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• W2015979443 abstract "Previous studies have revealed that activin A and transforming growth factor-β1 (TGF-β1) induced migration and morphological changes toward differentiation in bone marrow-derived cultured mast cell progenitors (BMCMCs). Here we show up-regulation of mouse mast cell protease-7 (mMCP-7), which is expressed in differentiated mast cells, by activin A and TGF-β1 in BMCMCs, and the molecular mechanism of the gene induction of mmcp-7. Smad3, a signal mediator of the activin/TGF-β pathway, transcriptionally activated mmcp-7. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, and heart and skeletal muscle, inhibited Smad3-mediated mmcp-7 transcription. MITF associated with Smad3, and the C terminus of MITF and the MH1 and linker region of Smad3 were required for this association. Complex formation between Smad3 and MITF was neither necessary nor sufficient for the inhibition of Smad3 signaling by MITF. MITF inhibited the transcriptional activation induced by the MH2 domain of Smad3. In addition, MITF-truncated N-terminal amino acids could associate with Smad3 but did not inhibit Smad3-mediated transcription. The level of Smad3 was decreased by co-expression of MITF but not of dominant-negative MITF, which resulted from proteasomal protein degradation. The changes in the level of Smad3 protein were paralleled by those in Smad3-mediated signaling activity. These findings suggest that MITF negatively regulates Smad-dependent activin/TGF-β signaling in a tissue-specific manner. Previous studies have revealed that activin A and transforming growth factor-β1 (TGF-β1) induced migration and morphological changes toward differentiation in bone marrow-derived cultured mast cell progenitors (BMCMCs). Here we show up-regulation of mouse mast cell protease-7 (mMCP-7), which is expressed in differentiated mast cells, by activin A and TGF-β1 in BMCMCs, and the molecular mechanism of the gene induction of mmcp-7. Smad3, a signal mediator of the activin/TGF-β pathway, transcriptionally activated mmcp-7. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, and heart and skeletal muscle, inhibited Smad3-mediated mmcp-7 transcription. MITF associated with Smad3, and the C terminus of MITF and the MH1 and linker region of Smad3 were required for this association. Complex formation between Smad3 and MITF was neither necessary nor sufficient for the inhibition of Smad3 signaling by MITF. MITF inhibited the transcriptional activation induced by the MH2 domain of Smad3. In addition, MITF-truncated N-terminal amino acids could associate with Smad3 but did not inhibit Smad3-mediated transcription. The level of Smad3 was decreased by co-expression of MITF but not of dominant-negative MITF, which resulted from proteasomal protein degradation. The changes in the level of Smad3 protein were paralleled by those in Smad3-mediated signaling activity. These findings suggest that MITF negatively regulates Smad-dependent activin/TGF-β signaling in a tissue-specific manner. Mast cells play a crucial role in inflammatory and immediate allergic responses. The multivalent binding of an antigen to receptor-bound IgE provides the trigger for activation of mast cells, leading to secretion of granules containing mast cell proteases (MCPs) 1The abbreviations used are: MCPsmast cell proteasesmMCPsmouse MCPsTGF-βtransforming growth factor-βMITFmicrophthalmia-associated transcription factorSCFstem cell factorBMCMCsbone marrow-derived cultured mast cell progenitorsbHLHLZbasic helix-loop-helix-leucine zipperRT-PCRreverse transcriptase-PCRHAhemagglutininWTwild typeG3PDHglyceraldehyde-3-phosphate dehydrogenasemmouse. (1Williams C.M. Galli S.J. J. Allergy Clin. Immunol. 2000; 105: 847-859Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 2Nadler M.J. Matthews S.A. Turner H. Kinet J.P. Adv. Immunol. 2000; 76: 325-355Crossref PubMed Google Scholar). MCPs are serine proteases, and nine mouse MCPs (mMCPs) have been described to date (3Miller H.R. Pemberton A.D. Immunology. 2002; 105: 375-390Crossref PubMed Scopus (198) Google Scholar). Mouse MCP-1, -2, -4, -5, and -9 are chymases, and mMCP-6 and -7 are tryptases (3Miller H.R. Pemberton A.D. Immunology. 2002; 105: 375-390Crossref PubMed Scopus (198) Google Scholar). Expression of mMCPs in mast cells varies between tissues, e.g. mmcp-7 mRNA is present in differentiated mast cells of the skin but not in mast cells of the intestinal mucosa (4McNeil H.P. Reynolds D.S. Schiller V. Ghildyal N. Gurley D.S. Austen K.F. Stevens R.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11174-11178Crossref PubMed Scopus (127) Google Scholar), whereas mmcp-2 mRNA is expressed in mast cells located in the mucosa of the stomach (5Jippo T. Tsujino K. Kim H.M. Kim D.K. Lee Y.M. Nawa Y. Kitamura Y. Am. J. Pathol. 1997; 150: 1373-1382PubMed Google Scholar). These observations suggest that gene expression of mMCPs is regulated by the local environment (6Kitamura Y. Morii E. Jippo T. Ito A. Int. J. Hematol. 2000; 71: 197-202PubMed Google Scholar), but currently little information is available on regulation of mMCP expression by local growth and differentiation factors (6Kitamura Y. Morii E. Jippo T. Ito A. Int. J. Hematol. 2000; 71: 197-202PubMed Google Scholar). mast cell proteases mouse MCPs transforming growth factor-β microphthalmia-associated transcription factor stem cell factor bone marrow-derived cultured mast cell progenitors basic helix-loop-helix-leucine zipper reverse transcriptase-PCR hemagglutinin wild type glyceraldehyde-3-phosphate dehydrogenase mouse. Activin and transforming growth factor-β (TGF-β) are members of the TGF-β superfamily and are multipotent growth and differentiation factors (7Mathews L.S. Endocr. Rev. 1994; 15: 310-325Crossref PubMed Scopus (274) Google Scholar, 8Luisi S. Florio P. Reis F.M. Petraglia F. Eur. J. Endocrinol. 2001; 145: 225-236Crossref PubMed Scopus (120) Google Scholar). These peptides are not only among the most potent cellular growth inhibitors, but they also regulate other diverse biological processes including early embryonic patterning and cell fate determination. Activin and TGF-β bind to two different types of serine/threonine kinase receptors, termed type I and type II. Type I receptor is activated by type II receptor upon ligand binding and mediates specific intracellular signals. Smads are the central signal mediators of the TGF-β superfamily (9Massagué J. Chen Y.G. Genes Dev. 2000; 14: 627-644PubMed Google Scholar, 10Moustakas A. Souchelnytskyi S. Heldin C.H. J. Cell Sci. 2001; 114: 4359-4369Crossref PubMed Google Scholar). Smad2 and -3 directly interact with type I receptors for activin and TGF-β and become activated through phosphorylation of the C-terminal SSXS motif. Smad2 and -3 then form heteromeric complexes with common partner Smad, Smad4, and translocate into the nucleus. Nuclear Smad complexes bind to transcriptional activators or co-repressors and regulate transcription of target genes (9Massagué J. Chen Y.G. Genes Dev. 2000; 14: 627-644PubMed Google Scholar, 10Moustakas A. Souchelnytskyi S. Heldin C.H. J. Cell Sci. 2001; 114: 4359-4369Crossref PubMed Google Scholar). Previous studies have shown that expression of activin A, a homodimer of inhibin/activin βA, is up-regulated in immune cells, including monocytes (8Luisi S. Florio P. Reis F.M. Petraglia F. Eur. J. Endocrinol. 2001; 145: 225-236Crossref PubMed Scopus (120) Google Scholar, 11Abe M. Shintani Y. Eto Y. Harada K. Kosaka M. Matsumoto T. J. Leukocyte Biol. 2002; 72: 347-352PubMed Google Scholar), macrophages (12Ogawa K. Funaba M. Mathews L.S. Mizutani T. J. Immunol. 2000; 165: 2997-3003Crossref PubMed Scopus (78) Google Scholar), and mast cells (13Funaba M. Ikeda T. Ogawa K. Abe M. Cell. Signal. 2003; 15: 605-613Crossref PubMed Scopus (29) Google Scholar), in response to activation. Activin A induced migration of human monocytes (14Petraglia F. Sacerdote P. Cossarizza A. Angioni S. Genazzani A.D. Franceschi C. Muscettola M. Grasso G. J. Clin. Endocrinol. Metab. 1991; 72: 496-502Crossref PubMed Scopus (72) Google Scholar) and increased gene expression and the activity of matrix metalloproteinase-2 in mouse peritoneal macrophages (12Ogawa K. Funaba M. Mathews L.S. Mizutani T. J. Immunol. 2000; 165: 2997-3003Crossref PubMed Scopus (78) Google Scholar). Furthermore, activin A and TGF-β1 also induced migration at lower concentrations and morphological changes and gene expression of mmcp-1 at higher concentrations in mouse mast cell progenitors (15Miller H.R. Wright S.H. Knight P.A. Thornton E.M. Blood. 1999; 93: 3473-3486Crossref PubMed Google Scholar, 16Funaba M. Ikeda T. Ogawa K. Murakami M. Abe M. J. Leukocyte Biol. 2003; 73: 793-801Crossref PubMed Scopus (51) Google Scholar). In this report, we show that activin and TGF-β up-regulate the mmcp-7 gene, which is transcriptionally regulated. Smad3 but not Smad2 is responsible for the transcriptional activation. Microphthalmia-associated transcription factor (MITF), which is a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, and heart and skeletal muscle (17Hodgkinson C.A. Moore K.J. Nakayama A. Steingrimsson E. Copeland N.G. Jenkins N.A. Arnheiter H. Cell. 1993; 74: 395-404Abstract Full Text PDF PubMed Scopus (947) Google Scholar, 18Oboki K. Morii E. Kataoka T.R. Jippo T. Kitamura Y. Biochem. Biophys. Res. Commun. 2002; 290: 1250-1254Crossref PubMed Scopus (42) Google Scholar, 19Takemoto C.M. Yoon Y.J. Fisher D.E. J. Biol. Chem. 2002; 277: 30244-30252Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), accelerates the breakdown of Smad3 protein by the proteosomal system. As a result, the Smad3-mediated transcription is inhibited in the presence of MITF. Our results suggest that the tissue-specific and negative regulation of Smad3 activity by MITF plays a role in the discrete control of activin and TGF-β activities in mast cells. Materials—Recombinant human activin A was kindly provided by Dr. A. F. Parlow through the National Pituitary and Hormone Distribution Program at NIDDK, National Institutes of Health. Purified TGF-β1 was purchased from BD Biosciences. Stem cell factor (SCF) was purchased from PeproTech EC (London, UK). Pokeweed mitogen was purchased from Seikagaku Kogyo (Tokyo, Japan). Mouse monoclonal anti-FLAG antibody (M2), anti-HA antibody (12CA5), and anti-Myc antibody (9E10) were obtained from Sigma, Roche Applied Science, and Santa Cruz Biotechnology, respectively. Lactacystin and MG-132 were obtained from Calbiochem. cDNA Constructs—Expression vectors for activin type II receptor (ActRII) (20Mathews L.S. Vale W.W. J. Biol. Chem. 1993; 268: 19013-19018Abstract Full Text PDF PubMed Google Scholar), ALK4 (21Willis S.A. Zimmerman C.M. Li L. Mathews L.S. Mol. Endocrinol. 1996; 10: 367-379Crossref PubMed Scopus (89) Google Scholar), and constitutively active ALK4 (ALK4(TE)) with Thr-206 replaced by Glu (21Willis S.A. Zimmerman C.M. Li L. Mathews L.S. Mol. Endocrinol. 1996; 10: 367-379Crossref PubMed Scopus (89) Google Scholar) were kindly provided by Dr. L. S. Mathews. The N-terminal HA-tagged constitutively active MEK1 (MEK1(ED)), with Ser-218 and -222 replaced by Glu and Asp, respectively (22Sugimoto T. Stewart S. Han M. Guan K.L. EMBO J. 1998; 17: 1717-1727Crossref PubMed Scopus (90) Google Scholar), cDNA was kindly provided by Dr. K. L. Guan. Expression vectors pEF-BOS (23Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5322Crossref PubMed Scopus (1502) Google Scholar) containing the whole coding region of wild-type (WT)-MITF or mi-MITF (24Tsujimura T. Morii E. Nozaki M. Hashimoto K. Moriyama Y. Takebayashi K. Kondo T. Kanakura Y. Kitamura Y. Blood. 1996; 88: 1225-1233Crossref PubMed Google Scholar) and reporter plasmids of mmcp-7 promoter (pP7-185 (25Ogihara H. Morii E. Kim D.K. Oboki K. Kitamura Y. Blood. 2001; 97: 645-651Crossref PubMed Scopus (38) Google Scholar)) and of mmcp-6 (pP6-171 (26Ogihara H. Kanno T. Morii E. Kim D.K. Lee Y.M. Sato M. Kim W.Y. Nomura S. Ito Y. Kitamura Y. Oncogene. 1999; 18: 4632-4639Crossref PubMed Scopus (31) Google Scholar)) were provided by Drs. Y. Kitamura and E. Morii; Dr. M. Whitman provided AR3-luc (27Hayashi H. Abdollah S. Qiu Y. Cai J. Xu Y.Y. Grinnell B.W. Richardson M.A. Topper J.N. Gimbrone Jr., M.A. Wrana J.L. Falb D. Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1164) Google Scholar) and FoxH3 (28Chen X. Rubock M.J. Whitman M. Nature. 1996; 383: 691-696Crossref PubMed Scopus (632) Google Scholar) cDNAs; TGF-β type II receptor (TβRII) cDNA (29Lin H.Y. Wang X.F. Ng-Eaton E. Weinberg R.A. Lodish H.F. Cell. 1992; 68: 775-785Abstract Full Text PDF PubMed Scopus (969) Google Scholar) was donated by Dr. H. F. Lodish; ALK5 cDNA (30ten Dijke P. Yamashita H. Ichijo H. Franzen P. Laiho M. Miyazono K. Heldin C.H. Science. 1994; 264: 101-104Crossref PubMed Scopus (511) Google Scholar) was donated by Dr. K. Miyazono; constitutively active ALK5 (ALK5(TD)) cDNA (31Wieser R. Wrana J.L. Massagué J. EMBO J. 1995; 14: 2199-2208Crossref PubMed Scopus (597) Google Scholar) was donated by Dr. X.-F. Wang; and HA-pcDNA3 and FLAG-pcDNA3 expression vectors (32Inohara N. Koseki T. Chen S. Wu X. Nunez G. EMBO J. 1998; 17: 2526-2533Crossref PubMed Scopus (283) Google Scholar) were from by Drs. N. Inohara and T. Koseki. A reporter plasmid of mmcp-9 promoter (mMCP-9-(-192)) was prepared as described previously (33Murakami M. Ikeda T. Ogawa K. Funaba M. Biochem. Biophys. Res. Commun. 2003; 311: 4-10Crossref PubMed Scopus (14) Google Scholar). The human Smad2, Smad3, and Smad4 cDNAs, which were provided by Dr. R. Derynck, were subcloned into the EcoRI and XbaI sites of HA-pcDNA3 or FLAG-pcDNA3 to produce N-terminal HA-tagged or FLAG-tagged proteins. The WT-MITF and mi-MITF cDNAs were subcloned into XbaI and EcoRI sites of 6Myc-pcDNA3 (34Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (871) Google Scholar) to produce a MITF protein containing six tandem copies of the Myc epitope at the N terminus. Cell Culture and Transfection—Bone marrow-derived cultured mast cell progenitors (BMCMCs) were cultured from the bone marrow cells of BALB/c mice as described previously (16Funaba M. Ikeda T. Ogawa K. Murakami M. Abe M. J. Leukocyte Biol. 2003; 73: 793-801Crossref PubMed Scopus (51) Google Scholar). More than 95% of the trypan blue-excluding viable cells were mast cells on the basis of staining with acid toluidine. L17 cells, a derivative of the mink lung epithelial cell line (Mv1Lu) (35Attisano L. Carcamo J. Ventura F. Weis F.M. Massagué J. Wrana J.L. Cell. 1993; 75: 671-680Abstract Full Text PDF PubMed Scopus (604) Google Scholar), obtained from Dr. J. Massagué, were cultured as described previously (21Willis S.A. Zimmerman C.M. Li L. Mathews L.S. Mol. Endocrinol. 1996; 10: 367-379Crossref PubMed Scopus (89) Google Scholar, 36Funaba M. Mathews L.S. Mol. Endocrinol. 2000; 14: 1583-1591Crossref PubMed Google Scholar). HepG2, COS7, and HEK293 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium with 10% calf serum. For transient transfection, cells in 24- or 6-well plates were transfected using PolyFect transfection reagent (Qiagen). RNA Isolation, Competitive RT-PCR—Total RNA from BMCMCs was isolated using an RNA isolation kit (RNeasy, Qiagen) according to the manufacturer's protocol. Recovered RNA was reverse-transcribed as described previously (12Ogawa K. Funaba M. Mathews L.S. Mizutani T. J. Immunol. 2000; 165: 2997-3003Crossref PubMed Scopus (78) Google Scholar). To examine the mRNA levels semi-quantitatively, competitive RT-PCR was performed as described previously (16Funaba M. Ikeda T. Ogawa K. Murakami M. Abe M. J. Leukocyte Biol. 2003; 73: 793-801Crossref PubMed Scopus (51) Google Scholar). For the mmcp-7 gene transcript (GenBank™ accession number NM031187), oligonucleotides encoding positions from 177 to 198 and 812 to 789 were used as the PCR primers. A competitor for mMCP-7 was made by a deletional mutation of the mMCP-7 PCR product, which deleted a cDNA segment between positions 199 and 493. The competitor was made by overlap extension PCR of the native PCR product, followed by purification using a Suprec-02 column (Takara, Tokyo, Japan). The PCR primers and competitor for G3PDH were as described previously (12Ogawa K. Funaba M. Mathews L.S. Mizutani T. J. Immunol. 2000; 165: 2997-3003Crossref PubMed Scopus (78) Google Scholar). A constant amount (2 × 10-5 amol for mMCP-7 and 5 × 10-4 amol for G3PDH) of the competitor template was co-amplified with the specific primers with reverse-transcribed samples or varying amounts of the target cDNA standard. The PCR products were separated on 2% agarose gels in 1× TAE buffer and visualized with ethidium bromide. Real Time Quantitative RT-PCR—Total RNA from BMCMCs was reverse-transcribed as described previously (12Ogawa K. Funaba M. Mathews L.S. Mizutani T. J. Immunol. 2000; 165: 2997-3003Crossref PubMed Scopus (78) Google Scholar). PCR was performed in a total volume of 25 μl with SYBR Green PCR Master Mix (Applied Biosystems), 2 μl of cDNA, and 200 nm of each primer. PCR primers were designed with the computer program Primer Express (Applied Biosystems), using parameters recommended by the manufacturer. For the mmcp-7 and g3pdh gene transcripts (GenBank™ accession numbers NM031187 and M32599, respectively), oligonucleotides encoding positions from 361 to 379 and 425 to 404 for mMCP-7 and those from 740 to 759 and 812 to 789 for G3PDH were used as the PCR primers. Reactions were carried out in an ABI-prism 7700 sequence detector (Applied Biosystems), using the following conditions: an initial denaturation step consisted of 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Levels of mMCP-7 and G3PDH expression in each sample were determined by using the relative standard curve method. A relative amount of DNA of mMCP-7 was expressed as a ratio to G3PDH DNA, and the mMCP-7 level in BMCMCs without TGF-β1, activin A, and SCF was 1. Reporter Assay—Luciferase assays were conducted as described previously (36Funaba M. Mathews L.S. Mol. Endocrinol. 2000; 14: 1583-1591Crossref PubMed Google Scholar, 37Funaba M. Zimmerman C.M. Mathews L.S. J. Biol. Chem. 2002; 277: 41361-41368Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Cells were transiently transfected with the indicated expression vectors, reporter construct, or a plasmid expressing β-galactosidase (pCMV-βGal). Equal amounts of DNA were transfected in each experiment and adjusted with pcDNA1 and pcDNA3, and cells were harvested 40 h after transfection. Luciferase activity was normalized to β-galactosidase activity, and the luciferase activity in the cell lysate transfected with empty vector was set at 1. Sequential Immunoprecipitation and Immunoblot—COS7 cells were transfected with HA-tagged Smad and Myc-tagged MITF. Twenty seven hours after transfection, cells were treated with or without 10 μm lactacystin or 50 μm MG-132 for 16 h. The cells were lysed in lysis buffer (20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 10% glycerol, 1mm phenylmethylsulfonyl fluoride, 1% aprotinin). After 30 min on ice, cell debris was removed by centrifugation at 600 × g for 5 min at 4 °C, and the supernatant was immunoprecipitated overnight at 4 °C with anti-HA antibody (12CA5) and incubated with protein G-agarose beads for 1 h at 4 °C. The beads were washed four times with lysis buffer, followed by elution in SDS-PAGE sample buffer. The immunoprecipitates were subjected to Western blotting with anti-HA antibody (12CA5) or anti-Myc antibody (9E10) as primary antibody, and the bands were visualized with ECL Plus reagent (Invitrogen). Mammalian Two-hybrid Assay—Mammalian two-hybrid assays were performed using the checkmate mammalian two-hybrid system (Promega), according to the manufacturer's protocol. In brief, HEK293 cells were co-transfected with the plasmid of interest, a plasmid expressing β-galactosidase (pCMV-βGal). Equal amounts of DNA were transfected in each experiment and adjusted with pBIND or pACT. Twenty seven hours after transfection, cells were treated with or without 10 μm lactacystin for 16 h. Luciferase activity was normalized to β-galactosidase activity, and the luciferase activity in the cell lysate transfected with empty vector was set at 1. Mouse mcp-7 Gene Is Transcriptionally Activated by Smad3—To investigate the effect of activin and TGF-β on BMCMCs, gene transcripts of mmcp-1, -7, and -9 were first examined by RT-PCR, which was conducted under nonsaturating conditions for the PCR products. The band intensities of mMCP-1 and mMCP-7 were reproducibly increased by treatment with activin A or TGF-β1 (data not shown). Activin A- and TGF-β1-induced gene expression of mMCP-1 has been described elsewhere (16Funaba M. Ikeda T. Ogawa K. Murakami M. Abe M. J. Leukocyte Biol. 2003; 73: 793-801Crossref PubMed Scopus (51) Google Scholar). To characterize mmcp-7 gene induction in response to activin A and TGF-β1 in detail, we semi-quantitatively measured levels of mmcp-7 gene transcript by competitive RT-PCR. Treatment of BMCMCs with activin A or TGF-β1 for 24 h clearly increased the gene transcript level of mmcp-7 (Fig. 1A). Consistent with a previous study (38Gu Y. Byrne M.C. Paranavitana N.C. Aronow B. Siefring J.E. D'Souza M. Horton H.F. Quilliam L.A. Williams D.A. Mol. Cell. Biol. 2002; 22: 7645-7657Crossref PubMed Scopus (51) Google Scholar), treatment with SCF also increased the mRNA level of mmcp-7. Co-treatment with SCF and activin A or TGF-β1 resulted in further accumulation of mmcp-7 mRNA (Fig. 1A). Real time quantitative RT-PCR analyses also revealed the stimulatory effect of activin A and TGF-β1 on mmcp-7 gene transcript (Fig. 1B). To examine whether the mmcp-7 gene induction by activin A and TGF-β1 is transcriptionally regulated, we performed transcriptional activation assays using the 5′-flanking region of mmcp-7 fused to the luciferase reporter gene (25Ogihara H. Morii E. Kim D.K. Oboki K. Kitamura Y. Blood. 2001; 97: 645-651Crossref PubMed Scopus (38) Google Scholar). As shown in Fig. 2A, transfection of type II (TβRII) and type I (ALK5) TGF-β receptor into HepG2 cells did not affect luciferase expression, whereas transfection of a constitutively active type I TGF-β receptor (ALK5(TD)) (31Wieser R. Wrana J.L. Massagué J. EMBO J. 1995; 14: 2199-2208Crossref PubMed Scopus (597) Google Scholar) increased luciferase expression 5.6-fold. Consistent with expression of a series of TGF-β receptor isoforms, expression of a constitutively active type I activin receptor (ALK4(TE)) (21Willis S.A. Zimmerman C.M. Li L. Mathews L.S. Mol. Endocrinol. 1996; 10: 367-379Crossref PubMed Scopus (89) Google Scholar) but not of a type II (ActRII) or type I (ALK4) activin receptor increased luciferase expression 6.3-fold. Transcriptional assays in NIH3T3 cells and L17 cells, a derivative of mink lung epithelial cells (35Attisano L. Carcamo J. Ventura F. Weis F.M. Massagué J. Wrana J.L. Cell. 1993; 75: 671-680Abstract Full Text PDF PubMed Scopus (604) Google Scholar), also showed increased luciferase expression only in cells expressing constitutively active type I receptors (ALK5(TD) and ALK4(TE)) (Fig. 2, B and C). These results suggest that TGF-β- and activin-induced mmcp-7 mRNA is transcriptionally regulated and that the receptor activation is responsible for transcriptional activation of mmcp-7. When individual Smad proteins were overexpressed in HepG2 cells (Fig. 2D), NIH3T3 cells (Fig. 2E), and L17 cells (Fig. 2F), Smad3, but not Smad2 or -4, increased luciferase expression. Overexpression of a constitutively active Smad3, which replaced the last three Ser to Glu (Smad3–3E) (39Chacko B.M. Qin B. Correia J.J. Lam S.S. de Caestecker M.P. Lin K. Nat. Struct. Biol. 2001; 8: 248-253Crossref PubMed Scopus (120) Google Scholar), tended to further increase luciferase expression as compared with that of Smad3, whereas overexpression of a constitutively active Smad2 (Smad2–2E) (36Funaba M. Mathews L.S. Mol. Endocrinol. 2000; 14: 1583-1591Crossref PubMed Google Scholar) did not affect luciferase expression. Smad3 Signaling Is Blocked by MITF—MITF is a transcription factor of the basic helix-loop-helix-leucine zipper (bHLHLZ) family, which controls gene expression of several mmcps (6Kitamura Y. Morii E. Jippo T. Ito A. Int. J. Hematol. 2000; 71: 197-202PubMed Google Scholar). The relationship between TGF-β signaling and the effect of MITF on mmcp-7 transcription was explored in HepG2 cells (Fig. 3A), NIH3T3 cells (Fig. 3B), and L17 cells (Fig. 3C). Co-expression of WT-MITF inhibited the luciferase expression induced by ALK5(TD), suggesting that MITF negatively regulates TGF-β-induced transcriptional activation of mmcp-7. Expression of WT-MITF also decreased Smad3-induced transcription of mmcp-7 in a dose-dependent manner (Fig. 4A). In contrast, overexpression of mi-MITF, a dominant-negative mutant that deletes one of four consecutive Arg in the basic domain (6Kitamura Y. Morii E. Jippo T. Ito A. Int. J. Hematol. 2000; 71: 197-202PubMed Google Scholar), had a weak effect on inhibition of the induced luciferase expression (Fig. 4A). We also examined the effect of MITF expression on transcriptional activation of AR3-luc, which contains regulatory sequences from the Xenopus mix2 gene (27Hayashi H. Abdollah S. Qiu Y. Cai J. Xu Y.Y. Grinnell B.W. Richardson M.A. Topper J.N. Gimbrone Jr., M.A. Wrana J.L. Falb D. Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1164) Google Scholar) and is activated by co-expression of the transcription factors FoxH3 and Smad2 (36Funaba M. Mathews L.S. Mol. Endocrinol. 2000; 14: 1583-1591Crossref PubMed Google Scholar, 37Funaba M. Zimmerman C.M. Mathews L.S. J. Biol. Chem. 2002; 277: 41361-41368Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Similar to the inhibitory effect on mmcp-7 transcription, WT-MITF inhibited FoxH3-dependent and Smad2-mediated AR3 transcription, whereas mi-MITF hardly affected transcriptional activity (Fig. 4B). These results suggest that functional MITF acts as a negative regulator for Smad2 and -3 signaling. Transcriptional inhibition of WTMITF was not a nonspecific event, since overexpression of WT-MITF but not mi-MITF increased luciferase expression when the 5′-flanking region of mmcp-6 fused to the luciferase gene was used as a reporter gene (Fig. 4C). In addition, when the 5′-flanking region of mmcp-9 fused to the luciferase gene was used, overexpression of ALK5(TD) and Smad3 neither increased luciferase expression nor affected WT-MITF-induced transcription of mMCP-9-luc (Fig. 4D). MITF has several isoforms. The mouse MITF gene contains at least five isoform-specific first exons, and exons 2–9 of all isoforms of MITF are the same (18Oboki K. Morii E. Kataoka T.R. Jippo T. Kitamura Y. Biochem. Biophys. Res. Commun. 2002; 290: 1250-1254Crossref PubMed Scopus (42) Google Scholar, 40Udono T. Yasumoto K. Takeda K. Amae S. Watanabe K. Saito H. Fuse N. Tachibana M. Takahashi K. Tamai M. Shibahara S. Biochim. Biophys. Acta. 2000; 1491: 205-219Crossref PubMed Scopus (122) Google Scholar, 41Takeda K. Yasumoto K. Kawaguchi N. Udono T. Watanabe K. Saito H. Takahashi K. Noda M. Shibahara S. Biochim. Biophys. Acta. 2002; 1574: 15-23Crossref PubMed Scopus (72) Google Scholar). Two isoforms of MITF, MITF-mc (19Takemoto C.M. Yoon Y.J. Fisher D.E. J. Biol. Chem. 2002; 277: 30244-30252Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and MITF-E (18Oboki K. Morii E. Kataoka T.R. Jippo T. Kitamura Y. Biochem. Biophys. Res. Commun. 2002; 290: 1250-1254Crossref PubMed Scopus (42) Google Scholar), inhibited Smad3-mediated mMCP-7 transcription and FoxH3-dependent and Smad2-mediated AR3-luc transcription (data not shown). MITF Binds to and Induces Breakdown of Smad Proteins—To explore the molecular basis of the inhibitory effect of MITF on Smad2 and -3 signaling, we examined the expression of Smads in response to co-expression of MITF in COS7 cells. Protein expression levels of Smad2, -3, and -4 were significantly decreased by co-expression of WT-MITF (Fig. 5, A–C). In contrast, mi-MITF hardly affected the protein level of Smad2, -3, or -4. The WT-MITF-induced decreases in Smad protein level were independent of TGF-β signaling; expression of ALK5(TD) had no effect on decreases in Smad2 and -3 when co-expressed with WT-MITF. The decreased protein levels of Smad associated with co-expression of WT-MITF were not nonspecific events, because the protein level of MEK1, a mitogen-activated protein kinase kinase, was relatively unchanged in response to WT-MITF expression (Fig. 5D). Next, we examined whether the decreased protein levels of Smad2 and -3 due to co-expression of WT-MITF resulted from proteasomal breakdown. Treatment with lactacystin (10 μm), which inhibits activity of 20 S proteasome (42Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1501) Google Scholar), effectively blocked the decreases in Smad2 and -3 proteins caused by co-expression of WT-MITF, resulting in constant protein levels of Smad2 and -3 irrespective of co-expression of WT-MITF (Fig. 6A). In addition, decreases in protein expression of Smad2 and -3 by co-expression of WT-MITF were also blocked by treatment with another proteasome inhibitor, MG-132 (50 μm) (Fig. 6B). These results suggest that expression of WT-MITF but not mi-MITF induces proteasome-dependent degradation of Smad2 and -3. Smad signaling is modulated by association" @default.
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• W2015979443 title "Transcriptional Activation of Mouse Mast Cell Protease-7 by Activin and Transforming Growth Factor-β Is Inhibited by Microphthalmia-associated Transcription Factor" @default.
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