Involvement of mi-Transcription Factor in Expression of α-Melanocyte-Stimulating Hormone Receptor in Cultured Mast Cells of Mice^1 2

The α-melanocyte-stimulating hormone (α-MSH)⁴ is essential for synthesis of melanin in melanocytes (1). In addition, there have been a number of pieces of evidence showing that α-MSH influences various inflammatory or immunological conditions by modulating function of lymphocytes (2, 3), neutrophils (4), or macrophages (5, 6, 7, 8). We recently reported that murine cultured mast cells (CMCs) expressed a receptor for α-MSH (MC1R) and that α-MSH inhibited histamine release from CMCs and the synthesis of proinflammatory cytokines in stimulated CMCs (9).

The microphthalmia (mi) locus of mice encodes a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of transcription factors (mi-transcription factor, MITF) (10, 11). The MITF encoded by the mutant mi allele (mi-MITF) deletes 1 of 4 consecutive arginines in the basic domain (10, 12, 13). Thus mi-MITF is defective in the DNA binding activity and the nuclear translocation, resulting in defective transactivation of target genes (14, 15). The mutant mice of mi/mi genotype show microphthalmia, deletion of pigment in both hair and eyes, osteopetrosis, and a decrease in the number of mast cells (16, 17, 18, 19, 20). In addition to decreased number of mast cells, the phenotype of mast cells is abnormal in mi/mi mice (20, 21, 22, 23). The expression of the mouse mast cell protease 6 (MMCP-6) (24), c-kit receptor tyrosine kinase (25), p75 nerve growth factor (NGF) receptor (26), MMCP-5 (27), granzyme B (Gr B) (28), tryptophan hydroxylase (TPH) (28), and MMCP-4 genes (29) are reduced in skin mast cells of mi/mi mice. The involvement of normal (+)-MITF (+-MITF) in the transactivation of these genes has been demonstrated in CMCs.

Because MITF regulates the expression of many genes in mast cells, we examined a possibility that MITF regulated MC1R expression using mi/mi CMCs, in which the transactivation by MITF was deficient. The subtracted cDNA library of (+/+ CMCs-mi/mi CMCs) contained the cDNA for MC1R, but the cDNA library obtained from mi/mi CMCs did not. Overexpression of +-MITF normalized the expression of the MC1R gene in mi/mi CMCs. +-MITF bound the neighboring two CANNTG motifs in the cloned promoter region of the MC1R gene and transactivated the MC1R gene through the two motifs. These results suggested +-MITF regulated the MC1R gene expression in mouse mast cells.

The original stock of C57BL/6-mi/+ mice was purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in our laboratory by consecutive backcrosses to our own inbred C57BL/6 colony. Female and male mi/+ mice were crossed together, and the resulting mi/mi mice were selected by their white coat color (16, 17). The original stock of VGA-9-tg/tg mice, in which the mice vasopressin-Escherichia coli β-galactosidase transgene was integrated at the 5′ flanking region of the mi (MITF) gene, were kindly given by Dr. H. Arnheiter (National Institutes of Health, Bethesda, MD). The integrated transgene was maintained by repeated backcrosses to our own inbred C57BL/6 colony. Female and male tg/+ mice were crossed together, and the resulting tg/tg mice were selected by their coat color (10).

CMCs were established from mi/mi, tg/tg, and control +/+ mice as described previously (29), and maintained in α-minimal essential medium (α-MEM; ICN Biomedicals, Costa Mesa, CA) supplemented with 10% PWM-stimulated spleen cell-conditioned medium (PWM-SCM) and 10% FCS (Nippon Biosupp Center, Tokyo, Japan). The helper virus-free packaging cell line (ψ2) was maintained in DMEM (ICN Biomedicals) supplemented with 10% FCS (30). The NIH/3T3 cell line was generously provided by Dr. S. A. Aaronson (National Cancer Institute, Bethesda, MD) and maintained in DMEM supplemented with 10% FCS. The murine mastocytoma cell line, FMA/3, was generously given by Dr. H. Hasegawa (Nishi-Tokyo University, Yamanashi, Japan) and maintained in α-MEM supplemented with 10% FCS.

The detailed process for the preparation of the subtracted cDNA library was described previously (28). ssDNA was prepared from the plasmid DNA from the +/+ CMCs cDNA library. Biotinylated RNA drivers were prepared from mi/mi CMCs by photobiotin (Vector Lab, Burlingame, CA). ssDNA prepared from the +/+ CMCs cDNA Library was hybridized with biotinylated RNA. After hybridization for 42 h, streptavidin was added into the mixture and extracted with phenol/chloroform/isoamyl alcohol. The aqueous phases were pooled. Streptavidin binding and phenol treatment were repeated once more. After repeating the subtraction process, the recovered ssDNA was converted to dsDNA. To detect MC1R, PCR was employed using +/+, mi/mi CMCs cDNA library, or the subtracted cDNA library (+/+-mi/mi) as a template. PCR was performed in a thermocycler (Astec, Tokyo, Japan) as follows: 94°C, 5 min; followed by 35 amplification cycles (94°C for 30 s; 60°C for 1 min; 72°C for 1 min). The sequence of the primers for MC1R are follows: the upper strand, GTG AGT CTG GTG GAG AAT GTG; and the lower strand, TTT TGT GGA GCT GGG CAA TGC (5). The PCR products were electrophoresed in 1.2% agarose gel containing ethidium bromide.

Total RNA was isolated from +/+, mi/mi, or tg/tg CMCs by the guanidine thiocyanate/phenol-chloroform extraction method and was treated with RNase-free DNase (Boehringer Mannheim, Mannheim, Germany) to remove genomic DNA. RNA (5.0, 0.5, and 0.05 μg) was reverse transcribed in 20 μl of the reaction mixture containing 20 U of avian myeloblastosis virus reverse transcriptase (Life Technologies, Gaithersburg, MD) and random hexamer. PCR was performed in the same conditions described above, and the PCR products were electrophoresed in 1.2% agarose gel containing ethidium bromide.

Bluescript KS(−) plasmid (pBS; Strategene, La Jolla, CA) containing the whole coding region of +-MITF or mi-MITF (pBS-+-MITF or pBS-mi-MITF, respectively) had been constructed in our laboratory (14, 15). A retroviral vector pM5Gneo (31), a derivative of myeloproliferative sarcoma virus vector, was a kind gift from Dr. W. Ostertag (Universitat Hamburg, Hamburg, Germany). The purified SmaI-HincII fragment from pBS-+-MITF or pBS-mi-MITF was introduced into the blunted EcoRI site of pM5Gneo. The resulting plasmids (hereafter called pM5Gneo-+-MITF or pM5Gneo-mi-MITF, respectively) were transfected into the packaging cell line (ψ2) (30) using the calcium phosphate method, and neomycin-resistant ψ2 cell clones were selected by culturing in DMEM containing 10% FCS and G418 (0.8 mg/ml, Life Technologies, Grand Island, NY). For the gene transfer, spleen cells obtained from mi/mi mice were incubated on irradiated (30 Gy) subconfluent monolayer of virus-producing ψ2 cells for 72 h in α-MEM supplemented with 10% PWM-SCM and 10% FCS. Neomycin-resistant CMCs were obtained by continuing the culture in α-MEM supplemented with 10% PWM-SCM, 10% FCS, and G418 (0.8 mg/ml) for 4 wk.

The isolation of the promoter region of the MC1R gene was performed with the Mouse Promoter Finder Kit (Clontech Laboratories, Palo Alto, CA) according to the manufacturer’s instructions. The isolated promoter region was cloned into pBS and sequenced.

The pEF-BOS expression vector was kindly provided by Dr. S. Nagata (Osaka University Medical School, Suita, Japan). The SmaI-HincII fragment of pBS-+-MITF or pBS-mi-MITF was introduced into the blunted XbaI site of pEF-BOS (BOS-+-MITF or BOS-mi-MITF, respectively). The luciferase gene subcloned into pSP72 (pSPLuc) was generously provided by Dr. K. Nakajima (Osaka University Medical School, Suita, Japan) (32). To construct reporter plasmids, a DNA fragment containing a promoter region and the first exon of the MC1R gene (nt −780 to +44) was cloned into the upstream of luciferase gene in pSPLuc. The deletion of MC1R promoter was produced using PCR and subcloned into pBS. A BamHI-HindIII fragment of the pBS-PCR product was cloned into pSPLuc. The mutation was introduced by PCR with mismatch primers. Deleted or mutated products were verified by sequencing.

NIH/3T3 cells (5 × 10⁵) were plated in a 10-cm plastic dish 1 day before the procedure. Cotransfection with 5 μg of reporter plasmids, 100 ng of effector plasmids, and 5 μg of an expression vector containing β-galactosidase gene was performed by the calcium phosphate precipitation method. The expression vector containing the β-galactosidase gene was used as an internal control. Because FMA/3 cells express the MITF gene by themselves (25), the reporter plasmids (5 μg) and the expression vector containing β-galactosidase gene were added to the cell suspension. For gene transfer, FMA/3 cells were electroporated by a single pulse (300 V, 950 μF) from a Gene Pulser II (Bio-Rad Laboratories, Richmond, CA). NIH/3T3 cells were harvested 48 h after transfection; FMA/3 cells were harvested 24 h after transfection. The cells were lysed with 0.1 M potassium phosphate buffer (pH 7.4) containing 1% Triton X-100. Soluble extracts were then assayed for luciferase activity with a luminometer LB96P (Berthold, Wildbad, Germany) and for β-galactosidase activity. Luciferase activity was normalized by β-galactosidase activity and total protein concentration.

The production and purification of GST-+-MITF or GST-mi-MITF fusion protein was described previously (14). Oligonucleotides were labeled with [α-³²P]dCTP by filling 5′-overhangs and used as probes of EMSA. DNA-binding assays were performed in a 20-μl reaction mixture containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 75 mM KCl, 1 mM DTT, 4% Ficoll type 400, 50 ng of poly(dI-dC), 25 ng of labeled DNA probe, and 1.0 μg of GST-+-MITF fusion protein. After incubation at room temperature for 15 min, the reaction mixture was subjected to electrophoresis at 14 V/cm on a 5% polyacrylamide gel in 0.25× TBE buffer (1 × TBE in 90 mM Tris-HCl, 64.6 mM boric acid, and 2.5 M EDTA, pH 8.3). The polyacrylamide gels were dried on Whatman 3 MM chromatography paper (Whatman, Maidstone, U.K.) and subjected to autoradiography.

We examined whether the subtracted cDNA library (+/+-mi/mi) contained the cDNA for MC1R. The PCR products obtained from the subtracted cDNA library (+/+-mi/mi) and from cDNA library of +/+ CMCs contained an amplified single band. But no amplified band was observed in the product from mi/mi cDNA library (Fig. 1,A). The expression of the MC1R gene in +/+, mi/mi, or tg/tg CMCs was also examined using semiquantitative RT-PCR. The expression of the MC1R gene was undetectable in not only mi/mi but also tg/tg CMCs. In contrast, +/+ CMCs expressed the MC1R gene (Fig. 1,B). Next, we introduced the cDNA encoding +-MITF or mi-MITF to mi/mi CMCs. The magnitude of mRNA expression of +-MITF was comparable to that of mi-MITF, and the expression level of the introduced +-MITF or mi-MITF was much greater than that of endogenous mi-MITF (data not shown). The expression of MC1R mRNA recovered to the normal level in mi/mi CMCs overexpressing +-MITF but did not in mi/mi CMCs overexpressing mi-MITF (Fig. 1 C).

The normalized expression of the MC1R gene in mi/mi CMCs overexpressing +-MITF indicated the involvement of +-MITF in the regulation of MC1R gene expression. To investigate the regulation mechanism, we cloned 780 bases of the 5′-upstream region of the MC1R gene (Fig. 2). The reporter plasmid that contained the luciferase gene under the control of the MC1R gene promoter (starting from nt −780; +1 shows the transcription initiation site) was constructed. There were five CANNTG motifs, which might be recognized by bHLH-Zip protein family of transcription factors. Thus we also constructed the reporter plasmid containing the deleted MC1R promoter starting from nt −483, −470, −295, −155, −45, or −25. These reporter plasmids were transfected into the FMA/3 mastocytoma cells, which constitutively expressed both +-MITF and MC1R mRNAs (data not shown). When the reporter plasmid containing the MC1R promoter starting from −780 was transfected, the luciferase activity was low. On the other hand, when the reporter plasmid containing the MC1R promoter starting from nt −483 was transfected, the luciferase activity increased about 60-fold (Fig. 3). The transfection of MC1R promoter starting from nt −470, −295, −155, or −45 did not show a significant increase of the luciferase activity. These studies suggested that the responsible region for the transactivation of the MC1R gene existed in the promoter starting from nt −483.

We sought to identify the motif that mediated the transactivation of the MC1R gene by +-MITF. The reporter plasmid containing MC1R promoter starting from nt −780, −483, −470, −295, −155, −45, or −25 was cotransfected with +-MITF into NIH/3T3 fibroblasts, which did not express endogenous +-MITF gene (26). Slightly increased luciferase activity was observed in the presence of +-MITF when the reporter plasmid containing MC1R promoter starting from nt −780 was transfected. On the other hand, significant luciferase activity (6.5-fold) was induced in the presence of +-MITF when the plasmid containing MC1R promoter starting from nt −483 (Fig. 4). Cotransfection of +-MITF did not induce a significant luciferase activity in the deleted promoter from nt −470, −295, −155, or −45. In contrast, cotransfection of mi-MITF did not increase a luciferase activity in any promoter constructs. These data suggested the importance of the promoter region from nt −483 to −471 for the transactivation of the MC1R gene by +-MITF. Because the CACATG motif from nt −477 to −472 exists close to the CATGTG motif from nt −466 to −461, the possibility that the latter motif also has a role in the transactivation of the MC1R gene still remained. The participation of the CATGTG motif in the transactivation could not be excluded using the deleted promoter constructs. Therefore, we cloned the reporter plasmids containing the MC1R gene promoter from nt −483 with the mutations in the CACATG motif and/or the CATGTG motif. The mutation in the CACATG motif (from nt −477 to −472) completely abolished the luciferase activity as seen in the assay with the deleted promoter from nt −470 (Fig. 5). The mutation in the CATGTG motif (from nt −466 to −461) also completely abolished the luciferase activity.

Because the transactivation assay using the mutated reporter plasmids indicated that both CACATG and CATGTG motifs mediated the transactivation ability by +-MITF, we examined the binding of +-MITF to the oligonucleotides containing these motifs. The purified +-MITF bound the oligonucleotide (Fig. 6, lane 2), and the specificity was demonstrated using the excess of nonlabeled oligonucleotide (lanes 3 and 4). The binding was inhibited by the competitor containing either the mutated CACATG motif (CACATG to CTCAAG) or the mutated CATGTG motif (CATGTG to CTTGAG) (lanes 5-8). But the magnitude of the inhibition by the competitor containing the mutated CACATG motif was significantly larger than that by the competitor containing the mutated CATGTG motif. The excess of the competitor containing the mutations in both the CACATG motif and the CATGTG motif did not affect the binding of +-MITF protein to the probe.

The expression of the MC1R gene was detected in neither mi/mi CMCs nor tg/tg CMCs although +/+ CMCs expressed the MC1R gene. The overexpression of +-MITF in mi/mi CMCs normalized the transcription of the MC1R gene, showing the involvement of +-MITF in the transactivation of the MC1R gene, whereas the overexpression of mi-MITF did not induce the expression of the MC1R gene. The genes that are deficient in mi/mi and tg/tg CMCs can be divided into two groups. One shows the equal severity in the transcriptional deficiency between mi/mi and tg/tg CMCs; the other shows transcriptionally more severe defect in mi/mi than in tg/tg CMCs (33). The former group includes MMCP-4, -5, and -6 genes, the latter includes c-kit, tryptophan hydroxylase, and granzyme B genes. In this context, the MC1R gene belongs to the former group. The transcriptional defect of the MC1R gene appears to result from the deficiency of +-MITF, but not from the negative effect of mi-MITF.

We sequenced the 5′ flanking region of the murine MC1R gene to analyze the transactivation mechanism by +-MITF. The transfection of the reporter plasmid starting from nt −483 of the MC1R promoter into FMA/3 cells resulted in the high luciferase activity. The results suggested that the promoter activity existed between nt −483 to −471 of the 5′ flanking region of the MC1R gene. But it was uncertain whether +-MITF, but not other transcription factors, transactivated the MC1R gene. Thus, we cotransfected both the reporter plasmid and the expression plasmid containing MITF cDNA to determine the role of +-MITF. When the reporter plasmid was cotransfected into NIH/3T3, cotransfection of +-MITF but not mi-MITF increased the luciferase activity, indicating the direct involvement of +-MITF in the transactivation of the MC1R gene. The luciferase assay with the deleted promoter constructs demonstrated the important role of the CACATG motif (nt −477 to −472) in the transactivation by +-MITF. Because the CATGTG motif (nt −466 to −461) located closely to the CACATG motif (nt −477 to −472), there is a possibility that the CATGTG motif is involved in the transactivation by +-MITF. The participation of the CATGTG motif could not be excluded using the deleted promoter constructs. Thus we examined the transactivation using the promoter constructs with the mutations in the CACATG motif and/or the CATGTG motif. The mutations in either the CACATG motif or the CATGTG motif completely abolished the transactivation by +-MITF. These results indicated the motifs did not work additively, but both motifs were indispensable for the transactivation of the MC1R gene. Because the binding of +-MITF protein to the oligonucleotide containing both the CACATG and the CATGTG motifs was significantly inhibited by the excess of the competitor containing either the mutated CACATG or the mutated CATGTG motif, +-MITF protein appeared to bind both the CACATG and the CATGTG motifs. But the magnitude of the binding of +-MITF protein to the CATGTG motif was different from that to the CACATG motif. This may reflect the difference of the binding affinities of +-MITF to the motifs. However, there could be another explanation that coactivators were required for efficient binding of +-MITF to a particular element of DNA. The neighboring two CANNTG motifs are reminiscent of the promoter of the c-kit (25), MMCP-6 (24), and MMCP-4 genes (29). Although there were two CANNTG motifs separated by only one nucleotide in the promoter region of the c-kit gene, +-MITF bound only the former motif. In the case of the MMCP-4 gene, +-MITF bound only the former motif, and the latter did not affect the transactivation by +-MITF. On the other hand, +-MITF bound both of the two motifs separated by 11 nucleotides in the promoter region of the MMCP-6 gene. Unlike the MC1R gene, both motifs in the promoter region of the MMCP-6 gene appeared to work additively for the transactivation by +-MITF.

Because there are a few but important similarities between mast cells and melanocytes (34), there is a possibility that MITF regulates the MC1R expression also in melanocytes. In fact, α-MSH has been known to increase the expression of MITF in melanocytes (35), and α-MSH also increases the expression of MC1R in melanocytes (36, 37). These findings seem to support the possibility that MITF regulates the MC1R gene expression in melanocytes. Because α-MSH increases the MITF expression and MITF induces the MC1R expression, the regulation of the MC1R gene seems to be a kind of positive feedback by α-MSH.

In conclusion, MITF regulates the expression of the MC1R gene in CMCs. α-MSH has been known to modulate inflammatory conditions through the MC1R on T cells, B cells, macrophages, neutrophils (38), and mast cells (9). Because MC1R signaling appears to inhibit proinflammatory effects and MITF transactivates the MC1R gene, MITF can promote antiinflammatory effects by regulating the expression of the MC1R gene. This would be the first suggestion of MITF promoting antiinflammatory effects.

We thank Dr. H. Arnheiter of the National Institutes of Health for VGA9-tg/tg mice, Dr. H. Hasegawa of the Nishi-Tokyo University for FMA/3 cell line, Dr. W. Ostertag of Universitat Hamburg for pM5Gneo, Dr. S. Nagata of Osaka University for pEF-BOS, and Dr. K. Nakajima of Osaka University for pSPLuc.