Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 26
Filter
Add more filters










Publication year range
1.
Oncogene ; 28(1): 52-62, 2009 Jan 08.
Article in English | MEDLINE | ID: mdl-18794805

ABSTRACT

The transcription factor E2F1 plays key roles in skin homeostasis, and is essential for normal keratinocyte proliferation and epidermal regeneration after injury. We have previously established that, in differentiating keratinocytes, E2F1 activity is controlled by nuclear export and subsequent degradation. These events are triggered by differentiation-induced stimulation of protein kinase C and p38 mitogen-activated protein kinase (MAPK). However, the mechanisms that induce E2F1 export from the nucleus and the role of p38 MAPK in this process are poorly understood. We now describe a novel regulatory pathway for E2F1, which involves phosphorylation by p38. We demonstrate that E2F1 forms complexes with active p38 through regions that exclude the N-terminus of this transcription factor, and that p38 activity is a major contributor to the phosphorylation status of E2F1 in keratinocytes. Using in vitro kinase assays, we identified Ser403 and Thr433 as the residues phosphorylated by p38. The biological significance of these observations is underscored by the inability of E2F1 mutants lacking one or both of these residues to be exported from the nucleus and degraded when keratinocytes receive differentiation stimuli, which results in impaired keratinocyte maturation.


Subject(s)
Cell Differentiation , E2F1 Transcription Factor/metabolism , Keratinocytes/cytology , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Cell Line , DNA Mutational Analysis , E2F1 Transcription Factor/genetics , Keratinocytes/metabolism , Mice , Phosphorylation , Sequence Deletion , Serine/genetics , Serine/metabolism , Threonine/genetics , Threonine/metabolism
2.
Oncogene ; 26(8): 1147-54, 2007 Feb 22.
Article in English | MEDLINE | ID: mdl-16924238

ABSTRACT

E2F factors modulate a plethora of cell functions, including proliferation, differentiation, DNA repair and apoptosis. We have shown that differentiation in primary epidermal keratinocytes leads to E2F1 downregulation via activation of protein kinase C and p38 mitogen-activated protein kinase. We now demonstrate that E2F1 downregulation in differentiating keratinocytes involves its ubiquitination, as well as proteasomal degradation subsequent to CRM1-dependent nuclear export. E2F1 nuclear export specifically in response to differentiation requires regions adjacent to the cyclin A-binding domain in the N-terminus of this protein. Significantly, inhibition of p38 interferes with nuclear export and degradation of E2F1 during differentiation, but has no effect on E2F1 in undifferentiated cells. Thus, induction of differentiation in epidermal keratinocytes activates a specific program for post-transcriptional downregulation of E2F1, which involves signaling through p38 and activation of nuclear export pathways.


Subject(s)
Cell Differentiation/genetics , Cell Nucleus/metabolism , E2F1 Transcription Factor/metabolism , Karyopherins/metabolism , Keratinocytes/cytology , Receptors, Cytoplasmic and Nuclear/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Active Transport, Cell Nucleus , Animals , Cell Nucleus/chemistry , Cells, Cultured , Cytoplasm/chemistry , Cytoplasm/metabolism , Down-Regulation , E2F1 Transcription Factor/analysis , E2F1 Transcription Factor/genetics , Enzyme Activation , Keratinocytes/chemistry , Keratinocytes/metabolism , Mice , Mutation , Proteasome Endopeptidase Complex/metabolism , Protein Structure, Tertiary , Signal Transduction , Ubiquitin/metabolism , Exportin 1 Protein
3.
Oncogene ; 25(3): 430-7, 2006 Jan 19.
Article in English | MEDLINE | ID: mdl-16116476

ABSTRACT

E2F transcription factors regulate proliferation, differentiation, DNA repair and apoptosis. Tight E2F regulation is crucial for epidermal formation and regeneration. However, virtually nothing is known about the molecular events modulating E2F during epidermal keratinocyte differentiation. Elucidation of these events is essential to understand epidermal morphogenesis, transformation and repair. Here we show that, in differentiating keratinocytes, Ca(2+)-induced protein kinase C (PKC) activation downregulates E2F1 protein levels. Further, we have identified PKC delta and eta as those isoforms specifically involved in induction of E2F1 proteasomal degradation. We also demonstrate that E2F1 downregulation by novel PKC isozymes requires activation of p38beta mitogen-activated protein kinase (MAPK). This is the first example of regulation in the E2F transcription factor family by activation of PKC and MAPK in the context of biologically significant differentiation stimuli in epithelia.


Subject(s)
Cell Differentiation , E2F1 Transcription Factor/metabolism , Keratinocytes/cytology , Protein Kinase C/metabolism , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Enzyme Activation , Keratinocytes/enzymology , Keratinocytes/metabolism , Mice
4.
J Biol Chem ; 276(26): 23531-8, 2001 Jun 29.
Article in English | MEDLINE | ID: mdl-11319226

ABSTRACT

The epidermis consists of a squamous epithelium continuously replenished by committed stem cells, which can either self-renew or differentiate. We demonstrated previously that E2F genes are differentially expressed in developing epidermis (Dagnino, L., Fry, C. J., Bartley, S. M., Farnham, P., Gallie, B. L., and Phillips, R. A. (1997) Cell Growth Differ. 8, 553-563). Thus, we hypothesized that various E2F proteins likely play distinct growth regulatory roles in the undifferentiated stem cells and in terminally differentiated keratinocytes. To further understand the function of E2F genes in epidermal morphogenesis, we have examined the expression, regulation, and protein-protein interactions of E2F factors in undifferentiated cultured murine primary keratinocytes or in cells induced to differentiate with Ca(2+) or BMP-6 (bone morphogenetic protein 6). We find similar patterns of E2F regulation with both differentiating agents and demonstrate a switch in expression from E2F-1, -2, and -3 in undifferentiated, proliferating cells to E2F-5 in terminally differentiated keratinocytes. Inhibition of keratinocyte proliferation by transforming growth factor-beta1 did not enhance E2F-5 protein levels, suggesting that this response is specific to differentiation rather than reversible cell cycle withdrawal. E2F-5 up-regulation is also accompanied by formation of heteromeric nuclear complexes containing E2F5, p130, and histone deacetylase (HDAC) 1. Overexpression of E2F5 specifically inhibited DNA synthesis in undifferentiated keratinocytes in an HDAC-dependent manner, suggesting that E2F-5.p130.HDAC1 complexes are likely involved in the permanent withdrawal from the cell cycle of keratinocytes responding to differentiation stimuli.


Subject(s)
Bone Morphogenetic Proteins/physiology , Calcium Signaling , Cell Cycle Proteins , Epidermis/growth & development , Keratinocytes/metabolism , Proteins , Transcription Factors/metabolism , Animals , Bone Morphogenetic Protein 6 , Bone Morphogenetic Proteins/pharmacology , Calcium/pharmacology , Cell Differentiation , Cell Division , Cells, Cultured , DNA/biosynthesis , DNA-Binding Proteins/metabolism , E2F Transcription Factors , E2F1 Transcription Factor , E2F5 Transcription Factor , Histone Deacetylase 1 , Histone Deacetylases/metabolism , Keratinocytes/cytology , Macromolecular Substances , Mice , Phosphoproteins/metabolism , Phosphorylation , Retinoblastoma-Like Protein p130 , Signal Transduction , Tissue Distribution , Transcription Factors/physiology
5.
Dev Biol ; 207(2): 257-70, 1999 Mar 15.
Article in English | MEDLINE | ID: mdl-10068462

ABSTRACT

The severe neurological deficit in embryos carrying null mutations for the retinoblastoma (Rb) gene suggests that Rb plays a crucial role in neurogenesis. While developing neurons undergo apoptosis in vivo neural precursor cells cultured from Rb-deficient embryos appear to differentiate and survive. To determine whether Rb is an essential regulator of the intrinsic pathway modulating terminal mitosis we examined the terminal differentiation of primary cortical progenitor cells and bFGF-dependent neural stem cells derived from Rb-deficient mice. Although Rb -/- neural precursor cells are able to differentiate in vitro we show that these cells exhibit a significant delay in terminal mitosis relative to wild-type cells. Furthermore, Rb -/- cells surviving in vitro exhibit an upregulation of p107 that is found in complexes with E2F3. This suggests that p107 may partially compensate for the loss of Rb in neural precursor cells. Functional ablation of Rb family proteins by adenovirus-mediated delivery of an E1A N-terminal mutant results in apoptosis in Rb-deficient cells, consistent with the interpretation that other Rb family proteins may facilitate differentiation and survival. While p107 is upregulated and interacts with the putative Rb target E2F3 in neural precursor cells, our results indicate that it clearly cannot restore normal E2F regulation. Rb-deficient cells exhibit a significant enhancement of E2F 1 and 3 activity throughout differentiation concomitant with the aberrant expression of E2F-inducible genes. In these studies we show that Rb is essential for the regulation of E2F 1 and 3 activity as well as the onset of terminal mitosis in neural precursor cells.


Subject(s)
Carrier Proteins , Cell Cycle Proteins , Neurons/metabolism , Retinoblastoma Protein/genetics , Stem Cells/metabolism , Transcription Factors/metabolism , Adenoviridae/genetics , Animals , Apoptosis , Cell Differentiation/genetics , Cells, Cultured , DNA-Binding Proteins/genetics , E2F Transcription Factors , E2F1 Transcription Factor , E2F3 Transcription Factor , Fibroblast Growth Factor 2/metabolism , Gene Expression Regulation, Developmental , Immunohistochemistry , Mice , Mice, Knockout , Mitosis , Nuclear Proteins/genetics , Protein Binding , Retinoblastoma-Binding Protein 1 , Retinoblastoma-Like Protein p107 , Transcription Factor DP1
6.
Genes Dev ; 12(8): 1092-8, 1998 Apr 15.
Article in English | MEDLINE | ID: mdl-9553039

ABSTRACT

Homozygous E2F-5 knockout embryos and mice have been generated. Although embryonic development appeared normal, newborn mice developed nonobstructive hydrocephalus, suggesting excessive cerebrospinal fluid (CSF) production. Although the CSF-producing choroid plexus displayed normal cellular organization, it contained abundant electron-lucent epithelial cells, consistent with excessive CSF secretory activity. Moreover, E2F-5 CNS expression in normal animals was largely confined to the choroid plexus. Cell cycle kinetics were not perturbed in homozygous knockout embryo fibroblasts. Thus, E2F-5 is not essential for cell proliferation. Rather, it affects the secretory behavior of a differentiated neural tissue.


Subject(s)
Choroid Plexus/physiology , Transcription Factors/physiology , Alleles , Animals , Cell Division , Choroid Plexus/metabolism , E2F5 Transcription Factor , Gene Deletion , Gene Targeting , Humans , Hydrocephalus/metabolism , Mice , Mice, Knockout , Transcription Factors/biosynthesis , Transcription Factors/genetics
7.
J Med Chem ; 41(4): 509-14, 1998 Feb 12.
Article in English | MEDLINE | ID: mdl-9484501

ABSTRACT

A group of racemic alkyl (or cycloalkyl) 1,4-dihydro-2,6- dimethyl-3-nitro-4-(2-, 3-, or 4-pyridyl)-5-pyridinecarboxylate isomers (6-14) were prepared using a modified Hantzsch reaction that involved the condensation of nitroacetone with an alkyl (or cycloalkyl) 3-aminocrotonate and 2-, 3-, or 4-pyridinecarboxaldehyde. Determination of their in vitro calcium channel-modulating activities using guinea pig ileum longitudinal smooth muscle (GPILSM) and guinea pig left atrium (GPLA) assays showed that the 2-pyridyl isomers acted as dual cardioselective calcium channel agonists (GPLA)/smooth muscle selective calcium channel antagonists (GPILSM). In contrast, the 3-pyridyl and 4-pyridyl isomers acted as calcium channel agonists on both GPLA and GPILSM. In the C-4 2-pyridyl group of compounds, the size of the C-5 alkyl (or cycloalkyl) ester substituent was a determinant of GPILSM antagonist activity where the relative activity profile was cyclopentyl and cyclohexyl > t-Bu, i-Bu, and Et > MeOCH2CH2 > Me. The point of attachment of the C-4 pyridyl substituent was a determinant of GPLA agonist activity where the potency order was generally 4- and 3-pyridyl > 2-pyridyl. (+)-Cyclohexyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-pyridyl)-5- pyridinecarboxylate [(+)-14a] was a less potent calcium antagonist (IC50 = 5.27 x 10(-6) M) than the (-)-enantiomer (IC50 = 7.48 x 10(-8) M) on GPILSM. In the GPLA assay, (+)-14a exhibited a much more potent agonist effect (EC50 = 8.45 x 10(-6) M) relative to the marginal agonist effect produced by (-)-14a. The C-4 2-pyridyl compounds (enantiomers) constitute a novel type of 1,4-dihydropyridine calcium channel modulator that could provide a new drug design concept directed toward the treatment of congestive heart failure, and for use as probes to study the structure-function relationships of calcium channels.


Subject(s)
Calcium Channel Agonists/chemical synthesis , Calcium Channel Blockers/chemical synthesis , Calcium Channels/physiology , Dihydropyridines/chemical synthesis , Heterocyclic Compounds/chemical synthesis , Muscle Contraction/drug effects , Muscle, Smooth/physiology , Myocardial Contraction/drug effects , Pyridines/chemical synthesis , Animals , Calcium Channel Agonists/chemistry , Calcium Channel Agonists/pharmacology , Calcium Channel Blockers/chemistry , Calcium Channel Blockers/pharmacology , Dihydropyridines/chemistry , Dihydropyridines/pharmacology , Guinea Pigs , Heart Atria , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/pharmacology , Ileum , In Vitro Techniques , Indicators and Reagents , Isomerism , Molecular Structure , Muscle, Smooth/drug effects , Pyridines/chemistry , Pyridines/pharmacology , Stereoisomerism , Structure-Activity Relationship
8.
Mech Dev ; 66(1-2): 13-25, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9376316

ABSTRACT

The E2F family of transcription factors consists of two subgroups termed E2F and DP. E2F is required for cell proliferation, and is necessary for fruit fly development. E2F activity is a target for regulation by the retinoblastoma gene family, which includes pRB, p107 and p130. Mutant RB-/-, RB-/-:p107-/- and p107-/-:p130-/- mice develop abnormally, probably as a result of dysregulation in the activity of E2F, indicating the importance of E2F in mammalian development. To investigate the role of E2F in murine development, we have examined the patterns of expression of E2F-1 through E2F-5, and DP-1 in the developing nervous system by in situ hybridization. E2F-1, E2F-2 and E2F-5 are first detected in the 9.5 days post-coitus (dpc) forebrain. Expression of these E2F forms extends caudally thereafter and includes the developing brain and the upper half of the 10.5 dpc spinal cord. By 11.5 dpc, these E2F factors are expressed throughout the central nervous system. In 12.5 dpc embryos, E2F-1, E2F-2 and E2F-5 are highly expressed in proliferating, undifferentiated neuronal precursors. As neurons differentiate and migrate to the outer marginal zones in the nervous system, expression of these E2F members is extinguished. In the developing retina, another neuronal tissue, E2F-1 expression is also confined to the proliferating, undifferentiated retinoblastic layer. In contrast, E2F-3 expression is up-regulated as retinoblasts differentiate into the ganglion cell layer. In non-neuronal tissues, high E2F-4 transcript levels are present in regions corresponding to proliferative chondrocytes, whereas E2F-2 and E2F-4 transcripts are very abundant in the thymic cortex, which contains immature thymocytes. We conclude that individual E2F forms are differentially regulated during the development of distinct tissues, and especially during neuronal development.


Subject(s)
Carrier Proteins , Cell Cycle Proteins , DNA-Binding Proteins , Gene Expression Regulation, Developmental , Nervous System/embryology , Transcription Factors/biosynthesis , Transcription Factors/genetics , 3T3 Cells , Amino Acid Sequence , Animals , Brain/embryology , Choroid Plexus/embryology , DNA, Complementary/isolation & purification , E2F Transcription Factors , E2F1 Transcription Factor , E2F3 Transcription Factor , E2F4 Transcription Factor , E2F5 Transcription Factor , Mice , Molecular Sequence Data , Organ Specificity/genetics , Retina/embryology , Retinoblastoma-Binding Protein 1 , Transcription Factor DP1
9.
Nat Genet ; 16(3): 283-8, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9207795

ABSTRACT

In both vertebrate and invertebrate development, cells are often programmed to adopt fates distinct from their neighbors. Genetic analyses in Drosophila melanogaster have highlighted the importance of cell surface and secreted proteins in these cell fate decisions. Homologues of these proteins have been identified and shown to play similar roles in vertebrate development. Fringe, a novel signalling protein, has been shown to induce wing margin formation in Drosophila. Fringe shares significant sequence homology and predicted secondary structure similarity with bacterial glycosyltransferases. Thus fringe may control wing development by altering glycosylation of cell surface and/or secreted molecules. Recently, two fringe genes were isolated from Xenopus laevis. We report here the cloning and characterization of three murine fringe genes (lunatic fringe, manic fringe and radical fringe). We find in several tissues that fringe expression boundaries coincide with Notch-dependent patterning centres and with Notch-ligand expression boundaries. Ectopic expression of murine manic fringe or radical fringe in Drosophila results in phenotypes that resemble those seen in Notch mutants.


Subject(s)
Gene Expression Regulation, Developmental/genetics , Glycosyltransferases , Membrane Proteins/genetics , Proteins/genetics , Xenopus Proteins , Amino Acid Sequence , Animals , Body Patterning/genetics , Cell Differentiation , DNA Probes , Drosophila Proteins , Drosophila melanogaster/genetics , Eye/cytology , Glucosyltransferases , In Situ Hybridization , Intercellular Signaling Peptides and Proteins , Intracellular Signaling Peptides and Proteins , Mice , Molecular Sequence Data , Mutation/genetics , Phenotype , Receptors, Notch , Wings, Animal/cytology , Xenopus/genetics
10.
Cell Growth Differ ; 8(5): 553-63, 1997 May.
Article in English | MEDLINE | ID: mdl-9149906

ABSTRACT

The E2F family of transcription factors includes five E2F and three DP forms. E2F is involved in the regulation of cell proliferation, but little is known about E2F function during vertebrate development. We have explored the regulation of E2F expression during mouse organogenesis by in situ hybridization. We find selective up-regulation of E2F-2, E2F-4, and E2F-5 transcripts in epidermis and intestinal epithelium at important developmental stages. E2F-4 transcript levels are high in early, undifferentiated single-cell-layer ectoderm, and later in 13.5-14.5-day-postcoitus (dpc) embryo epithelium, which contains several layers of proliferating cells. E2F-2 is up-regulated following the onset of E2F-4 expression and is first apparent in undifferentiated epithelium at 13.5-14.5 days of gestation. In contrast, E2F-5 transcripts are detected later in gestation, once the epidermis shows evidence of stratification. Stratification of the epidermis into basal, proliferating cells and suprabasal, terminally differentiating cells at 15.5-19.5 days of gestation coincides with expression of E2F-2 and E2F-4 in basal cells and of E2F-5 in suprabasal cells. Similarly, in intestinal epithelium, E2F-4 up-regulation in pseudostratified epithelium at 13.5 days of gestation precedes appearance of E2F-2 transcripts, in 14.5-dpc embryos, in the proliferating, intervillus epithelium. In 16.5-19.5-dpc embryos, no E2F-2 transcripts were detected at the tip of the developing villi, which contain terminally differentiating cells. In contrast, E2F-5 transcripts were limited to the upper half of the villi and were absent in the intervillus epithelium. This suggests that E2F-2 and E2F-4 may participate in maintaining epithelial cells in a proliferative, undifferentiated phenotype, whereas E2F-5 may be important to maintain the differentiated state. Thus, selective regulation of E2F forms occurs during murine epithelial development, irrespective of the ectodermal or endodermal origin of such epithelia.


Subject(s)
Carrier Proteins , Cell Cycle Proteins , DNA-Binding Proteins , Embryonic and Fetal Development , Gene Expression Regulation, Developmental , Transcription Factors/genetics , Amino Acid Sequence , Animals , E2F Transcription Factors , E2F2 Transcription Factor , E2F4 Transcription Factor , E2F5 Transcription Factor , Epithelium/embryology , Epithelium/metabolism , Female , Humans , Mice , Molecular Sequence Data , Pregnancy , Retinoblastoma-Binding Protein 1 , Sequence Alignment , Transcription Factor DP1 , Transcription Factors/biosynthesis
11.
Cell Growth Differ ; 6(2): 191-8, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7756178

ABSTRACT

The Rb family of proteins includes pRb, p107, and p130. These nuclear polypeptides associate with cyclins and transcription factors involved in the control of cell proliferation. This has suggested that members of the pRb family may modulate cell growth, at least in part, by regulating gene transcription. We have investigated the ability of p107 to modulate transcription and compared it with that of pRb. Whereas pRb inhibition of the c-myc promoter required the presence of E2F sites, p107 inhibition did not. Moreover, p107, but not pRb, repressed transcription from other promoters including fibronectin, herpes virus thymidine kinase, and a synthetic promoter containing a SV40 repeat activator motif upstream from the adenovirus major late-promoter TATA box. In contrast, the activity of the TATA-lacking promoters from the epidermal growth factor receptor and the cytoplasmic phospholipase A2 genes was unaffected by either p107 or pRb. Likewise, overexpression of p107 or pRb had no effect on the activity of a synthetic promoter lacking a TATA box and containing the SV40 repeat motif upstream from the terminal transferase gene initiator element. The domains in p107 required for transcriptional repression included the A segment of the pocket region and parts of the B segment, but not the spacer domain. In spite of their structural similarities, p107 and pRb may contribute to the control of cell proliferation by modulating the transcription of different genes.


Subject(s)
Carrier Proteins , Cell Cycle Proteins , DNA-Binding Proteins , Genes, Retinoblastoma , Multigene Family , Nuclear Proteins/physiology , Repressor Proteins/physiology , Transcription Factors/physiology , E2F Transcription Factors , Genes, myc , HeLa Cells , Humans , Promoter Regions, Genetic , Retinoblastoma-Binding Protein 1 , Retinoblastoma-Like Protein p107 , TATA Box , Transcription Factor DP1 , Tumor Cells, Cultured , Viral Proteins/physiology
12.
J Clin Endocrinol Metab ; 78(6): 1307-11, 1994 Jun.
Article in English | MEDLINE | ID: mdl-8200930

ABSTRACT

We have compared the expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) genes in various human tissues using a quantitative polymerase chain reaction technique. Tissues of three human subjects, obtained at autopsy, were analyzed. BNP transcripts could be detected in the central nervous system, lung, thyroid, adrenal, kidney, spleen, small intestine, ovary, uterus, and striated muscle. ANP transcripts could also be demonstrated in various human extracardiac tissues including several endocrine organs. In all peripheral tissues, the level of both natriuretic peptide transcripts was approximately 1-2 orders of magnitude lower than in cardiac ventricular tissues. This distribution is in marked contrast to the much lower level of ANP and BNP transcripts present in extracardiac rat tissues (generally less than 1/1000 of ventricles). These data suggest differential expression of the two natriuretic peptide genes in cardiac and extracardiac tissues in man. Furthermore, the presence of local synthesis of ANP and BNP in various peripheral organs suggests paracrine and/or autocrine function of these natriuretic peptides.


Subject(s)
Atrial Natriuretic Factor/biosynthesis , Gene Expression , Nerve Tissue Proteins/biosynthesis , Animals , Base Sequence , Brain/metabolism , Cardiomegaly/metabolism , DNA Primers , Female , Humans , Liver/metabolism , Lung/metabolism , Male , Middle Aged , Molecular Sequence Data , Natriuretic Peptide, Brain , Organ Specificity , Ovarian Neoplasms/metabolism , Ovary/metabolism , Pancreas/metabolism , Pituitary Gland/metabolism , Polymerase Chain Reaction/methods , Rats , Transcription, Genetic
13.
Mol Cell Biol ; 14(5): 3115-29, 1994 May.
Article in English | MEDLINE | ID: mdl-8164667

ABSTRACT

In contrast to skeletal muscle, the mechanisms responsible for activation and maintenance of tissue-specific transcription in cardiac muscle remain poorly understood. A family of hormone-encoding genes is expressed in a highly specific manner in cardiac but not skeletal myocytes. This includes the A- and B-type natriuretic peptide (ANP and BNP) genes, which encode peptide hormones with crucial roles in the regulation of blood volume and pressure. Since these genes are markers of cardiac cells, we have used them to probe the mechanisms for cardiac muscle-specific transcription. Cloning and functional analysis of the rat BNP upstream sequences revealed unexpected structural resemblance to erythroid but not to muscle-specific promoters and enhancers, including a requirement for regulatory elements containing GATA motifs. A cDNA clone corresponding to a member of the GATA family of transcription factors was isolated from a cardiomyocyte cDNA library. Transcription of this GATA gene is restricted mostly to the heart and is undetectable in skeletal muscle. Within the heart, GATA transcripts are localized in ANP- and BNP-expressing myocytes, and forced expression of the GATA protein in heterologous cells markedly activates transcription from the natural cardiac muscle-specific ANP and BNP promoters. This GATA-dependent pathway defines the first mechanism for cardiac muscle-specific transcription. Moreover, the present findings reveal striking similarities between the mechanisms controlling gene expression in hematopoietic and cardiac cells and may have important implications for studies of cardiogenesis.


Subject(s)
Atrial Natriuretic Factor/biosynthesis , DNA-Binding Proteins/metabolism , Gene Expression , Muscles/metabolism , Myocardium/metabolism , Promoter Regions, Genetic , Transcription Factors/metabolism , Transcription, Genetic , Amino Acid Sequence , Animals , Animals, Newborn , Atrial Natriuretic Factor/genetics , Base Sequence , Cell Nucleus/metabolism , Cells, Cultured , Consensus Sequence , Conserved Sequence , DNA Primers , DNA, Complementary/metabolism , DNA-Binding Proteins/biosynthesis , GATA4 Transcription Factor , HeLa Cells , Humans , Mice , Molecular Sequence Data , Natriuretic Peptide, Brain , Organ Specificity , Polymerase Chain Reaction , Rats , Rats, Sprague-Dawley , Sequence Homology, Amino Acid , Transcription Factors/biosynthesis , Transfection , Xenopus , Xenopus Proteins , Zinc Fingers
14.
Princess Takamatsu Symp ; 24: 250-63, 1994.
Article in English | MEDLINE | ID: mdl-8983080

ABSTRACT

The beta-type transforming growth factors (TGF beta) are potent inhibitors of cell proliferation. The mechanisms of TGF beta growth inhibition have been investigated. In skin keratinocytes, TGF beta 1 rapidly suppresses c-myc expression at the level of transcriptional initiation, and expression of c-myc was shown to be necessary for proliferation of these cells. Overexpression of c-myc, using an inducible construct, blocks growth inhibition by TGF beta 1. In 11.5 day p.c. lung bud organ cultures, TGF beta 1 inhibits tracheobronchial epithelial development, including branching morphogenesis. At this stage of development, the tracheobronchial epithelia express N-myc, but not c-myc, TGF beta 1 was shown to markedly inhibit N-myc expression in epithelia of the lung bud organ cultures. N-myc gene knockout experiments by others have shown that N-myc is required for branching morphogenesis of the tracheobronchial tree. The data indicate that suppression of expression of either N-myc or c-myc may play a role in TGF beta growth inhibition. To study the role of TGF beta 1 in normal mammary development and in mammary neoplasia, we have constructed three transgenic mouse lines that express a simian TGF beta 1S223/225 mutated to produce a constitutively active product under the control of the MMTV enhancer/promoter. Expression of the transgene was associated with marked suppression of the normal pattern of mammary ductal tree development in female transgenics from all three lines. However, during pregnancy, alveolar outgrowths developed from the hypoplastic ductal tree, and lactation occurred. Unlike many other transgenic mouse models in which expression of TGF alpha or oncogenes under control of the MMTV promoter leads to mammary epithelial hyperplasia and increased tumor formation, the MMTV-TGF beta 1 transgene causes conditional hypoplasia of the mammary ductal tree. No spontaneous tumors have been detected in the MMTV-TGF beta 1 transgenic animals, indicating that overexpression of TGF beta 1 in mammary epithelia does not enhance, and may actually suppress, early stages of carcinoma development. Other studies have shown that overexpression of TGF beta 1 in carcinoma cells enhances tumorigenicity and metastatic spread. We propose that TGF beta has a bifunctional role in carcinogenesis, retarding carcinoma development but enhancing progression once neoplastic transformation has occurred and the growth inhibitory response to TGF beta has been lost.


Subject(s)
Cell Division/physiology , Transforming Growth Factor beta/physiology , Animals , Cell Division/drug effects , Chick Embryo , Female , Gene Expression Regulation/drug effects , Genes, myc , Keratinocytes/cytology , Keratinocytes/drug effects , Mammary Glands, Animal/cytology , Mammary Glands, Animal/growth & development , Mammary Neoplasms, Experimental/physiopathology , Mammary Tumor Virus, Mouse/genetics , Mammary Tumor Virus, Mouse/physiology , Mice , Mice, Transgenic , Models, Biological , Pregnancy , Promoter Regions, Genetic , Recombinant Fusion Proteins/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/pharmacology , Transgenes
16.
Hypertension ; 20(5): 690-700, 1992 Nov.
Article in English | MEDLINE | ID: mdl-1385327

ABSTRACT

The cardiac natriuretic peptide family includes atrial natriuretic factor and brain or B-type natriuretic peptide, also known as iso-atrial natriuretic factor (isoANF). Although these peptides contribute to cardiovascular homeostasis, their respective roles remain unclear. To study regulation of atrial natriuretic factor and isoANF gene expression during progression of hypertension, we developed a quantitative polymerase chain reaction protocol to measure their transcript level in spontaneously hypertensive rat (SHR) hearts. At the onset of hypertension, atrial natriuretic factor transcripts in 5-week-old SHR were 50% of those of age-matched Wistar-Kyoto (WKY) rats, whereas the level of isoANF transcripts was similar in atria and twofold higher in ventricles. Because atria are the major sites of atrial natriuretic factor gene expression and ventricles contribute predominantly to cardiac isoANF synthesis, total atrial natriuretic factor messenger RNA (mRNA) in the hearts of 5-week-old SHR was about 50% of that in WKY rats, and total isoANF mRNA content was already higher than in control rats. In left ventricles and ventricular septa, progression of hypertension led to a maximal increase of twofold and fourfold in atrial natriuretic factor and isoANF mRNA levels, respectively, with no detectable change in right ventricles. In the atria of older SHR, atrial natriuretic factor and isoANF mRNA levels were comparable to those of age-matched controls. These data indicate that, although increased blood pressure stimulates both atrial natriuretic factor and isoANF gene expression, regulation of the two natriuretic peptide genes is not temporally coordinated in all cardiac compartments. Furthermore, isoANF mRNA is already induced in the ventricles at the onset of the hypertensive stage, and in older SHR, the isoANF gene is hyperresponsive to progression of hypertension compared with atrial natriuretic factor. Thus, isoANF might represent a very sensitive marker of cardiac changes in hypertension.


Subject(s)
Atrial Natriuretic Factor/genetics , Nerve Tissue Proteins/genetics , Polymerase Chain Reaction/methods , Rats, Inbred SHR/genetics , Transcription, Genetic , Animals , Base Sequence , Hypertension/genetics , Hypertension/physiopathology , Isomerism , Male , Molecular Probes/genetics , Molecular Sequence Data , Myocardium/metabolism , Natriuretic Peptide, Brain , RNA/metabolism , Rats , Rats, Inbred WKY
17.
J Biol Chem ; 267(24): 16951-6, 1992 Aug 25.
Article in English | MEDLINE | ID: mdl-1512237

ABSTRACT

Cultured mouse leukemia L1210 cells express the nucleoside-specific membrane transport processes designated es, ei, and cif. The es and ei processes are equilibrative, but may be distinguished by the high sensitivity of the former to 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine (NBMPR); the cif process is mediated by a Na+/nucleoside cotransporter of low sensitivity to NBMPR. Cells of an ei-deficient clonal line, L1210/MC5-1, were mutagenized, and clones were selected in soft agar medium that contained (i) NBMPR (an inhibitor of es processes), (ii) erythro-9-(2-hydorxy-3-nonyl)adenine (an inhibitor of adenosine deaminase), and (iii) arabinofuranosyladenine (a cytotoxic substrate for the three nucleotide transporters). The selection medium did not allow es activity and selected against cells that expressed the Na(+)-linked cif process. Cells of the L1210/B23.1 clonal isolate were deficient in cif transport activity, and inward fluxes of formycin B, a poorly metabolized analog of inosine, were virtually abolished by NBMPR in these cells. In the mutant cells, nonisotopic formycin B behaved as a countertransport substrate during influx of [3H]formycin B, and inward fluxes of the latter were competitively inhibited by purine and pyrimidine nucleosides. The transport behavior of L1210/B23.1 cells indicates that (i) the mutation/selection procedure impaired or deleted the Na(+)-linked cif process and (ii) es nucleoside transport activity is expressed in the mutant cells.


Subject(s)
Carrier Proteins/metabolism , Leukemia L1210/metabolism , Membrane Proteins/metabolism , Nucleosides/metabolism , Nucleosides/pharmacology , Adenine/analogs & derivatives , Adenine/metabolism , Adenosine/metabolism , Animals , Antiviral Agents/metabolism , Biological Transport/drug effects , Carrier Proteins/genetics , Cell Membrane/metabolism , Clone Cells , Formycins/metabolism , Kinetics , Membrane Proteins/genetics , Mice , Mutagenesis , Nucleoside Transport Proteins , Thioinosine/analogs & derivatives , Thioinosine/pharmacology , Thymidine/metabolism , Tumor Cells, Cultured , Vidarabine/metabolism
18.
Cancer Chemother Pharmacol ; 31(1): 71-5, 1992.
Article in English | MEDLINE | ID: mdl-1458562

ABSTRACT

Fludarabine phosphate (F-ara-AMP, Fludara) is rapidly converted in the circulation to fludarabine (F-ara-A) and is among the most effective single agents in the treatment of chronic lymphocytic leukemia. Although current treatment protocols are well tolerated, severe neurotoxicity was a consequence of high-dose F-ara-AMP regimens used in early phase I trials against adult acute leukemia. The present study showed that in mice implanted with leukemia L1210, fatal neurotoxicity, which initially manifested as hind-limb paralysis, was a consequence of high-dose F-ara-AMP treatment. However, the incidence of neurotoxicity was reduced by the coadministration of NBMPR-P, the 5'-phosphate of nitrobenzylthioinosine, a potent inhibitor of the es equilibrative nucleoside transport (NT) system. NBTGR-P, the 5'-phosphate of nitrobenzylthioguanosine (also a potent NT inhibitor) similarly prevented F-ara-AMP neurotoxicity in this experimental system. Treatment with F-ara-AMP/NBMPR-P combinations was more effective with respect to the fractional yield of "cured" mice than were the same treatment regimens without NBMPR-P.


Subject(s)
Leukemia L1210/drug therapy , Nervous System Diseases/chemically induced , Nervous System Diseases/prevention & control , Thioinosine/analogs & derivatives , Vidarabine/analogs & derivatives , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Drug Synergism , Female , Hindlimb , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Neoplasm Transplantation , Paralysis/chemically induced , Prodrugs/administration & dosage , Thioinosine/administration & dosage , Thioinosine/therapeutic use , Thionucleotides/administration & dosage , Vidarabine/pharmacology , Vidarabine/toxicity
19.
Mol Endocrinol ; 5(9): 1292-300, 1991 Sep.
Article in English | MEDLINE | ID: mdl-1837590

ABSTRACT

Cardiac myocytes secrete a family of natriuretic and diuretic peptides, including atrial natriuretic factor (ANF) and brain natriuretic peptide or the rat hormone isoANF. These peptides share structural and functional similarities, but their respective physiological roles have yet to be elucidated. Differential expression of natriuretic peptide genes may reflect distinct physiological or pathophysiological functions for these peptides. To test this hypothesis, we have determined the sites of expression of the ANF and isoANF genes in rat tissues using polymerase chain reaction amplification of ANF and isoANF transcripts. Like ANF mRNA, isoANF mRNA was detected in all heart compartments, and the transcription initiation sites of the isoANF gene, determined from primer extension experiments, were identical in atria and ventricles. In the adult heart, the ventricular isoANF mRNA concentration is only 3 times lower than in atria, and in sharp contrast to ANF, the isoANF gene is constitutively expressed in ventricles during postnatal development. Since ventricles are at least 20 times larger than atria, this implies that isoANF is mostly a ventricular hormone, whereas ANF is essentially an atrial hormone in normal adult hearts. Low levels of isoANF mRNA were found in few extracardiac tissues, including hypothalamus, brain, lung, and aorta. IsoANF transcripts were more abundant than ANF transcripts in aorta, whereas ANF and isoANF mRNA levels were similar in lung. In brain and hypothalamus, ANF transcripts were only 7- and 2-fold greater than isoANF transcripts. The presence of isoANF transcripts in brain and hypothalamus suggests that isoANF is the rat homolog of the human hormone brain natriuretic peptide. Thus, the expression of these two natriuretic peptide genes is not coordinated, suggesting that ANF and isoANF may play different physiological roles in cardiac and extracardiac tissues.


Subject(s)
Atrial Natriuretic Factor/genetics , Myocardium/metabolism , Nerve Tissue Proteins/genetics , RNA, Messenger/analysis , Animals , Base Sequence , DNA, Antisense , Gene Expression Regulation , Male , Molecular Sequence Data , Natriuretic Peptide, Brain , Oligonucleotides , Organ Specificity , Polymerase Chain Reaction , Rats , Transcription, Genetic , Ventricular Function
20.
J Biol Chem ; 266(10): 6308-11, 1991 Apr 05.
Article in English | MEDLINE | ID: mdl-2007583

ABSTRACT

Nucleoside permeation in L1210/AM cells is mediated by (a) equilibrative (facilitated diffusion) transporters of two types and by (b) a concentrative Na(+)-dependent transport system of low sensitivity to nitrobenzylthioinosine and dipyridamole, classical inhibitors of equilibrative nucleoside transport. In medium containing 10 microM dipyridamole and 20 microM adenosine, the equilibrative nucleoside transport systems of L1210/AM cells were substantially inhibited and the unimpaired activity of the Na(+)-dependent nucleoside transport system resulted in the cellular accumulation of free adenosine to 86 microM in 5 min, a concentration three times greater than the steady-state levels of adenosine achieved without dipyridamole. Uphill adenosine transport was not observed when extracellular Na+ was replaced by Li+, K+, Cs+, or N-methyl-D-glucammonium ions, or after treatment of the cells with nystatin, a Na+ ionophore. These findings show that concentrative nucleoside transport activity in L1210/AM cells required an inward transmembrane Na+ gradient. Treatment of cells in sodium medium with 2 mM furosemide in the absence or presence of 2 mM ouabain inhibited Na(+)-dependent adenosine transport by 50 and 75%, respectively. However, because treatment of cells with either agent in Na(+)-free medium decreased adenosine transport by only 25%, part of this inhibition may be secondary to the effects of furosemide and ouabain on the ionic content of the cells. Substitution of extracellular Cl- by SO4(-2) or SCN- had no effect on the concentrative influx of adenosine.


Subject(s)
Leukemia L1210/metabolism , Nucleosides/metabolism , Sodium/metabolism , Adenosine/metabolism , Animals , Biological Transport/drug effects , Formycins/metabolism , Furosemide/pharmacology , Mice , Nystatin/pharmacology , Thioinosine/analogs & derivatives , Thioinosine/pharmacology , Tumor Cells, Cultured
SELECTION OF CITATIONS
SEARCH DETAIL
...