bluuebunny/crossref_metadata_2025 huggingface dataset:
C/EBPβ (CCAAT/Enhancer Binding Protein) Controls Cell Fate Determination during Mammary Gland Development
Authors: Seagroves Tiffany N. , Lydon John P. , Hovey Russell C. , Vonderhaar Barbara K. , Rosen Jeffrey M.
Published: January 2014

Deletion of the transcription factor CCAAT/enhancer binding protein (C/EBP)β results in a severe inhibition of lobuloalveolar development in the mouse mammary gland. Because progesterone receptor (PR) is requisite for alveolar development, the expression of PR was investigated in C/EBPβ−/− mice. Unexpectedly, the number of PR-positive cells, as well as the levels of PR mRNA, were elevated 3-fold in the mammary glands of C/EBPβ−/− mice. Furthermore, in contrast to wild-type nulliparous mice, in which PR distribution shifted from a uniform to nonuniform pattern between 8–12 weeks of age, C/EBPβ−/− mice exhibited uniform PR distribution throughout all stages of mammary development analyzed. No change in C/EBPβ mRNA levels was observed in the mammary glands of PR−/− mice, suggesting that PR acts in a pathway either in parallel to or downstream of C/EBPβ. The overexpression and disrupted cellular distribution of PR in C/EBPβ−/− mice were coincident with a striking 10-fold decrease in cell proliferation after acute steroid hormone treatment, assayed by incorporation of bromodeoxyuridine. In wild-type mice, PR and bromodeoxyuridine-positive cells were adjacent to each other and rarely colocalized. No differences in the level or pattern of PR expression were observed in the uterus, suggesting that C/EBPβ influences PR in a mam-mary-specific fashion. Together, these data suggest that C/EBPβ may control cell fate decisions in the mammary gland through the appropriate temporal and spatial expression of molecular markers, such as PR, that induce the proliferation of alveolar progenitor cells via juxtacrine mechanisms.
5′-Heterogeneity of Glucocorticoid Receptor Messenger RNA Is Tissue Specific: Differential Regulation of Variant Transcripts by Early-Life Events
Authors: McCormick J. A. , Lyons V. , Jacobson M. D. , Noble J. , Diorio J. , Nyirenda M. , Weaver S. , Ester W. , Yau J. L. W. , Meaney M. J. , Seckl J. R. , Chapman K. E.
Published: January 2014

Glucocorticoid receptor (GR) gene expression is regulated in a complex tissue-specific manner, notably by early-life environmental events that program tissue GR levels. We have identified and characterized several new rat GR mRNAs. All encode a common protein, but differ in their 5′-leader sequences as a consequence of alternate splicing of, potentially, 11 different exon 1 sequences. Most are located in a 3-kb CpG island, upstream of exon 2, that exhibits substantial promoter activity in transfected cells. Ribonuclease (RNase) protection analysis demonstrated significant levels of six alternate exons 1 in vivo in rat, with differences between liver, hippocampus, and thymus reflecting tissue-specific differences in promoter activity. Two of the alternate exons 1 (exons 16 and 110) were expressed in all tissues examined, together present in 77–87% of total GR mRNA. The remaining GR transcripts contained tissue-specific alternate first exons. Importantly, tissue-specific first exon usage was altered by perinatal environmental manipulations. Postnatal handling, which permanently increases GR in the hippocampus, causing attenuation of stress responses, selectively elevated GR mRNA containing the hippocampus-specific exon 17. Prenatal glucocorticoid exposure, which increases hepatic GR expression and produces adult hyperglycemia, decreased the proportion of hepatic GR mRNA containing the predomin-ant exon 110, suggesting an increase in a minor exon 1 variant. Such tissue specificity of promoter usage allows differential GR regulation and programming.
Bcl-w Forms Complexes with Bax and Bak, and Elevated Ratios of Bax/Bcl-w and Bak/Bcl-w Correspond to Spermatogonial and Spermatocyte Apoptosis in the Testis
Authors: Yan Wei , Samson Michel , Jégou Bernard , Toppari Jorma
Published: January 2014

Bcl-w, a prosurvival member of the Bcl-2 family, is essential for spermatogenesis. However, the mechanisms by which Bcl-w participates in the regulation of apoptosis in the testis are largely unknown. To explore the potential role of Bcl-w in the regulation of apoptosis in the testis, the expression of Bcl-w mRNA and protein during testicular development and spermatogenesis, the dimerization with the proapoptosis members of the Bcl-2 family, and the responses to hormonal stimulation in vitro and apoptosis-inducing signals in vivo were investigated. Both Bcl-w mRNA and protein were detected in Sertoli cells, spermatogonia, and spermatocytes, as well as in Leydig cells. The steady-state levels of Bcl-w mRNA and protein were much higher in Sertoli cells than in spermatogonia and spermatocytes. In the adult rat testis, both Bcl-w mRNA and protein in Sertoli cells displayed a stage-specific expression pattern. Bcl-w could form complexes with Bax and Bak but not with Bad. Bax and Bak were immunohistochemically localized to the same cell types as Bcl-w, but with higher expression levels in spermatocytes and spermatogonia than in Sertoli cells. FSH could up-regulate Bcl-w mRNA levels in the seminiferous tubules cultured in vitro, whereas no effect was observed when testosterone was applied. Three animal models that display spermatogonial apoptosis induced by blockade of stem cell factor/c-kit interaction by a function-blocking anti-c-kit antibody, spermatocyte apoptosis induced by methoxyacetic acid, and apoptosis of spermatogonia, spermatocytes, and spermatids induced by testosterone withdrawal after ethylene dimethane sulfonate treatment were employed to check the changes of Bcl-w, Bax, and Bak protein levels during apoptosis of specific germ cells. In all three models, the ratios of Bax/Bcl-w and Bak/Bcl-w were significantly elevated. The present study suggests that Bcl-w is an important prosurvival factor of Sertoli cells, spermatogonia, and spermatocytes and participates in the regulation of apoptosis by binding proapoptotic factors Bax and Bak. The ratios of Bax/Bcl-w and Bak/Bcl-w may be decisive for the survival of Sertoli cells, spermatogonia, and spermatocytes.
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

Little 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

The 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

Most 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

Ectopic 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

The 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

Estrogen-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

The 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

The 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

GH 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

The 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

Insulin-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.
Transactivation of the Mouse Sulfonylurea Receptor I Gene by BETA2/NeuroD
Authors: Kim Ji-Won , Seghers Victor , Cho Jang-Hyeon , Kang Yup , Kim Soyeon , Ryu Yoonseok , Baek Kwanghee , Aguilar-Bryan Lydia , Lee Young-Don , Bryan Joseph , Suh-Kim Haeyoung
Published: January 2014

The sulfonylurea receptor 1 (SUR1) plays a key role in regulation of insulin secretion in pancreatic β-cells. In this study we investigated the mechanism for tissue-specific expression of the SUR1 gene. A −138/−20 fragment exhibited basal promoter activity while the −660/−20 fragment contained a regulatory element for tissue-specific expression of the mouse SUR1 gene. A pancreatic β-cell-specific transcription factor, BETA2 (β-cell E box transcription factor)/NeuroD, enhanced the promoter activity of the −660/−20 fragment in cooperation with E47. Coexpression of a dominant negative mutant of BETA2/NeuroD, BETA2(1–233), repressed the promoter activity of the −660/−20 fragment. BETA2/NeuroD bound specifically to the E3 element located at −141. The E3 sequence in a heterologous context conferred transactivation by BETA2/NeuroD in HeLa and HIT cells. Mutation of E3 eliminated the stimulatory effect of BETA2/NeuroD. Unlike BETA2/NeuroD, neurogenin 3 (ngn3) could not activate the E3 element in HeLa cells. Overexpression of ngn3 concomitantly increased expression of BETA2/NeuroD and SUR1 in HIT cells but not in HeLa cells. These results indicate that BETA2/NeuroD induces tissue-specific expression of the SUR1 gene through the E3 element. These results also suggest that E3 is specific for BETA2/NeuroD, and the stimulatory effect of ngn3 in HIT cells may require factors specifically expressed in HIT cells.
Regulation of Cholesterol Homeostasis by the Liver X Receptors in the Central Nervous System
Authors: Whitney Karl D. , Watson Michael A. , Collins Jon L. , Benson William G. , Stone Tammy M. , Numerick Mary Jo , Tippin Timothy K. , Wilson Joan G. , Winegar Deborah A. , Kliewer Steven A.
Published: January 2014

The nuclear oxysterol receptors liver X receptor-α [LXRα (NR1H3)] and LXRβ (NR1H2) coordinately regulate genes involved in cholesterol homeostasis. Although both LXR subtypes are expressed in the brain, their roles in this tissue remain largely unexplored. In this report, we show that LXR agonists have marked effects on gene expression in murine brain tissue both in vitro and in vivo. In primary astrocyte cultures, LXR agonists regulated several established LXR target genes, including ATP binding cassette transporter A1, and enhanced cholesterol efflux. In contrast, little or no effect on gene expression or cholesterol efflux was detected in primary neuronal cultures. Treatment of mice with a selective LXR agonist resulted in the induction of several LXR target genes related to cholesterol homeostasis in the cerebellum and hippocampus. These data provide the first evidence that the LXRs regulate cholesterol homeostasis in the central nervous system. Because dysregulation of cholesterol balance is implicated in central nervous system diseases such as Alzheimer’s and Niemann-Pick disease, pharmacological manipulation of the LXRs may prove beneficial in the treatment of these disorders.
NUBIScan, an in Silico Approach for Prediction of Nuclear Receptor Response Elements
Authors: Podvinec Michael , Kaufmann Michel R. , Handschin Christoph , Meyer Urs A.
Published: January 2014

Nuclear receptors (NRs) are transcription factors activated by a multitude of hormones, other endogenous substances, and exogenous molecules. These proteins modulate the regulation of target genes by contacting their promoter or enhancer sequences at specific recognition sites. The identification of these response elements is the first step toward detailed insight into the regulatory mechanisms affecting a gene. We have developed NUBIScan, a computer algorithm to predict DNA recognition sites for NRs in the regulatory regions of genes. The algorithm is based on weighted nucleotide distribution matrices and combines scores from both half-sites necessary for NR dimer binding. It provides more specific identification of functional sites than previous in silico approaches, as evidenced by scanning published regulatory regions of drug-inducible genes and comparing the obtained predictions with experimental results. In prospective analyses, NUBIScan consistently identified new functional NR binding sites in sets of large sequences, which had eluded previous analyses. This is exemplified by the detailed functional analysis of the flanking region of two genes. This approach therefore facilitates the selection of likely sites of gene regulation for subsequent experimental analysis.
Knockout of Pentraxin 3, a Downstream Target of Growth Differentiation Factor-9, Causes Female Subfertility
Authors: Varani Simona , Elvin Julia A. , Yan Changning , DeMayo Janet , DeMayo Francesco J. , Horton Heidi F. , Byrne Michael C. , Matzuk Martin M.
Published: January 2014

The ovulatory process is tightly regulated by endocrine as well as paracrine factors. In the periovulatory period, extensive remodeling of the follicle wall occurs to allow the extrusion of the oocyte and accompanying cumulus granulosa cells. Growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) are secreted members of the TGFβ superfamily that are expressed beginning in the oocyte of small primary follicles and through ovulation. Besides its critical role as a growth and differentiation factor during early folliculogenesis, GDF-9 also acts as a paracrine factor to regulate several key events in preovulatory follicles. By analyzing GDF-9-regulated expression profiles using gene chip technology, we identified TNF-induced protein 6 (Tnfip6) and pentraxin 3 (Ptx3 or PTX3) as novel factors induced by GDF-9 in granulosa cells of preovulatory follicles. Whereas Tnfip6 is induced in all granulosa cells by the LH surge, Ptx3 expression in the ovary is specifically observed after the LH surge in the cumulus granulosa cells adjacent to the oocyte. PTX3 is a member of the pentraxin family of secreted proteins, induced in several tissues by inflammatory signals. To define PTX3 function during ovulation, we generated knockout mice lacking the Ptx3 gene. Homozygous null (Ptx3−/−) mice develop normally and do not show any gross abnormalities. Whereas Ptx3−/− males are fertile, Ptx3−/− females are subfertile due to defects in the integrity of the cumulus cell-oocyte complex that are reminiscent of Bmp15−/−Gdf9+/− double mutant and BMP type IB receptor mutant mice. These studies demonstrate that PTX3 plays important roles in cumulus cell-oocyte interaction in the periovulatory period as a downstream protein in the GDF-9 signal transduction cascade.
Polyamines Modulate the Interaction between Nuclear Receptors and Vitamin D Receptor-Interacting Protein 205
Authors: Maeda Yutaka , Rachez Christophe , Hawel Leo , Byus Craig V. , Freedman Leonard P. , Sladek Frances M.
Published: January 2014

Nuclear receptors (NR) activate transcription by interacting with several different coactivator complexes, primarily via LXXLL motifs (NR boxes) of the coactivator that bind a common region in the ligand binding domain of nuclear receptors (activation function-2, AF–2) in a ligand-dependent fashion. However, how nuclear receptors distinguish between different sets of coactivators remains a mystery, as does the mechanism by which orphan receptors such as hepatocyte nuclear factor 4α (HNF4α) activate transcription. In this study, we show that HNF4α interacts with a complex containing vitamin D receptor (VDR)-interacting proteins (DRIPs) in the absence of exogenously added ligand. However, whereas a full-length DRIP205 construct enhanced the activation by HNF4α in vivo, it did not interact well with the HNF4α ligand binding domain in vitro. In investigating this discrepancy, we found that the polyamine spermine significantly enhanced the interaction between HNF4α and full-length DRIP205 in an AF-2, NR-box-dependent manner. Spermine also enhanced the interaction of DRIP205 with the VDR even in the presence of its ligand, but decreased the interaction of both HNF4α and VDR with the p160 coactivator glucocorticoid receptor interacting protein 1 (GR1P1). We also found that GR1P1 and DRIP205 synergistically activated HNF4α-mediated transcription and that a specific inhibitor of polyamine biosynthesis, α-difluoromethylornithine (DFMO), decreased the ability of HNF4α to activate transcription in vivo. These results lead us to propose a model in which polyamines may facilitate the switch between different coactivator complexes binding to NRs.
Physical Vapor Growth of Ultralong Microwires of Copper Phthalocyanine on 1-Butyl-3-methylimidazolium Tetrafluoroborate Ionic Liquid
Authors: Xiao Yan , Wang Kai-Kai , Liu Yong-Qiang , Wang Feng-Xia , Pan Ge-Bo
Published: January 2014

Ultralong microwires of CuPc were prepared by physical vapor deposition on [BMIM]BF4 ionic liquid. The as-prepared microwires are highly pure and single crystals. Moreover, this method is applicable to prepare microwires of NiPc and CoPc.
Photosubstitution of Dicyanoarenes by Hypervalent Allylsilicon Reagents via Photoinduced Electron Transfer
Authors: Matsuoka Daisuke , Nishigaichi Yutaka
Published: January 2014

Photosubstitution of dicyanoarenes with hypervalent allylsilicon reagents in the presence of anthracene efficiently proceeded to afford the allyl-substituted product in moderate to good yields. The efficiency for the present photoreaction was much higher than that for the tetracoordinate allylsilanes, because hypervalent allylsilicon reagents possess lower oxidation potential than the tetracoordinate ones, which resulted in more efficient photoinduced electron-transfer process.
Aminoglycoside resistance 16S rRNA methyltransferases block endogenous methylation, affect translation efficiency and fitness of the host
Authors: Lioy Virginia S. , Goussard Sylvie , Guerineau Vincent , Yoon Eun-Jeong , Courvalin Patrice , Galimand Marc , Grillot-Courvalin Catherine
Published: January 2014

In Gram-negative bacteria, acquired 16S rRNA methyltransferases ArmA and NpmA confer high-level resistance to all clinically useful aminoglycosides by modifying, respectively, G1405 and A1408 in the A-site. These enzymes must coexist with several endogenous methyltransferases that are essential for fine-tuning of the decoding center, such as RsmH and RsmI in Escherichia coli, which methylate C1402 and RsmF C1407. The resistance methyltransferases have a contrasting distribution—ArmA has spread worldwide, whereas a single clinical isolate producing NpmA has been reported. The rate of dissemination of resistance depends on the fitness cost associated with its expression. We have compared ArmA and NpmA in isogenic Escherichia coli harboring the corresponding structural genes and their inactive point mutants cloned under the control of their native constitutive promoter in the stable plasmid pGB2. Growth rate determination and competition experiments showed that ArmA had a fitness cost due to methylation of G1405, whereas NpmA conferred only a slight disadvantage to the host due to production of the enzyme. MALDI MS indicated that ArmA impeded one of the methylations at C1402 by RsmI, and not at C1407 as previously proposed, whereas NpmA blocked the activity of RsmF at C1407. A dual luciferase assay showed that methylation at G1405 and A1408 and lack of methylation at C1407 affect translation accuracy. These results indicate that resistance methyltransferases impair endogenous methylation with different consequences on cell fitness.
A long-term follow-up study of open reduction using Ludloff’s approach for congenital or developmental dislocation of the hip
Authors: Yamada K. , Mihara H. , Fujii H. , Hachiya M.
Published: January 2014

Objectives

There are several reports clarifying successful results following open reduction using Ludloff’s medial approach for congenital (CDH) or developmental dislocation of the hip (DDH). This study aimed to reveal the long-term post-operative course until the period of hip-joint maturity after the conventional surgical treatments.

Methods

A long-term follow-up beyond the age of hip-joint maturity was performed for 115 hips in 103 patients who underwent open reduction using Ludloff’s medial approach in our hospital. The mean age at surgery was 8.5 months (2 to 26) and the mean follow-up was 20.3 years (15 to 28). The radiological condition at full growth of the hip joint was evaluated by Severin’s classification.

Results

All 115 hips successfully attained reduction after surgery; however, 74 hips (64.3%) required corrective surgery at a mean age of 2.6 years (one to six). According to Severin’s classification, 69 hips (60.0%) were classified as group I or II, which were considered to represent acceptable results. A total of 39 hips (33.9%) were group III and the remaining seven hips (6.1%) group IV. As to re-operation, 20 of 21 patients who underwent surgical reduction after 12 months of age required additional corrective surgeries during the growth period as the hip joint tended to subluxate gradually.

Conclusion

Open reduction using Ludloff’s medial approach accomplished successful joint reduction for persistent CDH or DDH, but this surgical treatment was only appropriate before the ambulating stage. Cite this article: Bone Joint Res 2014;3:1–6.
Profilin-1 Promotes the Development of Hypertension-induced Artery Remodeling
Authors: Wang Yan , Zhang Jun , Gao Haiqing , Zhao Shaohua , Ji Xiang , Liu Xiangju , You Beian , Li Xiao , Qiu Jie
Published: January 2014

Hypertension is associated with the structural remodeling and stiffening of arteries and is known to increase cardiovascular risk. In the present study, we investigated the effects of overexpression and knock down of profilin-1 on the vascular structural remodeling in spontaneous hypertensive rats (SHRs) using an adenovirus injection to knock down or overexpress profilin-1 mRNA. As a control, blank adenovirus was injected into age-matched SHRs and Wistar-Kyoto rats (WKYs). We quantified arterial structural remodeling through morphological methods, with thoracic aortas stained with hematoxylin–eosin and picosirius red. Western blotting was performed to measure the protein expression of inducible nitric oxide synthase (iNOS) and p38 mitogen-activated protein kinase (p38), and peroxynitrite was quantified by immunohistochemical staining. Overexpression of profilin-1 significantly promoted aortic remodeling, including an increase in vessel size, wall thickness, and collagen content, whereas the knockdown of profilin-1 could reverse these effects. In addition, the expression of phosphorylated p38, iNOS and peroxynitrite was significantly upregulated in SHRs with profilin-1 overexpression along with an increased level of interleukin- 6 (IL-6). These changes could be reversed by knockdown of profilin-1. Our results demonstrate a crucial role for profilin-1 in hypertension-induced arterial structural remodeling at least in part through the p38–iNOS–peroxynitrite pathway.
Do Low-Skilled Migrants Contribute More to Home Country Income? Evidence from South Asia
Authors: Cooray Arusha
Published: January 2014

A recursive system is employed to investigate the indirect effect of out-migration on gross domestic product (GDP) through remittances in South Asia, namely Bangladesh, India, Nepal, Pakistan, and Sri Lanka. Out-migration is further disaggregated by skill level and country of destination (Middle East and other), and their effects on GDP through remittances are examined. The results suggest that migration and remittances have an important significant effect on the GDP of the countries under study. Of the skill categories, the unskilled category has the largest robust indirect effect on GDP. The effects of migration on GDP by country of destination suggest that migration to the Middle East has a robust and significant impact on GDP. There is some evidence of a combination between the altruistic and self-interested motives of migrants’ to remit.
Neurog1andNeurog2Control Two Waves of Neuronal Differentiation in the Piriform Cortex
Authors: Dixit Rajiv , Wilkinson Grey , Cancino Gonzalo I. , Shaker Tarek , Adnani Lata , Li Saiqun , Dennis Daniel , Kurrasch Deborah , Chan Jennifer A. , Olson Eric C. , Kaplan David R. , Zimmer Céline , Schuurmans Carol
Published: January 2014

The three-layered piriform cortex, an integral part of the olfactory system, processes odor information relayed by olfactory bulb mitral cells. Specifically, mitral cell axons form the lateral olfactory tract (LOT) by targeting lateral olfactory tract (lot) guidepost cells in the piriform cortex. While lot cells and other piriform cortical neurons share a pallial origin, the factors that specify their precise phenotypes are poorly understood. Here we show that in mouse, the proneural genesNeurog1andNeurog2are coexpressed in the ventral pallium, a progenitor pool that first gives rise to Cajal-Retzius (CR) cells, which populate layer I of all cortical domains, and later to layer II/III neurons of the piriform cortex. Using loss-of-function and gain-of-function approaches, we find thatNeurog1has a unique early role in reducing CR cell neurogenesis by temperingNeurog2's proneural activity. In addition,Neurog1andNeurog2have redundant functions in the ventral pallium, acting in two phases to first specify a CR cell fate and later to specify layer II/III piriform cortex neuronal identities. In the early phase,Neurog1andNeurog2are also required for lot cell differentiation, which we reveal are a subset of CR neurons, the loss of which prevents mitral cell axon innervation and LOT formation. Consequently, mutation ofTrp73, a CR-specific cortical gene, results in lot cell and LOT axon displacement.Neurog1andNeurog2thus have unique and redundant functions in the piriform cortex, controlling the timing of differentiation of early-born CR/lot cells and specifying the identities of later-born layer II/III neurons.