From the Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma 630-0101, Japan
Received for publication, August 16, 2002, and in revised form, October 16, 2002
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ABSTRACT |
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Fibroblast growth factor (FGF) signaling is
necessary for both proliferation and differentiation of lens cells.
However, the molecular mechanisms by which FGFs exert their
effects on the lens remain poorly understood. In this study, we show
that FGF-2 repressed the expression of lens-specific genes at the
proliferative phase in primary cultured lens cells. Using transfected
cells, we also found that the activity of L-Maf, a lens differentiation factor, is repressed by FGF/ERK signaling. L-Maf is shown to be phosphorylated by ERK, and introduction of mutations into the ERK
target sites on L-Maf promotes its stabilization. The stable L-Maf
mutant protein promotes the differentiation of lens cells from neural
retina cells. Taken together, these results indicate that FGF/ERK
signaling negatively regulates the function of L-Maf in
proliferative lens cells and that stabilization of the L-Maf protein is important for lens fiber differentiation.
The vertebrate lens consists of only two cell populations, and
because of this simple tissue organization, it provides an excellent
model of cell differentiation. The anterior surface of the lens is
covered by a simple epithelium, whereas the remainder and bulk of the
lens is composed of elongated lens fibers (1). During embryonic
development and throughout the life of the organism, fiber cells are
added via differentiation of the lens epithelial cells. The
proliferative epithelial cells in the anterior germinative zone move
across the lens equator to the posterior transitional zone followed by
withdrawal of cell cycling and differentiation into fiber cells. Fiber
cell differentiation is characterized by cell elongation and the
eventual degradation of all cellular organelles including the nucleus.
These morphological changes are accompanied by specific activation of
crystallin proteins as well as intermediate filament components such as
filensin and CP49 (reviewed in Ref. 2). Molecular genetic approaches
have also recently identified a number of signaling molecules and
transcription factors that are critical for lens development.
Fibroblast growth factors
(FGFs)1 regulate a diverse
range of biological activities including adhesion, migration,
proliferation, and differentiation (reviewed in Ref. 3). FGF signaling
is one of the most important regulators of differentiation from lens epithelial cells into fiber cells. Using lens explant cultures, it has
been demonstrated that FGF-1 and FGF-2 can stimulate both proliferation
of lens epithelial cells and differentiation of lens fiber cells (4,
5). In addition, in vivo gain-of-function studies have shown
that misexpression of FGF-1, -4, -8, or -9 under the control of the
lens-specific Several transcription factors, including Pax6, Sox2, Six3, c-Maf, and
L-Maf, are known to be important in lens development and in regulating
the expression of lens-specific genes such as crystallin genes
(10-17). Of these factors, L-Maf is one of the most attractive
candidates for mediating FGF signaling, because L-Maf has been
identified as a lens-specific regulator of avian To address the molecular mechanisms that determine how FGF
signaling regulates transcription of the lens-specific genes in lens
development, we investigated the effects of FGF signaling on the
regulation of L-Maf function. Here we show that FGF-2 promotes proliferation of lens cells in the early stages of primary culture but
represses expression of lens-specific genes at the same time points. In
addition, FGF/ERK signaling represses activity of an L-Maf-dependent reporter in the lens cells. We also found
that residues Thr-57 and Ser-65 of the L-Maf protein are important as
phosphorylation sites for extracellular-signal regulated kinase (ERK)
in vitro. Mutations at either of these two sites caused an
increased stability of the L-Maf protein, resulting in enhanced expression of Lens Cell Culture--
Lens cell cultures were prepared as
previously described (18). Lens cells from 14-day-old chick embryos
were maintained in Dulbecco's modified Eagle's medium, 10% fetal
bovine serum, 1% chicken serum. When the cells reached confluence,
they were harvested with trypsin and centrifuged to remove
nonproliferative lens fiber cells. The cells were then cultured at a
density of 1 × 105/30-mm dish to establish primary
cultures. Transfection with plasmids or treatment with FGF-2 (human
fibroblast growth factor-2, PeproTech House) were carried out 1 day
after establishment of the primary cultures.
Plasmid Construction--
Plasmids used to express FLAG
epitope-tagged L-Maf (pEFX3-fg-L-Maf) constructs in lens cells have
been described previously (14). pEFX-
Reporter constructs containing six copies of an
To construct the GAL4-Elk1 expression plasmid, the coding sequence for
the activation domain of human Elk1 (amino acids 307-428) (kindly
provided by Dr. R. Treisman (21)) was amplified by PCR, subcloned into
pCMX-GAL4 (kind gift from Dr. K. Umesono), and digested with
BamHI and EcoRV (pCMX-GAL4-Elk1).
Transfection and Reporter Assays--
Primary lens cultures in
12-well dishes were transfected with 0.5 µg of DNA using DEMRI-C
according to the manufacturer's protocol (Invitrogen). After
4 h of transfection, the medium was replaced by fresh 10% fetal
bovine serum, 1% chick serum medium. Cells were incubated for a
further 48 h, and then whole cell lysates were prepared in
extraction buffer (0.1 ml of 25 mM Tris-phosphate, pH 7.8, 15% glycerol, 2% CHAPS, 1% L- In Vitro Phosphorylation Analysis--
GST and GST-L-Maf
proteins were expressed in Escherichia coli DH5 Western Blot Analysis--
Nuclear extracts were prepared from
the lens cell cultures as described previously (18). An equal
amount of protein from each extract was subjected to 10% SDS-PAGE and
was subsequently transferred to a nitrocellulose membrane (Schleicher & Schuell). The membranes were incubated with the following primary
antibodies diluted in blocking buffer: anti-FLAG M2 monoclonal antibody
(Kodak, 1:8000), anti-GST (Amersham Pharmacia Biotech, 1:8000),
anti-L-Maf (1:1000 (14)), anti-ERK (Promega, 1:1000), anti-phospho-ERK (Promega, 1:1000), and anti- Proteasome Inhibitors--
The stability of the L-Maf was
analyzed by incubating the cells for 3 h in the presence of
proteasome inhibitor. The inhibitors used were LLnL
(Z-Leu-Leu-Leu-aldehyde, 20 µg/ml, BostonBiochem), MG132
(BostonBiochem, 50 µM), and
clasto-lactacystin- Northern Blot Analysis--
Northern blot analyses were carried
out as described previously (23). In brief, total RNA was isolated from
the cultured lens cells, and equal amounts of RNA were separated on
formaldehyde-agarose gels. After electrophoresis, RNA was blotted onto
Biodyne A nylon membranes (Pall) and hybridized with
digoxigenin-labeled DNA probes. mRNAs were detected with an
alkaline phosphatase-conjugated anti-DIG antibody (Roche Molecular
Biochemicals) and visualized by reaction with the chemiluminescent
substrate, disodium 3-(4-methoxysprio{1,2-dioxetane 3,2'(5'-chloro)tricyclo[3.3.1.13,7] decan}-4-yl)phenyl
phosphate (Applied Biosystems) (Tropix).
Immunostaining of Primary Cultured Cells--
Immunostaining of
primary cultured cells was carried out as described previously (19).
The cells were fixed and incubated with the appropriate antibody:
purified rabbit anti-L-Maf IgG (4), 1:1000; AlexaFluor-488 goat
anti-mouse IgG (Molecular Probes), 1:1000; AlexaFluor-594 goat
anti-rabbit IgG (Molecular Probes), 1:1000. An Olympus AX70 confocal
microscope was used to determine the localization of L-Maf,
Electrophoretic Mobility Shift Assays--
The probe used for
the electrophoretic mobility shift assay was
5'-CATTTCTGCTGACCACGTTGCCTTC-3' (c FGF-2 Stimulates Lens Epithelial Cell Proliferation but Represses
the Expression of Lens-specific Genes--
Previous studies had shown
that FGF-1 and FGF-2 promote proliferation and differentiation of lens
epithelial cells in explant cultures (4, 24). However, the molecular
mechanisms underlying FGF signaling in lens development are yet to be
elucidated. To address these mechanisms, we used primary
cultures enriched for proliferating lens epithelial cells. Initially,
we aimed to confirm whether FGF-2 regulates both proliferation and
differentiation of the lens cells. Cell counts were performed at
different times during culture with or without FGF-2 treatment. The
number of FGF-2-treated lens cells was greatly increased on days 1 and
2 compared with the untreated control cells (Fig.
1A). However, by days 3-6,
neither the FGF-2-treated nor untreated lens cells showed significant
proliferation. After 6 days of culture, we found abundant and large
lentoid bodies in the FGF-2-treated lens cell cultures (Fig.
1B). Lentoid bodies are clusters of differentiated fiber
cells. These results demonstrate that FGF-2 promotes both proliferation
and differentiation of primary cultured lens cells, which is consistent
with previous results using lens explants (4, 24).
Next we examined the effect of FGF-2 on the expression of marker genes
for lens fiber differentiation, crystallin genes and filensin gene.
Primary lens cells were incubated with or without FGF-2, and then total
RNA was isolated and subjected to Northern blot analysis. After day 3, expression of both crystallin and filensin genes was enhanced by the
addition of FGF-2 (Fig. 1C). Surprisingly, FGF-2 efficiently
reduced the expression of FGF Represses L-Maf-mediated Reporter Activity--
On the basis
of FGF-2 treatment, we then proposed that transcription factors
regulating the expression of crystallins should function under the
control of FGF-2. One of the attractive candidates for this regulation
is lens-specific Maf (L-Maf), which binds to chicken
We next examined the effect of FGF-2 on regulation of L-Maf at the
protein level. Since we could not detect L-Maf protein by
Western blot analysis (data not shown), we measured the transcriptional activity of L-Maf protein in primary cultured lens cells using a
reporter gene construct containing six copies of a 25-bp L-Maf binding
site in MEK1 Represses L-Maf-mediated Reporter Activity--
ERK1 and
ERK2, members of the mitogen-activated protein kinase (MAPK) family,
play major roles in a variety of developmental processes and biological
actions induced by FGF signaling (26, 27). Because activated ERK is
detected in lens in vivo (28), FGF-2 may regulate L-Maf
activity through ERK. To test this possibility, we first examined
whether FGF signaling involves the ERK pathway in primary cultured lens
cells. Because serum is a potent activator of the MAPK cascade, lens
cells were serum-starved for 4 h followed by FGF-2 treatment for
1 h. In the whole cell extracts of unstimulated cultured lens
cells, phosphorylated ERK was detected only as a weak band by Western
blot analysis with an anti-phospho-ERK antibody (Fig.
3A, lane 1). In
contrast, phosphorylation of ERK was increased in the FGF-2-treated
cells (Fig. 3A, lane 2). This phosphorylation of
ERK was clearly inhibited by addition of PD98059, a specific inhibitor
of MEK1, the ERK kinase (Fig. 3A, lane 3). These
results demonstrate that FGF-2 promotes phosphorylation of ERK through MEK1 in lens cells.
We next examined the effect of constitutively active MEK1 on the
activity of L-Maf in the primary cultured lens cells using the c Thr-57 and Ser-65 Are Important Amino Acids of the L-Maf Protein
for the Phosphorylation by ERK in Vitro--
We identified three
putative MAPK target sites in the L-Maf acidic region. To test whether
L-Maf is directly phosphorylated by ERK, we first performed in
vitro phosphorylation analysis. When incubated with purified ERK,
recombinant L-Maf protein was clearly phosphorylated (Fig.
4A). To examine
phosphorylation of L-Maf in living cells, we introduced an expression
plasmid encoding wild-type L-Maf into the COS-7 monkey kidney cell
line. Ectopically expressed L-Maf protein was detected as a 42-kDa band
in COS-7 cells by Western blot analysis using an anti-FLAG antibody
(Fig. 4C). A 34-kDa band, which is approximately the same
molecular size as the in vitro translated L-Maf protein,
appeared when the cell lysate was treated with calf intestine alkaline
phosphatase, indicating that L-Maf is phosphorylated in living cells.
To identify the ERK phosphorylation site, a series of L-Maf mutant
proteins was made with substitutions to alanine residues at the three
potential sites of Ser-14, Thr-57, or Ser-65 (designated as S14A, T57A, and S65A, respectively). The wild-type and mutant proteins were subjected to in vitro phosphorylation. Incorporation of
[ L-Maf Activity Is Regulated by Phosphorylation at Thr-57 and
Ser-65--
We next evaluated the effects of mutating the
phosphorylation sites on the transcriptional activation of L-Maf. The
mutation on Ser-14 did not significantly affect the transcriptional
activity of L-Maf (Fig. 5A),
whereas the T57A and S65A L-Maf mutants showed ~5- and 7-fold higher
levels of reporter activity than the wild-type protein, respectively.
We also examined the importance of Thr-57 and Ser-65 for the ability of
L-Maf to induce the expression of its in vivo target gene,
Unphosphorylated L-Maf Protein Is More Stable in Lens Cells
Compared with Wild-type L-Maf--
To elucidate how L-Maf function is
regulated by phosphorylation, we measured the DNA binding activity of
phosphorylated or unphosphorylated L-Maf protein by an electrophoretic
mobility shift assay using the L-Maf response element (c
We next examined the amounts of wild-type and mutant L-Maf proteins
expressed in primary cultured lens cells. Although the mutant
L-maf mRNA levels were equivalent to that of wild-type L-maf (Fig. 6B), both the T57A and S65A mutant
L-Maf proteins accumulated in the cultured lens cells (Fig.
6B, lanes 4 and 5), but the wild-type
L-Maf and S14A mutant protein did not (Fig. 6B, lanes
2 and 3). In addition, when proteasome-mediated
degradation was blocked by MG132, LLnL, or lactacystin, ectopically
expressed L-Maf protein accumulated in the cultured lens cells (Fig.
6C). We also compared the stability of the wild-type and
S65A mutant L-Maf proteins in cultured lens cells. Because wild-type
L-Maf protein is unstable in cultured lens cells, we used LLnL to
prevent its degradation. Cells were incubated in the presence of LLnL for 3 h, washed to remove the inhibitor and further incubated for
various periods. The wild-type L-Maf protein almost completely disappeared, even at 2 h (Fig. 6D), whereas the S65A
mutant L-Maf protein was detected consistently for at least 3 h
(Fig. 6D), indicating that the S65A mutant L-Maf protein is
more stable than the wild type.
To test the importance of MAPK signaling in this stability in cultured
lens cells, we examined the effect of the MEK1 inhibitor PD98059 on
degradation of the L-Maf protein. L-Maf protein was not detected in
untreated control lens cells but was detected in the PD98059-treated
cells (Fig. 6E), indicating that MEK1 signaling regulates
stability of the L-Maf protein. Taken together, these results indicate
that MEK1/ERK signaling regulates stability of the L-Maf protein
through Thr-57 or Ser-65 phosphorylation, and degradation of the L-Maf
protein is dependent on the proteasome pathway.
L-Maf Protein Accumulates in Differentiated Fiber Cells--
To
further investigate the connection between L-Maf protein and ERK
signaling, we examined expression patterns of the L-Maf protein and an
active form of ERK in in vitro differentiating cultured lens
cells. Previous studies have shown that primary cultured lens cells
differentiate into lens fiber cells and form lens-like bodies (lentoid
bodies) (29, 30), which appear to contain a few different cell types
(30). Relatively large fiber cells in the central region of the lentoid
bodies are surrounded by small cells, suggesting that the central cells
are differentiated into fiber cells, whereas the edge cells remains
undifferentiated. Double-immunostaining showed that L-Maf protein was
expressed only in differentiated fiber cells that expressed
Previous studies showed that FGF-1 and FGF-2 promote lens cell
proliferation and lens fiber differentiation in explant cultures (4,
31). Whether these two effects are achieved via identical or
distinct mechanisms remains to be clarified. Morphological changes
induced by FGF in fiber differentiation, such as cell elongation, are
blocked by a specific inhibitor against ERK signaling in explant
culture but FGF-induced To address the issue of how FGF-2 represses lens differentiation in the
early stages of culture, we focused on L-Maf, a protein that regulates
expression of crystallin genes and differentiation of the lens fiber
cells (14, 16, 17). By transcription assays using primary lens cell
cultures, we showed that L-Maf-dependent reporter gene
expression is repressed by FGF-2 treatment but is activated by the
addition of an FGFR inhibitor, SU5402. In addition, ERK can
phosphorylate L-Maf in vitro, and mutation at the ERK phosphorylation sites has a drastic effect on the activation of the
L-Maf-dependent reporter gene in primary cultured lens
cells, probably due to accumulation of the mutant L-Maf proteins. We also found that the mutant L-Maf proteins can induce lens fiber differentiation in primary cultured neural retina cells. These results
demonstrate that L-Maf is negatively regulated by FGF-2 through
phosphorylation by ERK at Thr-57 and Ser-65, resulting in degradation
of the L-Maf protein. Consistent with this idea, L-Maf protein is
undetected in proliferative lens epithelial cells but expressed in
differentiated fiber cells in primary cultured lens cells, as an
activated form of ERK is found in the lens epithelial cells (28).
Several transcription factors are regulated by phosphorylation not only
in regard to degradation but also their transcriptional activity.
Microphthalmia (Mi) is an essential transcription factor for melanocyte
development, which is phosphorylated by MAPK/ERK. This
phosphorylation triggers not only enhanced transcriptional activity of
the microphthalmia protein but also its degradation (32). Because we
normalized the luciferase activities against activities of the control
tk reporter gene in the transcription assays but not against amounts of
the wild- type or mutant L-Maf proteins, our results do not indicate
whether L-Maf phosphorylation also affects its transcriptional
activity. Several lines of evidence do, however, imply that
phosphorylated L-Maf is transcriptionally more active. First,
Benkhelifa and colleagues (33) recently reported that MafA, an L-Maf
homolog in quail, is phosphorylated by ERK at Ser-14 and Ser-65 and
that this phosphorylation is required for expression of QR1, a secreted
glycoprotein in neural retina cells. Secondly, FGF-2 stimulates
expression of lens-specific genes and characteristic morphological
changes of lens cells during the later phases of primary culture;
these FGF stimulative effects are also exerted by overexpression
of L-Maf in primary neural retina cultures (this study and Ref. 14),
implying that FGF-2 stimulates L-Maf activity. Therefore, FGF-2
regulates degradation of L-Maf protein during the early phase, and once
L-Maf is stabilized at a later phase (discussed below), FGF-2 regulates
transcriptional activity through phosphorylation by ERK at the same
sites, Thr-57 and Ser-65.
On the basis of our present study using primary cultured lens cells, we
propose that the stabilization of L-Maf protein is an important
mechanism for the differentiation of lens epithelial cells into lens
fiber cells. FGF-2 promotes proliferation of lens cells and represses
the expression of crystallin genes when lens cells proliferate. During
this period, L-Maf is negatively regulated by MEK1/ERK signaling under
the control of FGF-2, and repression of the L-Maf could prevent the
lens-specific genes from precocious activation. In fact, a
bromodeoxyuridine incorporation assay using primary lens cell cultures
shows that proliferative lens epithelial cells did not express the
L-Maf protein (data not shown), indicating that L-Maf
protein is unstable in proliferative cells. Once proliferating lens
epithelial cells begin to differentiate, they cease cell cycling and
initiate the late lens fiber differentiation events including enhanced
expression of lens-specific genes, such as crystallins, and elongation
of cell shape. Because L-Maf can activate lens-specific genes in chick
embryos (14) and promote lentoid body formation and elongation of
primary cultured neural retina cells (this study and Ref. 14), it is
probable that L-Maf is critical for this late event. Using frozen
sections, we examined L-Maf accumulation in the transitional zone in
which cells elongate and undergo the earliest morphological changes
associated with lens fiber
differentiation.2
Reciprocally, phosphorylated ERK is expressed throughout the proliferative population of epithelial cells in neonatal rat lens, extending into the transitional zone (28). Although it is known that
several transcriptional factors such as Pax6, Sox2, and Six3 also
regulate the expression of crystallin gene (13, 34, 35), L-Maf is the
first example of a differentiation factor that can be regulated
directly by FGF signaling in lens development.
Because FGF-2 is expressed widely in both lens epithelial cells and
fiber cells (1), there should be a certain mechanism that interrupts
FGF-2 signaling and prevents the L-Maf protein from degradation. McAvoy
and colleagues (1, 36) have reported that FGF receptors exhibit
distinct but overlapping expression patterns throughout lens
development. Therefore, the spatiotemporal pattern of the FGFRs may be
important in stabilizing the L-Maf protein. Another possibility
is that an antagonizing factor may function to block FGF-2 signaling
and degradation of the L-Maf protein. Sprouty2 is a potential
candidate; it is expressed in lens fiber rather than in lens epithelium
during chick development (37). Sprouty2 is induced by ectopic FGF8
protein within the neural tube (38), suggesting that Sprouty2 can
function in a negative feedback mechanism with FGF signaling.
Therefore, Sprouty2 may respond to FGF-2 signaling and block protein
degradation of L-Maf during differentiation of the lens fiber. Finally,
we also signal the possibility of a decline in the
proteasome-mediated pathway. Generally, target selection of
proteasome-mediated protein degradation is ruled by E3 ubiquitin
ligases, and each E3 enzyme has a specific target protein (39, 40).
Recent studies have shed some light on the connection between
ubiquitin-mediated protein degradation and development (reviewed in
Ref. 41) (42). Because our results suggest that degradation of the
L-Maf protein is mediated by the proteasome pathway, the ubiquitin E3
ligase specific to L-Maf may be repressed in an
FGF-dependent or -independent manner. Further studies are
needed to elucidate the molecular mechanisms underlying the
stabilization of the L-Maf protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A-crystallin promoter causes lens abnormalities and
the so-called microphthalamia (small lens) phenotype (6, 8). These FGFs
effectively induce cell cycle withdrawal, loss of the characteristic
cuboidal morphology, elongation of the cell shape, and accumulation of
-crystallin, resulting in premature differentiation of lens
epithelial cells into fiber cells. In addition, a truncated form of FGF
receptor (FGFR) lacking the cytoplasmic kinase domain inhibits fiber
cell differentiation when expressed in transgenic mice under the
control of a lens-specific promoter (7), and a secreted form of FGFR can cause a delay in fiber differentiation (9). Together, these studies
implicate FGFs as bifunctional molecules that regulate both
proliferation and differentiation of the lens cells. The transcription
factors that mediate FGF signaling in lens development and the
molecular mechanisms used by FGFs to regulate the transcriptional factors remain to be identified.
A-crystallin
expression (14), and c-maf-deficient mice have defective lens fiber differentiation and loss of crystallin gene expression (16, 17). There results show that Mafs are critical for
expression of crystallin genes and lens fiber differentiation. L-Maf is
a basic region/leucine zipper transcription factor and regulates
the expression of
A-crystallin by binding the lens-specific enhancer
element
CE2 (14, 18). In chicken, the expression of L-maf
is initiated in the lens placode and is subsequently restricted to the
developing lens (14, 19). In addition, ectopic expression of L-Maf
induces the expression of several crystallin and filensin genes (14)
and promotes the formation of lens-like structures called lentoid
bodies in retinal primary cultures. Furthermore, a dominant-negative
form of L-Maf inhibits crystallin gene expression and proper lens cell
differentiation (20). Thus, L-Maf is clearly important for crystallin
gene expression and for lens development.
-crystallin. These results indicate that FGF/ERK signaling controls terminal differentiation of the lens fiber cells
through regulation of L-Maf stability.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-gal containing the human
EF-1
promoter and
-galactosidase open reading frame inserts and
pCAGGS-GFP carrying the cytomegalovirus enhancer,
-actin basal
promoter, and green fluorescent protein open reading frame sequences
were used as controls for normalization (14). Single amino acid
mutations of the L-Maf protein were carried out by subcloning an
EcoRV-EagI fragment from pEFX3-fg-L-Maf, corresponding to the coding region of the N terminus of L-Maf, into
pBluescript vector (pBsk-L-Maf). Point mutations were generated on
pBsk-L-Maf using the QuikChange site-directed mutagenesis kit (Stratagene) using mutagenic oligonucleotide primers, as follows: S14A,
5'-GCT GCC CAC CGC CCC CCT CGC CAT CG-3'; T57A, 5'-CCC CTC TCA GCG CGC CGT GCT CCT CC-3'; S65A, 5'-CCG TGC CTT
CCG CGC CCA GTT TCT GC-3' (mutations are underlined).
Mutations were confirmed by sequence analysis and then subcloned back
into pEFX3-fg-L-Maf digested with EcoRV and EagI.
pCAGGS-DN-L-Maf, an expression plasmid for a dominant-negative form of
L-Maf, was described previously (20). A plasmid encoding the S218/222E
32-51 mutant of MEK1 was purchased from Stratagene.
CE2 core sequence
and the
-actin basal promoter (c
ALuc) were described previously
(14). tkLuc is a luciferase reporter plasmid carrying the viral
thymidine kinase gene promoter (base pairs
197 to +56) (18). For
UAS6
Luc, six copies of the upstream activation sequence fragment
were inserted into the
Luc plasmid (14) at the 5' end of the
-actin basal promoter and luciferase gene.
-phosphatidylcholine,
1% bovine serum albumin, 4 mM EGTA, 8 mM
MgCl2, 1 mM dithiothreitol, 0.4 mM
p-amidinophenyl methanesulfonyl fluoride
hydrochloride (APMSF, Wako)). The luciferase activities of the lysates
were measured with a microtiter plate luminometer (Dynex). Transfection efficiency in each dish was normalized by the
-galactosidase activity. All luciferase activity values represent mean ± standard deviation of results obtained from duplicates of three
independent transfection experiments.
strain
and then purified from the bacterial extracts using glutathione-Sepharose beads according to the manufacturer's
recommendations (Amersham Pharmacia Biotech). The recombinant L-Maf
protein was incubated with purified ERK (New England Biolabs) in kinase
buffer (50 mM Tris-HCl, 10 mM MgCl2
1 mM EGTA, 2 mM dithiothreitol, 0.01% Brij35)
at 30 °C for 3 h.
-crystallin (1:1 (22)). After three washes in PBST (phosphate-buffered saline containing 0.1% Tween 20),
membranes were incubated with the following secondary antibodies conjugated to horseradish peroxidase diluted in blocking buffer: anti-mouse IgG antibody (Dako, 1:1000), anti-rabbit IgG antibody (Dako,
1:1000). The signals were detected with an ECL kit (Amersham Pharmacia
Biotech).
-lactone (lactastatin, BostonBiochem,
10 µM). After the treatment, cells were washed to remove
inhibitors followed by further incubation in fresh Dulbecco's modified
Eagle's medium, 10% fetal bovine serum, 1% chick serum for various
periods. Then the whole cell extracts were subjected to Western blot analysis.
-crystallin, and phospho-ERK in lentoid bodies in primary cultured
lens cells.
A). Double-stranded
oligonucleotide probes were labeled at the 3' end using the Klenow
fragment and [
-32P]dATP. Then, 5 pmol of probe was
incubated with 1 pmol of L-Maf protein for 30 min at 30 °C in
reaction buffer (10 mM Hepes-KOH, pH 7.9, 0.1 mM EDTA, 2 mM dithiothreitol, 100 mM KCl, 5 mM MgCl2, bovine serum
albumin, 10% glycerol, 5 µg/µl poly (dI-dC)-poly (dI-dC)). The
reaction mixture was loaded onto a 4% polyacrylamide gel. The gel was
transferred to DEAE paper, dried, and subjected to autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
FGF-2 treatment of primary cultured lens
cells. A, primary cultured lens cells were
plated at a density of 1 × 105 cells/30-mm dish. One
day after plating, FGF-2 was added (100 ng/ml) and retained in the
medium during further culturing. At various stages of culture,
cells were counted. B, after 6 days of culture, the cells
were observed under phase contrast microscopy (b,
c) and immunostained with anti- -crystallin antibody
(b', c'). FGF-untreated (b,
b') and -treated (c, c') lens cells
differentiated into lens fiber cells and formed
-crystallin-positive
lentoid bodies (arrowheads). C, primary cultured
lens cells were cultured for the indicated periods of time in the
presence or absence of FGF-2 (100 ng/ml). Equal amounts of total RNA
were subjected to Northern blot analysis to examine the expression of
lens-specific genes. The expression was repressed in FGF-2-treated lens
cells at 1-2 days of culture.
-,
A-,
B1-crystallin, and filensin
genes at earlier culture points (Fig. 1C, days 1-2), which
coincided with the period of lens cell proliferation (Fig.
1A). These results suggest that FGF-2 represses lens-specific genes at early stages of culture when the lens epithelial cells are proliferating, whereas the same growth factor promotes lens
fiber differentiation to augment crystallin gene expression and lentoid
body formation at later stages of cell culture.
A-crystallin
enhancer (
CE2) and regulates expression of the
A-,
-,
B1-crystallin, and filensin genes (14, 18). In addition, a
dominant-negative form of L-Maf that lacks its acidic domain (DN-L-Maf
(20)) repressed expression of the
A-,
-,
B1-crystallin, and
filensin genes in primary cultured lens cells (Fig.
2A) and chick embryos (20),
showing that L-Maf is essential for the expression of the lens-specific
genes. To assess the possibility that FGF signaling regulates the
L-maf gene, we first examined the effect of FGF-2 on
expression of L-maf mRNA. Lens cell primary cultures
were incubated for 48 h with or without FGF-2, and then total RNA
was isolated and subjected to Northern blot analysis. L-maf
mRNA levels in the FGF-2-treated cells were comparable to those in
the untreated control cells (Fig. 2B), indicating that FGF-2
does not significantly affect transcription of the L-maf
gene.
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Fig. 2.
L-Maf-dependent reporter activity
is repressed by FGF-2 in primary cultured lens cells.
A, DN-L-Maf was transfected into primary cultured lens
cells, which were then incubated for 48 h, and then total RNA
isolated from the cultures was subjected to Northern blot analysis. 28 S rRNA was used as a control for the RNA concentration. B,
one day after plating, FGF-2 (50 ng/ml) was added to the medium. After
48 h of FGF-2 treatment, total RNA was subjected to Northern blot
analysis. C, schematic representation of the reporter
constructs, tkLuc, Luc, and c
ALuc is shown in the left
panel. Each reporter construct (300 ng/well) was transiently
transfected into lens cells with pEFX-
-gal (100 ng/well). FGF-2 was
then added into the medium (100 ng/ml), and the cells were incubated
for an additional 48 h. Luciferase activities were measured using
aliquots of cell extracts and were normalized to
-galactosidase
activity. The luciferase activities relative to that of tkLuc reporter
transfected cells were obtained from three independent experiments.
Each bar in the figure represents the mean ± S.D.
D, lens cells were transiently transfected with c
ALuc and
pEFX-
-gal. Following transfection, cultures were incubated in the
presence or absence of FGF-2 (5, 10, or 50 ng/ml) for 48 h. To
block FGF signaling, transfected lens cells were incubated for 24 h and then exposed to the FGFR inhibitor SU5402 (5, 10, or 20 µM) for another 24 h.
CE2,
-actin basal promoter, and the luciferase coding
sequence (c
ALuc, Fig. 2C). This construct was transiently transfected into the lens cells, which were incubated with or without
FGF-2 for 48 h. Luciferase activities provided a measure of
transcriptional activity. FGF-2 clearly repressed the expression of the
reporter gene (Fig. 2C), an effect that was
dose-dependent (Fig. 2D, left panel).
This repression was not due to a general effect on transcription
because a luciferase reporter driven by either a thymidine kinase
promoter or a
-actin basal promoter alone was not affected by the
addition of FGF-2 (tkLuc and
Luc, Fig. 2C). We also
examined the effect of a specific FGF receptor inhibitor, SU5402 (25).
The reporter activity significantly increased in the presence of SU5402
(Fig. 2D, right panel), indicating that
inhibition of FGF signaling promotes expression of the reporter gene.
Taken together, these results indicate that FGF signaling inhibits
L-Maf-dependent transcription in the primary cultured lens
cell, resulting in repression of the lens-specific genes at the early
culture stage.
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Fig. 3.
L-Maf-dependent reporter activity
is repressed by MEK1/ERK. A, primary cultured lens
cells were maintained in serum-free OPTIMEM I medium for 4 h and
then stimulated with FGF-2 (50 ng/ml) for 60 min. Prior to FGF-2
stimulation, cells were treated with 100 µM
phosphorylation inhibitor PD98059 for 1 h. Whole cell lysates were
separated by SDS-PAGE and electrotransferred onto a nitrocellulose
membrane. Western blot detection of ERK was performed using the
appropriate specific antibodies. B, primary cultured lens
cells were transiently transfected with the c ALuc reporter (300 ng/well) and an expression plasmid for a constitutively active MEK1 (75 ng/well). To block the phosphorylation of MEK1/ERK, cells were treated
with 100 µM PD98059. GAL4-Elk1 was used to evaluate this
system with its reporter, UAS6
Luc.
ALuc
reporter (Fig. 3B, left). Introduction of the
constitutively active MEK1 caused a decrease in expression of the
reporter gene. In contrast, MEK1 inhibitor PD98059 promoted expression
of the reporter gene (Fig. 3B), indicating that L-Maf
activity in lens cells is regulated through the MEK1/ERK pathway. We
also confirmed that the constitutively active MEK1 could activate Elk
considerably, an ERK-regulated transcription factor, using the
GAL4-Elk1 and UAS6
Luc reporter gene assay (Fig. 3B,
right), indicating that MEK1 is active in the cultured lens cells
and the inhibitory effect of MEK1 is not ubiquitous to all
transcription. These results indicate that MEK1/ERK mediates FGF-2
signaling to repress L-Maf-dependent transcription.
-32P]ATP was detected with the wild-type L-Maf
protein and mutant S14A, suggesting that Ser-14 is not involved in
phosphorylation by ERK (Fig. 4B). In contrast, T57A and S65A
mutants showed a significantly reduced phosphorylation signal. These
results implicate Thr-57 and Ser-65 as important in the phosphorylation
of L-Maf by ERK.
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Fig. 4.
Residues Thr-57 and Ser-65 of the L-Maf
protein are important for phosphorylation by ERK in
vitro. A, GST- L-Maf proteins were incubated
with purified ERK (New England Biolab) and [ -32P]ATP
at 30 °C for 3 h followed by autoradiography or Western blot
analysis with anti-GST antibody. B, mutation of amino acid
residues in the S14A, T57A, and S65A proteins is shown schematically.
Wild-type (WT) or mutant L-Maf protein fused to GST was
incubated with purified ERK followed by autoradiography or Western blot
analysis with anti-GST antibody. L-zip, leucine
zipper. C, COS-7 cells were transfected
with pEFX-fg-L-maf. After 48 h of transfection, nuclear proteins
were isolated and incubated with calf intestinal phosphatase followed
by Western blot analysis (WB) with anti-FLAG antibody.
-crystallin. When either T57A or S65A mutant L-Maf was introduced
into primary cultured retinal cells, expression of endogenous
-crystallin was significantly induced (Fig. 5C), and the
number of
-crystallin-positive cells increased 3-4-fold compared
with wild-type L-Maf (Fig. 5C, Table
I). To confirm that these mutational
effects involve MEK1/ERK signaling, constitutively active MEK1 was
cotransfected into cultured lens cells together with either wild-type
or S65A mutant L-Maf. The transcriptional activity of endogenous L-Maf
and overexpressed wild-type L-Maf were decreased by the co-expression
of constitutively active MEK1 (Fig. 5B), whereas S65A mutant
L-Maf was not sensitive to MEK1 signaling. Taken together, these
results indicate that the ERK phosphorylation sites at Thr-57 and
Ser-65 are important for the transcriptional activity of L-Maf and that
phosphorylation at these sites negatively regulates L-Maf function
in lens cells.
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Fig. 5.
Activity of L-Maf is regulated by
phosphorylation at Thr-57 and Ser-65. A, primary
cultured lens cells were co-transfected with c Aluc (300 ng/well) and
the wild-type or mutant L-Maf expression plasmids (5 ng/well).
Luciferase activities were measured 48 h after transfection.
B, primary cultured lens cells were co-transfected with
pEFX-L-Maf (5 ng/well) and constitutively active MEK1 (25, 50, or 75 ng/well). C, primary cultured neural retina cells were
transfected with equal amounts of the wild-type or mutant L-Maf
expression plasmids (1 µg/dish). Induction of
-crystallin
expression was analyzed 48 h after transfection.
Mutation of MAPK target sites of L-Maf promotes lens differentiation
A) that is
in
A-crystallin enhancer as a probe (Fig.
6A). The wild-type L-Maf protein phosphorylated by ERK showed DNA binding activity similar to
that of unphosphorylated L-Maf protein (Fig. 6A). We also
confirmed that mutant L-Maf proteins could bind to the L-Maf response
element (data not shown), indicating that the binding activity of L-Maf is not significantly affected by ERK phosphorylation.
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Fig. 6.
Stability of the L-Maf protein is regulated
by phosphorylation. A, GST-L-Maf proteins were
incubated in the presence or absence of purified ERK and subjected
subsequently to electrophoresis mobility shift assay. B,
primary cultured lens cells were transfected with either wild-type
(WT) or mutant L-Maf expression plasmids. After 48 h of
transfection, nuclear extracts were isolated, and equal amounts were
subjected to Western blot analysis using anti-L-Maf antibodies.
Northern blot analysis showed that expression levels of wild-type and
mutant L-maf mRNA were not affected. Green fluorescent
protein (GFP) was used as a control of transfection.
C, pEFX-L-Maf was transfected into lens cells, and the cells
were incubated for 3 h in the presence of proteasome inhibitors,
LLnL (20 µg/ml), MG132 (50 µM), or lactasystin (20 µM). Nuclear extracts were prepared and subjected to the
Western blot analysis with anti-L-Maf antibodies. DMSO,
dimethyl sulfoxide. D, wild-type or mutant L-Maf expression
plasmid was transfected into lens cells. The cells were treated for
3 h with the proteasome inhibitor LLnL (20 µg/ml), washed to
remove the inhibitor, and incubated further for various periods.
Nuclear extracts were prepared and subjected to Western blot analysis
with anti-L-Maf antibodies. E, pEFX-L-Maf was transfected
into lens cells, and the cells were incubated for 48 h in
the presence of PD98059 (100 µM). L-Maf expression was
analyzed by immunostaining.
-crystallin but not in epithelial cells (Fig.
7, A-C). In contrast, high
resolution confocal microscopy revealed that phosphorylated ERK is
found only at the edges of lentoid bodies but not in the central
regions (Fig. 7, D and E). These results indicate
that accumulation of L-Maf protein is involved in lens fiber
differentiation and the stability of L-Maf protein is negatively
regulated by an active form of ERK in the undifferentiated lens
epithelial cells.
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Fig. 7.
L-Maf protein is expressed in in
vitro differentiated fiber cells. Primary cultured lens
cells were incubated for 6 days. The cells were then immunostained with
an anti-phosphorylated ERK antibody (D, green) or
double-stained with anti-L-Maf antibodies (A,
red) and anti- -crystallin antibody (C,
green). Nuclei were counterstained with
4',6-diamidino-2-phenylindole (B and E).
LE and LF denote lens epithelial cells and lens
fiber cells, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-crystallin expression is not (28). This
suggests that FGFs exert their influences through at least two distinct
(e.g. ERK-dependent and -independent) molecular mechanisms. This study shows that after 1-2 days of FGF treatment, the
proliferation of lens cells is significantly enhanced, but the
expression of lens-specific genes is repressed in primary cell
cultures. Furthermore, during the later period of culture, expression
of lens-specific genes is enhanced, and lentoid body formation and
fiber cell elongation are promoted. Together, these results suggest
that FGF-2 has a binary function that is essential for both the
"early" events in which the lens cells proliferate and the
"late" events in which differentiation of the cells proceed.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Jun-ya Katoh for critical
reading of the manuscript and Dr. Hiroshi Itoh and colleagues in our
laboratory for helpful discussions and comments. We also thank Dr. Goro
Eguchi, Kumamoto University, for giving us hybridoma lines producing
monoclonal antibody against -crystallin.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Foundation for Nara Institute of Science and Technology.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.
Present address: Gilmer Hall, Rm. 253, University of Virginia,
Charlottesville, VA 22903.
§ To whom correspondence should be addressed. Tel.: 81-743-72-5550; Fax: 81-743-72-5559; E-mail: kyasuda@bs.aist-nara.ac.jp.
Published, JBC Papers in Press, October 18, 2002, DOI 10.1074/jbc.M208380200
2 H. Ochi and K. Yasuda, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
FGF, fibroblast
growth factor;
FGFR, FGF receptor;
ERK, extracellular signal-regulated
kinase;
MAPK, mitogen-activated protein kinase;
MEK, MAPK/ERK
kinase;
GST, glutathione S-transferase;
L-Maf, lens-specific
Maf;
-gal,
-galactosidase;
Luc, luciferase;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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