bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of PAX8-PPARγ1 Rearrangements in Both Follicular Thyroid Carcinomas and Adenomas
Authors: Marques Ana Rita , Espadinha Carla , Catarino Ana L. , Moniz Sónia , Pereira Teresa , Sobrinho Luís G. , Leite Valeriano
Published: January 2014

Recently, a translocation t(2;3)(q13;p25), leading to the formation of a chimeric PAX8-peroxisome proliferator-activated receptor (PPAR)γ1 oncogene, was detected in follicular thyroid carcinomas (FTC), but not in follicular thyroid adenomas (FTA), papillary thyroid carcinomas (PTC), or multinodular hyperplasias. However, previous cytogenetic studies have identified the t(2;3)(q13;p25) translocation also in some cases of FTA. In this study, we have combined RT-PCR with primers in exons 4–8 of PAX8 and in exon 1 of PPARγ1 with PPARγ immunohistochemistry to study PAX8-PPARγ1 oncogene activation in FTC (n = 9), FTA (n = 16), PTC (n = 9), anaplastic thyroid carcinomas (n = 4), and multinodular hyperplasias (n = 2). PAX8-PPARγ1 rearrangements were detected by RT-PCR in 5 of 9 (56%) FTC and in 2 of 16 (13%) FTA. By contrast, all cases of PTC, anaplastic thyroid carcinomas, and multinodular hyperplasia were RT-PCR-negative. Diffuse nuclear immunoreactivity for PPARγ was observed in 7 of 9 (78%) FTC, 5 of 16 FTA (31%), and 1 of 9 PTC (11%). Positivity was focal in 3 cases (1 FTC, 1 PTC, and 1 multinodular hyperplasia). Diffuse nuclear staining for PPARγ was present in RT-PCR- negative cases of FTC (n = 3), FTA (n = 3), and PTC (n = 1), suggesting that a different PAX8-PPARγ1 breakpoint, a rearrangement between PPARγ1 and a non-PAX8 partner, or overexpression of the native protein might be present.

Our findings that PAX8-PPARγ1 rearrangements are present in both follicular carcinomas and adenomas suggest that this oncogene is not a reliable marker to differentiate between FTC and FTA in fine-needle aspiration biopsies of follicular neoplasms of the thyroid.
A Mutation in the Follicle-Stimulating Hormone Receptor as a Cause of Familial Spontaneous Ovarian Hyperstimulation Syndrome
Authors: Montanelli Lucia , Delbaere Anne , Di Carlo Costantino , Nappi Carmine , Smits Guillaume , Vassart Gilbert , Costagliola Sabine
Published: January 2014

Ovarian hyperstimulation syndrome (OHSS) occurs mainly after excessive stimulation of the ovaries by exogenous gonadotropins administrated in the context of in vitro fertilization procedures (iatrogenic OHSS). Recently, spontaneous and recurrent occurrence of the disease (spontaneous OHSS) was shown in two families to be caused by mutations affecting the follitropin receptor (FSHr). The two mutant FSHr (T449I, D567N) harbor aminoacid substitutions in the serpentine portion of the receptor and display abnormally high sensitivity to the pregnancy hormone hCG, thus providing a satisfactory explanation to the phenotype. In addition, mutant D567N showed also increased sensitivity to thyrotopin (TSH) and displayed increase in basal (ligand-independent) activity. In this report, we describe a new familial case of recurrent OHSS. The affected women were heterozygous for a different mutation involving codon 449, where an alanine was substituted for threonine. Similar to D567N, the T449A FSHr mutant shows an increase of its sensitivity to both hCG and TSH, together with an increase in basal activity. Together with the two previous studies, this report shows that inappropriate stimulation of the FSHr by hCG is a cause of spontaneous OHSS.
Expression and Characterization of Recombinant Type 2 3α-Hydroxysteroid Dehydrogenase (HSD) from Human Prostate: Demonstration of Bifunctional 3α/17β-HSD Activity and Cellular Distribution
Authors: Lin Hsueh-Kung , Jez Joseph M. , Schlegel Brian P. , Peehl Donna M. , Pachter Jonathan A. , Penning Trevor M.
Published: January 2014

In androgen target tissues, 3α-hydroxysteroid dehydrogenase (3α-HSD) may regulate occupancy of the androgen receptor (AR) by catalyzing the interconversion of 5α-dihydrotestosterone (5α-DHT) (a potent androgen) and 3α-androstanediol (a weak androgen). In this study, a 3α-HSD cDNA (1170 bp) was isolated from a human prostate cDNA library. The human prostatic 3α-HSD cDNA encodes a 323-amino acid protein with 69.9%, 84.1%, 99.4%, and 87.9% sequence identity to rat liver 3α-HSD and human type 1, type 2, and type 3 3α-HSDs, respectively, and is a member of the aldo-keto reductase superfamily. The close homology with human type 2 3α-HSD suggests that it is either identical to this enzyme or a structural allele. Surprisingly, when the recombinant protein was expressed and purified from Escherichia coli, the enzyme did not oxidize androsterone when measured spectrophotometrically, an activity previously assigned to recombinant type 2 3α-HSD using this assay. Complete kinetic characterization of the purified protein using spectrophotometric, fluorometric, and radiometric assays showed that the catalytic efficiency favored 3α-androstanediol oxidation over 5α-DHT reduction. Using[ 14C]-5α-DHT as substrate, TLC analysis confirmed that the reaction product was[ 14C]-3α-androstanediol. However, in the reverse reaction, [3H]-3α-androstanediol was oxidized first to [3H]-androsterone and then to [3H]-androstanedione, revealing that the expressed protein possessed both 3α- and 17β-HSD activities. The 17β-HSD activity accounted for the higher catalytic efficiency observed with 3α-androstanediol. These findings indicate that, in the prostate, type 2 3α-HSD does not interconvert 5α-DHT and 3α-androstanediol but inactivates 5α-DHT through its 3-ketosteroid reductase activity. Levels of 3α-HSD mRNA were measured in primary cultures of human prostatic cells and were higher in epithelial cells than stromal cells. In addition, elevated levels of 3α-HSD mRNA were observed in epithelial cells derived from benign prostatic hyperplasia and prostate carcinoma tissues. Expression of 3α-HSD was not prostate specific, since high levels of mRNA were also found in liver, small intestine, colon, lung, and kidney. This study is the first complete characterization of recombinant type 2 3α-HSD demonstrating dual activity and cellular distribution in the human prostate.
Isolation and Characterization of a Pituitary Tumor-Transforming Gene (PTTG)
Authors: Pei Lin , Melmed* Shlomo
Published: January 2014

Pathogenesis of tumor formation in the anterior pituitary has been intensively studied, but the common mechanism involved in pituitary cell transformation and tumorigenesis remains elusive. In this study, we used mRNA differential display PCR to identify mRNAs that are differentially expressed in rat pituitary tumor cells compared with normal pituitary tissue. An mRNA exclusively expressed in pituitary tumor but not in normal pituitary was characterized. Using this pituitary tumor-specific PCR product as a probe to screen a cDNA library constructed from rat pituitary tumor GH4 cells, a cDNA of 974 bp was isolated. This cDNA encodes a novel protein of 199 amino acids, which contains no well characterized functional motifs. The mRNA of this cDNA is detected in normal adult testis and in embryonic liver, where the transcript is about 300 bp shorter and expressed at a much lower level than that detected from pituitary tumor cells. Overexpression of this protein in mouse 3T3 fibroblasts shows that it inhibits cell proliferation and induces cell transformation in vitro. Injection of transfected 3T3 cells into athymic nude mice resulted in tumor formation within 3 weeks in all animals. These results indicate that pituitary tumor cells express a unique and potent transforming gene (PTTG), which may play a role in pituitary tumorigenesis.
Angiotensin II and Potassium Regulate Human CYP11B2 Transcription through Common cis-Elements
Authors: Clyne Colin D. , Zhang Yin , Slutsker Liliya , Mathis J. Michael , White Perrin C. , Rainey William E.
Published: January 2014

Aldosterone synthase is a mitochondrial enzyme that catalyzes the conversion of 11-deoxycorticosterone to the potent mineralocorticoid aldosterone. The gene encoding aldosterone synthase, CYP11B2, is expressed in the zona glomerulosa of the adrenal cortex. Although the major physiological regulators of aldosterone production are angiotensin II (ANG II) and potassium (K+), the mechanisms by which these compounds regulate CYP11B2 transcription are unknown. Therefore we analyzed the human CYP11B2 5′-flanking region using a transient transfection expression system in the H295R human adrenocortical cell line. ANG II and K+ increased expression of a luciferase reporter construct containing 2015 bp of human CYP11B2 5′-flanking DNA. This response was mimicked by treatment with the calcium channel activator BAYK8644, whereas activation of the protein kinase C pathway with 12-o-tetradecanoylphorbol-13-acetate had no effect. Reporter gene activity was also increased after activation of cAMP-dependent pathways by (Bu)2cAMP. Deletion, mutation, and deoxyribonuclease I footprinting analyses of the CYP11B2 5′-flanking region identified two distinct elements at positions −71/−64 (TGACGTGA) and −129/−114 (CTCCAGCCTTGACCTT) that were both required for full basal reporter gene activity and for maximal induction by either cAMP or calcium-signaling pathways. The −71/−64 element, which resembles a consensus cAMP response element (CRE), bound CRE-binding proteins from H295R cell nuclear extracts as determined by electrophoretic mobility shift analysis. Analysis of the −129/−114 element using electrophoretic mobility shift analysis demonstrated binding of the orphan nuclear receptors steroidogenic factor 1 and chicken ovalbumin upstream promoter transcription factor. These data demonstrate that ANG II, K+, and cAMP-signaling pathways utilize the same SF-1 and CRE-like cis-elements to regulate human CYP11B2 expression.
Cross-Talk between Nuclear Factor-κB and the Steroid Hormone Receptors: Mechanisms of Mutual Antagonism
Authors: McKay Lorraine I. , Cidlowski John A.
Published: January 2014

Nuclear factor κB (NF-κB) is an inducible transcription factor that positively regulates the expression of proimmune and proinflammatory genes, while glucocorticoids are potent suppressors of immune and inflammatory responses. NF-κB and the glucocorticoid receptor (GR) physically interact, resulting in repression of NF-κB transactivation. In transient cotransfection experiments, we demonstrate a dose-dependent, mutual antagonism between NF-κB and GR. Functional dissection of the NF-κB p50 and p65 subunits and deletion mutants of GR indicate that the GR antagonism is specific to the p65 subunit of NF-κB heterodimer, whereas multiple domains of GR are essential to repress p65-mediated transactivation. Despite its repression of GR transactivation, p65 failed to block the transrepressive GR homologous down-regulation function. We also demonstrate that negative interactions between p65 and GR are not selective for GR, but also occur between NF-κB and androgen, progesterone B, and estrogen receptors. However, although each of these members of the steroid hormone receptor family is repressed by NF-κB, only GR effectively inhibits p65 transactivation. Further, in cotransfections using a chimeric estrogen-GR, the presence of the GR DNA-binding domain is insufficient to confer mutual antagonism to the p65-estrogen receptor interaction. Selectivity of p65 repression for each steroid receptor is demonstrated by IκB rescue from NF-κB-mediated inhibition. Together these data suggest that NF-κB p65 physically interacts with multiple steroid hormone receptors, and this interaction is sufficient to transrepress each steroid receptor. Further, the NF-κB status of a cell has the potential to significantly alter multiple steroid signaling pathways within that cell.
Structural Requirements for the Activation of the Human Growth Hormone Secretagogue Receptor by Peptide and Nonpeptide Secretagogues
Authors: Feighner Scott D. , Howard Andrew D. , Prendergast Kristine , Palyha Oksana C. , Hreniuk Donna L. , Nargund Ravi , Underwood Dennis , Tata James R. , Dean Dennis C. , Tan Carina P. , McKee Karen Kulju , Woods John W. , Patchett Arthur A. , Smith Roy G. , Van der Ploeg Lex H. T.
Published: January 2014

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 theβ 2-adrenergic receptor.
Tissue-Specific Imprinting of the Mouse Insulin-Like Growth Factor II Receptor Gene Correlates with Differential Allele-Specific DNA Methylation
Authors: Hu Ji-Fan , Oruganti Haritha , Vu Thanh H. , Hoffman Andrew R.
Published: January 2014

Imprinted genes may be expressed uniparentally in a tissue- and development-specific manner. The insulin-like growth factor II receptor gene (Igf2r), one of the first imprinted genes to be identified, is an attractive candidate for studying the molecular mechanism of genomic imprinting because it is transcribed monoallelically in the mouse but biallelically in humans. To identify the factors that control genomic imprinting, we examined allelic expression of Igf2r at different ages in interspecific mice. We found that Igf2r is not always monoallelically expressed. Paternal imprinting of Igf2r is maintained in peripheral tissues, including liver, kidney, heart, spleen, intestine, bladder, skin, bone, and skeletal muscle. However, in central nervous system (CNS), Igf2r is expressed from both parental alleles. Southern analysis of the Igf2r promoter (region 1) revealed that, outside of the CNS where Igf2r is monoallelically expressed, the suppressed paternal allele is fully methylated while the expressed maternal allele is completely unmethylated. In CNS, however, both parental alleles are unmethylated in region 1. The importance of DNA methylation in the maintenance of the genomic imprint was also confirmed by the finding that Igf2r imprinting was relaxed by 5-azacytidine treatment. The correlation between genomic imprinting and allelic Igf2r methylation in CNS and other tissues thus suggests that the epigenetic modification in the promoter region may function as one of the major factors in maintaining the monoallelic expression of Igf2r.
Insulin-Like Growth Factor-I Affects Perinatal Lethality and Postnatal Development in a Gene Dosage-Dependent Manner: Manipulation Using the Cre/loxP System in Transgenic Mice
Authors: Liu Jun-Li , Grinberg Alexander , Westphal Heiner , Sauer Brian , Accili Domenico , Karas Michael , LeRoith Derek
Published: January 2014

Insulin-like growth factor-I (IGF-I) is essential for cell growth, differentiation and postnatal development. A null mutation in igf-1 causes intrauterine growth retardation and perinatal lethality. The present study was designed to test the lower limit of igf-1 gene dosage that ensures survival and postnatal growth by using the Cre/loxP system. Mice with variable reductions in IGF-I levels were generated by crossing EIIa-cre transgenic mice and mice with loxP-flanked igf-1 locus (igf-1/flox). EIIa-cre mice express bacteriophage P1 Cre (causes recombination) recombinase under the adenovirus promoter EIIa, during early embryonic development before implantation, and cause genomic recombination of the igf-1/flox locus. Mice with the most extensive recombination die immediately after birth, while the survivors have significant growth retardation in proportion to the reduction in their igf-1 gene. Interestingly, this gene dosage effect on body weight was not very significant before weaning. However, when the young animals were weaned at 3 weeks, the igf-1 gene dosage was the only independent predictor of the weight gain between 3 and 6 weeks among the parameters tested. Although growth retarded, mice with Cre-induced partial igf-1 deficiency were fertile and gave birth to null mice. Thus Cre-induced genomic recombination using the EIIa promoter occurs during development and creates distinct phenotypes compared with the conventional null mutation. This variability allows for postnatal survival and will enable one to begin to explore the role of the endocrine vs. paracrine effects of IGF-I.
Characterization of the Thyroxine-Binding Site of Thyroxine-Binding Globulin by Site-Directed Mutagenesis
Authors: Buettner Christoph , Grasberger Helmut , Hermansdorfer Kristine , Chen Bingkun , Treske Bettina , Janssen Onno E.
Published: January 2014

The principal transport protein for T4 in human blood, thyroxine-binding globulin (TBG), binds T4 with an exceptionally high affinity (Ka = 1010m−1). Its homology to the superfamily of the serpins has recently been used in the design of chimeric proteins, providing experimental evidence that an eight-stranded β-barrel domain encompasses the ligand-binding site. We have now characterized the T4 binding site by site-directed mutagenesis. Sequence alignment of TBG from several species revealed a phylogenetically highly conserved stretch of amino acids comprising strands 2B and 3B of the β-barrel motif. Mutations within this region (Val228Glu, Cys234Trp, Thr235Trp, Thr235Gln, Lys253Ala, and Lys253Asp), designed to impose steric hindrance or restriction of its mobility, had no significant influence on T4 binding. However, binding affinity was 20-fold reduced by introduction of an N-linked glycosylation site at the turn between strands 2B and 3B (Leu246Thr) without compromising the proper folding of this mutant as assessed by immunological methods. In most other serpins, this glycosylation site is highly conserved and has been shown to be crucial for cortisol binding of corticosteroid-binding globulin, the only other member of the serpins with a transport function. The ligand-binding site could thus be located to a highly aromatic environment deep within theβ -barrel. The importance of the binding site’s aromatic character was investigated by exchanging phenylalanines with alanines. Indeed, these experiments revealed that substitution of Phe249 in the middle of strand 3B completely abolished T4 binding, while the substitution of several other phenylalanines had no effect.
Role of G Protein-Coupled Receptor Kinases on the Agonist-Induced Phosphorylation and Internalization of the Follitropin Receptor
Authors: Lazari Maria de Fatima M. , Liu Xuebo , Nakamura Kazuto , Benovic Jeffrey L. , Ascoli Mario
Published: January 2014

The experiments presented herein were designed to identify members of the G protein-coupled receptor kinase (GRK) family that participate in the agonist-induced phosphorylation and internalization of the rat FSH receptor (rFSHR). Western blots of human kidney 293 cells (the cell line used in transfection experiments) and MSC-1 cells (a cell line derived from Sertoli cells that displays many of the differentiated functions of their normal counterparts) reveal the presence of GRK2 and GRK6 in both cell lines as well as GRK4 in MSC-1 cells. Cotransfection of 293 cells with the rFSHR and GRK2, GRK4α, or GRK6 resulted in an increase in the agonist-induced phosphorylation of the rFSHR. Cotransfections of the rFSHR with GRKs or arrestin-3 enhanced the agonist-induced internalization of the rFHSR, and combinations of GRKs and arrestin-3 were more effective than the individual components. To characterize the involvement of endogenous GRKs on phosphorylation and internalization, we inhibited endogenous GRK2 by overexpression of a kinase-deficient mutant of GRK2 or Gαt, a scavenger of Gβγ. We also inhibited endogenous GRK6 by overexpression of a kinase-deficient mutant of GKR6. All three constructs were effective inhibitors of phosphorylation, but only the kinase-deficient mutant of GRK2 and Gαt inhibited internalization. The inhibition of internalization induced by these two constructs was less pronounced than that induced by a dominant-negative mutant of the nonvisual arrrestins, however. The finding that inhibitors of GRK2 and GRK6 impair phosphorylation, but only the inhibitors of GRK2 impair internalization, suggests that different GRKs have differential effects on receptor internalization.
Transcriptional Activation of c-fos Protooncogene by 17β-Estradiol: Mechanism of Aryl Hydrocarbon Receptor-Mediated Inhibition
Authors: Duan Renqin , Porter Weston , Samudio Ismael , Vyhlidal Carrie , Kladde Michael , Safe Stephen
Published: January 2014

17β-Estradiol (E2) induced c-fos protooncogene mRNA levels in MCF-7 human breast cancer cells, and maximal induction was observed within 1 h after treatment. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibited the E2-induced response within 2 h. The molecular mechanism of this response was further investigated using pFC2-CAT, a construct containing a −1400 to +41 sequence from the human c-fos protooncogene linked to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. In MCF-7 cells transiently transfected with pFC2-CAT, 10 nm E2 induced an 8.5-fold increase of CAT activity, and cotreatment with 10 nm TCDD decreased this response by more than 45%. α-Naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist, blocked the inhibitory effects of TCDD; moreover, the inhibitory response was not observed in variant Ah-nonresponsive MCF-7 cells, suggesting that the AhR complex was required for estrogen receptor cross-talk. The E2-responsive sequence (−1220 to −1155) in the c-fos gene promoter contains two putative core pentanucleotide dioxin-responsive elements (DREs) at− 1206 to −1202 and −1163 to −1159. In transient transfection assays using wild-type and core DRE mutant constructs, the downstream core DRE (at −1163 to −1159) was identified as a functional inhibitory DRE. The results of photo-induced cross-linking, gel mobility shift, and in vitro DNA footprinting assays showed that the AhR complex interacted with the core DRE that also overlapped the E2-responsive GC-rich site (−1168 to −1161), suggesting that the mechanism for AhR-mediated inhibitory effects may be due to quenching or masking at the Sp1-binding site.
The Nematode Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor (LGR) Protein Homologous to Vertebrate Gonadotropin and Thyrotropin Receptors is Constitutively Activated in Mammalian Cells
Authors: Kudo Masataka , Chen Thomas , Nakabayashi Koji , Yu Hsu Sheau , Hsueh Aaron J. W.
Published: January 2014

The receptors for LH, FSH, and TSH belong to the large G protein-coupled, seven-transmembrane (TM) protein family and are unique in having a large N-terminal extracellular (ecto-) domain containing leucine-rich repeats important for interactions with the large glycoprotein hormone ligands. Recent studies indicated the evolution of an expanding family of homologous leucine-rich repeat-containing, G protein-coupled receptors (LGRs), including the three known glycoprotein hormone receptors; mammalian LGR4 and LGR5; and LGRs in sea anemone, fly, and snail. We isolated nematode LGR cDNA and characterized its gene from the Caenorhabditis elegans genome. This receptor cDNA encodes 929 amino acids consisting of a signal peptide for membrane insertion, an ectodomain with nine leucine-rich repeats, a seven-TM region, and a long C-terminal tail. The nematode LGR has five potential N-linked glycosylation sites in its ectodomain and multiple consensus phosphorylation sites for protein kinase A and C in the cytoplasmic loop and C tail. The nematode receptor gene has 13 exons; its TM region and C tail, unlike mammalian glycoprotein hormone receptors, are encoded by multiple exons. Sequence alignments showed that the TM region of the nematode receptor has 30% identity and 50% similarity to the same region in mammalian glycoprotein hormone receptors. Although human 293T cells expressing the nematode LGR protein do not respond to human glycoprotein hormones, these cells exhibited major increases in basal cAMP production in the absence of ligand stimulation, reaching levels comparable to those in cells expressing a constitutively activated mutant human LH receptor found in patients with familial male-limited precocious puberty. Analysis of cAMP production mediated by chimeric receptors further indicated that the ectodomain and TM region of the nematode LGR and human LH receptor are interchangeable and the TM region of the nematode LGR is responsible for constitutive receptor activation. Thus, the identification and characterization of the nematode receptor provides the basis for understanding the evolutionary relationship of diverse LGRs and for future analysis of mechanisms underlying the activation of glycoprotein hormone receptors and related LGRs.
Regulation of Myosin-Bound Protein Phosphatase by Insulin in Vascular Smooth Muscle Cells: Evaluation of the Role of Rho Kinase and Phosphatidylinositol-3-Kinase-Dependent Signaling Pathways
Authors: Begum Najma , Duddy Noreen , Sandu Oana , Reinzie Jennifer , Ragolia Louis
Published: January 2014

In this study, we examined the molecular mechanism of myosin-bound protein phosphatase (MBP) regulation by insulin and evaluated the role of MBP in insulin-mediated vasorelaxation. Insulin rapidly stimulated MBP in confluent primary vascular smooth muscle cell (VSMC) cultures. In contrast, VSMCs isolated from diabetic and hypertensive rats exhibited impaired MBP activation by insulin. Insulin-mediated MBP activation was accompanied by a rapid time-dependent reduction in the phosphorylation state of the myosin-bound regulatory subunit (MBS) of MBP. The decrease observed in MBS phosphorylation was due to insulin-induced inhibition of Rho kinase activity. Insulin also prevented a thrombin-mediated increase in Rho kinase activation and abolished the thrombin-induced increase in MBS phosphorylation and MBP inactivation. These data are consistent with the notion that insulin inactivates Rho kinase and decreases MBS phosphorylation to activate MBP in VSMCs. Furthermore, treatment with synthetic inhibitors of phosphatidylinositol-3 kinase (PI3kinase), nitric oxide synthase (NOS), and cyclic guanosine monophosphate (cGMP) all blocked insulin’s effect on MBP activation. We conclude that insulin stimulates MBP via its regulatory subunit, MBS partly by inactivating Rho kinase and stimulating NO/cGMP signaling via PI3-kinase as part of a complex signaling network that controls 20-kDa myosin light chain (MLC20) phosphorylation and VSMC contraction.
Persistent Changes in Gene Expression Induced by Estrogen and Progesterone in the Rat Mammary Gland
Authors: Ginger Melanie R. , Gonzalez-Rimbau Maria F. , Gay Jason P. , Rosen Jeffrey M.
Published: January 2014

Epidemiological studies have consistently shown that an early full-term pregnancy is protective against breast cancer. We hypothesize that the hormonal milieu that is present during pregnancy results in persistent changes in the pattern of gene expression in the mammary gland, leading to permanent changes in cell fate that determine the subsequent proliferative response of the gland. To investigate this hypothesis, we have used suppression subtractive hybridization to identify genes that are persistently up-regulated in the glands of E- and progesterone (P)-treated Wistar-Furth rats 28 d after steroid hormone treatment compared with age-matched virgins. Using this approach, a number of genes displaying persistent altered expression in response to previous treatment with E and P were identified. Two markers have been characterized in greater detail: RbAp46 and a novel gene that specifies a noncoding RNA (designated G.B7). Both were persistently up-regulated in the lobules of the regressed gland and required previous treatment with both E and P for maximal persistent expression. RbAp46 has been implicated in a number of complexes involving chromatin remodeling, suggesting a mechanism whereby epigenetic factors responsible for persistent changes in gene expression may be related to the determination of cell fate. These results provide the first support at the molecular level for the hypothesis that hormone-induced persistent changes in gene expression are present in the involuted mammary gland.
TRs Have Common and Isoform-Specific Functions in Regulation of the Cardiac Myosin Heavy Chain Genes
Authors: Mansén Anethe , Yu Fushun , Forrest Douglas , Larsson Lars , Vennström Björn
Published: January 2014

TRα1 and TRβ mediate the regulatory effects of T3 and have profound effects on the cardiovascular system. We have analyzed the expression of the cardiac myosin heavy chain (MyHC) genesα and β in mouse strains deficient for one or several TR genes to identify specific regulatory functions of TRα1 and TRβ. The results show that TRα1 deficiency, which slows the heart rate, causes chronic overexpression of MyHCβ. However, MyHCβ was still suppressible by T3 in both TRα1- and TRβ-deficient mice, indicating that either receptor can mediate repression of MyHCβ. T3-dependent induction of the positively regulated MyHCα gene was similar in both TRα1- and TRβ-deficient mice. The data identify a specific role for TRα1 in the negative regulation of MyHCβ, whereas TRα1 and TRβ appear interchangeable for hormone-dependent induction of MyHCα. This suggests that TR isoforms exhibit distinct specificities in the genes that they regulate within a given tissue type. Thus, dysregulation of MyHCβ is likely to contribute to the critical role of TRα1 in cardiac function.
Floppy SOX: Mutual Induced Fit in HMG (High-Mobility Group) Box-DNA Recognition
Authors: Weiss Michael A.
Published: January 2014

The high-mobility group (HMG) box defines a DNA-bending motif of broad interest in relation to human development and disease. Major and minor wings of an L-shaped structure provide a template for DNA bending. As in the TATA-binding protein and a diverse family of factors, insertion of one or more side chains between base pairs induces a DNA kink. The HMG box binds in the DNA minor groove and may be specific for DNA sequence or distorted DNA architecture. Whereas the angular structures of non-sequence-specific domains are well ordered, free SRY and related autosomal SOX domains are in part disordered. Observations suggesting that the minor wing lacks a fixed tertiary structure motivate the hypothesis that DNA bending and stabilization of protein structure define a coupled process. We further propose that mutual induced fit in SOX-DNA recognition underlies the sequence dependence of DNA bending and enables the induction of promoter-specific architectures.
The Serine/Threonine Transmembrane Receptor ALK2 Mediates Müllerian Inhibiting Substance Signaling
Authors: Visser Jenny A. , Olaso Robert , Verhoef-Post Miriam , Kramer Piet , Themmen Axel P. N. , Ingraham Holly A.
Published: January 2014

Müllerian inhibiting substance (MIS or anti-Müllerian hormone) is a member of the transforming growth factor-β family and plays a pivotal role in proper male sexual differentiation. Members of this family signal by the assembly of two related serine/threonine kinase receptors, referred to as type I or type II receptors, and downstream cytoplasmic Smad effector proteins. Although the MIS type II receptor (MISRII) has been identified, the identity of the type I receptor is unclear. Here we report that MIS activates a bone morphogenetic protein-like signaling pathway, which is solely dependent on the presence of the MISRII and bioactive MIS ligand. Among the multiple type I candidates tested, only ALK2 resulted in significant enhancement of the MIS signaling response. Furthermore, dominant-negative and antisense strategies showed that ALK2 is essential for MIS-induced signaling in two independent assays, the cellular Tlx-2 reporter gene assay and the Müllerian duct regression organ culture assay. In contrast, ALK6, the other candidate MIS type I receptor, was not required. Expression analyses revealed that ALK2 is present in all MIS target tissues including the mesenchyme surrounding the epithelial Müllerian duct. Collectively, we conclude that MIS employs a bone morphogenetic protein-like signaling pathway and uses ALK2 as its type I receptor. The use of this ubiquitously expressed type I receptor underscores the role of the MIS ligand and the MIS type II receptor in establishing the specificity of the MIS signaling cascade.
Fibroblast Growth Factor Activation of the Rat PRL Promoter is Mediated by PKCδ
Authors: Jackson Twila A. , Schweppe Rebecca E. , Koterwas David M. , Bradford Andrew P.
Published: January 2014

Fibroblast growth factors play a critical role in cell growth, development, and differentiation and are also implicated in the formation and progression of tumors in a variety of tissues including pituitary. We have previously shown that fibroblast growth factor activation of the rat PRL promoter in GH4T2 pituitary tumor cells is mediated via MAP kinase in a Ras/Raf-1-independent manner. Herein we show using biochemical, molecular, and pharmacological approaches that PKCδ is a critical component of the fibroblast growth factor signaling pathway. PKC inhibitors, or down-regulation of PKC, rendered the rat PRL promoter refractory to subsequent stimulation by fibroblast growth factors, implying a role for PKC in fibroblast growth factor signal transduction. FGFs caused specific translocation of PKCδ from cytosolic to membrane fractions, consistent with enzyme activation. In contrast, other PKCs expressed in GH4T2 cells (α, βI, βII, andε ) did not translocate in response to fibroblast growth factors. The PKCδ subtype-selective inhibitor, rottlerin, or expression of a dominant negative PKCδ adenoviral construct also blocked fibroblast growth factor induction of rat PRL promoter activity, confirming a role for the novel PKCδ isoform. PKC inhibitors selective for the conventional α and β isoforms or dominant negative PKCα adenoviral expression constructs had no effect. Induction of the endogenous PRL gene was also blocked by adenoviral dominant negative PKCδ expression but not by an analogous dominant negative PKCα construct. Finally, rottlerin significantly attenuated FGF-induced MAP kinase phosphorylation. Together, these results indicate that MAP kinase-dependent fibroblast growth factor stimulation of the rat PRL promoter in pituitary cells is mediated by PKCδ.
Pituitary, Pancreatic and Gut Neuroendocrine Defects in Protein Tyrosine Phosphatase- Sigma-Deficient Mice
Authors: Batt Jane , Asa Sylvia , Fladd Chris , Rotin Daniela
Published: January 2014

The expression of receptor protein tyrosine phosphatase sigma (PTPσ) is developmentally regulated in neuronal and neuroendocrine tissues. We have previously shown that mice deficient in PTPσ demonstrate nervous system abnormalities, pituitary hypoplasia, increased neonatal mortality (60%), and death from a wasting syndrome at 2–3 wk of age (38%). We have now examined the role of PTPσ on pituitary, pancreas and enteroendocrine cytodifferentiation, hormone production, and development. The adenohypophyses of PTPσ(−/−) mice were small and exhibited reduced GH and PRL immunoreactivity. Cells containing TSH, LH, FSH, ACTH, pituitary-specific POU homeodomain factor (Pit-1), ER, and steroidogenic factor 1 were found in normal proportions and distributions. The diminished expression of GH and PRL was not associated with apoptosis of somatotrophs or lactotrophs. Pit-1-positive TSH-negative cells were detected, suggesting that impaired GH and PRL synthesis was not attributable to Pit-1 deficiency. In the knockout mice, pancreatic islets were hypoplastic with reduced insulin immunoreactivity, and there was also variable expression of gut hormones. Functionally, the GH deficiency was associated with hypoglycemia and death in the PTPσ(−/−) neonate and accordingly, ip administration of GH rescued the PTPσ(−/−) neonate and normalized the blood glucose. These data indicate that PTPσ plays a major role in differentiation and development of the neuroendocrine system.
DNA Binding-Independent Induction of IκBα Gene Transcription by PPARα
Authors: Delerive Philippe , De Bosscher Karolien , Vanden Berghe Wim , Fruchart Jean-Charles , Haegeman Guy , Staels Bart
Published: January 2014

PPARs are ligand-activated transcription factors that regulate energy homeostasis. In addition, PPARs furthermore control the inflammatory response by antagonizing the nuclear factor-κB (NF-κB) signaling pathway. We recently demonstrated that PPARα activators increase IκBα mRNA and protein levels in human aortic smooth muscle cells. Here, we studied the molecular mechanisms by which PPARα controls IκBα expression. Using transient transfection assays, it is demonstrated that PPARα potentiates p65-stimulated IκBα transcription in a ligand-dependent manner. Site-directed mutagenesis experiments revealed that PPARα activation of IκBα transcription requires the NF-κB and Sp1 sites within IκBα promoter. Chromatin immunoprecipitation assays demonstrate that PPARα activation enhances the occupancy of the NF-κB response element in IκBα promoter in vivo. Overexpression of the oncoprotein E1A failed to inhibit PPARα-mediated IκBα promoter induction, suggesting that cAMP response element binding protein-binding protein/p300 is not involved in this mechanism. By contrast, a dominant-negative form of VDR-interacting protein 205 (DRIP205) comprising its two LXXLL motifs completely abolished PPARα ligand-mediated activation. Furthermore, cotransfection of increasing amounts of DRIP205 relieved this inhibition, suggesting that PPARα requires DRIP205 to regulate IκBα promoter activity. By contrast, DRIP205 is not involved in PPARα-mediated NF-κB transcriptional repression. Taken together, these data provide a molecular basis for PPARα-mediated induction of IκBα and demonstrate, for the first time, that PPARα may positively regulate gene transcription in the absence of functional PPAR response elements.
Identification of Two Novel ACTH-Responsive Genes Encoding Manganese-Dependent Superoxide Dismutase (SOD2) and the Zinc Finger Protein TIS11b [Tetradecanoyl Phorbol Acetate (TPA)-Inducible Sequence 11b]
Authors: Chinn Anna M. , Ciais Delphine , Bailly Sabine , Chambaz Edmond , LaMarre Jonathan , Feige Jean-Jacques
Published: January 2014

ACTH is the major trophic factor regulating and maintaining adrenocortical function, affecting such diverse processes as steroidogenesis, cell proliferation, cell migration, and cell survival. We used differential display RT-PCR to identify genes that are rapidly induced by ACTH in the bovine adrenal cortex. Of 42 PCR products differentially amplified from primary cultures of bovine adrenocortical cells treated with 10 nm ACTH, six identified mRNAs that were confirmed by Northern blot analysis to be induced by ACTH. Four of these amplicons encoded noninformative repetitive sequences. Of the other two sequenced amplicons, one encoded a partial sequence for mitochondrial manganese-dependent superoxide dismutase (SOD2), an enzyme that is likely to protect adrenocortical cells from the cytotoxic effects of radical oxygen species generated during steroid biosynthesis. The second was identified as TIS11b (phorbol-12-myristate-13-acetate-inducible sequence 11b)/ERF-1/cMG, a member of the CCCH double-zinc finger protein family. SOD2 induction by ACTH was independent of extracellular steroid concentration or oxidative stress. SOD2 and TIS11b mRNA expressions were rapidly induced by ACTH, reaching a maximal level after 8 h and 3 h of treatment, respectively. These ACTH effects were mimicked by forskolin but appeared independent of cortisol secretion. Upon ACTH treatment, induction of TIS11b expression closely followed the previously characterized peak of vascular endothelial growth factor (VEGF) expression. Transfection of a TIS11b expression plasmid into 3T3 fibroblasts induced a decrease in the expression of a reporter gene placed upstream of the VEGF 3′-untranslated region, indicating that TIS11b may be an important regulator of VEGF expression through interaction with its 3′-untranslated region.
Evolution of Glycoprotein Hormone Subunit Genes in Bilateral Metazoa: Identification of Two Novel Human Glycoprotein Hormone Subunit Family Genes, GPA2 and GPB5
Authors: Hsu Sheau Yu , Nakabayashi Koji , Bhalla Alka
Published: January 2014

The canonical members of the human glycoprotein hormone subunit family of cystine knot-forming polypeptides include the common α-subunit, and four β-subunit genes, FSHβ, LHβ, TSHβ, and hCGβ. Using pairwise sequence analysis of the complete human genome, we have identified two novel glycoprotein hormone subunit-related genes. Based on unique sequence similarity to the α- and β-subunits of glycoprotein hormones, they were named glycoprotein-α2 (GPA2) and glycoprotein-β5 (GPB5), respectively. PCR analysis using a panel of human cDNAs from 14 different tissues demonstrated that GPB5 is similar to other β-subunits showing restricted tissue expression, mainly in pituitary and brain. In contrast, the GPA2 transcript is found in diverse tissues. Furthermore, immunoreactive GPA2 and GPB5 were detected in the anterior pituitary of mouse and frog, whereas the expression of GPA2 and GPB5 in transfected cells resulted in the secretion of recombinant polypeptides in conditioned medium. After GenBank searches in lower organisms, glycoprotein hormone β-subunit-related genes were identified from the genome of nematode Caenorhabditis elegans, hookworm Ancylostoma caninum, and Drosophila melanogaster. The evolutionary conservation of these invertebrate homologs can be seen in several key sequence characteristics, and the data suggest that the glycoprotein hormone β-subunit gene ancestor evolved before the emergence of bilateral metazoa, thus providing a better understanding of the evolution of this group of classic polypeptide hormones and their receptors. Studies of the complete inventory of genes homologous to glycoprotein hormone subunits in the human genome and lower organisms will allow future functional characterization and identification of their respective receptors.
RAS Mutations Are the Predominant Molecular Alteration in Poorly Differentiated Thyroid Carcinomas and Bear Prognostic Impact
Authors: Volante Marco , Rapa Ida , Gandhi Manoj , Bussolati Gianni , Giachino Daniela , Papotti Mauro , Nikiforov Yuri E.
Published: January 2014

Context

Poorly differentiated carcinomas represent an aggressive group of thyroid tumors with controversial classification placement and poorly understood pathogenesis. Molecular data in this group of tumors are extremely heterogeneous, possibly reflecting different inclusion criteria. Recently homogeneous diagnostic criteria have been proposed by our group (Turin proposal) that need to be complemented by detailed molecular characterization.

Objective

The objective of the study was to define a comprehensive molecular typing of poorly differentiated thyroid carcinomas classified following homogeneous diagnostic criteria.

Design

Sixty-five cases of poorly differentiated carcinoma selected following the Turin proposal have been screened for N-, K-, H-RAS, BRAF, RET/PTC1 and 3, and PAX8/PPARγ mutations-rearrangements using alternative techniques and in two different laboratories. Molecular data were compared with clinical pathological parameters and survival by univariate and multivariate analysis.

Results

RAS mutations in codon 61 were by far the most common genetic alteration in poorly differentiated carcinomas (23% of cases), with all mutation in NRAS except one in the HRAS gene. A single BRAF mutation was found in a poorly differentiated carcinoma with a residual component of a tall cell variant of papillary carcinoma. No KRAS, RET/PTC, or PAX8/PPARγ genetic alteration was detected. In this series, the presence of RAS mutations was a unique negative prognostic parameter at multivariate analysis.

Conclusions

The present study demonstrates that strictly classified poorly differentiated carcinomas are genetically homogeneous, RAS mutations being the almost exclusive genetic event. Moreover, the detection of RAS mutations might be clinically relevant for the prognostic stratification of these tumors.
Abrogation of TLR4 and CD14 Expression and Signaling in Human Adrenocortical Tumors
Authors: Kanczkowski Waldemar , Tymoszuk Piotr , Ehrhart-Bornstein Monika , Wirth Manfred P. , Zacharowski Kai , Bornstein Stefan R.
Published: January 2014

Context: Adrenocortical carcinoma (ACC) is a rare tumor with poor prognosis. The expression of innate immunity receptor Toll-like receptor (TLR)-4 was recently reported in various human tumors, and TLR4 was shown to regulate tumor immune escape processes, proliferation, and resistance to chemotherapeutical agents.

Objective: The aim of this study was to investigate TLR4 expression, signaling, and function in the process of tumorigenesis in the human adrenal cortex.

Measurements and Main Results: Real-time PCR analysis of human ACC (n = 8), adenoma (n = 8), and ACC cell lines (SW13, NCI-H295R, and HAC15) revealed a significant down-regulation of TLR4, MD2, and CD14 mRNA compared with normal human adrenal cortex and adrenocortical cells in primary culture. Furthermore, immunohistochemistry revealed an abrogation of TLR4 and CD14 expression in ACC but not adenoma tissues. Western blot analysis of MAPK, AKT, activator protein-1, and nuclear factor-κB signaling revealed that the ACC cell lines are unresponsive to lipopolysaccharide action. Restoration of TLR4 signaling by stable transfection of TLR4-CD14 plasmid into NCI-H295R cells sensitized them to lipopolysaccharide incubation as shown by nuclear factor-κB activation and decreased cell viability and induced apoptosis in these cells.

Conclusion: Our results demonstrate a significant reduction in the expression of TLR4 and CD14 and an inactivation of TLR4 signaling in ACCs. Furthermore, our data show that reintroduction of TLR4 expression in ACCs may provide a novel therapeutic strategy for adrenal cancer.