Simple epithelial keratins are dispensable for cytoprotection
in two pancreatitis models
Diana M.
Toivola1,
Hélène
Baribault2,
Thomas
Magin3,
Sara A.
Michie4, and
M. Bishr
Omary1
Departments of 1 Medicine and 4 Pathology, Palo Alto
Veterans Affairs Medical Center and Stanford University, Palo Alto
94304; 2 Deltagen, San Carlos, California 94205; and
3 University of Bonn, D-53117 Bonn, Germany
 |
ABSTRACT |
Pancreatic acinar cells express
keratins 8 and 18 (K8/18), which form cytoplasmic filament (CF) and
apicolateral filament (ALF) pools. Hepatocyte K8/18 CF provide
important protection from environmental stresses, but disruption of
acinar cell CF has no significant impact. We asked whether acinar cell
ALF are important in providing cytoprotective roles by studying keratin filaments in pancreata of K8- and K18-null mice. K8-null pancreas lacks
both keratin pools, but K18-null pancreas lacks only CF. Mouse but not
human acinar cells also express apicolateral keratin 19 (K19), which
explains the presence of apicolateral keratins in K18-null pancreas.
K8- and K18-null pancreata are histologically normal, and their acini
respond similarly to stimulated secretion, although K8-null acini
viability is reduced. Absence of total filaments (K8-null) or CF
(K18-null) does not increase susceptibility to pancreatitis induced by
caerulein or a choline-deficient diet. In normal and K18-null acini,
K19 is upregulated after caerulein injury and, unexpectedly, forms CF.
As in hepatocytes, acinar injury is also associated with keratin
hyperphosphorylation. Hence, K19 forms ALF in mouse acinar cells and
helps define two distinct ALF and CF pools. On injury, K19 forms CF
that revert to ALF after healing. Acinar keratins appear to be
dispensable for cytoprotection, in contrast to hepatocyte keratins,
despite similar hyperphosphorylation patterns after injury.
acute pancreatitis; acinar cell; caerulein; choline-deficient diet; keratin phosphorylation
 |
INTRODUCTION |
INTERMEDIATE
FILAMENT (IF) proteins are one of the three major cytoskeletal
components of mammalian cells, which also include microfilaments and
microtubules. In simple epithelia, as in hepatocytes and exocrine
pancreatic acinar cells, keratin polypeptides 8 (K8; a type II keratin)
and 18 (K18; a type I keratin) are the major IF proteins (21,
33). Biliary and pancreatic ductal cells also express, in
addition to K8/18, other type II (K7) and type I (K19) keratins.
Keratins form obligate noncovalent heteropolymers that consist of at
least one type I and one type II keratin. In pancreatic acinar cells,
K8/18 form a cytoplasmic (C) filamentous network and a distinct
apicolateral (AL) band of filaments (8, 36, 43).
One established keratin function in the skin, cornea, and liver is to
maintain cellular integrity (13, 14, 30, 38, 41), but
little if anything is known regarding keratin function in the pancreas.
Keratins in simple epithelia may also regulate the availability of
other abundant cellular proteins such as heat stress and 14-3-3 proteins (21).
It is well established that hepatocyte K8/18 play an important role in
hepatocyte cell resilience and in protecting the liver from damage
induced by a variety of toxins. This conclusion is based on
1) the extensive spontaneous liver hemorrhage noted in K8
null mice (6), 2) the significant hepatocyte
fragility when keratins are disrupted in transgenic mouse livers on
overexpression of dominant-negative K18 Arg89
Cys (called
K18C mice; Ref. 17), and 3) the increased
susceptibility of K18C (18, 44) and K8-null (
/
) mice
(26, 44) to toxin-induced liver injury. In the case of
K18C mice, the above findings are associated with keratin C filament
disruption in the liver. However, despite similar C filament disruption
in hepatocytes and pancreatic acinar cells of K18C mice, the K18C mouse
pancreas is histologically normal (17). In addition,
pancreata of K18C mice maintain their AL keratin filaments and do
not suffer significant consequences in terms of pancreatic stimulated
secretion or susceptibility to pancreatic injury that is induced via
two established pancreatitis mouse models (43). Similarly,
although K18C acinar cells had lower viability compared with cells
isolated from wild-type (WT) mice (68 vs. 93%, respectively), this
difference was significantly less dramatic than hepatocyte viability
differences on isolation from K18C (22%) vs. WT (95%) mice
(43). This raised the hypothesis that the remaining AL
filaments of acinar cells, which form more prominent bundles in acini
than in hepatocytes, may compensate functionally for the lack of
cytoplasmic filaments. Alternatively, acinar cells may use nonkeratin
proteins to serve keratin-like protective roles.
Here we tested the above hypotheses using K8
/
(5, 6)
and K18
/
mice (29) that reportedly had no pancreatic
histological abnormalities and, although not studied in detail, had
absent or normal pancreatic keratin filaments, respectively. We
challenged these mice, and their heterozygous and WT littermates, using
the two well-established pancreatitis models of caerulein (an analog to
CCK), which induces pancreatic secretion (16), and
choline/methionine-deficient diet supplemented with 0.5% ethionine (CD
diet) (25). We examined pancreatic histology and serology,
keratin and F-actin organization, and keratin phosphorylation
under basal conditions and on induction of pancreatitis. We also
examined the viability and secretagogue response of acini.
 |
MATERIALS AND METHODS |
Animals and primary antibodies.
Heterozygous (+/
) and homozygous (
/
) K8 or K18 mice and their WT
littermates were expanded and genotyped as described (5, 29). The targeted
/
mutation of K8 causes midgestational
lethality in homozygous mice, with bleeding into the embryonic liver in ~50% of the mice. The remaining K8
/
mice have a normal life span
but do suffer from colorectal hyperplasia, chronic colitis, mild serum
transaminase elevation, and female sterility (in an FVB/N background
strain; Ref. 5). The only reported pathological phenotype
of the K18
/
mice is spontaneous accumulation of Mallory bodies in
the liver as the mice age (29). The K8 and K18 +/
mice
do not have a phenotype (5, 29).
Human pancreatic tissues were provided by Dr. Vivek Mehta (Stanford
University) under a protocol that is approved by the Administrative Panel on Human Subjects in Medical Research. The primary antibodies (Ab) used were rat Troma I [anti-mouse (m)K8; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA], rat Troma II and
III (anti-mK18 and mK19, respectively; Ref. 7), mouse 4.62 [anti-human (h)K19; Sigma, St. Louis, MO], mouse KA4 (anti-hK19; provided by Dr. Robert Webster, Ciba Corning, Alameda, CA); rabbit 8592 (anti-h,mK8/18; Ref. 17); rabbit Endo B (anti-mK18;
provided by Dr. Robert Oshima, The Burnham Institute, La Jolla, CA);
anti-K16 and anti-K17 rabbit Ab (provided by Dr. Pierre Coulombe, Johns Hopkins University, Baltimore, MD); mouse RCK 105 (anti-K7; ICN Biochemicals, Aurora, OH), mouse 5B3 [anti-hK8 phospho-(p)S431 and
anti-mK8 pS436; Ref. 20], and mouse LJ4 (anti-hK8 pS73
and anti-mK8 pS79; Ref. 23).
Pancreatitis models.
All experiments included mice that were sex- and age matched. In the
caerulein model, mice were injected intraperitoneally with 50 µg/kg
caerulein (Sigma) or with carrier (0.9% NaCl) 7 times hourly and then
killed 12 h after the first injection. The number of animals in
each group was three for saline controls, six for caerulein (for +/
and
/
genotypes), and 12 for WT. Alternatively, mice were killed
1 h after a single injection, 1 h after six hourly
injections, or after 48, 120, or 240 h of recovery after 7 hourly
injections. All time points are given as hours after the first
injection. Food was withheld from the mice for 16-18 h before the
injections, but water was given ad libitum. In the CD diet
model, two types of experiments, lethality and serology/histology, were
performed. In both experiments, mice were fed (after a 12-h food
starvation) a powdered CD diet (Teklad, Madison, WI) supplemented with
0.5% DL-ethionine (Sigma) or were fed normal mouse chow
(Dean's Animal Feeds, Belmont, CA). For the serology/histology
experiment, mice (n = 3 for control diet, n = 7 for CD diet per genotype group; age = 6-7 wk) were killed 60 h after initiating the diet, followed
by serum and tissue isolation as detailed in Tissue processing,
histology grading, enzyme assays, and fluorescence and electron
microscopy. For the lethality experiment, mice (6 wk old,
n = 10-26 per genotype) were fed normal or CD diet
for 72 h and then were switched to normal diet and monitored for 7 additional days.
Tissue processing, histology grading, enzyme assays, and
fluorescence and electron microscopy.
Mice were killed by CO2 inhalation, and blood was drawn by
intracardiac puncture (0.3-1.0 ml) for subsequent serum
collection. The pancreas was then excised and divided into three or
more pieces for immediate fixation in 10% buffered formalin (pH 7.4;
Columbia Diagnostics, Springfield, VA), embedding in optimum cutting
temperature compound (Miles, Elkhart, IN), then freezing for
cryosectioning or snap-freezing in liquid N2 for subsequent
biochemical analyses. Formalin-fixed tissues were processed for
hematoxylin and eosin staining at Histo-tec Laboratory (Hayward, CA).
Pancreatitis severity was assessed by assigning a relative score for
vacuoles, cell death, edema, and inflammation on examination of the
hematoxylin and eosin-stained sections (0 = none, 1 = mild,
2 = moderate, and 3 = severe) by a pathologist (S. A. Michie) who did not know the mouse genotypes from which the sections
were obtained. Serum amylase and lipase were assayed using an Express
Plus instrument (Bayer, Tarrytown, NY). Immunofluorescence staining and
transmission electron microscopy (EM) were performed as described
(43), except that nuclear staining was also done with
YO-PRO-1 iodide or Toto-3 iodide (Molecular Probes, Eugene, OR; Refs.
19 and 24).
Protein preparation from whole tissue and by high-salt
extraction.
Pancreas tissue samples were homogenized with 600 µl of buffer
[0.187 M Tris · HCl (pH 6.8), 3% SDS, and 5 mM EDTA] per 25 mg of tissue using a Teflon homogenizer. Samples were heated (98°C, 5 min) and sheared with a 22-gauge needle, then centrifuged (2 min,
14,000 rpm) to remove undissolved tissue. The protein content of the
supernatant was determined using the BCA protein assay (Pierce,
Rockford, IL), and samples (15 µg/lane) were analyzed by SDS-PAGE
(22). Gels were stained with Coomassie blue or transferred to membranes for immunoblotting and visualization of Ab-bound species
by enhanced chemiluminescence. High-salt extraction (HSE), an
established method to obtain highly enriched keratin fractions (1), was done by homogenizing the pancreas with a Teflon
homogenizer in 1% Triton X-100 and 5 mM EDTA in PBS (pH 7.4),
containing 0.1 mM phenylmethylsulfonyl fluoride, 10 µM pepstatin, 10 µM leupeptin, and 25 µg/ml aprotinin (4°C, 2 min), followed by
centrifugation (10 min, 14,000 rpm, 4°C). The pellet was further
homogenized (4°C, 100 strokes) in high-salt buffer [10 mM
Tris · HCl (pH 7.6), 140 mM NaCl, 1.5 M KCl, 5 mM EDTA, and
0.5% Triton X-100] containing the above protease inhibitors. Samples
were incubated with rocking (4°C, 30 min) and centrifuged as above.
Pellets were dissolved with sample buffer and then analyzed by
SDS-PAGE.
Isolation and stimulated secretion of pancreatic acini.
Pancreatic acini were isolated from mice by CLSPA-collagenase
(Worthington, Lakewood, NJ) digestion as described (31,
43). The viability and stimulated secretion response were
determined by measuring the extent of amylase release from isolated
acini after incubation (10-60 min, 37°C) in the presence or
absence of 0.1 nM [Tyr(SO3H)27]-CCK-8 amide
(CCK8; Sigma). Samples were analyzed in duplicate, and experiments were
repeated three times. Amylase release into the incubation buffer was
measured using the Phadebas amylase test as described
(45), and values were calculated as percentage of
released/total amylase in the acini. The total amylase content was
determined by solubilization of the acini in SDS.
Statistical analysis.
Numerical data were compared using t-test or Wilcox test and
the JMP 3.1 program (DataViz, Trumbull, CT). Data are given as means ± SE. For the lethality experiments, results were analyzed using Fisher's exact test.
 |
RESULTS |
Keratin expression in normal, K8
/
, and K18
/
mice.
We examined keratin expression in the mouse models by immunoblotting
total pancreas extracts with Ab to K8, K18, or K19 and by visualizing
the keratin profile obtained after HSE. As predicted, keratins were not
detected in K8
/
mice (Fig. 1) due to
instability of type I keratins (K18 and K19) in the absence of type II
(K8). In K18
/
mice, K8 expression is significantly decreased, but not absent, likely because of relative stabilization by the low levels
of K19 (Fig. 1). K19 levels, relative to K8, increase significantly in
K18
/
mice as determined by immunoblotting (Fig. 1A) and
HSE (Fig. 1B). Assignment of the keratin bands in the HSE
(Fig. 1B) was confirmed by immunoblotting using
K8/18/19-specific Ab (not shown). The HSE profile (Fig. 1B)
indicates that there is no obvious upregulation of other keratins.
Hence, pancreata of WT mice express K8/18/19, but K8
/
mice
pancreata show no detectable keratins and K18
/
mice pancreata
express decreased K8 but increased K19 levels.

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 1.
Keratin (K) expression in pancreata of mice with
wild-type (WT), homozygous null ( / ), and heterozygous (+/ ) K8 and
K18 genotypes. Pancreata were homogenized in SDS-containing sample
buffer and analyzed by immunoblotting (A) with antibodies
(Ab) to K8 (Troma I), K18 (anti-Endo B), and K19 (Troma III)
B: extraction of keratins using a high-salt buffer as
described in MATERIALS AND METHODS. Note the relative
increase of K19 levels in K18 / mice (lane 5).
Mr, relative molecular mass.
|
|
Keratin and F-actin distribution within the exocrine pancreas.
Immunostaining of WT mouse pancreas revealed the unexpected result that
mouse acinar cells manifest weak but distinct K19 AL staining (Fig.
2, d and g) in
addition to the previously described ductal and centroacinar K19
localization (8). In contrast to mice, human acinar cells
do not express K19, although K19 is present in ductal and centroacinar
cells that appear more prominent in human compared with mouse pancreata
(Fig. 3). Immunostaining of pancreata
from K8
/
and K18
/
mice confirms the biochemical data shown in
Fig. 1. For example, K8
/
mice pancreata lack keratin filament
staining in acinar cells (Fig. 2, b, e, and
h), with scarce K19 staining in some ductal cells (Fig.
2e) that express small amounts of K7 (not shown), another
type II keratin. Staining and blotting with anti-K7, K16, and K17 Ab
showed no upregulation of these proteins in acinar cells of K8
/
or
K18
/
mice (not shown). In contrast to WT acinar cells, K18
/
acinar cells lack C keratins but show an intense AL K8 and K19 staining
(Fig. 2f). F-actin localizes similarly in all mouse
genotypes along the AL membranes (Fig. 2,
g-i). Double staining of F-actin and K19
confirms the AL localization of K19 and shows that K19 is localized on the cytoplasmic side of the actin band [Fig. 2g,
inset; note that actin (green) is more cortical than keratin
(red) staining]. Staining with anti-mK18 Ab showed C and AL filaments
in WT pancreata (i.e., similar staining to that in Fig. 2a;
not shown) but absent staining in K8
/
and K18
/
mice (not
shown).

View larger version (151K):
[in this window]
[in a new window]
|
Fig. 2.
Keratin and F-actin distribution in the exocrine pancreas
of WT, K8 / , and K18 / mice. K8 (a-c, red), K19
(d-i, red), and F-actin (g-i, green)
were stained with Troma I, Troma III, and FITC-phalloidin,
respectively. Nuclei (a-f, blue) were stained with
Toto-3. Frozen pancreas sections were fixed with acetone or with 0.5%
formaldehyde for keratin and F-actin staining, respectively. Double
labeling of F-actin and K19 was done after 0.5% formaldehyde fixation.
Panels a-c, d-f, and
g-i represent double labeling as indicated. A, acinar
cells; D, ductal or centroacinar cells; arrowheads in g,
F-actin staining in endothelial cells. Bar = 10 µm. Note that
the cytoplasmic filaments in WT mice in a are composed of
K18 (K18 staining is identical to K8; not shown) and K8, whereas K19 is
found preferentially in apicolateral (AL) filaments. K18 / mice
pancreata lack keratin cytoplasmic (C) filaments but retain the AL
filaments, at least in part, due to K19 partnering with K8. Double
staining of F-actin and K19 confirms the AL localization of K19 and
shows that K19 is localized on the cytoplasmic side of the actin band
(g, inset).
|
|

View larger version (141K):
[in this window]
[in a new window]
|
Fig. 3.
K19 distribution in normal mouse and human exocrine pancreas. Human
pancreatic biopsies were triple-stained with YO-PRO-1 (nuclear stain in
C) and Ab to human (h) K8/18 (A) and hK19
(B). Centroacinar cell cytoplasm (positive for anti-K19) and
nuclei are indicated by arrowheads (b and c), and
an outline of 3 acini is highlighted with a dotted line for
orientation. Bar = 10 µm. The schematic in D
highlights the species difference in human and mouse K19 expression.
Gray shading, K19; flat cells, ductal and centroacinar cells.
|
|
Ultrastructural examination of WT, K8
/
, and K18
/
mice pancreata
supported the staining results noted above. For example, EM of K8
/
mice pancreata showed no apparent keratin bundles (Fig.
4c) and absent immunogold
labeling using anti-K8/18 Ab (Fig. 4d), with normal
appearance of desmosomes (Fig. 4c). In contrast, K18
/
and WT mice keratin filament bundles were noted in proximity to the
lumen and next to normal-appearing desmosomes (Fig. 4; note the gold
particles in b and f). The keratins in
the K18
/
mice appeared less electron dense than those in WT mice
(Fig. 4; compare a and e). K8 and K18+/
mice
had no pancreatic ultrastructural differences compared with WT mice
(not shown). Together, C keratin filaments of WT mouse acinar cells
contain K8/18, whereas the AL filaments contain K8/18/19. K8
/
acinar cells do not express any detectable keratin, whereas K18
/
mice lack C filaments but retain AL filaments due to the presence of
K19. The differences of acinar cell keratins in WT, K8
/
, and
K18
/
mice do not affect desmosomes or F-actin distribution.

View larger version (188K):
[in this window]
[in a new window]
|
Fig. 4.
Ultrastructural assessment of WT, K8 / , and K18 / mice
pancreata. Thin sections were processed for conventional transmission
electron microscopy (EM; a, c, and e)
and for keratin immunogold EM labeling (b, d, and
f) using anti-K8/18 rabbit Ab 8592 and goat
anti-rabbit Ab conjugated to 10-nm gold particles. Normal rabbit Ab was
used as a negative control and manifested no gold particle labeling
(not shown; see also K8 / mouse staining in d, which
lacked keratin filaments). K, keratin filament bundles (note
intermediate filament decoration with gold particles in b
and f); D, desmosome; L, lumen; Z, zymogen granule;
Bar in a = 1 µm (for a, c, and
e); bar in b = 0.5 µm (for b,
d, and f).
|
|
Pancreatic histology, acini viability, and acini response to
stimulated secretion.
We compared the histology and isolated acini viability of K8
/
and
K18
/
mice with WT mice pancreata. K8
/
and K18
/
mice pancreata appeared histologically normal in young and older (up to 7 mo
old) mice (see, e.g., Fig. 5,
a-c). There were very few focal areas of
abnormal acinar cell organization in K8
/
and K18
/
mice of
unclear significance (not shown). Isolated acini from all mouse
genotypes excluded trypan blue similarly, but basal amylase secretion,
a more accurate reflection of cell leakiness and viability, showed some
differences. Hence basal amylase release at 30 min (37°C) was similar
in WT and K18
/
mice, whereas K8
/
mice showed a lower viability
(Table 1). However, acini from K18
/
and K8
/
mice released amylase in response to CCK8 similarly, thereby indicating that the acinar cells from these mice possess normal
functional signaling mechanisms for stimulated secretion. In addition,
more stringent collagenase digestion to isolate acinar cells did not
give any significant differences in cell viability when comparing WT
with K8
/
or K18
/
mice (not shown).

View larger version (117K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of CD diet and caerulein on the pancreatic
histological appearance in WT, heterozygous, K8 / , and K18 /
mice. Mice were allowed to eat the CD diet (CDD;
d-f) ad libitum for 60 h, whereas control mice
were fed standard mouse chow. Mice receiving the caerulein treatment
(g-i) were injected with caerulein (50 µg/kg ip)
7 times at hourly intervals and then were killed 12 h after the
first injection. Control mice for the caerulein model were injected
with saline (a-c). Indistinguishable results in control
mice of the CD diet and caerulein experiments were observed (not
shown). Tissues were stained with hematoxylin and eosin. D, dead cells;
E, edema; V, vacuoles.
|
|
Acute pancreatitis phenotypes in WT, K8
/
, and K18
/
mice.
To study the physiological effect of cytoplasmic or total acinar cell
keratin filament absence, we subjected WT, K8
/
, and K18
/
mice
and their heterozygous littermates to two established acute
pancreatitis models, namely the CD diet and caerulein injections. Feeding mice the CD diet for 72 h and then switching to normal chow for 7 days showed no significant lethality differences (Table 2). Histologically, the CD diet and
caerulein injections induced acute pancreatitis in all mouse types,
with vacuole formation and cell death in the CD diet model and coupled
with edema and mild inflammation in the caerulein model (Fig. 5 and
Table 3). However, the histological edema
and inflammation scores of the caerulein-induced damage in K8
/
mice
were less dramatic than WT mice (Fig. 5 and Table 3), despite more
prominent vacuolization for both null mouse types (Table 3). K8+/
mice also had a significantly lower inflammation score than WT mice
(Table 3). No hemorrhage was noted in the caerulein-treated mice. The
CD diet mice had similar increases in serum amylase and lipase (which
are markers of pancreatic injury) as the WT mice, with a trend toward
more prominent serum amylase elevations in K18
/
and less prominent amylase rises in K8
/
mice (Table 4).
There was a statistically significant amylase difference in untreated
K18
/
versus WT sera (P = 0.01) and a similar trend
in untreated K8
/
sera, which likely reflect mild underlying
hepatitis and/or necrosis, given that mouse hepatocytes also produce
amylase but not lipase (28). Therefore, absence of
pancreatic cytoplasmic or total keratins appears inconsequential in
terms of susceptibility to pancreatic injury and, if anything, may even
be somewhat protective. This is in sharp contrast to the marked
propensity for liver injury in mice that have cytoplasmic disruption or
total absence of keratin filaments (18, 26, 44).
View this table:
[in this window]
[in a new window]
|
Table 3.
Histological grading of vacuoles, cell death, edema, and inflammation
in acute pancreatitis mouse models
|
|
Keratin dynamics during acute pancreatitis.
Caerulein injections induce a rapid (within 1 h) breakdown of
WT and K18C pancreatic keratin and actin filaments (43).
Both C and AL keratin filaments almost completely disappear in WT
acinar cells (43), and a similar breakdown of K18
/
AL
filaments also occurs (not shown). We previously showed that acinar
cells of K18C and WT mice begin to recover 7 h after stopping
caerulein injections (i.e., the 12-h time point) and form bona fide
cytoplasmic filaments (43). Although C filaments are
basally absent in K18
/
acinar cells, they surprisingly do form on
recovery (Fig. 6, b and
f). The newly formed keratin C filaments are
composed of K8 and K19 in K18
/
mice (Fig. 6, b and
f) and of K8, K18, and K19 in WT mice (Fig. 6,
a and e; K18 staining was confirmed by Troma II
Ab staining, not shown). The K19 and K8 staining colocalizes, as
determined by double labeling, and recovery from caerulein injury in
K8
/
mice is not associated with induction of any acinar keratin
filaments (not shown). After 5 days, keratin C filaments in K18
/
mice pancreata decrease, with a concomitant increase of AL filaments,
whereas WT mice pancreata show widespread keratin C filaments (not
shown). After 10 days (240-h time point), keratin organization returns
in WT and K18
/
mice to the preinjury state (Fig. 6, c,
d, g, and h), with normal-appearing
histological architecture (not shown) and normal F-actin organization
(Fig. 6d, inset). Antibodies to K16, K17, or K7
did not manifest any altered staining in caerulein-treated mice (not
shown).

View larger version (86K):
[in this window]
[in a new window]
|
Fig. 6.
Keratin filament reorganization on caerulein-induced
pancreatitis in WT and K18 / mice. Mice were killed 48 or 240 h
(10 days) after caerulein injections, and frozen sections of the
isolated pancreata were fixed in acetone and then stained with
anti-K8/18 (Ab 8592; a-d), anti-K19 (Ab Troma III;
e-h), anti-K8 phospho(p)S436 (Ab 5B3;
i-l), and FITC-phalloidin (inset in d).
Initially, caerulein induces a dramatic acinar keratin filament
reorganization with loss of AL + C filament staining in WT mice
(43) and loss of AL filament staining in K18 / mice
(not shown). Note that K18 / mice, which normally lack C filaments
(see Fig. 2), express after 48 h de novo keratin C filaments that
stain with K8 and K19 Ab (b and f, respectively).
These C filaments disappear by 10 days in K18 / mice, with reversion
to basal state staining (d and h). Also note
keratin hyperphosphorylation during early recovery (i and
j), which reverts to baseline (near-absent) levels on
complete recovery (k and l).
|
|
Acinar cell keratin phosphorylation changes in caerulein-induced
pancreatitis.
We used phosphoepitope-specific keratin Ab to ask whether keratin
phosphorylation is altered upon pancreatic injury in WT and K18
/
mice. This is based on the strong correlation of keratin hyperphosphorylation with liver injury in vivo (21) and
the protective effect it has from hepatotoxic injury (19).
The cytoplasmic filaments that form on recovery from caerulein-induced
injury contain K8 that is newly phosphorylated on S436 (Fig. 6,
i and j) and S79 (not shown but very similar to
i and j). The K8 pS436 filaments make up a
distinct subpopulation of the overall keratin pool (not shown), and K8
pS436 becomes dephosphorylated by 240 h when acini are nearly
fully recovered (Fig. 6, k and l; i.e., similar
to the staining pattern before injury, with faint staining particularly
of some ductal cells; not shown). Immunoblot analysis of whole
pancreatic extracts from caerulein-treated WT and K18
/
mice (Fig.
7) confirmed the immunofluorescence data
shown in Fig. 6. Initially, keratins degrade in WT and K18
/
mice
pancreata starting a few hours after caerulein injection (e.g., see
multiple K8 reactive bands; Fig. 7). This is followed by increased
keratin synthesis, particularly K19 in WT and K18
/
mice (Fig. 7;
e.g., 48-h time point), during recovery.

View larger version (73K):
[in this window]
[in a new window]
|
Fig. 7.
Keratin expression during acute caerulein-induced
pancreatitis in WT and K18 / mice. Mice were given hourly injections
of 50 µg/kg ip caerulein and killed 5 (5 injections during the first
5 h), 48, 120, or 240 h (7 injections during the first 6 h each) after the first injection. Pancreata were then removed and
homogenized, followed by immunoblotting of the homogenates (15 µg/lane) with antibodies to K8 (Troma I), K18 (anti-Endo B), K19
(Troma III), K8 pS436 (5B3), and K8 pS79 (LJ4). Note the disappearance
(Coomassie-stained bands indicated by arrows) of some unknown proteins
(likely due to degradation) and the appearance of other proteins
(indicated by asterisks) during the early 5- and 48-h time points that
revert to baseline by 240 h.
|
|
In the case of the CD diet, K8
/
mice had no keratin or
phosphokeratin staining (not shown). WT and K18
/
mice that survived the CD diet had normal-appearing keratin filaments by day 10 and also had increased K8 pS79 and pS436 phosphorylation (not shown). Together, these results indicate that pancreatic keratins respond to
injury by reversible hyperphosphorylation as occurs in the liver.
However, acinar cells do not appear to be impacted by the absence or
disruption of keratins, in terms of an exaggerated injury response, as
occurs in hepatocytes.
 |
DISCUSSION |
The major findings of this study are the following: 1)
Absence of keratins is well tolerated in the pancreas, not only under basal conditions but more surprisingly in the context of two
pancreatitis injury models (Fig. 5 and Tables 1-4). This finding
is unexpected given that the same keratins (K8/18) in hepatocytes play
a major cytoprotective role, in that their absence or disruption
results in markedly fragile hepatocytes and in dramatically increased susceptibility to stress- or toxin-induced liver injury. This includes
liver exposure to griseofulvin, acetaminophen, pentobarbital, partial
hepatectomy, and microcystin (17, 18, 44). 2)
Contrary to previous reports (Ref. 8 and references
therein), K19 is expressed in mouse acinar cells, where it is
preferentially localized in the AL compartment and is not present in C
filaments that contain K8/18 (Figs. 1, 2, and 4). K19 acinar cell
expression provides one potential mechanism for stabilization of
keratin AL filaments in K18
/
and in K18 dominant-negative mice that
overexpress K18 Arg89
Cys. Mouse K19 acinar cell
expression is not conserved in human acinar cells (Fig. 3).
3) Findings of this study, coupled with earlier findings
(43), help define two keratin filament compartments in
mouse pancreatic acinar cells (summarized in Fig.
8): a C compartment that contains K8/18
and an AL compartment that contains K8/18/19. Both compartments are
absent in K8
/
mice, whereas the AL compartment remains intact in
conjunction with an absent or disrupted C compartment in K18
/
or
K18C mice, respectively. 4) Keratin filaments undergo a
dramatic and reversible reorganization in response to pancreatic injury
in vivo, in association with reversible keratin hyperphosphorylation (Figs. 6 and 7). Most notably, pancreatic K19 becomes overexpressed in
K18
/
mice, particularly after injury, and acquires the ability to
form C filaments that reorganize into their basal state AL distribution
on recovery.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 8.
Pancreatic acinar cell keratin filament phenotypes vary depending
on genotype. A schematic representation of keratin filament
organization highlights the normal situation of AL and C filaments in
WT mouse acini, absent keratin filaments in K8 / acini, absent C but
prominent AL bundles in K18 / acini (this study), and disrupted C
filaments with prominent AL bundles in transgenic mice that overexpress
a K18 dominant-negative mutation (Arg89 Cys)
(43). The equivalent Arg in epidermal keratins is commonly
mutated in human epidermal skin diseases (13,
14, 30). The AL bundles likely remain intact
in K18 / mice acinar cells due, at least in part, to the
preferential localization of K19 in that compartment.
|
|
K19 is "tailless" but not idle.
K19 is unique among the keratin family members since it has a very
short (13 amino acid) COOH-terminal non-
-helical segment (called the
"tail" in IF proteins) that is significantly longer in all other
keratins (4, 11, 40). It is expressed in stratified and
simple epithelia and in hair follicles and may serve as a skin stem
cell marker (32, 34). In simple epithelia, K19 is expressed in several organs, including the intestine, biliary, and
pancreatic ducts (21). In human pancreas, K19 is expressed in all epithelial cells in utero but is progressively lost from acinar
cells and remains expressed only in ductal cells (8). Our
data indicate that K19 is located not only in mouse pancreas ductal and
centroacinar cells as previously described (8) but also in
the AL compartment of acinar cells. This is supported by specific Troma
III Ab staining, complete colocalization with F-actin in terms of
ductal and acinar cell decoration (Fig. 2; Refs. 36,
37, and 43), and the retention of AL filaments in
acinar cells of K18
/
mice due to the presence of K19 (Figs. 1 and
4). The significance of the human and mouse species differences in K19
expression is unknown, and it remains to be determined if K19 is
present in acinar cells of other species, such as rats and pigs.
K19 distribution and assembly properties appear to be unique compared
with its sometimes coexpressed type I keratin K18. For example, K19 is
able to partner with K8 and generate ultrastructurally typical IF in
uterine epithelia (29), and K8/19 filaments can also be
formed in transfected cells (3, 4,
27, 46). However, purified K18 and K19 differ
in their kinetics and polymerization properties when paired with K8
(15), and so do K14 and K19 differ when paired with K5
(12). In addition, although K8/18/19 AL filaments were
similar to K8/19 filaments when comparing WT and K18
/
acinar cells
by immunofluorescence staining, the K8/19 AL filaments as noted by EM
were less electron dense compared with K8/18/19 filaments (Fig. 4). The
nearly exclusive AL distribution of K8/19 filaments in pancreatic
acinar cells (in contrast to the combined cytoplasmic and AL
distribution of K8/18 filaments) is reminiscent of K19 distribution in
polarized cultured intestinal Caco-2 cells (39). This
polarized distribution is not universal since normal mouse intestine
(not shown) and pancreatic ductal cells (Fig. 1) also contain
cytoplasmic K8/19 with or without associated K18 in WT and K18
/
mice, respectively. The polarized distribution of K19 is, however, not
static in that pancreatic injury results in overexpression of K19 (and
K18 in WT mice; Fig. 7), with the surprising formation of K8/19 and
K8/18/19 C filaments in K18
/
and WT mice, respectively (Fig. 6).
This C filament formation is associated with keratin
hyperphosphorylation (Figs. 6 and 7) and is transient in that as
recovery is completed the K19-containing C filaments ultimately reside
in the AL compartment. K19 C filament formation on acinar cell injury
and subsequent localization in the AL domain likely reflect two
independent processes. For example, increased K19 expression (which
peaks at 48 h; Fig. 7) correlates with C (and AL) filament
formation, but the C
AL translocation is likely posttranslational and
unrelated to increased K19 levels per se since this process occurs
after K19 expression levels peak (Figs. 6 and 7). To that end, K19 has
similar but also distinct phosphorylation properties compared with K18
(e.g., K19 has significantly higher basal phosphorylation and is far less sensitive to phosphatase types 1 and 2A inhibition; Ref. 46). Hence it is possible that phosphorylation and/or
modulation by associated proteins could account for the observed
translocation. An alternate hypothesis is that K19 C filaments may
degrade during the recovery phase, independent of any translocation per
se, thereby leaving the residual AL filaments intact.
Do identical keratins in pancreatic acinar cells and hepatocytes
have different cytoprotective roles despite their similar
hyperphosphorylation during injury?
A surprising finding of this study is that keratin absence in acinar
cells manifests a markedly different phenotype compared with
hepatocytes. For example, hepatocyte viability on isolation and
susceptibility to liver injury are significantly impacted by the lack
of keratins or disruption of cytoplasmic filaments (21).
In addition, baseline liver histology is slightly abnormal in
transgenic mice that lack K8/18 (i.e., in K8
/
mice; Ref. 26) or in transgenic mice with disrupted hepatocyte
keratins (2, 17). In contrast, baseline K8
/
mice
pancreatic histology is normal (Fig. 5), and K8
/
acini viability is
only marginally abnormal and their response to CCK-stimulated secretion
is intact (Table 1). In this context, the exocrine pancreas is unique, compared with the intestine and liver, given that lack of keratin filaments in all three organs results in an apparently normal pancreatic phenotype but abnormal liver and intestinal phenotypes (5, 6). Similarly, subjecting the pancreas of K18
/
or K8
/
mice to two well-established injury models still did not unmask
any significant cytoprotective role of keratins, as contrasted with
liver exposure to a variety of stresses. One potential explanation, which will require further testing using other pancreatic injury models, is that the type of injury we deployed does not involve K8/18
in the same fashion as the tested liver injury models. Although this
scenario is possible, the response to injury in terms of keratin
hyperphosphorylation was similar in the liver (21, 38, 42)
and pancreas (Figs. 6 and 7), at least in the context of K8 S436 and
S79 phosphorylation. This suggests that similar signaling mechanisms
are involved in the injuries subjected to both cell types, albeit
site-specific phosphorylation (aside from what we tested) or other
signaling cascades may be different when comparing the pancreatitis
models with the liver stress models. This raises the possibility that
acinar cell K8/18 may not have the same cytoprotective function as
hepatocyte K8/18 or that acinar cells have a keratin-like cytoprotective function that compensates for the disruption of cytoplasmic keratins in K18C mice (Ref. 43 and Fig. 8) or
for the lack of cytoplasmic or total keratins (this study). The only keratin-related mouse model that shows clear damage in the exocrine pancreas is the transgenic mouse model that overexpresses human K8
(9). These mice develop dysplasia, loss of acinar cell
architecture, acinar to ductal cell differentiation, inflammation,
fibrosis, and fat formation. Since these mice do not have a
liver-associated phenotype, it remains unclear at this stage whether
the observed pancreas changes reflect overexpression levels and/or
expression of a human (vs. mouse) protein. Ongoing experiments that
entail overexpression of mouse K8 should help address this question.
The mechanisms underlying caerulein and CD diet pancreatitis have been
studied, and several signaling cascades appear to be involved. For
instance, pancreatic tumor necrosis factor (TNF)-
levels increase on
caerulein and CD diet treatment, and the severity of injury in both
models is decreased by administration of antagonizing TNF receptor type
I or TNF antibodies or in TNF receptor type I null mice (reviewed in
Ref. 35). Interestingly, K8 and K18 bind to the
cytoplasmic domain of the TNF receptor type II in vitro, and
K8/18-deficient cultured epithelial cells or K8
/
and K18
/
mouse
livers are significantly more sensitive to TNF-associated death
(10). However, our data suggest that total (K8
/
) or cytoplasmic (K18
/
) absence of keratins renders mice slightly resistant or as susceptible, respectively, as WT mice in
caerulein-induced pancreatitis. Further studies are required to
understand the potential role of keratin-TNF receptor binding in the
pancreas and its implications in pancreatitis injury models.
 |
ACKNOWLEDGEMENTS |
We are very grateful to Vivek Mehta for providing normal pancreatic
tissue, Robert Oshima for the anti-mouse K18 antibody, Pierre Coulombe
for antibodies to K16 and K17, Evelyn Z. Resurreccion for assistance
with immunofluorescence staining, Nafisa Ghori for help with EM, Kris
Morrow and Phil Verzola for preparing the figures, and Steve Avolicino
(Histo-tec Laboratory, Hayward, CA) for the histology staining.
 |
FOOTNOTES |
This work was supported by Veterans Administration Merit and Career
Development Awards and a Postdoctoral Fellowship from The Academy of
Finland to D. M. Toivola.
Address for reprint requests: D. M. Toivola, VA Palo Alto
Health Care System, Mail Code 154J, 3801 Miranda Ave., Palo Alto, CA 94304.
Address for other correspondence: M. B. Omary, VA Palo Alto Health
Care System, Mail Code 154J, 3801 Miranda Ave., Palo Alto, CA 94304.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 17 July 2000; accepted in final form 15 August 2000.
 |
REFERENCES |
1.
Achtstaetter, T,
Hatzfeld M,
Quinlan RA,
Parmelee DC,
and
Franke WW.
Separation of cytokeratin polypeptides by gel electrophoretic and chromatographic techniques and their identification by immunoblotting.
Methods Enzymol
134:
355-371,
1986[ISI][Medline].
2.
Albers, KM,
Davis FE,
Perrone TN,
Lee EY,
Liu Y,
and
Vore M.
Expression of an epidermal keratin protein in liver of transgenic mice causes structural and functional abnormalities.
J Cell Biol
128:
157-169,
1995[Abstract].
3.
Bader, BL,
Magin TM,
Freudenmann M,
Stumpp S,
and
Franke WW.
Intermediate filaments formed de novo from tail-less cytokeratins in the cytoplasm and the in the nucleus.
J Cell Biol
115:
1293-1307,
1991[Abstract].
4.
Bader, BL,
Magin TM,
Hatzfeld M,
and
Franke WW.
Amino acid sequence and gene organization of cytokeratin no. 19, an exceptional tail-less intermediate filament protein.
EMBO J
5:
1865-1875,
1986[Abstract].
5.
Baribault, H,
Penner J,
Iozzo RV,
and
Wilson-Heiner M.
Colorectal hyperplasia and inflammation in keratin 8-deficient FVB/N mice.
Genes Dev
8:
2964-2973,
1994[Abstract].
6.
Baribault, H,
Price J,
Miyai K,
and
Oshima RG.
Mid-gestational lethality in mice lacking keratin 8.
Genes Dev
7:
1191-1202,
1993[Abstract].
7.
Boller, K,
Kemler R,
Baribault H,
and
Doetschman T.
Differential distribution of cytokeratins after microinjection of anti-cytokeratin monoclonal antibodies.
Eur J Cell Biol
43:
459-468,
1987[ISI][Medline].
8.
Bouwens, L.
Cytokeratins and cell differentiation in the pancreas.
J Pathol
184:
234-239,
1998[ISI][Medline].
9.
Casanova, ML,
Bravo A,
Ramirez A,
Morreale de Escobar G,
Were F,
Merlino G,
Vidal M,
and
Jorcano JL.
Exocrine pancreatic disorders in transgenic mice expressing human keratin 8.
J Clin Invest
103:
1587-1595,
1999[Abstract/Free Full Text].
10.
Caulin, C,
Ware CF,
Magin TM,
and
Oshima RG.
Keratin-dependent epithelial resistance to tumor necrosis factor-induced apoptosis.
J Cell Biol
149:
17-22,
2000[Abstract/Free Full Text].
11.
Eckert, RL.
Sequence of the human 40-kDa keratin reveals an unusual structure with very high sequence identity to the corresponding bovine keratin.
Proc Natl Acad Sci USA
85:
1114-1118,
1988[Abstract].
12.
Fradette, J,
Germain L,
Seshaiah P,
and
Coulombe PA.
The type I keratin 19 possesses distinct and context-dependent assembly properties.
J Biol Chem
273:
35176-35184,
1998[Abstract/Free Full Text].
13.
Fuchs, E,
and
Cleveland D.
A structural scaffolding of intermediate filaments in health and disease.
Science
279:
514-519,
1998[Abstract/Free Full Text].
14.
Fuchs, E,
and
Coulombe PA.
Of mice and men: genetic skin diseases of keratin.
Cell
69:
899-902,
1992[ISI][Medline].
15.
Hofmann, I,
and
Franke WW.
Heterotypic interactions and filament assembly of type I and type II cytokeratins in vitro: viscometry and determinations of relative affinities.
Eur J Cell Biol
72:
122-132,
1997[ISI][Medline].
16.
Jensen, RT,
Lemp GF,
and
Gardner JD.
Interaction of cholecystokinin with specific membrane receptors on pancreatic acinar cells.
Proc Natl Acad Sci USA
77:
2079-2083,
1980[Abstract].
17.
Ku, NO,
Michie SA,
Oshima RG,
and
Omary MB.
Chronic hepatitis, hepatocyte fragility, and increased soluble phosphoglycokeratins in transgenic mice expressing a keratin 18 conserved arginine mutation.
J Cell Biol
131:
1303-1314,
1995[Abstract].
18.
Ku, NO,
Michie SA,
Soetikno RM,
Resurreccion EZ,
Broome RL,
Oshima RG,
and
Omary MB.
Susceptibility to hepatotoxicity in transgenic mice that express a dominant-negative human keratin 18 mutation.
J Clin Invest
98:
1034-1046,
1996[Abstract/Free Full Text].
19.
Ku, NO,
Michie SA,
Soetikno RM,
Resurreccion EZ,
Broome RL,
and
Omary MB.
Mutation of a major keratin phosphorylation site predisposes to hepatotoxic injury in transgenic mice.
J Cell Biol
143:
2023-2032,
1998[Abstract/Free Full Text].
20.
Ku, NO,
and
Omary MB.
Phosphorylation of human keratin 8 in vivo at conserved head domain serine 23 and at epidermal growth factor-stimulated tail domain serine 431.
J Biol Chem
272:
7556-7564,
1997[Abstract/Free Full Text].
21.
Ku, NO,
Zhou XJ,
Toivola DM,
and
Omary MB.
The cytoskeleton of digestive epithelia in health and disease.
Am J Physiol Gastrointest Liver Physiol
277:
G1108-G1137,
1999[Abstract/Free Full Text].
22.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[ISI][Medline].
23.
Liao, J,
Ku NO,
and
Omary MB.
Stress, apoptosis, and mitosis induce phosphorylation of human keratin 8 at Ser-73 in tissues and cultured cells.
J Biol Chem
272:
17565-17573,
1997[Abstract/Free Full Text].
24.
Liao, J,
Lowthert LA,
Ku NO,
Fernandez R,
and
Omary MB.
Dynamics of human keratin 18 phosphorylation: polarized distribution of phosphorylated keratins in simple epithelial tissues.
J Cell Biol
131:
1291-1301,
1995[Abstract].
25.
Lombardi, B,
Estes LW,
and
Longnecker DS.
Acute hemorrhagic pancreatitis massive necrosis with fat necrosis induced in mice by DL-ethionine fed with a choline-deficient diet.
Am J Pathol
79:
465-480,
1975[Abstract].
26.
Loranger, A,
Duclos S,
Grenier A,
Price J,
Wilson-Heiner M,
Baribault H,
and
Marceau N.
Simple epithelium keratins are required for maintenance of hepatocyte integrity.
Am J Pathol
151:
1673-1683,
1997[Abstract].
27.
Lu, X,
and
Lane EB.
Retrovirus-mediated transgenic keratin expression in cultured fibroblasts: specific domain functions in keratin stabilization and filament formation.
Cell
62:
681-696,
1990[ISI][Medline].
28.
MacKenzie, PI,
and
Messer M.
Studies on the origin and excretion of serum-amylase in the mouse.
Comp Biochem Physiol B Biochem Mol Biol
54:
103-106,
1976[ISI].
29.
Magin, TM,
Schroder R,
Leitgeb S,
Wanninger F,
Zatloukal K,
Grund C,
and
Melton DW.
Lessons from keratin 18 knockout mice: formation of novel keratin filaments, secondary loss of keratin 7 and accumulation of liver-specific keratin 8-positive aggregates.
J Cell Biol
140:
1441-1451,
1998[Abstract/Free Full Text].
30.
McLean, WHI,
and
Lane EB.
Intermediate filaments in disease.
Curr Opin Cell Biol
7:
118-125,
1995[ISI][Medline].
31.
Menozzi, D,
Jensen RT,
and
Gardner JD.
Dispersed pancreatic acinar cells and pancreatic acini.
Methods Enzymol
192:
271-279,
1990[Medline].
32.
Michel, M,
Torok N,
Godbout MJ,
Lussier M,
Gaudreau P,
Royal A,
and
Germain L.
Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage.
J Cell Sci
109:
1017-1028,
1996[Abstract/Free Full Text].
33.
Moll, R,
Franke WW,
Schiller DL,
Geiger B,
and
Krepler R.
The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells.
Cell
31:
11-24,
1982[ISI][Medline].
34.
Narisawa, Y,
Hashimoto K,
and
Kohda H.
Immunohistochemical demonstration of keratin 19 expression in isolated human hair follicles.
J Invest Dermatol
103:
191-195,
1994[Abstract].
35.
Norman, J.
The role of cytokines in the pathogenesis of acute pancreatitis.
Am J Surg
175:
76-83,
1998[ISI][Medline].
36.
O'Konski, MS,
and
Pandol SJ.
Effects of caerulein on the apical cytoskeleton of the pancreatic acinar cell.
J Clin Invest
86:
1649-1657,
1990[ISI][Medline].
37.
O'Konski, MS,
and
Pandol SJ.
Cholecystokinin JMV-180 and caerulein effects on the pancreatic acinar cell cytoskeleton.
Pancreas
8:
638-646,
1993[ISI][Medline].
38.
Omary, MB,
and
Ku NO.
Intermediate filament proteins of the liver: emerging disease association and functions.
Hepatology
25:
1043-1048,
1997[ISI][Medline].
39.
Salas, PJ,
Rodriguez ML,
Viciana AL,
Vega-Salas DE,
and
Hauri HP.
The apical submembrane cytoskeleton participates in the organization of the apical pole in epithelial cells.
J Cell Biol
137:
359-375,
1997[Abstract/Free Full Text].
40.
Stasiak, PC,
Purkis PE,
Leigh IM,
and
Lane EB.
Keratin 19: predicted amino acid sequence and broad tissue distribution suggest it evolved from keratinocyte keratins.
J Invest Dermatol
92:
707-716,
1989[Abstract].
41.
Steinert, PM,
and
Bale SJ.
Genetic skin diseases caused by mutations in keratin intermediate filaments.
Trends Genet
9:
280-284,
1993[ISI][Medline].
42.
Stumptner, C,
Omary MB,
Fickert P,
Denk H,
and
Zatloukal K.
Hepatocyte cytokeratins are hyperphosphorylated at multiple sites in human alcoholic hepatitis and in a Mallory body mouse model.
Am J Pathol
156:
77-90,
2000[Abstract/Free Full Text].
43.
Toivola, DM,
Ku NO,
Ghori N,
Lowe AW,
Michie SA,
and
Omary MB.
Effects of keratin filament disruption on exocrine pancreas-stimulated secretion and susceptibility to injury.
Exp Cell Res
255:
156-170,
2000[ISI][Medline].
44.
Toivola, DM,
Omary MB,
Ku NO,
Peltola O,
Baribault H,
and
Eriksson JE.
Protein phosphatase inhibition in normal and keratin 8/18 assembly-incompetent mouse strains supports a functional role of keratin intermediate filaments in preserving hepatocyte integrity.
Hepatology
28:
116-128,
1998[ISI][Medline].
45.
Wank, SA,
Jensen RT,
and
Gardner JD.
Stimulation of secretion by secretagogues.
Methods Enzymol
192:
247-255,
1990[Medline].
46.
Zhou, X,
Liao J,
Hu L,
Feng L,
and
Omary MB.
Characterization of the major physiologic phosphorylation site of human keratin 19 and its role in filament organization.
J Biol Chem
274:
12861-12866,
1999[Abstract/Free Full Text].
Am J Physiol Gastrointest Liver Physiol 279(6):G1343-G1354