Expression of the 
5 Integrin Subunit Gene Promoter
Is Positively Regulated by the Extracellular Matrix Component
Fibronectin through the Transcription Factor Sp1 in Corneal
Epithelial Cells in Vitro*
Kathy
Larouche
,
Steeve
Leclerc,
Christian
Salesse§¶, and
Sylvain L.
Guérin¶
From the Oncology and Molecular Endocrinology Research Center, and
§ Ophthalmology Research Unit, CHUL/CHUQ and Laval
University, Ste-Foy, Québec G1V 4G2, Canada
Received for publication, April 6, 2000, and in revised form, August 8, 2000
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ABSTRACT |
The accumulation of fibronectin (FN) in response
to corneal epithelium injury has been postulated to turn on expression
of the FN-binding integrin
5
1. In this work, we
determined whether the activity directed by the 5 gene
promoter can be modulated by FN in rabbit corneal epithelial cells
(RCEC). The activity driven by chloramphenicol
acetyltransferase/5 promoter-bearing plasmids was
drastically increased when transfected into RCEC grown on FN-coated
culture dishes. The promoter sequence mediating FN responsiveness was
shown to bear a perfect inverted repeat that we designated the
fibronectin-responsive element (FRE). Analyses in electrophoretic
mobility shift assays provided evidence that Sp1 is the predominant
transcription factor binding the FRE. Its DNA binding affinity was
found to be increased when RCEC are grown on FN-coated dishes. The
addition of the MEK kinase inhibitor PD98059 abolished FN
responsiveness suggesting that alteration in the state of
phosphorylation of Sp1 likely accounts for its increased binding to the
5 FRE. The FRE also proved sufficient to confer FN
responsiveness to an otherwise unresponsive heterologous promoter.
However, site-directed mutagenesis indicated that only the 3' half-site
of the FRE was required to direct FN responsiveness. Collectively,
binding of FN to its 51 integrin
activates a signal transduction pathway that results in the
transcriptional activation of the 5 gene likely through
altering the phosphorylation state of Sp1.
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INTRODUCTION |
Corneal wounds account for a substantial proportion of all visual
disabilities and medical consultations for ocular problems in North
America. They can be superficial with damage limited to the epithelium
or associated with a deeper involvement of the epithelial basement
membrane and of the stromal lamella. Severe recurrent and persistent
corneal wounds are most commonly secondary to ocular diseases and
damage such as recurrent erosion, mild chemical burns, superficial
herpetic infections, neuroparalytic cornea, autoimmune diseases, and
stromal ulcerations due to viral or bacterial infections or to severe
burns (1). Despite currently available treatments, many of these
corneal wounds persist for weeks and months or else recur frequently
and can progress to corneal perforation.
Tissue repair requires cell migration, proliferation, and adhesion.
Cell adhesion and migration in turn require extracellular matrix
(ECM)1 synthesis and
assembly. ECM is a complex, cross-linked structure of proteins and
polysaccharides. It organizes the geometry of normal tissues.
Fibronectin (FN) is an ECM adhesion protein identified as a potential
wound healing agent because of its cell attachment, migration,
differentiation, and orientation properties (for a review see Refs.
2-4). In the unwounded rat eye, FN is observed by immunohistological
staining at the level of the corneal epithelium basement membrane
(5-7). Shortly after corneal injury, the basal cells that border the
injured area and stromal keratocytes start producing massive amounts of
FN (5, 8-11). FN promotes corneal cell migration both in
vivo (12, 13) and in vitro (14) by acting as a
temporary extracellular matrix to which corneal epithelial cells attach
as they migrate over the wounded area (13, 15). Once the wound is
re-epithelialized, the subepithelial immunohistological staining of FN
progressively decreases (5, 16-18).
The increase in FN expression that has been reported to occur during
corneal wound healing was postulated to be coordinated with the
expression of its major integrin receptor
51 (5), as has also been shown for
laminin and tenascin and their corresponding integrin receptor subunits
6 and 9, respectively (19-21). For instance, the integrin 51 was shown to be
present during corneal wound healing after radial keratectomy (22).
Direct evidence that FN can positively alter
51 integrin expression at both the
protein and mRNA levels has been provided through FN antisense expression studies performed in the epithelium-derived human colon carcinoma cell line Moser (23) as well as in murine AKR-2B fibroblasts (24). Other indirect evidence linking expression of
51 to that of FN has also emerged from
recent studies (25, 26).
As a consequence, it is not surprising that ECM, through its
interactions with membrane-bound integrins, exerts profound influences on the major cellular program of growth, differentiation, and apoptosis
by altering, through a number of signal transduction pathways, the
transcription of genes whose specific functions are linked to these
cellular functions. Binding of ECM components, such as FN, with their
corresponding integrin receptors will trigger the activation of
intracellular signaling mediators such as focal adhesion kinase,
mitogen-activated protein kinases (MAPKs), and Rho family GTPases (for
a review see Ref. 27). Activation of the MAPK signal transduction
pathway is of particular interest since it links integrin-mediated
signaling to transcriptional regulation of genes that are crucial for
cell growth and differentiation. The results presented hereby provided
evidence that, by acting on 5 gene expression, such a
route of signal transduction might alter cell adhesion properties as
well. The downstream cascade of family members that are activated
following transient activation of Ras GTP-binding proteins through
receptor tyrosine kinases include MAPK/ERK kinase (designated
MEK) and ERK1 (p44)/ERK2 (p42) (28). Activation of ERK1/ERK2
through phosphorylation causes their translocation to the nucleus,
where they have been reported to phosphorylate and activate distinct
transcription factors, such as ELK, c-Jun, and c-Myc (29-31), as well
as members of the ETS family (such as PEA3) (32).
In the present study, we demonstrated that FN can alter the
transcription of the 5 integrin subunit gene at the
promoter level. Such a FN responsiveness was shown to be determined by the binding of the transcription factor Sp1 to a target site that is
part of a perfect inverted repeat which, by itself, can confer FN
responsiveness to an otherwise unresponsive heterologous promoter. Most
of all, the FN-activated, integrin-mediated signal transduction pathway
appears to require activation of ERK1/ERK2 since the Sp1 DNA binding
affinity, and, as a consequence, the FN responsiveness of the
5 promoter were both found to be diminished by blocking their activation with the MEK kinase inhibitor PD98059. Together, these
results demonstrate the novel finding that the 5
integrin subunit, through activation of the MAPK pathway, can
autoregulate its own synthesis in a manner that is dependent on the
extracellular concentration of FN.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Media--
Rabbit corneal epithelial cells
(RCECs) were obtained from the central area of freshly dissected rabbit
corneas as described previously (33) and then grown to low (near 15%
coverage of the plates), intermediate (near 75% coverage), or high
cell density (100% coverage for more than 48 h) under 5%
CO2 in SHEM medium supplemented with 5% FBS and 20 µg/ml
gentamicin. When indicated, human plasma FN (obtained as described
previously (34)) or ECM gel (basement membrane matrice from
Engelbreth-Holm-Swarm mouse sarcoma, Fisher) was coated for 18 h
at 37 °C on the culture dishes at varying concentrations (FN, 1-16
µg per cm2; ECM, 10 µg per cm2). Coated
Petri dishes were washed twice with phosphate-buffered saline and
blocked at 37 °C with 2% bovine serum albumin in phosphate-buffered saline. Cells were then seeded and grown as above. Inhibition of
ERK1/ERK2 was performed by culturing subconfluent RCEC in the presence
of 10 µM of the MEK/kinase inhibitor PD98059 (Sigma) for
48 h before cells were harvested. Drosophila Schneider
cells (ATCC CRL-1963) were cultured at 28 °C without CO2
in Schneider medium (Sigma) supplemented with 10% FBS and 20 µg/ml gentamicin.
Plasmids and Oligonucleotides--
The plasmids
5
41, 592, 5178, and
5954, which all bear the chloramphenicol
acetyltransferase (CAT) reporter gene fused to DNA fragments from the
human 5 gene upstream regulatory sequence extending up
to 5' positions 41, 92, 178, and 954, respectively, but all
sharing a common 3' end located at position +23, have been described
previously (35). The recombinant plasmids bearing one or two sense
copies of either the 5 FRE or its mutant derivatives were created by inserting the corresponding double-stranded oligomers upstream from the basal promoter of the mouse p12 gene (into the unique
BamHI site) that has been previously mutated into its
Sp1-binding site (and designated p12.108/M (36)). The Sp1 expression
vector pPacSp1 was generously provided by Dr. Guntram Suske (Institute für Molecular Biology und Tumorforschung, Philipps
Universität Marburg, Germany), whereas the LacZ expression
plasmid pAC5/V5-His/LacZ was obtained from Invitrogen (Carlsbad, CA).
The double-stranded oligonucleotides used in the present study were
chemically synthesized using a Biosearch 8700 apparatus (Millipore).
They contained the DNA sequence from the human 5 promoter comprised between positions 82 and 56 and designated the
5 FRE (5'-GATCAGCCGGGAGTTTGGCAAACTCCTCCCC-3') or its
mutated derivatives (5FRE/m5',
5'-GATCAGCCGAAAAATTGGCAAACTCCTCCCC-3'; 5FRE/m3',
5'-GATCAGCCGGGAGTTTGGCAAACTAAAAAAC-3'; 5FRE/m5'+3', 5'-GATCAGCCGAAAAATTGGCAAACTAAAAAAC-3'), the DNA binding site for human HeLa CTF/NF-I in adenovirus type 2 (5'-GATCTTATTTTGGATTGAAGCCAATATGAG-3') (37), the high affinity binding
site for the positive transcription factor Sp1
(5'-GATCATATCTGCGGGGCGGGGCAGACACAG-3') (38), or the Sp1-binding site
(designated p12.A) identified in the basal promoter from the mouse p12
gene (5'-GATCCAGTGGGTGGAGCCTG-3') (36).
Transient Transfection and CAT Assay--
RCEC plated at either
low (5 × 104 cells per 35-mm tissue culture plates),
intermediate (5 × 105 cells per 35-mm tissue culture
plates), or high (1, 5 × 106 cells per 35-mm tissue
culture plates) cell density were transiently transfected using the
polycationic detergent LipofectAMINE (Life Technologies, Inc.) as
recommended by the manufacturer. Each LipofectAMINE-transfected plate
received 1.5 µg of the test plasmid and 0.5 µg of the human growth
hormone (hGH)-encoding plasmid pXGH5 (39). Drosophila Schneider cells were transfected according to the calcium phosphate precipitation procedure (36, 40) at a density of 1 × 106 cells per 60-mm culture plate.
Levels of CAT activity for all transfected cells were determined as
described (40) and normalized to the amount of hGH secreted into the
culture media and assayed using a kit for quantitative measurement of
hGH (Immunocorp, Montréal, Québec, Canada). Because the metallothionein-I promoter, which directs expression of hGH from
the pXGH5 plasmid, proved to be highly inefficient in
Drosophila cells, CAT activities from transfected Schneider
cells were normalized to the amount of -galactosidase encoded by the
plasmid pAC5/V5-His/LacZ and cotransfected along with the CAT
recombinant constructs. Each cell-containing plate therefore received
15 µg of the test plasmid, 4 µg of pAC5/V5-His/LacZ, and 1 µg of
pPAC (empty vector). In the cotransfection experiments performed with
the Sp1 expression plasmid, the empty pPAC was substituted for 1 µg
of pPacSp1. The value presented for each individual test plasmid
transfected corresponds to the mean of at least three separate
transfections done in triplicate. To be considered significant, each
individual value needed to be at least three times over the background
level caused by the reaction buffer used (usually corresponding to
0.15% chloramphenicol conversion). Standard deviation is also provided
for each transfected CAT plasmid.
Nuclear Extract Preparation--
Crude nuclear extracts were
prepared from RCEC grown solely on plastic or FN-coated culture dishes
and dialyzed against DNase I buffer (50 mM KCl, 4 mM MgCl2, 20 mM
K3PO4 (pH 7.4), 1 mM
-mercaptoethanol, 20% glycerol) as described (41) except that a
combination of protease inhibitors (pepstatin A (0.5 µg/ml),
leupeptin (5 µg/ml), chymostatin (5 µg/ml), antipain (5 µg/ml),
aprotinin (5 µg/ml), benzamidine (5 mM)) (all reagents
from Sigma) was added to all the buffers used in order to restrict
proteolysis. Extracts were kept frozen in small aliquots at 80 °C
until use.
Electrophoretic Mobility Shift Assays (EMSA) and Supershift
Experiments--
EMSAs were carried out using either the 27-bp
5 FRE or the high affinity Sp1 oligomer as 5'
end-labeled probes. Approximately 2 × 104 cpm labeled
DNA was incubated with crude nuclear proteins (as specified in the
figure legends) from RCEC grown on either untreated or FN-coated
culture dishes in the presence of 500 ng of poly(dI-dC)·poly(dI-dC) (Amersham Pharmacia Biotech) in buffer D (5 mM HEPES (pH
7.9), 10% glycerol (v/v), 25 mM KCl, 0.05 mM
EDTA, 0.5 mM dithiothreitol, 0.125 mM
phenyl methosulfonyl fluoride). Occasionally, crude nuclear extracts from human HeLa cells were also used in EMSA as a positive control for comparison purposes. Incubation proceeded at room temperature for 10 min upon which time DNA-protein complexes were separated by gel electrophoresis through 6% native polyacrylamide gels
run against Tris glycine buffer as described (42). Gels were dried and
autoradiographed at 80 °C to reveal the position of the shifted
DNA-protein complexes generated. Competitions in EMSA were performed
using 10 µg of crude nuclear proteins from RCEC grown in the presence
of FN at 8 µg per cm2 as above except that molar excesses
(100- and 500-fold) of synthetic double-stranded oligonucleotides
bearing the DNA sequence of the 5 FRE, the DNA-binding
site for human HeLa CTF/NF-I in adenovirus type 2 (37), the high
affinity binding site for the positive transcription factor Sp1 (38),
or the p12.A Sp1-binding site from the mouse p12 gene (36) were added
to the binding reaction prior to loading on the gel. Supershift
experiments in EMSA were conducted by first incubating varying amounts
(as specified in the figure legends) of crude nuclear proteins from
RCEC grown either with (8 µg per cm2) or without FN, in
the presence of 250 ng of poly(dI-dC)·poly(dI-dC), with either none
or 1 µl (corresponding to 1 µg) of a commercially engineered rabbit
antiserum raised against the transcription factor Sp1 (Santa Cruz
Biotechnology, Inc.) in buffer D. Then, 2 × 104 cpm
FRE-labeled probe was added, and incubation was extended for another 15 min at room temperature. Samples were finally loaded on high ionic
strength, 6% native polyacrylamide gels and run at 4 °C against
Tris glycine buffer as above. Formation of DNA-protein complexes was
revealed following autoradiography at 70 °C.
SDS-PAGE and Western Blot--
Crude nuclear proteins were
obtained from either HeLa cells (used as a positive control) or from
RCEC grown on culture dishes coated or not with FN (8 µg per
cm2) as detailed above. Protein concentration was evaluated
by the Bradford procedure and further validated following Coomassie
Blue staining of SDS-polyacrylamide fractionated nuclear proteins. One
volume of sample buffer (6 M urea, 63 mM Tris
(pH 6.8), 10% (v/v) glycerol, 1% SDS, 0,00125% (w/v) bromphenol
blue, 300 mM -mercaptoethanol) was added to 20 µg of
proteins before they were size-fractionated on a 10%
SDS-polyacrylamide minigel and transferred onto a nitrocellulose
filter. A full set of protein molecular mass markers (Life
Technologies, Inc.) was also loaded as a control to evaluate protein
sizes. The blot was then washed once in TS buffer (150 mM
NaCl, 10 mM Tris-HCl (pH 7.4)) and 4 times (5 min each at
22 °C) in TSM buffer (TS buffer plus 5% (w/v) fat free Carnation
milk and 0.1% Tween 20). Then, a 1:500 dilution of a rabbit monoclonal
antibody raised against the transcription factor Sp1 (Santa Cruz
Biotechnology, Inc.) was added to the membrane-containing TSM buffer
and incubation proceeded further for 4 h at 22 °C. The blot was
then washed in TSM buffer and incubated an additional 1 h at
22 °C in a 1:1000 dilution of a peroxidase-conjugated goat anti-mouse immunoglobulin G (Jackson ImmunoResearch). The membrane was
successively washed in TSM (4 times, 5 min each) and TS (twice, 5 min
each) buffers before immunoreactive complexes were revealed using
Western blot chemiluminescence reagents (Renaissance, PerkinElmer Life
Sciences) and autoradiographed.
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RESULTS |
The Activity Directed by the 5 Integrin Subunit Gene
Promoter Is Positively Regulated by Fibronectin--
Studies conducted
by Rajagopal et al. (23) and Huang et al. (24)
both provided evidence that the level of expression for the mRNA
encoding the 5 integrin subunit was positively modulated by the presence of the extracellular matrix component fibronectin. We
therefore exploited transient transfection of primary cultured RCEC
using recombinant plasmids bearing the CAT reporter gene fused to
various segments from the human 5 gene promoter (35) in
order to evaluate whether such an FN-dependent increase in 5 mRNA could be determined by discrete cis-acting
elements from the 5 gene upstream regulatory region. For
this purpose, a recombinant plasmid bearing the 5
promoter up to position 954 (5-954) inserted upstream
from the CAT reporter gene was transfected into RCEC plated either on
plastic or FN-coated culture dishes (2 µg/cm2) at varying
cell densities. As Fig. 1A
indicates, culturing RCEC on FN-coated Petris did not alter the
activity driven by the 5954 plasmid when transfected
at low cell density (near 15% coverage of the plates). However, at
both intermediate (near 75% coverage) and high (100% coverage for
more than 48 h) cell density, the activity of the 5
promoter was found to be 6.1- and 6.4-fold, respectively, higher when
cells are grown on FN-coated culture plates rather than solely on
plastic. Withdrawal of the serum contained into the culture medium
(which normally contains 5% FBS) prior to cell seeding on FN-coated
culture dishes had no statistical effect on the CAT activity directed
by 5954 (results not presented).
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Fig. 1.
5 promoter activity
in RCEC grown with or without FN. A, cell density
dependence of the 5 promoter FN responsiveness. RCEC
were plated at low (1 × 105 cells per Petri;
L), intermediate (5 × 105 cells per Petri;
I), and high cell density (1.5 × 106 cells
per Petri; H) on either uncoated or FN-coated (2 µg/cm2) tissue culture dishes. Cells were transiently
transfected 24 h later with the 5954 recombinant
plasmid and harvested 48 h post-transfection. CAT activities were
measured and normalized to hGH as described under "Experimental
Procedures." Each value is expressed as the ratio of the CAT activity
from RCEC grown on FN-coated Petri dishes over that of RCEC grown
solely on plastic. Standard deviation is provided for each individual
value. B, dose-dependent activation of the
5 promoter FN responsiveness in RCEC. RCEC (3 × 105 cells/Petri) were plated on culture dishes that have
been coated with either none or increasing concentrations of FN (1-16
µg/cm2). RCEC were then transfected with the
5954 recombinant construct and harvested 48 h
later. CAT activity was determined and normalized as described under
"Experimental Procedures." Each value is expressed as detailed in
A.
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The dose dependence of the 5 promoter FN responsiveness
was next evaluated by transfecting RCEC plated at an intermediate cell
density on culture dishes coated with either none or increasing concentrations of FN (from 1 to 16 µg/cm2). As shown on
Fig. 1B, the activity directed by the 5954
plasmid increased proportionally to the amount of FN coated on the
culture dishes, reaching a drastic 18-fold stimulation at 16 µg/cm2 FN. No further increase in 5
promoter function was observed at FN concentrations above 16 µg/cm2 (results not presented). We therefore conclude
that the activity of the human 5 promoter can be
drastically increased when RCEC are grown on FN-coated culture dishes
and that such a positive influence is obviously cell
density-dependent.
A Distinct Cis-acting Element from the Basal Promoter of the Human
5 Gene Mediates FN Responsiveness in RCEC--
Discrete
cis-acting regulatory elements are known to mediate many of the
regulatory effects that are triggered through signal transduction
pathways by binding trans-acting nuclear proteins with distinctive
regulatory properties. To determine more precisely the minimal
5 promoter sequence required to confer FN
responsiveness, CAT recombinant plasmids bearing various 5' deletions
of the 5 promoter were transfected into RCEC grown at
intermediate density on both plastic and FN-coated (2 µg/cm2) culture dishes. Neither the deletion of the
5 promoter down to position 178 nor 92 could prevent
the average 5-fold increase in 5 promoter activity
observed when RCEC are grown on FN-coated plates. However, the further
deletion of the 5 sequences down to position 41 almost
totally abolished the FN responsiveness of the 5
promoter. A detailed examination of this 41-bp sequence revealed the
presence of a perfect inverted repeat of the following sequence,
5'-GGAGTTTG-3' (Fig. 2B).
Therefore, FN responsiveness of the 5 promoter appears
to be determined by a short stretch of DNA sequence contained between
positions 41 and 92 relative to the 5 mRNA start
site.
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Fig. 2.
FN responsiveness directed by recombinant
constructs bearing 5' deletions of the
5 promoter in RCEC. A,
CAT activity directed by recombinant plasmids bearing 5' deletions of
the 5 promoter in RCEC grown with or without FN. RCEC
plated at an intermediate cell density (5 × 105 cells
per Petri) on either regular or FN-coated (2 µg/cm2)
culture dishes were transiently transfected, 24 h later, with CAT
recombinant plasmids bearing various lengths (up to positions 954,
178, 92, and 41 relative to the 5 mRNA start
site) from the human 5 gene promoter. CAT activity was
measured and expressed as the ratio of CAT + FN over CAT FN.
Standard deviation is provided for each value. B,
identification of a perfect inverted repeat in the 5
promoter segment that mediates FN responsiveness. The DNA sequence of a
perfect inverted repeat that has been designated as the
5 FRE and identified between positions 77 and 61
from the human 5 gene promoter is indicated in
bold capital letters. Arrows indicate the position of each
5 FRE half-sites.
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Influence of ECM Components Other Than FN on the Activity of the
5 Promoter--
Apart from FN, proteins such as
collagen IV, vitronectin, entactin, and laminin are also commonly found
in the extracellular matrix. We examined whether components from the
ECM other than FN can also alter the expression directed by the
5 promoter in RCEC. For this purpose, RCEC were grown to
intermediate cell density on either untreated or ECM-coated culture
dishes before they were transiently transfected with the
5954 plasmid. The ECM gel (basement membrane matrice
from Engelbreth-Holm-Swarm mouse sarcoma, Fisher) contains laminin,
collagen IV, entactin, and heparin sulfate proteoglycans but no FN. As
shown on Fig. 3A, the CAT
activity driven by 5954 is increased by only 2.5-fold
when RCEC are grown on ECM-coated dishes (10 µg/cm2). The
CAT activity directed by the 5 promoter was raised to 4-fold when both FN (2 µg/cm2) and the ECM gel (10 µg/cm2) are coated together on the culture dishes.
However, optimal promoter activation was obtained when FN (2 µg/cm2) was coated alone on the culture plates (7.6-fold
activation). Transient transfection of RCEC plated on ECM-coated
culture dishes with the recombinant plasmids bearing the various 5'
deletions of the 5 promoter identified the
ECM-responsive element somewhere between positions 178 and 954
(Fig. 3B). We conclude that components from the ECM other
than FN had only a moderate effect on the 5 promoter
activity and that their action is mediated through a cis-acting element
distinct from that which determines FN responsiveness in RCEC.
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Fig. 3.
Influence exerted by both ECM and FN on
5 promoter function. A,
5 promoter activity in RCEC grown on FN-, ECM-, or both
FN + ECM-coated dishes. RCEC were grown to mid-confluency either solely
on plastic () or on coated ((ECM (10 µg/cm2), FN (2 µg/cm2) or both ECM + FN; (10 and 2 µg/cm2,
respectively)) culture dishes prior to their transfection with the
5 recombinant plasmid 5954. Transfected
cells were harvested 48 h later, and CAT activity was determined
and normalized as described under "Experimental Procedures." Each
value is expressed as detailed in the legend to Fig. 1. B,
CAT activity directed by recombinant plasmids bearing 5' deletions of
the 5 promoter in RCEC grown with or without ECM. RCEC
plated at an intermediate cell density (5 × 105 cells
per Petri) for 24 h on either regular or ECM-coated (10 µg/cm2) culture dishes were transiently transfected with
the various 5' deletion constructs from the 5 promoter
(see Fig. 2A). CAT activity was measured and expressed as
the ratio of CAT + ECM over CAT ECM. Standard deviation is
provided for each value.
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The Transcription Factor Sp1 Binds Specifically to the
5 FRE in Vitro--
In order to determine whether the
FN responsiveness mediated by the 41/92 5 promoter
segment (which also contains the 82 to 56 inverted repeat
that has been designated as the fibronectin-responsive element (FRE))
depends on its recognition by nuclear transcription factors, EMSAs were
performed. For this purpose, the synthetic oligomer bearing the
5 FRE was 5' end-labeled and incubated with increasing
amounts of crude nuclear proteins (2, 5, 10, and 20 µg) from RCEC
grown either on plastic or FN-coated culture flasks (8 µg/cm2). As shown in Fig.
4A, three distinct DNA-protein
complexes (designated a, b, and c)
were observed upon autoradiography, complex a being the most
abundant at 5, 10, and 20 µg of proteins (a few other fast-migrating
complexes were also occasionally observed in EMSA but their formation
proved to be highly inconsistent). The signal corresponding to both
complexes a and c was usually found to be much
stronger in the crude extract prepared from RCEC grown on FN-coated
culture dishes. Specificity for the formation of these complexes was
then evaluated by competition experiments in EMSA using, as unlabeled
competitors, various double-stranded oligonucleotides bearing target
sequences for known transcription factors. Formation of both complexes
a and b could easily be competed off by a
100-fold molar excess of unlabeled FRE, whereas that of complex
c was partly prevented at a 100-fold molar excess but nearly
completely abolished at a 500-fold excess (Fig. 4B).
Formation of both complexes a and b could not be
prevented by an unrelated oligomer bearing the target sequence for HeLa
CTF/NF-I in adenovirus type 2. However, that of complex c
was efficiently prevented when a 500-fold molar excess of the NF1
oligomer was used suggesting that binding of a member of the NF1 family
of transcription factors likely accounts for the formation of this
complex. Most of all, an oligomer bearing the high affinity binding
site for the positive transcription factor Sp1 could compete for
formation of complexes a and b even as
efficiently as the FRE itself, a 100-fold molar excess being sufficient
to almost totally prevent their formation in EMSA (Fig. 4B).
As further evidence that Sp1 or any other member of this family (43) is
the major transcription factor binding the 5 FRE, a
synthetic oligomer bearing the target sequence for Sp1 that we
identified in the basal promoter from the mouse p12 gene (and
designated p12.A) (36) was also used as unlabeled competitor. This Sp1
site diverges from the Sp1 consensus by the lack of the central
C residue (Fig. 2B) which is substituted by a T in
the p12.A element. It is also relatively well preserved with the 3' half-site of the 5 FRE (9 out of 12 residues) since the
five G residues identified as critical for recognition of the p12.A element by Sp1 are also preserved in the 5 FRE (36) (see
Fig. 10). As shown in Fig. 4B, the p12.A element competed
nearly as well as the FRE for the formation of both complexes
a and b in EMSA. These results suggest that
formation of complexes a and b likely results
from the recognition of the labeled 5 FRE by distinct
members of the Sp1 family of transcription factors and that complex
c might result from the recognition of that same probe by a
member of the NF1 family. A detailed examination of the DNA sequence
from the 5 FRE indeed revealed the presence of a perfect
half-palindromic site for NF1 (TGGCA; see Figs. 2B and 10)
that has been previously reported to bind this transcription factor
(44, 45).
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Fig. 4.
Binding of nuclear proteins from RCEC to
the 5 FRE in
vitro. A, EMSA analysis of the nuclear
proteins from RCEC interacting with the 5 FRE. The
double-stranded oligonucleotide bearing the 5 FRE was 5'
end-labeled and incubated with varying concentrations (2-20 µg) of
crude nuclear proteins from RCEC grown on either not coated
(FN) or FN-coated (FN+) culture dishes (8 µg/cm2). The position of three DNA-protein complexes is
shown (a-c) along with that of the free probe
(U). P, labeled probe alone. B,
competitions in EMSA. The 5' end-labeled 5 FRE was
incubated with 10 µg of crude nuclear proteins from RCEC grown on
FN-coated culture dishes in the presence of either 100- or 500-fold
molar excess of various unlabeled double-stranded oligonucleotide
competitors (FRE, Sp1, NF1, and p12.A). U, free labeled
probe; P, labeled probe alone; C, labeled probe
with nuclear proteins but without unlabeled competitor.
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We next performed supershift experiments in EMSA to establish clearly
whether Sp1 was truly binding the FRE to yield complexes a
and b in EMSA. The experiment was conducted at three
different protein concentrations (5, 10, and 20 µg of crude nuclear
proteins) in the presence of either none or 1 µl (corresponding to 1 µg) of an antiserum raised against human Sp1. Again, formation of complex a but not that of complex c was found to
be much stronger when the extract from RCEC grown on FN-coated Petri
dishes was used at either 10 or 20 µg (but not at 5 µg; Fig.
5A), suggesting that Sp1
expression (or its corresponding DNA binding affinity) is increased
when RCEC are cultured on FN-coated dishes. The further addition of the
Sp1 antiserum resulted in a strong reduction of complex a
formation and yielded a new complex (a-Sp1Ab) with a lower
electrophoretic mobility resulting from the recognition of complex
a by the Sp1 antibody. The proportion of the signal supershifted by the Sp1 antibody was much stronger in the extract from
RCEC grown on FN-coated culture dishes (at both 10 and 20 µg but not
at 5 µg of proteins) than with RCEC grown solely on plastic,
providing further evidence that either Sp1 expression, or its DNA
binding affinity, is indeed increased in cells grown on FN-coated
culture dishes. As Fig. 5B indicates, no supershifted complex could be obtained when the Sp1 antiserum was substituted with
the non-immune serum, which is used as a negative control in such
experiments. Western blot analysis using the Sp1 antiserum as the
source of primary antibody revealed that RCEC express nearly the same
amount of Sp1 regardless of whether they are cultured on plastic or
FN-coated culture dishes (Fig. 5C), therefore providing evidence that an improved DNA binding affinity, rather than a variation
in the level of expression of Sp1, likely accounts for the increased
binding of Sp1 to the 5 FRE when RCEC are cultured on
FN-coated dishes. However, nearly five times more proteins from RCEC
were required in order to detect an Sp1 signal of equal strength to
that obtained using proteins from human HeLa cells (often used as a
positive control for Sp1 expression). This substantial difference did
not arise from a reduced affinity of the anti-human Sp1 antibody
directed against rabbit Sp1 in our experiments since EMSAs performed
using the high affinity Sp1 oligomer as labeled probe (Fig.
6A) also revealed a much
weaker shifted signal when crude nuclear extracts from RCEC are
selected, despite that equal amounts of proteins from both RCEC and
HeLa cells were used. Therefore, the reduced Sp1 binding observed in
nuclear extracts from RCEC likely suggests that Sp1 is expressed at a
much lower level in RCEC than in HeLa cells. Furthermore, Sp1 clearly
possesses a higher affinity for the Sp1 oligomer than for its target
site in the 5 FRE, since only a 100-fold molar excess of
the high affinity Sp1 oligomer is sufficient to totally prevent binding of Sp1 to the Sp1-labeled probe, whereas a 500-fold excess of the
5 FRE was required to almost totally prevent formation
of this complex in EMSA (Fig. 6B).
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Fig. 5.
In vitro binding of Sp1 to
the 5 FRE as revealed by
supershift analyses in EMSA and Western blots. A, the
nuclear protein yielding complex a corresponds to Sp1.
Varying amounts (5-20 µg) of crude nuclear proteins from RCEC grown
with (8 µg/cm2) or without FN were incubated with the
labeled FRE either alone or in the presence of 1 µl of the Sp1
antiserum. Formation of the shifted DNA-protein complexes was examined
by the EMSA as above. The position of both the Sp1-FRE (corresponding
to complex a) and the supershifted Sp1Ab-Sp1-FRE (identified
as a/Sp1Ab) complexes is provided, along with that of the
free probe (U). B, as controls, the FRE labeled
probe was incubated in the presence of either 1 µl of a non-immune
serum (NIS) or 1 µl of the Sp1 antiserum
(Sp1Ab) with or without crude nuclear proteins
(NP), and formation of DNA-protein complexes was evaluated
as above. U, unbound fraction of the labeled probe.
C, Western blot analysis of Sp1 in RCEC. Crude nuclear
proteins were obtained from RCEC grown on culture dishes coated with
(+FN, 8 µg/cm2) or without (FN)
fibronectin and tested in Western blot analyses using the Sp1 antiserum
as detailed under "Experimental Procedures." As a positive control,
increasing concentrations (5-20 µg) of a crude nuclear extract from
HeLa cells were also loaded next to those from RCEC on the SDS gel. The
molecular mass markers shown correspond to myosin (200 kDa) and
phosphorylase b (97.4 kDa). The position of the endogenous
Sp1 is indicated (Sp1 (95/106)).
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Fig. 6.
EMSA analyses of Sp1 in RCEC.
A, equal amounts (1, 2, and 5 µg) of nuclear proteins from
either RCEC (grown on culture dishes coated with 8 µg/cm2
FN) or HeLa cells were incubated in the presence of an oligomer bearing
the Sp1 high affinity binding site Sp1
(5'-GATCATATCTGCGGGGCGGGGCAGAC-ACAG-3'), and formation of the Sp1
complex was evaluated by EMSA as described under "Experimental
Procedures." The position of the Sp1 complex is indicated, as well as
that of three other complexes of which two likely correspond to
complexes b and c from Fig. 5, whereas the third
was shown in B as being nonspecific (ns).
B, approximately 5 µg of crude nuclear proteins from RCEC
grown on FN-coated dishes (8 µg/cm2) were incubated with
the high affinity Sp1-labeled probe with either none or increasing
concentrations (100- and 500-fold molar excess) of the unlabeled Sp1,
FRE, or NF1 oligomers. Formation of DNA-protein complexes was then
monitored by EMSA as in A. The position of the specific Sp1
complex is indicated along with that of a nonspecific complex
(ns). U, unbound fraction of the labeled
probe.
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The Inverted Repeat from the 5 Basal Promoter
Mediates Sp1-dependent FN Responsiveness--
To answer
whether the inverted repeat from the 5 FRE (Fig.
2B) is sufficient to confer FN responsiveness to a
heterologous promoter, a synthetic, double-stranded oligonucleotide
bearing the 5 sequence from 82 to 56 (designated as
FRE) was inserted upstream from the basal promoter of the mouse p12
gene. The p12 gene encodes a 12-kDa secretory protease inhibitor whose
expression is mainly restricted to the ventral prostate, the
coagulating gland, and the seminal vesicle (46). We have previously
shown that the basal promoter from the p12 gene, which extends from position 108 to +7 in plasmid p12.108, is constitutively expressed to
relatively high levels in most transfected cell types (36, 47).
However, to avoid any interference by the Sp1 site identified in the
middle of the p12 basal promoter, the FRE was inserted in a derivative
from p12.108 that bears mutations into the p12 Sp1 target site
(p12.108/M (36)) (Fig. 7A).
When transfected into mid-confluent RCEC, only a weak difference
(1.6-fold activation) was observed in the CAT activity directed by the
parental plasmid p12.108/M when 8 µg/cm2 FN was coated on
the culture plates (Fig. 7A). However, insertion of either
one (in plasmid p12/FRE) or two sense copies (in plasmid p12/2xFRE) of
an oligomer bearing the 82/56 5 FRE immediately upstream from the p12 basal promoter resulted in 6.1- and 8.8-fold increase in CAT activity, respectively. Mutations introduced in the 5'
half-site of the inverted repeat contained on the FRE (see under
"Experimental Procedures") had no statistically significant effect
on either the basal p12 promoter-driven activity when cells are grown
on plastic or on the FN responsiveness when they are cultured on
FN-coated culture dishes (32% reduction when compared with the level
directed by the wild-type p12/FRE) (Fig. 7B). On the other
hand, mutations that altered part of the 3' half-site of the FRE and
most of its downstream GC-rich sequence had no affect on the
unstimulated, p12 promoter basal activity but totally abolished FN
responsiveness when RCEC were cultured on FN-coated dishes. As
expected, mutating both the 3'- and 5' half-sites from the
5 FRE had the same effect as mutating the 3' half-site
alone. To confirm that the lack of FN responsiveness resulting from
mutating the 3' half-site of the 5 FRE was the
consequence of preventing Sp1 from properly interacting with its target
sequence in the FRE, competition experiments in EMSA were performed.
Crude nuclear proteins were prepared from mid-confluent RCEC grown on
FN-coated culture dishes and incubated with the 5
FRE-labeled probe in the presence of varying concentrations of
unlabeled oligonucleotides bearing the sequence from either the
wild-type FRE or any of its mutated derivatives. As shown in Fig.
7C, incubation of the labeled probe with nuclear proteins
from RCEC yielded the typical Sp1-FRE complex observed above
(also denoted complex a in both Figs. 4 and 5). As expected,
as little as a 100-fold molar excess of unlabeled wild-type FRE totally
prevented formation of this complex. Similarly, a 100-fold molar excess
of the 5' half-site-mutated FRE competed as well the unmutated FRE for
the formation of the Sp1 complex providing evidence that these mutated
positions did not interfere with the recognition of the oligomer by
Sp1. On the other hand, derivatives of the FRE bearing mutations in the
3' half-site (altering either the 3' half-site alone or in combination
with the 5' half-site) were totally inefficient in preventing formation
of the Sp1-FRE complex, even when used at a 500-fold molar excess,
therefore providing evidence that both mutated oligomers are unable to
bind Sp1. We therefore conclude that the inverted repeat identified in
the basal promoter of the 5 gene can confer
Sp1-dependent FN responsiveness to an otherwise
unresponsive heterologous promoter and that only the 3' repeat of the
FRE, along with its downstream GC-rich sequence, is required for this
effect to occur.
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Fig. 7.
The 5
FRE can confer FN responsiveness to the basal promoter of the
heterologous p12 gene. A, a synthetic oligonucleotide
bearing the 5 inverted repeat from position 82 to 56
(5 FRE) was inserted in either one (in plasmid p12/FRE)
or two sense copies (in plasmid p12/2xFRE) upstream from the basal
promoter of the mouse p12 gene (p12.108/M) (36). The recombinant
constructs were transiently transfected into midconfluent RCEC grown on
tissue culture dishes coated with either none (FN) or 8 µg/cm2 FN (FN+). CAT activity was measured and
expressed relative to the unstimulated level directed by the parental
plasmid p12.108/M. Arrows indicate the position of each of
the wild-type, unmutated 5 FRE inverted repeats.
B, double-stranded oligonucleotides bearing mutations in
either the 3' (in plasmid p12/FREm3') or the 5' (in plasmid p12/FREm5')
half-site from the 5 FRE, or both (in plasmid
p12/FREm5'+3'), were inserted in single-sense copies upstream from the
p12 promoter in p12.108/M. The CAT activity directed by each
recombinant construct was assessed following transient transfections in
mid-confluent RCEC grown on culture dishes coated or not with FN (8 µg/cm2) as in A. Arrows indicate
the position of each unmutated 5 FRE inverted repeat,
whereas mutation-bearing repeats are indicated by a ×. CAT activity
was measured as in A. C, formation of the
Sp1-5FRE DNA-protein complex (identified as complex a in
Fig. 5) was evaluated in EMSA by incubating the 5
FRE-labeled probe with crude nuclear proteins from mid-confluent RCEC
grown on FN-coated dishes (8 µg/cm2), in the presence of
unlabeled double-stranded oligonucleotides (both 100- and 500-fold
molar excess) bearing the DNA sequence of either the wild-type,
unmutated FRE or that of any of its mutated derivatives
(FREm3'; FREm5'; FREm3'+5'). The position of the
Sp1-FRE complex is provided, along with that of the free probe
(U). P, probe without nuclear proteins.
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As further evidence that FN responsiveness mediated by the
5 FRE was determined through its recognition by Sp1,
cotransfection experiments were therefore conducted into
Drosophila Schneider cells. These cells have been reported
to be deficient in producing this transcription factor, as well as many
others expressed in higher eukaryotes, which make them an ideal system
for studying gene expression or transcription factor functions (for a
review see Ref. 48). Both the FRE-bearing 592 and the
FRE-depleted 542 plasmids were cotransfected into
Schneider cells either alone or with a recombinant plasmid (pPacSp1, a
generous gift from Dr. Guntram Suske, Institute für Molecular
Biology und Tumorforschung, Philipps Universität Marburg,
Germany) containing the Sp1 cDNA under the control of the
Drosophila actin gene promoter and therefore ensuring high
levels of Sp1 expression in Schneider cells. Neither 541 nor 592 could determine high
basal promoter activity when individually transfected in Schneider
cells. However, when cotransfected along with pPacSp1, a dramatic
75-fold increase in promoter activity was observed with the
FRE-containing plasmid 592 but not with the
FRE-deleted plasmid 541 (Fig.
8A). The recombinant
5FRE/p12 promoter constructs were then transfected
either alone or with pPacSp1 into Schneider cells (Fig. 8B).
The parental plasmid p12.108/M, although encoding substantial amounts
of CAT in Schneider cells, responded only weakly (3.5-fold activation)
to the presence of Sp1. However, the further addition of the
5 FRE in p12/FRE resulted in a strong increase (52-fold)
in the CAT activity normally directed by p12.108/M. As with the
transfection experiments conducted in RCEC (see Fig. 7, A
and B), mutations introduced in the 5' half-site of the FRE
(in plasmid p12/FREm5') had only a modest effect on the Sp1-mediated
activation of the p12 promoter (Fig. 8B). On the other hand,
no Sp1-mediated activation could be observed upon mutating either the
3' half-site alone or both the 3' and 5' half-sites of the FRE (in the
plasmids p12/FREm3' and p12/FRE3' + 5', respectively). These results
are consistent with those of the competition experiment shown in Fig.
7C and provide clear evidence that Sp1 does bind to the 3'
half-site of the FRE in order to influence positively the activity of
its downstream promoter (in this case, either the 5 or
the p12 promoter).
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Fig. 8.
Transient transfection experiments in
Sp1-deficient Drosophila Schneider cells.
A, both the recombinant plasmids 541 and
592 were transiently transfected either alone or in
combination with the Sp1 expression plasmid pPacSp1 into
Drosophila Schneider cells. Cells were harvested 48 h
later, and CAT activity was determined and normalized as detailed under
"Experimental Procedures." B, same as in A
except that the recombinant plasmids p12.108/M and p12/FRE, or its
mutated derivatives p12/FREm3', p12/FREm5', and p12/FREm3'+m5' (see
under "Experimental Procedures"), were selected for the
cotransfection experiments.
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Activation of ERK1/ERK2 Mediates the FN Responsiveness of the
5 Promoter--
Culturing murine Swiss 3T3 or rat REF52
fibroblasts on substrata coated with either FN or with a synthetic
peptide containing the RGD sequence has been shown to result in the
activation of mitogen-activated protein kinases (MAPK) (49), such as
extracellular signal-regulated kinases (ERKs), which have been shown to
be recruited to the ECM ligand/integrin-binding site (50). Sp1 has been
recently recognized as one of the few target transcription factors
phosphorylated by ERK kinases (51-53). We have shown above that its
ability to interact with the 5 FRE is strongly increased
upon activation of the FN/51
integrin-mediated signal transduction. To determine whether the
Sp1-mediated FN responsiveness directed by the 5 FRE was
due to the activation of the Ras-Erk signaling pathway (27), RCEC grown
to intermediate cell density on culture dishes coated (8 µg/cm2) or not with FN were transiently transfected with
either the recombinant plasmids 592 or p12/FRE and
cultured with either none or 10 µM of the MEK-1 kinase
inhibitor PD98059. As expected, the CAT activity directed by the
transfected plasmid 592 was strongly increased
(9.9-fold increase) when RCEC were cultured on FN-coated dishes (Fig.
9A). However, the addition of
as little as 10 µM of the PD98059 inhibitor (many studies
have used doses 5-10-fold higher of this inhibitor (51-53)) totally
abolished this FN responsiveness, the level of CAT activity returning
to the unstimulated level. Identical results were also obtained with the recombinant plasmid p12/FRE, which bears one sense copy of the
5 FRE inserted upstream from the basal promoter of the
p12 gene (see Fig. 7, A and B). Again, the nearly
3-fold increase in the 5 FRE-mediated FN responsiveness
of the p12 promoter was totally abolished when cells were cultured in
the presence of the inhibitor (Fig. 9B). Crude nuclear
extracts were prepared from RCEC grown either on plastic or FN-coated
culture dishes in the presence of either none or 10 µM
PD98059 and then used in EMSAs. Upon incubation with the
5 FRE-labeled probe, a clear Sp1 signal that increased
severalfold when RCEC were grown on FN could be observed with nuclear
extracts from RCEC that have not been exposed to the MEK-1 inhibitor
(compare 1st and 3rd lanes in Fig.
9C). However, culturing RCEC in the presence of the
inhibitor totally abolished formation of the Sp1-5FRE
complex (Fig. 9C) even when cells were grown solely on
plastic. We therefore conclude that activation of the Ras-Erk signaling
pathway through the interaction of the 51
integrin with its ECM ligand FN accounts for the increase in
5 promoter activity when RCEC are grown on FN-coated
culture dishes and that this effect is most likely dependent on the
altered phosphorylation state of Sp1 by activated ERK1/ERK2.
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Fig. 9.
The integrin-mediated FN responsiveness is
abolished by the MEK/kinase inhibitor PD98059. A, the
recombinant plasmid 592 was transiently transfected
into RCEC grown on plastic (FN) or FN-coated
(+FN; 8 µg/cm2) culture dishes with either
none or 10 µM of the MEK/kinase inhibitor PD98059. Cells
were harvested 48 h later, and CAT activity was determined and
normalized as detailed under "Experimental Procedures."
B, same as in A except that the recombinant
plasmid p12/FRE (see Fig. 7) was substituted to 592
for the transfection experiments. C, the double-stranded
oligonucleotide bearing the 5 FRE was 5' end-labeled and
incubated with crude nuclear proteins (5 µg) from RCEC grown on
either none () or FN-coated (+) culture dishes (8 µg/cm2), in the presence of either none or 10 µM PD98059. Formation of the Sp1-FRE complex was then
monitored by EMSA as detailed in Fig. 4 except that the concentration
of the polyacrylamide gel was lowered to 4%. The position of the
Sp1-FRE complex is shown (Sp1) along with that of the free probe
(U). P, labeled probe alone.
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DISCUSSION |
Debridement of the corneal epithelium is known to promote wound
healing by stimulating migration and differentiation of both the basal
corneal epithelial cells that border the injured area and the precursor
cells from the corneal limbus. Induction of the migration process is
known to be influenced by the massive production of FN by both the
stromal keratinocytes and the basal cells that border the
injured area (5, 8-15). Moreover, recent studies provided evidence
that the level of expression for the mRNA encoding the
5 integrin subunit was positively modulated by the
presence of such FN (23, 24). The present study was therefore conducted
in order to investigate whether FN, through its
51 receptor-mediated signal transduction
pathway, can alter the transcriptional activity directed by the
promoter of the 5 integrin subunit gene. We provided
clear evidence that FN can indeed alter the transcriptional activity of
the 5 promoter by altering the DNA binding affinity of
the positive transcription factor Sp1 for a short 5
promoter segment located between positions 77 and 61 that has been
designated as the 5 FRE. The 5 FRE bears
a perfect inverted repeat of the following sequence, 5'-GGAGTTTG-3'. However, site-directed mutagenesis provided evidence that only the 3'
half-site (along with its nearby 3' GC-rich base pairs (TCCCC)) was
required for FN responsiveness to occur. This short stretch of sequence
from the 5 promoter was found to be highly homologous to
a 12-bp sequence from the murine acetylcholine receptor (AChR)
-subunit gene that was reported to be absolutely required for
muscle-specific expression of AChR- (54) (see Fig.
10). This cis-acting element, which is
comprised between positions 106 and 95, was postulated as being the
target sequence for the transcription factor myogenin (54). However,
myogenin has not been reported as a target protein that might be
subjected to differential phosphorylation by protein kinases.
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Fig. 10.
Sequence homology between the
5 FRE and other gene regulatory target
sequences. The DNA sequence from the human 5 FRE is
aligned with the Sp1-binding site identified in the promoter of the
mouse p12 gene (p12.A), the 16-bp repeat 3 element from the human LDLR,
and sequences from the murine AChR- subunit gene promoter that also
bears a binding site for the transcription factor myogenin
(MG) (underlined). Arrows indicate the
position of each 5 FRE half-inverted repeats, and
black dots indicate the position of those G residues whose
methylation by dimethyl sulfate interferes with the recognition
of the p12.A element by Sp1. The DNA sequences that show homology to
both the NF1 and Sp1 target sites are indicated.
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The substantial variations we have observed in the ability of Sp1 to
bind the 5 FRE in RCEC grown with or without FN might either result from alteration in the binding affinity of Sp1 or from
modification in the amount of Sp1 protein produced by RCEC under both
culture conditions. However, our inability to detect any significant
variations in the absolute amount of Sp1 between RCEC grown with or
without FN in Western blot analyses rather favors the former
hypothesis. Our results are consistent with those recently reported by
Alroy et al. (55) who could not see any variation in Sp1
protein levels despite an increased binding of Sp1 to the Neu
differentiation factor response element from the promoter of the
acetylcholine receptor 
upon stimulation with Neu differentiation
factors. Variations in the 5 promoter activity might
then be triggered by modifying the affinity of Sp1 for the FRE target
site by altering its state of phosphorylation through nuclear proteins
that belong to the MAPK family, such as ERK-1 (p44) and ERK-2 (p42).
Alteration of the state of phosphorylation for the transcription factor
Sp1 has been reported to alter, either positively or negatively, its
DNA-binding properties in vitro (52, 55, 56). Li et
al. (51) recently reported that Sp1 might also be a target for ERK
proteins. Indeed, they identified a 16-bp sequence (repeat 3) that
mediates responsiveness of the human low density lipoprotein receptor
(LDLR) to oncostatin M (OM) through a signal transduction pathway that
involves phosphorylation of ERK-1 and ERK-2 by the upstream kinases
MEK-1 and MEK-2. The sequence from the LDLR repeat 3 also bears an
intact copy of the GGAGTTT motif (on the non-coding strand) identified
in the 5 FRE (see Fig. 10). Most of all, it also
contains the GC-rich sequence (TCCCC) located downstream of the 3'
repeat that proved to be required for the FN responsiveness directed by
the 5 FRE. Interestingly, only Sp1 and the Sp1-related
protein Sp3 have been shown to bind repeat 3 (51). However, these
authors have been unable to detect any OM-induced alterations in Sp1
binding by EMSA or in the ratio of hyper- versus
hypophosphorylated Sp1 by Western blot analyses (51). As Fig.
5A reveals (and to some extent also Fig. 4A), no
significant changes in Sp1 binding to the 5 FRE could be
observed between nuclear extracts obtained from RCEC grown with or
without FN when only 5 µg of crude nuclear proteins were used.
However, raising the amount of proteins to either 10 or 20 µg clearly
revealed a much stronger binding of Sp1 to the FRE when RCEC are grown on FN-coated culture dishes, which is also supported by a more intense
supershift of the Sp1/FRE DNA-protein complex. Formation of this
DNA-protein complex is therefore likely to be
concentration-dependent and might explain why these authors
(57) could not detect any alterations in Sp1 binding. A recent study
conducted by Milanini et al. (53) also identified Sp1 as a
target of the p42/p44 MAPK pathway in the activation of the vascular
endothelial growth factor (VEGF) gene. However, transcriptional
activation of VEGF through this p42/p44 transduction pathway not only
requires binding of Sp1 to a GC-rich region from the VEGF promoter
located between positions
TOP
ABSTRACT
INTRODUCTION
