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Biomedical subjects

L Kedes

Publications and source records attributed to L Kedes.

At least 19 recordsLinked to original sources
PubMed2000

Proteasome-mediated degradation of the coactivator p300 impairs cardiac transcription.

The transcription of tissue-specific genes is controlled by regulatory factors and cofactors and is suppressed in cardiac cells by the antineoplastic agent doxorubicin. Here we show that exposure of cultured cardiomyocytes to doxorubicin resulted in the rapid depletion of transcripts for MEF2C, dHAND, and NKX2.5, three pivotal regulators of cardiac gene expression. Delivery of exogenous p300, a coactivator of MEF2C and NKX2.5 in cardiomyocytes, restored cardiac transcription despite the presence of doxorubicin. Furthermore, p300 also restored the accumulation of transcripts for MEF2C itself. Importantly, cardiocytes exposed to doxorubicin displayed reduced levels of p300 proteins. This was not due to alterations in the level of p300 transcripts; rather, and surprisingly, doxorubicin promoted selective degradation of p300 mediated by the 26S-proteasome machinery. Doxorubicin had no effect on the general level of ubiquitinated proteins or on the levels of beta-catenin, a protein known to be degraded by proteasome-mediated degradation. These results provide evidence for a new mechanism of transcriptional repression caused by doxorubicin in which the selective degradation of p300 results in reduced p300-dependent transcription, including production of MEF2C mRNA.

Animals↗
PubMed2000

Assessing the binding and endocytosis activity of cellular receptors using GFP-ligand fusions.

We have developed a simple scheme for characterizing ligand-receptor binding and post-binding activity on living cells. Our approach makes use of green fluorescent protein (GFP) as an auto-fluorescent tag to label protein ligands. We have constructed GFP-tagged ligands that can be expressed in bacteria as soluble fusion proteins. A cell-binding assay using fluorescence-activated cell sorting (FACS) demonstrates that GFP-tagged proteins retain their wild-type receptor-binding specificity. Using this assay, we measure ligand binding on unfixed cells and demonstrate receptor specificity using specific competitors. To determine the ability of receptor targets to internalize, we developed a second FACS-based assay to detect the rate and percentage of internalized ligand in living cells. Noninternalizing control ligands and fluorescence microscopy of treated cells confirm that our assay is reliable for determining receptor internalization activity.

Binding Sites↗
PubMed1999

Twist is a potential oncogene that inhibits apoptosis.

Oncogene activation increases susceptibility to apoptosis. Thus, tumorigenesis must depend, in part, on compensating mutations that protect from programmed cell death. A functional screen for cDNAs that could counteract the proapoptotic effects of the myc oncogene identified two related bHLH family members, Twist and Dermo1. Both of these proteins inhibited oncogene- and p53-dependent cell death. Twist expression bypassed p53-induced growth arrest. These effects correlated with an ability of Twist to interfere with activation of a p53-dependent reporter and to impair induction of p53 target genes in response to DNA damage. An underlying explanation for this observation may be provided by the ability of Twist to reduce expression of the ARF tumor suppressor. Thus, Twist may affect p53 indirectly through modulation of the ARF/MDM2/p53 pathway. Consistent with a role as a potential oncoprotein, Twist expression promoted colony formation of E1A/ras-transformed mouse embryo fibroblasts (MEFs) in soft agar. Furthermore, Twist was inappropriately expressed in 50% of rhabdomyosarcomas, a tumor that arises from skeletal muscle precursors that fail to differentiate. Twist is known to block myogenic differentiation. Thus, Twist may play multiple roles in the formation of rhabdomyosarcomas, halting terminal differentiation, inhibiting apoptosis, and interfering with the p53 tumor-suppressor pathway.

Animals↗
PubMed1999

Regulation of histone acetyltransferases p300 and PCAF by the bHLH protein twist and adenoviral oncoprotein E1A.

Histone acetyltransferases (HAT) play a critical role in transcriptional control by relieving repressive effects of chromatin, and yet how HATs themselves are regulated remains largely unknown. Here, it is shown that Twist directly binds two independent HAT domains of acetyltransferases, p300 and p300/CBP-associated factor (PCAF), and directly regulates their HAT activities. The N terminus of Twist is a primary domain interacting with both acetyltransferases, and the same domain is required for inhibition of p300-dependent transcription by Twist. Adenovirus E1A protein mimics the effects of Twist by inhibiting the HAT activities of p300 and PCAF. These findings establish a cogent argument for considering the HAT domains as a direct target for acetyltransferase regulation by both a cellular transcription factor and a viral oncoprotein.

Acetyltransferases↗
PubMed1999

A high-titer lentiviral production system mediates efficient transduction of differentiated cells including beating cardiac myocytes.

Human immunodeficiency virus (HIV, lentivirus) type-1 based vectors have a number of attractive features for gene therapy, including the ability to transduce non-dividing cells and long term transgene expression. We used a three-plasmid expression system to generate pseudotyped lentivirus-based vectors by transient transfection of human embryonic kidney 293T cells in the presence of sodium butyrate, which is known to activate the long terminal repeat-directed expression of HIV. Using this system we successfully generated versatile high titer lentivirus at titers of up to 2 x 10(8) transducing units/ml (TU/ml), and improved transduction efficiency in various cell types from seven to over twenty fold. We demonstrate its applicability of these vectors for the efficient transduction of non-dividing cells, including post mitotic beating rat cardiac myocytes and well-differentiated rat L6 myofibers. While both lentivirus-based and murine retrovirus-based vectors effectively transduced dividing cardiac fibroblasts and L6 muscle myoblasts in culture, lentivirus-based vectors also efficiently transduced cardiac myocytes and yielded titers of (6.3 +/- 1.2) x 10(5) TU/ml; however murine retrovirus-based vectors showed low transduction efficiency with titers reaching only (8.9 +/- 2.1) x 10(2) TU/ml. Furthermore, even 12 days after induction of differentiation of L6 myofibers, lentivirus-mediated transduction of beta-galactosidase (beta-Gal) at approximately 30-40% of the maximum expression levels achieved in replicating myoblasts. In contrast, the expression of beta-Gal following transduction of the myofibers by murine retrovirus-based vectors fell to less than 1% of an already reduced level of transduction in undifferentiated confluent myoblasts. These results demonstrate that lentivirus-based vectors can efficiently transduce both well-differentiated cardiac myocytes and differentiated myofibers. This appears to be an efficient method and provides a new tool for research and therapy for cardiovascular diseases.

Animals↗
PubMed1999

Acetylation of MyoD directed by PCAF is necessary for the execution of the muscle program.

p300/CBP and PCAF coactivators have acetyltransferase activities and regulate transcription, cell cycle progression, and differentiation. They are both required for MyoD activity and muscle differentiation. Nevertheless, their roles must be different since the acetyltransferase activity of PCAF but not of p300 is involved in controlling myogenic transcription and differentiation. Here, we provide a molecular explanation of this phenomenon and report that MyoD is directly acetylated by PCAF at evolutionarily conserved lysines. Acetylated MyoD displays an increased affinity for its DNA target. Importantly, conservative substitutions of acetylated lysines with nonacetylatable arginines impair the ability of MyoD to stimulate transcription and to induce muscle conversion indicating that acetylation of MyoD is functionally critical.

Acetylation↗
PubMed1999

Myogenic basic helix-loop-helix proteins and Sp1 interact as components of a multiprotein transcriptional complex required for activity of the human cardiac alpha-actin promoter.

Activation of the human cardiac alpha-actin (HCA) promoter in skeletal muscle cells requires the integrity of DNA binding sites for the serum response factor (SRF), Sp1, and the myogenic basic helix-loop-helix (bHLH) family. In this study we report that activation of the HCA correlates with formation of a muscle-specific multiprotein complex on the promoter. We provide evidence that proteins eluted from the multiprotein complex specifically react with antibodies directed against myogenin, Sp1, and SRF and that the complex can be assembled in vitro by using the HCA promoter and purified MyoD, E12, SRF, and Sp1. In vitro and in vivo assays revealed a direct association of Sp1 and myogenin-MyoD mediated by the DNA-binding domain of Sp1 and the HLH motif of myogenin. The results obtained in this study indicate that protein-protein interactions and the cooperative DNA binding of transcriptional activators are critical steps in the formation of a transcriptionally productive multiprotein complex on the HCA promoter and suggest that the same mechanisms might be utilized to regulate the transcription of muscle-specific and other genes.

Actins↗
PubMed1999

Murine tongue muscle displays a distinct developmental profile of MRF and contractile gene expression.

Few studies have addressed the molecular differences that exist between muscles of the body and those of the craniofacial apparatus. In this study, we characterize the molecular events associated with determination and differentiation of the tongue musculature. We assess the expression of myogenic regulatory factors as well as the developmentally regulated myosin heavy chain, (MHC), genes which serve as markers of differentiation. These results suggest that tongue and limb muscle form by distinct molecular pathways. The myoblasts that contribute to the formation of the tongue preferentially express Myf-5 during myoblast determination rather than MyoD. Subsequently, isolated regions of myogenin expression mark the differentiation of first, the small primary myofibers and later, the larger secondary myofibers. Analysis of differentiation markers demonstrates that the tongue muscle also assumes a unique profile of MHC expression as compared to that of the muscles of the body. Unlike the myoblasts of the developing limb, which express embryonic and neonatal forms of MHC and later express MHC-slow, the tongue myoblasts co-express MHC-embryonic, MHC-slow and MHC-fast isoforms from gestational age E12. Proteins for MHC embryonic and MHC fast isoforms are detected almost simultaneously. Interestingly, MHC-slow transcripts do not appear to be translated into a detectable MHC slow protein at any developmental stage assayed. These results provide further evidence to suggest that skeletal tongue muscle represents a myoblast lineage that develops differently than the limb.

Age Factors↗
PubMed1999

The myogenic regulatory circuit that controls cardiac/slow twitch troponin C gene transcription in skeletal muscle involves E-box, MEF-2, and MEF-3 motifs.

We have characterized the specific DNA regulatory elements responsible for the function of the human cardiac troponin C gene (cTnC) muscle-specific enhancer in myogenic cells. We used functional transient transfection assays with deletional and site-specific mutagenesis to evaluate the role of the conserved sequence elements. Gel electrophoresis mobility shift assays (EMSA) demonstrated the ability of the functional sites to interact with nuclear proteins. We demonstrate that three distinct transcription activator binding sites commonly found in muscle-specific enhancers (a MEF-2 site, a MEF-3 site, and at least four redundant E-box sites) all contribute to full enhancer activity but a CArG box does not. Mutation of either the MEF-2 or MEF-3 sites or deletion of the E-boxes reduces expression by 70% or more. Furthermore, the MEF-2 site and the E-boxes specifically bind, respectively, to MEF-2 and myogenic determination factors derived from nuclear extracts. EMSA assays using a MEF-3 containing oligonucleotide revealed indistinguishable separation patterns with extracts from myogenic cells and nonmyogenic cells. These data suggest that expression of the cTnC gene in slow-twitch skeletal muscle is sustained through complex interactions at the 3'Ile enhancer between muscle-specific and nontissue-specific transcription factors: either a myogenic bHLH complex or MEF-2 can activate transcription but only in the presence of a third transcriptional activator that appears not to be muscle specific. We conclude from these observations that the cTnC 3'Ile element is a composite enhancer that functions through the combined interactions of at least five regulatory elements and their cognate binding factors: three or four E-boxes, a MEF-2 site, and a MEF-3 site. The data support the notion that all of these sites contribute to enhancer function in cell systems in an additive way but that none are absolutely required for enhancer activity. The data imply that the levels of transcription of cTnC in myogenic tissues in which the activities of one of the transcriptional factors is lacking would be partially but not wholly suppressed. Our data support the critical role of E-box sites in conjunction with the adjacent elements. Hence, we assign CTnC gene regulation to the "ordinary" rather than to the "novel" category of transcriptional regulation during skeletal myogenesis.

Base Sequence↗
PubMed1998

Intramolecular regulation of MyoD activation domain conformation and function.

The MyoD family of basic helix-loop-helix (bHLH) proteins is required for myogenic determination and differentiation. The basic region carries the myogenic code and DNA binding specificity, while the N terminus contains a potent transcriptional activation domain. Myogenic activation is abolished when the basic region, bound to a myogenic E box, carries a mutation of Ala-114. It has been proposed that DNA binding of the MyoD basic region leads to recruitment of a recognition factor that unmasks the activation domain. Here we demonstrate that an A114N mutant exhibits an altered conformation in the basic region and that this local conformational difference can lead to a more global change affecting the conformation of the activation domain. This suggests that the deleterious effects of this class of mutations may result directly from defective conformation. Thus, the activation domain is unmasked only upon DNA binding by the correct basic region. Such a coupled conformational relationship may have evolved to restrict myogenic specificity to a small number of bHLH proteins among many with diverse functions yet with DNA binding specificities known to be similar.

Alanine↗
PubMed1997

A novel cardiac-restricted target for doxorubicin. CARP, a nuclear modulator of gene expression in cardiac progenitor cells and cardiomyocytes.

Doxorubicin (Dox), a cardiotoxic antineoplastic drug, disrupts the cardiac-specific program of gene expression (Kurabayashi, M., Dutta, S., Jeyaseelan, R., and Kedes, L. (1995) Mol. Cell. Biol. 15, 6386-6397; Jeyaseelan, R., Poizat, C., Wu, H. Y., and Kedes, L. (1997) J. Biol. Chem. 272, 5828-5832). To determine whether this drug might interfere with the function of cardiac-specific regulatory pathways, we used a differential display strategy to clone from neonatal rat cardiomyocyte candidate mRNAs that were rapidly sensitive to Dox. We report here the identification of a constitutively expressed, cardiac-restricted, nuclear protein whose mRNA level is exquisitely sensitive to Dox. Hence we have named this protein cardiac adriamycin-responsive protein (CARP). CARP mRNA is present at the earliest stages of cardiac morphogenesis. It was detected by in situ hybridization within the cardiogenic plate of 7. 5-day post coitum (p.c.) embryos, and in 8.5-day p.c. embryos CARP transcripts are present in uniformly high levels in the myocardium. Throughout cardiac development, CARP expression is specific for the myocardium; endocardial cushions and valves exhibit only background levels of signal. Transcript levels persist but gradually decrease in neonatal, 2-week-old, and adult hearts. There were no stages when CARP mRNA could not be detected. The pattern and timing of CARP mRNA expression, including transient expression in the tongue at 14.5 days p.c., coincides with that of Nkx2.5/Csx (a putative homolog of tinman, the Drosophila melanogaster gene responsible for cardiac development). The cloned full-length 1749 nucleotide CARP cDNA encodes a 319-amino acid 40-kDa polypeptide containing five tandem ankyrin repeats. CARP appears to be the rat homolog of a previously reported human single-copy gene (C-193; Chu, W., Burns, D. K., Swerlick, R. A., and Presky, D. H. (1995) J. Biol. Chem. 270, 10236-10245), whose mRNA is inducible by cytokines only in human endothelial cells. CARP appears to function as a negative regulator of cardiac-specific gene expression. Overexpression of CARP in cardiomyocytes suppresses cardiac troponin C and atrial natriuretic factor transcription. Cotransfection experiments in HeLa cells indicate that CARP inhibits Nkx2.5 transactivation of atrial natriuretic factor promoter. When fused to a GAL4 DNA-binding domain, CARP has transcriptional inhibitory properties in noncardiac cells. CARP thus represents the first example of a cardiac-restricted transcriptional regulatory protein that is sensitive to Dox.

Amino Acid Sequence↗
PubMed1997

Molecular mechanisms of doxorubicin-induced cardiomyopathy. Selective suppression of Reiske iron-sulfur protein, ADP/ATP translocase, and phosphofructokinase genes is associated with ATP depletion in rat cardiomyocytes.

Doxorubicin, a cardiotoxic antineoplastic, disrupts the cardiac-specific program of gene expression (Kurabayashi, M., Dutta, S., Jeyaseelan, R., and Kedes, L. (1995) Mol. Cell. Biol. 15, 6386-6397). We have now identified neonatal rat cardiomyocyte mRNAs rapidly sensitive to doxorubicin, or its congener daunomycin, including transcripts of nuclear genes encoding enzymes critical in production of energy in cardiomyocytes: ADP/ATP translocase, a heart- and muscle-specific isoform; Reiske iron-sulfur protein (RISP), a ubiquitously expressed electron transport chain component; and a muscle isozyme of phosphofructokinase. Loss of these mRNAs following doxorubicin or daunomycin is evident as early as 2 h and precedes significant reduction of intracellular ATP. ATP levels in control cardiomyocytes (17.9 +/- 2.9 nM/mg of protein) fall only after 14 h and reach residual levels of 10.4 +/- 0.9 nM (doxorubicin; p = <0.006) and 6.7 +/- 1.9 nM (daunomycin; p = <0. 001) by 24 h. Loss of mRNAs generating ATP was highly selective since mRNAs for other energy production enzymes, (cytochrome c, cytochrome b, and malate dehydrogenase), and genes important in glycolysis (pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase) were unaffected even at 24 and 48 h. The drugs had no effect on levels of ubiquitously expressed RISP mRNA in fibroblasts. These findings could link doxorubicin-induced damage to membranes and signaling pathways with 1) suppression of transcripts encoding myofibrillar proteins and proteins of energy production pathways and 2) depletion of intracellular ATP stores, myofibrillar degeneration, and related cardiotoxic effects.

Adenosine Diphosphate↗
PubMed1997

Two-step delivery of retroviruses to postmitotic, terminally differentiated cells.

Recombinant replication-defective retroviral vectors are currently the most commonly used vectors for introducing foreign genes into human cells in gene therapy protocols. Their genomes stably incorporate in the host chromosomes of mitotic cells, thus ensuring stable expression. However, the applications of retroviruses to gene therapy are limited by their inability to infect postmitotic cells such as muscle fibers. In an attempt to overcome such limitations, we have developed a novel two-step transduction protocol that allows integration and expression of retroviral genes in differentiated cells. We induced DNA synthesis in terminally differentiated cultured mouse myotubes derived from both established myogenic cell lines and from primary myoblasts. We infected the postmitotic cells with a recombinant replication-defective adenoviral vector encoding the SV40 large T antigen as a mitogen. Subsequently we transduced the adenovirus-infected cells with a Moloney retroviral vector bearing the LacZ gene. Histochemical analysis revealed the coincident expression of LacZ gene in those myotubes that had been induced to synthesize DNA.

Adenoviridae↗
PubMed1997

Differential expression of troponin C genes during tongue myogenesis.

Determination of muscle fiber type is related to the developmental stage of the tissue. Ordinarily the final distribution of fast and slow fibers in a muscle is determined postnatally. Tongue muscle, however, is composed solely of fast-twitch fibers that express only troponin C fast mRNA and fast (type II) myosin heavy chain (MHC) proteins in both the adult and the one-day-old mouse. The fiber-type determination of this muscle was examined during fetal development. Both troponin C fast and slow mRNAs were expressed at initial stages of tongue development at embryonic day 18. However, by embryonic day 16 the troponin C fast transcripts predominated. AT 17 days of embryonic development, TnC fast mRNA was 10 times more abundant than TnC slow, and at 18 days of development the TnC slow mRNA was barely detectable. The tongue muscle myotubes expressed fast, slow, and embryonic MHC isoforms during early embryonic development. At 18 days of gestation, the MHC isoform expressed by the majority of the myotubes was the fast isoform, whereas the slow isoform was present in very few fibers. RT-PCR analysis of the MHC transcripts present throughout tongue development demonstrated expression of the mdms or type IIx MHC in both late fetal and postnatal stages of development. In contrast, the type I/beta slow MHC mRNA was undetectable in the postnatal and adult tongue. The absence of TnC and MHC slow-isoform mRNAs in the newborn mouse tongue suggests that slow isoform genes become dominantly repressed with the TnC-F and MHC type IIx genes remaining transcriptionally active, giving rise to an unusually homogeneous fast-twitch phenotype. The tongue muscle fibers acquire their specific adult-type fiber characteristics during fetal development rather than postnatally.

Animals↗
PubMed1997

Differential roles of p300 and PCAF acetyltransferases in muscle differentiation.

PCAF is a histone acetyltransferase that associates with p300/CBP and competes with E1A for access to them. While exogenous expression of PCAF potentiates both MyoD-directed transcription and myogenic differentiation, PCAF inactivation by anti-PCAF antibody microinjection prevents differentiation. MyoD interacts directly with both p300/CBP and PCAF, forming a multimeric protein complex on the promoter elements. Viral transforming factors that interfere with muscle differentiation disrupt this complex without affecting the MyoD-DNA interaction, indicating functional significance of the complex formation. Exogenous expression of PCAF or p300 promotes p21 expression and terminal cell-cycle arrest. Both of these activities are dependent on the histone acetyltransferase activity of PCAF, but not on that of p300. These results indicate that recruitment of histone acetyltransferase activity of PCAF by MyoD, through p300/CBP, is crucial for activation of the myogenic program.

Acetyl-CoA C-Acyltransferase↗
PubMed1997

Cell-cycle-specific transcription termination within the human histone H3.3 gene is correlated with specific protein-DNA interactions.

In vitro studies using highly purified calf thymus RNA polymerase II and a fragment spanning the first intron of H3.3 as template DNA have demonstrated the existence of a strong transcription termination site consisting of thymidine stretches. In this study, nuclear run-on experiments have been performed to assess the extent to which transcription elongation is blocked in vivo using DNA probes corresponding to region 5' and 3' of the in vitro termination sites. These studies suggest that H3.3 expression is stimulated following the inhibition of DNA synthesis through the elimination of the transcription elongation block. Interestingly, both the in vivo and in vitro experiments have revealed that the transcriptional block/termination sites are positioned immediately downstream of a 73 bp region that has been over 90% conserved between the chicken and human H3.3 genes. The extreme conservation of this intronic region suggests a possible role in maintaining cis-acting function. Electrophoretic mobility shift experiments show that HeLa cell nuclear extracts contain protein factors that bind specifically to the region of transcription elongation block. Furthermore, we demonstrate a correlation between the protein binding activity and the transcriptional block in cells that have been either arrested at the initiation of S phase or were replication-interrupted by hydroxyurea. DNA footprinting experiments indicate that the region of protein binding is at the 3' end of the conserved region and overlaps with one of the three in-vitro-mapped termination sites.

Animals↗
PubMed1997

The anti-cancer agent distamycin A displaces essential transcription factors and selectively inhibits myogenic differentiation.

The anticancer drug, distamycin A, alters DNA conformation by binding to A/T-rich domains. We propose that binding of the drug to DNA alters transcription factor interactions and that this may alter genetic regulation. We have analyzed the effects of distamycin A upon expression of the muscle-specific cardiac and skeletal alpha-actin genes which have A/T-rich regulatory elements in their promoters. Distamycin A specifically inhibited endogenous muscle genes in the myogenic C2 cell line and effectively eliminated the myogenic program. Conversely, when 10T1/2C18 derived pleuripotential TA1 cells were induced to differentiate in the presence of distamycin A, adipocyte differentiation was enhanced whereas the numbers of cells committing to the myogenic program decreased dramatically. Using the mobility shift assay distamycin A selectively inhibited binding of two important transcription factors, SRF and MEF2, to their respective A/T-rich elements. The binding of factors Sp1 and MyoD were not affected. The inhibition of factor binding correlated with a repression of muscle-specific promoter activity as assayed by transient transfection assays. Co-expression of the myoD gene, driven by a distamycin A-insensitive promoter, failed to relieve the inhibition of these muscle-specific promoters by distamycin A. Additionally, SRF and MEF2 dependent promoters were selectively down regulated by distamycin A. These results suggest that distamycin A may inhibit muscle-specific gene expression by selectively interfering with transcription factor interactions and demonstrate the importance of these A/T-rich elements in regulating differentiation of this specific cell type.

Actins↗