Myogenesis and MyoD Down-regulate Sp1

Muscle cell differentiation caused a reduction of glucose transport, GLUT1 glucose transporter expression, and GLUT1 mRNA levels. A fragment of 2.1 kilobases of the rat GLUT1 gene linked to chloramphenicol acetyltransferase drove transcriptional activity in myoblasts, and differentiation caused a decrease in transcription. Transient transfection of 5′ and 3′ deletion constructs showed that the fragment −99/−33 of the GLUT1 gene drives transcriptional activity of the GLUT1 gene and participates in the reduced transcription after muscle differentiation. Electrophoretic mobility shift assays showed the binding of Sp1 protein to the fragment −102/−37 in the myoblast state but not in myotubes, and Sp1 was found to transactivate the GLUT1 promoter. Western blot analysis indicated that Sp1 was drastically down-regulated during myogenesis. Furthermore, the forced over-expression of MyoD in C3H10T1/2 cells mimicked the effects observed during myogenesis, Sp1 down-regulation and reduced transcriptional activity of the GLUT1 gene promoter.In all, these data suggest a regulatory model in which MyoD activation during myogenesis causes the down-regulation of Sp1, which contributes to the repression of GLUT1 gene transcription and, therefore, leads to the reduction in GLUT1 expression and glucose transport. Muscle cell differentiation caused a reduction of glucose transport, GLUT1 glucose transporter expression, and GLUT1 mRNA levels. A fragment of 2.1 kilobases of the rat GLUT1 gene linked to chloramphenicol acetyltransferase drove transcriptional activity in myoblasts, and differentiation caused a decrease in transcription. Transient transfection of 5′ and 3′ deletion constructs showed that the fragment −99/−33 of the GLUT1 gene drives transcriptional activity of the GLUT1 gene and participates in the reduced transcription after muscle differentiation. Electrophoretic mobility shift assays showed the binding of Sp1 protein to the fragment −102/−37 in the myoblast state but not in myotubes, and Sp1 was found to transactivate the GLUT1 promoter. Western blot analysis indicated that Sp1 was drastically down-regulated during myogenesis. Furthermore, the forced over-expression of MyoD in C3H10T1/2 cells mimicked the effects observed during myogenesis, Sp1 down-regulation and reduced transcriptional activity of the GLUT1 gene promoter. In all, these data suggest a regulatory model in which MyoD activation during myogenesis causes the down-regulation of Sp1, which contributes to the repression of GLUT1 gene transcription and, therefore, leads to the reduction in GLUT1 expression and glucose transport. The formation of skeletal muscle during embryogenesis involves, first, commitment of mesodermal stem cells to the myogenic lineage. Myoblast cells, although undifferentiated and capable of continued proliferation, differentiate when they receive the appropriate environmental signals, fuse, and form multinucleate myotubes. At the same time as this morphological differentiation, a battery of adult muscle-specific genes whose products are required for the unique contractile and metabolic properties of the muscle fiber are activated (1Lassar A. Münsterberg A. Curr. Opin. Cell Biol. 1994; 6: 432-442Crossref PubMed Scopus (143) Google Scholar, 2Olson E.N. Klein W.H. Genes 8: 1-8Crossref PubMed Scopus (606) Google Scholar). The factors that regulate the expression of muscle-specific genes following commitment to terminal differentiation are well established. The best characterized are the members of the myogenic basic helix-loop-helix (bHLH) 1The abbreviations used are: bHLH, basic helix-loop-helix; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; Id, differentiation inhibitor; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus; PBS, phosphate-buffered saline; bp, base pair(s). 1The abbreviations used are: bHLH, basic helix-loop-helix; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; Id, differentiation inhibitor; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus; PBS, phosphate-buffered saline; bp, base pair(s). protein family or MyoD family, that function as master regulators of muscle cell fate during development (2Olson E.N. Klein W.H. Genes 8: 1-8Crossref PubMed Scopus (606) Google Scholar, 3Edmondson D.G. Olson E.N. J. Biol. Chem. 1994; 268: 755-758Abstract Full Text PDF Google Scholar). Four members of the family have been cloned: MyoD (4Davis R.L. Weintraub H. Lassar A.B. Cell. 1987; 51: 987-1000Abstract Full Text PDF PubMed Scopus (2422) Google Scholar), Myf5 (5Braun T. Buschhausen-Denker G. Bober E. Tannich E. Arnold H.H. EMBO J. 1989; 8: 701-709Crossref PubMed Scopus (650) Google Scholar), myogenin (6Edmondson D.G. Olson E.N. Genes 3: 628-640Crossref PubMed Scopus (597) Google Scholar, 7Wright W.E. Sassoon D.A. Lin V.K. Cell. 1989; 56: 607-617Abstract Full Text PDF PubMed Scopus (924) Google Scholar), and Mrf4 (8Braun T. Bober E. Winter B. Rosenthal N. Arnold H.H. EMBO J. 1990; 9: 821-831Crossref PubMed Scopus (353) Google Scholar, 9Miner J.H. Wold B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1089-1093Crossref PubMed Scopus (344) Google Scholar, 10Rhodes S.J. Konieczny S.F. Genes 3: 2050-2061Crossref PubMed Scopus (560) Google Scholar). Each of these factors is expressed exclusively in skeletal muscle, and when expressed ectopically in a variety of non-muscle cell types, they activate the complete program of myogenic differentiation (2Olson E.N. Klein W.H. Genes 8: 1-8Crossref PubMed Scopus (606) Google Scholar). All the members of the myogenic bHLH family activate the transcription of muscle-specific genes by binding to the E-box consensus sequence (CANNTG) in muscle gene promoters and enhancers. However, not all muscle genes contain functional E boxes in their regulatory promoter regions, and myogenic bHLH proteins can also activate transcription of muscle-specific genes that lack E boxes in their control regions (3Edmondson D.G. Olson E.N. J. Biol. Chem. 1994; 268: 755-758Abstract Full Text PDF Google Scholar). As expected, from these data, other muscle-specific transcription factors have been described to function as intermediates in the activation of gene expression during myogenesis, such as the M-CAT binding factor (11Mar J.H. Ordahl C.P. Mol. Cell. Biol. 1990; 10: 4271-4283Crossref PubMed Scopus (146) Google Scholar), and the myocyte enhancer factor 2 (MEF2) (12Gosset L.A. Kelvin D.J. Sternberg E.A. Olson E.N. Mol. Cell. Biol. 1989; 9: 5022-5033Crossref PubMed Scopus (440) Google Scholar,13Yu Y.-T. Breitbart R.E. Smoot L.B. Lee Y. Mahdavi V. Nadal-Ginard B. Genes 6: 1783-1798Crossref PubMed Scopus (380) Google Scholar). Myogenesis is also associated with down-regulation of several growth-regulated myoblast proteins, including c-Fos (14Trouche D. Grigoriev M. Lenormand J.L. Robin P. Leibovitch S.A. Sassone-Corsi P. Harel-Bellan A. Nature. 1993; 363: 79-82Crossref PubMed Scopus (77) Google Scholar), β- and γ-actins (15Hayward L.J. Schwarts R.J. J. Cell Biol. 1986; 102: 1485-1493Crossref PubMed Scopus (104) Google Scholar, 16Paterson B.M. Eldridge J.D. Science. 1984; 224: 1436-1438Crossref PubMed Scopus (68) Google Scholar), and the differentiation inhibitor (Id) (17Benezra R. Davis R.L. Lockshon D. Turner D.L. Weintraub H. Cell. 1990; 61: 49-59Abstract Full Text PDF PubMed Scopus (1785) Google Scholar). In contrast to the wealth of information regarding the mechanisms that activate genes participating in the myotube phenotype, relatively little is known regarding regulatory sequences or factors involved in the control of the repression of the muscle embryo genes during muscle cell differentiation. Thus, it has been described that an activating transcription factor site is required for the expression of the Id2A gene in muscle cells, and that the binding of nuclear factors to the activating transcription factor site is decreased during myogenic differentiation (18Kurabayashi M. Dutta S. Kedes L. J. Biol. Chem. 1994; 269: 31162-31170Abstract Full Text PDF PubMed Google Scholar). Glucose transporter expression is developmentally regulated in skeletal muscle (19Santalucı́a T. Camps M. Castelló A. Muñoz P. Nuel A. Testar X. Palacı́n M. Zorzano A. Endocrinology. 1992; 130: 837-846Crossref PubMed Scopus (0) Google Scholar, 20Castelló A. Cadefau J. Cussó R. Testar X. Hesketh J.E. Palacı́n M. Zorzano A. J. Biol. Chem. 1993; 268: 14998-15003Abstract Full Text PDF PubMed Google Scholar). Thus, during fetal and early postnatal life, GLUT1 is highly expressed in heart and skeletal muscles. Postnatal life is characterized by GLUT1 repression in muscle, which is concomitant with the induction of GLUT4 expression (19Santalucı́a T. Camps M. Castelló A. Muñoz P. Nuel A. Testar X. Palacı́n M. Zorzano A. Endocrinology. 1992; 130: 837-846Crossref PubMed Scopus (0) Google Scholar). Similarly, it has been reported that myogenesis leads to induction of GLUT4 expression and repression of GLUT1 expression (21Mitsumoto Y. Burdett E. Grant A. Klip A. Biochem. Biophys. Res. Commun. 1991; 175: 652-659Crossref PubMed Scopus (124) Google Scholar, 22Mitsumoto Y. Klip A. J. Biol. Chem. 1992; 267: 4957-4962Abstract Full Text PDF PubMed Google Scholar). Based on the fact that congenital hypothyroidism partially blocks GLUT1 repression associated with neonatal life (23Castelló A. Rodrı́guez-Manzaneque J.C. Camps M. Pérez-Castillo A. Testar X. Palacı́n M. Santos A. Zorzano A. J. Biol. Chem. 1994; 269: 5905-5912Abstract Full Text PDF PubMed Google Scholar) and that denervation up-regulates GLUT1 in skeletal muscle (20Castelló A. Cadefau J. Cussó R. Testar X. Hesketh J.E. Palacı́n M. Zorzano A. J. Biol. Chem. 1993; 268: 14998-15003Abstract Full Text PDF PubMed Google Scholar, 24Block N.E. Menick D.R. Robinson K.A. Buse M.G. J. Clin. Invest. 1991; 88: 1546-1552Crossref PubMed Scopus (79) Google Scholar, 25Coderre L. Monfar M.M. Chen K.S. Heydrick S.J. Kurowski T.G. Ruderman N.B. Pilch P.F. Endocrinology. 1992; 131: 1821-1825Crossref PubMed Scopus (66) Google Scholar, 26Henriksen E.J. Rodnick K.J. Mondon C.E. James D.E. Holloszy J.O. J. Appl. Physiol. 1991; 70: 2322-2327Crossref PubMed Scopus (17) Google Scholar), it is likely that thyroid hormones and muscle innervation play a role in the regulation of muscle GLUT1 expressionin vivo. However, the detailed mechanisms that contribute to GLUT1 repression during perinatal development and myogenesis are largely unknown. Here we have examined the mechanisms that repress GLUT1 expression during myogenesis. 125I-labeled protein A and α-32P-dCTP from was from was from and was from was from and used from Dulbecco's modified Eagle's and from rat skeletal muscle cell was by B. Nadal-Ginard C3H10T1/2 cells with MyoD from V. The the of the rat GLUT1 sequence was from M. of was from was from N. PubMed Scopus Google Scholar). was by of the after the the glucose transporter of of was used a and was with protein in in to was used a in in and of the with the was A for or the Western blot in which was in the was the as described by and P. N. Biochem. 1987; PubMed Scopus Google Scholar). All was in the of and to the of was on a and on The in and in was with by to the of to the of of and to blot was as reported P. Testar X. Palacı́n M. Zorzano A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The rat for GLUT1 was a fragment from M. of and was with by was by a fragment the rat GLUT1 promoter from to to the transcription site of was with in of the with in the of the and with The GLUT1 promoter fragment was in the of and fragment and to the 5′ by with with and with and with and and with and and The was by a the fragment from a fragment of the GLUT1 promoter in The fragment was the site of The 3′ deletion constructs by with and with cells in for 2 in with fetal and transfection was by the PubMed Scopus Google Scholar). of of deletion acetyltransferase of control and of was to the and was the cells and with of in for with DMEM, and with complete for the myotubes, the was to differentiation with fetal after the with the The cells 2 with and by in of and The cells by in a for and the was in of The cells by of and in a for the was The activity of of was by of and the for At the of the and Mol. Cell. Biol. PubMed Scopus Google Scholar) The activity was an activity was as described J. T. A Scholar). of the nuclear protein was as described by R. R.E. in 175: 652-659Crossref PubMed Scopus (124) Google Scholar, 22Mitsumoto Y. Klip A. J. Biol. Chem. 1992; 267: 4957-4962Abstract Full Text PDF PubMed Google Scholar). A reduction in GLUT1 mRNA was also in with myoblast cells in for of found in The reduction in GLUT1 protein and mRNA in which in the of protein or not the for the repression of GLUT1 expression, or in the of for time and the of GLUT1 mRNA 2 that the of GLUT1 mRNA was and myoblast and myotube cells 2 the reduction in GLUT1 mRNA during muscle cell differentiation is not to in the of GLUT1 myoblast or myotube cells with a fragment of the GLUT1 gene promoter to the gene with the gene showed of to by cells 2 with the in caused a in activity Furthermore, activity in was reduced with in 2 that the fragment of the GLUT1 gene information that is to transcriptional activity in the myoblast and that repression in to myogenesis. the involved in the transcriptional activity of the GLUT1 5′ deletion constructs of the GLUT1 promoter to the gene and in myoblast and myotube cells from to of the GLUT1 gene caused in activity and deletion from to caused of a The transcriptional activity of the was and a decrease was after deletion from to However, deletion of a base to to a reduction in the transcriptional The repression of transcriptional activity to myogenesis was in the although found in the The the to the transcription site S.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of the of the fragment of the GLUT1 promoter which 3′ of the transcription constructs by 3′ deletion of base and caused a reduction in transcriptional activity for the transcriptional these myogenesis reduced the transcriptional activity of all constructs data that the fragment −99/−33 is for the transcriptional activity of the GLUT1 promoter. Furthermore, this with the fragment the to to muscle cell The −99/−33 fragment of the GLUT1 gene consensus Sp1 and nuclear proteins to the fragment −99/−33 of the GLUT1 a fragment the sequence −102/−37 was and assays in the of nuclear from or A of was in nuclear of and to myoblasts, and and in in myotubes. In and in in was the to a in from and from binding of nuclear factors from and to the GLUT1 fragment was with of nuclear from or after of differentiation and on a The of the formation was examined by of the of the −102/−37 fragment used as a A is the that the binding of the assays in the of an of assays in the of which the Sp1 and an site in a and found in myoblast in the of the sequence to the site or with which the not we on the of the and of myoblast Based on the of a Sp1 site in we binding of factors to this site for and assays in the of an of a Sp1 site these and in the of of the Sp1 Furthermore, the in the of an of in which the Sp1 site was to and Sp1 protein to a family of transcription factors T. G. J.L. R. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar, K.A. R. Biochem. Sci. 1986; Full Text PDF Scopus Google Scholar), and the formation of in is to the of or in the D.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Based on in the or of or The of to the the formation of and in a In of caused a of and the binding of Sp1 shift assays also this assays in the of an to the of a of and to a the as a was as and In these Sp1 protein was also with fragment −102/−37 that Sp1 a with fragment which a to and the formation of this was in the of an of In of and a a A was observed when Sp1 was with that Sp1 in from to the GLUT1 promoter. Sp1 the transcriptional activity of the GLUT1 or with the and the for Sp1 caused a of the GLUT1 promoter activity in In Sp1 activated the GLUT1 promoter activity in the transcriptional activity in was in have found that Sp1 protein to the GLUT1 gene promoter in but not in myotubes. the of the mechanisms we the of Sp1 protein in nuclear from and Sp1 protein was observed in Western blot as with an of and which is in with R. Science. 1986; PubMed Scopus Google Scholar, P. S. Mol. Cell. Biol. PubMed Scopus Google Scholar). The of Sp1 protein in was in of Sp1 protein in for of found in was the of the transcription factor was in from or not The best characterized factors that regulate the terminal differentiation of the muscle cells are the members of the MyoD MyoD a role in the regulation of GLUT1 gene expression during myogenesis, we the of the over-expression of MyoD in C3H10T1/2 The expression of MyoD in these cells caused a reduction in the transcriptional activity of the GLUT1 promoter as by transfection of the activity in C3H10T1/2 and and expressed as and assays the of highly in nuclear from C3H10T1/2 cells that showed mobility to and from Furthermore, these with an the consensus sequence for Sp1 binding and with not over-expression of MyoD caused the of the binding to the Sp1 and the formation of Furthermore, Western blot assays of Sp1 protein from nuclear from C3H10T1/2 and cells indicated a down-regulation of Sp1 protein after MyoD over-expression suggest a role of MyoD in the down-regulation of Sp1 associated with myogenesis. In this we have that GLUT1 is in muscle cells during differentiation as a of in transcriptional activity of the GLUT1 which to the fragment −99/−33 and the fragment 5′ to the transcription site the Furthermore, the of GLUT1 mRNA and the transcriptional activity of the GLUT1 promoter reduced in to this that the in the transcriptional activity of the GLUT1 promoter are to for the GLUT1 In the −99/−33 of the GLUT1 we have the binding of Sp1 to the GLUT1 gene this to from a functional Sp1 in transfection the transcriptional activity of GLUT1 promoter. In contrast to the that Sp1 is a we have found that myogenesis leads to a reduction in the formation of a Sp1, which is to a down-regulation of Sp1 also that MyoD over-expression Sp1 expression in cells, which is in to a reduction in the transcriptional activity of the GLUT1 Based on we the model in to the transcriptional activity of the GLUT1 gene is in myoblasts, in to a expression of the Muscle cell differentiation is associated with activation of MyoD transcription which as master regulators to activation of muscle-specific In activation leads to the repression of Sp1 expression in muscle In Sp1 down-regulation causes of the transcriptional activity of the GLUT1 gene and, therefore, leads to GLUT1 down-regulation and to a of glucose transport. A that an is required for the expression of factor of MyoD in muscle cells (18Kurabayashi M. Dutta S. Kedes L. J. Biol. Chem. 1994; 269: 31162-31170Abstract Full Text PDF PubMed Google Scholar). Based on we that Sp1 down-regulation contributes to the repression of found during myogenesis and which is known to in the activation of myogenic transcription factors (17Benezra R. Davis R.L. Lockshon D. Turner D.L. Weintraub H. Cell. 1990; 61: 49-59Abstract Full Text PDF PubMed Scopus (1785) Google Scholar, D. Mol. Cell. Biol. 1991; PubMed Scopus Google Scholar). on the regulation of the GLUT1 gene have exclusively on the functional role of enhancer found in the GLUT1 The enhancer has been kilobases of the transcription the is in the of the gene T. T. T. Y. M. S. Y. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). the activation of the transcription in to or T. T. T. Y. M. S. Y. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, M. T. T. S. M. H. M. Y. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we have the properties of the promoter of the rat GLUT1 the of a in the fragment that is in and in myotubes. data also suggest that the fragment to the or the participates in the repression of GLUT1 transcription during myogenesis. In this it has been reported that factors such as or the formation of and gene transcription A. G. M. EMBO J. PubMed Scopus Google J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of these factors play a role the in the regulation of the GLUT1 gene during myogenesis unknown. Furthermore, we have the fragment −99/−33 of the GLUT1 gene that is for the transcriptional activity of the GLUT1 promoter. have also the of found in or myotube nuclear and that to the −99/−33 we have and that are to Based on the of these to the Sp1 binding the of these to and the fact that they a to Sp1, and that is a in the of an Sp1, we that Sp1 participates in the formation of these In we have found that Sp1 the transcriptional activity of the GLUT1 gene in transfection data, with the fact that is a expression of Sp1 protein in nuclear from and that Sp1 to the fragment −99/−33 in myoblasts, the that Sp1 GLUT1 transcription in muscle the binding of Sp1 protein to the GLUT1 promoter with a GLUT1 gene expression in a variety of T. J. X. M. and A. The transfection of Sp1 to a of the GLUT1 of transcription that was that in suggest a of Sp1 in myotube In this it has been that Sp1 with several The protein protein Sp1 and as a in transcription T. G. J.L. R. 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Biol. 1991; PubMed Scopus Google Scholar). these we have found in this that myogenesis the expression of Sp1 in muscle Furthermore, the that MyoD causes down-regulation of Sp1 in transfection In we that activation of MyoD transcription factors causes down-regulation of Sp1 and this the mechanisms that to GLUT1 repression and of the differentiation program in the muscle The mechanisms Sp1 down-regulation in to MyoD transcription factors are in R. for also J. and V. for of assays and J. for