bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hypoxia-Inducible Factor-1 (HIF-1) Up-Regulates Adrenomedullin Expression in Human Tumor Cell Lines during Oxygen Deprivation: A Possible Promotion Mechanism of Carcinogenesis
Authors: Garayoa Mercedes , Martínez Alfredo , Lee Sunmin , Pío Rubén , An Won G. , Neckers Len , Trepel Jane , Montuenga Luis M. , Ryan Heather , Johnson Randall , Gassmann Max , Cuttitta Frank 
Published: January 2014

AbstractLittle is known about the molecular mechanisms that control adrenomedullin (AM) production in human cancers. We demonstrate here that the expression of AM mRNA in a variety of human tumor cell lines is highly induced in a time-dependent manner by reduced oxygen tension (1% O2) or exposure to hypoxia mimetics such as desferrioxamine mesylate (DFX) or CoCl2. This AM expression seems to be under hypoxia-inducible factor-1 (HIF-1) transcriptional regulation, since HIF-1α and HIF-1β knockout mouse cell lines had an ablated or greatly reduced hypoxia AM mRNA induction. Similarly, inhibition or enhancement of HIF-1 activity in human tumor cells showed an analogous modulation of AM mRNA. Under hypoxic conditions, immunohistochemical analysis of tumor cell lines revealed elevated levels of AM and HIF-1α as compared with normoxia, and we also found an increase of immunoreactive AM in the conditioned medium of tumor cells analyzed by RIA. AM mRNA stabilization was shown to be partially responsible for the hypoxic up-regulated expression of AM. In addition, we have identified several putative hypoxia response elements (HREs) in the human AM gene, and reporter studies with selected HREs were capable of enhancing luciferase expression after exposure to DFX. Furthermore, transient coexpression of HIF-1α resulted in an augmented transactivation of the reporter gene after DFX treatment. Given that most solid human tumors have focal hypoxic areas and that AM functions as a mitogen, angiogenic factor, and apoptosis-survival factor, our findings implicate the HIF-1/AM link as a possible promotion mechanism of carcinogenesis.
Thyroid Hormone Regulation of Hepatic Genes in Vivo Detected by Complementary DNA Microarray
Authors: Feng Xu , Jiang Yuan , Meltzer Paul , Yen Paul M. 
Published: January 2014

AbstractThe liver is an important target organ of thyroid hormone. However, only a limited number of hepatic target genes have been identified, and little is known about the pattern of their regulation by thyroid hormone. We used a quantitative fluorescent cDNA microarray to identify novel hepatic genes regulated by thyroid hormone. Fluorescent-labeled cDNA prepared from hepatic RNA of T3-treated and hypothyroid mice was hybridized to a cDNA microarray, representing 2225 different mouse genes, followed by computer analysis to compare relative changes in gene expression. Fifty five genes, 45 not previously known to be thyroid hormone-responsive genes, were found to be regulated by thyroid hormone. Among them, 14 were positively regulated by thyroid hormone, and unexpectedly, 41 were negatively regulated. The expression of 8 of these genes was confirmed by Northern blot analyses. Thyroid hormone affected gene expression for a diverse range of cellular pathways and functions, including gluconeogenesis, lipogenesis, insulin signaling, adenylate cyclase signaling, cell proliferation, and apoptosis. This is the first application of the microarray technique to study hormonal regulation of gene expression in vivo and should prove to be a powerful tool for future studies of hormone and drug action.
Distinct Functions of the Two Protein Tyrosine Phosphatase Domains of LAR (Leukocyte Common Antigen-Related) on Tyrosine Dephosphorylation of Insulin Receptor
Authors: Tsujikawa Kazutake , Kawakami Naoto , Uchino Yukiko , Ichijo Tomoko , Furukawa Tatsuhiko , Saito Haruo , Yamamoto Hiroshi 
Published: January 2014

AbstractMost receptor-like, transmembrane protein tyrosine phosphatases (PTPases), such as CD45 and the leukocyte common antigen-related (LAR) molecule, have two tandemly repeated PTPase domains in the cytoplasmic segment. The role of each PTPase domain in mediating PTPase activity remains unclear; however, it has been proposed that PTPase activity is associated with only the first of the two domains, PTPase domain 1, and the membrane-distal PTPase domain 2, which has no catalytic activity, would regulate substrate specificity. In this paper, we examine the function of each PTPase domain of LAR in vivo using a potential physiological substrate, namely insulin receptor, and LAR mutant proteins in which the conserved cysteine residue was changed to a serine residue in the active site of either or both PTPase domains. LAR associated with and preferentially dephosphorylated the insulin receptor that was tyrosine phosphorylated by insulin stimulation. Its association was mediated by PTPase domain 2, because the mutation of Cys-1813 to Ser in domain 2 resulted in weakening of the association. The Cys-1522 to Ser mutant protein, which is defective in the LAR PTPase domain 1 catalytic site, was tightly associated with tyrosine-phosphorylated insulin receptor, but failed to dephosphorylate it, indicating that LAR PTPase domain 1 is critical for dephosphorylation of tyrosine-phosphorylated insulin receptor. This hypothesis was further confirmed by using LAR mutants in which either PTPase domain 1 or domain 2 was deleted. Moreover, the association of the extracellular domains of both LAR and insulin receptor was supported by using the LAR mutant protein without the two PTPase domains. LAR was phosphorylated by insulin receptor tyrosine kinase and autodephosphorylated by the catalytic activity of the PTPase domain 1. These results indicate that each domain of LAR plays distinct functional roles through phosphorylation and dephosphorylation in vivo.
The CpG Island Promoter of the Human Proopiomelanocortin Gene Is Methylated in Nonexpressing Normal Tissue and Tumors and Represses Expression
Authors: Newell-Price John , King Peter , Clark Adrian J. L. 
Published: January 2014

AbstractEctopic secretion of ACTH, from sites such as small cell lung cancer (SCLC), results in severe Cushing’s syndrome. ACTH is cleaved from POMC. The syndrome may occur when the highly tissue-specific promoter of the human POMC gene (POMC) is activated. The mechanism of activation is not fully understood. This promoter is embedded within a defined CpG island, and CpG islands are usually considered to be unmethylated in all tissues. We demonstrate that much of this CpG island is methylated in normal nonexpressing tissues, in contrast to somatically expressed CpG island promoters reported to date, and is specifically unmethylated in expressing tissues, tumors, and the POMC-expressing DMS-79 SCLC cell line. A narrow 100-bp region is free of methylation in all tissues. E2F factors binding to the upstream domain IV region of the promoter have been shown to be involved in the expression of POMC in SCLC. We show that these sites are methylated in normal nonexpressing tissues, which will prevent binding of E2F, but are unmethylated in expressing tissue. Methylation in vitro is sufficient for silencing of expression, which is not reversed by treatment with Trichostatin A, suggesting that inhibition of expression may be mediated by means other than recruitment of histone deacetylase activity. The DMS-79 cells lack POMC demethylating activity, implying that the methylation and expression patterns are likely to be set early or before neoplastic transformation, and that targeted de novo methylation might be a potential therapeutic strategy.
Involvement of Hepatocyte Nuclear Factor-4 in the Expression of the Growth Hormone Receptor 1A Messenger Ribonucleic Acid in Bovine Liver
Authors: Jiang Honglin , Lucy Matthew C. 
Published: January 2014

AbstractThe GH receptor 1A mRNA (GHR 1A mRNA) is one of the major GHR mRNA variants that differ in the 5′-untranslated region. The GHR 1A mRNA is unique because it is exclusively expressed in liver. The objective of the present study was to understand the mechanism for the liver-specific expression of the GHR 1A mRNA in the bovine. Twenty-six kilobases of 5′-flanking region of the bovine GHR gene was cloned and sequenced. The first exon (exon 1A) that corresponded to the 5′-untranslated region of the GHR 1A mRNA was 15,250 bp upstream from exon 2 in the GHR gene. The major transcription start site for the GHR 1A mRNA was 19 bp downstream from a putative TATA box. Transient transfection analyses of the 5′-flanking region of exon 1A in liver cell lines vs. nonliver cell lines did not reveal a positively regulatory region responsible for the liver-specific expression of the GHR 1A mRNA perhaps because the liver cell lines do not recapitulate the in vivo hepatic environment. A putative regulatory region was then found by deoxyribonuclease I footprinting analyses of the proximal 5′-flanking region of exon 1A with nuclear extracts from bovine liver tissue. This regulatory region contained a putative binding site for the liver-enriched transcription factor hepatocyte nuclear factor-4 (HNF-4). Binding of HNF-4 in bovine liver to this putative HNF-4 binding site was confirmed by electrophoretic mobility shift assays. Overexpression of HNF-4 enhanced the transcriptional activity of the 5′-proximal region of exon 1A in various cell lines. Mutation of the HNF-4 binding site abolished the transactivation. In addition, the HNF-4 mRNA was found to be primarily expressed in liver and absent in most nonhepatic tissues in the bovine. Collectively, these observations suggest that the liver-enriched transcription factor HNF-4 plays a role in the expression of GHR 1A mRNA in bovine liver.
Allosteric Modulation of Estrogen Receptor Conformation by Different Estrogen Response Elements
Authors: Wood Jennifer R. , Likhite Varsha S. , Loven Margaret A. , Nardulli Ann M. 
Published: January 2014

AbstractEstrogen-regulated gene expression is dependent on interaction of the estrogen receptor (ER) with the estrogen response element (ERE). We assessed the ability of the ER to activate transcription of reporter plasmids containing either the consensus vitellogenin A2 ERE or the imperfect pS2, vitellogenin B1, or oxytocin (OT) ERE. The A2 ERE was the most potent activator of transcription. The OT ERE was significantly more effective in activating transcription than either the pS2 or B1 ERE. In deoxyribonuclease I (DNase I) footprinting experiments, MCF-7 proteins protected A2 and OT EREs more effectively than the pS2 and B1 EREs. Limited protease digestion of the A2, pS2, B1, or OT ERE-bound receptor with V8 protease or proteinase K produced distinct cleavage products demonstrating that individual ERE sequences induce specific changes in ER conformation. Receptor interaction domains of glucocorticoid receptor interacting protein 1 and steroid receptor coactivator 1 bound effectively to the A2, pS2, B1, and OT ERE-bound receptor and significantly stabilized the receptor-DNA interaction. Similar levels of the full-length p160 protein amplified in breast cancer 1 were recruited from HeLa nuclear extracts by the A2, pS2, B1, and OT ERE-bound receptors. In contrast, significantly less transcriptional intermediary factor 2 was recruited by the B1 ERE-bound receptor than by the A2 ERE-bound receptor. These studies suggest that allosteric modulation of ER conformation by individual ERE sequences influences the recruitment of specific coactivator proteins and leads to differential expression of genes containing divergent ERE sequences.
Inhibitory Effects of 1α,25-Dihydroxyvitamin D3 on the G1–S Phase-Controlling Machinery
Authors: Jensen Simon Skjøde , Madsen Mogens Winkel , Lukas Jiri , Binderup Lise , Bartek Jiri 
Published: January 2014

AbstractThe nuclear hormone 1α,25-dihydroxyvitamin D3 induces cell cycle arrest, differentiation, or apoptosis depending on target cell type and state. Although the antiproliferative effect of 1α,25-dihydroxyvitamin D3 has been known for years, the molecular basis of the cell cycle blockade by 1α,25-dihydroxyvitamin D3 remains largely unknown. Here we have investigated the mechanisms underlying the G1 arrest induced upon 1α,25-dihydroxyvitamin D3 treatment of the human breast cancer cell line MCF-7. Twenty-four-hour exposure of exponentially growing MCF-7 cells to 1α,25-dihydroxyvitamin D3 impeded proliferation by preventing S phase entry, an effect that correlated with appearance of the growth-suppressing, hypophosphorylated form of the retinoblastoma protein (pRb), and modulation of cyclin-dependent kinase (cdk) activities of cdk-4, -6, and -2. Time course immunochemical and biochemical analyses of the cellular and molecular effects of 1α,25-dihydroxyvitamin D3 treatment for up to 6 d revealed a dynamic chain of events, preventing activation of cyclin D1/cdk4, and loss of cyclin D3, which collectively lead to repression of the E2F transcription factors and thus negatively affected cyclin A protein expression.While the observed 10-fold inhibition of cyclin D1/cdk 4-associated kinase activity appeared independent of cdk inhibitors, the activity of cdk 2 decreased about 20-fold, reflecting joint effects of the lower abundance of its cyclin partners and a significant increase of the cdk inhibitor p21CIP1/WAF1, which blocked the remaining cyclin A(E)/cdk 2 complexes.Together with a rapid down-modulation of the c-Myc oncoprotein in response to 1α,25-dihydroxyvitamin D3, these results demonstrate that 1α,25-dihydroxyvitamin D3 inhibits cell proliferation by targeting several key regulators governing the G1/S transition.
Salt-Inducible Kinase Is Involved in the ACTH/cAMP-Dependent Protein Kinase Signaling in Y1 Mouse Adrenocortical Tumor Cells
Authors: Lin Xing-zi , Takemori Hiroshi , Katoh Yoshiko , Doi Junko , Horike Nanao , Makino Ariko , Nonaka Yasuki , Okamoto Mitsuhiro 
Published: January 2014

AbstractThe involvement of salt-inducible kinase, a recently cloned protein serine/threonine kinase, in adrenal steroidogenesis was investigated. When Y1 mouse adrenocortical tumor cells were stimulated by ACTH, the cellular content of salt-inducible kinase mRNA, protein, and enzyme activity changed rapidly. Its level reached the highest point in 1–2 h and returned to the initial level after 8 h. The mRNA levels of cholesterol side-chain cleavage cytochrome P450 and steroidogenic acute regulatory protein, on the other hand, began to rise after a few hours, reaching the highest levels after 8 h. The salt-inducible kinase mRNA level in ACTH-, forskolin-, or 8-bromo-cAMP-treated Kin-7 cells, mutant Y1 with less cAMP-dependent PKA activity, remained low. However, Kin-7 cells, when transfected with a PKA expression vector, expressed salt-inducible kinase mRNA. Y1 cells that overexpressed salt-inducible kinase were isolated, and the mRNA levels of steroidogenic genes in these cells were compared with those in the parent Y1. The level of cholesterol side-chain cleavage cytochrome P450 mRNA in the salt-inducible kinase-overexpressing cells was markedly low compared with that in the parent, while the levels of Ad4BP/steroidogenic factor-1-, ACTH receptor-, and steroidogenic acute regulatory protein-mRNAs in the former were similar to those in the latter. The ACTH-dependent expression of cholesterol side-chain cleavage cytochrome P450- and steroidogenic acute regulatory protein-mRNAs in the salt-inducible kinase-overexpressing cells was significantly repressed. The promoter activity of the cholesterol side-chain cleavage cytochrome P450 gene was assayed by using Y1 cells transfected with a human cholesterol side-chain cleavage cytochrome P450 promoter-linked reporter gene. Addition of forskolin to the culture medium enhanced the cholesterol side-chain cleavage cytochrome P450 promoter activity, but the forskolin-dependently activated promoter activity was inhibited when the cells were transfected with a salt-inducible kinase expression vector. This inhibition did not occur when the cells were transfected with a salt-inducible kinase (K56M) vector that encoded an inactive kinase. The salt-inducible kinase’s inhibitory effect was also observed when nonsteroidogenic, nonAd4BP/steroidogenic factor-1 -expressing, NIH3T3 cells were used for the promoter assays. These results suggested that salt-inducible kinase might play an important role(s) in the cAMP-dependent, but Ad4BP/steroidogenic factor-1-independent, gene expression of cholesterol side-chain cleavage cytochrome P450 in adrenocortical cells.
Regulation of PIT-1 Expression By Ghrelin and GHRP-6 Through the GH Secretagogue Receptor
Authors: García Angel , Alvarez Clara V. , Smith Roy G. , Diéguez Carlos 
Published: January 2014

AbstractGH secretagogues are an expanding class of synthetic peptide and nonpeptide molecules that stimulate the pituitary gland to secrete GH through their own specific receptor, the GH-secretagogue receptor. The cloning of the receptor for these nonclassical GH releasing molecules, together with the more recent characterization of an endogenous ligand, named ghrelin, have unambiguously demonstrated the existence of a physiological system that regulates GH secretion. Somatotroph cell-specific expression of the GH gene is dependent on a pituitary-specific transcription factor (Pit-1). This factor is transcribed in a highly restricted manner in the anterior pituitary gland. The present experiments sought to determine whether the synthetic hexapeptide GHRP-6, a reference GH secretagogue compound, as well as an endogenous ligand, ghrelin, regulate pit-1 expression. By a combination of Northern and Western blot analysis we found that GHRP-6 elicits a time- and dose-dependent activation of pit-1 expression in monolayer cultures of infant rat anterior pituitary cells. This effect was blocked by pretreatment with actinomycin D, but not by cycloheximide, suggesting that this action was due to direct transcriptional activation of pit-1. Using an established cell line (HEK293-GHS-R) that overexpresses the GH secretagogue receptor, we showed a marked stimulatory effect of GHRP-6 on the pit-1 −2,500 bp 5′-region driving luciferase expression. We truncated the responsive region to −231 bp, a sequence that contains two CREs, and found that both CREs are needed for GHRP-6-induced transcriptional activation in both HEK293-GHS-R cells and infant rat anterior pituitary primary cultures. The effect was dependent on PKC, MAPK kinase, and PKA activation. Increasing Pit-1 by coexpression of pCMV-pit-1 potentiated the GHRP-6 effect on the pit-1 promoter. Similarly, we showed that the endogenous GH secretagogue receptor ligand ghrelin exerts a similar effect on the pit-1 promoter. These data provide the first evidence that ghrelin, in addition to its previously reported GH-releasing activities, is also capable of regulating pit-1 transcription through the GH secretagogue receptor in the pituitary, thus giving new insights into the physiological role of the GH secretagogue receptor on somatotroph cell differentiation and function.
Regulation of Steroidogenesis and the Steroidogenic Acute Regulatory Protein by a Member of the cAMP Response-Element Binding Protein Family
Authors: Manna Pulak R. , Dyson Matthew T. , Eubank Darrell W. , Clark Barbara J. , Lalli Enzo , Sassone-Corsi Paolo , Zeleznik Anthony J. , Stocco Douglas M. 
Published: January 2014

AbstractThe mitochondrial phosphoprotein, the steroidogenic acute regulatory (StAR) protein, is an essential component in the regulation of steroid biosynthesis in adrenal and gonadal cells through cAMP-dependent pathways. In many cases transcriptional induction by cAMP is mediated through the interaction of a cAMP response-element binding protein (CREB) family member with a consensus cAMP response element (CRE; 5′-TGACGTCA-3′) found in the promoter of target genes. The present investigation was carried out to determine whether a CRE-binding protein (CREB) family member [CREB/CRE modulator (CREM) family] was involved in the regulation of steroidogenesis and StAR protein expression. Transient expression of wild- type CREB in MA-10 mouse Leydig tumor cells further increased the levels of (Bu)2cAMP-induced progesterone synthesis, StAR promoter activity, StAR mRNA, and StAR protein. These responses were significantly inhibited by transfection with a dominant-negative CREB (A-CREB), or with a CREB mutant that cannot be phosphorylated (CREB-M1), the latter observation indicating the importance of phosphorylation of a CREB/CREM family member in steroidogenesis and StAR expression. The CREB/CREM-responsive region in the mouse StAR gene was located between −110 and −67 bp upstream of the transcriptional start site. An oligonucleotide probe (−96/−67 bp) containing three putative half-sites for 5′-canonical CRE sequences (TGAC) demonstrated the formation of protein-DNA complexes in EMSAs with recombinant CREB protein as well as with nuclear extracts from MA-10 or Y-1 mouse adrenal tumor cells. The predominant binding factor observed with EMSA was found to be the CREM protein as demonstrated using specific antibodies and RT-PCR analyses. The CRE elements identified within the− 96/−67 bp region were tested for cAMP responsiveness by generating mutations in each of the CRE half-sites either alone or in combination. Although each of the CRE sites contribute in part to the CREM response, the CRE2 appears to be the most important site as determined by EMSA and by reporter gene analyses. Binding specificity was further assessed using specific antibodies to CREB/CREM family members, cold competitors, and mutations in the target sites that resulted in either supershift and/or inhibition of these complexes. We also demonstrate that the inducible cAMP early repressor markedly diminished the endogenous effects of CREM on cAMP-induced StAR promoter activity and on StAR mRNA expression. These are the first observations to provide evidence for the functional involvement of a CREB/CREM family member in the acute regulation of trophic hormone-stimulated steroidogenesis and StAR gene expression.
Acyl-Coenzyme A Dehydrogenases Are Localized on GLUT4-Containing Vesicles via Association with Insulin-Regulated Aminopeptidase in a Manner Dependent on Its Dileucine Motif
Authors: Katagiri Hideki , Asano Tomoichiro , Yamada Tetsuya , Aoyama Toshifumi , Fukushima Yasushi , Kikuchi Masatoshi , Kodama Tatsuhiko , Oka Yoshitomo 
Published: January 2014

AbstractInsulin-regulated aminopeptidase (IRAP, also termed vp165) is known to be localized on the GLUT4-containing vesicles and to be recruited to the plasma membrane after stimulation with insulin. The cytoplasmic region of IRAP contains two dileucine motifs and acidic regions, one of which (amino acid residues 55–82) is reportedly involved in retention of GLUT4-containing vesicles. The region of IRAP fused with glutathione-S-transferase [GST-IRAP(55–82)] was incubated with lysates from 3T3-L1 adipocytes, leading to identification of long-chain, medium-chain, and short-chain acyl-coenzyme A dehydrogenases (ACDs) as the proteins associated with IRAP. The association was nearly abolished by mutation of the dileucine motif of IRAP. Immunoblotting of fractions prepared from sucrose gradient ultracentrifugation and vesicles immunopurified with anti-GLUT4 antibody revealed these ACDs to be localized on GLUT4-containing vesicles. Furthermore, 3-mercaptopropionic acid and hexanoyl-CoA, inhibitors of long-chain and medium-chain ACDs, respectively, induced dissociation of long-chain acyl-coenzyme A dehydrogenase and/or medium-chain acyl-coenzyme A dehydrogenase from IRAP in vitro as well as recruitment of GLUT4 to the plasma membrane and stimulation of glucose transport activity in permeabilized 3T3-L1 adipocytes. These findings suggest that ACDs are localized on GLUT4-containing vesicles via association with IRAP in a manner dependent on its dileucine motif and play a role in retention of GLUT4-containing vesicles to an intracellular compartment.