(Received for publication, July 25, 1994; and in revised form, December 26, 1994)
From the
Immortalized hepatic cell lines obtained from laboratory animals or patients with defects in copper metabolism in the liver provide new approaches to examine related metabolism and toxicity. We established a series of hepatic cell lines from the liver of Long-Evans Cinnamon (LEC) rats, using recombinant adenovirus which expresses SV40 large T. Cells from the LEC rats were cultured and accumulated larger amounts of copper than did control cells, when the concentrations of copper in the culture medium exceeded 5 µM. The secretion of ceruloplasmin (CP) from the cultured cells was not reduced in hepatocytes from LEC cells, as compared with the control cells. As accumulation of copper did not affect CP secretion, CP production was not likely to be affected by the accumulation of copper in LEC rat hepatocytes. The production of holo-CP was further investigated by transfection of human CP cDNA and detection of human holo-CP by immunological procedures and use of a monoclonal antibody (mAb CP60) which recognizes human holo-CP but not human apo-CP and rat CP. Hepatocytes from the LEC rats processed and secreted holo-CP into the medium, even with excess copper present in the medium. These observations suggest that the genetic defect in LEC rats did not alter biosynthetic and secretory pathways of CP and that the intracellular copper concentration did not regulate the synthesis and processing of CP in the cultured hepatocytes. Low ceruloplasmin levels are observed in most, but not all, patients with Wilson disease, as well as in LEC rats. Our results do suggest that the copper transporting ATPase encoded in the Wilson disease gene is not a integral part of the biochemical mechanism of copper incorporation into apoprotein. The cell lines and immunological procedures we used are expected to add to information on biologically important process related to copper metabolism and to CP biosynthesis.
Ceruloplasmin (CP) ()is a blue-copper oxidase found
in theserum of all vertebrate species where it accounts for greater
than 95% of circulating plasma copper(1, 2) . The
function of the holoprotein includes roles in copper transport, iron
metabolism, antioxidant defense, tissue angiogenesis,
coagulation(3, 4, 5, 6, 7) ,
and oxidation of LDL (8) . Properties of the protein have been
studied extensively(9, 10, 11) . However,
little is known of the mechanisms by which the expression of this gene
is regulated.
Low levels of serum CP have been observed in neonates, cases of nutritional copper deficiency, Menkes disease, toxic milk mice, and severe liver diseases(12) , as well as Wilson disease (13) and LEC rats (14) . A large amount of copper accumulates in the liver of LEC rats, and most of the animals die due to liver failure(15) . Accumulation of copper in the liver is associated with low levels of CP in serum and with increased levels of metallothionein in the liver(16) . Genetic analyses revealed that these traits are inherited in an autosomal recessive manner. Cox and colleagues (17) obtained evidence that the LEC rat has a mutation in the Wilson disease gene. Accordingly, studies on copper metabolism in the rat need to be done to determine the biochemistry related to the hepatotoxicity of copper, the effect of chelating agents on the development of hepatocyte injury by the metal, the role of metallothioneins in hepatocytes, and synthesis and secretion of CP by hepatocytes, etc.
Cultured mammalian cell lines or primary cultured hepatocytes are useful to study copper uptake(18) , toxicity and resistance(19) , and the effect of copper on cellular metabolism(20) . Immortalized or established hepatic cell lines obtained from laboratory animals or patients with defects in copper metabolism of the liver are expected to provide new approaches for studying copper metabolism and toxicity in hepatocytes. We established a series of hepatic cell lines from the liver of LEC rats, and we examined the biological processes related to copper metabolism and CP biosynthesis in the hepatocytes. We obtained evidence that copper accumulates in long-term cultured hepatocytes from LEC rats; however, the secretion of CP and the biosynthesis of holo-CP from apo-CP is not affected in these cells, regardless of their copper content.
All clones of cultured hepatocytes secreted CP, and there were no differences between the LEC and LEA hepatocytes in amounts of CP secreted into the culture medium (Table 1). The amount of rat holo-CP in the cultured medium was estimated, using SDS-PAGE and immune blot, according to the description of Sato and Gitlin (34) and Yamada et al.(35) . Ceruloplasmin secreted into the culture medium by LEC and LEA hepatocytes was purified by immune precipitation and analyzed by SDS-PAGE, with or without heat treatment. Both LEC (Fig. 1, lanes 3 and 5) and LEA (Fig. 1, lanes 2 and 4) hepatocytes secreted holo-CP and apo-CP. The ratio of apo- to holo-CP in culture medium from LEC hepatocytes was similar to that from LEA hepatocytes. Rates of synthesis and secretion of CP were assessed by metabolic labeling of the cells followed by immune precipitation, SDS-PAGE, and autoradiogram analysis of CP associated with cells and present in culture medium (Fig. 2). However, no differences were detected between the hepatocytes from LEC rats and LEA rats. Elution profiles from the Mono Q column (Pharmacia Biotech Inc.) of CP in medium in LEC and LEA hepatocytes could not be distinguished (data not shown).
Figure 1:
Immune blot analysis of rat
ceruloplasmin secreted by the long-term cultured hepatocytes from LEC
and LEA rats. Long-term cultured hepatocytes from LEC and LEA rats were
maintained at confluence on 25-cm flasks containing
complete Williams' medium E at 37 °C in a 5% CO
environment. The confluent monolayers were rinsed three times
with PBS and incubated for 24 h with Williams' medium E in the
absence of fetal calf serum at 37 °C in 5% CO
. After
the incubation, cell culture medium (5 ml) was removed, centrifuged at
1000
g, and incubated with Sepharose 4B containing
anti-rat rabbit IgG at 37 °C for 60 min in an end to end rotor. The
gel suspension was centrifuged at 4 °C for 5 min at 1000
g. The gel was washed three times with ice-cold PBS and mixed
with an equal volume of 2
sample buffer. An aliquot of
suspension was subjected to electrophoresis on 7.5% polyacrylamide gels
containing 0.1% SDS, with or without prior heat treatment at 100 °C
for 3 min. Proteins were transferred onto nitrocellulose filters at 4
°C for 8 h at 40 V in 20 mM Tris-HCl and 200 mM glycine, pH 8.3, and CP was detected by anti-rat CP IgG. Apo-CP is
indicated by a black arrow, and holo-CP was indicated by a white arrow. Lane 1, purified rat CP with heat treatment; lane 2, culture medium from LEA hepatocytes with heat
treatment; lane 3, culture medium from LEC hepatocytes with
heat treatment; lane 4, culture medium from LEA hepatocytes
without heat treatment; lane 5, culture medium from LEC
hepatocytes without heat treatment.
Figure 2:
Pulse-labeling of biosynthesized
ceruloplasmin from long-term cultured hepatocytes with
[S]methionine. Long-term cultured hepatocytes
from LEC and LEA rats were maintained at confluence on 6-well tissue
culture plates containing complete Williams' medium E at 37
°C in 5% CO
. The monolayers were rinsed three times
with PBS and incubated for 60 min in methionine-free Dulbecco's
modified Eagle's medium at 37 °C in 5% CO
, then
the monolayers were incubated with 2.56 MBq/ml
[
S]methionine for 10 min and chased for 0, 15,
30, 60, 120, and 180 min with 1000-fold excess L-methionine.
Culture medium (1 ml) was removed and mixed with 100 µl of PBS
containing 1% Tween 20, 4% bovine serum albumin. The monolayers were
washed three times with ice-cold PBS and collected by a rubber
policeman. Cells were suspended in ice cold PBS in the presence of 1
mM phenylmethylsulfonyl fluoride, 0.1% SDS, 0.1% Triton X-100,
0.4% bovine serum albumin, and lysed by freeze-thawing. The cell
culture medium and the lysate were centrifuged at 1000
g for 5 min. The supernatants were incubated with Sepharose 4B
containing anti-rat CP rabbit IgG at 37 °C for 60 min.
Immunoprecipitates from the cell lysates or culture medium were
analyzed by SDS-PAGE and fluorography, as described(50) . A, culture medium from LEA hepatocytes; B, culture
medium from LEC hepatocytes; C, cell lysate of LEA
hepatocytes; D, cell lysate of LEC
hepatocytes.
Figure 3:
Accumulation of copper in the long-term
cultured hepatocytes from LEC and LEA rats. The long-term cultured
hepatocytes from LEC and LEA rats were maintained at confluence on
24-well tissue culture plates containing complete Williams'
medium E at 37 °C in 5% CO. The monolayers were rinsed
three times with PBS and incubated for 24 to 120 h with various
concentrations of copper in complete Williams' medium E at 37
°C in 5% CO
. The monolayers were rinsed three times
with PBS, and cells were lysed with 100 µl of 1 N NaOH.
The cell lysates were dissolved in 900 µl of distilled water.
Amounts of copper in the lysates were determined, as described under
``Experimental Procedures.'' Amounts of copper in all lysates
from LEC rats (open circles) and LEA rats (open
squares) were plotted. A, amounts of copper in cells
exposed to increasing concentrations of copper in the medium after 72 h
of culture; B, amounts of copper in cells after various times
of incubation in the presence of 80 µM of copper. Four
different clones from LEC rat and LEA rat, respectively, were analyzed.
The value represents mean ± S.D. obtained from six wells from
the same cell line (the vertical bar represents 1 S.D.). The
values from one clone are linked by solid lines (LEC) or by dotted lines (LEA).
Thus, copper accumulated in both LEA and LEC cells; however, contents of copper in the latter were significantly higher in the former, regardless of copper concentration (5 to 80 µM) or time of exposure (24 to 120 h). The mean copper content of LEC cells of different clones (54.23 ± 8.79 nmol/mg of protein, n = 25) was statistically significantly higher than that of LEA cells (13.98 ± 5.23 nmol/mg of protein, n = 20) (p = 0.0001).
The toxicity of copper was examined by culturing the cells with increasing concentrations of copper in culture medium and the subsequent count of viable cells. The viability of the LEC cells did not differ from that of LEA cells until the copper concentration in the medium increased to 6.4 mM (data not shown). Concentrations of copper-histidine never exceeded 6.4 mM, in any experiment.
Effect of copper on CP synthesis was examined by culturing the cells with increasing concentrations of copper and the subsequent measurement of CP in medium. The amounts of CP produced by the cultured cells from LEC and LEA rats remained unchanged regardless of the concentrations of copper in the medium (Table 1).
Figure 4: Immune blot analysis of human ceruloplasmin secreted by long-term cultured hepatocytes transfected with human ceruloplasmin cDNA. The expression vector containing human CP cDNA was introduced into long-term cultured hepatocytes using Lipofectin, as described under ``Experimental Procedures.'' The transfected cells were cultured in complete Williams' medium E in the presence of 360 µM G418 for 2 weeks. Individual colonies in monolayer cultures were manually removed and transferred to 24-well plates. The culture medium (1 ml) was removed and incubated at 37 °C for 60 min with Sepharose 4B containing mAb CP60. The gels were treated as described in the legend in Fig. 2. The CP was detected by anti-human CP goat IgG. Lane 1, culture medium from transfected hepatocytes from LEA rats, without heat treatment; lane 2, culture medium from transfected hepatocytes from LEA rats with heat treatment; lane 3, culture medium from transfected hepatocytes from LEA rats without heat treatment; lane 4, culture medium from transfected hepatocytes from LEC rats with heat treatment; lane 5, culture medium from HepG2 cells without heat treatment; lane 6, culture medium from HepG2 cells with heat treatment. Apo-CP is indicated by a black arrow, and holo-CP is indicated by a white arrow.
Quantitative assay of human holo-CP by EIA showed that the ratios of holo- to apo-CP in cultured medium obtained from these transfected cells were variable and depended on the clones; however, there were no differences in amounts of holo-CP secreted between cells from the LEC and LEA rats (Table 1).
When the transfected and cloned cells were cultured for 72 h in complete Williams' medium E containing 80 µM copper, cultured hepatocytes from LEC rats contained more copper (58.97 ± 10.31 nmol/mg of protein, n = 12) than did these cells from LEA rats (17.70 ± 10.08 nmol/mg of protein, n = 8), an event noted in the previous experiment on LEC and LEA hepatocytes without transfection. However, the production of human holo-CP was not altered by changes in copper concentrations in the medium (Table 1). These results indicate that the content of intracellular copper did not alter the processing of exogenously expressed CP.
Cultured hepatocytes from LEC cells accumulate larger amounts of copper than do the LEA rat cells. Loading of cultured hepatocytes from both LEC and LEA rats with copper provides hepatocytes with excess amounts of copper, and the amounts of copper in hepatocytes from LEC rats are similar to those observed in vivo(15) . Under these conditions, the amounts of rat CP secreted by the cultured cells do not change. It has been shown that CP gene expression is not regulated by the concentration of intracellular copper in copper-deficient rats(36) , neonates of toxic milk mice, and adult toxic milk mice(37) . Studies on primary cultured rat hepatocytes demonstrated that copper content in the cells has no effect on CP gene expression or on CP biosynthesis(36) . In addition in primary cultured hepatocytes from mice, CP production was not regulated by intracellular copper levels(38) . Our investigation on intrinsic CP production indicated that the production of CP by the cultured hepatocytes from LEC and LEA rats was not influenced by intracellular copper contents. Therefore, expression of the CP gene may not have been altered by the increase of intracellular content of copper in hepatocytes from LEC rats. Previous studies on LEC rats in vivo showed that the levels of CP mRNA in the liver were the same as those in LEA rats(39) . It seems likely that the CP gene expression in LEC rat cells as well as in LEA rats is not regulated by the intracellular copper concentration. These features are different in that intracellular iron concentrations regulate levels of ferritin and transferrin receptor, transcriptionally and post-transcriptionally(40) . Transferrin expression may be regulated in a similar manner(41) . Serum transferrin is a protein analogous to CP and is reduced in the presence of hemochromatosis (42) .
The gene for Wilson disease has been identified(43, 44, 45) . This gene in humans encodes a predicted 1,411 amino acid protein(43) , a member of a cation-transporting P-type ATPase subfamily, and is highly homologous to the Menkes disease gene product(46, 47, 48) . Although the intracellular localization of the membrane-bound ATPase has not been elucidated, the nonfunctional copper transporter leads to defective biliary excretion of copper in humans. Partial deletion of the Wilson disease gene in the LEC rat has been identified(17) . Thus, the hepatocyte cell lines established in our study likely carried the mutation, and, if so, then these cell lines can serve as biochemical and genetic models of Wilson disease. Phenotype of cultured hepatocytes from LEC rats is accumulation of copper; therefore, a defect in copper transport likely exists in these cells. In patients with Wilson disease, accumulation of copper in hepatocytes is associated with a reduction in excretion of copper into bile. Our results suggest that biochemical abnormalities in LEC rats are linked to a mechanism to excrete copper from hepatocytes. It is likely that the genetic defect in the Wilson disease gene directly affects the excretion of copper in hepatocytes, in vivo and in vitro, the result being copper accumulation.
The incorporation of copper into apo-CP is apparently impaired in LEC rats (14) as well as in patients with Wilson disease(12) . Our investigation has raised the question as to whether copper transporting ATPase encoded in the Wilson disease gene is an integral part of the biochemical mechanism of copper incorporation into apoprotein. To address this question we transfected human CP cDNA under the control of an exogenous promoter, and synthesis of holoceruloplasmin was monitored by immunological detection of holo-CP. The processing of exogenously expressed CP was apparently not influenced by the increasing concentrations of copper in cells from LEC cells or from LEA rats. Based on these investigations, we tentatively conclude that the hepatocytes can synthesize holo-CP from apo-CP, even after the loading of copper.
Our results suggest that the copper-transporting ATPase is not an integral part of the mechanism to incorporate copper into apoprotein. The limitation in this study is that one is looking at cultured hepatocytes and assuming that the cells reflect genetic abnormalities in LEC rats. The hepatocytes cultured in monolayer lost the tertiary structure, and this loss of the three-dimensional structure may alter biochemical mechanisms related to copper transport, e.g. distribution of the Wilson disease gene product in hepatocytes means that transformation of cells with SV40 may change the pattern of expression of hepatocytes specific genes related to copper metabolism. Menkes disease gene is not normally expressed in hepatocytes(46, 48) , and expression of this gene in transformed hepatocytes may alter the phenotype. In addition, growth of SV40 transformed cells in culture was rapid, and copper metabolism in rapidly growing cells differs from that in cells which grow slowly(49) . These changes in copper metabolism in cultured cells may contribute to normalization of the copper incorporation into apo-CP in cultured cells from LEC rats. However, these cultured cells do provide models of abnormal copper metabolism in hepatocytes by virtue of carrying a defined defect in the copper-transporting ATPase encoded in the Wilson disease gene.