| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Departments of Medicine*
andPathology,
University of Graz, Graz, Austria
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Male Swiss Albino mice (strain Him OF1 SPF) were obtained from the Institute of Laboratory Animal Research, University of Vienna School of Medicine, Himberg, Austria, housed with a 12-hour light-dark cycle and permitted ad libitum consumption of water and a standard mouse diet (Marek, Vienna, Austria). Experiments were performed with 2-month-old mice weighing 25 to 30 g. The experiments described in this article were approved by the local ethics committee and followed the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" (National Academy of Sciences) as published by the National Institutes of Health (NIH publication 86-23, revised 1985). Cholic acid (CA) was obtained from Aldrich (Steinheim, Germany), ursodeoxycholic acid (UDCA) was kindly provided by the Falk Foundation (Freiburg, Germany).
Animal Experiments
Common Bile Duct Ligation (CBDL)
All surgical procedures were performed under sterile conditions. To study the effects of obstructive cholestasis on hepatic CK expression, the common bile duct was ligated under general anesthesia (10 mg Avertin intraperitoneally) close to the liver hilum immediately below the bifurcation and dissected between the ligatures as described previously.20 Cholecystectomy was performed after ligation of the cystic duct. Controls underwent a sham operation with exposure, but without ligation of the common bile duct and removal of the gallbladder. The livers were excised under general anesthesia 3 and 7 days after surgery, respectively. Aliquots of liver tissue were frozen in liquid nitrogen for molecular analysis and immunohistochemistry or fixed in 4% neutral buffered formaldehyde solution and paraffin-embedded for light microscopy and in situ hybridization. Serum samples from each mouse were stored at -70°C for analysis of AST/ALT, alkaline phosphatase, and total bile acid levels.
Bile Acid Feeding
To study the differential effects of bile acids on CK expression and phosphorylation, mice were fed a diet supplemented with toxic CA or hydrophilic nontoxic UDCA in different concentrations (0.1%, 0.5%, or 1%) for 7 days.21
Determination of mRNA Copy Numbers by Competitive Reverse Transcriptase-Polymerase Chain Reaction
mRNA copy numbers for CK 8, CK 18, and glyceraldehyde-3-phosphate dehydrogenase were determined by competitive reverse transcriptase-polymerase chain reaction as previously described by Zatloukal and colleagues.16
In Situ Hybridization (ISH)
Synthesis of 35S-Labeled Mouse CK 8 Probe
Labeled sense and antisense transcripts were synthesized in a
20-µl reaction mixture containing 1 µg of linearized plasmid, 10x
transcription buffer (0.4 mol/L Tris-HCl, pH 8.0, 60 mmol/L
MgCl2, 20 mmol/L spermidine, 100 mmol/L
dithiothreitol), 20 mmol/L dithiothreitol, rNTP-labeling mixture (1
mmol/L each of ATP, CTP, GTP; Boehringer Mannheim, Mannheim, Germany),
40 U Inhibit-ACE (5'
3' Inc, Boulder, CO), 120 µCi
-35S UTP (Amersham, Buckinghamshire, UK), and
20 U of either SP6 or T7 RNA polymerase (Boehringer Mannheim). After an
incubation period of 2 hours at 37°C, DNA was digested with 2 U DNase
(RNase-free, Boehringer Mannheim) for 10 minutes at 37°C and the
reaction was stopped with 2 µl of 0.5 mol/L
ethylenediaminetetraacetic acid (EDTA), pH 8.0. Unincorporated
nucleotides were removed using a MicroSpin S-200 HR column (Pharmacia,
Uppsala, Sweden). Fifty µl of hydrolysis buffer (80 mmol/L
NaHCO3, 120 mmol/L
Na2CO3, 120 mmol/L
dithiothreitol) were added to 50 µl of the eluted sample and
hydrolysis was performed at 60°C for 45 minutes to obtain an average
sample size of 150 bp. After addition of 5 µl of stopping solution
(0.2 mol/L Na acetate, 10 mmol/L dithiothreitol, 1% glacial acetic
acid) the sample was precipitated with LiCl/isopropanol. The washed
pellet was resuspended in 100 µl of 50% formamide containing 25
mmol/L dithiothreitol.
Pretreatment of Paraffin Sections
Four-µm paraffin sections mounted on silanized glass slides were deparaffinized and postfixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 minutes at room temperature. After rinsing with Tris-buffered saline (TBS) (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl) sections were treated with 0.2 mol/L of HCl for 10 minutes. After washing in TBS sections were incubated in 20 µg/ml of proteinase K (Sigma Chemical Company, St. Louis, MO) in TBS containing 2 mmol/L of CaCl2 for 15 minutes at 37°C. The reaction was stopped with TBS for 5 minutes at 4°C. After treatment with 0.5% acetic anhydride in 100 mmol/L Tris, pH 8.0, for 10 minutes, sections were rinsed with TBS, dehydrated in graded ethanol, and air-dried.
Hybridization and Autoradiography
For hybridization the labeled sample (1 x 106cpm per section) was diluted in 50 µl of hybridization buffer containing 12.5 mmol/L phosphate buffer, pH 6.8, 12.5 mmol/L Tris, 0.4 mol/L NaCl, 3 mmol/L EDTA, 1.25x Denhardt, 50% formamide, 12.5% dextran sulfate, 0.1 mol/L dithiothreitol, 100 nmol/L S-rATP (Boehringer Mannheim), 60 ng t-RNA, and 30 ng poly(A). The sections were hybridized with the diluted probe overnight at 50°C in a humid chamber containing 2x standard saline citrate (0.3 mol/L NaCl, 30 mmol/L Na citrate, pH 7.0) and 50% formamide. Thereafter, sections were washed with formamide washing buffer (10 mmol/L phosphate buffer, pH 6.8, 10 mmol/L Tris-HCl, pH 7.7, 0.3 mol/L NaCl, 5 mmol/L EDTA, 0.1x Denhardt, 0.07% ß-mercaptoethanol, and 50% formamide) at 45°C once for 1 hour and once for 2 hours, followed by washing in 10 mmol/L Tris-HCl, pH 7.4, 0.5 mol/L NaCl, 2.5 mmol/L EDTA, and 0.07% ß-mercaptoethanol two times for 15 minutes. After RNase A treatment (20 µg/ml, Boehringer Mannheim) in the same buffer for 30 minutes at 37°C, washing was continued overnight in the formamide washing buffer at 37°C. On the next day there was a high-stringent wash in 2x standard saline citrate and 0.07% ß-mercaptoethanol for 30 minutes at 45°C and in 0.1x standard saline citrate and 0.07% ß-mercaptoethanol for 30 minutes at 45°C. After dehydration in graded ethanol, air-dried sections were coated with Ilford K2 photoemulsion (Ilford Ltd., Mobberly, Cheshire, UK). After 1 to 3 weeks of exposure slides were developed with Kodak D19 developer (Eastman Kodak, Rochester, NY) and counterstained with hematoxylin and eosin.
Western Blotting of CK 8 and CK 18
Snap-frozen liver tissue was homogenized in buffer containing 10 mmol/L NaH2PO4, pH 7.4, 5% sodium dodecyl sulfate, and 10% ß-mercaptoethanol. Protein concentration was determined using the Bradford method.22 Samples (20 µg protein per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.23 Equal loading amounts of protein were confirmed by Coomassie blue staining. For immunoblotting proteins were electrotransferred to nitrocellulose membranes (BioRad, Hercules, CA).24 After blocking with 3% nonfat milk in PBS, CKs were detected using a monoclonal mouse antibody against CK 8 (Ks 8.7; Progen, Heidelberg, Germany) in a dilution of 1:500 and a monoclonal mouse antibody to CK 18 (K18.04, Progen) in a dilution of 1:1000. In addition, ß-actin was detected using a monoclonal mouse anti-ß-actin antibody (Sigma) in a dilution of 1:5000. After washing in PBS, blots were incubated with horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulins (DAKO, Glostrup, Denmark) in a dilution of 1:1000. Specific binding was detected using the enhanced chemiluminescence (ECL) Western blotting detection system (Amersham) and exposing the blots to Trimax XDA Plus films (3M Imation, White City, OR). Band intensities were determined with a Docu Gel V video densitometer (MWG-Biotech, Munich, Germany) and RFLP-Scan Software (Scanalytics, Billerica, MA). Accuracy of the ECL method for quantification of CK protein levels was determined by standard curves (not shown).
Immunofluorescence Microscopy
To study alterations of the IF network, immunofluorescence microscopy was performed using the polyclonal rabbit CK antibody 50K160 recognizing CK 8 and CK 1825,26 as well as the monoclonal mouse anti-K7 (Monosan, Uden, Netherlands) and anti-K19 (Amersham) recognizing CK 7 and CK 19, respectively. In addition, the monoclonal antibody LJ4 (kindly provided by Bishr Omary, Palo Alto, CA) directed against CK 8 phosphorylated at Ser 79 was used.16 Immunofluorescent specimens were analyzed with a MRC 600 (BioRad) laser-scanning confocal device attached to a Zeiss Axiophot microscope. The fluorescent images were collected using the confocal photomultiplier tube as full frame (768 x 512 pixels). For dual labeling, separate excitation wavelengths (488 nm for fluorescein isothiocyanate; 568 nm for tetramethylrhodamine B isothiocyanate) from a krypton/argon ion laser were used.
Histology
At the time of harvesting, mouse livers were fixed in 4% neutral buffered formaldehyde solution and embedded in paraffin. Hematoxylin and eosin-stained sections were coded and examined by two pathologists (HD, KZ), who were blinded in regard to the treatment groups.
Routine Serum Biochemistry and Bile Acid Measurements
Serum biochemistry (ALT and AST) was performed by routine clinical
chemistry testing on a Hitachi 717 analyzer (Boehringer Mannheim).
Alkaline phosphatase and total serum bile acid levels were determined
for assessment of the degree of cholestasis. For
determination of total serum bile acid
levels a commercially available 3-hydroxysteroid dehydrogenase assay
(Merck, Darmstadt, Germany) was used. Tests were performed in duplicate
(Tables 1 and 2)
.
|
|
In each group five animals were studied at each time point. Data are reported as arithmetic means ± SEM. Statistical analysis was performed using Student’s t-test as appropriate, or analysis of variance with posttesting when three or more groups were compared. A P value <0.05 was considered significant.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Obstructive cholestasis (CBDL) for 3 days led to biliary type of
hepatocytic necroses (resembling bile infarcts), predominantly in
acinar zones 1 and 2 that were infiltrated by variable numbers of
neutrophils. Interlobular bile ducts were elongated with dilated lumina
and irregular epithelium. The surrounding portal tissue was edematous
and infiltrated by neutrophils. No pronounced ductular reaction was
observed. Necroses were more prominent in 7-day-ligated than in
3-day-ligated mouse livers. In agreement with the histological findings
serum transaminases, alkaline phosphatase, and serum bile acid levels
were significantly elevated in comparison to controls (for details see
Table 1
).
CBDL resulted in a significant increase of CK 8 and CK 18 mRNA levels
after 3 and 7 days (Figure 1A)
. CK 8 and
CK 18 proteins were also increased after 3 and 7 days of CBDL (Figure 1B)
compared to sham-operated animals, whereas ß-actin expression
remained constant (Figure 1B)
. After CBDL, increased CK 8 mRNA was
observed particularly in hepatocytes surrounding bile infarcts (Figure 1C)
, in hepatocytes in acinar zone 1, and also to a minor degree in
bile duct epithelial cells (Figure 1D)
as revealed by in
situ hybridization. Increased CK 8 and CK 18 protein expression
after CBDL was also detected by immunofluorescence microscopy (Figure 1, E and F)
. CBDL resulted in an increased density of the cytoplasmic
IF network predominantly around bile canaliculi (Figure 1F
,
arrowheads). Because neoexpression of CK 7 and CK 19 has previously
been described in hepatocytes in human cholestatic liver
disease,27
additional immunostainings using antibodies
against CK 7 and CK 19 were performed on CBDL livers showing expression
only in the apical submembraneous cytoplasm of cholangiocytes but
negative staining of hepatocytes for these CKs (not shown).
|
To further discriminate between mechanical (eg, increased
canalicular pressure as present in CBDL) and toxic effects (eg, changes
in bile acid pool composition) of cholestasis, mice were either fed a
diet supplemented with potentially toxic CA or hydrophilic nontoxic
UDCA. Feeding of CA (0.1%, 0.5%, 1%) for 7 days led to disseminated
liver cell necroses and a significant increase in the number of mitotic
hepatocytes. Feeding of 1% CA-supplemented diet was, in addition,
associated with dilatation of interlobular bile ducts. Serum
transaminase, alkaline phosphatase, and serum bile acid levels were
also significantly elevated in CA-fed mice in comparison to controls
(for details see Table 2
). Feeding of UDCA did not result in liver cell
necroses, increased mitotic figures, or elevated serum enzyme levels,
but led to a dilatation of medium-sized and larger interlobular bile
ducts.
Feeding of CA stimulated CK 8 and CK 18 mRNA and protein expression in
a dose-dependent manner (Figure 2, A and B)
. After 7 days of CA (0.1%, 0.5%, and 1%, respectively) feeding CK
8 and CK 18 mRNA levels were significantly increased. To determine
whether the increase in CK 8 and CK 18 steady-state mRNA levels
resulted in a comparable increase in protein, immunoblotting was
performed with liver homogenates from CA-fed and control animals
(Figure 2B)
. CA feeding (1%) for 7 days resulted in a significant
increase of CK 8 and CK 18 proteins compared to control animals.
Interestingly, the increase of CK 8 always exceeded that of CK 18 and
the dissociation between CK 8 and CK 18 was particularly prominent in
mice fed 1% CA-containing diet. In situ hybridization
revealed a marked increase of the CK-8 mRNA-related signal in
hepatocytes, and, to a lesser degree, also in bile duct epithelial
cells from CA-fed mice (Figure 2D)
. CK 8 and CK 18 overexpression in
CA-fed animals was also detected by immunofluorescence microscopy in
tissue sections. Staining with a polyclonal antibody against CK 8 and
CK 18 revealed in CA-fed mice a cytoplasmic IF skeletal meshwork with
increased density, particularly at the periphery of enlarged
hepatocytes and around bile canaliculi (Figure 2F)
. Hepatocytes of
CA-fed mice did not express CK 7 and CK 19 (not shown). Feeding the
nontoxic UDCA had no significant effect on CK 8 and CK 18 mRNA and
protein levels (data not shown) and on IF structure at any dose studied
(Figure 3C)
.
|
|
Because mutations of CK phosphorylation sites predispose mice to
toxic liver injury, and cell stress induces CK
phosphorylation14,15
we studied the influence of
obstructive cholestasis and bile acid feeding on the phosphorylation
status of CK 8. Overexpression of CKs after CBDL was accompanied by CK
phosphorylation in cells surrounding bile infarcts and hepatocytes in
acinar zone 1 as revealed by a CK 8 phosphoepitope antibody (Figure 3, A and B)
. Marked IF phosphorylation was also observed in clusters of
hepatocytes in CA-fed animals (Figure 3D)
. In contrast, UDCA-fed
animals did not show phosphorylation of the CK-IF network (Figure 3C)
.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
The CK-IF network is important for the structural integrity of hepatocytes,6 but CK proteins in addition seem to have important nonmechanical functions, including protection from toxic stress, as demonstrated by increased sensitivity of CK-8 knockout (CK 8(-/-)) mice to DDC toxicity and of mice overexpressing a dominant-negative human CK 18 serine 52-alanine mutant to griseofulvin toxicity.6,14,16 Comparative studies with CK 8(-/-)- and CK-18 knockout (CK 18(-/-))-mice demonstrated the overall importance of CK 8 in MB formation and in protection from toxic stress as indicated by enhanced mortality and lack of MB formation in DDC-intoxicated CK 8(-/-) mice.11,16 Although hepatocytes of both CK 8(-/-) and CK 18(-/-) mice lacked a cytoplasmic IF network, CK 18(-/-) animals did not show an increased mortality after DDC feeding but developed MBs even earlier compared to intoxicated wild-type mice.16 Interestingly, feeding of potentially toxic CA (but not of the nontoxic hydrophilic UDCA) not only induced hepatic CK expression but resulted in a more pronounced increase of CK 8 in comparison to CK 18 and thus disturbance of the 1:1 CK 8:CK 18 ratio that is essential for correct IF formation. In addition, animals of the experimental groups with the highest bile acid levels showed the most pronounced increase of CK 8 expression, which further underlines the significance of CK 8 in toxic liver injury. Isolated CK 8 could either be degraded or aggregated and thus be the substrate of MB formation.29
Increased CK synthesis and phosphorylation occurred in hepatocytes in acinar zone 1 and in the vicinity of bile infarcts that are areas with probably the highest bile acid concentrations and resulting toxic stress. Phosphorylation of CKs could represent a cellular defense mechanism and may support hepatocytes in handling bile acid-induced toxic stress, because CK phosphorylation decreases intracellular adenosine triphosphate levels and may enable the hepatocyte to maintain a phosphate reserve.6 The lack of effects of nontoxic UDCA on CK 8 and CK 18 expression and phosphorylation further supports the concept of a toxic stress response. The detailed mechanisms of how stress-induced CK phosphorylation may protect against certain types of liver injury, however, remains an open question.6
An alternative, although less attractive, explanation for hepatic up-regulation of CK-IFs in CA-fed mice could be a role of the CK-IF network in transcytosis and targeting of bile acid transporters to the canalicular membrane, similar to the role of other components of the cytoskeleton.30-34 IFs are closely associated with the canalicular membrane and provide a structural scaffold of the canaliculus, which is believed to be important for its formation and maintenance.1,2,7,9 However, evidence for the involvement of CK-IF in the regulation of bile flow is only fragmentary and the detailed role of CK-IF in bile formation has not yet been established.1,6 CK 8(-/-) and transgenic mice with disrupted CK 8/18 network because of ectopic CK 14 expression showed markedly decreased bile flow and bile acid secretion.12,35 Because canalicular secretion is the rate-limiting step in bile formation,1,3 it is likely that this defect is located at the canalicular membrane. Interestingly, the livers of these mice are unable to handle higher bile acid concentrations, as shown by a decreased Tmax in response to increased bile acid load,12,35 which is consistent with defective vesicular targeting of bile acid transporters to the bile canalicular membrane. In addition, a possible role for CKs in transcellular vesicle transport has recently been discussed based on the finding of abnormally dispersed zymogen granules in pancreatic acinar cells in mice expressing human CK 8 or a dominant-negative mutant transforming growth factor-ßII receptor.36 The authors speculated that the observed pancreatic insufficiency in these mice could be because of a defect in the processing and/or secretion of zymogen granules, suggesting that CKs may also be involved in vesicular targeting. In addition, recent studies demonstrated binding affinity of CK 18 for bilirubin, and the authors speculated that CK 18 may play a role as a membrane reservoir in the transport and secretion of bile pigments.37 Together with our finding of CK 8 and CK 18 overexpression leading to an increased density of the CK-IF network and a thickened pericanalicular sheath in livers exposed to increased bile acid levels, a direct or indirect role of the CK-IFs in vesicular transport and targeting or bile secretion could be possible. However, the lack of effect of UDCA (known to stimulate bile secretion) on CK expression in the present study argues against a direct role of CKs in bile secretion.38,39
This study provides the first evidence that bile acids lead to marked alterations of the CK-IF network. We could demonstrate that up-regulation of CK 8 and CK 18 in cholestatic conditions is not merely because of mechanical stress but mediated by potentially toxic bile acids. Furthermore, toxic bile acids induce CK phosphorylation, which could reflect a stress response against the toxic effects of bile acids.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by grants 7401-MOB from the Austrian Science Foundation (to K. Z.), 8522 and 7171 from the Jubilee Funds of the Austrian National Bank (to M. T.), and the Joseph Skoda Prize from the Austrian Society of Internal Medicine (to M. T.).
Presented in part at the annual meeting of the American Association for the Study of the Liver Diseases, November 1999, Dallas, Texas, and published in abstract form (Hepatology 1999, 30:464A).
Accepted for publication December 17, 2001.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  M. T. Flowers, M. P. Keller, Y. Choi, H. Lan, C. Kendziorski, J. M. Ntambi, and A. D. Attie Liver gene expression analysis reveals endoplasmic reticulum stress and metabolic dysfunction in SCD1-deficient mice fed a very low-fat diet Physiol Genomics, May 1, 2008; 33(3): 361 - 372. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Fickert, U. Stoger, A. Fuchsbichler, T. Moustafa, H.-U. Marschall, A. H. Weiglein, O. Tsybrovskyy, H. Jaeschke, K. Zatloukal, H. Denk, et al. A New Xenobiotic-Induced Mouse Model of Sclerosing Cholangitis and Biliary Fibrosis Am. J. Pathol., August 1, 2007; 171(2): 525 - 536. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Fickert, A. Fuchsbichler, H.-U. Marschall, M. Wagner, G. Zollner, R. Krause, K. Zatloukal, H. Jaeschke, H. Denk, and M. Trauner Lithocholic Acid Feeding Induces Segmental Bile Duct Obstruction and Destructive Cholangitis in Mice Am. J. Pathol., February 1, 2006; 168(2): 410 - 422. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Zhong, Q. Zhou, D. M. Toivola, G.-Z. Tao, E. Z. Resurreccion, and M. B. Omary Organ-specific stress induces mouse pancreatic keratin overexpression in association with NF-{kappa}B activation J. Cell Sci., May 1, 2004; 117(9): 1709 - 1719. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G.-Z. Tao, D. M. Toivola, B. Zhong, S. A. Michie, E. Z. Resurreccion, Y. Tamai, M. M. Taketo, and M. B. Omary Keratin-8 null mice have different gallbladder and liver susceptibility to lithogenic diet-induced injury J. Cell Sci., November 15, 2003; 116(22): 4629 - 4638. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Fickert, M. Trauner, A. Fuchsbichler, C. Stumptner, K. Zatloukal, and H. Denk Bile Acid-Induced Mallory Body Formation in Drug-Primed Mouse Liver Am. J. Pathol., December 1, 2002; 161(6): 2019 - 2026. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
