Departments of 1 Medicine, 2 Surgery, 3 Environmental Science and Engineering, and 6 Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599; and 4 Department of Cell Biology, Duke University, Durham, North Carolina 27710; and 5 Departments of Medicine and Pathology, University of Southern California, Los Angeles, California 90033
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ABSTRACT |
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Using the differential PCR
display method to select cDNA fragments that are differentially
expressed after hepatic stellate cell (HSC) activation, we have
isolated from activated HSCs a cDNA that corresponds to rat
B-crystallin. Northern blots confirmed expression of
B-crystallin in culture-activated HSCs but not in quiescent HSCs.
Western blot analysis and immunocytochemical staining confirmed
expression of
B-crystallin protein in activated but not quiescent
HSCs.
B-crystallin is induced as early as 6 h after plating
HSCs on plastic and continues to be expressed for 14 days in culture.
Expression of
B-crystallin was also induced in vivo in activated
HSCs from experimental cholestatic liver fibrosis. Confocal microscopy
demonstrated a cytoplasmic distribution of
B-crystallin in a
cytoskeletal pattern. Heat shock treatment resulted in an immediate
perinuclear redistribution that in time returned to a normal
cytoskeletal distribution. The expression pattern of
B-crystallin
was similar to that of HSP25, another small heat shock protein, but
differed from the classic heat shock protein HSP70. Therefore,
B-crystallin represents an early marker for HSC activation.
differential polymerase chain reaction display; gene expression; liver fibrosis
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INTRODUCTION |
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IN LIVER FIBROSIS, THE SYNTHESIS and deposition of several extracellular matrix proteins are increased. Increased extracellular matrix production alters the normal architecture of the liver and inhibits its functions. The perisinusoidal hepatic stellate cell (HSC) is largely responsible for this increase in extracellular matrix deposition. The HSC after a fibrogenic stimulus is transformed from a quiescent vitamin A-storing cell type to that of an activated cell type (see Ref. 11 for review). Accompanying HSC activation are numerous changes in cellular morphology and in the pattern of gene expression. These changes include a loss of the stored retinoids, an increase in rough endoplasmic reticulum, enhanced cell proliferation, and dramatic increases in synthesis and deposition of several extracellular matrix proteins, of which type I collagen is predominant. HSC activation is observed in several in vivo models of experimentally induced liver fibrosis, including alcohol-induced liver fibrosis (52) and cholestatic liver injury induced by ligation of the common bile duct (43). Several key features of HSC activation can be recapitulated by culturing the cells on plastic, and this offers a convenient model system to study HSC activation.
Differential PCR display (DD-PCR) (33) provides a
convenient and sensitive method to assess differential gene expression after HSC activation. This method allows for the detection of mRNAs
that are either increased or decreased after cellular changes. Using
this technique, we (14) have previously demonstrated that the expression of intercellular adhesion molecule-1 is induced after
activation of the HSC, both in vitro and in vivo. Using DD-PCR as the
initial screening method, we now report that expression of
B-crystallin is induced after both in vitro and in vivo activation of HSCs.
B-crystallin is a major component of the vertebrate eye lens but is
also present at lower levels in other tissues, such as the heart,
skeletal muscle, kidney, placenta, skin, brain, peripheral nerves,
spinal cord, retina, and liver (8, 16, 20).
B-crystallin belongs to the family of small heat shock proteins and
displays a molecular chaperone activity under stress conditions
(8, 24, 32). The expression of
B-crystallin in the HSC
was similar to that of HSP25, another small heat shock protein.
Expression of both proteins was induced after HSC activation, but heat
shock did not result in an appreciable increase in protein levels. On the other hand, expression of HSP70, the classic heat shock protein, was not induced after HSC activation, whereas heat shock resulted in
persisted induction of HSP70 protein and transient induction of HSP70
mRNA in cultured HSCs. Therefore, expression of
B-crystallin is
induced early and persistently after HSC activation.
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MATERIALS AND METHODS |
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HSC isolation and culture. HSCs were isolated from adult male Sprague-Dawley rats (>400 g) using sequential pronase-collagenase digestion followed by arabinogalactan (Larcoll, Sigma Chemical, St. Louis, MO) density gradient centrifugation as previously described (48). For isolation of HSCs from cholestatic rats, a mixture of 0.02% type IV collagenase and 0.04% type I collagenase (Boehringer Mannheim, Indianapolis, IN) was used during the perfusion. Isolated HSCs were cultured in DMEM (GIBCO BRL, Gaithersburg, MD) supplemented with 10% FCS in a 5% CO2-95% air atmosphere. All animal procedures were performed under the guidelines set by the University of North Carolina Institutional Animal Care and Use Committee and the Institutional Care and Use Committee of the University of Southern California and are in accordance with the Guide for the Care and Use of Laboratory Animals [DHEW Publication No. (NIH) 85-23, revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20505].
Bile duct ligation. Male Wistar rats (body wt, 550-600 g) underwent common bile duct ligation and transection as previously described (14). To serve as controls, another group of animals underwent sham operations.
DD-PCR analysis.
Total RNA was obtained from freshly isolated quiescent HSCs and from
culture-activated HSCs using acidified phenol as previously described
(48). The DD-PCR method was performed essentially as
described by Liang and Pardee (33). Briefly, first-strand cDNA was generated in a reaction containing 2 µg of total RNA, 20 µmol/l dNTPs, 1 µmol/l of a poly-T primer (5'-TTTTTTTTTTTGC-3'), 50 mmol/l Tris · HCl, pH 8.3, 75 mmol/l KCl, 3 mmol/l
MgCl2, and 10 mmol/l dithiothreitol (DTT). The reaction
mixture was heated at 65°C for 5 min and then at 37°C for 10 min.
Murine leukemia virus RT (200 U; GIBCO BRL) was added, and the reaction
was incubated at 37°C for an additional 50 min. One-tenth of the
first-strand cDNA synthesis reaction was subjected to PCR amplification
using the poly-T primer described above along with a 10-nucleotide
primer (5'-GTTCCATACG-3') in a reaction mixture containing 2 µmol/l
dNTPs, 1 µmol/l of each primer, 10 mmol/l KCl, 0.01 mCi
[-35S]-dATP (1,000 Ci/mmol; ICN Biomedicals, Costa
Mesa, CA), and 2.5 U of Taq DNA polymerase (Boehringer
Mannheim). The PCR reaction was performed with 40 cycles at 94°C for
30 s, 40°C for 2 min, and 72°C for 30 s followed by an
extension incubation at 72°C for 10 min followed by a 4°C
incubation using a GeneAmp PCR system 9600 (Perkin-Elmer Cetus,
Emeryville, CA). An aliquot of the PCR reaction was run in a 4%
sequencing gel and dried without fixing. After autoradiography at room
temperature for 24 h, the autoradiogram was carefully aligned with
the dried gel, and differentially expressed bands were excised. The DNA
was eluted from the gel by incubating the gel slice in sterile,
distilled water for 10 min, followed by boiling for 15 min. Afterwards,
the DNA was precipitated by adding 100 µg/ml glycogen, 0.1 vol of 3 mol/l sodium acetate, and 2.5 vol of 100% ethanol and incubating at
20°C for 1 h. The sample was centrifuged at room temperature
for 10 min, and the pellet was washed with 75% ethanol and dried. The
eluted DNA fragment was suspended in sterile distilled water, and an
aliquot was reamplified using the same primers, PCR conditions, and
amplification parameters as described above. PCR amplification products
were visualized by electrophoresing an aliquot of the PCR reaction in a
5% nondenaturing polyacrylamide gel, and DNA bands were visualized by
ethidium bromide staining. The PCR products were cloned into the TA
cloning vector (Invitrogen, San Diego, CA) following the
manufacturer's recommended protocol. Cloned PCR fragments were
sequenced using the Sequenase DNA sequencing kit version 2 (United
States Biochemical, Cleveland, OH). A computer search was performed
with the nucleotide sequence of each clone using the National Center
for Biotechnology Information BLAST network service.
Northern blot analysis.
Northern blot analysis was performed using total RNA as previously
described (48). Random primer radiolabeled probes were generated from gel-purified, cloned PCR cDNA fragments of
B-crystallin and a cDNA insert of human HSP70 (13)
using a random primer radiolabeling kit (RediPrime kit, Boehringer Mannheim).
Western blot analysis.
Whole cell extracts were prepared using Dignam C buffer
(49). Protein samples (20 µg/lane) were electrophoresed
in a 10% acrylamide gel and electrophoretically transferred to a
nitrocellulose membrane. The membrane was blocked using 5% nonfat milk
in TBS-T (25 mmol/l Tris · HCl, pH 8.0, 144 mmol/l NaCl, and
0.75% Tween 20) at room temperature for 30 min then incubated with
either B-crystallin, HSP25, or HSP70 antibodies (1:1,000) (StressGen Biotechnology, Victoria, BC, Canada), each diluted in 5% nonfat milk
in TBS-T at room temperature for 2 h. Afterwards, the membrane was
washed two times in TBS-T for 5 min. Alkaline phosphatase-conjugated anti-rabbit antibody diluted 1:1,000 in 5% nonfat milk in TBS-T (Santa
Cruz Biotechnology, Santa Cruz, CA) was incubated with the membrane at
room temperature for 2 h and then washed as described above.
Signals were detected using 5-bromo-4-chloro-3-indolyl phosphate and
nitro blue tetrazolium as the substrate. In the Western blot
confirming that the phosphoserine antibody recognizes rat phosphoserine
proteins, 50 µg of whole cell extract were obtained from Rat-1 cells,
and the proteins were electrophoretically separated in a 10%
SDS-polyacrylamide gel. The proteins were transferred to a
polyvinylidine difluoride membrane (Immobilon-P, Millipore, Bedford,
MA). The membrane was blocked at room temperature for 1 h using
3% BSA in TBS-T (10 mmol/l Tris · HCl, pH 7.5, 50 mmol/l NaCl,
and 0.1% Tween 20). The membrane was probed with phosphoserine antibody (1:500 in 3% BSA in TBS-T) at room temperature for 1 h
and washed in TBS-T twice at room temperature for 20 min. The secondary
antibody, anti-rabbit horseradish peroxidase (HRP) (1:1,000 in 3% BSA
in TBS-T) was incubated with the membrane at room temperature for 30 min and washed as described above. Signals were detected using the
enhanced chemiluminescence (ECL) detection kit (Amersham Life Sciences,
Arlington Heights, IL).
Immunoprecipitations.
The immunoprecipitation assays were performed as previously described
(49). Briefly, whole cell extracts were made from culture-activated HSCs by lysing cell pellets in RIPA buffer (20 mmol/l
Tris · HCl, pH 7.5, 150 mmol/l NaCl, 2 mmol/l EDTA, 1% sodium
deoxycholate, 1% Triton X-100, and 0.25% SDS). Extracts were
precleared using normal rabbit serum overnight at 4°C. Immune complexes were collected by adding protein A agarose and briefly centrifuged at room temperature. The supernatant was collected, phosphoserine antibody (5 µg; Zymed Laboratories, South San
Francisco, CA) or B-crystallin antibody (2 µl; StressGen
Biotechnology) was added, and the reaction mixture was incubated at
4°C overnight. Immune complexes were collected, as described above,
boiled for 5 min in 2× SDS buffer (100 mmol/l Tris · HCl, pH
6.8, 200 mmol/l DTT, 4% SDS, 0.2% bromophenol blue, and 20%
glycerol), and electrophoresed in a 10% SDS-polyacrylamide gel. In
addition, cellular extracts (10 µg/lane) obtained from quiescent and
culture-activated HSCs were electrophoresed in the gel. Samples were
electrophoretically transferred to a nitrocellulose membrane, and the
membrane was blocked at room temperature for 30 min using 5% nonfat
milk in TBS-T (25 mmol/l Tris · HCl, pH 8.0, 144 mmol/l NaCl,
and 0.75% Tween 20). The membrane was probed with
B-crystallin
antibody (1:1,000; StressGen Biotechnology) at room temperature for
2 h and then washed three times in TBS-T. The membrane was
incubated in anti-rabbit HRP (1:1,000, Santa Cruz Biotechnology) at
room temperature for 30 min and washed as described above. Signals were
detected using the ECL detection kit (Amersham Life Sciences).
Immunocytochemistry.
Freshly isolated HSCs were seeded (5 × 104
cells/well) in six-well dishes that contained coverslips. After 14 days
in culture, the cells were fixed in methanol and washed three times
with wash solution (PBS containing 1% goat serum, 1% BSA, and 0.2%
Tween 20). The cells were then incubated with wash solution containing 0.5% Triton X-100 at room temperature for 10 min to permeabilize the
cells. Afterward, the cells were washed three times and then incubated
at room temperature for 3 h with the primary antibody diluted in
wash solution. Primary antibodies and dilutions used were
B-crystallin, 1:1,000 (StressGen Biotechnologies); smooth muscle
-actin, 1:100 (DAKO, Capinteria, CA); vimentin, 1:100 (Sigma
Chemical); paxillin, 1:100 (Santa Cruz Biotechnology);
-tubulin,
1:100 (Santa Cruz Biotechnology); glial fibrillar acidic protein
(GFAP), 1:250 (BioGenex, San Ramon, CA); and foiden, 1:100 (gift from
Dr. Keith Burridge, University of North Carolina, Chapel Hill). The
cells were washed three times and then incubated with FITC-conjugated
anti-rabbit antibody or rhodamine-conjugated anti-mouse antibody (each
diluted 1:200 in wash solution) at room temperature for 1 h. The
cells were washed three times, and the coverslips were mounted on glass
slides using Vectashield mounting medium (Vector Laboratories,
Burlingame, CA). To costain for
B-crystallin and F-actin, the cells
were incubated with the primary and secondary antibodies to detect for
B-crystallin, as described above, then incubated with 165 nmol/l
rhodamine phalloidin (Molecular Probes, Eugene, OR) diluted in PBS at
room temperature for 20 min. The cells were washed three times and
mounted on glass slides as described above. Cells were visualized using
confocal microscopy.
RT-PCR.
HSCs were isolated from the livers of sham-operated and bile
duct-ligated rats, and total RNA was isolated from the cells as
described above. First-strand cDNA was generated using 2 µg of RNA in
a reaction mixture containing 10 mmol/l oligo(dT)15 primer,
50 mmol/l Tris · HCl, pH 8.3, 1 mmol/l of each dNTP, 75 mmol/l
KCl, 3 mmol/l MgCl2, 10 mmol/l DTT, and 25 U RNase
inhibitor (Boehringer Mannheim). The reaction mixture was incubated at
68°C for 2 min, then at 4°C for 2 min during which murine leukemia virus RT (600 U; GIBCO BRL) was added. The reaction mixture was incubated at 42°C for 60 min. The synthesized cDNA was used in a PCR
reaction using specific sets of primers for B-crystallin or
-actin, as an internal control, based on previously published sequences for
B-crystallin (Ref. 3; GenBank accession
no. X60352) and
-actin (54). The nucleotide sequences
for the primers used for
B-crystallin amplification were as follows: sense strand, 5'-CTGACCTCTTCTCTACAGCCACT-3' (nt 101-123) and
antisense strand, 5'-CGTGCACCTCAATCACGTCTCC-3' (nt 301-280). The
nucleotide sequence for the
-actin primers were as follows: sense
strand, 5'-GAGCTATGAGCTGCCTGACG-3' (nt 801-820) and antisense
strand, 5'-AGCACTTGCGGTCCACGATG-3' (nt 1045-1026). Each PCR
reaction contained 1 µmol/l of each specific primer pair, 10 mmol/l
Tris · HCl, pH 8.3, 200 µmol/l of each dNTP, 2 mmol/l
MgCl2, 50 mmol/l KCl, and 2.5 U Taq DNA
polymerase. The PCR reaction was cycled as follows: an initial
denaturation of the cDNA sample at 94°C for 3 min, followed by 23 cycles at 94°C for 45 s, 55°C for 30 s, 72°C for 90 s, and a final extension incubation at 72°C for 10 min.
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RESULTS |
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DD-PCR reveals that B-crystallin is differentially expressed
after HSC activation.
Using the DD-PCR technique, we have isolated a bank of cDNA fragments
whose expression is either induced or suppressed after HSC activation
in culture. The nucleotide sequence of one of the differentially
expressed cDNA fragments that we isolated, ~495 bp in size, was 97%
homologous to the 5' and 3' portions of the rat
B-crystallin cDNA
(GenBank accession no. X60352). This isolated cDNA fragment encompassed
nt 46-657 of the reported
B-crystallin cDNA sequence. To confirm
that
B-crystallin is differentially expressed after HSC activation,
we performed Northern blot analysis using total RNA obtained from
quiescent and culture-activated HSCs.
B-crystallin expression was
detected in activated HSCs but not in quiescent HSCs (Fig.
1A). The size of the
B-crystallin transcript was ~1200 nt, similar to that reported for
B-crystallin in other non-lens tissue (18). Western
blot analysis was used to demonstrate differential expression of
B-crystallin protein after HSC activation. The presence of
B-crystallin protein was detected in activated HSCs but not in
quiescent HSCs (Fig. 1B).
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B-crystallin is expressed early after HSC activation.
To assess when
B-crystallin is induced during HSC activation, cells
were isolated, plated on plastic, and incubated for various time
periods. The cells were fixed and stained immunocytochemically for
B-crystallin. Within 6 h after plating HSCs,
B-crystallin was detected and displayed a focal expression pattern (Fig.
2). Over time, the cellular pattern of
expression became increasingly diffused, and after 7 days in culture
the localization pattern became cytoskeletal like in appearance.
Expression of
B-crystallin persisted at high levels throughout 14 days of culture.
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Cellular distribution of B-crystallin demonstrates cytoskeletal
pattern.
B-crystallin has been shown to colocalize with several cytoskeletal
proteins in other cell types. These include associations with actin and
desmin in cells derived from the lens and heart (2, 6, 34,
56), GFAP, vimentin, and desmin in astrocytoma cells
(45), and associations with vimentin and peripherin in cellular extracts (10). We attempted to colocalize
B-crystallin with several cytoskeletal proteins in the activated
HSC. HSCs were isolated and plated for 14 days. The cells were
trypsinized and seeded onto glass coverslips and grown for 2 days, then
fixed and costained for
B-crystallin and smooth muscle
-actin
(Fig. 3, A-C),
F-actin (Fig. 3, D-F), vimentin (Fig. 3,
G-I), paxillin (Fig. 3,
J-L), or
-tubulin (Fig. 3,
M-O). In addition, we performed costaining
experiments for
B-crystallin and GFAP or foiden (data not shown).
Although the pattern of
B-crystallin expression is cytoskeletal in
nature, we were unable to show colocalization with the different
cytoskeletal proteins that we examined.
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Cellular distribution of B-crystallin changes after heat shock
treatment of HSCs.
B-crystallin is a member of the small heat shock protein family
(8). These proteins function to protect other proteins in
the cell from detrimental effects of excess heat and stress. To assess
the cellular location of
B-crystallin after heat shock, culture-activated HSCs were subjected to heat shock and then
immunocytochemically stained for
B-crystallin after different time
periods following heat shock. Dramatic changes were noted in the
distribution pattern for
B-crystallin after heat shock. Within
1 h after heat shock recovery the location of
B-crystallin
became perinuclear. After 2 h of heat shock recovery, the cellular
distribution of
B-crystallin became globular in appearance, and
within 24 h, the normal cellular distribution pattern for
B-crystallin was restored (Fig. 4). This cellular redistribution pattern after heat shock represents a
typical response for heat shock proteins (10).
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B-crystallin is not phosphorylated in activated HSC.
It has been shown that
B-crystallin becomes phosphorylated after
stress conditions (17). Three specific serine residues are
targeted for phosphorylation. To assess whether culture-induced activation of HSCs results in serine phosphorylation of
B-crystallin, we performed immunoprecipitations using extracts
obtained from culture-activated HSCs. Extracts were immunoprecipitated
with an
B-crystallin antibody or a phosphoserine antibody and the immunoprecipitated proteins probed for
B-crystallin. When the
B-crystallin antibody was used for immunoprecipitation,
B-crystallin was detected in the immunoprecipitated proteins (Fig.
5A). However, when the
extracts were immunoprecipitated with the phosphoserine antibody,
B-crystallin was not detected in the immunoprecipitated proteins (Fig. 5B). As a control, cellular extracts from
quiescent and culture-activated HSCs were included in the Western blot
showing expression of
B-crystallin in activated HSCs and not in
quiescent HSCs. In addition, cellular extracts from Rat-1 cells were
probed using the phosphoserine antibody to confirm that the antibody recognized rat phosphoserine proteins (Fig. 5C). This data
demonstrates that
B-crystallin is not phosphorylated in
culture-activated HSCs.
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Expression of B-crystallin is increased in HSCs isolated after
fibrogenic stimulus in vivo.
To determine if
B-crystallin gene expression is induced after a
fibrogenic stimulus in vivo, bile duct ligations in rats were
performed.
B-crystallin gene expression in HSCs was assessed by
RT-PCR. Only low levels of
B-crystallin mRNA were detected in HSCs
isolated from sham-operated rats, whereas HSCs isolated from bile
duct-ligated rats demonstrated significantly higher levels of
B-crystallin mRNA (Fig. 6). Together,
these data demonstrate that
B-crystallin expression is
induced in HSCs when activated in vitro by culture and in vivo after a
fibrogenic stimulus.
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Expression of B-crystallin and HSP25 in HSC is similar but
significantly differs from that of HSP70.
Because
B-crystallin belongs to the family of small heat shock
proteins, we compared the expression pattern of
B-crystallin to
HSP25, another small heat shock protein family member, and to HSP70,
the classic heat shock protein. Western blot analysis using proteins
isolated from quiescent and culture-activated HSCs showed that
expression of
B-crystallin and HSP25 is induced after HSC activation
in culture (Fig. 7). After a 1-h heat
treatment of activated HSCs at 42°C followed by recovery at 37°C,
protein expression of either
B-crystallin or HSP25 was not
significantly increased following heat treatment. On the other hand,
expression of HSP70 was not induced after HSC activation in culture but
was persistently induced for 24 h after the heat shock (Fig. 7).
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DISCUSSION |
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To gain a better understanding of the molecular events that take
place after HSC activation, we have been using the DD-PCR technique
(33) to isolate genes that are either activated or suppressed after HSC activation in culture. In this study, we demonstrated that expression of B-crystallin is induced within 6 h after plating the cells and thus represents one of the
earliest induced genes identified after HSC activation. The
transcription factor KLF6, previously referred to as Zf9, has also been
shown to be an early induced gene after HSC activation in vivo
(47). We also demonstrated that
B-crystallin gene
expression is induced in HSCs that are activated in vivo after
cholestatic liver injury induced by common bile duct ligation. After
1 h of heat treatment, the cellular distribution of
B-crystallin changes from a cytoskeletal type of pattern to a
perinuclear localization within activated HSCs. Within 24 h of
heat recovery, the cellular distribution of
B-crystallin returns to
its normal localization pattern. A similar redistribution response for
B-crystallin after heat shock has been described in other cell types
(8, 10). This response is believed to represent molecular
chaperone activity in protecting cellular proteins from heat stress.
B-crystallin along with
A-crystallin constitute the subunit
proteins for
-crystallin.
-Crystallin represents >30% of the major soluble protein in vertebrate eye lens (8).
Expression of
B-crystallin is generally restricted to the eye lens;
however, low levels of expression have been reported in the spleen,
thymus, and retina (30, 51, 56).
B-crystallin, on the
other hand, is expressed in a variety of tissues, including the heart,
skeletal muscle, kidney, placenta, skin, brain, peripheral nerves,
spinal cord, retina, and liver (8, 16, 20, 21, 36). On the basis of structural and nucleotide sequence homology,
B-crystallin belongs to the small heat shock protein family, which includes the heat
shock proteins HSP20, HSP25, HSP27, and HSP30 (8, 32). We
compared the expression pattern of
B-crystallin with the small heat
shock protein HSP25 and with the classic heat shock protein HSP70 in
quiescent and culture-activated HSCs and after heat shock in activated
HSCs. The expression pattern was similar between the small heat shock
proteins
B-crystallin and HSP25. Both proteins were not expressed in
quiescent HSCs, but expression was induced after HSC activation.
Following heat shock, protein levels of these small heat shock proteins
did not appreciably increase. In contrast, expression of HSP70 was not
detected in quiescent or activated HSCs. HSP70 mRNA expression was
found to be transiently increased after heat shock, although HSP70
protein levels remained elevated 24 h after the heat shock.
Therefore, expression of small heat shock proteins differs from the
classic heat shock protein HSP70 in HSCs. Although others (16,
32) have reported that
B-crystallin is induced after heat
treatment, we did not observe heat induced
B-crystallin expression
in activated HSCs. Constitutive expression of
B-crystallin has been
reported in other cell types in the absence of stress
(31). It is possible that the level of expression within
the activated HSC is at maximum expression and may not be able to be
further increased. Because HSCs are generally activated during stress,
as seen in the oxidative stress conditions that occur during chronic
alcohol consumption, the cell may be preprogrammed to cope with
continued stress by constitutively expressing the small heat shock
proteins, including
B-crystallin and HSP25. HSP70 expression may be
reserved for dealing with additional, transient stress conditions to
which the cell may be exposed. Alternatively, the different classes of
heat shock proteins may serve to protect cells from different types of
stress conditions.
Increased expression of B-crystallin has been shown to be induced by
high oxidative activity and thermal and osmotic stress and following
treatment of cells with tumor necrosis factor-
(TNF-
) (7,
12, 20, 39). We found
B-crystallin expression to be induced
early in culture. Cultured HSCs are exposed to higher oxidative
conditions than these cells normally are exposed to in the liver. This
may explain the early and high expression levels of
B-crystallin
when these cells are cultured. Both bile duct ligation and alcoholic
liver disease represent high oxidative stress conditions in the liver
during which
B-crystallin expression has also been shown to increase
(Fig. 6) (16). Interestingly, administration of ethanol
before but not during stress in rats can inhibit the increased
stress-induced
B-crystallin expression (16).
Phosphorylation of B-crystallin increases after stress
(17) when specific serine residues are phosphorylated by
both cAMP-dependent (50, 53) and cAMP-independent
mechanisms (27). The p38 mitogen-activated protein kinase
(MAPK) (29) as well as p44/p42 MAPK and MAPK-activated protein kinase 2 (28) have been shown to phosphorylate
B-crystallin.
B-crystallin has also been reported (25,
26) to possess an autokinase activity. Interestingly, we did not
detect serine phosphorylation of
B-crystallin in culture-activated
HSCs (Fig. 5). Evidently, the stress conditions of culture are not
sufficient to induce phosphorylation of
B-crystallin. The biological
effects of phosphorylation are poorly understood. It has been shown
(55) that phosphorylation decreases oligomer size;
however, the consequence of phosphorylation does not affect chaperone
activity of the protein measured by suppression of thermal-induced
aggregation of low B- or
-crystallins.
Heat shock proteins are implicated in a variety of medical conditions,
including ischemia, stroke, cardiovascular disease, cancer,
inflammation, trauma, aging, and autoimmunity, among others (5,
21-23, 35, 36, 41). B-crystallin has been shown
(4) to protect cells against ultraviolet-induced protein
aggregation. The small heat shock proteins, including
B-crystallin,
have also been shown to confer thermoresistance to cells
(19) and provide protection against TNF-
(39), oxidative stress (39), and ischemic
injuries (9). These proteins have also been shown (40) to protect cells against apoptosis mediated by
staurosporine and Fas ligand. Resistance to TNF-
-mediated cell death
is at least partially due to increased glutathione levels
(1). Small heat shock proteins have been shown
(1) to increase cell survival due to oxidative stress by
decreasing intracellular reactive oxygen species in a
glutathione-dependent manner. The small heat shock proteins also
inhibit protease activity (44). This may be useful to
prevent endogenous cellular proteases from attacking partially or
transiently denatured proteins during stress.
Associations of B-crystallin have been observed with several
cytoskeletal components, including actin (6, 34, 56), desmin (2, 34), peripherin (10), vimentin
(10, 45), and GFAP (45). It is believed that
these associations protect against ischemic stress conditions and
heat-induced protein aggregation in a chaperone-like manner. In fact,
B-crystallin inhibits heat-induced aggregation of actin
(17). The molecular chaperone activity of the small heat
shock proteins is more pronounced at increased temperatures (10,
46). It is possible that we did not observe
B-crystallin
colocalization with the cytoskeletal proteins we examined since our
studies were performed at normal, unstressed temperatures. The small
heat shock proteins have also been shown to associate with membranes
(15).
The demonstration that expression of both HSP25 and B-crystallin
increases after HSC activation represents two additional heat shock
proteins whose expression is induced following HSC activation.
Expression of HSP45, a collagen-specific molecular chaperone, has been
also been shown to increase after HSC activation. HSP45 synthesis
parallels collagen synthesis during normal development in a
tissue-specific, temporal, and spatial manner (38). In addition, HSP45 gene expression is increased after a fibrogenic stimulus. HSP45 gene expression follows the increased expression of
types I and III collagen and is expressed in the activated HSC
(37, 42). Our study demonstrates that not all heat shock proteins are similarly regulated after HSC activation. Whereas expression of
B-crystallin, HSP25, and HSP45 is increased after HSC
activation, expression of HSP70 is not increased during activation but
is induced following heat shock in the activated HSC. This suggests
significantly different roles for these stress proteins in the cell.
HSP45 has been shown to be a collagen-specific molecular chaperone;
however, the roles of HSP25 and
B-crystallin in the HSC remain
unknown. Analysis of the functions of these proteins in other cell
types suggest that these proteins probably act as molecular chaperones
protecting the HSC from cellular stress.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute on Alcohol Abuse and Alcoholism Grants AA-10459 (R. A. Rippe) and AA-06603 (H. Tsukamoto), National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34987 (R. A. Rippe and D. A. Brenner), and the Tissue Culture Core Facility of the University of Southern California Research Center for Liver Disease (Grant P30-DK-48522).
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. A. Rippe, CB# 7038, Division of Digestive Diseases, Dept. of Medicine, Univ. of North Carolina, Chapel Hill, NC 27599-7038.
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 1 February 2000; accepted in final form 29 June 2000.
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