(Received for publication, November 15, 1996, and in revised form, April 3, 1997)
From We already identified two distinct laminin Laminins constitute a family of glycoproteins specific to the
basement membranes, which are associated with a variety of biological activities, including cell adhesion, spreading, migration, and differentiation (1, 2). Seven laminin variants with tissue-specific distributions have been reported thus far (2-4). All these molecules are heterotrimers comprising an Laminin-5 ( Further evidence for the major adhesion role of laminin-5 was provided
by studies on junctional epidermolysis bullosa
(JEB),1 an inherited
skin-blistering disease, in which a defective expression of laminin-5
causes disadhesion of integuments with a cleavage plane of the skin
blisters lying within the lamina lucida of the dermal-epidermal
junction (18). Genetic mutations of the three genes encoding laminin-5
have recently been identified in distinct clinical variants of JEB
patients (19-23, 24 and references therein). Laminin-5 is also the
target for autoantibodies in cicatricial pemphigoid, an acquired
autoimmune disease characterized by mucosal subepidermal blistering.
The epitopes have been localized on the carboxyl-terminal globular
domain of the laminin Recently, we have isolated and characterized the cDNA encoding the
To gain insight into the regulation of the laminin All recombinant DNA technology was performed according to
standard procedures (27).
Restriction enzymes and other DNA modification
enzymes were purchased from New England Biolabs (Ozyme, France). The
Sequenase 2.0 sequencing kit was obtained from Pharmacia (Biotech St.
Quentin-Yvelines, France). Radioactive chemicals were purchased from
Amersham (Les Ulis, France). Oligonucleotide primers were synthesized
by Eurogentec (Liége, Belgium). The genomic DNA library was made
with SV 129 D3 embryonic stem cell DNA and constructed in The genomic library was screened
by plaque hybridization with the Two µg of purified phage DNA were
digested with the appropriate restriction enzymes. The DNA fragments
were separated by electrophoresis on a 0.7% agarose gel and then
transferred to nylon membranes (Hybond N, Amersham). The genomic DNA
clone The DNA fragment located upstream to the 5 One pmol of the antisense
oligonucleotide BR was 5 Mouse PAM212 keratinocytes
(kindly provided by Dr. S. H. Yuspa, NIH) were cultured in
Eagle's minimum essential medium supplemented with 10% fetal calf
serum. NIH-3T3 fibroblasts (American Type Culture Collection, CRL 1658)
were grown in Dulbecco's modified Eagle's medium supplemented with
10% bovine serum. Plasmid DNAs were purified using the silica columns
from Qiagen (Hylden, Germany), and transfections were carried out in
24-well dishes using LipofectAMINETM (Life Technologies,
Inc.) as detailed elsewhere (28). Each DNA construct was tested in
triplicate wells in three separate experiments. Transfection efficiency
of pGalA and its derivative constructs was determined by cotransfection
of plasmid pGL2-promoter (Promega) and that of construct pLucB and its
derivative constructs by cotransfecting with plasmid p Since the laminin The genomic clone
From these results we conclude that the genomic clone
To determine whether the
The 5
To assess whether the genomic DNA region
upstream to the transcriptional start site of
To verify whether the DNA sequences upstream to the
We have analyzed the organization of the distinct regions of the
genomic DNA located 5 Acute phase reactant consensus sequences and NF-IL-6 binding sites have
been found in the two lama3 promoters. The acute phase sequences were shown in the flanking region of genes coding for proteins induced in response to trauma or injury (34, 35). The
transcription factor NF-IL-6 activates IL-6-mediated induction of
several acute-phase proteins. NF-IL-6, constitutively expressed at a
low level, is rapidly up-regulated by inflammatory cytokines such as
IL-1, tumor necrosis factor, and IL-6 (34-36). Healing of skin wound
includes two major early stages: 1) migration of inflammatory cells to
the wound site following platelet aggregation and blood coagulation and
2) migration and proliferation of keratinocytes, fibroblasts, and
endothelial cells, leading to re-epithelialization and granulation
tissue formation. Several lines of evidence suggest a role for
laminin-5 in these steps of wound healing. Indeed, it has been
hypothesized that T lymphocyte migration into the epidermis may involve
the interaction of the T cell integrin The second step, concerning migration of keratinocytes, seems also to
involve the regulation of lama3 gene since wounding up-regulates synthesis of laminin-5 and its receptor Herlitz JEB is characterized by a reduced adherence of the squamous
epithelia from the underlying mesenchyme due to deficient expression of
one of the genes encoding laminin-5 (39). We recently reported that
retroviral vector-mediated transduction of laminin-5 cDNA restores
synthesis of functional laminin-5 and induces changes in focal adhesion
and motility in Herlitz JEB keratinocytes (28). This study is the first
step for an application of in vivo gene transfer for the
treatment of the JEB genodermatosis. However, although retroviral
vectors are highly effective for transfer of genes into tissues
in vivo, several limitations, including extinction of the
viral promoter activity, tissue specificity, and insert size capacity,
have hampered their use (40). The PA fragment identified here should
allow the identification of regulatory sequences useful in the
targetting of cDNA constructs aimed at the complementation defects
of laminin-5 chains in JEB and in physiological situations as wound
healing and cell migration.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y08738 and Y08850. We thank Z. Djabari and G. Spennato
for skilled technical assistance and C. Minghelli for artwork.
U385 INSERM,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
3A
and
3B chain isoforms which differ in their amino-terminal ends and
display different tissue-specific expression patterns. In this study we have investigated whether these two different isoforms are products of
the same laminin
3 (lama3) gene and transcribed from one
or two separate promoters. Genomic clones were isolated that encompass the sequences upstream to the 5
ends of both the
3A and the
3B
cDNAs. Sequence analysis of the region upstream to the
3A open
reading frame revealed the presence of a TATA box and potential binding
sites for responsive elements. By primer extension analysis, the
transcription start site of the
3B mRNA isoform was defined. The
sequences upstream to the
3B mRNA transcription start site do
not contain a TATA box near the transcription initiation sites, but
AP-1, AP-2, and Sp1 consensus binding site sequences were identified.
The genomic regions located immediately upstream of the
3A and
3B
transcription start sites were shown to possess promoter activities in
transfection experiments. In the promoter regions, response elements
for the acute phase reactant signal and NF-interleukin 6 were found,
and their possible relevance in the context of inflammation and wound
healing is discussed. Our results demonstrate that the
lama3 gene produces the two polypeptides by alternative
splicing and contains two promoters, which regulate the production of
the two isoforms
3A and
3B.
,
, and
chain which are known to exist in at least 10 genetically distinct forms (3, 5, 6).
3
3
2) is a unique laminin variant substantially
truncated within the short arm domains compared with the other laminin
chains (7-11). Laminin-5 is secreted into the basement membrane by the
basal cells of stratified and transitional epithelia with predominant
secretory or protective functions (12). In the skin, the protein
colocalizes with the anchoring filaments of the lamina lucida of the
dermal epidermal basement membrane zone and is concentrated beneath the
hemidesmosomes, suggesting a role in the adhesion between the basal
keratinocytes and the underlying mesenchyme (13). Accumulating data
from in vitro and in vivo studies demonstrate
that laminin-5 is the major adhesion ligand of keratinocytes. This
protein binds to integrin
3
1 in focal adhesions (14) and
interacts with hemidesmosomes via
6
4 to form a stable anchorage
complex (15-17). The interaction of laminin-5 with the integrins
regulates associations of both focal adhesions and the hemidesmosomes
with actin filaments and intermediate filaments, respectively, through
distinct transmembrane signal pathways (14).
3 chain (25). This is consistent with the
observation that treatment with monoclonal antibody BM165, which is
directed against the G domain of the laminin
3 chain, prevents
adhesion of keratinocytes in vitro and in vivo
(13) and with the experimental evidences for a role of the G domain of
laminin-5 in nucleation and structural integrity of hemidesmosomes
(26).
chain of the mouse laminin-5, and we have identified two distinct
isoforms
3A and
3B which differ in their amino-terminal domains
(11). The
3B polypeptide bears two globular domains and an epidermal
growth factor-like domain that substitute the short amino-terminal
domain of the
3A isoform. In situ hybridization analysis
on mouse embryos demonstrated a restricted tissue-specific distribution
for the two isoforms. The
3A isoform is widely distributed in
squamous and transitional epithelia and more predominantly in the skin
and hair follicles, whereas the
3B counterpart, which is weakly
expressed in epidermis, is readily detected in the lung epithelium and
in neuroepithelia. The structural differences in the amino-terminal
domains of the laminin
3A and
3B chain isoforms and their
specific expression patterns raise questions on possible distinct
physiological roles for the two polypeptides.
3 chain isoforms,
we have isolated and analyzed the noncoding regions of genomic DNA
located upstream to the DNA sequences coding for the
3A and
3B
polypeptides. We have shown that the laminin
3A and
3B mRNAs
are produced by alternative splicing of the lama3 gene
transcripts. We also demonstrate that the transcription of lama3 is regulated by two distinct promoters specific for
the
3A and
3B DNA sequences.
GEM12.
This library was kindly provided by J-M. Garnier (laboratory of P. Chambon, Strasbourg, France).
-32P-labeled cDNA
probes PR6 and MR10 encoding the 5
domains of laminin
3A and
3B
chains, respectively (11). Two nonoverlapping phages,
A (15 kb) and
B (20 kb), were isolated and further analyzed.
A was hybridized to the oligonucleotide primer pairs
BL and AR. Oligonucleotide BL,
5
-ACAGGTGACTCATGCCAG-3
, corresponds to the cDNA sequences encoding the 3
region of the amino-terminal domain of the mouse
3B
polypeptide and oligonucleotide AR,
5
-GGTACCACCTCACTGCAGGC-3
, is located within the 5
region of the
cDNA sequences specific to the
3A chain isoform. The
B phage
DNA was hybridized to the oligonucleotide primer BR,
5
-CTGGACTGCCTCACAGACAATCTCACCCTTACTTC-3
, which is complementary to a
sequence located 28-56 bp downstream to the translation initiation
site of the mouse
3B mRNA.
to
3A and
3B
mRNAs
3A
exon was amplified from the
A DNA by polymerase chain reaction using
the primer pairs AR and BL. The amplification
product (1.0 kb) was initially introduced into pTag vector (R & D
Systems, Oxon, UK) to yield clone pMA. After digestion of phage
B
DNA with restriction enzymes, the EcoRV/EagI
2.7-kb DNA fragment hybridizing with the oligonucleotide BR
probe was cloned into SmaI/EagI-digested
pBluescriptII SK plasmid to obtain clone pMB. Positive clones were
sequenced on both strands. For subsequent tests of promoter activity,
recombinant plasmids were constructed by subcloning the inserts of pMA
and pMB into reporter vectors. pMA insert was excised by
KpnI digestion and subcloned into KpnI-digested
pGalbasic (CLONTECH, Ozyme, France) to produce
plasmid pGalA. Deletion constructs of pGalA were obtained by digestion
with SnaB1 (multicloning site of pTag
vector)/BglII, blunted, and self-ligated to obtain the
construct DSB. To obtain the construct DEA, pGalA was digested by
EcoRI within the multicloning site of pTag vector and
AflII, blunted, and self-ligated. Construct DSM was obtained
by digesting pGalA with SnaBI within the multicloning site
of pTag vector and MscI, blunted, and self-ligated. To
obtain the construct DSAP, pGalA was digested with SnaBI and
ApaI, blunted, and self-ligated. To produce pLucB, pMB
insert was excised by SmaI/EagI digestion
followed by a blunt reaction and subcloned into
SmaI-digested pGL2-basic (Promega Corp.,
Charbonniéres, France). This construction was digested with
EcoRV and KpnI, blunted, and self-ligated to
produce the construct KE. Construct AE was obtained by digesting KE
with KpnI and AflII, blunted, and self-ligated. Construct KE was digested with AflII and EagI,
blunted, and self-ligated to obtain the construct KA.
3B by
Primer Extension Analysis
-end-labeled using
[
-32P]ATP (3000 Ci/mmol) and T4 polynucleotide kinase.
The radioactive primer (2 × 105 dpm) was added to 10 µg of mouse lung total RNA in 25 µl of hybridization buffer (5 mM PIPES, pH 6.4, 0.5 mM NaCl, 1.0 mM EDTA, and 80% formamide), heated for 5 min at 85 °C,
and annealed for 16 h at 58 °C. The primer was then extended in
a reverse transcriptase reaction for 60 min at 42 °C using reverse
transcription buffer with 5 mM dNTPs, 25 units of RNase
inhibitor, and 50 units of murine leukemia virus reverse transcriptase.
RNase A (2 µl of a 0.5 µg/µl solution in 0.5 M EDTA)
was added, and incubation was continued at 37 °C for 15 min. After
ethanol precipitation, the extended products were fractionated on a 6%
acrylamide, 7 M urea sequencing gel in parallel with the
products of a double-stranded sequencing reaction.
Gal-promoter
(CLONTECH). The values of
-galactosidase
expression data were normalized to the measured luciferase activity.
Enzyme assays were performed using either the Promega luciferase assay
system or the CLONTECH
-galactosidase reporter
system. The enzyme activities were measured using a luminometer (Berthold Biolumat LB9500C). We measured the activity of pGL2-basic and
pGalbasic vectors, which contain no insert, to determine background activity.
Isolation of Genomic Clones
3A and
3B
chain isoforms harbor identical carboxyl-terminal domains (11), we
suspected these polypeptides to be encoded by the same gene. To
determine the genomic structure of the 5
regions of
3A and
3B
DNA sequences, we screened a mouse genomic DNA library using
radioactive cDNA probes specific for the 5
-terminal domains of
each polypeptide. Two positive clones, respectively,
A and
B,
were isolated and further characterized by Southern blot analysis.
A was digested with the restriction enzymes
SmaI and HindIII. A restriction
HindIII DNA fragment of 6.4 kb was found to hybridize to
both the mouse cDNA sequences specific to the 3
end of
3B and
to those specific to the 5
end of
3A (Fig.
1A). The restriction map of
the 6.4-kb fragment is shown in Fig. 1B. Sequencing of the
genomic HindIII 6.4-kb subclone revealed that this DNA
fragment contains a 2-kb intron located 5
to the 3
end of the
3B-specific exon sequences (230 bp) and DNA sequences specific to
the
3A isoform (200 bp). These two DNA sequences are separated by a
1.035-kb segment (PA) of untranscribed genomic DNA. A 3.0-kb intron
bridges the
3A-specific DNA sequences to 38 bp that constitute the
5
terminus of the DNA sequences common to both the
3A and
3B
isoforms.
Fig. 1.
A, Southern blot analysis of the 15-kb
genomic clone A. DNA from clone
A was digested with
SmaI (lane 1), HindIII (lanes 2 and 3), or SmaI/HindIII
(lane 4) electrophoresed, transferred to a nylon membrane,
and hybridized to oligonucleotide BL (lanes 1 and 2) and AR (lanes 3 and
4) as probes. One HindIII band of the same size
(6.4 kb) appeared in the Southern analysis (arrow). B, organization of the 6.4-kb HindIII fragment
obtained from the genomic clone
A within the mouse lama3
gene. Filled boxes represent the exons, open
boxes represent the introns, and the cross-hatched box
represents the 1035-bp 5
-untranscribed region of
3A (PA). H, HindIII; S, SmaI.
[View Larger Version of this Image (10K GIF file)]
A contains a
DNA fragment of the lama3 gene encoding domains of laminin chains
3A and
3B (Fig. 1A and see also Fig.
4B). The exons specific to the
3B polypeptide are located
5
to the unique exon encoding the
3A amino-terminal domain, which
is located 5
to the sequences coding for the carboxyl-terminal
domains common to the two isoforms.
Fig. 4.
A, Southern blot analysis of the 20-kb
genomic clone B. DNA from clone
B was digested with
EcoRV (lane 1) or EcoRV plus EagI (lane 2), electrophoresed, transferred to a
nylon membrane, and hybridized to oligonucleotide BR
(complementary to sequence located 28-56 bp downstream from the
translation initiation site of the mouse
3B mRNA) as probe. The
DNA fragment of 2.7 kb in size (PB) was generated by double digestion
with EcoRV and EagI (arrow).
B, schematic representation of the regulatory regions of the
lama3 gene. The relative localization on the
lama3 gene of the genomic fragment PB, which contains the
noncoding DNA sequences 5
to the
3B coding region, and that of the
genomic fragment PA, which corresponds to the noncoding DNA sequences
5
to the
3A coding region, are indicated by cross-hatched
boxes. The exact physical distance between PA and PB on the
genomic DNA (indicated by a broken line) is not determined.
The location of the cDNA probes MicaR10 and PR6 used to screen the
genomic library, and that of the genomic clones
A and
B, are
represented. R, EcoRV; E,
EagI; and H, HindIII.
[View Larger Version of this Image (8K GIF file)]
-Flanking Region of the
3A
Exon
3A and
3B mRNAs are
produced by transcription from different promoters or derive from a
unique primary transcript, the 1.0-kb genomic fragment (PA), located upstream to the
3A-specific exon, was polymerase chain
reaction-amplified from
A using pairs of primers
BL/AR and sequenced (Fig.
2). Analysis of the nucleotide sequences
revealed a TATA box 27 bp upstream to the transcription start site
defined previously (11). No potential splice acceptor sites could be
identified, suggesting that this DNA fragment is not an intron. Careful
inspection of the DNA sequence detected the presence of canonical
cis-acting binding sites characteristic of eucaryotic promoters (Fig.
2). Two sequences identical to the consensus sequence for AP-1 sites and three for AP-2 sites are located upstream to the
3A coding sequences. A consensus sequence for binding of the ubiquitous Sp1
factor is identified (29). Two acute phase-reactant consensus sequences
are found (30) as well as two NF-IL-6 binding sites.
Fig. 2.
Nucleotide sequence and identification of
putative cis-acting elements in the 5-flanking region of
3A
mRNA. Nucleotide numbering is relative to the first nucleotide
of the codon for the initiation methionine (ATG). Asterisks
represent the location of the 5
end of the
3A cDNA described
previously (11). Position and polarity of the oligonucleotides used to
amplify the 1-kb fragment are indicated by arrows. The
putative TATA box and the splicing donor site between the
3B-specific exon and the
3A promoter (CagGTAGGG) are represented
in bold; the vertical arrow delineates the
junction. The cis-acting elements containing consensus sequences, as
well as the relevant restriction sites are underlined.
[View Larger Version of this Image (53K GIF file)]
-Flanking Region of the
3B
Exons
end of the
3B mRNA was determined by primer
extension analysis using the synthetic oligonucleotide BR
(see "Experimental Procedures"). The adenine residue of the
translational start site (ATG) is numbered +1. The primer extension
yields a single product of 126 nucleotides, placing the transcription
initiation site 90 bp upstream to the ATG (Fig.
3). The genomic clone
B (20 kb) was
analyzed by Southern blot using the primer BR as a
radioactive probe. The sequence complementary to the oligonucleotide
BR corresponds to the 5
end of the
3B cDNA
sequence, starting 25 bp upstream to an EagI restriction
site. A unique 2.7-kb EcoRV/EagI fragment hybridized to the probe (Fig.
4A, arrow). A
schematic representation of the lama3 gene is depicted in
Fig. 4B showing the relative location of
3A and
3B
domains as well as the genomic
A and
B clones. The 2.7-kb
EcoRV/EagI fragment was subcloned, and the corresponding sequence is presented in Fig.
5. No TATA box motif was detected near
the transcription initiation site. A putative TATA box site (TAATATA)
is identified upstream at position
265. In addition,
computer-assisted sequence analysis of the 5
-flanking region of the
lama3 gene identified AP-1, AP-2, and Sp1 sites. Nine acute
phase reactant consensus sequence and two NF-IL-6 binding sites were
also localized.
Fig. 3.
Mapping of the 5 end of mouse
3B
mRNA. The autoradiogram shows a primer extension on
3B
mRNA isolated from 10 µg of total mouse lung along with dideoxy
sequencing reaction of pMB. The left-hand lane contains the
primer-extended cDNAs obtained. The same primer was used for both
extension and sequencing reactions.
[View Larger Version of this Image (77K GIF file)]
Fig. 5.
Sequence of the 2.7-kb
EcoRV/EagI genomic fragment comprising the
5-noncoding DNA sequence of the mouse lama3 gene. The
transcription initiation site identified by primer extension assays is
indicated by an asterisk (nucleotide
90). Position and
polarity of the synthetic oligonucleotide BR used in the
primer extension analysis is indicated (arrow). The ATG
translational start site and the putative TATA box are in
bold. The adenine residue of the first ATG is numbered +1
and is located 25 bp upstream to the previously published translation
initiation site (accession number X84014). The cis-acting elements
containing the consensus sequences for the AP-1, AP-2, and Sp1
transcription factors, the acute phase reactant signal
(APS), and NF-IL-6 are underlined.
[View Larger Version of this Image (66K GIF file)]
3A has a functional
promoter activity, we inserted different portions of fragment PA into
the pGalbasic vector. The resulting constructs were transfected into
PAM 212 mouse keratinocytes (Fig. 6).
Construct DSB, which contains a 904-bp DNA sequence upstream to the
transcription initiation site, yielded a
-galactosidase activity
comparable to that of the construct pGalA. Further deletion within the
5
end of the PA sequences (plasmid DEA) drastically affected the
promoter activity. However, as the PA sequences were further deleted,
-galactosidase activity was restored since the DSM construct exerted
an activity comparable to that of the intact PA DNA fragment. A
deletion to position
160 (DSAP construct) abolished the promoter
activity. These results suggest the presence of positive acting cis
elements in the region between
961 and
515 bp and implies the
existence of a negative acting cis element located between
515 and
358 bp. To test the cell type specificity of the promoter activity of
the fragment PA, the different constructs were also transfected into
NIH-3T3 fibroblasts, which do not produce laminin-5. Although the
expression levels were lower than those observed in experiments
with PAM 212 cells, significant promoter activity and similar
modulation of expression was observed in these fibroblasts. It should
be noted, however, that the most deleted construct DSAP showed higher activity in NIH-3T3 cells than in PAM 212 keratinocytes.
Fig. 6.
Promoter activity of the PADNA
fragment. Left, schematic representation of the reporter
gene constructs DSB, DEA, DSM, and DSAP obtained by deletions of the 5
region of the PA fragment. Right,
-galactosidase activity
detected upon transfection of the constructs in PAM 212 (open
boxes) and NIH-3T3 (filled boxes) cells. The values are
the means of triplicates from three independent experiments. All values
are expressed as a percent of the activity of the construct pGalA
(100%) in PAM 212 keratinocytes after subtraction of the background
activity observed with pGalbasic vector.
[View Larger Version of this Image (13K GIF file)]
3B open reading
frame of lama3 gene exert a promoter activity, the 2.7-kb EcoRV/EagI DNA fragment was cloned into the
vector pGL2-basic to obtain the construct pLucB. After transfection
into the PAM 212 keratinocytes and NIH-3T3 fibroblasts, pLucB showed
significant promoter activity in both cell lines (Fig.
7). Deletions of pLucB fragment were also
examined. The constructs KE and AE show a slight increase in promoter
activity in PAM 212 keratinocytes, indicating the possible removal of
upstream inhibitory sequences. KE demonstrated a reduced activity in
NIH-3T3 cells compared with pLucB and AE, which may confirm the
presence of cis-acting regulatory motifs within portion
2.6 kb and
1.6 kb. The construct KA, which lacks the transcription initiation
site and the proximal 5
-flanking DNA sequence of
3B, had no
promoter activity.
Fig. 7.
Promoter activity of the PB DNA
fragment. Left, schematic representation of the reporter
gene constructs pLucB, KE, AE, and KA obtained by deletions of the 5
region of the PB fragment. Right, luciferase activity
observed in PAM 212 (open boxes) and NIH-3T3 (filled
boxes) cells transfected with the different reporter genes. The
values are the means of triplicates from three independent experiments.
All values are expressed as a percent of the activity of the construct
pLucB (100%) in PAM 212 keratinocytes after subtraction of the
background activity observed with pGL2-basic vector.
[View Larger Version of this Image (11K GIF file)]
to the DNA sequences encoding for the domains
specific to the
3A and
3B laminin chains. Our results demonstrate
that the two laminin
3 isoforms are both encoded by the
lama3 gene and that their transcription is regulated by distinct promoters. The promoter PA lies within the 1-kb DNA segment located upstream to the 198-bp exon specific to the
3A isoform. This
DNA sequence contains a TATA box 27 bp upstream to the transcription initiation site of the
3A mRNA (11) and several consensus
binding sites for transcription factors. The promoter activity of this DNA fragment was demonstrated by cloning in a reporter gene and transfecting into cultured keratinocytes and fibroblasts. The promoter
PB localizes at the 5
end of the lama3 gene, within a
2.7-kb DNA fragment flanking in 5
the coding region of the
3B DNA
sequences. This DNA fragment does not contain any TATA box proximal to
the transcription initiation site of the
3B mRNA. However, when
inserted in a reporter gene, it induces luciferase activity both in
keratinocyte and fibroblast cell lines. Several genes that are
transcribed from alternative promoters have been described. The
corresponding transcripts differ in their 5
-untranslated regions but
encode identical polypeptides. In few cases, the use of alternative
promoters results in protein products harboring different sequence
domains with tissue-specific distributions (31). For instance, the
1(IX) and
1(XVIII) collagen genes both contain two distinct
promoters that regulate a tissue-specific expression of polypeptide
variants harboring different amino-terminal domains (32, 33). Thus far,
the lama3 gene is unique among the laminin genes because the
presence of multiple promoters has not been described in this family of
genes. This may reflect an important distinct role for the laminin
3A and
3B isoforms.
3
1 with laminin-5 in the
epidermal basement membrane (37). IL-6, which is the major systemic
alarm signal produced by almost all injured tissues, is produced by T
lymphocytes which are recruited during the inflammation process. Thus,
the presence of NF-IL-6 binding sites on the
3A and
3B promoters
suggests a regulation of the lama3 gene by the IL-6 cytokine
during wound healing.
3
1, the molecules being deposited in the leading edge of the migratory tongue
of wound epithelium (9, 38). Moreover, an increase in laminin-5
mRNA levels is observed at low cell densities in vitro,
in migrating and proliferating keratinocytes, as occurs at the wound
edge.
*
This work was supported by grants from the Association pour
la Recherche sur le Cancer, Ligue du Var contre le cancer,
INSERM-Caisse Nationale d'Assurance Maladie de Travailleurs
Salarie's, Fondation pour la Recherche Médicale (France),
Ministére de l' Education Nationale de l' Enseiguement
Supérieur et de la Récherche Actions Concertées
Coordoneés-Sciences du Viraut, E. E. C. Biomed 2, and Association
Française contre les Myopathies.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: INSERM U385
U. F. R. 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: JEB, junctional
epidermolysis bullosa; kb, kilobase(s); bp, base pair(s); IL,
interleukin; PIPES, 1,4-piperazinediethanesulfonic acid.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.