Three Activator Protein-1-binding Sites Bound by the Fra-2·JunD
Complex Cooperate for the Regulation of Murine Laminin
3A
(lama3A) Promoter Activity by Transforming Growth
Factor-
*
Thierry
Virolle
,
Marie-Noëlle
Monthouel
§,
Zied
Djabari
,
Jean-Paul
Ortonne
¶,
Guerrino
Meneguzzi
, and
Daniel
Aberdam
From
INSERM U385, Faculté de Médecine,
06107 Nice Cedex 2 and the ¶ Service de Dermatologie,
Hôpital L'Archet, 06002 Nice Cedex 1, France
 |
ABSTRACT |
Several lines of evidence suggest a role for
laminin-5 in skin wound healing. We report here that transforming
growth factor-
(TGF-
), which elicits various responses during
cutaneous healing, stimulates transcription of the mouse laminin
3A
(lama3A) gene. To identify the TGF-
-responsive elements
(TGF
-REs) on the lama3A promoter, we have generated a
series of 5'-deletions of the promoter upstream of the
-galactosidase reporter gene. Transient cell transfection assays
using mouse PAM212 keratinocytes revealed that TGF
-REs lie between
nucleotides
297 and
54 relative to the transcription start site.
Insertion of the TGF
-RE in front of the unresponsive minimal SV40
promoter conferred TGF-
inducibility. Computer analysis of the
promoter sequence identified three canonical activator protein-1 (AP-1)
sites located at nucleotides
277 (AP-1A),
125 (AP-1B), and
69
(AP-1C). Site-directed mutagenesis of either the AP-1A or AP-1C site
did not drastically alter the basal activity of the lama3A
promoter, but reduced TGF-
responsiveness by 50%. Simultaneous
mutation of these two AP-1 sites resulted in a 65% decline in the
response to TGF-
, suggesting a cooperative contribution of each site
to the overall promoter activity. In contrast, mutation of the AP-1B
site markedly reduced the basal activity of the lama3A promoter, indicating that this AP-1 site is essential for gene expression. Mobility shift assays demonstrated specific binding of
Fra-2 and JunD to the AP-1 sites, suggesting for the first time a
possible regulatory function for the Fra-2·JunD AP-1 complex in a
basal keratinocyte-specific gene.
 |
INTRODUCTION |
Laminin-5 is the major adhesion ligand present in the basement
membranes of stratified squamous epithelia (1, 2). In the skin, this
adhesive protein is secreted by the basal keratinocytes and colocalize
with the anchoring filaments of the lamina lucida of the dermal
epidermal junction (3-5). Laminin-5 binds to integrin
3
1 in focal adhesions and interacts with
hemidesmosomes via
6
4 to form a stable
anchorage complex (6, 7). Laminin-5 is a heterotrimeric glycoprotein
composed of the
3A,
3, and
2 polypeptide chains that are
products of different genes. Mutations in the genes encoding laminin
3 (LAMA3),
3 (LAMB3), and
2
(LAMC2) have been shown to underlie the Herlitz or
non-Herlitz forms of junctional epidermolysis bullosa, characterized by
blister formation and erosions of the skin and mucosas that frequently
lead to neonatal death (8-13).
Several lines of evidence suggest a role for laminin-5 in the
re-epithelialization of wound skin repair. Laminin-5 is found at the
epidermal-dermal junction at sites and times that coincide with
actively migrating or rapidly proliferating basal keratinocytes (14,
15). Moreover, enhancement of laminin-5 transcription is observed at
low cell densities in vitro in migrating and proliferating keratinocytes, similar to what happens at the wound edge (15). Although
the function of laminin-5 in wound healing remains to be clarified, its
major dual contribution would be to allow migration and adhesion of
keratinocytes during the wound process. In addition, accumulating data
suggest that laminin-5 might be associated with growth and migration of
cancer cells (16-19). Recently, cloning of the cDNAs encoding the
3 chain of mouse laminin-5 has identified two distinct polypeptides
(
3A and
3B) that derive from a single alternatively spliced gene
(20-22). Genomic organization analysis of the murine lama3
gene revealed that the
3A chain is transcribed by an independent
internal promoter (21).
In this study, we investigated the transcriptional regulation of the
laminin
3A (lama3A) gene by
TGF-
.1 Indeed, it has been
shown earlier that TGF-
up-regulates the adhesion protein laminin
3A mRNA level (23). TGF-
, a member of a large superfamily of
cytokines, is generally acknowledged to be the cytokine with the
broadest range of activities in injured tissue repair and tumor
progression (24). It affects nearly every aspect of tissue repair.
Indeed, TGF-
is the most potent known stimulator of chemotaxis since
it promotes the migration of the majority of cell types that
participate in the repair processes. TGF-
regulates the
transcription of a wide spectrum of matrix proteins, increasing their
production while decreasing their proteolysis and modulating their
interactions with cellular integrin receptors (25). Matrix production
by keratinocytes is also regulated by TGF-
(26-28).
Using transient transfection, site-directed mutagenesis, and nuclear
protein binding assays, we have mapped a TGF-
-responsive region
between nt
297 and
54 of the lama3A promoter. Computer analysis of this sequence identified three canonical AP-1 sites. Insertion of this region upstream of the unresponsive minimal SV40
promoter conferred TGF-
inducibility. Site-directed mutagenesis of
either AP-1 site suggested a cooperativity among these AP-1 sites in
the laminin
3A promoter. Mobility shift assays demonstrated specific
binding to the three AP-1 sites. Moreover, antibody supershift analyses
have identified JunD and Fra-2 proteins binding to these sites.
 |
EXPERIMENTAL PROCEDURES |
Cell Cultures--
Mouse PAM212 keratinocytes (kindly provided
by Dr. S. H. Yuspa, NCI, National Institutes of Health, Bethesda,
MD) were cultured in Eagle's minimal essential medium supplemented
with 10% fetal calf serum (Life Technologies, Inc.).
Plasmid Constructs--
The mouse lama3A promoter
construct pGalA (21) contains 1 kilobase of genomic lama3A
sequence extending upstream from the transcription initiation site. The
deletion panel
4.2 to
6.5 was created using the exonuclease III
system Erase-a-Base (Promega). The 5'-deletion constructs were
confirmed by sequencing and restriction digest analysis. To create
SM and
SAP, pGalA was digested with EcoRI/MscI, and SnaBI/ApaI,
respectively, blunted with Klenow polymerase I, and religated. The
PA20,
PA23,
PA24, and
PA25 constructs were generated by
polymerase chain reaction using the oligonucleotides PA20 (sense,
5'-GTCTGGCGCTGTCTGCA-3'), PA23 (sense, 5'-TGTGGGCTTTTCCCTGACTCAGC-3'),
PA24 (sense, 5'-CCTGACTCAGCCTGTGATTT-3'), PA25 (sense,
5'-GATTTACAGCTGCTCTAATT-3'), and PA22 (antisense, 5'-CGTGCAAACCTCTGTGTTC-3'), located at
244,
291,
279,
265, and
21 base pairs, respectively, of the transcription initiation site of
murine laminin
3A. These polymerase chain reaction fragments were
cloned into pUag (R&D Systems) and then extracted from the vector by
digestion with BamHI and HindIII and ligated into
BglII/HindIII-digested pGalbasic empty expression
vector (CLONTECH). The SV-RE4 construct was created
by first synthesizing complementary oligonucleotides representing four
consecutive copies of the sequences in the lama3A promoter
between bases
279 and
265 (sense, 5'-CCTGACTCAGCCTGT-3'). These
oligonucleotides were annealed and cloned into pUag, extracted from the
vector by SmaI/XhoI digestion, and inserted into
SmaI/XhoI-digested pGal-promoter, in front of the
heterologous SV40 promoter. The SV-MS construct was prepared by first
digesting the
SM construct by SmaI, and then the
SmaI digestion fragment was introduced in the
SmaI site of the pGal-promoter vector in front of the SV40 promoter (CLONTECH). To create mutA, mutB, mutC,
mutAB, mutAC, mutBC, and mutABC constructs, two point mutations were
introduced into each of the three putative AP-1-binding sites AP-1A,
AP-1B, and AP-1C of the pGalA construct with the Quick-Change kit
(Stratagene) using the complementary mutant primers for AP-1A (sense,
5'-GGCCAGGGTGTGGGCTTTTCCCTGCCTTAGCCTGTGATTTACAGCTGCTC-3'; and antisense;
5'-GAGCAGCTGTAAATCACAGGCTAAGGCAGGGAAAAGCCCACACCCTGGCC-3'), for AP-1B (sense,
5'-GCTCTCTCTGCCTGTGTAGGCTGCCTTATGTGTGAAGTTTAAAGGTGGGGC-3'; and antisense,
5'-GCCCCACCTTTAAACTTCACACATAAGGCAGCCTACACAGGCAGAGAGAGC-3'), and for AP-1C (sense,
5'-CGCAGACAGCCTTCTTTCCCTCCTGCGTTAGGCAGGCCCGGGCACTGCAGGAAG-3'; and antisense,
5'-CTTCCTGCAGTGCCCGGGCCTGCCTAACGCAGGGAAAGAAGGCTGTCTGCG-3').
Transient Transfections--
Plasmid DNAs were purified using
silica columns from QIAGEN (Hylden), and transfections were carried out
in 24-well dishes using LipofectAMINETM (Life Technologies,
Inc.) as detailed elsewhere (21). Each DNA construct was tested in
triplicate wells in at least three separate experiments. The
transfection efficiency of pGalA and its derivative constructs was
determined by cotransfection of the plasmid pGL2-control (Promega,
France). The values of
-galactosidase expression data were
normalized to the measured luciferase activity. The enzyme activities
were measured using a luminometer (Berthold Biolumat LB9500C). The
activity of the promoterless vector pGalbasic, which contain no insert,
was measured to determine background activity. In the TGF-
induction
assays, 4 h post-transfection, the cells were treated with or
without 100 pM recombinant TGF-
1 (R&D Systems) for an
additional 16-20 h.
Electrophoretic Mobility Shift Assay--
AP-1 DNA binding
activity was analyzed in total cell extracts made from untreated or
TGF-
1 (100 pM)-treated PAM212 cells in Totex lysis
buffer (20 mM Hepes, pH 7.9, 350 mM NaCl, 20%
glycerol, 1% Nonidet P-40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol, 0.1% phenylmethylsulfonyl fluoride, and 0.1%
aprotinin) as described (29). Supernatants (15,000 × g, 15-min centrifugation) were collected. An in
vitro binding reaction of AP-1 in a total volume of 25 µl was
performed by incubation of 5 µg of whole cell extract in a binding
buffer containing 10 mM Hepes, pH 7.8, 50 mM
KCl, 2 mM dithiothreitol, 1 mM EDTA, 5 mM MgCl2, 10% glycerol, 3 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, 2 mg/ml poly(dI-dC), and 2 mg/ml bovine serum albumin. After 15 min of preincubation on ice,
200,000 cpm of 32P-end-labeled oligonucleotide probe was
added and incubated at 25 °C for 20 min. Then DNA-protein complexes
were resolved by electrophoresis on 4% polyacrylamide gels in 10 mM Tris, 9 mM sodium acetate/acetic acid, and
275 mM EDTA for 1 h at 300 V; dried; and subjected to
autoradiography. When indicated, an excess of unlabeled competitor
oligonucleotides was added during preincubation. For antibody
supershift assays, nuclear extracts were preincubated with 0.3 µl of
antisera against members of the Jun and Fos families described
elsewhere (30, 31).
 |
RESULTS |
TGF-
Stimulates lama3A Transcription--
The structure of the
murine lama3A promoter and several of its regulatory DNA
elements is depicted in Fig. 1.
Full-length pGalA, which contains 1 kb of promoter region upstream of
the
-galactosidase reporter gene, was transfected into PAM212 cells and examined for responsiveness to TGF-
. A dose-responsive increase in lama3A activity was observed with maximal stimulation
(4.5-fold) at 100 pM TGF-
(Fig.
2). A series of 5'-deletions of the
lama3A promoter were examined to delineate the minimal
region responsive to TGF-
. TGF-
responsiveness was significant
(3.0-4.5-fold) for pGalA (nt
1096),
2.4M (nt
839),
4.2M (nt
619),
6.5M (nt
505), and
SM (nt
297) constructs, but
decreased to 1.5-fold upon deletion from nt
297 to
244 (
PA20)
(Fig. 3, A and C). This slight induction was lost from nt
244 to
94 (
SAP). It therefore appeared that an essential TGF-
-responsive element (TGF
-RE) was located between nt
297 and
244 of the
lama3A promoter. However, since the basal activity
drastically decreased for
SAP, it remained to be determined whether
the loss of stimulation was due to the low basal activity or to the
presence of additional sequences allowing secondary TGF-
responsiveness between nt
244 and
94.

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Fig. 1.
Sequence organization of the mouse
lama3A gene promoter. Nucleotide numbering is relative
to the first nucleotide of the transcription initiation site previously
described (21). The relevant regulatory AP-1 motifs are
underlined, and the restriction sites used for cloning are
shown. The bent arrows represent the positions of
the 5'-ends of the deletion constructs. The TATA box and the
transcription initiation site are double-underlined.
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Fig. 2.
Effect of TGF- on mouse lama3A
promoter transcriptional activity. Cells (4 × 104) were transfected with 0.2 µg of promoter-reporter
plasmid pGalA and 0.125 µg of SV40-luciferase DNA (pGL3-control,
Promega) as an internal control. After removing the transfection
medium, increasing concentrations of TGF- in serum-free medium were
added to the cells and incubated for 16-20 h at 37 °C prior to
extraction of cell extracts. -Galactosidase activity was measured as
relative light units and normalized by luciferase activity of
cotransfected pGL2-promoter. -Fold induction was calculated by
comparing the normalized -galactosidase activities of
TGF- -treated cells and untreated control cells.
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Fig. 3.
Deletion analyses and transcriptional
activation of the mouse lama3A gene promoter by TGF- in
transient transfection assays of PAM212 cells. A,
full-length and deletion lama3A promoter-reporter constructs
were transiently transfected into PAM212 cells and stimulated with 100 pM TGF- 1 for 16-20 h as described under "Experimental
Procedures" and as described in the legend of Fig. 2. The numbers on
the left indicate the 5'-ends of the constructs relative to the
transcription initiation site. The values are the means of triplicates
from at least three independent experiments. B, progressive
5'-deletion constructs of SM were transfected as described for
A. The AP-1A binding site is underlined.
C, -fold induction was calculated by comparing the
normalized -galactosidase ( gal) activities of
TGF- -treated cells and untreated control cells.
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Delineation of TGF-
-responsive Elements within the lama3
Promoter--
To more precisely map the potential TGF
-RE that
should reside between
SM and
PA20, additional 5'-deletions of
SM constructs were created (Fig. 3, B and C).
Similar induction with TGF-
treatment was obtained with the
SM
(nt
297),
PA23 (nt
291), and
PA24 (nt
279) constructs,
whereas a decrease in induction was observed with
PA25 (nt
265).
This result indicates that a TGF-
-responsive element is located
between nt
279 and
265 of the lama3A promoter. Examination of the deleted region that reduced TGF-
responsiveness revealed the presence of one potential AP-1-binding site (Figs. 1 and
3B).
To assess whether the region between nt
279 and
265 was sufficient
for activation by TGF-
, four copies of this sequence were linked to
a heterologous unresponsiveness SV40 promoter (construct SV-RE4) (Fig.
4). Since no TGF-
induction was
observed in SV-RE4, we conclude that the 15-base pair sequence
containing AP-1 (nt
279) is necessary but not sufficient to confer
TGF-
responsiveness.

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Fig. 4.
Functional analysis of putative
TGF -responsive elements using the heterologous promoter. A
synthetic oligonucleotide spanning four identical copies of the region
from nt 279 to 265 of the lama3A promoter was cloned
upstream of the SV40 promoter linked to the -galactosidase gene,
generating the plasmid SV-RE4. The construct SV-MS carries the
MscI/SmaI fragment ( 297/ 54) in front of the
SV40 promoter- -galactosidase plasmid. The -galactosidase
activities are represented as -fold stimulation of TGF- -treated
cells over untreated cells. The pGL3-control cotransfectant served as
an internal transfection control. Other conditions were the same as
described in the legend of Fig. 2. pGalprom,
pGal-promoter.
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As suggested previously, additional TGF
-RE sequences must reside
downstream of the AP-1-binding site. To assess whether the region
between nt
297 and the TATA box was sufficient for activation by
TGF-
, the MscI/SmaI DNA fragment from pGalA
(from nt
297 to
54) was linked to a heterologous SV40 promoter
(construct SV-MS). This construct was able to fully restore the TGF-
induction of
-galactosidase transcription in PAM212 cells as
compared with
SM, whereas no induction was observed with the
pGal-promoter vector alone (Fig. 4). Thus, the sequence of this region
of the lama3A promoter is sufficient to confer TGF-
responsiveness to a previously non-inducible promoter. Examination of
the TGF-
-responsive region indicated the presence of three potential
AP-1-binding sites located at nt
277 (AP-1A),
125 (AP-1B), and
69
(AP-1C) (Fig. 1). Site-directed mutagenesis was used to sequentially
modify these putative AP-1 sites within the context of the pGalA
promoter fragment because this region retains high basal activity and
TGF-
responsiveness (Fig. 3A). The mutated constructs
were then used in transient transfection experiments in parallel with
their wild-type counterparts. As shown in Fig.
5, the mutation of AP-1B (mutB) drastically reduced the promoter activity, whereas mutation of each of
the other AP-1 sites (mutA and mutC) still maintained 60% of the pGalA
basal activity. However, elimination of each AP-1 site reduced TGF-
responsiveness to half the level seen with the wild-type promoter
construct pGalA. Simultaneous mutation of both AP-1A and AP-1C (mutAC)
abated by 65% the inducible activity by TGF-
, whereas simultaneous
mutations of the three AP-1 sites (mutABC) completely abolished TGF-
responsiveness, as did dual mutants including the AP-1B mutation (mutAB
and mutBC) (Fig. 5). These results indicate that the integrity of each
AP-1 site of the lama3A promoter is necessary for full
transcriptional induction. Furthermore, since half the response to
TGF-
is retained within single AP-1 mutants, the full
transcriptional induction of the lama3A promoter by TGF-
requires cooperation among the three AP-1 sites.

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Fig. 5.
Mutation of AP-1 sites modulates the
lama3A promoter response to TGF- . A,
full-length pGalA and variants in which one (mutA, mutB, and mutC), two
(mutAB, mutAC, and mutBC), or three (mutABC) AP-1 sites have been
inactivated by mutation were transfected into PAM212 cells in the
presence or absence of TGF- . Constructs were tested for activity
exactly as described in the legend of Fig. 2. B, the
relative induction of each construct by TGF- is represented and
compared with that obtained by pGalA. gal,
-galactosidase.
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AP-1 Proteins Bind to the lama3 TGF-
-responsive Element--
To
confirm the interaction of nuclear proteins with the AP-1 sites,
electrophoretic gel mobility shift assays were performed using
radiolabeled lama3A TGF
-RE sequences corresponding to
each of the three AP-1 sites and flanking regions, as detailed in Table I. AP-1Am, AP-1Bm, and AP-1Cm are mutants
of the AP-1A, AP-1B, and AP-1C sites, respectively. Fig.
6 shows a gel mobility shift experiment
in which unstimulated and TGF-
-stimulated mouse keratinocyte total
cell extracts were incubated with 32P-labeled AP-1A, AP-1B,
or AP-1C oligonucleotides in the absence or presence of excess
nonradioactive competitors. Our results indicate that, although these
regions bound protein complexes isolated from both control and
TGF-
-treated keratinocytes, TGF-
treatment induced a significant
increase in DNA binding within 1 h for the AP-1A, AP-1B, and AP-1C
oligonucleotides (Fig. 6, A-C). Protein binding was
specifically competed in a dose-dependent manner by
addition of 20- and 50-fold excesses of homologous DNA, but not by a
50-fold excess of the corresponding homologous mutant AP-1
sequence or heterologous Sp1 oligonucleotide competitors. These data
indicate that AP-1 complexes bind to each of the three TGF
-RE
elements of the lama3A promoter. Although the AP-1A and AP-1C oligonucleotides were specifically competed by 20-fold (data not
shown) and 50-fold (Fig. 6, A and C,
respectively) molar excesses of every AP-1 oligonucleotide, the
AP-1B band shift was not displaced by a 50-fold molar excess of either
AP-1A or AP-1C competitor (Fig. 6B).
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Table I
Sequence of 1ama3A AP-1 sites and double-stranded oligonucleotides
employed in gel mobility shift assays
AP-1Am, AP-1Bm, and AP-1Cm are mutants of AP-1A, AP-1B, and AP-1C in
which the A at position 3 and the C at position 6 of the consensus
sequence were changed to C and T, respectively (underlined in each
oligonucleotide). Sp1 has commercial (Promega) consensus binding sites.
Consensus binding sites of all oligonucleotides are underlined.
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Fig. 6.
Binding of nuclear proteins to the
lama3A AP-1-binding sites. A-C, binding of
nuclear proteins to the AP-1A, AP-1B, and AP-1C binding sites,
respectively. 32P-Labeled oligonucleotides were incubated
with nonstimulated (NS) or TGF- -stimulated total cell
extracts in the absence (0x) or presence of a 20- or 50-fold
molar excess of unlabeled competitors. none indicates a lane
in which the total cell extract was omitted. Arrows indicate
the free probe (bottom) and the specific retarded bands
(top). For details, refer to Table I and "Experimental
Procedures."
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To characterize further the protein complexes binding to the TGF
-RE
region of the murine lama3A promoter, total cell extracts from untreated and TGF-
-treated PAM212 cultures were incubated prior
to DNA/protein interactions with antibodies specific for each member of
both the Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and
Fra-2) family proteins and visualized by gel mobility shift assay.
Since identical results were obtained from untreated and
TGF-
-treated cells, we illustrated the data from treated cells. As
shown in Fig. 7, the antibodies against
JunD and Fra-2 (and to a much less extent, JunB) induced a supershift of the labeled DNA probes (brackets). These results indicate
that JunD and Fra-2 participate in the formation of the complex that binds to the three AP-1 sites of the lama3A promoter.

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Fig. 7.
Identity of nuclear factors that interact
with the lama3A promoter AP-1 sites. TGF- -treated
PAM212 total cell extracts were incubated with labeled AP-1A
(A) or AP-1B (B) or 32P-labeled AP-1C
(C) oligonucleotides in the presence of nonimmune serum
(NI) or antibodies against JunD (D), JunB
(B), c-Jun (c), c-Fos (c), FosB
(B), Fra-1, or Fra-2. The brackets indicate the
supershifted complexes.
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 |
DISCUSSION |
This study was designed to investigate the molecular mechanism of
lama3A transcriptional induction by TGF-
. TGF-
is
known to regulate many different gene transcripts of the extracellular matrix, thereby modulating cell adhesion, migration, and proliferation, under both physiological and pathological conditions (24). Our results
define three AP-1-binding sites in the mouse lama3A promoter that cooperate to confer TGF-
responsiveness since (a)
their presence is needed in front of unresponsive SV40 promoter
sequences to fully restore TGF-
inducibility; (b)
mutations of the AP-1A, AP-1B, and AP-1C sites significantly abate the
TGF-
-promoter transactivation in PAM212 cells; and (c)
simultaneous mutations of the three AP-1 sites abolish this
transactivation. Furthermore, the AP-1B site is crucial for
lama3A promoter activity since mutating its sequence almost
completely abolished the basal transcriptional activity of the
lama3 promoter. Since the AP-1B site is not cross-competed on band shift assays by a 50-fold molar excess of either AP-1A or AP-1C
oligonucleotide competitors, the AP-1 protein heterodimer binds the
AP-1B site with higher affinity than the two other AP-1 sites. These
results suggest that the AP-1B site is preferentially bound by the AP-1
proteins under basal activity and must cooperate with the two other
AP-1 sites to allow full transcriptional basal and TGF-
-stimulated
activities.
As reviewed recently (32), many promoters of keratinocyte-specific
genes contain cis-acting elements that are capable of binding AP-1 heterodimer complexes. In the epidermis, the AP-1 heterodimer serves as an activator of gene expression in all the keratinocyte layers, i.e. keratin K5 in the basal layer;
human papillomavirus in the suprabasal compartment; loricrin and
profilaggrin in the granular layer; and finally, keratin K1,
involucrin, and transglutaminase 1 in the spinous/granular layer (32).
Therefore, it is intriguing how the cell coordinately regulates genes
through identical DNA elements. Since AP-1 consists of proteins from
the Jun and Fos families that associate as homodimers (Jun·Jun) or heterodimers (Fos·Jun), one clue might be the composition of the AP-1
dimers, meaning that a certain combination of Fos·Jun complexes may
be necessary to provide different levels of transcriptional activity,
depending on the stage of keratinocyte differentiation. It should be
noted that laminin
3A transcripts are produced only by proliferating
basal keratinocytes in vivo (33) and in vitro (15). We have shown here that the lama3A promoter AP-1 sites are preferentially bound by the Fra-2·JunD complex and that the binding is significantly increased by TGF-
stimulation of PAM212 keratinocytes as compared with untreated cells. Recently,
immunohistological studies on Fos/Jun factors have shown that the
distribution of AP-1 proteins in the mouse epidermis is
compartmentalized in vivo, suggesting that distinct AP-1
complexes act in the various layers of the skin (34). In accordance,
Fra-2 and JunD are coexpressed only in the basal layer of mouse skin,
but are absent in the spinous (for Fra-2) and granular (for JunD)
layers of the epidermis, implying that the particular Fra-2·JunD
heterodimer is selectively down-regulated in the early stages of
epidermal differentiation (34). All the epidermal genes investigated so
far are regulated by different AP-1 family members, i.e.
JunB/JunD for HPV18 (35), JunB/Fra-1 and JunD/Fra-1 for involucrin
(36), and c-Jun/c-Fos for profilaggrin (37). These keratinocyte genes
are expressed in the suprabasal compartments of the epidermis. Since
the lama3A gene is expressed exclusively in the basal cells
of most stratified epithelia, it will be important to determine the
potential role of Fra-2 and JunD in the regulation of basal
keratinocyte-specific genes in general and of the lama3A
gene in particular.
AP-1 has been shown to be the transcriptional mediator of several
TGF-
-responsive genes, including TGF-
itself (40), plasminogen activator inhibitor type-1 (41), c-jun (42),
2(I)-collagen (43), osteocalcin (44), retinoic acid receptor (45),
and clusterin (46). However, the nature of the AP-1 complexes that are
induced by TGF-
has not been determined, except partially for the
2(I)-collagen promoter, which is bound by c-Jun in keratinocytes and
JunB in fibroblasts (47). AP-1-binding sites are not the exclusive
cis-acting elements bound in response to TGF-
stimulation; Sp1 has also been reported to participate in the
regulation of human
2(I)-collagen (48), p15 (49), and
p21waf1 (50) gene expression by TGF-
. In other promoters
(51), a TGF-
-responsive consensus sequence has been identified as a
nuclear factor 1-binding motif, but the corresponding transcription
factors that bind to these sequences remain unidentified.
In conclusion, we have used a combination of techniques to identify the
regulatory elements important for the transcriptional induction of the
lama3A gene by TGF-
. We have demonstrated a cooperative
contribution of three canonical AP-1-binding sites to the stimulation
of the promoter activity and that the AP-1B site is essential for basal
gene expression. These AP-1-binding sites contain almost exclusively
the JunD and Fra-2 proteins, which suggests for the first time a
possible regulatory function of the Fra-2·JunD AP-1 complex in a
basal keratinocyte-specific gene. Our results therefore strongly
support the idea that AP-1 proteins play a central role in the
transcriptional regulation of epidermal gene expression (32). As for
many other genes, the same regulatory elements are involved in both
basal and stimulated transcriptional activities. These studies
represent the initial steps toward the identification of the signaling
pathways involved in TGF-
-mediated transcriptional activation of
laminin
3A under physiological and pathological conditions, such as
wound healing and carcinogenesis.
 |
ACKNOWLEDGEMENTS |
We thank D. Lallemand (Institut Pasteur,
Paris) for generous gifts of antibodies to Jun and Fos family members
and Dr. S. H. Yuspa for providing the PAM212 cells. We are
grateful to Dr. R. Buscà for critical reading of this
manuscript.
 |
FOOTNOTES |
*
This work was supported in part by EEC Biomed-2 Grant
BMH4-CT972062, by the ssociation pour la Recherche sur le Cancer Grant 5003, and by grants from the Ligue du Var contre le Cancer, INSERM, and
the Fondation pour la Recherche Médicale.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y08738.
§
Recipient of a Ligue Nationale Contre le Cancer predoctoral
fellowship.
To whom correspondence should be addressed: INSERM U385, UFR
de Médecine, Av. de Valombrose, 06107 Nice Cedex 2, France. Tel.:
33-04-93-37-77-18; Fax: 33-04-93-81-14-04; E-mail:
Aberdam{at}unice.fr.
1
The abbreviations used are: TGF-
,
transforming growth factor-
; TGF
-RE, TGF-
-responsive element;
nt, nucleotide(s); AP-1, activator protein-1.
 |
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