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J Biol Chem, Vol. 273, Issue 47, 31252-31261, November 20, 1998
From the Departments of Like other sHSPs, Current understanding of the cellular functions of molecular chaperones
is based largely on the roles of the HSP60 and HSP70 chaperone families
in protein translocation, folding, and assembly (11, 12). Members of
these families play vital roles in normal growth and development and
prevent stress-induced denaturation of other proteins. A key element of
their function is their ability to prevent improper protein
associations by binding to transiently exposed hydrophobic protein
surfaces. Although sHSPs are also believed to be important for normal
cell growth and differentiation (for example, HSP27 protects cells
during stress by preserving actin microfilaments and preventing
apoptotic cell death (13-15)), little information is available
regarding the cellular functions of this family in general and
Because of its location on the optic axis of the eye, the lens is
exposed to solar ultraviolet radiation (UVA and UVB). UV irradiation
generates photooxidative stress within the tissue and is thought to be
one of the factors contributing to deleterious age- and
cataract-induced changes in the lens. Much of the UV radiation entering
the eye is blocked by the cornea, but about 0.001 W/cm2 of
UVA reaches the lens epithelium. Chronic exposure of animal models to
this irradiance produces lens opacities in vivo (16). UVA-induced lens opacification is thought to result from the generation of reactive oxygen species such as singlet oxygen, superoxide, hydrogen
peroxide, and hydroxyl radicals (16). Since Recent studies on mice deficient in Cell Culture--
Mouse and human lens epithelial cell cultures
were used in this study. The wild type mouse strain was the 129SvJ
mouse. Transfection--
A full-length cDNA clone containing the
complete coding sequence of the Western Blotting--
The antibodies used for Western blot
analysis of Stress Conditions--
Cells were exposed to levels of UVA
radiation comparable to in vivo levels (24). Cells were
washed twice with PBS containing CaCl2 and
MgCl2, and a layer of PBS was added to the controls and
cells to be irradiated. UVA radiation at 365 nm was obtained from a
Hg-Xe arc lamp using a monochromator, focusing lens, and beam turner to
give a fluence rate of 0.007 W/cm2 and a total fluence of
37.8 J/cm2 unless indicated otherwise (25). Cells were
either processed immediately or incubated for up to 20 h after
irradiation in normal tissue culture medium. In some experiments, UVB
radiation (302 nm, total fluence 0.02 J/cm2) or heat
treatment (45 °C, 30 min) was also used as a stress inducer.
TUNEL Assay--
Cells were fixed in 4% paraformaldehyde, pH
7.4, for 30 min, permeabilized for 30 min in 0.1% Triton X-100/PBS at
room temperature, and washed for 20 min in PBS as described previously
(26). Apoptotic nuclei were detected using a TUNEL-labeling reaction
according to the manufacturer's instructions (Boehringer Mannheim).
This reaction utilizes fluorescein-labeled nucleotides that are
incorporated at the 3'-OH ends of DNA molecules by the enzyme terminal
deoxynucleotidyltransferase. In some cases, fixed and permeabilized
cells were pretreated with DNase I (Boehringer Manneheim, 50 µg/ml in
10 mM Tris, pH 7.6) for 10 min at room temperature as a
positive control. In each experiment, some samples were labeled with
the mix lacking the terminal deoxynucleotidyltransferase enzyme, as a
negative control. To determine the proportion of apoptotic cells in a
culture, cells were co-labeled with 4',6-damidino-2-phenylindole
(DAPI), a DNA-binding fluorophore which fluoresces in the UV region.
TUNEL-positive nuclei and DAPI-labeled nuclei were visualized using a
fluorescence microscope (Axioskop; Zeiss). Images were recorded on a
cooled CCD camera (Photometrix), and the number of TUNEL-positive
nuclei and total nuclei were counted using image analysis software
(Metamorph; Universal Imaging Corp.).
Long Term Survival--
HLE B-3 cells overexpressing varying
levels of Cell Growth--
Wild type or Cell Cycle Distribution--
Wild type or Localization of To study the effect of Mouse Lens Epithelial Cultures--
The absence of
In primary cell cultures, capsule epithelial explants from
The finding that primary cultures of
The inhibition of cell growth observed in
Lens epithelial cells are exposed to UVA radiation (~0.001
W/cm2) in vivo on a daily basis, and UVA
radiation has been found to produce lens opacities in animal models. We
therefore examined the effect of UVA radiation (0.007 W/cm2
for 90 min, giving a total fluence of 37.8 J/cm2) on
apoptosis in primary cultures of wild type and
To quantitate the effect of UVA exposure on apoptosis of wild type and
Cultures were exposed to UVA radiation and incubated in medium for
different times after exposure. Four hours after UVA exposure, the
UVA-treated Human Lens Epithelial Cultures--
The HLE B-3 cells (passage 12)
used for transfection had no detectable levels of
Fig. 7B shows the expression of
Three cell lines expressing different levels of
HLE B-3 cells have a high growth potential, and a large number of cells
can easily be obtained. Because the number of cells is not limiting in
these cultures, cell death can be measured by a variety of techniques.
Flow cytometry was used to measure the number of dead cells in cultures
exposed to UVA radiation. The plots of forward light scatter and side
scatter were compared for
The effect of expression of
The cellular localization of We studied Although the The effect of The slower growth of When lens epithelial cultures were irradiated with UVA, Previous work has shown that UVA radiation induces apoptosis 4 h
after exposure (35). This process, termed immediate apoptosis, may
increase membrane permeability, Ca2+ influx, and the
Ca2+-dependent proteolysis of an endogenous
endonuclease that subsequently degrades the genomic DNA (35-37). The
mechanism by which UVA radiation induces cell death probably involves
the generation of reactive oxygen species (38). Recent studies suggest
that sHSP expression decreases the intracellular levels of reactive
oxygen species and increases the cellular content of the antioxidant
glutathione (39). It will be interesting to determine whether
prevention of apoptosis by The present study suggested that F-actin filaments were protected from
the damaging effects of UVA radiation in cells constitutively expressing The structure and solubility of HSP27 in mammalian cells has been shown
to be dependent on the physiologic state of the cells (30-32). Heat
shock treatment causes a redistribution of HSP27 and Chaperone proteins such as We thank Dr. J. Mark Petrash for the cDNA
clone of *
This work was supported by National Institutes of
Health Grants R01-EY05681 (to U. P. A.), R01-EY09852 (to S. B.),
Core Grant EY02687, and an unrestricted grant from Research to Prevent
Blindness (RPB) to the Department of Ophthalmology and Visual Sciences, a RPB Robert E. McCormick Scholar award (to U. P. A.), and a RPB career
development award (to S. B.).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.
¶
To whom correspondence should be addressed: Dept. of
Ophthalmology and Visual Sciences, Washington University School of
Medicine, 660 S. Euclid Ave., Campus Box 8096, St. Louis, MO 63110. Tel.: 314-362-7167; Fax: 314-362-3638; E-mail:
andley{at}seer.wustl.edu.
The abbreviations used are:
sHSP, small heat
shock protein; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline; DAPI, 4',6-damidino-2-phenylindole; W, watt; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.
The Molecular Chaperone 
A-Crystallin Enhances Lens Epithelial
Cell Growth and Resistance to UVA Stress*
§¶,,
, and**
Ophthalmology and
Visual Sciences, § Biochemistry and Molecular Biophysics,
** Cell Biology and Physiology, Washington University School of
Medicine, St. Louis, Missouri 63110, and National
Eye Institute, National Institutes of Health,
Bethesda, Maryland 20892
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
A-Crystallin (A) is a member of
the small heat shock protein (sHSP) family and has the ability to
prevent denatured proteins from aggregating in vitro. Lens
epithelial cells express relatively low levels of A, but in
differentiated fiber cells, A is the most abundant soluble protein.
The lenses of A-knock-out mice develop opacities at an early age,
implying a critical role for A in the maintenance of fiber cell
transparency. However, the function of -crystallin in the lens
epithelium is unknown. To investigate the physiological function of
A in lens epithelial cells, we used the following two systems: A
knock-out (A(
/)) mouse lens epithelial cells and human lens
epithelial cells that overexpress A. The growth rate of
A(/) mouse lens epithelial cells was reduced by 50% compared
with wild type cells. Cell cycle kinetics, measured by
fluorescence-activated cell sorter analysis of propidium iodide-stained
cells, indicated a relative deficiency of A(/) cells in the
G2/M phases. Exposure of mouse lens epithelial cells to
physiological levels of UVA resulted in an increase in the number of
apoptotic cells in the cultures. Four hours after irradiation the
fraction of apoptotic cells in the A(/) cultures was increased
40-fold over wild type. In cells lacking A, UVA exposure modified
F-actin, but actin was protected in cells expressing A. Stably
transfected cell lines overexpressing human A were generated by
transfecting extended life span human lens epithelial cells with the
mammalian expression vector construct pCI-neoA. Cells overexpressing
A were resistant to UVA stress, as determined by clonogenic
survival. A remained cytoplasmic after exposure to either UVA or
thermal stress indicating that, unlike other sHSPs, the protective
effect of A was not associated with its relocalization to the
nucleus. These results indicate that A has important cellular
functions in the lens over and above its well characterized role in refraction.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-Crystallin, a member of the small heat shock protein
(sHSP)1 family of molecular
chaperones, is an aggregate of two polypeptides, A and B, that
share 55% sequence identity. The two ~20-kDa subunits form soluble
aggregates with an average molecular mass of 800 kDa. Originally
regarded as lens-specific, the expression of these proteins (especially
B) has since been observed in other tissues. The extralenticular
expression of -crystallins and their ~40% sequence identity with
Drosophila HSP27 (1, 2) suggest that during evolution
-crystallin may have been recruited as a refractive protein in the
lens through a gene-sharing mechanism involving duplication of an
ancestral sHSP gene (3).
A and B polypeptides can act as molecular
chaperones in vitro, preventing protein aggregation induced by heat and other stresses (4). The two subunits are differentially distributed within the lens with A maximally expressed in the lens
fibers and B highest in the epithelium (3). Of the two subunits,
A expression is considered to be more lens-specific but only B
expression is induced by stress (3, 5). At present, the tertiary and
quaternary structures, in vivo functions, and mechanism of
chaperone action of the -crystallin aggregates are largely enigmatic.
-Crystallin is the most abundant soluble protein in the lens and
plays an important role in establishing the refractive index of the
cytoplasm. However, the extralenticular expression of -crystallins, their autokinase activity, phosphorylation patterns, association with
neurodegenerative diseases, and ability to protect against heat shock
argue for a more generalized cellular function of -crystallins, over
and above their role in image formation (3, 5-10).
-crystallins in particular.
-crystallin has been
found to prevent both UV- and singlet oxygen-induced aggregation of
proteins in vitro, it may also protect lens proteins from
photooxidative changes in vivo (17, 18). Additionally, studies on cultured mammalian cells indicate that the overexpression of
A in mammalian cells confers increased thermoresistance on these
cells, suggesting a relationship between the in vitro
molecular chaperone activity and in vivo thermoprotection
(19). Although the physiological significance of its thermoprotective
property is not known, A may promote the survival of cells (such as
those in the lens epithelium) that are proximal to sources of
environmental stress.
A have demonstrated a critical
role for this protein in the maintenance of lens transparency (20).
Lenses of A(/) mice are smaller in size and develop opacities
as a result of the presence of inclusion bodies in the fiber cells. The
inclusion bodies contain both B-crystallin and HSP25 (21). This
observation suggests that one function of A in the lens may be to
ensure the solubility of other important chaperones. In the current
work, we have investigated the in vivo functions of A
using lens epithelial cells from normal and A(/) mice. We have
also investigated the effect of overexpression of A in extended-life
span human lens epithelial cells. Our results demonstrate a critical
role for A in lens cell growth. We also show that a photoprotective
phenotype can be conferred on lens epithelial cells in culture by the
expression of A-crystallin.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
AKO-127 A(/) mice were made by targeted disruption of
the A gene (20). Mouse lenses were obtained from 6- to 12-week-old
mice. Capsule epithelia of wild type and A(/) lenses were
dissected and cultured in 20% fetal bovine serum in Eagle's minimum
essential medium in 24-well tissue culture plates (Falcon). Cells were
passaged using trypsin-EDTA and plated in 35-mm plates. In some
experiments, cells were grown on sterile glass coverslips (with or
without collagen coating) to facilitate microscopic examination.
Cultures were fed twice weekly. Human lens epithelial cells with
extended life span (HLE B-3 cells) have been described previously (22). They were derived from an infant human lens epithelial culture by
Ad12-SV40 hybrid virus infection and propagated through at least 11 passages. After passage 11, HLE B-3 cells cease to produce A (as
determined by Western blot) (23). Therefore, A cDNA can be
reintroduced into these cells by cDNA transfection, and stably
transfected cell lines expressing different amounts of A can be
generated. The HLE B-3 cells were cultured in 20% fetal bovine serum
in Eagle's minimum essential medium according to published procedures
(22, 23).
A gene was taken from the vector
pDirect (18). The A cDNA was cloned into the XbaI and
SalI site of the mammalian expression vector, pCI-neo
(Promega). This vector utilized the cytomegalovirus
immediate-early enhancer/promoter region for strong constitutive
expression of the A gene. The neor marker allowed for
stable transfectants to be isolated. The pCI-neoA was stably
transfected into HLE B-3 cells by standard calcium phosphate protocols.
Two days post-transfection, the cells were trypsinized and subcultured
1:3, and geneticin was added for colony selection. Geneticin-resistant
colonies were isolated, and individual colonies were expanded into mass
cultures. Cultures were examined by Western blot for the expression of
A and compared with mock-transfected cells (cells transfected with
vector lacking A insert). The transfection efficiency as determined
by Western blot analysis was found to be 10-20% in different
transfection assays.
A were a polyclonal antiserum to bovine lens
-crystallin (at a dilution of 1:1000) or a monoclonal antiserum to
bovine A (at a dilution of 1:100). For Western blotting analysis of
B, a polyclonal antiserum raised against the 21-amino acid
C-terminal peptide of human B was used at a dilution of 1:1000.
Immune complexes were detected using 125I-protein A. Western blotting was used to examine the expression of A and B in
primary mouse lens epithelial cultures, to confirm the lack of
endogenous A in untransfected HLE-B3 cells after passage 11 and, in
stably transfected HLE B-3 cells, to select those cell lines expressing
the highest levels of A. The latter were used to determine the
effect of physiological levels of UVA stress as a function of A
expression level.
A and untransfected or mock-transfected controls were used
to compare the effect of A expression on long term cell survival of
cells exposed to UVA stress. Cell survival was determined by the
relative colony-forming ability of the cells when plated at low
densities (25). Here, survival is defined as the long term reproductive
potential of the cells and does not refer to individual cell death.
Cells were exposed to various fluences of UVA radiation. Immediately
after irradiation, cells were trypsinized, and approximately 8 × 103 cells per ml were suspended in culture medium. An
aliquot of the suspension was counted using a Coulter counter, and
cells were diluted to 200 cells/ml. Colony-forming ability was
determined by plating 200, 100, and 50 cells per well in a 24-well
plate containing 1 ml of tissue culture medium. The cultures were
allowed to incubate undisturbed for 7-10 days. Colonies formed were
stained with 0.5% methylene blue and counted. The surviving fraction
at each UVA fluence was calculated as the ratio of the colony-forming abilities of the irradiated and non-irradiated controls.
A(/) lens epithelial
cells in passage 1 or 2 were subcultured in 24-well plates (2 × 103 cells per well) for a period of 2 weeks. Cultures were
fed twice weekly. Cell numbers were measured on specific days after
trypsinization using a Coulter counter (27).
A(/) cells
(106 cells) were washed with PBS, and cell pellets were
labeled for 30 min on ice with propidium iodide (50 µg/ml) in 0.1%
sodium citrate containing 0.3% Nonidet P-40 and 20 µg/ml
ribonuclease A, pH 8.3. The percentage of cells in each phase of the
cell cycle was determined by a flow cytometer (Becton-Dickinson
FACScan) using the Cell Quest software. Flow cytometry was also used to
determine the effect of UVA radiation on cell death in HLE B-3 cells
overexpressing various levels of A-crystallin. Cells exposed to UVA
radiation were labeled with propidium iodide and a plot of forward
light scatter (cell size) versus the side scatter (cell
surface area) was determined using the FACScan.
A and F-actin--
Immunofluorescence was
carried out as described previously (26). Cells were fixed in 4%
paraformaldehyde/PBS for 30 min at room temperature and then
permeabilized in 0.1% Triton X-100 for 30 min. Nonspecific binding was
blocked by incubation in 10% normal goat serum for 30 min. To
visualize the distribution of A, cells were incubated overnight at
4 °C in a 1:100 dilution of a monoclonal antibody raised against
bovine A. A lissamine rhodamine-conjugated goat anti-mouse IgG was
used as the secondary antibody. To visualize the organization of
F-actin, fixed and permeabilized cells were incubated in a 1:50
dilution in PBS of a fluorescein phalloidin (Molecular Probes Inc.)
methanolic stock solution (100 units/ml of methanol). Cells were
stained with fluorescein phalloidin for 20 min, washed 3 times for 5 min in PBS, and viewed. Lens epithelial cells were viewed using a Zeiss
LSM 410 confocal microscope equipped with an argon-krypton laser.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
A expression on lens epithelial cell
function, cultured human and mouse lens epithelial cells were utilized.
Since primary cultures of human cells cannot be propagated for more
than three passages, we chose to use the SV40-immortalized human lens
epithelial cell line HLE B-3, as a model system. HLE B-3 cells do not
express A after passage 11. This allows the effect of A
expression to be examined in transfection experiments against a null
background. However, one limitation of this approach is that late
passage HLE B-3 cells may differ from normal epithelial cells in ways
other than the level of A expression. For this reason we also chose
to examine an alternative murine model. Gene knock-out technology has
been used recently to create A-deficient mice (20), and primary
cultures of lens capsule-epithelial explants from these animals were
therefore used in the present studies.
A is the major soluble
protein of lens fiber cells. Immunofluorescence microscopy of lens
slices from wild type mice confirmed the high levels of expression of
A in the lens fiber cells (data not shown). In these preparations
the level of A immunostaining in the lens epithelium was comparably
high. In A(/) lenses, A immunofluorescence was undetectable
in either the fibers or epithelium, confirming the efficacy of the A
gene deletion (data not shown). The lack of A immunofluorescence in
the A(/) lenses also demonstrated the specificity of the
monoclonal antibody used in these studies. The differences in A
expression between wild type and A(/) lenses were conserved in
primary cell cultures derived from these lenses (Fig.
1, A and B,
respectively). Interestingly, although there was strong A
immunostaining in primary epithelial cell cultures from wild type
lenses, A was not evenly dispersed in the cytoplasm. In wild type
cells, A was localized in discrete cytoplasmic patches of 5-20 µm
in diameter (Fig. 1A). As expected, there was no evidence of
A expression in A(/) cells (Fig. 1B). Although
both cell types contained well organized actin stress fibers and had
similar overall morphologies, the A(/) cells were consistently
larger than wild type cells. This observation was confirmed
independently by FACS analysis (data not shown).
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Fig. 1.
Distribution of A and actin in primary
cultures of mouse wild type and A(/) lens epithelial cells.
A (red) was localized using an antibody to bovine A;
F-actin (green) was localized using fluorescein-phalloidin,
and cellular morphology was imaged using differential interference
contrast (blue). A, epithelial cells from wild
type lenses. In these cultures, A was often concentrated in discrete
patches (arrows) in the cytoplasm. B, epithelial
cells from A(/). There was no detectable A immunofluorescence
in these cells. Scale bars = 30 µm.
A protein from primary epithelial cultures derived
from A(/) lenses was confirmed by Western blot analysis (Fig.
2). In wild type cultures, a prominent
immunoreactive band was detected at the expected molecular mass of
~21 kDa. This band was absent from the primary culture of
A(/) cells. A second, higher molecular weight band, at ~24
kDa in the wild type cultures, may represent the A insert protein
(28). In the lens, -crystallin exists as a high molecular weight
complex of two polypeptides, A and B. Because A and B can
each act as a molecular chaperone (4) and because the expression of
A has been shown to influence the expression and distribution of
B (20), we also tested the cultured cells for the presence of B
(Fig. 2). Approximately equal amounts of B were present in the
A(/) and wild type cultures.
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Fig. 2.
Western blot analysis of A and B
expression in cultured lens epithelial cells from wild type and
A(/) lenses. Cell lysates were prepared from 1.4 × 105 wild type or 2.7 × 105 A(/)
lens epithelial cells and analyzed by SDS-polyacrylamide gel
electrophoresis and Western blotting. Left panel, the
primary antibody used was a monoclonal antiserum to bovine A.
WT, lysates of wild type lens epithelial cell cultures. The
higher molecular weight band represents the A insert protein;
A(/), lysates of A(/) lens epithelial cell
cultures. Note the absence of any immunoreactive bands in the lysates
from A(/) lens epithelial cells, demonstrating the specificity
of the antiserum in Western blotting. Right panel, the
primary antibody used was a polyclonal antiserum raised to the 21-amino
acid C-terminal peptide of human B, which does not cross-react with
A. WT, cell lysate obtained from wild type cells.
A(/), lysates obtained from A(/) lens
epithelial cell cultures. Note that both wild type and A(/)
lens epithelial cells continued to express B-crystallin in
culture.
A(/) and wild type mouse lenses showed lens epithelial cell outgrowth within 3-4 days (Fig. 3,
A and B). Cells harvested from wild type cells
and A(/) cells showed similar attachment efficiencies (50-60%) when subcultured. When cell growth was monitored over a
period of 11 days, A(/) grew significantly more slowly than wild type controls. In medium containing 10 or 20% serum, the growth
rate for A(/) cells was approximately 50% that of wild type
cells (Fig. 3, C and D). In 5% serum, cell
growth was substantially diminished for both the A-deficient and
wild type cells (Fig. 3E).
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Fig. 3.
Cell growth analysis of primary cultures of
wild type and A(/) lens epithelial cells in culture media
containing different amounts of fetal bovine serum. A
and B, phase contrast images of cell outgrowth from
explanted capsule/epithelia from wild type (A) or
A(/) lenses (B) in Eagle's minimum essential
medium containing 20% serum. Note the smaller size and higher cell
density in the cultures of wild type cells. First passage cells were
cultured in minimum essential medium containing different
concentrations of fetal bovine serum, trypsinized, and counted in a
Coulter counter at the indicated times. 
, wild type cells; 
,
A(/) cells. C 20% serum; D, 10% serum;
E, 5% serum. Scale bars in A and
B = 100 µm.
A(/) lens epithelial
cells had a slower growth rate than wild type cells suggested that the
molecular chaperone A may affect cell cycle kinetics. To explore
this further, propidium iodide-labeled cells from primary cultures of
wild type and A(/) lens epithelial cells were analyzed by flow
cytometry. Compared with wild type cells, where 15% of the cells were
in the G2/M phases, only 2% of A(/) cells were in
the G2/M phases (Fig. 4).
Since the number of cells in S phase was similar for both cell types
(5-6%), this result indicates that A(/) cells are blocked
after S phase of the cell cycle.
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Fig. 4.
Cell cycle distribution using propidium
iodide labeling and FACS analysis. Plots of number of cells
(counts) versus fluorescence (FL2-A)
were used to determine the proportion of cells in G1, S,
and G2/M phases of the cell cycle. Top, wild
type; bottom, A(/) mouse lens epithelial cells.
Data are representative of three different experiments. Note that the
proportion of cells in the G2/M phases decreased from
~15% in wild type to 2% in the A(/) cells.
A(/) cultures (Fig.
3) could also be explained by increased levels of apoptosis in these
cultures. In this regard it is interesting to note that other sHSPs and
HSP70 have been found to protect cells from apoptosis induced by a
variety of stresses. To determine whether the rate of apoptosis
differed between wild type and A(/) lens epithelial cells, we
used a TUNEL assay of cells cultured under normal or UVA stress
conditions. TUNEL-labeled nuclei were not observed in untreated wild
type control cells (Fig. 5A)
but were present at low levels (0.2%) in untreated A(/) cells
(Fig. 5C).
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Fig. 5.
The effect of UVA irradiation on TUNEL
labeling and actin organization in wild type and A(/) mouse
lens epithelial cell cultures. Epithelial cells were exposed to
37.8 J/cm2 UVA radiation (365 nm), incubated in normal
medium for 4 h, and processed for TUNEL labeling
(green). F-actin was visualized using Texas Red phalloidin
(red). A, wild type cells showed no
TUNEL-positive nuclei. B, wild type cells exposed to UVA
radiation and then incubated for 4 h had very few TUNEL-positive
cells but showed the formation of actin rings around the nuclei,
arrows. C, non-irradiated A(/) cells had
very low levels of TUNEL labeling. D, A(/) cells
exposed to UVA radiation and then incubated for 4 h in normal
media showed high levels of TUNEL labeling (arrows).
Scale bars = 25 µm.
A(/) lens epithelial cells. Four hours after UVA irradiation, the major morphological change in wild type cells was the formation of rings of
F-actin around the cell nuclei (Fig. 5B). Previous studies on skin fibroblasts exposed to UVA radiation have shown a similar redistribution of actin into perinuclear rings (29). TUNEL-positive nuclei were rarely observed in irradiated wild type cells. In contrast,
TUNEL-positive nuclei were commonly observed in irradiated A(/) cultures (Fig. 5D). A characteristic of the
TUNEL-positive A(/) cells was that actin was either absent
from these cells or clumped around the apoptotic nuclei (Fig.
5D).
A(/) cells, we counterstained the cultures with DAPI. This
allowed the total number of cells in any field to be counted and the
proportion of TUNEL-positive cells to be determined. In A(/)
cultures, TUNEL-positive cells were occasionally observed even under
normal conditions. UVA radiation induced apoptotic cell death in
A(/) cells with a maximal effect observed 4 h after UVA
treatment. The effect on wild type cells was significantly less (Fig.
6). The A(/) cells had
significantly higher number of TUNEL-positive cells 4 h after UVA
exposure. This phenomenon was not observed in the wild type cells (Fig.
6).
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Fig. 6.
Effect of UVA exposure and subsequent
incubation on the proportion of TUNEL-positive cells in primary
cultures of wild type and A(/) lens epithelial cells.
Cells were TUNEL-labeled and stained with DAPI to enable a count of the
total number of cells in each microscopic field. The number of
TUNEL-positive cells and DAPI-stained cells were counted in 10 randomly
chosen fields from three or more cultures at each time point.
A(/) cells had 40 times as many apoptotic cells as
the wild type UVA-treated cells. The number of apoptotic cells increased from 0.21 ± 0.1% for the wild type to 8.05 ± 0.08% for the A(/) cells. Twenty hours after UVA exposure,
TUNEL-positive nuclei in the A(/) cells had decreased.
A-crystallin by
Western blot analysis (Fig. 7A). Cells were transfected
with increasing amounts of pCI-neoA DNA (0.5-40 µg) and, on
Western blotting, showed a concentration-dependent increase
in A expression (Fig. 7A). Untransfected or
mock-transfected cells had no detectable levels of A.
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Fig. 7.
Western blot analysis of the expression of
A in HLE B-3 cells. Top, cells were transfected with
different concentrations of pCI-neoA construct. Lane 1, 1 µg; lane 2, 7 µg; lane 3, 23 µg; lane
4, 80 µg; lane 5, 23 µg DNA without A
insert; lane 6, untransfected cells. The primary
antibody was a polyclonal antiserum to bovine lens -crystallin which
only detects the A polypeptide in Western blots. The secondary
antibody was 125I-Protein A. Bottom, Western
blot analysis of the expression of A in cell lines expanded from
geneticin-resistant clones. Lanes 1-7, representative
clonal cell lines that tested positive for the expression of A;
lane 8, a mock transfected clonal cell line; lane
9, untransfected HLE B-3 cells; lane 10, native human
-crystallin.
A in seven individual
cell lines expanded from the original geneticin-resistant clones.
Western blots of lysates from each of these cell lines revealed a
single immunoreactive band (~21 kDa) which co-migrated with native
A. Localization of A was also visualized by confocal
immunofluorescence microscopy of several clonal cell lines. Two
representative cell lines are shown in Fig.
8. In clone 65A, a cell line that
expresses very low levels of A (0.1 ng of A/µg of total
protein), A is distributed throughout the cytoplasm of some cells,
whereas other cells in the culture apparently do not express A (Fig.
8A). In clone 6A, expressing 1.8 ng of A/µg, all
cells strongly expressed the protein (Fig. 8B). The combined
Western blot and immunofluorescence analysis allowed the selection of
HLE B-3 clonal cell lines expressing distinctly different levels of
A. Several of these cell lines were used to investigate the effects
of A expression on resistance to UVA stress.
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Fig. 8.
Confocal micrographs of A
immunofluorescence in stably transfected human lens epithelial cell
lines. Cell lines were generated by transfection of HLE B-3 cells
with pCI-neoA (see text for details). A immunofluorescence is
shown in red, and fluorescein-phalloidin staining is shown
in green. A, clone 65A, one of the low
A-expressing clones, which was found to express 0.1 ng of human
A-crystallin per µg of cellular protein by quantitative Western
blot analyses. Note that not all cells expressed A to the same
level, and A-crystallin was not uniformly expressed in all parts of
the cytoplasm of a given cell. B, clone 6A, expressing
1.8 ng of human A per µg of cellular protein was one of the high
A expressing clones. Note that A was uniformly distributed at
high levels in all cells and was excluded from nuclei
(arrow). Scale bars = 25 µm.
A were exposed to
increasing fluences of UVA radiation. Cell survival was measured by
their ability to form colonies when plated at low densities. When
compared with the mock-transfected cells (plasmid without the insert)
or untransfected cells, the survival level of UVA-exposed HLE B-3 cells
expressing A increased significantly (Fig.
9A). Moreover, the cell line
expressing the highest level of A, cell line 9A (expressing 2.7 ng of A/µg of cellular protein), was the most resistant to UVA
stress. Compared with untransfected cells, the survival of clone 9A
increased by 90-fold.
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Fig. 9.
Effect of expression of
A-crystallin on cell survival of HLE B-3 cells exposed to UVA
radiation. A, cell survival as determined by clonogenic
survival. Cells from mock-transfected (), untransfected (
), and
clonal cell lines 6A (), 8A (
), and 9A (
) were
irradiated with increasing fluences of UVA radiation, and the surviving
fraction was determined. Data points are the means of 12 replicates of
a representative experiment for which the standard deviation was
±10%. Cell lines 6A, 8A, and 9A expressed 1.8, 1.4, and 2.7 ng of human A per µg of cellular protein, respectively.
B, plots of forward light scatter (FSC-H)
versus side scatter (SSC-H) for UVA-exposed cell
lines as determined by flow cytometry. The white area on the
lower left corner of the plots shows the proportion of dead
cells in the culture, which are smaller in size. Top, clonal
cell line 24A expressing 0.5 ng of A per µg of cellular
protein; bottom, mock-transfected cell line. Note that the
relative proportion of dead cells as indicated by the size of the white
area was significantly higher in the mock-transfected cells. The narrow
band in mock-transfected cells (bottom panel) indicates a
highly concentrated area of dead cells.
A-overexpressing cells and
mock-transfected cells (Fig. 9B). Forward light scatter
indicated by the white area on the left of the plot was significantly
greater in mock-transfected cells than in A-overexpressing cells.
Clone 24A expressing 0.5 ng of A/µg of cellular protein had a
significantly smaller proportion of dead cells than mock-transfected
cells (Fig. 9B). These results indicate that, even at low
levels, expression of A had a protective effect against UVA stress.
A-crystallin on UVA radiation-induced
apoptotic cell death as measured by TUNEL assay is shown in Fig.
10. The basal level of apoptosis was
higher in the mock-transfected cells than in A-overexpressing cells
(Fig. 10, A and C). After UVA exposure, cells
without A-crystallin had a significantly higher number of
TUNEL-labeled cells than those overexpressing A-crystallin (Fig. 10,
B and D). Quantitative analysis indicated that
the mock-transfected or untransfected cells had 6-10-fold more
TUNEL-positive nuclei than the A-overexpressing cells. These observations show that as was observed in the case of A(/) and
wild type mouse lens epithelial cells, A expression in transfected HLE B-3 cells also prevents UVA-induced apoptosis.
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Fig. 10.
Merged confocal images of TUNEL and Texas
Red phalloidin staining after UVA-induced apoptosis in human lens
epithelial cells. Cells were exposed to 37.8 J/cm2 UVA
radiation (365 nm), incubated in 20% fetal bovine serum-Eagle's
minimum essential medium for 4 h, and then processed for TUNEL
labeling. A, mock-transfected cells not exposed to UVA had a
small but significant number of TUNEL-positive cells
(arrow). B, mock-transfected cells exposed to UVA
radiation and then incubated for 4 h had a large number of
TUNEL-positive cells (arrows). C, lens epithelial
cells from A-overexpressing cell line 6A had no TUNEL-positive
cells. D, A-overexpressing cell line 6A exposed to UVA
radiation and then incubated for 4 h had sparse TUNEL-positive
cells as compared with mock-transfected cells exposed to UVA. F-actin
fluorescence, shown in red, remained bright in these cells.
Note that F-actin staining was absent in many of the UVA-irradiated
TUNEL-positive cells. Scale bars = 25 µm.
A-crystallin in clonal cell line 9A
was examined after exposure to different stresses. A-crystallin was
visualized by immunofluorescence with a monoclonal antiserum to
A-crystallin as the primary antibody and lissamine rhodamine goat
anti-mouse IgG as the secondary antibody. F-actin was labeled using
fluorescein-phalloidin. Confocal microscopy showed that UVA, UVB, or
heat (45 °C for 30 min) treatment did not change dramatically the
cellular localization of A-crystallin in the clone 9A (data not
shown). These results indicate that unlike the reported change in
localization of B and HSP27 to the nucleus during heat shock or
other stresses (30-32), A remains cytoplasmic after these treatments.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
A and B are synthesized as major components of all
vertebrate lenses (3). Although A is highly expressed in lens fiber cells, it is also readily detected in lens epithelial cells. In the
mouse lens epithelium, for example, A transcripts are present at
embryonic day 12.5 and continue to be expressed during embryonic development and postnatally (33). In the present study, we confirmed that A is expressed in the lens epithelium in situ and
demonstrated that A is also expressed in primary and secondary
cultures of lens epithelial cells.
A(/) mouse lens epithelial cells under normal
conditions of growth and after exposure to physiological levels of
stress induced by UVA radiation. Our main finding was that, in the
absence of A, apparently normal levels of B were insufficient to
sustain normal cell growth or to protect cells from UVA-induced apoptosis. The A(/) lens epithelial cells grew at a rate 50% slower than that of wild type cells. Because the fiber cells that comprise the bulk of the lens are derived from the epithelium, the
lower growth rate of A(/) epithelial cells may explain the
observation that A-deficient lenses weigh ~30% less than wild
types (20).
A(/) cultures had the same number of cells in the
S phase of the cell cycle as wild type, they had 6-fold fewer cells in
the G2/M phases of the cell cycle, suggesting a cell cycle
block after the S phase. Decreased growth of A(/) cells may be
due to a direct interaction of A with cell cycle regulators.
Interestingly, other chaperone proteins, such as GrpE and HSP90, are
essential for cell growth (11, 12). It remains to be tested if the
effects of A on growth depend upon its ability to be phosphorylated
and/or its autokinase activity (8, 9, 34).
A on cell growth could also be mediated by proteins,
such as B, that are normally complexed to A. Interestingly, B
phosphorylation is increased in A(/) lenses (21). In
A(/) lenses, B is located in cytoplasmic inclusion bodies,
raising the possibility that the A(/) phenotype may be due to
a functional absence of B. However, B inclusion bodies are not
present in the epithelium of A(/) lenses, and in the present
study, we found B was expressed in apparently normal amounts in both
wild type and A(/) cells. These data suggest that the
decreased growth rates observed in A(/) cells were not due to
the absence of B.
A(/) lens epithelial cells may be
explained in part by increased levels of apoptosis in these cells. In
this study, the levels of apoptosis in A(/) cells were low but
significantly greater than in wild type cells. Thus, even in the
absence of external stress, the expression of A appeared to enhance
cell viability. This observation was subsequently confirmed by our
studies on HLE B-3 cells. Mock-transfected or untransfected HLE B-3
cells had significantly higher level of apoptosis than the
A-overexpressing clonal cell lines. These findings suggest that A
plays a critical role in preventing apoptosis in these cells.
A(/)
cells had 40 times more apoptotic cells than wild type cells. The
A(/) cells still expressed B but were not protected from UVA-induced apoptotic death, suggesting a specific role for A in
protecting cells from apoptosis. Remarkably, A overexpression in HLE
B-3 cells also protected against UVA-induced cell death as measured by
TUNEL labeling (apoptosis), clonogenic assay, and FACS analysis.
Overexpression of the small heat shock protein, HSP27, protects mouse
fibrosarcoma cells against apoptosis induced by various treatments,
indicating that sHSPs can act as inhibitors of apoptosis generated
through a variety of signal transduction pathways (14).
A depends upon its action on
intracellular reactive oxygen species or GSH levels.
A. Apoptotic A(/) cells had either no actin
labeling or had clumps of actin surrounding the degraded DNA. This
observation is consistent with in vitro studies showing that
the strong actin depolymerizing effect of cytochalasin D could be
blocked by -crystallin (40). HSP27 protects cells from stress
inducers such as heat, arsenite, or hydrogen peroxide by stabilizing
the microfilament network (41). This protective effect of HSP27 is
regulated by a mitogen and stress-sensitive signaling pathway involving
p38 mitogen-activated protein kinase (42). Previous studies have shown
that UVA disruption of actin in lens epithelial cells has deleterious
effects on their integrity and function (43, 44). Absorption of UVA
radiation by tryptophan photoproducts, riboflavin, or other cellular
metabolites can result in the formation of singlet oxygen and free
radicals (45) that are expected to damage cellular components such as
actin. In the present study, a comparison of wild type and
A(/) cells suggests that A expression enhances cell
survival by preventing the actin depolymerization effect of UVA stress.
B from the
cytoplasm into the nucleus/insoluble portion of the cell, concomitant
with an increase in aggregate size. Cells made thermotolerant by a
previous, mild heat shock do not exhibit this relocalization. It is
also known that during recovery from heat shock, the HSP27 gradually
returns to its cytoplasmic location. The present study demonstrated
that, unlike the effect of heat shock and other toxic treatments on the
cellular localization of HSP27 and B-crystallin, there was no
dramatic effect of stress on the cellular localization of A.
A are likely to be involved in specific
growth regulation and protection from environmental stress. Our
experiments provide strong evidence that this is the case in the lens.
We have shown that A(/) cells have a decreased growth
potential. Our studies also indicate that A expression protects
epithelial cells from UVA-induced apoptosis. Identifying the cell cycle
proteins and apoptotic pathways with which A interacts are topics
for further investigation.
ACKNOWLEDGEMENTS
A-crystallin; Dr. Paul Fitzgerald for the monoclonal
antibody to A-crystallin; and Dr. Timothy P. Fleming for helpful
suggestions on the transfection studies.
FOOTNOTES
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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