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Regular Articles |
From the Departments of Pathology*
and
Medicine,
University of Graz, Graz, Austria;
and the Department of Medicine,
Palo Alto VA
Medical Center and Stanford University, Palo Alto, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-internexin, desmin, vimentin, glial filaments, and
the nuclear lamins1,2
also belong to the IF family. The
diverse cell biological functions of IFs are still poorly understood.
However, a diversity of human diseases is associated with severe
alterations of IFs. A common pathological feature of many IF-related
diseases is the accumulation of intracytoplasmic inclusions
consisting of modified IF proteins, for
example in neurodegenerative diseases such as amyotrophic lateral
sclerosis, Parkinson’s disease, and Lewy body
dementia3-6
; in neuromuscular disorders (eg, spheroid
body myositis7
); and the formation of Mallory bodies (MBs)
in alcoholic hepatitis (AH) and other liver disorders (eg,
non-alcoholic steatohepatitis, Wilson’s disease, primary biliary
cirrhosis, Indian childhood cirrhosis, hepatocellular
neoplasms8-11
). Although the underlying pathogenetic
mechanisms are as yet unclear, posttranslational modifications of IF
proteins, such as phosphorylation, limited proteolysis, and
cross-linking, may play a major role. For example, the presence of
hyperphosphorylated neurofilament epitopes in some neuronal
inclusion bodies12-15
and of abnormally phosphorylated
desmin in muscle fibers16
were reported. Furthermore, a
possible association of cytokeratin hyperphosphorylation with the
formation of MBs in hepatocytes, a hallmark of AH, was suggested by
in vitro and animal studies performed by our own group and
others.17-20 AH follows chronic alcohol abuse and occurs in 20 to 40% of heavy drinkers. Although reversible at the beginning, most cases of AH progress to irreversible cirrhosis. Besides the amount of alcohol ingested per day, a variety of other factors such as dietary habits, genetic factors influencing alcohol metabolism, viral infections, and additional toxins or drugs seem to determine the extent of liver damage. Classical morphological features of AH are liver cell ballooning and necrosis, inflammation, steatosis, and the formation of cytokeratin-containing MBs, which is associated with severe derangement (ie, diminution or even loss) of the hepatocyte cytokeratin IF network.8-11,22-26 Diverse animal models have been generated to study in more detail and under defined conditions mechanisms involved in the pathogenesis of this alcoholic liver disease. Experimental long-term intoxication of mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or griseofulvin (GF) mimics the particular hepatocellular alterations associated with AH, ie, ballooning of hepatocytes, accumulation of MBs, and alterations of the cytokeratin IF network.27-31 These animal models are valuable not only for investigating the effects of long-term (chronic) intoxication (ie, for 2 to 4 months) but also for assessing the time course of alterations finally leading to MB formation and cytokeratin filament derangement. In addition to elucidating mechanisms involved in the pathogenesis of AH, these DDC and GF animal models may provide insight into biology and pathology of cytokeratins.
Cytokeratins are a multigene family consisting of at least 21 catalogued proteins.2,32 According to sequence homology and biochemical properties they can be divided into two subgroups, the type I and type II cytokeratins, which form obligatory noncovalent heteropolymers that spontaneously assemble into 10-nm filaments.2,33 Epithelia express characteristic patterns of type I and type II heteropolymers depending on their tissue origin and state of differentiation. For example, glandular and secretory simple type epithelia express cytokeratin 8 (CK8) and CK18 as their major cytokeratin pair with variable amounts of CK7, CK19, and CK20. Normal adult hepatocytes express CK8/18, which are equivalent to Endo A (CK8) and Endo B (CK18), originally described as cytokeratins A and D, in mouse hepatocytes.34-36
Cytokeratins undergo several posttranslational modifications, such as phosphorylation, glycosylation, acetylation, and transglutaminase-induced cross-linking, that are likely to be involved in regulating their function.37-41 In the case of CK8/18, phosphorylation has been extensively studied in cultured cells and in vitro experiments. Phosphorylation of cytokeratins and other IF proteins plays important roles in the cell,41 including regulation of filament disassembly and reorganization, particularly during mitosis, solubility, interaction with other proteins, and determining localization within specific compartments of the cell. With regard to CK8/18, phosphorylation occurs exclusively on serine residues and is increased during mitosis, apoptosis, growth factor stimulation, and different forms of cell stress. Various kinases seem to be involved in the phosphorylation of specific sites on either CK8 or CK18.39-50 Several phosphorylation sites at the amino- and carboxy-terminal regions (termed head and tails, respectively) of cytokeratin proteins have been identified, such as serine 23 (Ser23), Ser73, and Ser431 in CK8 as well as Ser52 and Ser33 in CK18. Most if not all phosphorylation sites are located within these head and tail domains. Accumulating evidence indicates that they are important for filament assembly and interaction with other proteins. Recently, monoclonal and polyclonal antibodies selectively recognizing the various phospho-epitopes on CK8 or CK18 have been generated.46-50 These antibodies provide ideal tools for studying the phosphorylation states of the various sites and their dynamics and topographical distribution in tissues and/or cultured cells.
Although many studies suggest a role for CK8/18 hyperphosphorylation in the pathogenesis of AH based primarily on the MB mouse models, there is no report about altered CK8/18 phosphorylation patterns in human AH compared to human control livers. We therefore investigated frozen tissue sections of liver biopsies with AH and human control livers using antibodies to four different phospho-epitopes on CK8/18 and determined the presence and distribution of the various CK8/18 phospho-epitopes. In addition, DDC-fed mice at different stages of intoxication were also studied to examine the time course of hepatocyte cytokeratin alterations under standardized experimental conditions.
In this report, we show that human and murine MBs contain CK8/18 phosphorylated at multiple sites. Moreover, phosphorylation levels and intracellular distribution of phosphorylated CK8/18 are altered in hepatocytes of human AH as well as DDC-intoxicated livers in comparison with control livers. This is the first demonstration that phosphorylation of hepatocyte CK8/18 is associated with human AH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human liver biopsy material was obtained from nine patients with histologically confirmed AH. Parts of the specimens were snap-frozen within 2 minutes after biopsy and stored in liquid nitrogen until analysis by immunofluorescence microscopy. Normal (control) liver parenchyma was obtained from the non-neoplastic margins of nine cases of surgically resected liver tumors.
Animal Model and Experimental Design
Male Swiss Albino mice (35 g body weight; strain Him OF1 SPF;
Institute of Laboratory Animal Research, University of Vienna School of
Medicine, Himberg, Austria) were fed either a standard (control) diet
(Altromin, Marek, Vienna, Austria) or a diet containing 0.1% DDC
(Aldrich, Steinheim, Germany) for time periods ranging from 1 day to
2.5 months. After 2.5 months of continuous DDC feeding, some animals
were allowed to recover on standard (control) diet for 1 month. Some of
the recovered mice were then reintoxicated with DDC for 3 days. Figure 1
shows details of the experimental
design. Mice were killed by cervical dislocation and the livers were
snap-frozen in methylbutane precooled with liquid nitrogen for
immunohistochemistry and preparation of cytoskeletal proteins.
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Double-label immunofluorescence microscopy was performed on
cryostat sections of liver tissue (3 µm thick, fixed in acetone at
-20°C for 10 minutes) as described previously.51
The
antibodies directed against various epitopes (phosphorylated and
nonphosphorylated) on CK8/18 are summarized in Table 1
. The antibodies to phospho-CK8 (5B3,
LJ4) or phospho-CK18 (8250, 3055) have been characterized
recently.46-50
The antibodies Ks 8.7, Ks 18.04 (Progen,
Heidelberg, Germany), and 50K16052
(also termed CK
antibodies) were used to detect total CK8 and/or CK18 independent of
phosphorylation state. As secondary antibodies, Cy2-conjugated goat
anti-mouse IgG (Amersham, Buckinghamshire, UK) and tetramethylrhodamine
isothiocyanate (TRITC)-conjugated swine anti-rabbit Ig (Dako, Glostrup,
Denmark) were used. For control of background staining, the primary
antibodies were omitted or replaced by IgG isotype control (Dako).
Immunofluorescent stained specimens were analyzed with a MRC 600
(Bio-Rad, Richmond, CA) laser-scanning confocal device attached to a
Zeiss Axiophot microscope. The fluorescent images were collected using
the confocal photomultiplier tube at full frame (768 x 512
pixels). For dual labeling, separate excitation wavelengths (488 nm for
Cy2, 568 nm for TRITC) from a krypton/argon ion laser were used.
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Cytoskeletal proteins were prepared from frozen mouse liver tissue by high salt-detergent extraction as described previously.38 The following protease inhibitors were included in all buffers: 200 µmol/L pefabloc SC (Merck, Darmstadt, Germany), 50 mmol/L sodium fluoride (Merck), 0.3 µmol/L aprotinin (Boehringer Mannheim, Mannheim, Germany), 100 µmol/L leupeptin (Boehringer Mannheim), 1 µmol/L pepstatin (Boehringer Mannheim), 200 µmol/L sodium vanadate (Sigma, St. Louis, MO), and 1 mmol/L sodium pyrophosphate (Fluka, Buchs, Switzerland). Equal amounts of protein were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis53 and electrophoretically transferred onto nitrocellulose membranes (0.2 µm pore size, Schleicher & Schuell, Dassel, Germany) with 0.1 A for 18 hours.54 The membranes were blocked with 5% nonfat milk in TBS-T (10 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl containing 0.1% Tween-20) and incubated with the primary antibodies (5B3, 1:2000; LJ4, 1:1000; 8250, 1:2000 supplemented with 200 µg/ml nonphosphorylated peptide to prevent binding to the nonphosphorylated epitope) overnight at 4°C. After washing with TBS-T for 1 hour, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Dako) for 2 hours, followed by a final washing step in TBS-T for 1 hour. Antibody binding was detected using enhanced chemiluminescence (ECL, Amersham).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In human control liver, CK antibodies that recognize cytokeratin
independent of its phosphorylation state (Figure 2, a, c, e, and g)
revealed a filamentous
pattern with increased density at the cell periphery. Antibodies to
phospho-CK8 (5B3) and phospho-CK18 (8250, 3055) preferentially stained
this peripheral rim of cytokeratin filament bundles in the majority of
hepatocytes (Figure 2)
. No staining with the phospho-CK8 antibody LJ4
(Figure 2, c and d)
was obtained, suggesting that, in contrast to the
other epitopes on CK8 and CK18, no phosphorylation of this site exists
in human control liver.
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revealed in some hepatocytes a derangement and diminution of the
cytokeratin IF cytoskeleton and accumulation of CK8/18-containing MBs.
All phospho-CK8 and phospho-CK18 antibodies intensely decorated MBs.
However, LJ4 did react only with a subset of human MBs independent of
their size (eg, Figure 3, c and d
). In comparison with the staining
pattern of phospho-CK8 antibodies in control liver, a shift in
phospho-CK8 staining from the cell periphery to a pronounced staining
of the cytoplasmic cytokeratin IF network occurred in AH (compare
Figures 2 and 3
). Moreover, immunostaining with LJ4, which was absent
in control liver, became strongly positive in AH (Figure 3, c and d)
.
The results obtained with antibodies to phospho-CK18 (8250, 3055;
Figure 3, e
-h) were similar but less conspicuous than that with
phospho-CK8 antibodies (5B3, LJ4). The immunofluorescence results are
summarized in Table 2
.
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Prolonged feeding of mice with a DDC-containing diet has been
widely used as an animal model for experimental induction of MBs with
cytokeratin IF alterations similar to those associated with AH. We
compared the phosphorylation patterns of CK8/18 in control mice, in
mice at different stages of DDC intoxication (ie, short-term treatment
for 1 to 7 days, and long-term treatment for 2.5 months), in mice that
were allowed to recover for 1 month on control diet after 2.5 months of
DDC feeding, and in mice reintoxicated with DDC for 3 days after an
initial DDC intoxication and subsequent recovery period (see Materials
and Methods and Figure 1
). Immunofluorescence microscopy was performed
on frozen liver sections with antibodies that were used in Figure 2
,
except for the 3055 antibody, which does not cross-react with mouse
CK18.
In livers of mice fed a control diet, hepatocytes displayed a regular
cytokeratin IF network stained with CK antibodies (Figure 4, a, c, and e)
, whereas phospho-CK8
antibodies 5B3 and LJ4 did not stain (Figure 4, b and d)
, except for a
few single cells (<0.1%, not shown). Phospho-CK18 antibody 8250
preferentially decorated the cytokeratin filaments at the cell
periphery (Figure 4, e and f)
. However, after only 1 day of DDC
intoxication, a dramatic increase in phosphorylation, particularly of
CK8, was obvious. The staining pattern of phospho-CK8 antibody 5B3
resembled that revealed by CK antibodies, ie, staining of the
cytoplasmic cytokeratin network with accentuation of the cell periphery
and around the bile canaliculi (Figure 5, a and b)
. In contrast the distribution of LJ4 reactivity was patchy,
with clusters of 2 to 5 cells showing bright cytoskeletal staining and
other cells displaying only slight reactivity at the cell periphery
(Figure 5, c and d)
. Immunostaining with anti-phospho-CK18 (8250) was
similar to 5B3 but slightly weaker. Because CK18 was phosphorylated at
a low basal level in control mouse liver, the increase in
phosphorylation on DDC intoxication was not as conspicuous as that
observed for CK8. The red aggregates in hepatocytes and sinusoidal
spaces (eg, Figures 5–8
, e and f) represent autofluorescent porphyrin
deposits, which accumulate in the liver on DDC intoxication. These
aggregates were also seen in the corresponding negative controls where
the first antibodies had been omitted (not shown). After 2.5 months of
DDC intoxication, mouse hepatocytes showed the characteristic
derangement of the cytokeratin IF system as revealed by
immunostaining with CK antibodies. The staining changes included
loosening or loss of the (immunostainable) cytokeratin IF network and
accumulation of MBs (Figure 6)
. As in AH,
phospho-CK8/18 antibodies (5B3, LJ4, 8250) brightly decorated MBs and
the cytokeratin IF network of numerous hepatocytes (Figure 6)
. Although
phospho-cytokeratins were obviously integrated in the cytokeratin IF
network, in some hepatocytes they preferentially accumulated in MBs. In
these hepatocytes phospho-CK8 antibodies strongly reacted with MBs but
did not stain the cytokeratin network adjacent to the MBs (eg, asterisk
in Figure 6, c and d
). With LJ4, however, occasional MBs independent of
their size were negative or only weakly stained (not shown).
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. Residual alterations
were the presence of few hepatocytes without detectable cytokeratin
network but with small MBs mostly at the cell periphery (arrowheads in
Figure 7
). Phosphorylation of CK8/18 returned to normal, ie, no
staining of the cytokeratin filaments with phospho-CK8 antibodies (5B3,
LJ4) and weak staining of the cytokeratins predominantly at the cell
periphery with anti-phospho-CK18 (8250). However, 5B3 and 8250 staining
was still present in MBs (Figure 7, a, b, e, and f)
, whereas LJ4 did
not recognize residual MBs (Figure 7, c and d)
. On reintoxication, a
profound increase in CK8/18 phosphorylation was observed within 3 days
(Figure 8, a
-f). Cytokeratin filaments
were strongly stained by 5B3, LJ4, and 8250 antibodies. Newly formed
MBs present at the intersections of the cytokeratin IF network were
also brightly stained with 5B3 and 8250 (Figure 8)
. As already observed
in mice fed DDC for 2.5 months, there was preferential staining of MBs
with antibodies to phospho-CK8, whereas the residual cytokeratin IF
network of the hepatocytes that contained the MBs remained unstained.
The phospho-CK8 antibody LJ4 did not stain the majority of these small,
apparently newly formed MBs. Hence, phosphorylation of CK8 at this
particular site (ie, phospho-Ser73, which is recognized by LJ4)
displays a different response to DDC in that it appears late in the
process of MB formation, as seen in reintoxicated mice, but disappears
early, as shown in recovered mice. A summary of the immunofluorescence
results is shown in Table 3
.
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. A clear-cut increase in CK8
phosphorylation was detectable within 1 week of DDC intoxication and
persisted for the whole DDC intoxication period (2.5 months). On
withdrawal of DDC and recovery, CK8 phosphorylation level was reduced
but rose again on DDC reintoxication. These immunoblotting results are
in line with the immunofluorescence data in that dramatic and
site-specific CK8 hyperphosphorylation in response to DDC intoxication
was noted. Moreover, the immunostaining with phospho-CK8 antibodies of
the cytokeratin IF network as seen in immunofluorescence microscopy as
well as the presence of hyperphosphorylated CK8 in the cytoskeletal
insoluble fraction as analyzed by Western blotting, suggested that
phosphorylated CK8 was able to form IF structures in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using a panel of monoclonal antibodies that are specific for various
phosphorylation sites on CK8 and CK18, we report here on marked
phosphorylation of cytokeratins at multiple sites in human toxic liver
injury, namely AH, and accumulation of phosphorylated cytokeratin
material in MBs. The strong reactivity of MBs and, to a variable
extent, of the cytokeratin IF cytoskeleton of hepatocytes with all
phospho-cytokeratin-specific antibodies tested implies involvement of
different protein kinases and/or phosphatases in this disease. The
phosphorylation sites recognized by these antibodies have been
characterized in previous studies, giving rise to speculation about the
mechanisms leading to their phosphorylation and the biological
consequences. For example, phosphorylation of CK8 at Ser73 as detected
by LJ4 occurs preferentially in response to stress situations but is
also seen during apoptosis and mitosis.46
Although
phosphorylation of IF proteins generally results in their disassembly
and increased solubility, phosphorylation of Ser73 is significantly
associated with the insoluble cytokeratin fraction.46
Furthermore, phosphorylation-induced disassembly of cytokeratin
proteins may indirectly support their aggregation with other MB
components, because MBs form not from cytokeratin IF but from
disassembled cytokeratin proteins (Zatloukal K, Stumptner C,
Lehner M, Deuk H, Baribault H, Eshkind LG, Franke WW, manuscript
submitted). In this context it is interesting that we observed in AH as
well as in DDC-treated mice a subpopulation of hepatocytes where
phosphorylated cytokeratin accumulated in MBs but was not detectable in
the cytokeratin IF network adjacent to the MBs. Based on the observed
immunoreactivities of MBs with the antibodies 5B3, 3055, and 8250, it
is likely that a variety of protein kinases, such as ERK1/2, which
phosphorylates CK8 at Ser431,49
or PKC and cdc2 kinases,
which can phosphorylate CK18 at Ser5247
and
Ser33,50
respectively, are activated in AH and in DDC-fed
mice (for summary of results, see Tables 2 and 3
). This is in line with
and extends previous reports on the effect of ethanol on the activation
of several protein kinases, such as PKC, JNK, and p42/44 MAPK, that are
involved in different signal transduction pathways.61,62
Because of the complexity of alterations induced by ethanol and DDC
intoxication, one must take into account that besides the cytokeratin
IFs, a variety of other cellular proteins are affected as well and thus
could contribute to the observed cytoskeletal changes.
To further dissect this complex situation and to get more insights into the course of cytokeratin phosphorylation during intoxication, we analyzed mice after different periods of DDC feeding, recovery from intoxication, and re-exposure to DDC. Phosphorylation of cytokeratin was already detectable at 1 day after commencement of DDC feeding; thus it is one of the earliest changes seen after intoxication that precedes structural alterations of the cytokeratin IF network. In contrast to the phosphorylation of Ser33 on CK18 (as detected by 8250) and Ser431 on CK8 (as detected by 5B3), which was seen in the majority of hepatocytes and, therefore, could reflect a cellular response to the metabolism of DDC, phosphorylation of Ser73 on CK8 (as detected by LJ4) was found in only a few cells. The distribution pattern of LJ4-reactive hepatocytes, typically as cell doublets, suggests that some of these cells represent daughter cells generated by mitosis. This is also supported by the previous observations that the LJ4 epitope is generated during mitosis in addition to stress situations. In mice intoxicated long-term, the difference in the staining patterns of the antibodies 8250, 5B3, and LJ4 was less clearly revealed. Here, scattered groups of hepatocytes showed phosphorylation of the cytokeratin network to a variable extent. Furthermore, MBs were stained, as in human AH, by all three antibodies. There were, however, some MBs that did not stain with LJ4. Differences in the phosphorylation pattern of MBs were more clearly revealed in reintoxicated mice where newly formed MBs could be studied.63 In these mice 8250 and 5B3 strongly reacted with small MB gran- ules at the intersections of the cytokeratin IF network, representing the earliest detectable phase of MB formation. The phosphorylation of the epitopes recognized by these two antibodies may, therefore, participate in the initiation of MB formation. In contrast to 8250 and 5B3, LJ4 did not react with newly formed MBs, which shows that phosphorylation of Ser73 on CK8 (the epitope recognized by LJ4) is not involved in early MB formation. In more advanced stages of MB formation, however, phosphorylation of the LJ4 epitope could contribute to the growth of MBs, since we observed in later phases of reinduction as well as in the mice intoxicated with DDC for 2.5 months a subpopulation of hepatocytes with large MBs where LJ4 reacted with the MB but did not stain the residual cytokeratin network adjacent to the MB.
Further differences in the immunoreactivity profile of MBs at different phases of development were seen in mice at 1 month after recovery from intoxication. In contrast to 8250 and 5B3, which decorated small MB remnants at the cell periphery, LJ4 did not bind to MBs in these livers. This indicates that some protein kinases were still active even 1 month after cessation of intoxication whereas others, eg, stress-activated kinases, returned to baseline levels. Another mechanism that can be responsible for the observed dissociation of phospho-epitopes is that the phosphorylation sites in MBs have different half-lives of the bound phosphate. It is known that protein phosphatases are important regulators of steady-state cytokeratin phosphorylation.20,40,60,64 Furthermore, different turnover rates were identified for the various phosphorylation sites on CK8 and CK18.48 In contrast to CK8, which showed a higher steady-state phosphorylation in a variety of situations, including chronic intoxication of mice with GF or stimulation of PKC in cultured hepatocytes, CK18 had a lower steady state phosphorylation level because of rapid dephosphorylation by protein phosphatases.17,19,64
In summary, the applied phospho-cytokeratin antibodies proved to be a powerful tool, which allowed for the first time investigation of cytokeratin phosphorylation in human AH. The identification of the diverse phosphorylated serines on CK8 and CK18 in different phases of MB formation will help to elucidate the signals responsible for the activation of the corresponding protein kinases in future studies. The characterization of the signal transduction pathways will not only provide new insights into the pathogenesis of alcoholic liver disease, but also identify potential targets for new therapeutic approaches.
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Acknowledgements |
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Footnotes |
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Supported by the grants of the Austrian Science Foundation S7401-MOB (to K. Z.) and 7628-MED (to H. D.) and by a U.S. Veterans Affairs Merit and Career Development Award (to M. B. O.).
Accepted for publication September 4, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-related kinase associates with, and phosphorylates cytokeratin 8 and 18. J Cell Biol 1992, 117:583-593This article has been cited by other articles:
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