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L Kedes

Publications and source records attributed to L Kedes.

At least 73 records · Page 4Linked to original sources
PubMed1993

Regulation of the human cardiac/slow-twitch troponin C gene by multiple, cooperative, cell-type-specific, and MyoD-responsive elements.

The cardiac troponin C (cTnC) gene produces identical transcripts in slow-twitch skeletal muscle and in heart muscle (R. Gahlmann, R. Wade, P. Gunning, and L. Kedes, J. Mol. Biol. 201:379-391, 1988). A separate gene encodes the fast-twitch skeletal muscle troponin C and is not expressed in heart muscle. We have used transient transfection to characterize the regulatory elements responsible for skeletal and cardiac cell-type-specific expression of the human cTnC (HcTnC) gene. At least four separate elements cooperate to confer tissue-specific expression of this gene in differentiated myotubes; a basal promoter (between -61 and -13) augments transcription 9-fold, upstream major regulatory sequences (between -68 and -142 and between -1319 and -4500) augment transcription as much as 39-fold, and at least two enhancer-like elements in the first intron (between +58 and +1028 and between +1029 and +1523) independently augment transcription 4- to 5-fold. These enhancers in the first intron increase myotube-specific chloramphenicol acetyltransferase activity when linked to their own promoter elements or to the heterologous simian virus 40 promoter, and the effects are multiplicative rather than additive. Each of the major myotube regulatory regions is capable of responding directly or indirectly to the myogenic determination factor, MyoD.A MyoD expression vector in 10T1/2 cells induced constructs carrying either the upstream HcTnC promoter elements or the first intron of the gene 300- to 500-fold. Expression was inhibited by cotransfection with Id, a negative regulator of basic helix-loop-helix transcription factors. The basal promoter contains five tandem TGGGC repeats that interact with Sp1 or an Sp1-like factor in nuclear extracts. Mutational analysis of this element demonstrated that two of the five repeat sequences were sufficient to support basal level muscle cell-specific transcription. Whereas the basal promoter is also critical for expression in cardiac myocytes, the elements upstream of -67 appear to play little or no role. Major augmentation of expression in cardiomyocytes is also provided by sequences in the first intron, but these are upstream (between +58 and +1028). The downstream segment of the first intron has no enhancer activity in cardiomyocytes. A specific DNA-protein complex is formed by this C2 cell enhancer with extracts from C2 cells but not cardiomyocytes. These observations suggest that tissue-specific expression of the HcTnC gene is cooperatively regulated by the complex interactions of multiple regulatory elements and that different elements are used to regulate expression in myogenic and cardiac cells.

Animals↗
PubMed1993

Parathyroid hormone-parathyroid hormone related protein receptor messenger RNA is present in many tissues besides the kidney.

Parathyroid hormone (PTH) acts on a large number of cells derived from many different tissues that are not traditional targets (kidney and bone) for their action. Also, the acute exposure of many of these cells to PTH resulted in the generation of cAMP. These observations are consistent with the presence of PTH receptors on these cells. However, there is no evidence that the cells that are not traditional targets for PTH, express the receptor of the hormone. The cloning of the PTH-PTH related protein (PTH-PTHrP) receptor provided the tool to examine whether these cells contain the mRNA for this receptor. Poly A+RNA from a variety of rat tissues was probed with a 1,200-bp fragment of the cDNA of the PTH-PTHrP receptor by the Northern blot technique. We found that mRNA for the PTH-PTHrP receptor is present in the heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis. Transcripts of 2.4 kb were found in all these tissues with the strongest expression in the kidney. In addition, smaller RNAs were detected in the kidney (approximately 1.8 kb) and testis (1.5 kb). These results indicate that many cell types express the PTH-PTHrP receptor gene. The data provide a possible explanation for the direct effects of PTH on so many cells and for the understanding of the harmful effects of chronic excess of PTH on the function of many organs in chronic renal failure.

Animals↗
PubMed1993

Tissue-specific restriction of skeletal muscle troponin C gene expression.

Expression of the skeletal muscle troponin C (TnC) gene is confined to fast-twitch skeletal muscle fibers (Gahlmann et al., 1988) and appears to be subject to an unexpected form of regulation. Unlike enhancers of other muscle genes, the TnC enhancer and basal promoter are muscle cell-specific only when linked to each other. We identified a strong classical enhancer element within the 5'-flanking sequence of this gene at -1.5 kb and a basal promoter near the transcription start site. Both elements are required for the transcriptional activity of TnC test constructs in myogenic cells. When the TnC enhancer was linked to the SV40 early basal promoter, or the TnC basal promoter was linked to the SV40 enhancer, each supported expression in non-muscle cells. Nuclear factors from both muscle and non-muscle cells bind to one CTF/NF1 binding site and to two functionally related MEF2-like A/T-rich binding sites in the enhancer element. It is currently unknown whether modifications of these nuclear factors, differences in their concentrations, or their interaction with additional factors restrict human fast-twitch TnC expression to skeletal muscle cells. However, it appears that the human fast-twitch skeletal troponin C gene is restricted in non-muscle cells in a distinctive way requiring communication between its enhancer and basal promoter.

Base Sequence↗
PubMed1992

Myocardial activation of the human cardiac alpha-actin promoter by helix-loop-helix proteins.

The cardiac alpha-actin gene is expressed in both heart and skeletal muscle. In skeletal myogenic cells, the 177-base-pair promoter of the human cardiac alpha-actin (HCA) gene requires three transcription factors for activation: Sp1, serum response factor (SRF), and MyoD. However, MyoD is undetectable in heart. To search for a functional equivalent of MyoD, we analyzed the transcriptional regulation of the HCA promoter in primary cultures of rat cardiac myocytes. The same DNA sequence elements recognized by SRF, Sp1, and MyoD and required for HCA transcription in skeletal muscle cells were also found to be necessary for expression in cardiomyocytes. Overexpression of Id, a negative regulator of basic helix-loop-helix proteins, selectively attenuated expression of the HCA promoter. Cardiomyocyte nuclei contain a protein complex that specifically interacts with the same required sequence (E box) in the HCA promoter that is bound by MyoD in skeletal myogenic cells. Furthermore, these complexes contain a peptide that is a member of the E2A family of basic helix-loop-helix proteins. Cardiomyocyte nuclei appear to be enriched for a protein that can bind to the E-box site as dimers with the E12 protein. These results suggest that a member of the basic helix-loop-helix family, together with SRF and Sp1, activates the HCA promoter in heart. Alternative strategies for myocardial transcription of HCA are discussed.

Actins↗
PubMed1992

The human skeletal alpha-actin gene is regulated by a muscle-specific enhancer that binds three nuclear factors.

The tissue-specific distal promoter of the human skeletal alpha-actin gene (-1282 to -708) induces transcription in myogenic cells approximately 10-fold and, with the most proximal promoter domain (-153 to -87), it synergistically increases transcription 100-fold (Muscat and Kedes 1987). We report here that it is a short fragment of the distal promoter, the distal regulatory element (DRE) from -1282 to -1177 that functions as a muscle-specific, composite enhancer. An internal deletion in the DRE (delta -1282/-1151) in the context of the full-length 2000 bp promoter, resulted in a 10-fold reduction in transcription. Three distinct nuclear proteins, DRF-1, DRF-2, and DRF-3, interact specifically with the DRE between positions -1260 and -1193. A site specific mutation that abolishes DRF-2 binding also results in a 10-fold reduction in transcriptional activity. The DRF-2 nuclear protein has characteristics similar to those of the muscle-specific regulatory factor, MEF-2 (Buskin and Hauschka 1989; Gossett et al., 1989). Like the MEF-2 binding site in the muscle creatine kinase enhancer, the critical DRF-2 binding site is also an A/T-rich sequence element. The DRF-2 nuclear protein binds equally well to the MCK MEF-2 binding site and to the A/T-rich regulatory element of the skeletal muscle fast-twitch troponin C gene (Gahlmann and Kedes 1990). Furthermore, this troponin C site competes in vivo for DRF-2 driven expression of the skeletal alpha-actin gene in C2 cells. The DRF-2 site alone, however, does not activate transcription in muscle cells when linked to the SV40 promoter. We conclude that the DRF-2 binding element is a MEF-2 binding site that is required but insufficient for regulation of muscle-specific skeletal alpha-actin gene expression by the DRE. Thus, muscle-specific regulation of the human skeletal alpha-actin gene appears to require interactions between the other elements of the composite DRE enhancer with the protein:DNA complex formed by DRF-2.

Actins↗
PubMed1991

Adrenergic regulation of the skeletal alpha-actin gene promoter during myocardial cell hypertrophy.

The skeletal alpha-actin gene is expressed in fetal rat heart and is induced during norepinephrine (NE)-stimulated hypertrophy in cultures of neonatal rat cardiac myocytes. Here we report that NE positively regulates the human skeletal alpha-actin gene promoter in transiently transfected neonatal rat cardiac myocytes. NE increased expression from the full-length promoter by 2.4-fold. A DNA region required for NE responsiveness but not for tissue-specific expression was located between base pair -2000 and base pair -1300. Distinct regions required for cardiac myocyte expression were located between -1300 to -710 and -153 to -87. None of these elements separately conferred tissue specificity or adrenergic responsiveness on a heterologous promoter, although the intact promoter from -2000 to -36 conferred both when cloned in its correct position and orientation. Additional elements in the basal promoter (-87 to +187) were required for maximal NE responsiveness. The NE induction was mediated by the beta-adrenergic receptor in high-density cultures (3-4 x 10(6) cells per 60-mm dish), as was induction of hypertrophy, contractility, and endogenous skeletal alpha-actin gene expression. The beta-adrenergic agonist isoproterenol was as potent as NE in inducing expression. Furthermore, beta-adrenergic antagonists inhibited the effects on skeletal alpha-actin gene expression but alpha 1-adrenergic antagonists did not. The alpha 1-adrenergic system was intact in these high-density cultures, since the effects of NE on the expression of another contractile protein gene, alpha-myosin heavy chain, were blocked by alpha 1- but not by beta-adrenergic antagonists. In these high-density cultures, cell contact and intermyocardiocyte bridging were prevalent. When cardiac myocytes were plated at a low density, minimizing cell contact, NE induction of skeletal alpha-actin gene expression and hypertrophy was mediated by the alpha 1-adrenoceptor. Factors related to cell communication may influence the pathways mediating NE-regulated gene transcription during cardiac myocyte hypertrophy.

Actins↗
PubMed1990

Cloning, structural analysis, and expression of the human slow twitch skeletal muscle/cardiac troponin C gene.

The two isoforms of troponin C that are differentially expressed in slow and fast twitch skeletal muscle are encoded by single copy genes. We are analyzing the mechanisms that control their highly restricted pattern of differential expression. The structure of the human fast twitch troponin C isoform gene has been reported (Gahlmann, R. and Kedes, L. (1990) J. Biol. Chem. 265, 12520-12528). Here we describe the isolation, nucleotide sequence, and localization of a regulating promoter element sufficient to impart expression of the human slow twitch skeletal muscle troponin C gene which is also the isoform expressed in heart. The 3.0-kilobase gene is composed of 6 exons and 5 introns. Introns and 5'-flanking sequences between the human and mouse slow troponin C genes are highly conserved. The gene is transcribed from the same start site in skeletal and cardiac muscle. A consensus TATA box is located 29 base pairs upstream of the transcriptional start site but no canonical CAAT box was observed. Cell transfection experiments provided evidence that promoter elements that are responsible for a cell type-specific pattern of gene expression are located in the 5'-flanking sequences. Constructs comprising 4.0 kilobases of 5'-flanking sequences, attached upstream of the chloramphenicol transferase gene as reporter, were expressed at high levels in differentiated cells of three myogenic cell lines (C2, L8, and H9c2(2-1)) and also at high levels in undifferentiated C2 and H9c2(2-1) cells. Chloramphenicol acetyltransferase activity was not detected in either WI38 cells or monkey kidney cells, CV-1. 5'-Deletion constructs were assayed for expression in differentiated H9c2(2-1) and C2 cells. Sequences between base pairs -67 and +24 were sufficient for high level expression in these cell lines.

Amino Acid Sequence↗
PubMed1990

Cloning, structural analysis, and expression of the human fast twitch skeletal muscle troponin C gene.

The gene encoding human fast skeletal muscle troponin C (TnC) was cloned, mapped, and sequenced. The locations of intron positions in this gene were compared to those in the related genes for mouse slow skeletal TnC and vertebrate and nonvertebrate calmodulins. We detected strikingly similar purine-rich DNA sequences on the coding strand in the basal promoter of the genes for fast and slow troponin C and chicken calmodulin II which may represent conserved regulatory elements in genes of the vertebrate troponin C/calmodulin gene family. We mapped the transcriptional start site of the gene and analyzed the expression of TnC test genes in the myogenic cell lines C2, L8, and H9c2(2-1) and in the human fibroblast line HuT12. Constructs comprising 4.7 or 6.2 kilobase pairs of 5'-flanking sequence (including the genuine transcriptional start site) upstream of the chloramphenicol acetyltransferase gene as reporter expressed the hybrid gene in C2 cells but not in nonmuscle cells. Surprisingly, no expression was found in cell lines L8 and H9c2(2-1) despite the fact that all three muscle cell lines vigorously express the endogenous TnC fast mRNA after differentiation. The discrepancy between the expression of endogenous genes and the test gene in these cell lines indicates different requirements for regulatory elements in different myogenic cells.

Amino Acid Sequence↗
PubMed1990

Coordinate reciprocal trends in glycolytic and mitochondrial transcript accumulations during the in vitro differentiation of human myoblasts.

Changes in the mRNA levels during mammalian myogenesis were compared for seven polypeptides of mitochondrial respiration (the mitochondrial DNA-encoded cytochrome oxidase subunit III, ATP synthase subunit 6, NADH dehydrogenase subunits 1 and 2, and 16S ribosomal RNA; the nuclear encoded ATP synthase beta subunit and the adenine nucleotide translocase) and three polypeptides of glycolysis (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and triose-phosphate isomerase). Progressive changes during the conversion from myoblasts to myotubes were monitored under both atmospheric oxygen (normoxic) and hypoxic environments. Northern analyses revealed coordinate, biphasic, and reciprocal expression of the respiratory and glycolytic mRNAs during myogenesis. In normoxic cells the mitochondrial respiratory enzymes were highest in myoblasts, declined 3- to 5-fold during commitment and exist from the cell cycle, and increased progressively as the myotubes matured. By contrast, the glycolytic enzyme mRNAs rose 3- to 6-fold on commitment and then progressively declined. When partially differentiated myotubes were switched to hypoxic conditions, the glycolytic enzyme mRNAs increased and the respiratory mRNAs declined. Hence, the developmental regulation of muscle bioenergetic metabolism appears to be regulated at the pretranslational level and is modulated by oxygen tension.

Cell Differentiation↗
PubMed1990

Regulation of contractile protein gene family mRNA pool sizes during myogenesis.

During myogenesis, muscle contractile protein gene expression is induced and the products are used to assemble the contractile apparatus characteristic of striated muscle. The different muscle proteins are accumulated in a fixed stoichiometric ratio related to their organization in the contractile apparatus. We have examined the relationship between contractile protein gene expression and the maintenance of stoichiometry at different stages of human myogenesis. Essentially all of the known components of adult human skeletal muscle thick and thin filaments have been cloned in the form of cDNAs and used to generate isoform-specific DNA probes. The expression of fast, slow, and cardiac isoforms was measured in human myogenic primary culture and in fetal and adult human skeletal muscle. We observed that neither fast nor slow nor cardiac isoforms are coordinately regulated at the level of comparative transcript accumulation throughout myogenesis. Thus, the stoichiometry of contractile protein levels cannot be explained by coordination of expression in each of these isoform classes. However, we find that the stoichiometry of mRNA accumulation of each gene family is very similar among three developmental stages: myotubes, fetal skeletal muscle, and adult skeletal muscle. This is consistent with the possibility that the maintenance of stoichiometry between the contractile proteins could be largely regulated by the total accumulation of mRNA from each of these gene families.

Actins↗
PubMed1990

cDNA sequence, tissue-specific expression, and chromosomal mapping of the human slow-twitch skeletal muscle isoform of troponin I.

Troponin I (TnI) is a myofibrillar protein involved in the calcium-mediated regulation of striated muscle contraction. Three isoforms of TnI are known and each is expressed in a muscle fiber-type-specific manner. TnI-fast and TnI-slow are expressed exclusively in fast-twitch and slow-twitch skeletal muscle myofibers, respectively, while a third isoform, TnI-card, is expressed in both the atrium and the ventricle of the heart. An explanation of the myofiber-type-restricted expression of the troponin I multigene family will further aid in understanding how various types of striated muscle fibers are established. To initiate the study of TnI isoform gene expression, we have isolated a full-length cDNA representing the human slow-twitch skeletal muscle isoform of troponin I. Sequence comparisons demonstrate that the TnI-slow protein is highly conserved between species. Therefore, the cDNA was used as a probe to investigate the tissue-specific and developmental regulation of the TnI-slow gene in both rodent and human myogenic cells. TnI-slow message appears to be restricted to muscle tissue containing slow-twitch skeletal muscle myofibers. TnI-slow gene expression is induced in differentiated cultures of primary human muscle cells and several (but not all) myogenic cell lines. In addition, a human-specific probe prepared from the 3' untranslated region of the cDNA has been used to probe a panel of human/mouse somatic cell hybrid lines, resulting in the assignment of the human TnI-slow gene to the q12----qter region of chromosome 1. The locus is designated TNNI1.

Amino Acid Sequence↗
PubMed1990

Doxorubicin selectively inhibits muscle gene expression in cardiac muscle cells in vivo and in vitro.

The anthracycline antibiotic doxorubicin produces a characteristic myopathy in cardiac muscle that limits its use in cancer therapy. We have shown in cultured neonatal rat cardiac muscle cells that doxorubicin treatment resulted in a rapid, selective decrease in the expression of muscle-specific genes, which preceded other changes characteristic of doxorubicin cardiomyopathy. Doxorubicin selectively and dramatically decreased the levels of mRNA for the sarcomeric genes, alpha-actin, troponin I, and myosin light chain 2, as well as the muscle-specific, but nonsarcomeric M isoform of creatine kinase. However, doxorubicin did not affect nonmuscle gene transcripts (pyruvate kinase, ferritin heavy chain, and beta-actin). Actinomycin D, an inhibitor of DNA-dependent RNA polymerase, did not show a similar selective decrease of muscle-specific mRNAs but, rather, produced a nonspecific, dose-dependent decrease of muscle and nonmuscle transcripts. The doxorubicin effect on muscle gene expression was limited to cardiac muscle; cultured skeletal myocytes were resistant to the effects of doxorubicin at 100-fold greater doses than those causing changes in mRNA levels in cardiac muscle cells. These effects of doxorubicin were reproduced in vivo; rats injected with doxorubicin showed a dose-dependent decrease in the levels of mRNAs for alpha-actin, troponin I, myosin light chain 2, and M isoform of creatine kinase in cardiac but not skeletal muscle. These selective changes in gene expression in cardiocyte cultures and cardiac muscle precede classical ultrastructural changes and may explain the myofibrillar loss that characterizes doxorubicin cardiac injury.

Actins↗
PubMed1990

Muscle-specific expression of the cardiac alpha-actin gene requires MyoD1, CArG-box binding factor, and Sp1.

Expression of the human cardiac alpha-actin gene (HCA) depends on the interactions of multiple transcriptional regulators with promoter elements. We report here that the tissue-specific expression of this promoter is determined by the simultaneous interaction of at least three specific protein-DNA complexes. The myogenic determinant gene MyoD1 activated the transcription of transfected HCA-CAT promoter constructs in nonmuscle cells, including CV-1 and HeLa cells. Gel mobility-shift and footprinting assays revealed that MyoD1 specifically interacted with a single consensus core sequence, CANNTG, at -50. Previously characterized sites interact with a protein identical with or related to the serum response factor (SRF) at -100 and Sp1 at -70. All three elements must be intact to support transcription in muscle cells: site-specific mutation within any one of these three elements eliminated transcriptional expression by the promoter. Furthermore, expression of the promoter in embryonic Drosophila melanogaster cells that lack MyoD1 and Sp1 is strictly dependent on all three sites remaining intact and on the presence of exogenously supplied Sp1 and MyoD1. These experiments suggest that the presence of three sequence-specific binding proteins, including MyoD1, and their intact target DNA sequences are minimal requirements for muscle-specific expression of the HCA gene.

Actins↗
PubMed1990

The c-fos cyclic AMP-responsive element conveys constitutive expression to a tissue-specific promoter.

The c-fos and cardiac alpha-actin promoters share homologous 5' protein binding elements that are essential for serum-inducible and tissue-specific expression, respectively. Additional elements, auxiliary proteins or factor modifications, must distinguish the individual transcriptional responses of these two promoters. An element in the c-fos basal promoter that is normally responsible for transient stimulation of the fos gene in response to Ca2+ or cyclic AMP (CRE) may be able to modulate the expression of the upstream elements. We report here that this element, when inserted into the cardiac alpha-actin promoter, conveys constitutive expression to this otherwise highly restricted promoter. Additional data support the proposal that the CRE binding protein creates an alternative pathway whereby upstream regulatory elements in the cardiac alpha-actin promoter can activate transcription in a manner which circumvents the requirement for a tissue-specific environment.

Actins↗
PubMed1989

Sequence and expression of human myosin alkali light chain isoforms.

In order to initiate the study of the functional differences between myosin alkali light chain isoforms and to investigate the mechanisms of their differential expression, we have isolated cDNA clones for two human alkali light chain isoforms. Here we report DNA sequence and RNA blotting analyses that demonstrate that these cDNAs represent transcripts encoding human MLC3F and MLC1Sb. The sequence of the human MLC1Sb cDNA offers the first fully characterized example of a slow-fiber skeletal muscle alkali light chain isoform from any species. The sequence analysis of these two cDNAs allows an examination of evolutionarily conserved features of mammalian alkali light chain genes. Examination of the genomic organization of the human alkali light chain isoform genes revealed that, in contrast with some strains of mice, both are single copy genes. RNA blot analysis conclusively demonstrates that the human skeletal muscle MLC1Sb gene is also expressed in the heart ventricle but not the atria. In addition, we examined the expression of alkali light chain isoforms during the in vitro differentiation of a variety of human and rodent myogenic cells and found striking variation in the pattern of alkali light chain isoform gene expression in different myogenic cells.

Amino Acid Sequence↗