Knockdown and Effect of Lactational Hormones
on CTR-1 and Ceruloplasmin
Michael Wan
Department of Chemistry and Biochemistry
California State University Fullerton
Abstract
A series of experiments were performed to understand what mediates the transport of
copper into and across the mammary gland during lactation. This was achieved by
attempting to determine 1) the effects of lactational hormones (prolactin, insulin, and
dexamethazone) on the expression of the copper transporter CTR1 and on the production
and secretion of ceruloplasmin by mammary epithelial cells 2) the effects of knocking
down CTR1 expression on uptake of copper by mammary epithelial cells. Copper uptake
studies were performed with radioactive 64 Cu introduced into PMC42 cells. SDS PAGE
and Western Blotting allowed us to characterize CTR1 and ceruloplasmin expression
while Real Time PCR allowed us to quantify the expression of mRNA. The results
suggest: 1) CTR1 may not be involved in copper uptake; 2) lactational hormones appear
to increase the expression of CTR1 at the protein level. The effects of lactational
hormones on ceruloplasmin at the protein level and CTR1 at the mRNA level remain
unclear and require further investigation.
Introduction
Copper is an essential nutrient for that is required in the growth and development
of all living organisms. In mammals, copper is absorbed primarily in the small intestine
via CTR1 (Linder, Azam, 1996). CTR1 is a transmembrane protein/channel that is
responsible for cellular copper uptake and transport of copper across the plasma
membrane (Sinani et al., 2007), (Nose 2006). It exists as a trimer (105 kDa) and is highly
specific for Copper (Guo et al., 2004). Like all essential metals, copper is potentially
toxic at increased concentrations. Thus, homeostatic mechanisms have evolved to avoid
copper toxicity while providing sufficient copper for metabolic requirements. As copper
enters the blood, it is immediately bound to albumin and transcuprein - blood plasma
proteins (Linder, Azam, 1996). Copper bound to the plasma proteins is then transported
and deposited to organs, namely the liver. In the liver, copper is incorporated into
ceruloplasmin and secreted back into the blood bound to ceruloplasmin where it is then
ready for distribution to target organs.
Ceruloplasmin is one of the major copper transporting proteins and holds
the majority of copper (90-95%) in human blood plasma, conducting most of the delivery
to tissues in the body (Takahashi et al., 1984). It is an enzyme synthesized in the liver
containing 6 atoms of copper bound tightly at defined sites in its structure. It has a
molecular weight of approximately 132 kDa, is composed of 1046 amino acids.
Ceruloplasmin binds copper in the liver, and traffics it to necessary tissues and organs in
the body. During lactation, about 50% of copper ions entering the blood are transported
to the mammary glands (in rats) where it crosses the mammary epithelial cells and enters
the milk (Linder et al., 1998). Although it is still unclear how copper is donated to CTR1,
ceruloplasmin is suspected to be the copper donating protein (Zatulovsky et al., 2007).
Decreased serum ceruloplasmin levels are characteristic in the genetic disorder Wilson
disease, also known as hepatolenticular degeneration (Scheinberg, Gitlin, 1952). There is
a disruption of normal copper metabolism and copper deposition in tissues (Gitlin, 1975).
To study the copper components in milk we used a mammary epithelial
cell model, PMC-42 cells, grown on a Transwell membrane as a monolayer with tight
junctions. PMC-42 cells are very similar to normal breast epithelial tissue in cell
composition and physiological responses (Krishnan, Cleary 1990). They are polarized
and mimic the basal and apical sides of mammary epithelial cells. The basal (blood) side
is where copper is delivered on different plasma proteins, such as albumin and
transcuprein (Git et al., 2008). Medium is collected on the apical, or milk secretion side
and we analyze the secretions. Mammary explants obtained from virgin female mice
were also used as a model system to test for the effect of lactational hormone on CTR1
and ceruloplasmin. Based on findings from Whitehead et al., “[PMC42] cells were shown
to respond to hormonal stimuli in a manner similar to breast epithelial tissue, such as the
appearance of lipid droplets upon treatment with prolactin or stimulation of growth by
hydrocortisone, in marked contrast to other breast tumor cell lines inhibited by this
glucocorticoid” (Whitehead et al., 1984). Culture media were also analyzed after 48 and
96 hours post-incubation.
Methods
PMC 42 cell culture / Copper transport study
PMC 42 cells were trypsinized and 100 ul of siCTR and non-targeting siRNA (as
negative control) were added onto Transwells treated with Matrigel for cell polarization.
The cells were incubated with RPMI containing 10% Fetal Bovine Serum at 37 C.
Radioactive 64 Cu (5uM)–His (50uM) was delivered on the basal side 48-90h post
transfection with siRNAs,. Apical and cellular secretions were counted with gamma
counter.
Quantifying expression of CTR1 mRNA
CTR1 mRNA was isolated using RNABee solution and performing phenol chloroform
extraction. The supernatant aqueous layer was collected, and isopropanol was added to it
and allowed to sit overnight. cDNA was synthesized by mixing isolated RNA with
reverse transcriptase, deoxynucleoside triphosphates, and random primer. The expression
of CTR1 was quantified by performing Real Time PCR (50 cycles in triplicates with a
fluorescent reporter probe), relating the expression to 18S ribosomal RNA as an internal
control.
Determining protein expression of CTR1knockdown
siCTR (100nM) cells on the apical membrane were cut out of the Transwells. 500 ul of
lysis buffer (10mM tris, 2% SDS, pH 7.2) was added, and the samples were incubated
with shaking for 5 minutes. 5 ul 4x sample treatment buffer was mixed with 20 ul of
sample and then loaded into 12.5% acrylamide gel. Gel electrophoresis was performed
with 25 ul loaded in each well, and the gel was transferred onto a membrane with transfer
buffer (192 mM glycine, 25 mM Tris base, 150ml MeOH, 0.375g SDS, ddH2O). The
membrane was blocked overnight with 5% dry milk (1g). 2 ul of CTR1 primary antibody
(rabbit anti-CTR1 from Jack Kaplan) was added afterwards and left to shake over night.
It was then washed with TTBS 3 times (100ml 1x TBS + 100µL Tween 20) and 2 ul of
CTR1 secondary antibody (Sigma monoclonol antirabbit antibody produced in mouse)
was added and allowed to shake overnight. The membrane was washed twice with 1x
TTBS. The membrane was then placed in developing solution [50 ml development buffer
(pH 9.4), 500 ul BCIP + 500 ul NBT] in the dark and allowed to develop. After bands
appeared, the membrane was placed in ddH2O.
Mammary Explants
Mammary tissue from virgin female mice (6-8 weeks old) was harvested and sliced. They
were then grown on wells plated with collagen and immersed in DMEM medium with
and without lactational hormones (insulin, prolactin, dexamethazone). The wells were
then incubated at 37 C for 96 hours. Secretions were collected from the apical side at 48
hours and 96 hours after incubation. Tissues that had been grown were weighed, and a
40% solution was made for each sample with SDS tank buffer (18g Tris base, 86.4g
glycine, 60 ml 10% SDS, 30 ml 2% NaN3, H2O). The samples were then homogenized
to prepare for gel electrophoresis. The concentrations of secretions were measured based
on the Bradford protein assay. 200 ul of Bradford reagent was combined with water and
BSA in multiples of 10 ul beginning at 0 ul and ending at 70 ul. The absorbance values at
595 nm were obtained with a spectrophotometer, and a standard curve was generated
based on the known concentrations of BSA. 50 ul of each sample was combined with 750
ul of water and 200 ul of Bradford reagent, where the absorbance at 595 nm was taken.
Based on the absorbance values plotted against the standard curve, the concentrations
were determined.
Determining protein expression of CTR1 and CP from mammary explants
5 ul 4x sample treatment buffer was mixed with 20 ul of sample (either from mammary
tissue or secretions) and then loaded into 12.5% acrylamide gel (7% for ceruloplasmin).
Gel electrophoresis was performed with 25 ul loaded in each well, and the gel was
transferred onto a membrane with transfer buffer (192 mM glycine, 25 mM Tris base,
150ml MeOH, 0.375g SDS, ddH2O). The membrane was blocked overnight with 5% dry
milk (1g). Depending on the study, 2 ul of CTR1 primary antibody (rabbit anti-CTR1
from Jack Kaplan) or 2 ul of CP primary antibody (Biomatic mouse CP AB) was added
afterwards and left to shake over night. It was then washed with TTBS 3 times (100ml 1x
TBS + 100µL Tween 20) and 2 ul of CTR1 secondary antibody (Sigma monoclonol
antirabbit antibody produced in mouse) was added and allowed to shake overnight. The
membrane was washed twice with 1x TTBS. The membrane was then placed in
developing solution [50 ml development buffer (pH 9.4), 500 ul BCIP + 500 ul NBT] in
the dark and allowed to develop. After bands appeared, the membrane was placed in
ddH2O.
Results
CTR1 Knockdown: Effectiveness and Effect on Copper Uptake from Cu-His
The CTR1 gene was knocked down using siRNA specific for CTR1 and compared to
treatment with non-specific siRNA. We were able to study the expression of CTR1 at the
mRNA and protein level as well as the effect of CTR1 on copper uptake by mammary
epithelial cells.
Figure 1
Relative expression of CTR1 mRNA after treatment of PMC42 cell monolayers with non-specific and CTR1-specific siRNA. The
data represent means +/- SD for 5-6 determinations. Knockdown was with siCTR-1 (100nM). There was no statistically significant
difference between the negative control and siCTR1. There was also no CTR1 knockdown at the mRNA level.
Figure 2
1
2
3
siNeg
4
5
6
7
siCTR
Samples from above were loaded onto a gel and transferred to a membrane in order to detect and observe protein expression. Negative
control samples were loaded into lanes 2, 3, 4, and CTR knockdown samples were loaded in lanes 5, 6, 7. There was no significant
difference in the amount of CTR1 protein expression.
Figure 3
300000
250000
200000
siCTR
siNEG
150000
100000
50000
0
Apical
Cellular
Effect of CTR1 Knockdown: Copper uptake by PMC42 cell monolayers, in cells where CTR1 mRNA knockdown was successful
(comparing siRNA negative cells - purple; with CTR1 siRNA treated cells - blue). No differences in uptake of 64Cu into cells or
transfer to the apical medium were observed.
Effect of Lactational Hormones on CTR1 mRNA and Protein Expression
mRNA expression of CTR1
Real Time PCR was performed on mouse mammary explants in order to observe the
mRNA expression of CTR1. The amount of mRNA present in samples treated with and
without lactational hormones allowed us to determine if CTR1 responded to the
treatment.
Figure 4
Effect of lactational hormones on CTR-1 in mouse mammary explants
2.5
Relative CTR-1 mRNA
2.0
1.5
1.0
0.5
0.0
Non-Hormone
Hormone
Effect of lactational hormones on CTR1 mRNA expression in mouse mammary explants based upon data
from Real Time -PCR. The results did not show any statistical difference in CTR1 mRNA expression in the
presence of lactational hormones.
Figure 5
0.9
0.8
Relative CTR-1 mRNA
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Non-hormone
-0.1
Hormone
Another set of data using Real Time PCR on CTR1 in mouse mammary explants to observe the effect of
lactational hormones on CTR1 mRNA. We saw a decrease in CTR1 expression at the mRNA level due to
hormone treatment.
Protein expression of CTR1
Western blotting was performed in order to see if there was increased protein expression
of CTR1 in the presence of lactational hormones. The amount of CTR1 protein
expression reflects the effect that lactational hormones on it.
Figure 6
Mouse
4
-
+
Mouse Mouse Mouse
3
2
1
-
+
-
+
-
+
106
81
48
36
28
21
Western blot attempting to observe difference in CTR1 protein expression in mouse mammary explants,
comparing treatment with (+) and without (-) lactational hormones in four different mice. The results were
inconclusive as it is somewhat difficult to compare the bands. The bands were approximately where the
CTR1 trimer size should be (105 kDa).
Figure 7
Mouse
2
+
Mouse
1
-
+
106
81
48
36
28
21
CTR1 protein detection and expression through western blotting reveals that there is increased CTR1
protein expression in mouse mammary explants when treated with lactational hormonal, compared to nonhormonal treatment in two mice. CTR1 was once again detected by bands at the 105 kDa mark, where the
trimer should exist at. This replication saw obvious increase in CTR1 expression with hormonal treatment.
Effect of Lactational Hormones on Ceruloplasmin Protein Expression
Figure 8
9
8
7
6
5
4
3
2
1
202
120
Lane
1
2
3
4
5
6
7
8
9
Sample
1:10 dilution of mouse plasma as control
Mouse A 48 hour treatment without hormone
Mouse A 48 hour treatment with hormone
Mouse B 48 hour treatment without hormone
Mouse B 48 hour treatment with hormone
Mouse A 96 hour treatment without hormone
Mouse A 96 hour treatment with hormone
Mouse B 96 hour treatment without hormone
Mouse B 96 hour treatment with hormone
98
49
Western blot for ceruloplasmin in mouse mammary explant secretions. The desired band
size of 130 kDa was not present. Non-specific bands (probably albumin) were detected
after long exposure to substrate.
Discussion
Our findings have shown that there was no statistically significant difference in
the expression of CTR1 mRNA treated with siCTR1 compared with the nonspecific
siRNA, indicating no CTR1 knockdown. Likewise, there was no significant difference in
the amount of CTR1 protein expression based on our immunoblot. There was also no
difference in uptake by PMC42 cells with decreased CTR1 expression (successful
knockdown). This suggests that CTR1 may not be involved in copper uptake. However,
data from other studies show otherwise. Findings demonstrated that there was increased
copper uptake and accumulation with increased expression of mouse CTR1 (Lee et al.,
2001), which suggests that CTR1 functions as a Cu transporter in mammals and is the
“limiting molecule for the Cu uptake system”. Further reinforcement for CTR1 being
involved in copper uptake was shown in a similar study by the overexpression of CTR1
in which increased copper uptake was observed and also named CTR1 “the limiting
factor in copper uptake” (Dancis et al., 1994).
Our data also shows that although lactational hormones appear to increase the
expression of CTR1 at the protein level, there is conflicted data with regards to the
expression at the mRNA level. Studies performed by Kelleher & Lönnerdal suggest that
elevated amounts of CTR1 was not affected by short term hormonal (prolactin) treatment
in the mammary epithelial cells, but rather by proteosomal degradation (Petris et al.,
2002).
Our data on the expression of mouse ceruloplasmin in the presence of lactational
hormones were also inconclusive and appear to be the first to be tested. Ceruloplasmin
was not detected by immunoblotting. Non-specific bands, however, were detected. We
assume the band to be albumin, which has a size of 65 kDa. Further testing is required.
Conclusion
Overall, the experiment was a success in that the effects on CTR1 and
ceruloplasmin were observed in the presence of lactational hormones. Although results
regarding the effects of lactational hormones on ceruloplasmin at the protein level and
CTR1 at the mRNA level remain unclear and require further investigation, it leaves room
to refine the techniques used and open other possibilities for future work such as
quantifying ceruloplasmin in the mammary tissue and further knockdown studies.
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