Murine Laminin alpha 3A and alpha 3B Isoform Chains Are Generated by Usage of Two Promoters and Alternative Splicing*

(Received for publication, November 15, 1996, and in revised form, April 3, 1997)

Olivier Ferrigno Dagger , Thierry Virolle Dagger , Marie-Florence Galliano Dagger , Nathalie Chauvin Dagger , Jean-Paul Ortonne Dagger §, Guerrino Meneguzzi Dagger and Daniel Aberdam Dagger

From Dagger  U385 INSERM, Faculté de Médecine, 06107 Nice Cedex 2 and § Service de Dermatologie, Hôpital L'Archet, 06002 Nice Cedex 1, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

We already identified two distinct laminin alpha 3A and alpha 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 alpha 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 alpha 3A and the alpha 3B cDNAs. Sequence analysis of the region upstream to the alpha 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 alpha 3B mRNA isoform was defined. The sequences upstream to the alpha 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 alpha 3A and alpha 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 alpha 3A and alpha 3B.


INTRODUCTION

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 alpha , beta , and gamma  chain which are known to exist in at least 10 genetically distinct forms (3, 5, 6).

Laminin-5 (alpha 3beta 3gamma 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 alpha 3beta 1 in focal adhesions (14) and interacts with hemidesmosomes via alpha 6beta 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).

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 alpha 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 alpha 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).

Recently, we have isolated and characterized the cDNA encoding the alpha  chain of the mouse laminin-5, and we have identified two distinct isoforms alpha 3A and alpha 3B which differ in their amino-terminal domains (11). The alpha 3B polypeptide bears two globular domains and an epidermal growth factor-like domain that substitute the short amino-terminal domain of the alpha 3A isoform. In situ hybridization analysis on mouse embryos demonstrated a restricted tissue-specific distribution for the two isoforms. The alpha 3A isoform is widely distributed in squamous and transitional epithelia and more predominantly in the skin and hair follicles, whereas the alpha 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 alpha 3A and alpha 3B chain isoforms and their specific expression patterns raise questions on possible distinct physiological roles for the two polypeptides.

To gain insight into the regulation of the laminin alpha 3 chain isoforms, we have isolated and analyzed the noncoding regions of genomic DNA located upstream to the DNA sequences coding for the alpha 3A and alpha 3B polypeptides. We have shown that the laminin alpha 3A and alpha 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 alpha 3A and alpha 3B DNA sequences.


EXPERIMENTAL PROCEDURES

All recombinant DNA technology was performed according to standard procedures (27).

Materials

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 lambda GEM12. This library was kindly provided by J-M. Garnier (laboratory of P. Chambon, Strasbourg, France).

Genomic Library Screening

The genomic library was screened by plaque hybridization with the alpha -32P-labeled cDNA probes PR6 and MR10 encoding the 5' domains of laminin alpha 3A and alpha 3B chains, respectively (11). Two nonoverlapping phages, lambda A (15 kb) and lambda B (20 kb), were isolated and further analyzed.

Southern Analysis

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 lambda 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 alpha 3B polypeptide and oligonucleotide AR, 5'-GGTACCACCTCACTGCAGGC-3', is located within the 5' region of the cDNA sequences specific to the alpha 3A chain isoform. The lambda 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 alpha 3B mRNA.

Isolation of Genomic DNA Fragments 5' to alpha 3A and alpha 3B mRNAs

The DNA fragment located upstream to the 5' alpha 3A exon was amplified from the lambda 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 lambda 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.

Determination of the Transcription Initiation Site of alpha 3B by Primer Extension Analysis

One pmol of the antisense oligonucleotide BR was 5'-end-labeled using [gamma -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.

Analysis of Promoter Activities

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 pbeta Gal-promoter (CLONTECH). The values of beta -galactosidase expression data were normalized to the measured luciferase activity. Enzyme assays were performed using either the Promega luciferase assay system or the CLONTECH beta -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.


RESULTS

Isolation of Genomic Clones

Since the laminin alpha 3A and alpha 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 alpha 3A and alpha 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, lambda A and lambda B, were isolated and further characterized by Southern blot analysis.

The genomic clone lambda 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 alpha 3B and to those specific to the 5' end of alpha 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 alpha 3B-specific exon sequences (230 bp) and DNA sequences specific to the alpha 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 alpha 3A-specific DNA sequences to 38 bp that constitute the 5' terminus of the DNA sequences common to both the alpha 3A and alpha 3B isoforms.


Fig. 1. A, Southern blot analysis of the 15-kb genomic clone lambda A. DNA from clone lambda 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 lambda 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 alpha 3A (PA). H, HindIII; S, SmaI.
[View Larger Version of this Image (10K GIF file)]

From these results we conclude that the genomic clone lambda A contains a DNA fragment of the lama3 gene encoding domains of laminin chains alpha 3A and alpha 3B (Fig. 1A and see also Fig. 4B). The exons specific to the alpha 3B polypeptide are located 5' to the unique exon encoding the alpha 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 lambda B. DNA from clone lambda 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 alpha 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 alpha 3B coding region, and that of the genomic fragment PA, which corresponds to the noncoding DNA sequences 5' to the alpha 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 lambda A and lambda B, are represented. R, EcoRV; E, EagI; and H, HindIII.
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Characterization of the 5'-Flanking Region of the alpha 3A Exon

To determine whether the alpha 3A and alpha 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 alpha 3A-specific exon, was polymerase chain reaction-amplified from lambda 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 alpha 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 alpha 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 alpha 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 alpha 3B-specific exon and the alpha 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.
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Characterization of the 5'-Flanking Region of the alpha 3B Exons

The 5' end of the alpha 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 lambda 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 alpha 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 alpha 3A and alpha 3B domains as well as the genomic lambda A and lambda 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 alpha 3B mRNA. The autoradiogram shows a primer extension on alpha 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.
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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.
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Promoter Activities

To assess whether the genomic DNA region upstream to the transcriptional start site of alpha 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 beta -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, beta -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, beta -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.
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To verify whether the DNA sequences upstream to the alpha 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 alpha 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.
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DISCUSSION

We have analyzed the organization of the distinct regions of the genomic DNA located 5' to the DNA sequences encoding for the domains specific to the alpha 3A and alpha 3B laminin chains. Our results demonstrate that the two laminin alpha 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 alpha 3A isoform. This DNA sequence contains a TATA box 27 bp upstream to the transcription initiation site of the alpha 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 alpha 3B DNA sequences. This DNA fragment does not contain any TATA box proximal to the transcription initiation site of the alpha 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 alpha 1(IX) and alpha 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 alpha 3A and alpha 3B isoforms.

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 alpha 3beta 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 alpha 3A and alpha 3B promoters suggests a regulation of the lama3 gene by the IL-6 cytokine during wound healing.

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 alpha 3beta 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.

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.


FOOTNOTES

*   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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y08738 and Y08850.


   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.

ACKNOWLEDGEMENTS

We thank Z. Djabari and G. Spennato for skilled technical assistance and C. Minghelli for artwork.


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