from Hepa 1-6 cells was used in the PrimeScriptTM
one-step RT–PCR kit (Takara Bio) to amplify cDNAs.
Primer sequences for the genes were as follows: Commd1,
5 -CAAGCTGCAGTCATCTCCAA-3 and 5 -GTTGAGTGCCGTGACTGAGA-3 ; Ctr1, 5 -GACCACCTCAGCCTCACACT3 and 5 -GGCATGGAATTGTAGCGAAT-3 ; Atox1, 5 -TCAACAAGCTGGGAGGAGTG-3 and 5 -ACATGGAAGCTTGCAGGGAG-3 ; CCS, 5 -TATCGATGAGGGGGAAGATG3 and 5 -CGCTCCTCCCAGATAGTGAG-3 ; Cox17, 5 -TAGTCGGAGTTTGGGAGCTT-3 and 5 -ATTCACAAAGTAGGCCACCAC-3 ; and β-actin, 5 -GTGGGCCGCTCTAGGCACCAA-3 and 5 -CTCTTTGATGTCACGCACGATTTC-3 . Reactions were run on a Takara Thermal Cycler Personal (Takara)
for one cycle of 50 ◦C for 30 min (reverse transcription) and 94 ◦C
for 2 min (denaturation), followed by 21–28 cycles of 94 ◦C for
30 s, 60 ◦C for 30 s and 72 ◦C for 30 s. The reaction mixture was
run on 5.0 % non-denaturing polyacrylamide gels at 100 V for
50 min. The gels were stained with SYBR-safe DNA gel stain
(Invitrogen) and PCR products were visualized and determined
with a fluorescence image analyser (LAS-1000 plus; FujiFilm).
The relative RNA expression of each gene to the control was
calculated.
55
transfected with COMMD1 siRNA or control siRNA were either
exposed or not to 10 μM Cu(CH3 COO)2 plus 30 μM GSH for
24 h, and then either treated or not treated with BCS, for an
additional 24 h. Cisplatin is a substrate of Atp7b similar to
Cu(I) [26] and in the present experiment, cisplatin was used to
evaluate the effects of COMMD1 dysfunction on Atp7b activity.
Hence, cells that were transfected with COMMD1 siRNA or
control siRNA were either exposed or not to 25 μM cisplatin
(Nippon Kayaku) and then either treated or not treated with
100 μM GSH-E (GSH ethyl ester; Sigma–Aldrich) for 24 h to
sequester cisplatin. Treated Hepa 1-6 cells were rinsed three
times with PBS, fixed with acetone at −20 ◦C for 10 min,
and permeabilized with 0.1 % Triton X-100 (Wako) in PBS
for 5 min at room temperature (26 ◦C). Cells were blocked
with 3 % (w/v) BSA (Sigma–Aldrich) in PBS for 30 min and
incubated with 0.5 μg/ml anti-Atp7b antibody (H-94; Santa
Cruz Biotechnology) in 1 % (w/v) BSA/PBS for 1 h at room
temperature. After discarding the primary antibody solution from
the wells, 0.75 μg/ml FITC-tagged secondary antibody [goat anti(rabbit Ig)–FITC; Chemicon/Millipore] in 1 % BSA/PBS was
applied for 1 h, then 5 μM BODIPY® (boron dipyrromethene)TR ceramide (a Golgi marker; Invitrogen) in 1 % BSA/PBS
was applied for an additional 30 min. After washing out excess
antibodies and marker, cells on the coverslips were mounted
with Crystal Mount (Biomeda) and viewed with a confocal laser
microscope (LSM510, Carl Zeiss).
Western blotting
For the detection of COMMD1 protein, Hepa 1-6 cells were
seeded on a six-well plastic plate at 1 × 105 cells/well and were
pre-incubated for 24 h. The pre-incubated cells were transfected
with 100 nM COMMD1 siRNA or control siRNA for 24 h. For the
detection of the Atp7b protein, Hepa 1-6 cells were treated with
control siRNA, COMMD1 siRNA or Atp7b siRNA at 100 nM
for 24 h. The cells were exposed to 10 μM Cu(CH3 COO)2 plus
30 μM GSH for 24 h after the siRNA transfection. Then, the cells
were treated with 1.0 mM BCS for an additional 24 h.
The cells were lysed in PBS containing 1 % (v/v) Triton X100, 0.1 % SDS, 1 mM EDTA and protease inhibitor cocktail
(Roche) for 1 h on ice. The protein samples were subjected
to SDS/PAGE (12.5 % gels) and transferred on to PVDF
membrane at 20 V for 60 min. The membrane was blocked
overnight with 5 % (w/v) skimmed milk powder in TBS-T
(Tris-buffer saline containing Tween 20; 25 mM Tris/HCl, pH 7.5,
containing 0.9 % NaCl and 0.05 % Tween 20) at 4 ◦C. The
membrane was incubated with a rabbit anti-COMMD1 antibody
(1:1000; Proteintech Group), a rabbit anti-Atp7b antibody (1:1000, Novus Biologicals) or a anti-β-actin antibody
(1:5000) in TBS-T for 1 h, and then washed three times with
TBS-T. The membrane was then incubated with the secondary
antibody (1:2500, GE Healthcare) in TBS-T containing 2 % (w/v)
skimmed milk powder, and washed six times with TBS-T. The
blots were detected with Immobilon Western chemiluminescent
HRP (horseradish peroxidase) substrate (Millipore) by a lumino
image analyser, LAS-1000plus (FujiFilm) according to the
manufacturer’s instructions.
Cytotoxicity
Immunocytochemistry
Hepa 1-6 cells were seeded on to a collagen-coated coverslip,
placed into a six-well plate at 105 cells/well and pre-incubated
for 24 h. The pre-incubated cells were transfected with 100 nM
COMMD1 siRNA or control siRNA for 24 h. Cells that were
Hepa 1-6 cells were seeded on to a 96-well plate at
8 × 103 cells/well and were pre-incubated for 24 h. The preincubated cells were transfected with 100 nM COMMD1 siRNA,
Atp7b siRNA (GenBank® accession number NM_007511:
5 -CGUCUGUCAUGAACCUGCAGCAGAU-3 , as a positive
c The Authors Journal compilation c 2010 Biochemical Society
56
T. Miyayama and others
control) or control siRNA for 24 h. Cells that were transfected
with siRNA were exposed to cisplatin at a concentration of 25 μM
for 24 h. Cytotoxicity was assessed by performing a CellTiter
96® AQueous One solution cell proliferation assay (Promega)
according to the manufacturer’s instructions. The experiments
were repeated four times.
Statistics
Results are presented as means +
− S.D. Statistical analysis
involved one-way ANOVA followed by a Student’s t test. The
level of significant difference was set at P < 0.05 and P < 0.01, as
indicated.
RESULTS
Cu concentration and distribution in Hepa 1-6 cells
COMMD1 mRNA expression was reduced with the introduction
of siRNA targeting COMMD1 to 11.5 % of that with control
siRNA expression in Hepa 1-6 cells 24 h after transfection
(Figure 1A). The knockdown efficacy in the present study was
comparable with that of a previous report using a canine hepatic
cell line [27]. COMMD1 protein expression in Hepa 1-6 cells
was also silenced with the introduction of the siRNA targeting
COMMD1 (Figure 1B). The silencing effects were maintained for
72 h in the COMMD1-knockdown cells. Although the viability
of transfected cells was slightly reduced, to around 90 % that
of non-transfected cells, there were no significant differences
in cytotoxicity between COMMD1 siRNA and control siRNA
transfected cells (results not shown). Thus we used this protocol
to evaluate the effects of COMMD1 knockdown in the present
study.
As COMMD1 has been suggested to play a role in Cu
excretion, Cu concentration in COMMD1 siRNA or control
siRNA transfected cells was determined 20 h after changing the
medium to one that did not contain excess Cu, to evaluate changes
in Cu excretion. We first determined that no significant differences
in basal Cu incorporation were observed between COMMD1 and
control siRNA transfected cells in the absence of Cu (results not
shown); Cu concentration in Hepa 1-6 cells without Cu treatment
5
was 0.98 +
− 0.18 ng/10 cells (Figure 2). At 20 h after treatment
with Cu, the Cu concentration in control siRNA transfected cells
5
was 1.40 +
− 0.64 ng/10 cells, which was not significantly different
from that in Cu-untreated cells, suggesting that the increased
Cu concentration with Cu treatment at 10 μM returned to the
normal physiological level after 20 h. However, Cu concentration
5
in COMMD1-knockdown cells was 6.06 +
− 2.51 ng/10 cells, 4.3fold higher than that in control siRNA transfected cells (Figure 2).
This indicates that COMMD1 contributes to Cu excretion from
the cells.
There were no morphological differences between COMMD1
siRNA and control siRNA transfected cells (Figures 3A–3C).
Fluorescence of the Cu(I)–CS1 complex was primarily localized
in the cytoplasm of Hepa 1-6 cells (Figures 3D–3F). This suggests
that Hepa 1-6 cells accumulate Cu in cytoplasm on COMMD1
knockdown; the fluorescence intensity was significantly increased
by COMMD1 knockdown in Hepa 1-6 cells (compare Figures 3D
and 3E). There are no differences between control cells and BCStreated COMMD1-knockdown cells (compare Figures 3D and
3F). Thus the Cu concentration recovered to normal levels upon
the treatment with the Cu(I) chelator BCS.
Two Cu peaks were found in the elution profile of the cytosolic
fraction of Hepa 1-6 cells (Figure 4A). The peak appearing at
c The Authors Journal compilation c 2010 Biochemical Society
Figure 1
Determination of COMMD1 mRNA and protein expression
(A) The relative level of COMMD1 mRNA expression in COMMD1 siRNA-treated cells compared
with control siRNA-treated cells. Statistical differences between control and COMMD1 siRNA
transfected groups are indicated as **(P < 0.01). Values are expressed as means +
− S.D. for
three independent experiments. (B) Protein levels of COMMD1 in COMMD1 siRNA-treated cells
compared with control siRNA-treated cells at the indicated time points. β-Actin levels are shown
as a loading control.
the retention time of 11.7 min corresponds to Cu/Zn–SOD1 and
that appearing at 13.2 min corresponds to Cu bound to MTs,
based on the chromatographic behaviour as described previously
[25]. In the elution profile of the cytosolic fraction of COMMD1knockdown cells, the peak intensity of Cu bound to MT appearing
at the retention time of 13.2 min was specifically increased,
whereas that of Cu bound to SOD1 was not altered (Figure 4B).
Peak area calculations indicated that approx. 80 % of the Cu
that accumulated in the COMMD1-knockdown cells was of the
species bound to MT.
mRNA expression of Cu-regulating genes in Hepa 1-6 cells
The mRNA expression of two MT isoforms, MT-I and MT-II,
in COMMD1-knockdown cells was significantly increased to
392.7 +
− 33.2 % and 319.0 +
− 23.4 % respectively relative to that
in control siRNA-transfected cells (Figure 5). Although the two
MT isoforms could not be separated by the speciation analysis
(Figure 4) due to structural similarity, mRNA expression suggests
coincidental increases in both MT-I and MT-II protein expression.
Roles of COMMD1 in Atp7b function
57
Figure 2 Intracellular Cu concentration in COMMD1 or control siRNAtreated cells
Cu concentration was measured by ICP-MS using a microconcentric nebulizer at m /z 65 and a
standard addition method. Results are the means +
− S.D. of the intracellular Cu concentration in
cells without siRNA and Cu treatment, with control siRNA and Cu treatment, or with COMMD1
siRNA and Cu treatment as indicated for three independent experiments. *P < 0.05 compared
with the control siRNA-treated groups.
Figure 4 Elution profile of Cu in cell supernatant from COMMD1 or control
siRNA-treated cells
A 5.0-μl aliquot of the supernatants from (A) control siRNA- and (B) COMMD1 siRNA-treated
cells was applied to a narrow-bore gel-filtration column. Cu in the eluate was monitored at m /z
65 (by HPLC–ICP-MS). Peaks labelled with SOD1 and MTs correspond to Cu/Zn-SOD1 and
the two MT isoforms respectively.
Figure 3 Cu imaging in Hepa 1-6 cells treated with COMMD1 siRNA, control
siRNA and BCS
Hepa 1-6 cells were transfected with control siRNA (A and D) or COMMD1 siRNA (B
and E) for 24 h. Then, the cells were exposed to 10 μM Cu(CH3 COO)2 plus 30 μM GSH.
COMMD1-knockdown cells were additionally exposed to BCS (C and F). After the BCS treatment,
the cells were treated with CS1, and the fluorescence of the Cu(I)–CS1 complex in the cells was
detected by confocal microscopy (D, E and F). A differential interference contrast image of the
cells is also presented (A, B and C).
The mRNA expression of the major transporter for Cu
influx, Ctr1, was not altered by COMMD1-knockdown (results
not shown). Similarly, the mRNA expression of Atox1, a Cu
chaperone involved in the transport of Cu from Ctr1 to Atp7b, was
not changed by the knockdown (results not shown). There were no
significant changes in the expression of the other Cu chaperones,
CCS and Cox17, which are involved in the transport of Cu
from Ctr1 to SOD1 and mitochondria respectively, compared
Figure 5
MT mRNA expression in COMMD1 siRNA-treated cells
The mRNA expression of two major MT isoforms, MT-I and MT-II, was determined by
qRT-PCR in COMMD1 siRNA-treated cells and is expressed as the percentage of that in
control siRNA-treated cells (all qRT-PCR results were normalized to the expression of β-actin).
Results are means +
− S.D. for three independent experiments. Statistical differences between
control and COMMD1 siRNA-treated groups are indicated (**P < 0.01).
with the control siRNA treatment (results not shown). These
results suggest that the increase in intracellular Cu concentration
resulting from COMMD1-knockdown induces no changes in the
mRNA expression of Cu-regulating genes except for the MT
isoforms.
c The Authors Journal compilation c 2010 Biochemical Society
58
Figure 6
T. Miyayama and others
Atp7b localization depending on Cu concentration in COMMD1 siRNA- or control siRNA-treated cells
The cells were transfected with siRNA targeting COMMD1 (B, D and F) or control siRNA (A, C and E) for 24 h. Cells transfected with COMMD1 siRNA or control siRNA were either exposed or not
exposed to 10 μM Cu(CH3 COO)2 plus 30 μM GSH, and then either treated or not treated with BCS as indicated. Treated cells were stained with an anti-Atp7b antibody (green) and the Golgi marker,
BODIPY® -TR ceramide (red).
Intracellular localization of Atp7b with Cu and Cu chelator
treatment in Hepa 1-6 cells
Atp7b localized in the perinuclear region in the absence of
Cu and BCS treatment in control siRNA-transfected cells,
and its distribution merged with the Golgi-marker-stained
region, suggesting that Atp7b localized on the TGN (transGolgi network) (Figure 6A). COMMD1-knockdown induced
no apparent changes in Atp7b distribution (Figure 6B). Cu
treatment caused cytoplasmic punctate distributions of Atp7b in
cytoplasm of control siRNA- and COMMD1 siRNA-transfected
cells (Figures 6C and 6D); COMMD1 knockdown did not affect
Atp7b localization (Figure 6D). However, when Cu concentration
was decreased by treatment with the Cu(I)-specific chelator BCS,
Atp7b returned to the perinuclear TGN region in the control
siRNA-transfected cells (Figure 6E), but Atp7b was still seen
in the cytoplasmic vesicles after BCS treatment in COMMD1knockdown cells (Figure 6F). In summary, the decrease in
COMMD1 function did not affect the movement of Atp7b from
TGN to the cytoplasmic vesicles, in order to efflux Cu when
Cu was present in excess, whereas the return of Atp7b from the
cytoplasmic vesicles to the TGN was obstructed by COMMD1
depletion when Cu concentration was decreased back to basal
levels. Atp7b protein expression levels was also determined
by Western blotting; however, the fact that it was not altered by
COMMD1-knockdown (results not shown) suggests that the
reduction of COMMD1 function affected only the return of
the Atp7b to the TGN, resulting in Cu accumulation.
c The Authors Journal compilation c 2010 Biochemical Society
Figure 7
Effect of COMMD1-knockdown on the amount of Atp7b protein
Hepa 1-6 cells were treated with control, COMMD1 or Atp7b (as a positive control) siRNA. The
cells were exposed to 10 μM Cu(CH3 COO)2 plus 30 μM GSH after the siRNA transfection.
Then, the cells were treated with BCS. Cell lysates were used for Western blotting with a rabbit
anti-Atp7b antibody (upper panel) or an anti-β-actin antibody (lower panel).
Effect of COMMD1 knockdown on the Atp7b protein amount and the
Atp7b function
The amount of Atp7b protein in Hepa 1-6 cells was reduced by
Atp7b siRNA treatment (Figure 7, right-hand lane) and therefore
this treatment can be used as a positive control. Similarly, Atp7b
protein expression was also decreased by the COMMD1 siRNA
treatment (Figure 7, centre lane).
The viability of control siRNA-transfected cells was 72.0 %
with cisplatin treatment at 25 μM (Figure 8). The toxic effect
of cisplatin was enhanced (31.0 %) by the knockdown of
Atp7b, which contributes to the efflux of cisplatin. Cells treated
Roles of COMMD1 in Atp7b function
59
efflux cisplatin and the reduction of its function results in an
increase in cisplatin toxicity.
Intracellular localization of Atp7b with cisplatin and cisplatin
chelator treatment in Hepa 1-6 cells
Figure 8 Effect of COMMD1- or Apt7b-knockdown on cytotoxicity induced
by cisplatin
Hepa 1-6 cells transfected with the indicated siRNA were exposed to cisplatin (CDDP) for
24 h. The experiments were repeated four times and results are means +
− S.D. *P < 0.05 and
**P < 0.01 compared with the control.
with COMMD1 siRNA also showed significant decreases in
viability after cisplatin treatment (47.3 %); however, treatment
with COMMD1 siRNA was less effective than treatment with
Atp7b siRNA at this dose. As observed in Figure 6, this suggests
that COMMD1 may be required to recruit Atp7b to efficiently
Figure 9
To determine whether cisplatin toxicity is related to Atp7b redistribution, Atp7b localization was also evaluated by the immunocytochemistry in the COMMD1-knockdown cells. COMMD1knockdown induced no apparent changes in the Atp7b distribution
(Figures 9A and 9B). The cisplatin treatment caused studded
distributions of Atp7b in cytoplasm of both control and COMMD1
siRNA-transfected cells (Figures 9C and 9D). When the cisplatin
concentration was decreased by the treatment with GSH-E, Atp7b
was observed in the perinuclear region in the control siRNAtransfected cells (Figure 9E). On the other hand, Atp7b was still
stained in the cytoplasmic vesicles after the GSH-E treatment
in COMMD1-knockdown cells (Figure 9F). To summarize the
observations, the decrease in COMMD1 function did not affect
the migration of Atp7b from the TGN to the cytoplasmic vesicles
to efflux cisplatin, whereas the recruitment of Atp7b from the cytoplasmic vesicles back to the TGN was obstructed by COMMD1
depletion, when the cisplatin concentration was decreased.
DISCUSSION
It was reported previously that Bedlington terriers with a
deletion in exon 2 of COMMD1 presented with abnormal
Atp7b localization depending on cisplatin concentration in COMMD1 or control siRNA-treated cells
Hepa 1-6 cells were transfected with 100 nM COMMD1-targeting siRNA (B, D and F) or control siRNA (A, C and E) siRNA. Cells transfected with COMMD1 or control siRNA were either exposed or
not exposed to 25 μM cisplatin and then either treated or not treated with 100 μM GSH-E for an additional 24 h, as indicated. Treated cells were stained with an anti-Atp7b antibody (green) and the
Golgi marker, BODIPY® -TR ceramide (red).
c The Authors Journal compilation c 2010 Biochemical Society
60
T. Miyayama and others
hepatic accumulation of Cu resulting from an impediment in
the biliary excretion of Cu [28], and that a canine hepatic cell
line and a human embryonic kidney epithelial cell line with
COMMD1 gene silencing showed increases in intracellular Cu
concentration [27]. Although there was no significant difference in
Cu concentration between control and COMMD1 siRNA-treated
groups immediately after Cu exposure, a significant difference
was observed 20 h after Cu exposure in a mouse hepatoma cell
line (Figure 2). This suggests that COMMD1 may also play a role
in the excretion of Cu into bile in mice.
We observed that the marker Cu(I)–CS1 complex was localized
in the cytoplasm of Hepa 1-6 cells (Figure 3). The speciation study
revealed that upon COMMD1-knockdown approx. 80 % of Cu in
the cell was bound to MT and the rest existed in the insoluble
fraction of cells; such a speciation study can provide quantitative and qualitative results for Cu, as it is limited to Cu in
the soluble fraction. On the other hand, the Cu-specific imaging
can visualize the Cu distribution in the whole cell, including
insoluble fraction despite the technique being less quantitative
than speciation studies. Thus the elemental imaging and speciation
studies are complementary techniques. It was reported previously
that late endosomes and lysosomes act as a Cu pool for the
maintenance of cellular Cu homoeostasis in HEK-293T cells
[HEK (human embryonic kidney)-293 cells expressing the large
T-antigen of SV40 (simian virus 40)], HeLa and U2OS cell lines
[11]. The vesicles, i.e. late endosomes and lysosomes, may be the
candidate locations for the Cu in insoluble fraction of Hepa 1-6
cells. Indeed, recently Cu in vesicles was detected by CS1 [29].
As mentioned above, the Cu accumulated upon COMMD1knockdown was predominantly bound to MT. This phenomenon
is complementary with a previous study revealing that MT
was transcriptionally induced by COMMD1-knockdown [27].
There were no significant changes in the expression of Curegulating genes other than MT. However, it has been shown
that the post-translational regulation of CCS, depends on Cu
concentration [30]. That study revealed that the elevation of Cu
concentration increased the ratio of holoCCS to apoCCS, and that
holoCCS was more efficiently degraded than apoCCS in the 26S
proteosome in order to maintain SOD1 activity and intracellular
Cu concentration. From the results of our speciation analysis in the
present study, the amount of Cu bound to SOD1 was not changed
by COMMD1-knockdown suggesting that the SOD1 activity was
maintained even in COMMD1-knockdown cells. Hence, the posttranslational regulation of CCS may occur in these cells upon
COMMD1-knockdown, and Cu released from degraded holoCCS
may induce MT and be sequestered by MT. However, it is still
unclear how Cu transcriptionally induces MT expression.
Atp7b resides in the TGN membrane under basal Cu
conditions. When intracellular Cu concentration is increased,
Atp7b translocates to intracellular vesicles and the vesicles
move to the cell periphery to excrete Cu [31]. In this Cu
excretion process, performed by Atp7b, it is suggested that
COMMD1 co-operates with Atp7b to facilitate the excretion.
It is known that the translocated Atp7b in the intracellular
vesicles, which occurs upon the increase in Cu concentration, is
relocated back to the TGN membrane after the Cu concentration
returns to the normal physiological level [32]. It was reported
previously that the Cu-dependent translocation of Atp7b is
independent of COMMD1-knockdown in HEK-293T cells and
COMMD1 markedly decreased the stability of newly synthesized
ATP7B [33]. Our observations are consistent with these previous
results (Figures 6A–6D and Figure 7). The results of our
immunocytochemistry showed that the relocation of Atp7b to
the TGN membrane, after the Cu concentration was returned
to normal physiological level with the Cu(I) chelator BCS,
c The Authors Journal compilation c 2010 Biochemical Society
was disrupted by COMMD1-knockdown (Figures 6E and 6F).
Therefore we propose that the role of COMMD1 in the Cu
excretion process, is to facilitate the recruitment of Atp7b from
the vesicle to the TGN membrane, i.e. COMMD1 is required to
shuttle Atp7b after the Cu returns to normal physiological level.
Such continuous translocation and recruitment of Atp7b between
the TGN membrane and the intracellular vesicles may be needed
to efficiently excrete Cu.
It has been reported that the Cu transporters, Ctr1 and Atp7b,
are involved in the transport of cisplatin in cultured cells and
tumour samples [34,35]. Indeed, COMMD1-knockdown reduced
the Atp7b expression and function resulting in an increase in the
intracellular Cu concentration and the cytotoxicity to cisplatin
(Figure 2 and Figure 8). It has been reported that cisplatin is
a substrate of Atp7b [26] and as such triggers the migration of
Atp7b, resulting in the efflux of cisplatin [36]. As a result of the
reduction of the Atp7b-shuttle upon COMMD1 dysfunction, Cu is
unable to be incorporated into the Golgi apparatus, and then the Cu
is sequestered by MT. This may be the reasons why the Bedlington
terriers show similar clinical manifestations to animals bearing an
Atp7b mutation. In fact, the abnormal accumulation of Cu in the
MT-bound form in the liver, the low ceruloplasmin activity in
serum, and the low biliary Cu excretion are clinical symptoms
common to Bedlington terriers, Wilson disease patients, LEC rats
and toxic-milk mice [37].
In conclusion, a novel function of COMMD1, a protein relevant
to canine Cu toxicosis, has been proposed. Although COMMD1knockdown did not affect Cu incorporation, it inhibited Cu
excretion, resulting in Cu accumulation. Cu accumulating in the
cells predominantly existed in the form bound to MT. Moreover,
COMMD1-knockdown reduced the amount of Atp7b protein
and abolished the relocation of Atp7b from the periphery of
the cell to the TGN membrane when the concentrations of the
substrates of Atp7b, such as Cu(I) and cisplatin, returned to normal
or physiological levels (Figures 3D, 3E and 6). These results
suggest that COMMD1 has a dual function in the interaction
with Atp7b. Namely, COMMD1 maintains the amount of Atp7b
protein and facilitates recruitment of Atp7b from the vesicle to the
TGN membrane, i.e. COMMD1 is required to shuttle Atp7b. In
addition, multiple functions of COMMD1 have been suggested:
it has been implicated in the inhibition of NF-κB (nuclear factor
κB) [38] or the interaction with XIAP (X-linked inhibitor of
apoptosis) [39], a suppressor of apoptosis. Hence further studies
on COMMD1 will provide valuable and novel insights into Cu
homoeostasis and cell metabolism regulated by Cu.
AUTHOR CONTRIBUTION
All experiments were conceived and designed by Takamitsu Miyayama, Daisuke Hiraoka
and Yasumitsu Ogra. The experiments using the ICP-MS and confocal laser microscopy
were performed by Takamitsu Miyayama, Daisuke Hiraoka and Fumika Kawaji. Biochemical
experiments were performed by Daisuke Hiraoka, Takamitsu Miyayama and Fumika Kawaji.
The experiments using cisplatin were performed by Takamitsu Miyayama and Daisuke
Hiraoka. Synthesis of the Cu(I)-specific probe was carried out by Emi Nakamura and
Noriyuki Suzuki. The paper was prepared by Yasumitsu Ogra with input from Takamitsu
Miyayama, Daisuke Hiraoka and Noriyuki Suzuki.
ACKNOWLEDGEMENTS
The authors thank Showa Denko for providing the narrow-bore column and the HPLC
system.
FUNDING
This work was supported by a Grants-in-Aid from the Ministry of Education, Culture,
Sports, Science and Technology, Japan [grant numbers 09J04232 (to T.M.) and 19390033
to Y.O.)]; and by the Agilent Technologies Foundation, U.S.A.
Roles of COMMD1 in Atp7b function
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Received 9 February 2010/6 April 2010; accepted 30 April 2010
Published as BJ Immediate Publication 30 April 2010, doi:10.1042/BJ20100223
c The Authors Journal compilation c 2010 Biochemical Society