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D C Dean

Publications and source records attributed to D C Dean.

At least 37 records · Page 2Linked to original sources
PubMed1999

zfh-1, the Drosophila homologue of ZEB, is a transcriptional repressor that regulates somatic myogenesis.

zfh-1 is a member of the zfh family of proteins, which all contain zinc finger and homeodomains. The roles and mechanisms of action of most family members are still unclear. However, we have shown previously that another member of the family, the vertebrate ZEB protein, is a transcriptional repressor that binds E box sequences and inhibits myotube formation in cell culture assays. zfh-1 is downregulated in Drosophila embryos prior to myogenesis. Embryos with zfh-1 loss-of-function mutation show alterations in the number and position of embryonic somatic muscles, suggesting that zfh-1 could have a regulatory role in myogenesis. However, nothing is known about the nature or mechanism of action of zfh-1. Here, we demonstrate that zfh-1 is a transcription factor that binds E box sequences and acts as an active transcriptional repressor. When zfh-1 expression was maintained in the embryo beyond its normal temporal pattern of downregulation, the differentiation of somatic but not visceral muscle was blocked. One potential target of zfh-1 in somatic myogenesis could be the myogenic factor mef2. mef2 is known to be regulated by the transcription factor twist, and we show here that zfh-1 binds to sites in the mef2 upstream regulatory region and inhibits twist transcriptional activation. Even though there is little sequence similarity in the repressor domains of ZEB and zfh-1, we present evidence that zfh-1 is the functional homologue of ZEB and that the role of these proteins in myogenesis is conserved from Drosophila to mammals.

Animals↗
PubMed1999

Independent repressor domains in ZEB regulate muscle and T-cell differentiation.

ZEB is a zinc finger-homeodomain protein that represses transcription by binding to a subset of E-box sequences. ZEB inhibits muscle differentiation in mammalian systems, and its Drosophila orthologue, zfh-1, inhibits somatic and cardiac muscle differentiation during Drosophila embryogenesis. ZEB also binds to the promoter of pivotal hematopoietic genes (including those encoding interleukin-2, CD4, GATA-3, and alpha(4)-integrin), and mice in which ZEB has been genetically targeted show thymic atrophy, severe defects in lymphocyte differentiation, and increased expression of the alpha(4)-integrin and CD4. Here, we demonstrate that ZEB contains separate repressor domains which function in T lymphocytes and muscle, respectively. The most C-terminal domain inhibits muscle differentiation in mammalian cells by specifically blocking the transcriptional activity of the myogenic factor MEF2C. The more N-terminal domain blocks activity of hematopoietic transcription factors such as c-myb, members of the ets family, and TFE-III. Our results demonstrate that ZEB has evolved with two independent repressor domains which target distinct sets of transcription factors and function in different tissues.

Animals↗
PubMed1999

Studies on cytochrome P-450-mediated bioactivation of diclofenac in rats and in human hepatocytes: identification of glutathione conjugated metabolites.

The nonsteroidal anti-inflammatory drug diclofenac causes a rare but potentially fatal hepatotoxicity that may be associated with the formation of reactive metabolites. In this study, three glutathione (GSH) adducts, namely 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified by liquid chromatography-tandem mass spectrometry analysis of bile from Sprague-Dawley rats injected i.p. with a single dose of diclofenac (200 mg/kg). These adducts presumably were formed via hepatic cytochrome P-450 (CYP)-catalyzed oxidation of diclofenac to reactive benzoquinone imines that were trapped by GSH conjugation. In support of this hypothesis, M1, M2, and M3 were generated from diclofenac in incubations with rat liver microsomes in the presence of NADPH and GSH. Increases in adduct formation were observed when incubations were performed with liver microsomes from phenobarbital- or dexamethasone-treated rats. Adduct formation was inhibited by polyclonal antibodies against CYP2B, CYP2C, and CYP3A (40-50% inhibition at 5 mg of IgG/nmol of CYP) but not by an antibody against CYP1A. Maximal inhibition was obtained when the three inhibitory antibodies were used in a cocktail fashion (70-80% inhibition at 2.5 mg of each IgG/nmol of CYP). These data suggest that diclofenac undergoes biotransformation to reactive metabolites in rats and that CYP isoforms of the 2B, 2C, and 3A subfamilies are involved in this bioactivation process. With respect to CYP2C isoforms, rat hepatic CYP2C7 and CYP2C11 were implicated as mediators of the bioactivation based on immunoinhibition studies using antibodies specific to CYP2C7 and CYP2C11. Screening for GSH adducts also was carried out in human hepatocyte cultures containing diclofenac, and M1, M2, and M3 again were detected. It is possible, therefore, that reactive benzoquinone imines may be formed in vivo in humans and contribute to diclofenac-mediated hepatic injury.

Animals↗
PubMed1999

Interaction of diclofenac and quinidine in monkeys: stimulation of diclofenac metabolism.

The cytochrome P-450 (CYP)3A4-mediated metabolism of diclofenac is stimulated in vitro by quinidine. A similar effect is observed in incubations with monkey liver microsomes. We describe an in vivo interaction of diclofenac and quinidine that leads to enhanced clearance of diclofenac in monkeys. After a dose of diclofenac via portal vein infusion at 0.055 mg/kg/h, steady-state systemic plasma drug concentrations in three male rhesus monkeys were 87, 104, and 32 ng/ml, respectively (control). When diclofenac was coadministered with quinidine (0.25 mg/kg/h) via the same route, the corresponding plasma diclofenac concentrations were 50, 59, and 18 ng/ml, representing 57, 56, and 56% of control values, respectively. In contrast, steady-state systemic diclofenac concentrations in the same three monkeys were elevated 1.4 to 2.5 times when the monkeys were pretreated with L-754,394 (10 mg/kg i.v.), an inhibitor of CYP3A. Further investigation indicated that the plasma protein binding (>99%) and blood/plasma ratio (0.7) of diclofenac remained unchanged in the presence of quinidine. Therefore, the decreases in plasma concentrations of diclofenac after a combined dose of diclofenac and quinidine are taken to reflect increased hepatic clearance of the drug, presumably resulting from the stimulation of CYP3A-catalyzed oxidative metabolism. Consistent with this proposed mechanism, a 2-fold increase in the formation of 5-hydroxydiclofenac derivatives was observed in monkey hepatocyte suspensions containing diclofenac and quinidine. Stimulation of diclofenac metabolism by quinidine was diminished when monkey liver microsomes were pretreated with antibodies against CYP3A. Subsequent kinetic studies indicated that the K(m) value for the CYP-mediated conversion of diclofenac to its 5-hydroxy derivatives was little changed (75 versus 59 microM), whereas V(max) increased 2.5-fold in the presence of quinidine. These data suggest that the catalytic capacity of monkey hepatic CYP3A toward diclofenac metabolism is enhanced by quinidine.

Animals↗
PubMed1998

Interferon regulatory factor-2 is a transcriptional activator in muscle where It regulates expression of vascular cell adhesion molecule-1.

Previously, we have suggested that vascular cell adhesion molecule-1 (VCAM-1) and its integrin receptor alpha4beta1 mediate cell-cell interactions important for skeletal myogenesis. Expression of the receptors subsequently subsides in muscle after birth. Here, we examine the mechanism regulating VCAM-1 gene expression in muscle. An enhancer located between the TATA box and the transcriptional start site is responsible for VCAM-1 gene expression in muscle-this element is inactive in endothelial cells where VCAM-1 expression is dependent on nuclear factor kappaB sites and inflammatory cytokines. We identify interferon regulatory factor-2 (IRF-2), a member of the interferon regulatory factor family, as the enhancer-binding transcription factor and show that expression of IRF-2 parallels that of VCAM-1 during mouse skeletal myogenesis. IRF-2 is not dependent upon cytokines for expression or activity, and it has been shown to act as a repressor in other nonmuscle cell types. We show that the basic repressor motif located near the COOH-terminal of IRF-2 is not active in muscle cells, but instead an acidic region in the center of the molecule functions as a transactivating domain. Although IRF-2 and VCAM-1 expression diminishes on adult muscle fiber, they are retained on myogenic stem cells (satellite cells). These satellite cells proliferate and fuse to regenerate muscle fiber after injury or disease. We present evidence that VCAM-1 on satellite cells mediates their interaction with alpha4beta1(+) leukocytes that invade the muscle after injury or disease. We propose that VCAM-1 on endothelium generally recruits leukocytes to muscle after injury, whereas subsequent interaction with VCAM-1 on regenerating muscle cells focuses the invading leukocytes specifically to the sites of regeneration.

Animals↗
PubMed1998

Rb interacts with histone deacetylase to repress transcription.

Previously, we found that Rb can actively repress transcription of cell cycle genes by binding and inactivating transcription factors at the promoter. Here, we demonstrate that Rb can also repress transcription of endogenous cell cycle genes containing E2F sites through recruitment of histone deacetylase, which deacetylates histones on the promoter, thereby promoting formation of nucleosomes that inhibit transcription. These two mechanisms of repression by Rb are selective-some promoters and transcription factors are blocked by this recruitment of histone deacetylase, whereas others are resistant to histone deacetylase activity and are repressed directly by inhibition of transcription factors.

Adenoviridae↗
PubMed1998

Structural requirements for the activation of the human growth hormone secretagogue receptor by peptide and nonpeptide secretagogues.

Antibodies raised against an intracellular and extracellular domain of the GH secretagogue receptor (GHS-R) confirmed that its topological orientation in the lipid bilayer is as predicted for G protein-coupled receptors with seven transmembrane domains. A strategy for mapping the agonist-binding site of the human GHS-R was conceived based on our understanding of ligand binding in biogenic amine and peptide hormone G protein-coupled receptors. Using site-directed mutagenesis and molecular modeling, we classified GHS peptide and nonpeptide agonist binding in the context of its receptor environment. All peptide and nonpeptide ligand classes shared a common binding domain in transmembrane (TM) region 3 of the GHS-R. This finding was based on TM-3 mutation E124Q, which eliminated the counter-ion to the shared basic N+ group of all GHSs and resulted in a nonfunctional receptor. Restoration of function for the E124Q mutant was achieved by a complementary change in the MK-0677 ligand through modification of its amine side-chain to the corresponding alcohol. Contacts in other TM domains [TM-2 (D99N), TM-5 (M213K, S117A), TM-6 (H280F), and extracellular loop 1 (C116A)] of the receptor revealed specificity for the different peptide, benzolactam, and spiroindolane GHSs. GHS-R agonism, therefore, does not require identical disposition of all agonist classes at the ligand-binding site. Our results support the hypothesis that the ligand-binding pocket in the GHS-R is spatially disposed similarly to the well characterized catechol-binding site in the beta2-adrenergic receptor.

Amino Acid Sequence↗
PubMed1997

c-Myb and Ets proteins synergize to overcome transcriptional repression by ZEB.

The Zfh family of zinc finger/homeodomain proteins was first identified in Drosophila where it is required for differentiation of tissues such as the central nervous system and muscle. ZEB, a vertebrate homolog of Zfh-1, binds a subset of E boxes and blocks myogenesis through transcriptional repression of muscle genes. We present evidence here that ZEB also has an important role in controlling hematopoietic gene transcription. Two families of transcription factors that are required for normal hematopoiesis are c-Myb and Ets. These factors act synergistically to activate transcription, and this synergy is required for transcription of at least several important hematopoietic genes. ZEB blocks the activity of c-Myb and Ets individually, but together the factors synergize to resist this repression. Such repression imposes a requirement for both c-Myb and Ets for transcriptional activity, providing one explanation for why synergy between these factors is important. The balance between repression by ZEB and transcriptional activation by c-Myb/Ets provides a flexible regulatory mechanism for controlling gene expression in hematopoietic cells. We demonstrate that one target of this positive/negative regulation in vivo is the alpha4 integrin, which play a key role in normal hematopoiesis and function of mature leukocytes.

Antigens, CD↗
PubMed1997

ZEB, a vertebrate homolog of Drosophila Zfh-1, is a negative regulator of muscle differentiation.

A number of genes, spanning the evolutionary scale from yeast to mammals, that are involved in spatial and temporal patterning during development contain zinc finger and homeodomain motifs. One such zinc finger/homeodomain protein is Drosophila Zfh-1, a member of the zfh family of Drosophila genes, which is expressed in muscle precursors and is critical for the proper development of muscle. Here we demonstrate that a vertebrate homolog of Zfh-1 (ZEB) is a negative regulator of muscle differentiation. We show that ZEB binds to a subset of E boxes in muscle genes and functions by actively repressing transcription. One target of this repression is the members of the MEF-2 family, which synergize with proteins of the myogenic basic helix-loop-helix family (bHLH) (myoD, myf-5, myogenin and MRF-4) to induce myogenic differentiation. As muscle differentiation proceeds, myogenic bHLH proteins accumulate to levels sufficient to displace ZEB from the E boxes, releasing the repression and allowing bHLH proteins to further activate transcription. This mechanism of active transcriptional repression distinguishes ZEB from other negative regulators of myogenesis (Id, Twist and I-mfa) that inhibit muscle differentiation by simply binding and inactivating myogenic factors. The relative affinity of ZEB versus myogenic bHLH proteins varies for E boxes in different genes such that ZEB would be displaced from different genes at distinct times as myogenic bHLH proteins accumulate during myogenesis, thus providing a mechanism to regulate temporal order of gene expression.

Animals↗
PubMed1997

Cell anchorage regulates apoptosis through the retinoblastoma tumor suppressor/E2F pathway.

Epithelial cells are dependent upon adhesion to extracellular matrix for survival. We show that loss of beta1 integrin receptor contact with extracellular matrix signals the inhibition of G1 cyclin-dependent kinase activity. This loss of cyclin-dependent kinase activity leads to accumulation of the hypophosphorylated (active) form of the retinoblastoma tumor suppressor protein (Rb). We present evidence that in epithelial cells deprived of matrix contact, the growth suppression signal elicited by hypophosphorylated Rb opposes stimulatory signals from serum growth factors, leading to a cell cycle conflict that triggers apoptosis. This apoptotic pathway is modulated by Bcl-2 through a novel mechanism that regulates Rb phosphorylation. We present evidence that the Rb-dependent apoptotic pathway functions in vivo in the apoptosis of the prostate glandular epithelium following castration.

Animals↗
PubMed1996

A receptor in pituitary and hypothalamus that functions in growth hormone release.

Small synthetic molecules termed growth hormone secretagogues (GHSs) act on the pituitary gland and the hypothalamus to stimulate and amplify pulsatile growth hormone (GH) release. A heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPC-R) of the pituitary and arcuate ventro-medial and infundibular hypothalamus of swine and humans was cloned and was shown to be the target of the GHSs. On the basis of its pharmacological and molecular characterization, this GPC-R defines a neuroendocrine pathway for the control of pulsatile GH release and supports the notion that the GHSs mimic an undiscovered hormone.

Amino Acid Sequence↗
PubMed1996

The Rb family contains a conserved cyclin-dependent-kinase-regulated transcriptional repressor motif.

Progression through the cell cycle is dependent on the sequential expression of cyclins, which combine with cyclin-dependent kinases (cdks) to form active kinases. The transition from G1 to S phase is dependent on D cyclins in complex with cdk4 or cdk6 and cyclin E complexed with cdk2. One target of G1 cyclins is the retinoblastoma susceptibility protein (Rb). Rb is a transcriptional repressor that is selectively targeted to genes through interaction with the E2F family of cell cycle transcription factors. Rb is a member of a family of proteins that include p107 and p130. The three proteins share a region known as the pocket that is important for binding E2F and is also the binding site for oncoproteins from DNA tumor viruses that inactivate Rb. We have found that two conserved domains within the Rb pocket (A and B) interact to form a transcriptional repressor motif (K. N. B. Chow and D. C. Dean, Mol. Cell. Biol. 16:4862-4868, 1996). Here we demonstrate that p107 also has an A-B repressor motif, and using domain swapping and coimmunoprecipitation assays, we compare A and B from Rb and p107. Finally and most importantly, we demonstrate that the A-B interaction which forms the repressor motif is blocked by G1 cdk phosphorylation, thereby blocking repressor activity. This A-B repressor motif is then the first example of a cdk-regulated transcriptional repressor.

Animals↗
PubMed1996

Transcriptional repression and growth suppression by the p107 pocket protein.

p107 is a member of the pocket family of proteins that includes the retinoblastoma tumor suppressor. Overexpression of p107 arrests cells in G1, suggesting that it is important for cell cycle control. This growth suppression is mediated at least in part through the interaction of p107 with a member of the E2F family of cell cycle transcription factors, and this interaction can be disrupted by oncoproteins from DNA tumor viruses such as adenovirus E1a that bind p107. Not only does the binding of p107 to E2F inactivate E2F, but also we show that when p107 is tethered to the promoter through binding to E2F it functions as a general transcriptional repressor. This general repressor activity was also evident when p107 was fused to the DNA binding domain of Gal4 so that it could be directly targeted to the promoter in an E2F-independent fashion. Using p107 mutants, we compared the regions of the protein required for transcriptional repression and cell growth suppression. We found that the pocket domain is sufficient for inactivation of E2F, general repressor activity, and most of the growth suppressor activity. Binding of conserved region 1 from Ela to p107 blocked interaction with E2F, but it did not affect general repressor activity, demonstrating that binding and inactivation of E2F and general repressor activity are distinguishable properties of p107. Within the pocket, two conserved domains, A and B, were sufficient for growth suppression and transcriptional repressor activity. Surprisingly, we found that these two domains were fully functional when they were coexpressed as separate proteins, and we present results suggesting that the domains may interact at the promoter to form an active pocket.

Base Sequence↗
PubMed1996

Domains A and B in the Rb pocket interact to form a transcriptional repressor motif.

The retinoblastoma protein (Rb) is a tumor suppressor that regulates progression from the G1 phase to the S phase of the cell cycle. Previously, we found that Rb is a transcriptional repressor that is selectively targeted to promoters through an interaction with the E2F family of cell cycle transcription factors--when Rb is tethered to a promoter through E2F, it not only blocks E2F activity, it also binds surrounding transcription factors, preventing their interaction with the basal transcription complex, thus resulting in a dominant inhibitory effect on transcription of cell cycle genes. Here we examine the repressor motif of Rb. The two domains in the Rb pocket, A and B, which are conserved across species and in the Rb-related proteins p107 and p130, are both required for repressor activity. The nonconserved spacer separating A and B is not required. Although neither A nor B alone had any repressor activity, surprisingly, repressor activity was observed when the domains were coexpressed on separate proteins. Transfection assays suggest that one domain can recruit the other to the promoter to form a repressor motif that can both interact with E2F and have a dominant inhibitory effect on transcription. Using coimmunoprecipitation and in vitro binding assays, we show that A and B interact directly and that mutations which disrupt this interaction inhibit repressor activity. The Rb pocket was originally defined as the binding site for oncoproteins from DNA tumor viruses such as adenovirus E1a. We present evidence that E1a interacts with a site formed by the interaction of A and B and that this interaction with A and B induces or stabilizes the A-B interaction.

Adenovirus E1A Proteins↗
PubMed1996

Identification of a new G-protein-linked receptor for growth hormone secretagogues.

The potential application of small molecules in GH therapy has recently become a topic of increasing interest. The spiroindoline MK-0677, the benzolactam L-692,429, and the peptides, GHRP-6 and hexarelin, have been shown to possess potent and selective GH-secretory activity in several species including human. Moreover, these synthetic GH secretagogues act on a signal transduction pathway distinct from that of GHRH. A specific high affinity binding site in porcine and rat anterior pituitary membranes that mediates the activity of these secretagogues has now been identified. The binding affinity of these structurally diverse secretagogues is tightly correlated with GH-secretory activity. The binding is Mg(2+)-dependent, is inhibited by GTP-gamma-S, and is not displaced by GHRH and somatostatin. The receptor is distinct from that for GHRH and has the properties of a new G-protein-coupled receptor. It is speculated that these GH secretagogues mimic an unidentified natural hormone that regulates GH secretion in concert with GHRH and somatostatin.

Animals↗
PubMed1995

Mechanism of active transcriptional repression by the retinoblastoma protein.

The retinoblastoma tumour-suppressor protein (Rb) belongs to a family that share a motif known as the pocket. The pocket was originally identified as the region of Rb required for binding to oncoproteins from DNA tumour viruses, which disrupt the binding of Rb to the E2F family of cell-cycle transcription factors (referred to collectively here as E2F). Rb switches E2F sites from positive to negative elements, suggesting that Rb-E2F is an active complex that blocks transcription. Here we report that Rb is selectively recruited to promoters through E2F, where it in turn inactivates surrounding transcription factors by blocking their interaction with the basal transcription complex. We suggest that this repressor activity is essential for inhibiting promoters that contain enhancers in addition to E2F sites.

Carrier Proteins↗
PubMed1995

Control of vascular cell adhesion molecule-1 gene promoter activity during neural differentiation.

Here we demonstrate that vascular cell adhesion molecule-1 (VCAM-1) is expressed in the developing central nervous system on neuroepithelial cells, which are the precursors of neurons and glia. As these cells differentiate, VCAM-1 is restricted to a subset of the glial population. An understanding of mechanisms responsible for this restricted pattern could provide insights into how lineage-specific gene expression is maintained during neural differentiation. As a model of neural differentiation, we turned to the P19 embryonic carcinoma cell line, which in response to retinoic acid will differentiate along a neural pathway. We show that VCAM-1 expression on the differentiating P19 cells resembles that in the central nervous system. Transfection of VCAM-1 gene promoter constructs into P19 cells revealed that the VCAM-1 gene is controlled sequentially by negative and positive elements during differentiation. We present evidence that early during differentiation, POU proteins block VCAM-1 gene activity; however, later in differentiation coincident with the appearance of VCAM-1 the pattern of POU proteins changes and the VCAM-1 gene promoter is activated. This activation is mediated through the NF kappa B/rel complex p50/p65, which forms during P19 cell differentiation.

Animals↗