*Molecular and Cellular Nephrology, Institute of Medical Sciences and Department of Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan; Division of Nephrology and Endocrinology University of Tokyo School of Medicine, Tokyo, Japan; and Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan.
Correspondence to Dr. Toshio Miyata, Molecular and Cellular Nephrology, Institute of Medical Sciences, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan. Phone: 81-463-93-1121 ext. 2713; Fax: 81-463-93-1938; E-mail: t-miyata{at}is.icc.u-tokai.ac.jp
ABSTRACT. The cDNA of a new human mesangium-predominant gene,megsin, a novel member of the serpin superfamily, has recentlybeen cloned. This study investigates the regulatory mechanismsof megsin gene expression. A genomic clone of the human megsingene was obtained by screening bacterial artificial chromosome(BAC) library with the megsin cDNA. The analysis for exon-intronjunctions of megsin genomic DNA demonstrated that the gene contained8 exons and 7 introns, spanned 20 kbp, and that the genomicstructure of the serpin superfamily was highly conserved. Fluorescencein situ hybridization (FISH) revealed that the megsin gene islocalized in chromosome 18q21.3, close to the other serpin genes.The transcriptional start site, located by primer extensionanalysis, was 391 bp upstream from the start codon. The sequenceand reporter analyses on 4021-bp-long 5'-flanking region ofmegsin gene demonstrated a consensus promoter segment withinthis region and a relatively strong promoter activity in humanmesangial cells and A431, a human tumor cell line recently reportedto express a novel serpin identical with megsin. Moreover, thisstudy utilized site-directed and deletion mutagenesis analyses,and electrophoretic mobility shift assay identified one positiveregulatory motif, an incomplete activator protein-1 (AP-1) bindingmotif (CTGATTCAC) within the -120 to -112 region. This cis-actingelement in the 5'-flanking region of megsin is involved in theactivation of the megsin gene in mesangial cells.
Mesangial cells play an important role in maintaining the structureand function of the glomerulus and are thus involved in thepathogenesis of glomerular diseases (1,2). It is therefore importantto identify and characterize the genes that are expressed predominantlyby mesangial cells. Rapid large-scale DNA sequencing and computerizeddata processing allowed us to recently identify a new humanmesangium predominant gene, megsin (3). This methodology hasalso been successful in the identification of tubules-specificgenes (4,5). The full-length cDNA clone of megsin as well asits novel coded 380 amino acid protein exhibited a sequencesimilarity with those of the serine protease inhibitor (serpin)superfamily. This family includes plasminogen activator inhibitortype 2 (PAI-2), the squamous cell carcinoma antigen (SCCA),monocyte/neutrophil elastase inhibitor, and maspin. It has beenstrongly conserved over the last 500 million years (6). Expressionof megsin has also been shown to be upregulated in a varietyof renal disorders with mesangial cell injury in humans (7,8)and in a mesangioproliferative glomerulonephritis model in rats(9). Of interest, overexpression of megsin led to progressivemesangial matrix expansion and an increase in the number ofmesangial cells (10).
Several other genes, coding mainly for ion channels or transporters,are also expressed exclusively in renal tubular cells (11–16).The mechanisms responsible for their tissue specificity remainunknown at this stage. Elucidation of the transcriptional regulationof megsin should help us understand its tissue specificity andprovide important insights into the mechanisms of cell type-dependentgene expression.
In the present study, we further characterize the structure,organization, and sequence of the mesangium dominant gene, megsin.We demonstrated that it is located in chromosome 18q21.3, closeto other serpin genes. Finally, we identify, within the promoterregion, a positive regulatory element involved in the activationof megsin gene transcription.
Isolation and Characterization of Human Megsin Gene
To isolate genomic DNA containing human megsin gene, bacterialartificial chromosome (BAC) library was screened with fragmentsof 5'- and 3'-untranslated regions of megsin. One positive clone(F581) was partially digested with BamHI, subcloned into theBamHI site of the pBluescript SK(-) (Stratagene, La Jolla, CA)for DNA sequencing analysis of 5'-flanking region of human megsin.
To analyze exon-intron junctions of megsin gene, the F581 cloneand Genome Walker human genomic libraries (Clontech, Palo Alto,CA) were utilized.
Cell Cultures
Primary cultured human mesangial cells were purchased from Clontech.Cells were grown in Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 10% fetal bovine serum, 100IU/ml penicillin, 100 µg/ml streptomycin, and 200 µg/mlL-glutamine. A431 cells, human tumor cell line derived formepidermoid carcinoma, were purchased from Dai-Nihon PharmacologicalCo. (Osaka, Japan). Primary cultured human dermal fibroblastsand umbilical vein endothelial cells (HUVEC) were purchasedfrom Kurabo Co. (Tokyo, Japan). Human cell lines, includingFL (from amnion), HeLa (from adenocarcinoma), and Chang liver(from liver) cells, were also purchased from ATCC (AmericanType Culture collection, Manassas, VA).
Fluorescence In Situ Hybridization (FISH)
DNA from bac BAC clone F581 encoding human megsin gene was labeledwith digoxigenin dUTP by nick translation. Labeled probe wascombined with sheared human DNA and hybridized to normal metaphasechromosomes derived from phytohemagglutinin stimulated peripheralblood lymphocytes in a solution containing 50% formamide, 10%dextran sulfate, and 2 x SSC. Specific hybridization signalswere detected by incubating the hybridized slides in fluoresceinatedanti-digoxigenin antibodies followed by counterstaining withDAPI for one-color experiments. Probe detection for two colorexperiments was accomplished by incubating the slides in fluoresceinatedanti-digoxigenin antibodies and Texas red avidin followed bycounterstaining with DAPI.
Primer Extension Analysis
Ten micrograms of total RNA from cultured human mesangial cellwere then mixed with 2.5 pmol of 5' 32P-labeled antisense primercorresponding to nucleotide +67 to +86 in the 5' untranslatedregion of megsin (5'-AGG CTG TCC AAA GGT GCA GC-3'), denaturedby heating at 65°C for 30 min, then annealed at room temperaturefor 90 min. Primer extension proceeded at 37°C for 60 minwith 200 U/µl of SuperScript II RNase H- reverse transcriptase(Life Technologies BRL, Rockville, MD) in a buffer (50 mM Tris-HCl[pH 8.3], 75 mM KCl, 3mM MgCl2,10 mM DTT, 0.25 mM dNTPs). Afterethanol precipitation, the products were analyzed on 8% acrylamide-7M urea sequence gel. The subcloned 5' flanking region was sequencedusing the same primer by the Tth polymerase Sequencing PRO DNASequencing Kit (TOYOBO, Kyoto, Japan) and electrophoresed onthe same gel.
Construction of Reporter Plasmids
To assess human megsin gene promoter activity, enzyme restrictionor PCR-amplified fragments of megsin gene were subcloned intopGL3-Basic vector (Promega, Madison, WI). Enzyme restrictionfragments corresponding to nucleotides -4021, -2542, -1874,-1451, and -1052 to +130 in megsin transcript were generatedby digestion of pBluescript SK(-) contained megsin gene fromBAC clone, F581, with both BamHI, StuI, SacI, PstI, or KpnIand XbaI. A series of deletion mutants corresponding to nucleotides-1052 to +130 in megsin were prepared by PCR with a pair ofprimers spanning a region to be studied. These mutants contained5'-flanking region of megsin from -834, -240, or -72 to +130.For the deleted fragment, which was deleted between -139 bpand -85 bp, the restriction fragments corresponding to nucleotides-240 to +130 in megsin was digested with EcoRV and HpaI andwas then self-ligated.
For the site-directed mutations of AP-1 (from CTGATTCAC to CAGAATCTC)or Oct-1 (from CTGATTCACATACA to CTGATTCACGCACA) binding motifsin megsin gene, site-directed mutagenesis of the potential transcriptionalregulatory domain was performed utilizing Quick Change site-directedmutagenesis kit (Stratagene). The deletion mutant of AP-1 bindingmotif was constructed by PCR-based technique. The mutationswere verified by a direct dideoxy nucleotide sequencing.
Transfection and Luciferase Assay
Various kinds of human cells were transfected with 1 µgof megsin promoter-inserted pGL3-Basic vector containing fireflyluciferase gene using LipofectAMINE PULS (Life Technologies-BRL)according to the manufacturer’s protocol. Transfectionefficiencies were normalized by co-transfection with 5 ng ofpRL CMV vector containing Renilla luciferase gene (Promega).At the completion of culture incubations, cell media were aspiratedand the cells washed once in phosphate-buffered saline PBS thenlysed in cell lysis buffer. The cell lysates were centrifuged,and the supernatant was analyzed for the promoter activitiesusing the dual-luciferase reporter assay system (Promega) anda Lumat LB 9507 luminometer (EG & G Berthold, Bad Wildbad,Germany).
In some experiments, the transfected cells were subsequentlytreated with PDTC (pyrrolidine dithiocarbamate; Sigma, DorsetUK) for 24 h and then analyzed for the promoter activity asdescribed above.
Electrophoretic Mobility Shift Assays (EMSA)
Nuclear extract from cultured human mesangial cells was obtainedas described by Dignam et al. (17). Binding reactions were carriedout on ice for 15 min in a mixture containing 10% glycerol,5 mM MgCl2, 1 mM EDTA, 25 mM dithiothreitol, 50 mM KCl, 10 mMHepes-KOH (pH 7.8), 2 µg of poly dI-dC, and 10 µgof nuclear extract, followed by further reaction with 80,000cpm of 32P-end-labeled oligonucleotide probe at room temperaturefor 20 min. The complexes were subjected to electrophoresisin 4.5% polyacrylamide gels in running buffer containing 50mM Tris-HCl, pH 8.5, 0.4 M glycine, and 2 mM EDTA. In this study,we used DNA fragment containing incomplete AP-1 binding motifof megsin (-127 bp to -108 bp; see Figure 5A) and AP-1 consensusoligonucleotide (Promega) as the probe.
Figure 5. Transcriptional activity of deletion mutants of megsin promoter. A431 (A) or human mesangial cells (B) were transfected with pGL3-Basic firefly luciferase vector containing various-length 5'-flanking region of megsin gene. Transfection efficiencies were normalized by co-transfection with pRL CMV Renilla luciferase vector. Representative data from the three experiments are shown. Each experiment was performed in quadruplicate. Data are shown as the mean ± SD of relative luciferase activity.
For competition studies, unlabeled competitor oligonucleotidewas added by 100-fold molar excess to the reaction mixture.For super shift assay, anti-c-Fos antibody (Santa Cruz Biotechnology,Santa Cruz, CA) was used.
Isolation and Characterization of the HumanMegsin Gene
We have previously characterized a cDNA encoding human megsin(3). The 5'-flanking region and the exon-intron junctions ofthe human megsin gene were obtained by a PCR-based method withprimers designed from megsin cDNA sequences.
The structure and organization of the human megsin gene areshown in Figure 1. The megsin gene contains 8 exons and 7 intronsand spans approximately 20 kbp. The exons range in size from51 bp to 1141 bp, and exon-intron boundaries follow the GT/AGrule. Comparison of nucleotide sequences flanking exon-intronboundaries of the human megsin gene with those of another memberof the serpin superfamily, PAI-2 (18), show that these sequencesare well conserved, indicating a close phylogenetic relationship(Figure 1).
Figure 1. Structure and organization of the human megsin gene.
The 4021-bp-long 5'-flanking region of the human megsin genehas also been determined (GeneBank accession No. AF234618).Primer extension analysis located the transcriptional startsite at -35bp downstream of a consensus TATA and at 391 bp upstreamof the translational start site (Figure 2). We demonstrate thatthe first intron is approximately 6.5 kbp long and is locatedbetween the transcriptional and translational start sites (Figure 1).
Figure 2. Identification of the transcription start site of the human megsin gene. A primer extension analysis was performed with 10 µg of total RNA isolated from human cultured mesangial cells. The transcription start site (+1) and TATA box (-35) are indicated by arrows.
Chromosomal Localization of Human Megsin Gene
We performed fluorescence in situ hybridization utilizing asprobe a megsin genomic DNA fragment, obtained from BAC cloneF581 (Fig.ure3). The initial experiment resulted in the specificlabeling of the long arm of a group E chromosome believed tobe chromosome18 on the basis of size, morphology, and bandingpattern. In a second experiment, a biotin-labeled probe specificfor the centromere of chromosome 18 was cohybridized with cloneF581. This experiment resulted in the specific labeling of thecentromere (red) and the long arm (green) of chromosome 18.Measurements of ten specifically labeled chromosomes 18 demonstratedthat F581 is located at 68% of the distance from the centromereto the telomere of chromosome arm 18q, an area that correspondsto band 18q21.3. Out of total of 80 examined metaphase, 76 exhibitedthe specific labeling.
Figure 3. Localization of the human megsin gene to chromosome 18q21.3. The green dots indicated by arrows show the hybridization signals of the megsin gene probe to a metaphase chromosome spread. The red bands show binding of a specific probe for the centromere of chromosome 18.
Functional Analysis of Megsin Promoter
The promoter activity of the isolated 5'-flanking region ofhuman megsin was further investigated. We transfected the fireflyluciferase reporter vector (pGL3-Basic) containing the 4151-bp-long5'-flanking region (-4021bp to +130 bp), by lipofection intoprimary cultured cells, including human mesangial cells, dermalfibroblasts, and HUVEC. After normalization for Renilla luciferaseactivity, the firefly luciferase activity was statistically(mesangial cells versus dermal fibroblasts, P = 0.003; mesangialcells versus HUVEC, P = 0.006) higher in cultured human mesangialcells than in other primary cultured cells (Figure 4A).
Figure 4. Megsin promoter activity in human primary cultured cells (A) and malignant cell lines (B). Various kinds of human cells were transfected with pGL3-Basic firefly luciferase vector containing the 4151-bp-long 5'-flanking region of megsin gene (-4021 bp to +130 bp). Transfection efficiencies were normalized by co-transfection with pRL CMV Renilla luciferase vector. Representative data from the three experiments are shown. Each experiment was performed in quadruplicate. Data are shown as the mean ± SD of relative luciferase activity. HMC, human mesangial cells; HDF, human dermal fibroblasts; HUVEC, human umbilical vein endothelial cells.
Another group (19) has recently identified in a malignant A431cell line a new serpin with megacaryocyte maturating activity.The amino acid sequence of the protein is identical with thatof megsin. We thus transfected the firefly luciferase gene intothe A431 cell line as well as in other human cell lines (FL,HeLa, Chang liver cells). Of interest, the firefly luciferaseactivity was also statistically increased (A431 versus FL, P= 0.001; A431 versus HeLa or Chang liver cells, P = 0.007) inthe A431 than in the others cells (Figure 4B). Altogether thesedata shows that the megsin promoter region is mainly activein cells expressing megsin.
To identify the regulatory element required for megsin expression,we generated various lengths of the megsin 5'-flanking regionand subcloned them in a pGL3-basic vector. The slow growth ofhuman cultured mesangial cells and their low yield in termsof the number of available cells led us to utilize A431 cells.The results of these experiments were subsequently confirmedin a limited number of human glomerular cell assays. As shownin Figure 5, the deletion from -4021 bp to -240 bp did not decreasethe activity, whereas the deletion to -72 bp decreased the activityto approximately 5% of the initial promoter activity. The similarlevel of decrease in the promoter activity was observed in thedeletion mutant, which was deleted between -139 bp and -85 bp(Figure 6B).
Figure 6. Identification of the cis-acting element on megsin transcription. Comparison of the nucleotide sequences between the consensus activator protein-1 (AP-1) binding motif and the -120 to -112 region of megsin gene (A). We mutated the incomplete AP-1 and Oct-1 binding motifs in megsin gene and assessed its promoter activity in A431 cells (B) and human mesangial cells (C). pGL3-Basic, luciferase vector alone; -240bp-Basic, pGL3-Basic containing the 370-bp-long 5'-flanking region of megsin gene (-240 to +130 bp); -72bp-Basic, pGL3-Basic containing the 202-bp-long 5'-flanking region of megsin gene (-72 to +130 bp); -139 to -85 (Del)-Basic, -240bp-Basic with the deletion from -139 bp to -85 bp; AP-1(Mut)-Basic, --40bp-Basic with the site-directed mutation of AP-1; AP-1(Del)-Basic, -240bp-Basic with the deleted mutation of AP-1; Oct-1(Mut)-Basic, -240bp-Basic with the site-directed mutation of Oct-1.
Regulatory Element for Megsin Expression
The motif search with the -139 to -85 region of human megsinrevealed several potential transcription regulatory motifs,including AP-1 (Figure 6A), Oct-1, and TCF11 binding motifs.To identify the regulatory element within this region for transcriptionalactivation of megsin, we mutated the highly conserved transcriptionfactor binding motifs, AP-1 and Oct-1, and assessed its promoteractivity (see Materials and Methods).
When A431 cells were transfected with pGL-Basic vector containing-240 to +130 region, both site-directed and deleted mutationsfor AP-1 led to the decrease in the transcriptional activationto approximately 50% of the initial promoter activity (Figure 6B).In contrast, Oct-1 mutation did not change the activity.These observations were confirmed in cultured human mesangialcells (Figure 6C).
Specific DNA-protein complexes were identified by oligonucleotidecorresponding to the incomplete AP-1 site (Figure 7). A competitiveinhibition assay relying on the massive addition of unlabeledAP-1 consensus oligonucleotide blocked the binding of the labeledprobe. In EMSA using antibody to AP-1 binding transcriptionalfactor, c-fos, the specific DNA-protein complexes further shifted,demonstrating the binding of incomplete AP-1 site with AP-1transcriptional factor.
Figure 7. Transcriptional activity of an incomplete AP-1 binding motif in megsin promoter. (A) Electrophoretic mobility shift assays (EMSA) was performed with nuclear extracts from human mesangial cells. Specific DNA-protein complexes (indicated by an arrow) were identified by the incomplete AP-1 site oligonucleotide of megsin promoter. The specific band was competed by unlabeled AP-1 consensus oligonucleotide and further shifted (indicated by an arrowhead) by antibody to AP-1 binding transcriptional factor, c-Fos. (B) AP-1 activator, PDTC, upregulates the transcriptional activity of megsin promoter. Human mesangial cells were transfected with megsin promoter (-240 bp to +130 bp) containing an incomplete AP-1 site, treated with various concentrations of PDTC for 24 h, and then assessed for its promoter activity by luciferase assay.
Treatment with antioxidant PDTC, which dominantly activatesAP-1, was performed to assess whether or not the AP-1 activatorupregulates the activity of megsin promoter (-240 bp to +130bp) containing the incomplete AP-1 site. Our luciferase assaysshowed that PDTC indeed upregulated the promoter activity incultured human mesangial cells in a dose-dependent manner (Figure 7B).
In the present study, we report the structure, organization,and sequence of the megsin gene. The megsin gene shows a similaritywith other human chromosome 18 serpins (18) regarding the numberof exon/intron and the positioning of exon-intron boundaries.The 18q21.3 locus has been previously shown to contain the genesencoding other serpins such as PAI-2, maspin, squamous cellcarcinoma antigen 1 (SCCA1), and SCCA2 (20). We speculated thatthe megsin gene is located in the 18q21.3 locus because themegsin gene has a genetic organization similar to that of theother sepin genes and that the latter genes clusters in thisregion. The localization of the human megsin gene on chromosome18q21.3 was indeed confirmed by our in situ hybridization experiments.
Megsin is expressed almost exclusively in mesangial cells. Still,two novel serpins with an identical cDNA/protein have also beenidentified either in malignant A431 cell lines (19) or in dendriticcells present in the human thymus (21). Still, megsin expressionin two cell types tested in our laboratory is significantlylower than in mesangial cells (data not shown). The presenceof megsin in A431 may reflect cancerous derepression. A431 cellsprovide us with alternative tools to study the regulatory mechanismsof megsin gene expression.
We further determined the sequence of the promoter region anddiscovered a significant positive regulatory element. In the5'-flanking region of megsin gene (-4021 bp to +130 bp), wedemonstrated the existence of a cis-acting element that activatesthe transcription of megsin gene in both A431 and mesangialcells. The use of the luciferase assay allowed us to demonstratethat the reporter construct with the 5'-flanking region of megsingene is able to stimulate luciferase gene expression in bothcell types. Although the promoter activity of megsin gene wasstronger in A431 and mesangial cells, the reporter constructwith the 4151-bp-long 5'-flanking region also induced a milderluciferase expression in other cell types. This observationsuggests that the cell type-specific transcription regulationis not located solely in this promoter region.
Sequence analysis revealed various potential transcription factorbinding sites, including an incomplete AP1 binding site (22,23),a c-Myb binding site (24,25), an Oct-1 binding site (26), andan NF-B binding motif (27,28) within the 5'-flanking regionof megsin gene. A detailed analysis of the promoter region revealeda cis-acting element within the -240 to -72 region, which isable to activate the transcription of megsin; the element hashighly conserved transcription factor binding motifs such asAP-1, Oct-1, and TCF11. Our site-directed and deletion mutagenesisanalyses and EMSA identified one positive regulatory motif,an incomplete AP-1 binding motif (CTGATTCAC) within -120 to-112 region. PDTC, a dominant activator of AP-1, activates themegsin promoter (-240 bp to +130 bp), suggesting that AP-1 isa good candidate as a transcription factor of megsin among themolecules described above. In spite of these results, the 5'-flankingregion of megsin gene tested in this study was not stimulatedby the pathogenic mediators, e.g., TGF- and PDGF. This observationindicates that the cell type-specific transcription of megsinis not regulated solely by this promoter region. Extensive investigationof more upstream of megsin promoter region as well as intronsshould be necessary for understanding of the inducible transcriptionregulation of megsin.
AP-1 is known to regulate the gene expression of various cytokines,chemotactic proteins, adhesion molecules, matrix proteins, andproteases involved in inflammation, immunologic responses, celldifferentiation, and growth control. AP-1 DNA-binding activitiesin mesangial cells are regulated by the microenvironment (29),and Hernandez-Presa et al. (30) showed activated AP-1 in mesangialcells of immune complex glomerulonephritis rats utilizing Southwesternhistochemical methods. Previous studies demonstrated that c-Jun/AP-1activation is essential to the induction of matrix metalloproteinase-9(MMP-9) by IL-1 or thrombin in cultured mesangial cells (31–33).We reported that megsin is upregulated in mesangioproliferativediseases in both humans and rats (3,7–9) and that overexpressionof megsin induces progressive mesangial cell proliferation andexpansion (10). These findings together with our results suggestthat AP-1 may regulate matrix turnover by stimulating proteinasesand protease inhibitors such as megsin in parallel.
Interestingly, the megsin promoter region includes one YB-1binding site at -1588 bp to -1599 bp (GGG ATT GGT TAA). Mertenset al. (34) demonstrated that YB-1 is a major, cell type-specifictransactivator of MMP-2 gene expression in glomerular mesangialcells. Still, our studies with deletion mutants of megsin promotervectors have shown that the YB-1 binding site is not importantfor megsin gene transcription. They suggest that the differentcell type-specific transcriptional regulation between megsinand MMP-2 relies on a different mechanism. Combinations of tissue-restrictedand widely expressed factors sometimes mediate tissue-specificexpression (35–38). However, the relatively low cell typespecificity of this promoter region makes the tissue-specificregulation of megsin by these ubiquitous transcription factorsunlikely. The cis-acting element demonstrated in this studymight act in coordination with some tissue-specific enhancersand/or silencers in other regions such as introns.
In conclusion, we provide a detailed analysis of the genomicstructure and the transcriptional regulation of a new humanmesangium-predominant gene, megsin. The megsin gene is composedof eight exons and is localized to human chromosome 18q21.3,close to the other serpin genes. We have isolated the promoterregion of human megsin gene and identified a positive cis-regulatoryelement. Our results demonstrate that the promoter region of-120 to -112 containing an incomplete AP-1 binding motif isimportant in the transcriptional activation of megsin. The actualsequence of the cis-elements identified in the megsin promoterby this study is a first step toward the elucidation of themolecular mechanism of mesangium-predominant gene regulation.Further studies such as cloning of a responsible transcriptionalfactor are required to completely understand the mesangium-predominantgene regulation of megsin.
Acknowledgments
We thank Dr. C. van Yepersele de Strihou (Universite Catholiquéde Louvain, Belgium) and Dr. M. Kitamura (Jikei University,Japan) for their helpful discussions. This study was supportedby grants from the Ministry of Education, Culture, Sports, Scienceand Technology of Japan to RI and KK (13671129 and 13307032),from Research on Human Genome and Tissue Engineering of theJapanese Ministry of Health Labor and Welfare to TM (H12-genome-22),from the Tokyo Biochemical Research Foundation to RI, from SankyoFoundation of Life Science to RI, and from Suzuken MemorialFoundation to RI.
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Received for publication March 11, 2002.
Accepted for publication July 25, 2002.
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Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan.
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Abstract
Introduction
Tth polymerase Sequencing PRO DNA Sequencing Kit (TOYOBO, Kyoto, Japan) and electrophoresed on the same gel. 
B binding motif (27,28) within the 5'-flanking region of megsin gene. A detailed analysis of the promoter region revealed a cis-acting element within the -240 to -72 region, which is able to activate the transcription of megsin; the element has highly conserved transcription factor binding motifs such as AP-1, Oct-1, and TCF11. Our site-directed and deletion mutagenesis analyses and EMSA identified one positive regulatory motif, an incomplete AP-1 binding motif (CTGATTCAC) within -120 to -112 region. PDTC, a dominant activator of AP-1, activates the megsin promoter (-240 bp to +130 bp), suggesting that AP-1 is a good candidate as a transcription factor of megsin among the molecules described above. In spite of these results, the 5'-flanking region of megsin gene tested in this study was not stimulated by the pathogenic mediators, e.g., TGF-
and PDGF. This observation indicates that the cell type-specific transcription of megsin is not regulated solely by this promoter region. Extensive investigation of more upstream of megsin promoter region as well as introns should be necessary for understanding of the inducible transcription regulation of megsin. 
