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Originally published In Press as doi:10.1074/jbc.M206796200 on October 3, 2002

J. Biol. Chem., Vol. 277, Issue 51, 49903-49910, December 20, 2002
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MafA Is a Glucose-regulated and Pancreatic beta -Cell-specific Transcriptional Activator for the Insulin Gene*

Kohsuke KataokaDagger §, Song-iee HanDagger , Setsuko ShiodaDagger , Momoki Hirai, Makoto Nishizawa||, and Hiroshi HandaDagger

From the Dagger  Frontier Collaborative Research Center, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan, the  Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Tokyo 277-8562, Japan, and the || Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037

Received for publication, July 8, 2002, and in revised form, October 2, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The insulin gene is specifically expressed in beta -cells of the Langerhans islets of the pancreas, and its transcription is regulated by the circulating glucose level. Previous reports have shown that an unidentified beta -cell-specific nuclear factor binds to a conserved cis-regulatory element called RIPE3b and is critical for its glucose-regulated expression. Based on the sequence similarity of the RIPE3b element and the consensus binding sequence of the Maf family of basic leucine zipper transcription factors, we here identified mammalian homologue of avian MafA/L-Maf, an eye-specific member of the Maf family, as the RIPE3b-binding transcriptional activator. Reverse transcription-PCR analysis showed that mafA mRNA is detected only in the eyes and in pancreatic beta -cells and not in alpha -cells. MafA protein as well as its mRNA is up-regulated by glucose, consistent with the glucose-regulated binding of MafA to the RIPE3b element in beta -cell nuclear extracts. In transient luciferase assays, we also showed that expression of MafA greatly enhanced insulin promoter activity and that a dominant-negative form of MafA inhibited it. Therefore, MafA is a beta -cell-specific and glucose-regulated transcriptional activator for insulin gene expression and thus may be involved in the function and development of beta -cells as well as in the pathogenesis of diabetes.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin is the only polypeptide hormone that critically regulates blood glucose levels and is produced exclusively by beta -cells of the islets of Langerhans in the pancreas. The molecular mechanism of the beta -cell-restricted expression of insulin has been extensively studied (for reviews, see Refs. 1 and 2), which led to the identification of various cis-regulatory elements on its promoter region. The most important among them are the conserved E1, A3, and RIPE3b/C1 elements (3-6). The islet-restricted transcription factors, Beta2/NeuroD and Pdx1/IPF1/STF1/IDX1/GSF/IUF1, which bind to the E1 and A3 elements, have also been isolated (7-10). Gene disruption experiments in mice have revealed that both Beta2 and Pdx1 play critical roles in insulin gene expression as well as in islet development and function (11-13). Furthermore, mutations in beta2 and pdx1 genes are found in some population of patients with type 2 diabetes (14-16).

The third regulatory element, RIPE3b, has also been shown to play a critical role in beta -cell-specific insulin gene transcription as well as in its glucose-regulated expression (4, 17). Previous studies have identified a beta -cell-restricted RIPE3b-binding factor, called the RIPE3b1 activator, that appears in response to glucose in pancreatic beta -cell nuclear extracts (18). However, despite growing evidence of its importance in the regulation of insulin gene transcription, it remains to be cloned.

We and others have previously determined the consensus DNA-binding sequences of the Maf family of transcription factors as the 13- and 14-base pair palindromic sequences TGCTGACTCAGCA and TGCTGACGTCAGCA, which we have called Maf recognition elements (MAREs)1 (19, 20). By searching the GenBankTM data base, we were previously able to make a list of putative Maf-regulated genes that have MARE-related sequences in their regulatory regions, including the insulin gene (19). Later, some of them, for example, erythroid lineage-specific genes (alpha - and beta -globin and porphobilinogen deaminase) and eye-specific genes (opsin and crystalline genes) were actually found to be downstream targets of Maf family members (see below).

Maf family proteins belong to the basic leucine zipper family of transcription factors, and the originally identified member, v-Maf, is an oncoprotein encoded by the avian transforming retrovirus AS42 (21). To date, several maf-related genes have been identified in various species including human, mouse, rat, chicken, quail, frog, and zebrafish. The Maf family members are divided into two groups depending on their molecular sizes (i.e. the large Maf and small Maf proteins).

In both mammalian and chicken genomes, three small maf genes, mafK, mafF, and mafG, have been identified (22, 23). Their products lack a transactivator domain, but they activate transcription when they form heterodimers with members of CNC ("cap'n'collar"), another basic leucine zipper family (24). For example, by forming heterodimers with the erythroid-specific CNC member p45, they constitute the erythroid-specific transactivator NF-E2 and activate transcription of the alpha - and beta -globin and porphobilinogen deaminase genes (25, 26).

In contrast to the small Mafs, the large Maf proteins contain a transactivator domain in their amino terminus and activate transcription as homodimers (27, 28). In chicken and quail, the c-maf, mafB, and mafA/L-maf genes have been identified as large maf members (28-31). In mammals, c-maf, mafB, and nrl have been identified (32-34), but a homologue of mafA/L-maf has not yet been cloned. Nrl shows retina-specific expression and regulates opsin gene expression and retinal development (33, 35, 36); its mutation causes retinal degeneration in humans and mice (37, 38). Chicken MafA/L-Maf, on the other hand, is exclusively expressed in lens and plays a critical role in the regulation of crystalline genes and lens development (29).

As we have mentioned previously, insulin genes of various species, such as human, mouse, and rat, contain conserved MARE-related sequence in their promoters (19), and we have noticed that the MARE sequence overlaps with that of the RIPE3b element. In this report, we show that the RIPE3b1 factor contains a previously unidentified mammalian member of the large Maf family. Through GenBankTM data base searches and PCR amplification, we have isolated human and mouse homologues of the mafA gene and demonstrated that MafA is the RIPE3b1 factor.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Preparation of Nuclear Extracts-- MIN6 cells (39) were a generous gift from Dr. Jun-ichi Miyazaki (Osaka University). beta TC6 and alpha TC1 clone 9 (hereafter alpha TC1) cells were purchased from American Tissue Culture Collection. MIN6 and beta TC6 cells were grown in Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum, and alpha TC1 cells were grown in F12K medium supplemented with 10% fetal calf serum. When controlling the glucose concentration, the medium was changed to Dulbecco's modified Eagle's medium containing various concentrations of glucose supplemented with 10% fetal calf serum. Nuclear extracts were prepared as described by Schreiber et al. (40).

Gel Mobility Shift Analysis-- For the gel mobility shift analysis, nuclear extracts were incubated in DNA-binding buffer (final concentration 10 mM HEPES (pH 7.9), 100 mM NaCl, 2 mM EDTA, 8 mM dithiothreitol, 5% glycerol) with 1 µg of poly(dI-dC)(dI-dC) on ice for 15 min. Then 0.5 ng of 32P-labeled probe was added, and the extracts were further incubated at room temperature for 15 min. The reaction mixture was subjected to electrophoresis in a 0.5× Tris borate-EDTA buffer plus 6% polyacrylamide gel at 4 °C. The oligonucleotide probe containing the human RIPE3b element was made by annealing two 32-mer oligonucleotides, 5'-GATCCGGAAATTGCAGCCTCAGCCCCCAGCCA-3' and 3'-GATCTGGCTGGGGGCTGAGGCTGCAATTTCCG-3'.

The core sequences of the mutated 32-mer RIPE3b-oligonucleotides used as competitors are shown in Fig. 2B. The complete sequences of MARE-related oligonucleotides were described in Ref. 19.

Anti-v-Maf serum was previously raised against a nearly full-length recombinant chicken v-Maf protein (41). Anti-c-Maf (M-153), anti-c-Maf (N-15), anti-MafB (E-20), anti-Nrl (N-19), anti-MafK (C-16), anti-MafK/F/G (C-18), and anti-c-Fos (K-25) sera were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-c-Jun serum (42) was a generous gift from Dr. Kazuhiro Chida (Tokyo University). Anti-MafA-specific serum was obtained by immunizing rabbits with a keyhole limpet hemocyanin-conjugated synthetic peptide (REPSPAQAGPGAAKGC) corresponding to the carboxyl-terminal part of mouse MafA.

Cloning of Human and Mouse mafA-- By searching the GenBankTM data base, we found a human genome contig (NT 023684) on chromosome 8q24 and several human expressed sequence tags that were most homologous to avian mafA. From the nucleotide sequence information, we designed primers (5'-agaacggTCCCGGGCGATGGCCGCGGAG-3' and 5'-agaacggcGTCCGGCGCCTACAGGAAGAAG-3') and cloned the entire open reading frame of this gene by PCR. Human genomic DNA was used as a template because the chicken and quail mafA genes contain no introns. The same primers were used for cloning the mouse mafA homologue from mouse genomic DNA (Balb/c strain) by PCR. The PCR products were cloned into a pGEM-T-easy vector (Promega) to generate pGEM-T-easy/h-mafA and pGEM-T-easy/m-mafA.

Fluorescent in Situ Hybridization-- A 2-kb DNA fragment encompassing the human mafA locus was cloned by inverse PCR (43) from NcoI-digested and self-ligated HeLa genomic DNA (pT7blue/h-mafA-inv-NcoI) using nested primers (first PCR, 5'-GGTTCAGCTCGCGCACCGACATGG-3' and 5'-GTGCAGCAGCGGCACATTCTGGAG-3'; second PCR, 5'-TTCTGCTTGAGCCGGATGACCTCC-3' and 5'-AGTCCTGCCGCTTCAAGCGGGTGC-3') and was used as a probe to determine the chromosomal localization of the human mafA gene by fluorescent in situ hybridization as described previously (44).

RNA Analyses-- Total RNA was isolated from cultured cells and mouse tissues (from a 7-week-old male, ICR strain) using the TRIZOL Reagent (Invitrogen). Reverse transcription (RT)-PCR analysis was performed using the Titan One-Tube RT-PCT System (Roche Molecular Biochemicals). The primers used were as follows: mafA, 5'-CGCAGGCCACCACGTGCGCTTGGAGGAG-3' and 5'-CTGCGCTGGCGAGGGCTCCCGAGGGAAG-3'; insulin, 5'-CTTAGTGACCAGCTATAATCAGAGACC-3' and 5'-GGGCCTTAGTTGCAGTAGTTCTCCAGC-3'; glucagon, 5'-TCTACACCTGTTCGCAGCTTCAGTCCC-3' and 5'-CCACTACGGTTACCAGGTGGTCATGTC-3'.

Northern blot analysis was performed as described (21). The mafA-specific probe (pSae-hA) was constructed by deleting the BssHII-PmlI fragment from pGEM-T-easy/h-mafA to remove the GC-rich and CAC repeat region. To obtain the insulin probe, insulin cDNA was cloned from MIN6 total RNA by RT-PCR using the specific primers 5'-AGGAATTCAACATGGCCCTGTTGGTGC-3' and 5'-CCTGGTGTTTTATCACAAGCTTCATAC-3'.

Western Blotting-- Aliquots of nuclear extracts were separated by 10% SDS-polyacrylamide gel electrophoresis and were transferred onto an Immobilon membrane (Millipore Corp.). The membrane was then stained with anti-MafA (500× dilution) or anti-c-Maf (M-153; Santa Cruz Biotechnology) (2000× dilution) and with biotinylated anti-rabbit IgG serum and streptavidin-horseradish peroxidase conjugate (Amersham Biosciences).

Immunofluorescent Staining-- MIN6 cells grown on collagen-coated cover glass (IWAKI) were fixed with phosphate-buffered saline containing 3% formaldehyde at room temperature for 15 min and were treated with 0.25% Triton X-100 in phosphate-buffered saline for an additional 15 min. The cells were then stained with anti-c-Maf (M-153) serum (200× dilution), Alexa Fluor 488-labeled anti-rabbit IgG serum (Molecular Probes, Inc., Eugene, OR), and 4,6-diamidino-2-phenylindole.

Luciferase Assay-- To construct the reporter plasmid h-ins-p-luc, the human insulin promoter region was amplified by PCR using primers (5'-caggtaCCCCGCCCTGCAGCCTCCAGCTC-3' and 5'-agaagcTTCTGATGCAGCCTGTCCTGGA-3') from human genomic DNA. The fragment was inserted into the KpnI-HindIII site of the pGL2-basic plasmid (Promega) after digesting with KpnI and HindIII. Mutations were introduced into the RIPE3b/MARE element by site-directed overhang extension PCR mutagenesis (45) using the primers 5'-TGCAGCCgactaCCCCAGCCATCTGCC-3' and 5'-ATGGCTGGGGtagtcGGCTGCAATTTC-3'. pG4×5/TATA/luc plasmid that contains five copies of the Gal4-binding sequence was a gift from Dr. Kazuhiko Igarashi (Hiroshima University).

The expression plasmid for mouse MafA was constructed by inserting a NotI fragment excised from pGEM-T-easy/m-mafA into a pHygEF2 mammalian expression vector. To construct hemagglutinin (HA)-tagged human mafA, a double-stranded oligonucleotide encoding the HA epitope tag sequence (5'-tctagacgcgtccATGGGGTACCCATACGATGTTCCAGATTACGCAGGAATTC-3') was inserted into the 5'-EcoRI site (T7 side) of pGEM-T-easy/h-mafA. The resultant HA-h-mafA fragment (XbaI-SpeI) was inserted into pHygEF2 to obtain pHygEF2/HA-h-mafA.

To construct the expression vector for the repressor form of MafA (pHygEF2/HA-SID-h-mafA), the EcoRI-PmlI fragment of pHygEF2/HA-h-mafA encoding the amino-terminal putative transactivation domain of MafA (amino acids 1-219) was replaced by a DNA fragment encoding the Sin3 interaction domain (SID) of Mxi1 (amino acids 1-70) (46-48).

cDNA fragments containing the entire open reading frames of mouse Pdx1 and Beta2 were amplified from MIN6 total RNA using RT-PCR (primers for pdx1: 5'-agaggccTGCCGGCTGCCACCATGAAC-3' and GCTCACCCTCAGACTGCTGTCCTCACC-3'; primers for beta2: 5'-gggaggcctTGGAAACATGACCAAATCATA-3' and 5'-agaccgcgGCCTCTAATCGTGAAAGATGGC-3') and were inserted into pGEM-T-easy vector (Promega). A double-stranded oligonucleotide encoding the FLAG tag (5'-ctagtaccATGGATTACAAGGATGACGACGATAAGGGAGG-3' and 5'-CCTCCCTTATCGTCGTCATCCTTGTAATCCATggta-3') was then inserted into the SpeI-StuI sites. The resultant FLAG-pdx1 and FLAG-beta2 fragments were excised by NotI digestion and were inserted into the NotI site of the pHygEF2 vector.

NIH3T3 cells grown in a 35-mm dish were transfected with a total of 1.5 µg of plasmids (0.4 µg of luciferase plasmid, a total of 1.0 µg of expression plasmids, and 0.1 µg of pEF-Rluc (48)) using 10 µl of Polyfect reagent (Qiagen). Cells were harvested 24 h after transfection. MIN6 cells grown in a 35-mm dish were transfected with a total of 1.0 µg of plasmids (0.3 µg of luciferase plasmid, 0.6 µg of expression plasmid, and 0.1 µg of pEF-Rluc) using 8 µl of LipofectAMINE and 6 µl of PLUS reagent (Invitrogen). Twenty-four hours after transfection, the medium was changed to Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 0 or 20 mM of glucose, and the cells were incubated for another 24 h. The firefly and Renilla luciferase activities were measured using the Dual Luciferase Assay System (Promega).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Similarity of the DNA Binding Specificities of RIPE3b1 and Maf-- As we have previously described (19), the conserved RIPE3b element in the promoter region of the insulin genes of human, mouse, and rat has a nucleotide sequence that is very similar to the consensus MARE sequence (Fig. 1A). This finding prompted us to examine whether RIPE3b-binding complexes in pancreatic beta -cells contain Maf family members.


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  		Fig. 1.   Detection of the RIPE3b-binding factor in beta -cell lines. A, sequence similarity of MARE and the RIPE3b element. The conserved RIPE3b elements of the insulin gene promoters of humans, mice, and rats are aligned with the consensus MARE sequence. The numbers are the nucleotide positions from the transcriptional start site. B, gel mobility shift analysis of beta - and alpha -cell nuclear extracts. Nuclear extracts were prepared from MIN6, beta TC6, and alpha TC1 cells grown in the absence (-) or presence (+) of 20 mM glucose for 24 h and were subjected to gel mobility shift analysis using a 32P-labeled 32-mer oligonucleotide containing the RIPE3b element. The arrow indicates the glucose-responsive DNA-protein complex specifically detected in beta -cell lines. C, binding sequence specificity of the RIPE3b-binding factor. MIN6 nuclear extracts were analyzed by gel mobility shift assay using the RIPE3b element probe in the absence or presence of a 100-fold molar excess of the unlabeled oligonucleotides indicated at the top. The core sequences of the competitor oligonucleotides are shown at the right. The arrow indicates the DNA-protein complex of the RIPE3b1 activator, and the asterisks indicate nonreproducibly appearing DNA-binding complexes.

To explore this possibility, we prepared nuclear extracts from insulin-producing mouse beta -cell lines, MIN6 and beta TC6, grown in the presence or absence of 20 mM glucose, and subjected them to gel mobility shift analysis using an oligonucleotide probe containing the human RIPE3b element. As shown in Fig. 1B (lanes 1-4), a DNA-protein complex that appeared in the presence of glucose was detected both in MIN6 and beta TC6 nuclear extracts. In contrast, we did not detect this complex in nuclear extracts prepared from the non-beta -cell lines alpha TC1 (alpha -cell) (lane 5), NIH3T3 (embryonic fibroblast) (see Fig. 2C), C2C12 (myoblast), and RAW264 (monocyte) (data not shown). Therefore, this beta -cell-specific complex seemed to be identical to the previously reported RIPE3b1 complex (49, 50).


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  		Fig. 2.   Identification of the RIPE3b1 activator as MafA. A, reactivity of the RIPE3b1 activator with anti-Maf antisera. MIN6 nuclear extracts were subjected to gel mobility shift analysis in the presence of the various antisera indicated at the top. The arrow indicates the RIPE3b1 activator. B, gel mobility supershift analysis. An antiserum raised against a MafA-derived peptide (lane 2) was included in the gel mobility shift analysis of the MIN6 nuclear extract. The arrowheads indicate the supershifted DNA-protein complexes of the RIPE3b1 activator. C, DNA binding activity of recombinant MafA. Nuclear extracts were prepared from NIH3T3 cells transfected with expression plasmids for enhanced green fluorescent protein (EGFP; lane 2) or mouse MafA (lane 3) and were tested for binding to the RIPE3b element. MIN6 nuclear extracts were also tested for comparison (lane 1). The arrow indicates the RIPE3b1 activator in MIN6 cells. The arrowhead indicates the specific DNA-protein complex that appeared in MafA-expressing NIH3T3 cells.

We next tested the DNA binding specificity of this complex. As shown in Fig. 1C, a 100-fold molar excess of unlabeled human and mouse/rat RIPE3b-containing oligonucleotides specifically competed for the binding of this factor to the probe (lanes 1-3). The DNA binding specificity of the complex, as determined by the addition of excess cold oligonucleotides bearing various nucleotide substitutions (mut-A to mut-D) (lanes 4-7), was very similar to what has been previously reported (49, 50). These results again confirm the identity of this complex as the previously reported RIPE3b1 complex. Furthermore, the complex formation was inhibited by the addition of oligonucleotides containing the consensus MARE sequence (Fig. 1C, #1, lane 8) or a related sequence that can bind to Maf family members (#11 and #7, lanes 9 and 10) but not of oligonucleotides that do not bind to Maf (#17 and #23, lanes 11 and 12). Therefore, the RIPE3b1 factor showed similar DNA binding specificity to the Maf family of transcription factors (19).

Identification of the RIPE3b1 Factor as MafA-- We then tested whether the RIPE3b1 complex contains Maf family members using a gel mobility shift analysis with a series of anti-Maf antibodies. As shown in Fig. 2A, the RIPE3b1 complex was supershifted by the addition of antisera that broadly react with large Maf members (anti-v-Maf and anti-c-Maf (M-153), lanes 1-4), whereas anti-small Maf antisera (anti-MafK and anti-MafK/F/G) did not react with the RIPE3b1 complex (lanes 8 and 9). The addition of antisera that broadly react with Fos and Jun family members, possible heterodimeric partners for Maf family proteins (19, 20, 22, 28), had no effect (lanes 10 and 11). These results clearly indicate that RIPE3b1 does contain a large Maf. However, none of antisera that specifically react with c-Maf (anti-c-Maf N-15), MafB, or Nrl affected the RIPE3b1 complex (lanes 5-7). These results strongly suggest that the RIPE3b1 complex contains an unidentified member of the large Maf family that reacts with anti-v-Maf and anti-c-Maf (M-153) sera.

Among the large maf family members identified in vertebrates to date, c-maf, mafB, and nrl genes have been isolated in mammals, but a homologue of avian mafA has not yet been identified. By searching the GenBankTM data base, we found a human genome contig on chromosome 8q24 and several human expressed sequence tags that were most homologous to avian mafA. From this nucleotide sequence information, we cloned the entire open reading frame of this gene and its mouse homologue (human and mouse mafA genes) by PCR. From the deduced amino acid sequence of mouse MafA, we raised a MafA-specific antiserum by immunizing a rabbit with a MafA-derived peptide and added it into the gel mobility shift analysis. As shown in Fig. 2B, the RIPE3b1 complex was supershifted by the addition of the anti-MafA serum. We also confirmed that recombinant human and mouse MafA reacted with anti-v-Maf and anti-c-Maf (M-153) sera but not with anti-c-Maf (N-15), MafB, or Nrl sera (data not shown). Furthermore, transfection of an MafA expression vector into NIH3T3 cells resulted in the appearance of a DNA-protein complex of very similar mobility to the RIPE3b1 complex (Fig. 2C). These results clearly indicate that MafA is a component of the RIPE3b1 factor.

Structure of Human and Mouse MafA-- Nucleotide sequence analyses of human and mouse mafA (GenBankTM accession numbers AB086960 and AB086961) have revealed that they encode proteins of 351 (human) and 359 (mouse) amino acids that exhibit the highest homology to avian MafA (Fig. 3, A and B) (29, 30). As shown in Fig. 3B, the carboxyl-terminal basic domain and leucine zipper of human and mouse MafA that should serve as the DNA-binding and the dimerization domains, respectively, are completely conserved and are highly similar to other Maf family members. They also contain an amino-terminal acidic amino acid/serine/threonine/proline-rich domain, which may serve as a transcriptional activation domain (27). The middle part of MafA is relatively divergent but contains clusters of glycine and histidine as do all of the other large Maf members except Nrl.


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  		Fig. 3.   Human and mouse MafA. A, comparison of the deduced amino acid sequences of human and mouse MafA with chicken MafA/L-Maf. The repeated residues in the leucine zipper region are indicated by asterisks. B, schematic comparison of large Maf proteins. The primary structures of human, mouse, and chicken MafA are shown together with the other large Maf proteins identified in humans (c-Maf, MafB, and Nrl). The sequence identities in the basic domain and leucine zipper are also indicated. H and G, stretches of histidine and glycine residues, respectively.

We determined the chromosomal location of the human mafA gene by fluorescent in situ hybridization. Specific hybridization signals were observed on chromosome 8q24.3, and no other hybridization sites were detected (Fig. 4). This result is consistent with the map position (8q24) of the mafA-containing human genome contig (NT 023684).


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  		Fig. 4.   Chromosome mapping of human mafA gene. The biotinylated DNA probe for the human mafA gene was hybridized to chromosomes with replication bands prepared from normal donors. After hybridization and washing, hybridization signals were amplified using rabbit anti-biotin (Enzo) and fluorescein-labeled goat anti-rabbit IgG (Enzo). The chromosomes were counterstained with propidium iodide.

Restricted Expression of mafA in Eye and Pancreatic beta -Cells-- We next prepared total RNA from MIN6, beta TC6, and alpha TC1 cells and examined the expression of mafA mRNA by RT-PCR analysis. As is clearly shown in Fig. 5A, mafA mRNA expression was detected in two insulin-producing beta -cell lines, MIN6 and beta TC6, but not in the glucagon-producing alpha TC1 cell line. Moreover, we could not detect mRNAs for retina-specific nrl and for c-maf and mafB in MIN6 and beta TC6 cells (data not shown), although c-maf and mafB are known to be expressed in a wide variety of tissues (28). Therefore, among the large Maf family members, insulin-producing beta -cells selectively express mafA.


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  		Fig. 5.   mRNA expression of mafA. A, RT-PCR analyses. Total RNA (1 µg, bottom panel) prepared from MIN6, beta TC6, and alpha TC1 cells was subjected to RT-PCR analysis using specific primers for mafA, insulin, and glucagon. The aliquots of the amplified fragments were separated by agarose gel electrophoresis and were visualized by ethidium bromide staining. B, tissue distribution of mafA mRNA in mice. One microgram of total RNA (lower panel) isolated from various tissues of 7-week-old male mice was analyzed by RT-PCR using mafA-specific primers (upper panel).

We also examined tissue distribution of mafA mRNA expression in adult mouse by RT-PCR. As shown in Fig. 5B, we could not detect mafA mRNA in any tissues we have examined except the eye. Expression of mafA in the eye makes sense in that avian mafA/L-maf is specifically expressed in the lens (29, 30). Although mafA is expressed in beta -cell lines as shown above, we could not detect mafA mRNA in the pancreas, probably because of the very low abundance of beta -cells in whole pancreas or, alternatively, because of degradation of the RNA source.

Glucose-regulated Expression of MafA Protein and mafA mRNA-- In order to further confirm that MafA is the RIPE3b1 factor, we examined the expression of MafA protein in beta - and alpha -cells. Nuclear extracts prepared from MIN6, beta TC6, and alpha TC1 cells grown in the presence or absence of glucose were analyzed by Western blotting using anti-MafA antiserum. As shown in Fig. 6A, a protein of 48 kDa was specifically detected in MIN6 and beta TC6 nuclear extracts (lanes 2 and 4). A protein of this molecular weight was also detected using anti-v-Maf and anti-c-Maf (M-153) antisera (see Fig. 6B and data not shown), indicating that the 48-kDa protein is MafA. MafA protein was barely detectable when MIN6 and beta TC6 cells were grown in the absence of glucose (lanes 1 and 3), and it was not present in an alpha TC1 nuclear extract even when the cells were grown in the presence of glucose (lane 5). These expression profiles correlated quite well with the appearance of the RIPE3b1 DNA-binding complex (see Fig. 1B), again supporting the idea that MafA is the RIPE3b1 factor.


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  		Fig. 6.   Glucose-regulated expression of MafA protein and mRNA in beta -cell lines. A, Western blot analysis of beta - and alpha -cell nuclear extracts using anti-MafA serum. Nuclear extracts were prepared from MIN6, beta -TC6, and alpha TC1 cells grown in the absence (-) or presence (+) of 20 mM of glucose for 24 h and were separated by SDS-PAGE followed by Western blotting using anti-MafA serum. MafA protein (48 kDa) is indicated by an arrow. The asterisk indicates a nonspecific band. B, regulation of expression of mafA mRNA and protein by glucose. A nuclear extract and total RNA were prepared from MIN6 cells grown in the presence of 0-20 mM glucose for 24 h. The nuclear extract was subjected to Western blot analysis using anti-c-Maf (M-153) serum (top panel), and the total RNA was subjected to Northern blot analyses using 32P-labeled mafA (second panel) or insulin (third panel) probes. The amount and integrity of the RNA was verified by agarose gel electrophoresis and ethidium bromide staining (bottom panel).

Furthermore, we found that the expression level of MafA protein (Fig. 6B, top panel) and mafA mRNA (second panel) in MIN6 cells was dependent on glucose concentration, which correlated well with the insulin mRNA level (third panel). Induction of MafA protein expression by glucose was also evident with immunofluorescent staining of MafA in MIN6 cells (Fig. 7) and in beta TC6 cells (data not shown). MafA protein was detected in the nucleus when these cells were grown in the presence of glucose.


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  		Fig. 7.   Nuclear localization of MafA. MIN6 cells grown in the absence or presence of glucose were fixed and stained with anti-c-Maf (M-153) antiserum and Alexa Fluor 488-labeled anti-rabbit IgG antiserum (upper panels) and with 4,6-diamidino-2-phenylindole (lower panels).

Activation of Insulin Promoter by MafA-- We next investigated the effect of MafA on insulin gene promoter activity. A luciferase reporter plasmid containing the human insulin promoter was transfected into NIH3T3 cells together with increasing amounts of the MafA expression plasmid. The insulin promoter activity was very low in NIH3T3 cells as compared with the activity of pG4×5/TATA/luc that contains five copies of the Gal4-binding sequence and TATA-box (Fig. 8A). Co-expression of Pdx1 or Beta2, which are expressed in beta -cells and have been shown to bind and activate the insulin promoter, resulted in dose-dependent activation. When MafA was expressed, the insulin promoter activity was greatly enhanced in a dose-dependent manner, indicating that MafA is an efficient transcriptional activator of the insulin promoter.


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  		Fig. 8.   Activation of insulin promoter by MafA. A, luciferase assay. 0.4 µg of luciferase reporter plasmid driven by the human insulin promoter (h-insulin promoter-luc) was transfected into NIH3T3 cells with increasing amounts (0.2, 0.5, and 1.0 µg) of expression plasmids for MafA, Pdx1, or Beta2. The luciferase activity of pG4×5/TATA/luc was also indicated for comparison of relative activity of the insulin promoter. B, insulin promoter activity in MIN6 cells. Reporter plasmids containing wild-type (WT) or mutated (mut MARE) insulin promoters or multiple Gal4-binding sites (pG4×5/TATA/luc) were transfected into MIN6 cells together with an empty expression plasmid or expression plasmid for repressor MafA (HA-SID-ND-MafA). SID, the Sin3 interaction domain of the Mxi1 transcriptional repressor. The transfected cells were grown in the presence or absence of glucose for 24 h and were assayed for luciferase activity.

When the insulin promoter reporter plasmid was transfected into MIN6 cells, its promoter activity was significantly high relative to pG4×5/TATA/luc, and was regulated by glucose (Fig. 8B), reflecting endogenous insulin gene transcription. Mutation of the RIPE3b/MARE element resulted in a decrease of both the basal and glucose-induced activities of the promoter, which has been reported previously (49, 50), and confirmed the importance of the RIPE3b/MARE in basal and glucose-regulated insulin gene expression. In order to evaluate the possible role of endogenous MafA protein on insulin promoter activity, we constructed a dominant-negative mutant of MafA (HA-SID-ND-MafA), whose amino-terminal putative activation domain was replaced with the heterologous transcriptional suppressor domain of Mxi1 (SID). SID is known to actively suppress transcription by interacting with Sin3 protein and by recruiting the histone deacetylase complex (46-48). As shown in Fig. 8B, expression of this mutant MafA protein in MIN6 cells resulted in a significant decrease in insulin promoter activity both in the presence and absence of glucose in the culture medium, suggesting an essential role of MafA in the insulin gene transcription.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this report, we first demonstrated that the RIPE3b1 activator is related to a large Maf family member in its DNA binding specificity and its reactivity to a series of anti-Maf antisera. We then molecularly cloned human and mouse homologues of the mafA gene and showed that MafA is the RIPE3b1 activator. Anti-MafA-specific antiserum reacted with the RIPE3b1 activator in gel mobility shift analyses, and mafA mRNA was detected only in insulin-producing beta -cell lines and the eye, but not in other cell lines and tissues as far as we have examined. Consistent with the previously suggested role of RIPE3b1 factor in glucose-regulated insulin gene expression, glucose induced the expression of mafA mRNA and protein in beta -cell lines.

Previous reports have demonstrated that the RIPE3b1 activator is a protein of 37-49 kDa (49, 50), which is consistent with our estimation of the molecular size of the MafA protein (48 kDa) by Western blotting analysis. During preparation of this manuscript, Olbrot et al. (51) reported that they purified a 47-kDa RIPE3b1-binding protein from MIN6 cells and identified it as MafA, which is again consistent with our observations. Olbrot et al. (51) reconstituted the RIPE3b1 factor from the purified 47-kDa protein alone in gel mobility shift assay. Therefore, despite the fact that the MafA protein possibly forms heterodimers with other basic leucine zipper proteins, including the other large Maf proteins, we believe that the RIPE3b1 factor is a homodimer of MafA. This idea is supported by our observations that the DNA binding specificity of the RIPE3b1 factor is very similar to that of the Maf homodimer (Fig. 1C) and that recombinant MafA protein had very similar mobility to the RIPE3b1 complex on gel shift analysis (Fig. 2C). Moreover, none of mRNAs for c-maf, mafB, and nrl were detected in MIN6 and beta TC6 cells by RT-PCR analysis (data not shown).

Olbrot et al. (51) detected mafA mRNA in mouse islet and insulinoma cell lines but not in glucagon-producing alpha TC1 cells, which is consistent with our results (Fig. 5). Recently, Planque et al. (58) have shown that mafA mRNA is expressed in pancreas and lens of quail embryo, which suggests the conserved expression profiles and roles of MafA in birds and mammals. Planque et al. (58) have reported that c-maf mRNA is detected in alpha TC cells and that Pax6 and the large Maf family members synergistically activate the glucagon gene transcription. Therefore, interestingly, distinct members of the large Maf family (MafA and c-Maf) are expressed in distinct types of pancreatic islet cells (beta - and alpha -cells) and regulate insulin and glucagon gene transcription, respectively. It is worth examining whether the large Maf proteins are also expressed in islet delta - and gamma -cells and whether they are involved in transcription of somatostatin and pancreatic polypeptide genes.

Previous studies have identified a set of transcription factors that specifically bind to the cis-regulatory elements on the insulin promoter, including Beta2 and Pdx1, as well as Isl1 and Lmx1 (7-10, 54, 55). It has also been shown that these transcription factors synergistically activate the insulin promoter (54). Furthermore, some of them have been shown to physically interact (56, 57). In contrast, MafA alone strongly activated the insulin gene promoter in NIH3T3 cells (Fig. 8A), and we did not see additional enhancement of MafA transcriptional activity with co-expression of Beta2 or Pdx1 as far as we have examined.2 Together with our (Fig. 8B) and previous (3-6) findings that mutation of the RIPE3b element greatly reduces the basal and glucose-induced insulin promoter activity in beta -cells, MafA seems to be one of the most important transcriptional activators for efficient insulin gene expression.

Most of the previously identified transcription factors required for insulin gene expression have also been shown to be involved in development of beta -cells as well as the other islet cells. For example, targeted deletion of beta2 in mice resulted in a decreased number of beta -cells and the failure of mature islets to develop (11), and pdx1 gene disruption caused ablation of the pancreas (12, 13). MafA may also play important roles in beta -cell development and maintenance of beta -cell function as well as in expression of insulin. Generation of mafA knockout mice should elucidate the role of mafA in the development of beta -cells and its functional relationship with the other islet-specific transcription factors. Furthermore, taking into account that mutations in beta2 and pdx1 genes are associated with type 2 diabetes mellitus in humans (14-16), a defect in MafA function may also cause diabetes in humans as well as in model animals. However, as far as we know, the chromosome location of the human mafA gene (8q24.3) is not assigned as a susceptibility locus of both type 1 and type 2 diabetes.

It has been suggested that tyrosine phosphorylation of the RIPE3b1 activator stimulates its DNA binding activity (52). Although avian MafA has been shown to be phosphorylated on serine residues at the amino-terminal transactivator domain (53), there is no evidence of tyrosine phosphorylation of MafA to date. It is worth examining whether the MafA protein is tyrosine-phosphorylated and what effect that would have on its DNA binding activity, and such post-translational regulation of MafA remains to be examined. As we have shown here, expression of MafA is regulated by glucose at least at the level of transcription (Fig. 6), which might in turn be responsible for glucose-regulated insulin gene expression. However, the glucose concentration may also regulate MafA activity by other mechanisms, such as post-transcriptional and/or post-translational modifications, which, if any, may help to elucidate the molecular mechanism of insulin transcription activation by glucose.

    ACKNOWLEDGEMENTS

We thank Kunio Yasuda and Kiyo Sakagami for helpful discussions; Kiyomi Yoshitomo-Nakagawa, Keiko Watanabe, and Michiko Tatsuno for technical assistance; and Kozue Ando and Hiroki Narimatsu for tissue RNA preparation. We are also grateful to Jun-ichi Miyazaki (Osaka University) for the MIN6 cell line, Kazuhiro Chida (Tokyo University) for anti-c-Jun serum, and Kazuhiko Igarashi (Hiroshima University) for the pG4×5/TATA/luc plasmid.

    FOOTNOTES

* This work was supported by Grants-in-Aid for Scientific Research on Priority Areas and for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture in Japan and a grant from the Ministry of Health, Labor and Welfare in Japan (to K. K.).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: Frontier Collaborative Research Center, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan. Tel.: 81-45-924-5799; Fax: 81-45-924-5834; E-mail: kkataoka@bio.titech.ac.jp.

Published, JBC Papers in Press, October 3, 2002, DOI 10.1074/jbc.M206796200

2 K. Kataoka, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: MARE, Maf-recognition element; HA, hemagglutinin; SID, Sin3 interaction domain; RT, reverse transcription; contig, group of overlapping clones.

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