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Abstract
The functions of hepatic tissues rely heavily on polarization of the hepatocyte,
a differentiation process characterized by targeting of cellular components to different
plasma membrane domains. The molecular mechanisms underlying specific delivery
of newly synthesized membrane proteins and their subsequent metabolisms are not
fully understood. Specifically, how or if protein glycosylation affects membrane
protein targeting is still remained controversial. In the present study, we utilized
human hepatoma cell lines as a model to study the hepatic polarization.
Well-differentiated hepatic cells such as Hep G2 and HuH-7 develop
characteristic actin-rich spheroid structures at sites of cell-cell contact that resemble
both structurally and functionally with the bile canaliculi (BC) found in vivo.
Junctional complexes such as tight junctions were found surrounding the in vitro BC
domain. In Hep G2 cells cultured for 3 day about 33% of BC exhibited profound
accumulation of aminopeptidase N (APN) at the BC compartment, while Na,KATPase was found only on the basolateral plasma membrane. Addition of
pharmacological reagents that inhibited membrane protein glycosylation appeared to
perturb the distribution of some membrane proteins but not others. Specifically,
tunicamycin treatments increased targeting of APN to BC, resulting in twice as many
APN-positive BC than the control condition. Interestingly, deglycosylation appeared
to misguide Na/K-ATPase from basolateral localization to the canalicular structures,
while the same treatments had no effect on the distribution of dipeptidyl peptidase IV
(DPPIV), another BC domain marker. The altered membrane protein targeting
appeared to cause changes of some hepatic secretory activities. of the appeared to
By using flurescein diacetate (FDA) and 3 KDa dextran as excretion marker
we found that the secretary activity of FDA in Hep G2 cells was decreased under
tunicamycin treatment.
Introduction
Polarized epithelial and hepatic cells have distinct membrane domains that are
separated by tight junctions in order to maintain the specific lipid and protein
composition of each domain. Development and maintain of this membrane polarity is
critical to the function of the cells. Correct targeting of specific membrane proteins is
essential for generating and maintaining of this polarity (Zegers et al., 1998; Caplan,
1997; Wilson, 1997; Yeaman et al., 1999).
The plasma membrane of hepatocytes can differentiate into three functionally
and structurally distinct domains: the sinusoidal membrane that faces the circulation,
the lateral membrane that involves in cell-cell and cell-substratum interactions, and
the apical domain (or bile canalicular domain) that, with its well developed microvilli
and cell junction complexes, forms bile canaliculi (BC; Phillips et al., 1987;
Nathanson and Boyer, 1991; Arias et al., 1993; Crawford, 1996; Erlinger, 1996). This
membrane polarity is essential for functional integrity of the liver (Hubbard, 1991;
Rodriguez-Boulan and Powell, 1992; Talamini et al., 1997). Strict regulation over
targeting of membrane proteins toward appropriate membrane domain would play
important role in the development and maintenance of this membrane polarity.
Various model systems were used for hepatic studies: perfused liver (Hems et
al., 1966; Mortimore and Schworer, 1979), hepatocytes couplets/triplets (Graf et al.,
1984; Sakisaka et al., 1988; Boyer, 1997), or fractionated membrane vesicles (Meier,
1989; Barr et al., 1995). Recently, better hepatic polarity could be maintained by
plating primary cultured-hepatocytes in sandwiched collagen gels (LeCluyse et al.,
1994). Another approach has also been made using various hepatoma cell lines
(Chang et al., 1983; Venetianer et al., 1983; Gatmaitan et al., 1983; Chiu et al., 1990;
Sormunen et al., 1993), including cell hybrids (Ihrke et al., 1993; Konieczko et al.,
1998), that are capable of undergoing BC formation in vitro.
Human hepatoma cell lines can be further classified into well- and poorlydifferentiated types based on morphology, hepatocyte-associated enzymes, HBV
genome expression (Aden et al., 1979; Chang et al., 1987), plasma protein secretion
(Knowles et al., 1980; Chang et al., 1983) and major histocompatability complex
class II molecules (Sung et al., 1989). Several well-differentiated human hepatoma
cells, such as Hep G2, Hep 3B and HuH-7, polarize and form BC-like structures in
vitro (Chiu et al., 1990; Sormunen et al. 1993). These BC-like structures are located
specifically at sites of cell-cell contact and typically exhibited as characteristic void
spheres containing high concentration of actin filaments. Such structures were
seldom found in the poorly differentiated hepatoma cells, such as HA22T/VGH and
SK-HEP-1 (Chiu et al., 1990; Sormunen et al., 1993).
Our data showed that pharmacological reagents that affect protein
glycosylation appeared to perturb distribution of various membrane proteins in Hep
G2 cell line. Specifically, addition of tunicamycin to these hepatic cells resulted in
sorting of a basolateral membrane protein, Na,K-ATPase, to the BC domain.
Na,K-ATPase is a transmembrane enzyme that moves Na+ out of the cell and
K+ in utilizing ATP as the driving force (Skou et al., 1992). It is found in the cell of
all higher eukaryotes including Drosophila but not in lower eukaryotes such as yeast.
The enzyme is composed of two subunits, a larger  subunit and a smaller  subunit
(Lingrnl, 1992). The  subunit is a polytopic membrane protein with 10 membranespanning domains and responsible for the catalytic activity. The  subunit is a type II
membrane glycoprotein with only one membrane-spanning domain at N-terminal.
Apical mislocation of Na,K-ATPase has been implicated as a feature of human
polycystic kidney disease (PKD; Wilson et al., 1991; Carone et al., 1994; Ogborn et
al., 1993, 1995). No similar clinical observation in the liver has been reported yet.
Effects of glycosylation or mislocation of membrane proteins on hepatic secretary
function are not fully understood either. In this study, we have tested the secretary
function with Fluorescein diacetate (FDA) and 3 KDa dextran in our hepatoma cell
models under the treatment of glycosylation inhibitor. We found that FDA secretion
was decreased when cells were treated with tunicamycin.
Discussion
Effects of glycosylation on protein targeting to different membrane domains in
polarized cells are still controversial. In epithelial cells, studies showed that Nglycans might serve as apical sorting signals for membrane proteins (Fiedler et al.,
1995; Scheiffele et al., 1995). Early studies in hepatic cells using tunicamycin, a
glycosylation inhibitor, demonstrated that when N-linked glycosylation is blocked,
most nonglycosylated forms of the proteins accumulate and aggregate in the ER
(Olden et al., 1982; Yeo et al., 1989). While other studies indicated that
transportation of some surface glycoproteins does not influenced by N-linked
glycosylation (Ploegh et al., 1981; Varki, 1993). Here we found that deglycosylation
may cause the mis-guided targeting to the BC domain (equilibrium to apical domain
in epithelial cells).
Previous studies using chimeric  subunit derived from Na,K-ATPase and an apical
homologous ion pump H,K-ATPase found that the fourth transmembrane domain of
both enzyme contain specific sorting signal (Caplan et al., 1997; Muth et al., 1998).
In this study, we demonstrated that inhibition of glycosylation might affect the
targeting of Na,K-ATPase. This was resulted more likely from a specific targeting
signal of the N-linked glycosylation than a general effect of glycosylation inhibitors.
Because targeting of DPPIV was not affected by the same treatment. And APN
showed a change in quantity instead of the change of pattern found in Na,K-ATPase.
Treatments with different glycosylation inhibitors may give us another hint. The
degree of Na,K-ATPase mistargeting seemed to be correlated with the degree of
deglycosylation. Other studies showed that disruption of glycosylation and disulfide
bond formation in  subunit might affect the structural and functional maturation of
subunit as well as the complete heterodimeric enzyme (Ahmed et al., 1997;
Zamofing et al., 1989). But we did not find the accumulation of this ion pump in ER
or other cell compartments.
FDA excretion was drastically reduced by inhibition of glycosylation while transport
3 KDa dextran was only slightly reduced. Dextrans were used as extracellular
reporter molecules for the diffusion selectivity of the domain boundary (Ihrke et al.,
1993). But our data showed that some 3 KDa dextran accumulated in the cells. This
indicated that at least some dextran move to BC through transcytosis pathway like
FDA instead of extracellular transport and might be counted for the slight decline in
the number of dextran-positive BC. FDA would not pass the domain boundary for it
was a charged molecule.
Materials and Methods
Cells
HuH-7 and Hep G2 hepatoma cell lines were used in this investigation.
Standard cell culture protocols were followed. Cells were cultured with Dulbecco’s
Modified Eagle Medium (GIBCO BRL) supplemented with 10% fetal bovine serum,
2mM L-glutamine, 0.1mM non-essential amino acid and incubated at 37oC in the
presence of 5% CO2.
Pharmacology treatment
Cells were cultured for 48 hr and then pharmacological reagents were added
into the culture medium for 24 hr. The concentration of the reagents used were:
Tunicamycin 20 g/ml, Castanspermine 10 g/ml and 1-Deoxymannojirimycin 5 mM
(Sigma).
Western blot analysis
Cell lysate separated by 10% SDS-PAGE were transfer to a nitrocellular paper
(Bio-Rad). After confirmation of the presence of proteins by Ponceous S staining,
standard Western blot analysis was performed using anti-Na,K-ATPase Beta antibody
(Affinity Bioreagents Inc.). The membrane was incubated with primary antibody for
1 hr at 37oC and with secondary antibody conjugated with Houseradish peroxidase
(Bio-Rad) for 30 min at 37oC. The blotting signal was detected by SuperSignal
Chemiluminescent substrate (Pierce) and recorded by Hyperfilm (AmershamPharmacia).
Secretary assay
Fluorescein Diacetate
Cells grown on coverslips were incubated with fluorescein diacetate at a final
concentration of 5 g/ml for 15 min at 37oC, and then washed three times with PBS.
The cells were fixed and stain with 1 unit/ml Rh-Ph (Molecular Probes) according the
potocal listed below.
Specimens were observed and photographed on a Nikon
inverted fluorescence microscope with MetaMorph imaging system (Universal
Imaging Corp, West Chester, USA)
3 KDa dextran
Cells grown on coverslips were washed serum-free medium once and
incubated with 3 KDa dextran (Molecular Probes) at a final concentration of 1 mg/ml
for 15 min at 37oC. Cells were wased with PBS before observation.
Immunostaining
For immunofluorescence staining, cells were typically cultured on a 22 x 22
mm square coverslip which is pretreated with 6N HCl and 95% ethanol, and coated
with 200 g/ml poly-L-lysine (MW 70-150 KDa, Sigma) as previously described (Lin
and Forscher, 1993; Lin and Forscher, 1995; Lin et al., 1996). Cells were fixed with
4% paraformaldehyde/2 mM EGTA/400 mM sucrose/PBS at RT for 15 min, then
permeabilized with 0.5% Triton X-100 in the fix solution for 5 min. The samples
were then incubated with 5 mg/ml BSA/PBS, then with primary antibodies at RT for
1hr. The concentrations of primary antibodies utilized were:1:100 anti-Na,K-ATPase
Beta antibody . After extensive PBS washes, fluorophore-conjugated secondary
antibodies (Jackson Immuno Research, West Grove, PA) were added at the
concentrations recommended by the manufacturer at room temperature for 1 h. About
1 unit/ml Rh-Ph was used for F-actin staining. The stained samples were mounted
using an anti-photobleaching medium containing 20 mM n-propyl-gallate (Sigma) in
80% glycerol/20% PBS, then observed under a Leica TCS-NT confocal microscope
(Leica Lasertechnik GmbH, Heidelberg, Germany). All images were recorded in a
digital platform for data analysis and image processing.
Results
Bile canaliculi development and distribution of membrane marker proteins in HuH-7
hepatoma cell line
Previous studies in our lab found that BC-like structures were found in vitro in a welldifferentiated hepatoma cell line Hep G2. These structures were full of microvilli
thus F-actin rich. Membrane marker proteins were expressed on specific membrane
domains as observed in vivo.
For examples, Na,K-ATPase was located on the
basoleteral domain while aminopeptidase N (APN) was on the BC domain. Like Hep
G2 cells, when staining F-actin with Rh-Ph, HuH-7 cells cultured for 72 hr showed
spheroid BC-like structures at cell-cell contact sites (Fig. 1A, B).
By using a
monoclonal antibody 9B2 generated by Chiu et. al that recognized APN, we
performed immunostaining on HuH-7 cells. APN signal in HuH-7 cells was found in
some of the BC membrane as well as vesicle-like structures in the cytoplasm (Fig.
1A). This expression pattern was the same as that in Hep G2 cells (Fig. 1B). Na,KATPase localization in HuH-7 cells revealing with anti-Na,K-ATPase beta subunit
antibody was on the basolateral membrane. This was identical to the pattern found in
Hep G2 cells (Fig. 1C, D).
Pharmacology reagent that inhibit N-linked glycosylation afftected some protein
targeting in Hep G2 cells
Hep G2 cells cultured for 48 hr were treated with tunicamycin (TM), a glycosylation
inhibitor, for 24 hr before staining with specific antibodies against different
membrane proteins. Tunicamycin inhibits N-linked glycosylation of glycoproteins in
higher organisms by blocking the first step (synthesis of dollchol pyrophosphate N-
acetylglucosamine) in the biosynthesis of the lipid-linked oligosaccharide precursor.
After inhibition of N-linked glycosylation, APN-positive BC increased from about
50% to over 75% (Fig. 2A). Na,K-ATPase, the basolateral membrane protein, had a
significant change in its distribution.
The Na,K-ATPase signal was on the BC
domain and evenly distributed in the cytoplasm after tunicamycin treatment (Fig.2B).
We also tested another BC domain protein DPPIV. We found that targeting of
DPPIV was not affected by inhibition of glycosylation (Fig. 2C).
Na,K-ATPase targeting was affected by different glycosyaltion inhibitors
Na,K-ATPase showed the most obvious distribution change under tunicamycin
treatment. So we tested two other glycosylation inhibitors their effects on Na,KATPase targeting.
Castanspermine (Cas) inhibits mammalian glucosidase I and
mammalian lysosomal alpha-glucosidase thus blocks the first step in the processing of
the N-linked precursor oligosaccharide to high-mannose oligosaccharide.
1-
Deoxymannojlrimycin (dMM) binds to the active site of mammalian Golgi alphamannosidase and inhibits its activity. When Hep G2 cells were treated with dMM
(Fig 3, dMM), some Na,K-ATPase expressed on the basolateral membrane as control
(Fig. 3, CTL & dMM) while some signal showed up in the cytoplasm.
Some
ATPase-positive BC could also be found. After Cas treatment, Na,K-ATPase was
found in every BC while the ATPase signal on basolateral membrane declined.
Vesicle-like staining pattern could also be found within cytoplasm (Fig. 3, Cas).
Na,K-ATPase distribution after tunicamycin treatment was also showed here for
comparison (Fig. 3, TM). Note that ATPase signal appeared strongly in BC and
evenly in cytoplasm. No specific signal could be found on the basolateral membrane.
Tumicamycin showed no effects on Na,K-ATPase targeting in HuH-7 cell line
HuH-7 hepatoma cell line share many similar properties with Hep G2 cell line as
shown in Fig. 1. But surprisingly, we found that Na,K-ATPase distribution was not
changed in HuH-7 cells after treated with tunicamycin (Fig. 4A). The ATPase signal
was mainly on the basolateral membrane and no signal was found in BC domain.
Total cell lysate for Western blot analysis was prepared from the cells under the same
treatment. The lysate separated with SDS-PAGE was transferred to NC paper and
blot with anti-Na,K-ATPase beta subunit antibody. The fully glycosylated form of
the protein was around 50 KDa and the core-protein has a molecular weight of 32
KDa. The cell treated with tunicamycin had a band shift to 32 KDa indicating that the
inhibition of glycosylation was successful (Fig. 4B). A major band at 50 KDa in drug
treated cells was no surprise for Na,K-ATPase has important function on maintain the
ion gradient thus may have a longer half-life.
Inhibition of glycosylation affected some secretary activities in hepatic cells
Correct targeting of membrane proteins is important to maintain specific function in
polarized cells. Since we found that inhibition of glycosylation affected some protein
targeting in Hep G2 cells. We were interested to know if glycosylation inhibitor
affected the secretary function in hepatic cells. Fluorescine diacetate (FDA) and 3
KDa were used to access the transcytosis activity of the cells. Cells cultured for 48 hr
were treated with tunicamycin for 24 hr before loaded with different secretary
markers. About half of the BC in the cells without inhibition of glycosylation could
functionally secreted FDA (Fig 5, control). But only about 20% BC showed FDA
signal after tunicamycin treatment (Fig5, +tunicamycin). The ratio of 3 KDa dextranpositive BC in control cells was about the same range as FDA (Fig. 6, control).
Instead of obvious decrease in FDA secretion, tunicamycin treated cells showed only
slight decline in the secretion of 3 KDa dextran (Fig. 6, +tunicamycin). There was
some dextran accumulate in the cytoplasm. The ratio of fluorescent BC was shown in
Fig. 7. Over 500 of total BC were counted in each sample. The secretary activity of
FDA was only one-half under tunicamycin treatment. The same drug treatment just
slightly decrease the secretion of 3 KDa dextran.
Figure 1. Formation of BC among well-differentiated hepatic cells.
(A) Hep G2 and (B) HuH-7 cells cultured for 72 h were stained with Rh-Ph then
observed under a confocal microscope. Canalicular structures typically contained
high concentration of F-actin and exhibited as spheroid structures at sites of cell-cell
contact (arrows). (C) Poorly-differentiated SK-HEP-1 cells cultured for the same
period contained no discernible BC.
Figure 2. Tight junction formation in well-differentiated hepatic cells.
(A, B, C) Hep G2 cells cultured for 72 h were stained for F-actin (A) and ZO-1
proteins (B, C), an essential component of tight junctions. Different optical sections
through the BC were performed by confocal microscopy (diagram) to assess both the
horizontal (B) and vertical (C) orientations of the tight junction (black circle)
surrounding the BC (spheroid). (D) The tight junction formation of 72 h-cultured
Hep G2 cells was also examined by thin-sectioned transmitted electron microscopy.
Note many microvilli present in the lumen of BC (arrows). Junction complexes were
found along the cell contact between two neighboring cells (1 and 2). Several
regions of the junctional complex exhibited features indicative of membrane fusion
(arrowheads, inset), the hallmark for tight junction formation. Bars = 0.5 m and 0.05
m (inset).
Figure 3. Different membrane markers were localized at specific membrane domains.
(A, B, C) All hepatoma cells examined contained the membrane protein APN, as
revealed by immunofluorescence staining, but their distributions varied. APN
proteins of poorly-differentiated HA22T/VGH cells resided mainly on the plasma
membrane (arrowheads). In well-differentiated HuH-7 and Hep G2 cells, APN was
targeted to the membrane of BC domain (arrows). There was also punctate staining
in the cytoplasmic compartments (double arrowheads), indicating the presence of
APN-containing vesicles. (C, E) 72 h-cultured Hep G2 cells were stained with RhPh (D) and anti- subunit Na,K-ATPase antibody (E). The distribution of this
basolateral membrane marker (arrowheads) was devoid of BC domain (arrows). Bar
= 5 m.
Figure 4. Deglycosylation affectd targeting of some membrane proteins but not
others.
Hep G2 cells were cultured for 48 hr and then treated with tunicamycin for 24 hr. (A)
Cell were stained with anti-APN antibody 9B2 (stained green) and Rh-Ph (stained
red). After tunicamycin treatment, the percentage of APN-positive BC increased
form 50% to over 75%. (B) Na,K-ATPase (stained green) showed typical basolateral
staining pattern in control cells. After tunicamycin treatment, the majority of Na,KATPase proteins were relocated to BC; there were also more Na,K-ATPase proteins
evenly distributed in the cytoplasm than the control condition. (C) On the other hand,
the distribution of DPPIV, expressed mainly at the BC domain, exhibited no
significant difference after tunicamycin treatments compared with the control.
Figure 5. Different deglycosylation treatments resulted in different degrees of
protein mistargeting.
Hep G2 cells cultured for 48 hr were treated with mock solution (A) , dMM (B) , Cas
(C) , and tunicamycin (D) for the subsequent 24 hr, and then stained with anti-
subunit Na,K-ATPase antibody (left column and green channel of the right column),
together with Rh-Ph (red channel). (A) In the control condition, Na,K-ATPase was
found along the basolateral domain (arrowheads, A), but devoid of BC (double
arrows). (B) Mild deglycosylation by addition of dMM relocated a portion of Na,K-
ATPase to some of the BC domain (arrows), but not others (double arrows). There
was still profound Na,K-ATPase signal along the basolateral membrane (arrowheads).
(C) Treatments with Cas resulted in mis-targeting of Na,K-ATPase to every BC
observed (arrows), staining along the basolateral membrane was further reduced.
There were also vesicle-like punctate stainings in the cytoplasm (double arrowheads).
(D) Complete N-linked deglycosylation by tunicamycin also caused relocation of
Na,K-ATPase to the BC domain (arrow) and Na,K-ATPase-positive vesicles (double
arrowheads), without any discerible signal found on the basolateral membrane. Note
also the presence of increased Na,K-ATPase proteins evenly distributed in the cytosol
after the deglycosylation treatments. Bar = 5 m
Figure 6.
Western blot analysis of Na,K-ATPase proteins after deglycosylation
treatments.
Total cell lysates were colleccted from 72 hr-cultured Hep G2 cells treated during the
last 24 hr with 20 g/ml tunicamycin. The proteins were subjected to 10% SDSPAGE separation and blotted with anti- subunit Na,K-ATPase antibodies. The native
fully glycosylated  subunit Na,K-ATPase proteins were about 50 KDa. Note the
presence of a 32 KDa band after the tunicamycin treatment. This molecular weight
was about the size of the non-glycosylated core protein, suggesting that glycosylation
of the newly synthesized  subunit Na,K-ATPase was successfully inhibited by the
drug treatments. Bar = 5 m
Figure 7.
Bulk flow of fluid-phase transcytosis transport was affected by
deglycosylation treatments.
72 hr-cultured Hep G2 cells were treated during the last 24 hr with 20 g/ml
tunicamycin. These cells were loaded with FDA at 37oC for 15 min to measure the
bulk flow of fluid-phase transcytosis transport into the BC (stained green), before
fixed and stained with Rh-Ph to reveal the localization of BC (stained red). In the
control condition (A), note about 45% of BC were able to secrete FDA (arrows),
while the other half show no significant FDA filling (arrowheads) in the 3d-cultured
Hep G2 cells. The percentage of FDA-positive BC (arrow) decreased to only 22%
after exposure to tunicamycin. Bar = 5 m
Figure 8. Paracellular transport of the hepatic cells was insensitive to tunicamycin
treatments.
72 hr-cultured Hep G2 cells were treated during the last 24 hr with 20 g/ml
tunicamycin. 3 KDa fluorescein-label dextran was added to the culture medium at
37oC for 15 min to measure the paracellular transport of this compound into the BC
(stained green). The cell were then fixed and stained with Rh-Ph to reveal the
localization of BC (stained red). In the control condition (A), about 52% of BC were
filled with fluorescent dextran (arrows). Tunicamycin treatments caused no
significant change as measured by this secretory assay. Bar = 5 m
Figure 9. Statistics of various canalicular secretory assays
Pooled data of canalicular secretory assays as measured by FDA and 3 KDa dextran.
Note the percentage of BC capable of secreting FDA decreased from 45% in the
control condition (FDA-CTL) to 22% after the deglycosylation treatments (FDA-tuni).
Excretion of 3 KDa dextran was not affected by the same pharmacological
intervention. There were 52% and 45% dextran-positive BC in the control (Dextran-
CTL) and drug addtion contdition (Dextran-tuni), respectively. A total of 500 BC
were counted in each condition.
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