1: J Biol Chem. 2003 Nov 7;278(45):44230-7. Epub 2003 Aug 26. Diabetes and the accompanying hyperglycemia impairs cardiomyocyte calcium cycling through increased nuclear O-GlcNAcylation. Clark RJ, McDonough PM, Swanson E, Trost SU, Suzuki M, Fukuda M, Dillmann WH. Department of Medicine, University of California San Diego, La Jolla, California 92093, USA. Diabetic cardiomyopathy is characterized by impaired cardiac contractility leading to poor myocardial performance. We investigated the role that the hexosamine pathway, and especially altered nuclear O-Glc-NAcylation, plays in the development of diabetic cardiomyopathy. Incubating neonatal rat cardiomyocytes in high glucose (25 mM) resulted in prolonged calcium transients when compared with myocytes incubated in normal glucose (5.5 mM), which is consistent with delayed myocardial relaxation. High glucose-treated myocytes also exhibited reduced sarcoendoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) mRNA and protein expression, decreased SERCA2a promoter activity, and increased O-GlcNAcylation of nuclear proteins compared with myocytes treated with normal glucose. Exposure of myocytes to 8 mM glucosamine or an adenovirus expressing O-GlcNAc-transferase (OGT) resulted in prolonged calcium transient decays and significantly reduced SERCA2a protein levels, whereas treatment with an adenovirus encoding O-GlcNAcase (GCA) resulted in improved calcium transients and SERCA2a protein levels in myocytes exposed to high glucose. Effects of elevated glucose or altered O-GlcNAcylation were also observed on essential transcription factors involved in cardiomyocyte function. High glucose-treated myocytes (with or without OGT adenovirus) exhibited increased levels of O-GlcNAcylated specificity protein 1 compared with control myocytes, whereas infecting high glucose-treated myocytes with GCA adenovirus reduced the degree of specificity protein 1 Glc-NAcylation. Treatment of myocytes with 25 mM glucose, 8 mM glucosamine, or OGT adenovirus also significantly reduced levels of myocytes enhancer factor-2A protein compared with control myocytes, whereas infection with GCA adenovirus resulted in improved myocytes enhancer factor-2 expression. Our results suggest that the hexosamine pathway, and O-GlcNAcylation in particular, is important in impaired cardiac myocyte function and the development of diabetic cardiomyopathy. PMID: 12941958 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: J Exp Med. 2003 Jun 2;197(11):1441-52. Orphan nuclear receptor Nur77 is involved in caspase-independent macrophage cell death. Kim SO, Ono K, Tobias PS, Han J. Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037, USA. Activation-induced cell death in macrophages has been observed, but the mechanism remains largely unknown. Activation-induced cell death in macrophages can be independent from caspases, and the death of activated macrophages can even be triggered by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD). Here, we show that this type of macrophage death can occur in the septic mouse model and that toll-like receptor (TLR)-2 or TLR4 signaling is required in this process. We conclude that Nur77 is involved in the macrophage death because Nur77 expression correlates with cell death, and cell death is reduced significantly in Nur77-deficient macrophages. The extracellular signal-regulated kinase pathway, which is downstream of TLR2 or TLR4, and myocyte-specific enhancer binding factor 2 (MEF2) transcription factor activity, which is up-regulated by zVAD, are required for Nur77 induction and macrophage death. Reporter gene analysis suggests that Nap, Ets, Rce, and Sp1 sites in the Nur77 promoter are regulated by TLR4 signaling and that MEF2 sites in the Nur77 promoter are regulated by zVAD treatment. MEF2 transcription factors are constitutively expressed and degraded in macrophages, and zVAD increases MEF2 transcription factor activity by preventing the proteolytic cleavage and degradation of MEF2 proteins. This paper delineates the dual signaling pathways that are required for Nur77 induction in macrophages and demonstrates a role of Nur77 in caspase-independent cell death. PMID: 12782711 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Mol Immunol. 2002 Sep;39(1-2):25-30. Synergistic interaction of MEF2D and Sp1 in activation of the CD14 promoter. Park SY, Shin HM, Han TH. Room 5313, Department of Molecular Cell Biology and Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 300 Chunchundong, Jangangu, Suwon, South Korea. The expression of CD14, a monocyte receptor for the bacterial lipopolysaccharide (LPS), is upregulated during monocytic cell differentiation. Although a Sp1 site at -110bp of the CD14 promoter was shown to be critical for activation of the promoter during the differentiation, how the Sp1 site is regulated has not been well understood. We have recently reported that expression of MEF2D protein increases during the differentiation of HL60 promyeloid cells to monocyte and that the upregulation of the protein is required for CD14 expression during the differentiation [Mol. Immunol. 36 (1999) 1209]. However, there is no obvious MEF2 binding site in the critical region of the CD14 promoter. In this study, which aimed to determine the regulatory role of MEF2D in monocytic cell differentiation, MEF2D was found to form a complex with Sp1 in U937 promyeloid cells. Transient transfection experiments showed that co-expression of MEF2D and Sp1 synergistically activated the CD14 promoter. The results support a model in which increased MEF2D protein during monocytic cell differentiation activates the CD14 promoter through interaction with Sp1. PMID: 12213324 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Biochem J. 2002 Oct 15;367(Pt 2):369-80. Expression of creatine kinase isoenzyme genes during postnatal development of rat brain cerebellum: evidence for transcriptional regulation. Shen W, Willis D, Zhang Y, Schlattner U, Wallimann T, Molloy GR. Department of Biological Sciences, University of Delaware, 117 Wolf Hall, Newark, DE 19716, U.S.A. Transcription and accumulation of brain-type creatine kinase (CKB) mRNA and its protein was examined during postnatal development of rat brain cerebellum, the brain region containing highest CKB mRNA in the adult. CKB protein was extremely low at day 1, increased about 10-fold until week 4 and remained constant until week 10. This time course was paralleled by cerebellar CKB mRNA, which was also extremely low at day 1 and increased 5-fold during the first 3 weeks and then remained constant. High levels of CKB protein were also detected in cultured primary cerebellar granular neurons. Nuclear run-on assays directly showed that CKB mRNA accumulation during postnatal cerebellar development was due to increased transcription. When compared with cerebrum and whole brain, cerebellar CKB mRNA accumulation during postnatal development was temporally delayed. Analysis of myocyte enhancer factor (MEF)-2 and Sp1, factors known to initiate or sustain CKB transcription in tissues other than brain, revealed that MEF-2 in cerebellum was low at week 1 but increased 3.5-fold by week 7, while Sp1 remained unchanged. The increase in CKB protein during cerebellar postnatal development was coincident with that of the ubiquitous mitochondrial CK protein and mRNA, indicating that a functional phosphocreatine energy shuttle probably exists for efficient ATP regeneration in the cerebellum. This should be beneficial for the many energy-demanding requirements during cerebellar development, as indicated by the observed temporal co-expression of CKB with myelin basic protein, which is involved in axon myelination by oligodendrocytes. PMID: 12093362 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Cardiovasc Res. 2001 Apr;50(1):3-6. Comment on: Cardiovasc Res. 2001 Apr;50(1):24-33. On the trail of cardiac specific transcription factors. Flesch M. Publication Types: Comment Editorial PMID: 11282072 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: J Biol Chem. 1998 Sep 25;273(39):25371-80. Combinatorial cis-acting elements control tissue-specific activation of the cardiac troponin I gene in vitro and in vivo. Di Lisi R, Millino C, Calabria E, Altruda F, Schiaffino S, Ausoni S. Department of Biomedical Sciences, University of Padova, 35121 Padova, Italy. The cardiac troponin I gene is one of the few sarcomeric protein genes exclusively expressed in cardiac muscle. We show here that this specificity is controlled by a proximal promoter (-230/+16) in transfected cardiac cells in culture, in the adult hearts, and in transgenic animals. Functional analysis indicates that MEF2/Oct-1, Sp1, and GATA regulatory elements are required for optimal gene activation because selective mutations produce weak or inactive promoters. MEF2 and Oct-1 transcription factors bind to the same A/T-rich element. A mutation that blocks this binding markedly reduces gene activation in vivo and in vitro, and overexpression of MEF2A, MEF2C, and MEF2D in noncardiac cells transactivates the cardiac troponin I promoter. Disruption of these elements inactivates the cardiac troponin I promoter in cultured cardiac cells but has a less important role in transfected adult heart. Moreover, nuclear extracts from an almost pure population of adult cardiac cells contain much lower levels of GATA binding activity compared with fetal cardiac cells. These findings point to a differential role of GATA factors in the maintenance of gene expression in the adult heart as compared with the activation of cardiac genes in fetal cardiomyocytes. Overexpression of GATA family members transactivates the cardiac troponin I promoter, and GATA-5 and GATA-6 are stronger transactivators than GATA-4, a property apparently unique to the cardiac troponin I promoter. Transgenic mice carrying the -230/+126 base pair promoter express beta-galactosidase reporter gene in the heart both at early stages of cardiogenesis and in the adult animals. These results indicate that the ability of the cardiac troponin I proximal promoter to target expression of a downstream gene in the heart is also maintained when the transgene is integrated into the genome. PMID: 9738004 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: J Cell Biochem. 1998 Sep 1;70(3):366-75. Collaborative interactions between MEF-2 and Sp1 in muscle-specific gene regulation. Grayson J, Bassel-Duby R, Williams RS. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8573, USA. Previous investigations have demonstrated synergistic interactions in vivo between CCAC and A/T-rich nucleotide sequence motifs as functional components of muscle-specific transcriptional enhancers. Using CCAC and A/T-rich elements from the myoglobin and muscle creatine kinase (MCK) gene enhancers, Sp1 and myocyte-specific enhancer factor-2 (MEF-2) were identified as cognate binding proteins that recognize these sites. Physical interactions between Sp1 and MEF-2 were demonstrated by immunological detection of both proteins in DNA binding complexes formed in vitro by nuclear extracts in the presence of only the A/T sequence motif, by coprecipitation of recombinant MEF-2 in the presence of a glutathione-S-transferase-Sp1 fusion protein bound to glutathione beads, and by a two-hybrid assay in Saccharomyces cerevisiae. The interaction with Sp1 in vitro and in vivo is specific for MEF-2 and was not observed with serum response factor, a related MADS domain protein. Forced expression of Sp1 and MEF-2 in insect cells otherwise lacking these factors promotes synergistic transcriptional activation of a promoter containing binding sites for both proteins. These data expand the repertoire of functional and physical interactions between lineage-restricted (MEF-2) and ubiquitous (Sp1) transcription factors that may be important for myogenic differentiation. PMID: 9706874 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: J Biol Chem. 1998 Mar 13;273(11):6402-9. A novel site, Mt, in the human desmin enhancer is necessary for maximal expression in skeletal muscle. Gao J, Li Z, Paulin D. Laboratoire de Biologie Mol culaire de la Differentiation Cellulaire, Universite Paris VII, 25 rue du Dr. Roux, Paris cedex 15, France. Previous investigations have shown that expression of the muscle-specific intermediate filament desmin gene in skeletal muscle is controlled in part by a 5' muscle-specific enhancer. This enhancer activity can be divided into myoblast-specific and myotube-specific activation domains. The myotube-specific region contains a MyoD and MEF2 sites, whereas the myoblast-specific region contains Sp1, Krox, and Mb sites. In the present study, we designed mutations in the conserved portion of the myotube-specific region; transfection analysis of these mutations showed that a novel site located between the MyoD and MEF2 sites, named Mt (GGTATTT), is required for full transcriptional activity of the desmin enhancer in skeletal muscle. Although gel mobility shift assays demonstrate that myotube, myoblast, fibroblast, and HeLa nuclear extracts contain a nuclear factor that binds specifically to Mt, four copies of the Mt site function as the native enhancer only in myotubes. Functional synergism among the MyoD, MEF2, and Mt sites in myotubes has been demonstrated. These results show that the novel Mt site cooperates with MyoD and MEF2 to give maximal expression of the desmin gene. PMID: 9497371 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Mol Cell Biol. 1995 Aug;15(8):4272-81. Regulation of Gax homeobox gene transcription by a combination of positive factors including myocyte-specific enhancer factor 2. Andres V, Fisher S, Wearsch P, Walsh K. Division of Cardiovascular Research, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135, USA. Homeobox-containing genes play an essential role in basic processes during embryogenesis and development, but little is known about the regulation of their expression. To elucidate regulatory networks that govern homeobox gene expression, we defined the core promoter of the mouse Gax homeobox gene and characterized its interactions with cellular proteins. Transient transfection experiments revealed Gax promoter activity in several cell types. Deletion analysis defined a 138-bp minimal promoter fragment between positions -125 and +13 relative to the transcription initiation site. Mutagenesis and protein-DNA binding assays suggested that at least three positive factors interact with this fragment and are required for transcriptional activity. One of these factors, HRF-1, recognizes a cis element consisting of an inverted palindromic motif. A second factor is Sp1, that binds to a G/C-rich element. The third is the MADS box factor referred to as MEF2 or RSRF. Mutations in the MEF2/RSRF site had the greatest effect on transcription in cell types that expressed the highest levels of endogenous MEF2 activity. Conversely, overexpression of MEF2A transactivated the Gax promoter more efficiently in cells lacking endogenous MEF2. These data provide evidence for a direct transcriptional link between members of the MADS and homeobox families of transcription factors. PMID: 7623821 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------