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B-Crystallin in the Trabecular Meshwork Is Inducible by Transforming Growth Factor-ß
1 From the Department of Anatomy II, University of Erlangen–Nürnberg, Erlangen, Germany; and the 2 Department of Biochemistry, University of Nijmegen, Nijmegen, The Netherlands.
Abstract
PURPOSE. Because in glaucomatous eyes transforming growth factor-ß (TGF-ß)
and 
B-crystallin are increased in the anterior eye segment, the
effect of TGF-ß1 and TGF-ß2 on the expression of B-crystallin
and its corresponding mRNA was studied in human trabecular meshwork
(TM) cells.
METHODS. Monolayer cultures of "cribriform" and "corneoscleral" TM cells
of 5 human donors (12–73 years of age) were treated with either 1.0
ng/ml TGF-ß1, TGF-ß2, or 5 x 10-7 dexamethasone
(DEX) for 12 to 96 hours. Induction of B-crystallin and the related
mRNA was investigated by western and northern blot analyses. For
comparison, human foreskin fibroblasts (HFF) and NIH 3T3 cells were
treated in the same way as the TM cells.
RESULTS. An increase of B-crystallin mRNA was observed after treatment of TM
cells with TGF-ß1 and TGF-ß2, whereas DEX had no effect. In the
cribriform TM cells with a high basal level, the enhancement ranged
between 2 and 3 times; whereas in the corneoscleral TM cells
B-crystallin mRNA increased between 5 and 6 times. Using western
blot analysis, the increase of B-crystallin expression in the
cribriform TM cells was only small compared with the significant
increase in the corneoscleral TM cells. Treatment of HFF and NIH 3T3
cells with TGF-ß did not induce B-crystallin mRNA.
CONCLUSIONS. This is the first time to show that B-crystallin is not only induced
by stress factors but also by TGF-ß in TM cell cultures. The
difference in induction of mRNA and protein seems to be dependent on
B-crystallin concentration before
treatment.
Alpha B-Crystallin, a member of the small heat shock
protein family (HSP), can act as a molecular chaperone, preventing
aggregation and unfolding of proteins in response to
stress.1
Constitutively the protein is expressed not only
in lens cells2
but also in a variety of nonlenticular
cells.3
4
5
6
7
8
Most of these cells show only minor mitotic
capacity and are exposed to physiological, mechanical, osmotic, or
oxidative stress over a long period. In the eye, constitutive
expression of B-crystallin has been found in the trabecular meshwork
(TM). Histologic sections revealed the presence of B-crystallin only
in cells adjacent to the inner wall of Schlemm’s canal but not in the
inner portions of the TM.9
10
11
In the present study we
showed that at the electronmicroscopic level of B-crystallin is
present only in cribriform TM cells and not in cells of the lamellated
portion of the TM or in endothelial cells lining Schlemm’s canal. In
many glaucomatous eyes B-crystallin is increased in the TM and is
also present in inner corneoscleral and uveal TM cells.12
Elevated levels of B-crystallin have also been described in various
neurodegenerative disorders such as Creutzfeld–Jacob or Alexander’s
disease.13
14
15
The reason for the accumulation of
B-crystallin under these pathologic conditions is unknown.
In vitro human TM cells accumulate B-crystallin in response to
oxidative stress and heat shock.10
Oxidative damage is
considered an important factor in the pathogenesis of
glaucoma,16
17
18
but direct evidence for this assumption is
still lacking. On the other hand, it has been shown that the aqueous
humor of many glaucomatous eyes exhibits increased amounts of
transforming growth factor-ß (TGF-ß).19
Moreover, it
is known that glucocorticoid treatment is causative for some forms of
glaucoma. It has been shown that hormone responsiveness is a
characteristic of small HSP genes (e.g., human hsp27 is
estrogen-responsive and is expressed in several estrogen-sensitive
human tissues and breast tumors).20
21
22
23
Accumulation of
B-crystallin in response to the glucocorticoid hormone dexamethasone
(DEX) has been demonstrated in murine NIH 3T3 cells.24
In
the present study we investigated whether treatment with DEX or TGF-ß
results in the induction of B-crystallin in TM cells.
Materials and Methods
Electronmicroscopic Investigation
In previous studies we have shown that localization of
B-crystallin staining is similar in human and monkey
eyes.9
Sufficient morphology for ultrastructural
B-crystallin demonstration was only obtained in primate eyes fixed
immediately after enucleation. These studies were therefore only
performed in monkey eyes. Cynomolgus monkey eyes were kindly provided
by the primate center in Marburg (Behring Werke, Marburg, Germany). All
animals were kept and treated in agreement with the Helsinki Convention
on the Use of Animals in Research and conform to the ARVO Statement for
the Use of Animals in Ophthalmic and Vision Research. The animals were
killed in conjunction with other nonocular protocols. The eyes were
immediately enucleated and 2-mm–wide sections of the anterior eye
segment were placed in 4% paraformaldehyde and 0.1% glutaraldehyde
for 4 hours. Cryoprotection was performed by washing in ice-cold 30%
dimethylformamide twice for 30 minutes. The samples were then
incubated in methylbutan for 30 minutes and immediately frozen in
liquid nitrogen. Low-temperature embedding was performed with a EM-CPC
(Leica, Germany, Bensheim) using Lowicryl HM20 (Polysciences Europe,
Eppelheim, Germany) according to the instruction manual of the
manufacturer. After UV-polymerization, ultrathin sagittal sections were
prepared using a Reichert microtome (Ultracut OmU3).
Sections were mounted on Ni-grids, air-dried, and preincubated with 2%
bovine serum albumin in phosphate-buffered saline (PBS, pH 7.4) for 20
minutes, followed by incubation with the primary antibody (rabbit anti
B-crystallin 1:100)4
for 2 hours. After washing six
times for 10 minutes each, labeling was performed using goat
anti-rabbit IgG 10 nm gold particles and the sections washed again.
Uranyl-acetate–stained sections were viewed with a Zeiss EM 902
electronmicroscope (Zeiss, Oberkochen, Germany).
Tissue Culture
Cell cultures derived from eyes of 5 human donors (12, 49, 57, 57,
and 73 years of age, obtained 4–8 hours postmortem) were prepared,
grown, and classified as described previously.9
11
Cells
derived from the cribriform and outer corneoscleral meshwork were
termed cribriform, cells derived from the inner corneoscleral and uveal
portion were termed corneoscleral trabecular cells. Confluent TM cells
were distinguished from each other and from adjacent scleral spur and
ciliary muscle cells by their morphology and immunohistochemical
staining as described previously.11
In brief, confluent cells of the third passage being confluent
for 7 days were stained with antibodies against -smooth muscle (sm)
actin (mouse, clone No. 1A4 Ig G2a anti-bovine; Dakopatts, Hamburg,
Germany) diluted 1:150, desmin (mouse, clone D33 anti-human IGG1;
Dakopatts) diluted 1:10, and B-crystallin (rabbit)4
diluted 1:100. For staining with antibodies against -sm actin and
desmin, cells were fixed with ice-cold methanol for 3 minutes; for
demonstration of B-crystallin the fixation was performed with 4%
paraformaldehyde for 15 minutes, followed by two washes with PBS
containing 0.1% Triton X-100. All antibodies were incubated overnight
at 4°C. After washing in PBS, cells were incubated with
fluorescein-labeled swine anti-mouse and swine anti-rabbit
immunoglobulins (Dakopatts) diluted 1:20. Slides were viewed and
photographed with a Leitz Aristoplan microscope (Leitz, Wetzlar,
Germany).
Confluent cribriform and corneoscleral TM cells of passage 3 were incubated for 12, 24, 48, or 96 hours in serum-free medium supplemented with 1.0 ng/ml TGF-ß1 (Boehringer Mannheim, Mannheim, Germany), 1.0ng/ml TGF-ß2 (Boehringer Mannheim), 5 x 10-7 M DEX (Sigma, Deisenhofen, Germany). The treated cells were compared with cultures incubated under identical conditions but without TGF-ß or DEX in the medium.
To test whether the effect of induction of B-crystallin mRNA is due
to the HCl that is used to activate TGF-ß, cribriform cells were
either treated with 1 µM HCl or a combination of 1.0 ng/ml TGF-ß2
and 10 mg/ml anti–TGF-ß2 (RD-Systems, Wiesbaden, Germany).
Additionally we treated primary human foreskin fibroblasts (HFF; kindly provided by the Department of Virology, Erlangen) and NIH 3T3 fibroblasts with DEX, TGF-ß1, or TGF-ß2, in the same way as the TM cells for 24 hours, 48 hours, and 7 days.
RNA Isolation and Northern Blot Analysis
Total RNA was isolated from 35-mm petri dishes by the
guanidinium thiocyanate–phenol–chloroform extraction method (RNA
isolation kit, Stratagene, Heidelberg, Germany). RNA (15 µg) was
denatured and size-fractionated by gel electrophoresis in 1% agarose
gels containing 2.2 M formaldehyde. The RNA was then vacuum-blotted
onto a nylon membrane (Boehringer Mannheim) and cross-linked (1600
µJ, Stratalinker; Stratagene). To assess the amount and quality of
the RNA, the membrane was stained with methylene blue. Images were
taken with the Lumi-Imager (Boehringer Mannheim). Prehybridizations
were performed at 68°C for 1 hour in Dig Easy Hyb (Boehringer
Mannheim). Hybridizations were at 68°C overnight in prehybridization
solution containing 50 ng/ml B-crystallin–specific 450-bp antisense
riboprobe. Riboprobes were synthesized by using a combined polymerase
chain reaction (PCR). Briefly, a DNA fragment was amplified from cDNA
of TM cells using the reaction conditions and primers described
previously,11
except that the downstream primer
(MWG-Biotech, Ebersberg, Germany) contained the sequence for the
T7-promoter. After purification with a Quiagen (Hilden, Germany) PCR
Purification Kit, 1 µg DNA was used as a template for in vitro
transcription using the digoxigenin labeling RNA Kit from Boehringer
Mannheim. Labeling efficiency was checked by direct detection of the
labeled RNA probe with anti–digoxigenin–alkaline phosphatase. After
hybridization, the membrane was washed twice with 2x SSC, 0.1% sodium
dodecyl sulfate (SDS) at room temperature, followed by two washes in
0.1x SSC, 0.1% SDS, for 15 minutes at 68°C. After hybridization and
posthybridization washes, the membrane was washed for 5 minutes in
washing buffer (100 mM maleic acid, 150 mM NaCl; pH 7.5; 0.3%
Tween-20) and incubated for 60 minutes in blocking solution (100 mM
maleic acid, 150 mM NaCl, pH 7.5, 1% blocking reagent, Boehringer
Mannheim). Anti–digoxigenin–alkaline phosphatase (Boehringer
Mannheim) was diluted 1:10,000 in blocking solution, and the membrane
incubated for 30 minutes. The membrane was washed four times, 15
minutes per wash, in washing buffer. The membrane was equilibrated in
detection buffer (100 mM Tris–HCl, 100 mM NaCl, pH 9.5) for 10
minutes. For chemiluminescence detection CDP-star (Boehringer Mannheim)
was diluted 1:100 in detection buffer, and the filter incubated for 5
minutes at room temperature. After air-drying, the semidry membrane was
sealed in a plastic bag. Chemiluminescence was detected with the
Lumi-Imager workstation. Exposure times ranged between 10 minutes and 1
hour. The quantification was performed with Lumi Analyst (Boehringer
Mannheim).
Western Blot Analysis
Cells grown on tissue culture dishes were washed twice with PBS
(pH 7.2), collected, and lysed in SDS sample buffer for gel
analysis.25
The samples for gel analysis were boiled for 5
minutes, and protein content was measured using BCA protein assay
reagent (Pierce, Rockford, IL). For analysis of the proteins (2 µg),
a 5% SDS–polyacrylamide gel electrophoresis (PAGE) for stacking gel
and 12% SDS–PAGE for separating gel were used. After electrophoresis
the proteins were transferred with semidry blotting onto a polyvinyl
difluoride membrane (Boehringer Mannheim). The membrane was incubated
with PBS containing 0.1% Tween-20 (PBST, pH 7.2) and 5% bovine serum
albumin for 1 hour. The primary antibody (B-crystallin diluted
1:4000)4
was then added and allowed to react overnight at
room temperature. After washing three times in PBST, an alkaline
phosphatase–conjugated swine–anti-rabbit antibody (diluted 1:20,000;
Dianova, Hamburg, Germany) was added for 30 minutes. Visualization of
the alkaline phosphatase was achieved using chemiluminescence. CDP-star
was diluted 1:100 in detection buffer, and the filter was incubated for
5 minutes at room temperature. After air-drying the semidry membrane
was sealed in a plastic bag. Chemiluminescence was detected with the
Lumi-Imager workstation. Exposure times ranged between 1 and 5 minutes.
The quantification was performed with Lumi Analyst.
Results
Immunoelectronmicroscopy
Immunogold labeling for B-crystallin was seen only in the
3 to 5 layers of cribriform cells (Fig. 1)
. The endothelial cells lining Schlemm‘s canal as well as the TM cells
covering the corneoscleral and uveal trabecular lamellae were
completely unstained.
|
B-crystallin, whereas most
of the corneoscleral cells remained unstained (Figs. 2
A, 2B). These staining differences were seen in all cultures derived
from all five donors. None of the cultures stained for desmin, only
single cells in both TM cultures stained for -sm actin.
|
B-Crystallin mRNAB-crystallin mRNA in the 0.8-kb band (3 times; Fig. 3
), whereas the 1.1-kb band appeared only slightly increased.
Additionally, a band at approximately 2.6 to 2.9 kb hybridized with the
B-crystallin probe.
|
B-crystallin mRNA. There were, however, differences in the
responsiveness of the two cell types. In the cribriform TM cells the
increase ranged between 2 and 3 times (Fig. 3)
, whereas the
corneoscleral TM cells, which had a 3 times lower basal level than the
cribriform TM cells, showed a much higher increase (6–8 times) of
B-crystallin mRNA after 12-hour treatment with TGF-ß1 or TGF-ß2.
In both cell types the increase was mainly in the 0.8-kb band, whereas
the 1.1-kb band nearly disappeared. Additionally, in cribriform and
corneoscleral TM cell northern blot analysis, the B-crystallin probe
hybridized with the 2.6- to 2.9-kb band after treatment with TGF-ß.
Treatment of the corneoscleral or cribriform TM cells for 24, 48, or
even 96 hours with TGF-ß1 and TGF-ß2 showed the same results (Fig. 3
, Table 1
).
|
B-crystallin mRNA levels after 24 hours (Fig. 4)
.
|
B-crystallin mRNA (data not shown).
Treatment with DEX for 12, 24, 48, and 96 hours had no effect on
B-crystallin mRNA expression, either in the cribriform or in the
corneoscleral cells (Fig. 3
, Table 1
).
Effect of TGF-ß and DEX on B-Crystallin Expression
Using western blot analysis, the cribriform TM cells showed a 6
times higher amount of B-crystallin than the corneoscleral TM cells
(Fig. 5)
.
|
B-crystallin in the
corneoscleral cells, whereas the increase in the cribriform TM cells
was only small (Fig. 5)
. In the corneoscleral TM cells B-crystallin
increased after 12 hours of treatment with TGF-ß1 and TGF-ß2
between 4.5 and 6.8 times, whereas in the cribriform TM cells
the increase ranged between 2 and 3 times. After 96 hours of treatment,
the increase in the corneoscleral and cribriform TM cells was in the
same range.
Treatment with DEX for 12 to 96 hours had no effect on the amount of
B-crystallin, either in cribriform or in corneoscleral TM cells.
Treatment of NIH 3T3 cells and HFFs
NIH 3T3 and HFFs showed a clear difference in their response to
DEX and TGF-ß when compared with TM cells. In NIH 3T3 cells treatment
with TGF-ß1 and TGF-ß2 for 24 hours, 48 hours, and 7 days did not
induce B-crystallin mRNA. On the other hand, B-crystallin mRNA
was induced by treatment with DEX. This induction was seen after 24
hours of DEX treatment and increased after 48 hours and 7 days of
treatment (Fig. 6)
.
|
B-crystallin mRNA (Fig. 7)
. Untreated and treated HFFs showed a faint hybridization of the
antisense B-crystallin probe at 0.8 kb.
|
The induction of B-crystallin has so far only been shown in
various cell cultures after heat shock, oxidative damage, and osmotic
or mechanical stress.26
27
28
The present study demonstrates
for the first time that TGF-ß, a growth factor known to be increased
in the aqueous humor of a number of glaucomatous eyes, stimulates
expression of B-crystallin in TM cells. In both cultures of
trabecular cells, B-crystallin mRNA and protein expression were not
induced by treatment with DEX. On the other hand, B-crystallin mRNA
and protein expression were induced by TGF-ß treatment, which had no
effect on NIH 3T3 fibroblasts or on HFF cell lines.
Before treatment, western blot analysis showed clear differences in the
amount of B-crystallin between corneoscleral and cribriform TM
cells. Cribriform TM cells constitutively express the protein
B-crystallin, whereas in corneoscleral TM cells the protein is
virtually absent. These results confirmed the immunohistochemical
differences between these two cell types. By northern blot analysis we
have demonstrated that parallel to the protein expression, the amount
of B-crystallin mRNA is high in cribriform TM cells but very low or
even undetectable in corneoscleral TM cells.
Stimulation at the mRNA level is paralleled by a similar effect at the
protein level. As can be concluded from Table 1
, treatment of
corneoscleral TM cells with growth factors TGF-ß1 and -ß2 had a
pronounced effect on B-crystallin mRNA and protein levels, whereas
in the cribriform TM cells the increase was only small. These findings
indicate that high constitutive levels of B-crystallin result in
lowered induction of mRNA and protein.
Our findings lead to the assumption that TGF-ß induces
B-crystallin mRNA expression in TM cells at the transcriptional
level. For HSP70 and HSP90 it has previously been shown that TGF-ß
acts in this way.29
Induction of B-crystallin during
heat shock, hypertonic stress, cadmium exposure, and treatment with
TNF- is also thought to be mediated at the level of
transcription.30
In the 5'flanking region of the human
B-crystallin gene a considerable number of cis-regulatory
sequences have been described, including binding sites for the heat
shock transcription factor 1 (HSF1)31
32
and an alkaline
phosphatase-1–like consensus sequence.30
In rat
astrocytes two different transcriptional regulation mechanisms of
B-crystallin mRNA were shown.30
After cadmium exposure,
an increase in B-crystallin mRNA level and activation of HSF1 were
demonstrated. With hypertonic stress B-crystallin mRNA was induced,
but no activation of HSF1 took place. The elucidation of the exact
mechanism of B-crystallin induction by TGF-ß awaits further
experiments.
It is not known why TGF-ß1 and TGF-ß2 stimulate B-crystallin
expression in TM cells but not in fibroblasts, and it is unknown why
treatment with DEX induces B-crystallin in NIH 3T3 cells but not in
TM cells or HFFs. In a number of glaucomatous eyes nearly the entire TM
but not the adjacent fibroblasts are stained with antibodies against
B-crystallin, reflecting differences in stimulation of protein
expression between the two cell types also in vivo.12
Because TGF-ß has been shown to be increased in the aqueous humor of
glaucomatous eyes,19
this increase might be involved in
the increase of B-crystallin levels in glaucomatous TM.
Acknowledgements
We thank Sandra Hartmann, Angelika Pach, Gerti Link, and Marco Gößwein for expert technical assistance.
Footnotes
Supported by Grant BIOMED PL 961593 of the European Commission to HB and ELD, grant SFB 539 (Glaukome) der Deutschen Forschungsgemeinschaft Bonn, and the Academy of Science, Mainz, Germany.
Submitted for publication February 3, 1999; accepted March 29, 1999.
Proprietary interest category: N.
Corresponding author: Elke Lütjen–Drecoll, Department of Anatomy II, University of Erlangen–Nürnberg, Universitätsstr. 19, D-91054 Erlangen, Germany. E-mail: anat2.gl@anatomie.uni-erlangen.de
References
-Crystallin can function as molecular chaperone Proc Natl Acad Sci USA 89,10449-10453B subunit of lens-specific protein -crystallin is present in other ocular and non-ocular tissues Biochem Biophys Res Commun 158,319-325[Medline][Order article via Infotrieve]
B-crystallin in the anterior segment of primate and bovine eyes Curr Eye Res 12,871-876[Medline][Order article via Infotrieve]
B-crystallin in non-lenticular tissues J Histochem Cytochem 38,31-39[Abstract]
B-crystallin in the rat determined with a sensitive immunoassay system Biochim Biophys Acta 1074,201-208[Medline][Order article via Infotrieve]
B-Crystallin in the mammalian inner ear Otorhinolaryngology 60,121-125
B-Crystallin in the primate ciliary muscle and trabecular meshwork Eur J Cell Biol 71,165-169[Medline][Order article via Infotrieve]
B-crystallin in response to heat shock and oxidative stress Invest Ophthalmol Vis Sci 37,2402-2413B-crystallin and trabecular meshwork inducible glucocorticoid response protein in normal and glaucomatous trabecular meshwork Invest Ophthalmol Vis Sci 39,517-525B-crystallin in central nervous system, glia and neurons in pathologic conditions Am J Pathol 140,345-356[Abstract]
B-crystallin, ubiquitin and stress-response protein-27 Acta Neuropathol 84,443-448[Medline][Order article via Infotrieve]
B-Crystallin is present in reactive glia in Creutzfeldt-Jakob disease Acta Neuropathol 83,324-327[Medline][Order article via Infotrieve]
B-Crystallin expression in mouse NIH 3T3 fibroblasts: glucocorticoid responsiveness and involvement in thermal protection Mol Cell Biol 13,1824-1835B-crystallin is a small heat shock protein Proc Natl Acad Sci USA 88,3652-3656B-crystallin expression Exp Eye Res 54,461-470[Medline][Order article via Infotrieve]
B-crystallin mRNA in rat skeletal muscle to mechanical tension and denervation Biochem Biophys Res Commun 181,1323-1330[Medline][Order article via Infotrieve]
B-crystallin in astrocytes: analysis of HSF and AP1 activation by different types of physiological stress J Cell Sci 109,1029-1039[Abstract]
B-crystallin gene and preferential promoter function in lens Genomics 7,594-601[Medline][Order article via Infotrieve]
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