bluuebunny/crossref_metadata_2025 huggingface dataset:
Hyperglycemia Acutely Increases Monocyte Extracellular Signal-Regulated Kinase Activity in Vivo in Humans
Authors: Ceolotto Giulio , Gallo Alessandra , Sartori Michelangelo , Valente Roberto , Baritono Elisabetta , Semplicini Andrea , Avogaro Angelo
Published: January 2014

Glycemic spikes may negatively affect the long-term prognosis of patients with diabetes. Extracellular signal-regulated kinases (ERKs) are intracellular mediators of cell proliferation, and they can be activated in response to high glucose levels. However, the modifications of their activity in response to hyperglycemia have been poorly investigated, in vivo, in humans. Thus, we sought to determine in circulating monocytes: 1) the role of hyperglycemia in ERKs activity and phosphorylation, and 2) whether hyperglycemia affects mitogen-activated protein kinase kinase (MEK) activity and mitogen-activated protein phosphatase-1 (MKP-1) expression. These goals were performed in five normal subjects. Baseline monocyte ERKs activity was 60 ± 5 pmol/min·mg protein; when exogenous hyperglycemia was induced, both monocyte ERKs activity (81 ± 11 pmol/min·mg protein; P < 0.05) and phosphorylation significantly increased (P < 0.01). MEK activity was significantly increased by hyperglycemia (1251 ± 136 vs. 2000 ± 42 cpm; P = 0.0017), whereas no changes were observed in MKP-1 expression. We conclude that hyperglycemia acutely stimulates ERKs activity and phosphorylation in human monocytes by the MEK pathway in vivo. These findings may be relevant in understanding the negative role of acute hyperglycemia on monocyte pathophysiology.
Evidence against a Rate-Limiting Role of Proinsulin Processing for Maximal Insulin Secretion in Subjects with Impaired Glucose Tolerance and β-Cell Dysfunction1
Authors: Stumvoll Michael , Fritsche Andreas , Stefan Norbert , Hardt Elke , Häring Hans
Published: January 2014

In subjects with impaired glucose tolerance (IGT) insulin secretion is impaired. Increased proinsulin/insulin (PI/I) ratios suggest that there is also reduced processing of proinsulin to insulin in this condition. The PI/I ratio in the insulin secretory granule is ideally assessed by plasma measurements in response to acute stimulation of insulin secretion. In the present study we tested the hypothesis that maximal stimulation of insulin secretion results in exhaustion of the proinsulin conversion pathway to insulin. We therefore determined the PI/I ratio in 11 normal glucose-tolerant subjects (NGT) and 11 subjects with IGT in response to glucose (squarewave hyperglycemic clamp, 10 mmol/L), glucagon-like peptide-1 (GLP-1; primed-continuous infusion), and arginine given during the continued GLP-1 infusion. In IGT, insulin levels were significantly lower during the first phase (144 ± 20 vs. 397 ± 119 pmol/L; P = 0.02), at the end of the GLP infusion (2142 ± 350 vs. 5430 ± 1091 pmol/L; P = 0.002), and in response to arginine (3983± 375 vs. 8663 ± 1430 pmol/L; P = 0.005). In response to glucose, the minimum PI/I ratio was significantly higher in IGT (3.4 ± 0.6%) than in NGT (1.4 ± 0.5%; P = 0.02), suggesting defective proinsulin processing in this condition. In subjects with IGT, the PI/I ratio decreased significantly after GLP-1 priming (1.7 ± 0.2%; P = 0.02) and after arginine given during GLP-1 (1.4 ± 0.2%; P = 0.007) and was not significantly different from those values in NGT (1.3 ± 0.2% and 1.3 ± 0.2%, respectively; both P = NS). In conclusion, during maximal stimulation of insulin secretion in subjects with IGT, the PI/I ratio in plasma decreased significantly and was not different from that in normal controls. This strongly argues against the hypothesis that defective processing of proinsulin to insulin represents a major component of the β-cell dysfunction in IGT.
Analysis of the P3 Promoter of the Human Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor Gene in Pseudohypoparathyroidism Type 1b1
Authors: Minagawa Masanori , Watanabe Tomoyuki , Kohno Yoichi , Mochizuki Hiroshi , Hendy Geoffrey N. , Goltzman David , White John H. , Yasuda Toshiyuki
Published: January 2014

Hypocalcemia and hyperphosphatemia caused by PTH resistance are the only discernible abnormalities in pseudohypoparathyroidism type 1b (PHP-1b). Because of the selective resistance toward PTH, inactivating mutations in its receptor, the PTH/PTH-related peptide receptor (PTHR1), were thought to be responsible for PHP-1b. However, gene abnormalities responsible for PHP-1b have not been identified in the coding region and well conserved promoters (P1 and P2) of the PTHR1 gene. The purpose of the present study was to analyze the structure of the P3 promoter, the main promoter of the human PTHR1 gene in kidney, in patients with PHP-1b. Southern analysis of genomic DNA from lymphoblastoid cell lines of eight nonfamilial patients with PHP-1b revealed neither gross rearrangements nor methylation abnormalities in the P3 promoter region of the PTHR1 gene. Sequencing revealed no abnormalities in the P3 promoter region, although one patient was homozygous for an (AAAG)n polymorphic variant.

In conclusion, despite the selective resistance toward PTH in the kidney, which mainly uses the PTHR1 P3 promoter, PHP-1b in eight cases is not associated with structural abnormalities in this promoter. This study also indicates that inactivation of the P3 promoter is not achieved by methylation as tested in patients’ genomic DNA from lymphoblastoid cell lines. The influence of alterations in the polymorphic A-rich repeat sequence on promoter activity warrants further study.
Changes in Non-22-Kilodalton (kDa) Isoforms of Growth Hormone (GH) after Administration of 22-kDa Recombinant Human GH in Trained Adult Males1
Authors: Wallace Jennifer D. , Cuneo Ross C. , Bidlingmaier Martin , Lundberg Per Arne , Carlsson Lena , Boguszewski Cesar Luiz , Hay John , Boroujerdi Massoud , Cittadini Antonio , Dall Rolf , Rosén Thord , Strasburger Christian J.
Published: January 2014

GH is being used by elite athletes to enhance sporting performance. To examine the hypothesis that exogenous 22-kDa recombinant human GH (rhGH) administration could be detected through suppression of non-22-kDa isoforms of GH, we studied seventeen aerobically trained males (age, 26.9 ± 1.5 yr) randomized to rhGH or placebo treatment (0.15 IU/kg/day for 1 week). Subjects were studied at rest and in response to exercise (cycle-ergometry at 65% of maximal work capacity for 20 min). Serum was assayed for total GH (Pharmacia IRMA and pituitary GH), 22-kDa GH (2 different 2-site monoclonal immunoassays), non-22-kDa GH (22-kDa GH-exclusion assay), 20-kDa GH, and immunofunctional GH. In the study, 3 h after the last dose of rhGH, total and 22-kDa GH concentrations were elevated, reflecting exogenous 22-kDa GH. Non-22-kDa and 20-kDa GH levels were suppressed. Regression of non-22-kDa or 20-kDa GH against total or 22-kDa GH produced clear separation of treatment groups. In identical exercise studies repeated between 24 and 96 h after cessation of treatment, the magnitude of the responses of all GH isoforms was suppressed (P < 0.01), but the relative proportions were similar to those before treatment. We conclude: 1) supraphysiological doses of rhGH in trained adult males suppressed exercise-stimulated endogenous circulating isoforms of GH for up to 4 days; 2) the clearest separation of treatment groups required the simultaneous presence of high exogenous 22-kDa GH and suppressed 20-kDa or non-22-kDa GH concentrations; and 3) these methods may prove useful in detecting rhGH abuse in athletes.
Insulin-Like Growth Factor (IGF)-Binding Protein-3 (IGFBP-3) Binds to Fibronectin (FN): Demonstration of IGF-I/IGFBP-3/FN Ternary Complexes in Human Plasma1
Authors: Gui Yaoting , Murphy Liam J.
Published: January 2014

We used a yeast two-hybrid system to identify binding partners for insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3). A partial complementary DNA encoding the carboxyl-terminal of fibronectin (FN), including the cell binding site, the heparin-binding domain, and the fibrin-binding domain, was identified in a screen of a human placental complementary DNA library. The interaction of IGFBP-3 with FN and the 40-kDa heparin-binding carboxyl-terminal fragment of FN was confirmed using Western ligand blotting. Both glycosylated and nonglycosylated IGFBP-3 bound to FN with a Kd of approximately 0.3 nmol/L. IGF-I and IGFBP-1 had no effect on IGFBP-3 binding to FN. Competitive inhibition of IGFBP-3 binding to FN was observed in the presence of IGFBP-5 and heparin. The binding affinity of the immobilized IGFBP-3/FN complex for [125I]IGF-I (Kd = 0.8 nmol/L) was similar to that of IGFBP-3 alone. The presence of IGF-I/IGFBP-3/FN ternary complexes in human plasma was demonstrated by coimmunoprecipitation of IGFBP-3 and [125I]IGF-I with anti-FN monoclonal antibody. These data indicate that FN may have a role in the transportation of IGFBP-3 and IGF-I in the circulation and the sequestration of these proteins in tissues.
The Antral Mucosa as a New Site for Endocrine Tumors in Multiple Endocrine Neoplasia Type 1 and Zollinger-Ellison Syndromes1
Authors: Bordi C. , Corleto V. D. , Azzoni C. , Pizzi S. , Ferraro G. , Gibril F. , Delle Fave G. , Jensen R. T.
Published: January 2014

Carcinoid tumors were identified in the antro-pyloric mucosa of four patients with multiple endocrine neoplasia type 1 (MEN-1)/Zollinger-Ellison syndrome, accounting for 8.7% of 46 patients with this condition examined by endoscopy and histology. In contrast, no tumors were found in the antral biopsies from 124 cases of sporadic Zollinger-Ellison syndrome (P < 0.001), indicating a prominent role for the MEN-1 gene defects in tumor development. Immunohistochemically the tumors did not express the hormones produced by antral endocrine cells (gastrin, somatostatin, serotonin). In contrast, two of them were diffusely immunoreactive for the isoform 2 of the vesicular monoamine transporter (VMAT-2), a marker specific for the gastric nonantral enterochromaffin-like (ECL) cells. In one of these patients a second antral VMAT-2-positive carcinoid was seen 21 months after the first diagnosis. The other two antral carcinoids were unreactive for VMAT-2. Multiple ECL cell tumors were found in the gastric body-fundus mucosa of the two patients with VMAT-2-positive, but not in those with VMAT-2-negative, antral carcinoids. In one case, the former tumors were diagnosed 22 months after the detection of the antral tumor. We conclude that the antral mucosa is an additional tissue that may harbor endocrine tumors in MEN-1 syndrome. These tumors did not express the phenotype of normal antral endocrine cells and, in at least two cases, were identified as ectopic ECL cell carcinoids.
A Unique Exonic Splicing Mutation in the Human Androgen Receptor Gene Indicates a Physiologic Relevance of Regular Androgen Receptor Transcript Variants1
Authors: Hellwinkel Olaf José-Carlos , Holterhus Paul-Martin , Struve Dagmar , Marschke Christine , Homburg Nicole , Hiort Olaf
Published: January 2014

In a patient with partial androgen insensitivity syndrome (AIS), we identified a single inherited presumably silent nucleotide variation (AGC -> AGT) in exon 8 (codon 888) of the AR gene. However, in the patient’s genital skin fibroblasts, a considerably shortened transcript of 5.5 kb (normal: 10.5 kb) was detected, which misses a part of exon 8 and a prominent portion of the 3′-untranslated region. The translation product includes eight missense amino acids from codon 886 onward followed by a premature stop codon. As shown by in vitro expression analysis, the mutant protein lacks any residual function. However, reverse transcribed PCRs and sequence data indicate the existence of two additional splicing variants of 6.4 kb and 7.8-kb length both in patient and normal control genital skin fibroblasts. These splicing variants comprise the complete coding region but a shortened 3′-untranslated region. Thus, a distinct alternative pre-messegner RNA-processing event leading to two additional transcripts occurs generally in genital skin fibroblasts. In addition, this process partially prevents aberrant splicing in the patient and produces a small fraction of normal, functionally intact AR-protein that could explain the partial masculinization in this patient.

This first report of an exonic splicing mutation in the AR-gene indicates a physiologic relevance of the regular AR-messenger RNA variants with shortened 3′-untranslated regions and their functional translation products in human genital development.
Metabolic and Steroidogenic Alterations Related to Increased Frequency of Polycystic Ovaries in Women with a History of Gestational Diabetes1
Authors: Koivunen Riitta M. , Juutinen Jaana , Vauhkonen Ilkka , Morin-Papunen Laure C. , Ruokonen Aimo , Tapanainen Juha S.
Published: January 2014

The prevalence of polycystic ovaries (PCO) and clinical, endocrine, and metabolic features were investigated in women with previous gestational diabetes (GDM). Thirty-three women with a history of GDM and 48 controls were studied. Glucose and insulin secretion capacity was evaluated by means of the oral glucose tolerance test (OGTT), and insulin action was determined by means of a euglycemic insulin clamp. Compared with control women, women with previous GDM more often had significantly abnormal OGTT, a higher prevalence of PCO (39.4% vs. 16.7%; P = 0.03), higher serum concentrations of cortisol, dehydroepiandrosterone, and dehydroepiandrosterone sulfate and a greater area under the glucose curve.

Women with previous GDM showed a lowered early phase insulin response to glucose and impaired insulin sensitivity, which was accounted for mainly by decreased glucose nonoxidation. They also demonstrated a significantly lower fasting serum C peptide/insulin ratio than the controls, indicating that women with previous GDM have impaired hepatic insulin extraction, which tended to be more marked among women with PCO. This may explain why women with PCO and previous GDM were significantly more hyperinsulinemic than women with normal ovaries.

In conclusion, our data demonstrate that women with previous GDM often have PCO and abnormal OGTT. They are insulin resistant as a result of lowered glucose nonoxidation and show inappropriately low insulin responses to glucose, reflecting impaired β-cell function. They also have higher adrenal androgen secretion, which may be associated with abdominal obesity.
Delineation of the Molecular Defects in the AIRE Gene in Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy Patients from Southern Italy
Authors: Meloni Alessandra , Perniola Roberto , Faà Valeria , Corvaglia Enrico , Cao Antonio , Rosatelli Maria Cristina
Published: January 2014

In this study, we have carried out molecular analysis of the AIRE (autoimmune regulator) gene in 11 patients (from 8 families) affected by autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, originating from a restricted area of Southern Italy (the Salento peninsula in Puglia). Of the 16 mutant AIRE alleles from the 8 probands studied, 12 carried a missense mutation (W78R in 9, P539L in 2, and P252L in 1), 2 carried the Q358X nonsense mutation, and 2 carried the 1058delT frameshift mutation. All these mutations except the 1058delT are novel. Each of the detected mutations either predicts a premature termination of the protein or results in a nonconservative amino acid change, most likely adversely affecting the function of the protein. The W78R missense mutation is relatively common in these patients, having been detected (in homozygosity or compound heterozygosity) in 6 of the 8 probands tested, indicating the presence of a founder effect. The results of this study contribute to the delineation of the molecular pathology of the AIRE gene and enhance our ability to perform a molecular diagnosis in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients from Southern Italy.
Peroxisome Proliferator Activated Receptor γ Expression Is Reduced in the Colonic Mucosa of Acromegalic Patients
Authors: Bogazzi Fausto , Ultimieri Federica , Raggi Francesco , Costa Aurelio , Gasperi Maurizio , Cecconi Elisabetta , Mosca Franco , Bartalena Luigi , Martino Enio
Published: January 2014

Acromegalic patients have an increased prevalence of colonic polyps, due to the elevated serum insulin like growth factor (IGF)-I levels. The mechanisms underlying this process are poorly understood. Peroxisome proliferator activated receptor (PPAR)γ, a nuclear receptor that regulates adipocyte differentiation, is highly expressed in the human colonic mucosa, where it induces growth inhibition and cell differentiation. In the present study we evaluated the expression of PPARγ in the bioptic samples of colonic mucosa from 22 patients with acromegaly (Acro) and 13 normal subjects (Controls) matched for age and sex. Among Acro patients, 10 had active, untreated disease (AcroUntr), 6 were in remission after surgery (AcroRem), and 6 had active disease under treatment with somatostatin analogs (AcroSMSa). Serum GH and IGF-I (mean ± SD) levels were as follows: AcroUntr: GH, 36 ± 40 μg/l; IGF-I, 769 ± 298 μg/l; AcroRem: GH, 1.0 ± 1.3μ g/l; IGF-I, 248 ± 96 μg/l; AcroSMSa: GH, 7.8 ± 12 μg/l; IGF-I, 429 ± 221 μg/l; Controls: GH, 0.8 ± 0.7 μg/l; IGF-I, 203 ± 67μ g/l. In the Controls the level of expression of PPARγ mRNA was 15230 ± 891 transcripts. Patients with AcroUntr had 2750 ± 688 transcripts of PPARγ. (p < 0.0001) vs. Controls); patients AcroSMSa had 4629 ± 1286 transcripts of PPARγ. (p = NS vs AcroUntr, p = 0.0002 vs Controls); patients with AcroRem had 8261 ± 2481 transcripts of PPARγ (p = 0.008 vs AcroUntr, p = NS vs AcroSMSa, p = 0.007 vs Controls). A significant inverse correlation was found between serum IGF-I concentrations and the level of expression of PPARγ (r = 0.43, p = 0.03). In conclusion, patients with active Acro have a reduced expression of PPARγ in the colonic mucosa, which appears be related to the increased serum IGF-I levels and might lead to an increased prevalence of colonic polyps.
Long-Term Follow-Up of Prolactinomas: Normoprolactinemia after Bromocriptine Withdrawal
Authors: Passos Vanessa Q. , Souza Jean J. S. , Musolino Nina R. C. , Bronstein Marcello D.
Published: January 2014

Bromocriptine (BRC) and other dopamine agonist drugs are the first-choice treatment for prolactinomas. However, the major disadvantage is the need for prolonged therapy. We retrospectively studied 131 patients [62 microprolactinoma (MIC), 69 macroprolactinoma (MAC)], who achieved serum prolactin (PRL) normalization during BRC use. Twenty-seven percent of them (31% MIC and 69% MAC) underwent previous surgery. Twenty-seven patients (20.6%: 25.8% MIC and 15.9% MAC) persisted with normoprolactinemia after a median time of 44 months of BRC withdrawal. The median time of BRC use was 47 months. There were no statistically significant differences regarding age, gender, BRC initial dose, length of BRC use, tumor size, pregnancy during treatment, previous surgery, or radiotherapy among patients who persisted with normoprolactinemia and those who did not, using both univariate and multivariate analysis. BRC-induced prolactinoma cell alterations are highly controversial; and so, whether the mechanism of PRL normalization after BRC withdrawal is related to BRC use or whether it is attributable to natural history is a matter for debate. A periodic assessment of PRL levels during BRC (and other dopamine-agonist drugs) withdrawal is recommended to avoid the unnecessary maintenance of therapy in a subset of patients with prolactinomas.
Genomic Priming of the Antisecretory Response to Estrogen in Rat Distal Colon throughout the Estrous Cycle
Authors: O'Mahony Fiona , Alzamora Rodrigo , Chung Ho-Lam , Thomas Warren , Harvey Brian J.
Published: January 2014

The secretion of Cl− across distal colonic crypt cells provides the driving force for the movement of fluid into the luminal space. 17β-Estradiol (E2) produces a rapid and sustained reduction in secretion in females, which is dependent on the novel protein kinase Cδ (PKCδ) isozyme and PKA isoform I targeting of KCNQ1 channels. This sexual dimorphism in the E2 response is associated with a higher expression level of PKCδ in female compared with the male tissue. The present study revealed the antisecretory response is regulated throughout the female reproductive (estrous) cycle and is primed by genomic regulation of the kinases. E2 (1–10 nm) decreased cAMP-dependent secretion in colonic epithelia during the estrous, metestrous, and diestrous stages. A weak inhibition of secretion was demonstrated in the proestrous stage. The expression levels of PKCδ and PKA fluctuated throughout the estrous cycle and correlated with the potency of the antisecretory effect of E2. The expression of PKCδ and PKA were up-regulated by estrogen at a transcriptional level via a PKCδ-MAPK-cAMP response element-binding protein-regulated pathway indicating a genomic priming of the antisecretory response. PKCδ was activated by the membrane-impermeant E2-BSA, and this response was inhibited by the estrogen receptor antagonist ICI 182,780. The 66-kDa estrogen receptor-α isoform was present at the plasma membrane of female colonic crypt cells with a lower abundance found in male colonic crypts. The study demonstrates estrogen regulation of intestinal secretion both at a rapid and transcriptional level, demonstrating an interdependent relationship between both nongenomic and genomic hormone responses.
A Rat Model of Epilepsy in Women: A Tool to Study Physiological Interactions Between Endocrine Systems and Seizures
Authors: Scharfman Helen E. , Malthankar-Phatak Gauri H. , Friedman Daniel , Pearce Patrice , McCloskey Daniel P. , Harden Cynthia L. , MacLusky Neil J.
Published: January 2014

Epilepsy in women is influenced by endocrine status and antiepileptic drugs (AEDs), but without an animal model, the effects of endocrine variables and AEDs cannot be easily dissociated from the influence of epilepsy itself. Animal models have had limited utility because experimentally-induced seizures typically result in reproductive failure. This study was conducted to develop an improved animal model. The muscarinic convulsant pilocarpine was used to elicit status epilepticus (SE) in adult female Sprague-Dawley rats. The selective estrogen receptor modulator raloxifene was administered 30 min before pilocarpine. An anticonvulsant barbiturate, pentobarbital, was injected 5–10 min after the onset of SE, and at least once thereafter to minimize acute convulsions. Mortality, morbidity, estrous cyclicity, and the ultimate success of the procedure (i.e. induction of recurrent, spontaneous seizures) were monitored. The combination of raloxifene and pentobarbital led to significantly improved estrous cyclicity compared to previous methods. Animals treated with raloxifene and pentobarbital became epileptic, as defined by the recurrence of spontaneous convulsions in the weeks after SE. The results of this study provide an improved animal model to examine the interactions between seizures and ovarian hormone secretion. The results also suggest that treatment of SE with raloxifene may benefit women with SE.
Minireview: Epigenetic Changes in Ovarian Cancer
Authors: Balch Curt , Fang Fang , Matei Daniela E. , Huang Tim H.-M. , Nephew Kenneth P.
Published: January 2014

Epigenetic aberrations, including DNA methylation, histone modifications, and micro-RNA dysregulation, are now well established in the development and progression of ovarian cancer, and their gradual accumulation is associated with advancing disease stage and grade. Epigenetic aberrations are relatively stable, associated with distinct disease subtypes, and present in circulating serum, representing promising diagnostic, prognostic, and pharmacodynamic biomarkers. In contrast to DNA mutations and deletions, aberrant gene-repressive epigenetic modifications are potentially reversible by epigenetic therapies, including inhibitors of DNA methylation or histone-modifying enzymes. Although epigenetic monotherapies have not shown activity against solid tumors, including ovarian cancer, preclinical studies suggest they will be effective when used in combination with one another or with conventional chemotherapeutics, and combinatorial epigenetic therapy regiments are being examined in cancer clinical trials. A greater understanding of the role of epigenetics in ovarian neoplasia will provide for improved interventions against this devastating malignancy.
Evidence of a Role for Insulin-Like Growth Factor Binding Protein (IGFBP)-3 in Metabolic Regulation
Authors: Yamada P. M. , Mehta H. H. , Hwang D. , Roos K. P. , Hevener A. L. , Lee K. W.
Published: January 2014

Endocrinology, published October 6, 2010, 10.1210/en.2010-0672
Osteogenic Protein-1 Up-Regulation of the Collagen X Promoter Activity Is Mediated by a MEF-2-Like Sequence and Requires an Adjacent AP-1 Sequence
Authors: Harada Shun-ichi , Sampath T. Kuber , Aubin Jane E. , Rodan Gideon A.
Published: January 2014

Bone morphogenetic proteins induce chondrogenesis and osteogenesis in vivo. To investigate molecular mechanisms involved in chondrocyte induction, we examined the effect of osteogenic protein (OP)-1/bone morphogenetic protein-7 on the collagen X promoter. In rat calvaria-derived chondrogenic C5.18 cells, OP-1 up-regulates collagen X mRNA levels and its promoter activity in a cell type- specific manner. Deletion analysis localizes the OP-1 response region to 33 bp (−310/−278), which confers OP-1 responsiveness to both the minimal homologous and heterologous Rous sarcoma virus promoter. Transforming growth factor-β2 or activin, which up-regulates the expression of a transforming growth factor-β-inducible p3TP-Lux construct, has little effect on collagen X mRNA and on this 33-bp region. Mutational analysis shows that both an AP-1 like sequence (−294/−285, TGAATCATCA) and an A/T-rich myocyte enhancer factor (MEF)-2 like sequence (−310/−298, TTAAAAATAAAAA) in the 33-bp region are necessary for the OP-1 effect. Gel shift assays show interaction of distinct nuclear proteins from C5.18 cells with the AP-1-like and the MEF-2- like sequences. OP-1 rapidly induces nuclear protein interaction with the MEF-2-like sequence but not with the AP-1 like sequence. MEF-2-like binding activity induced by OP-1 is distinct from the MEF-2 family proteins present in C2C12 myoblasts, in which OP-1 does not induce collagen X mRNA or up-regulate its promoter activity. In conclusion, we identified a specific response region for OP-1 in the mouse collagen X promoter. Mutational and gel shift analyses suggest that OP-1 induces nuclear protein interaction with an A/T-rich MEF-2 like sequence, distinct from the MEF-2 present in myoblasts, and up-regulates collagen X promoter activity, which also requires an AP-1 like sequence.
Characterization of Receptor Interaction and Transcriptional Repression by the Corepressor SMRT
Authors: Li Hui , Leo Christopher , Schroen Daniel J. , Chen J. Don
Published: January 2014

SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor) are two related transcriptional corepressors that contain separable domains capable of interacting with unliganded nuclear receptors and repressing basal transcription. To decipher the mechanisms of receptor interaction and transcriptional repression by SMRT/N-CoR, we have characterized protein-protein interacting surfaces between SMRT and nuclear receptors and defined transcriptional repression domains of both SMRT and N-CoR. Deletional analysis reveals two individual nuclear receptor domains necessary for stable association with SMRT and a C-terminal helix essential for corepressor dissociation. Coordinately, two SMRT domains are found to interact independently with the receptors. Functional analysis reveals that SMRT contains two distinct repression domains, and the corresponding regions in N-CoR also repress basal transcription. Both repression domains in SMRT and N-CoR interact weakly with mSin3A, which in turn associates with a histone deacetylase HDAC1 in a mammalian two-hybrid assay. Far-Western analysis demonstrates a direct protein-protein interaction between two N-CoR repression domains with mSin3A. Finally we demonstrate that overexpression of full-length SMRT further represses basal transcription from natural promoters. Together, these results support a role of SMRT/N-CoR in corepression through the utilization of multiple mechanisms for receptor interactions and transcriptional repression.
Inhibition of the T-Type Ca2+ Current by the Dopamine D1 Receptor in Rat Adrenal Glomerulosa Cells: Requirement of the Combined Action of the Gβγ Protein Subunit and Cyclic Adenosine 3′,5′-Monophosphate
Authors: Drolet Patrick , Bilodeau Lyne , Chorvatova Alzbeta , Laflamme Liette , Gallo-Payet Nicole , Payet Marcel D.
Published: January 2014

Modulation of ionic Ca2+ currents by dopamine (DA) could play a pivotal role in the control of steroid secretion by the rat adrenal glomerulosa cells. In the present study, we report that DA decreases the T-type Ca2+ current amplitude in these cells. The use of pharmacological agonists and antagonists reveals that this effect is mediated by activation of the D1-like receptors. Modulation by cAMP is complex inasmuch as preincubation of the cells with 8-Br-cAMP or the specific adenylyl cyclase inhibitor, 2′,3′-dideoxyadenosine, have no effect per se, but prevent the DA-induced inhibition. The inhibitory effect of DA was abolished by addition of GDPβS to the pipette medium but not by pertussis toxin. If a cell is dialyzed with medium containing Gαs-GDP, the inhibitory effect is reduced and cannot be recovered by the addition of GTPγS, indicating that the αs is not involved, but rather the βγ-subunit. Indeed, DA-induced inhibition was mimicked by Gβγ in the pipette and 8-Br-cAMP in the bath. Similarly, Gβγ release from the activation of the AT1 receptor of angiotensin II did affect the current amplitude only in the presence of 8-Br-cAMP in the bath. The mitogen-activated protein kinase cascade, which can be activated by receptors coupled to Gs, was not involved as shown by the lack of activation of p42mapk by DA and the absence of effect of the mitogen-activated protein kinase inhibitor, PD 098059, on the DA-induced inhibition. Because the binding of Gβγ-subunits to various effectors involves the motif QXXER, we therefore tested the effect of the QEHA peptide on the inhibition of the T-type Ca2+ current induced by DA. The peptide, added to the medium pipette (200 μm), abolished the effect of DA. We conclude that the presence of the Gβγ and an increase in cAMP concentration are both required to inhibit the T-type Ca2+ current in rat adrenal glomerulosa cells.
The Role of Interleukin-2 in Combination Adenovirus Gene Therapy for Head and Neck Cancer
Authors: O’Malley Bert W. , Sewell Duane A. , Li Daqing , Kosai Ken-ichiro , Chen Shu-Hsia , Woo Savio L.C. , Duan Ling
Published: January 2014

Interleukin-2 (IL-2) gene therapy alone and in combination with the herpes thymidine kinase gene (tk) was used to evaluate immunological responses and antitumor effects in head and neck cancer. Established floor of mouth squamous cell carcinomas in C3H/HeJ mice were directly injected with recombinant adenoviral vectors carrying both therapeutic and control genes. One week after adenoviral gene transfer, only the animals treated with combination IL-2+tk or tk alone demonstrated significant tumor regression. Residual tumors were harvested for microscopic evaluation and immunohistochemistry staining, which revealed a predominance of CD8+ lymphocytes in the tumor beds of the animals treated with IL-2. To evaluate the systemic immune effects of IL-2, animals treated with single or combination gene therapy received a second site challenge with parental tumor cells or a heterologous but syngeneic sarcoma cell line. Mice treated with combination IL-2 and tk demonstrated a protective systemic immunity specific to the parental tumor cell line, whereas no systemic immune response was evident in mice receiving IL-2 alone. In a separate experiment, a range of concentrations of the adenovirus IL-2 vector were used to treat established tumors. Even with the maximal single-dose adenovirus concentration, IL-2 alone was ineffective as a single therapy. These results support the use of adenovirus-mediated gene transfer of IL-2 as an effective immunotherapy when used adjuvantly with the tk “suicide gene”.
Identification of a Functional Androgen-Response Element in the Exon 1-Coding Sequence of the Cystatin-Related Protein Gene crp2
Authors: Devos A. , Claessens F. , Alen P. , Winderickx J. , Heyns W. , Rombauts W. , Peeters B.
Published: January 2014

Two hormone-responsive segments, one in the region of the promoter and one in intron 1, are identified in two homologous androgen-regulated and differentially expressed rat genes encoding the cystatin-related proteins (CRPs). Footprint analysis with the androgen receptor (AR) DNA-binding domain on the promoter-containing fragments reveals an AR-binding site downstream of the transcription start point in the crp2 gene (ARBSd/crp2,+ 40/+63). It displays an androgen response element-like sequence motif 5′-AGAAGAaaaTGTACA-3′ and overlaps with the ATG translation start codon. A double-stranded oligonucleotide containing this sequence forms a DNA-protein complex with the full-length AR synthesized by vaccinia, as seen in band shift assays. Additional AR-binding sites, ARBSu/crp1 and ARBSu/crp2, occur 5′ upstream of the transcription start point and are located at an identical position (−142/−120) in crp1 and crp2. The AR affinity for these two slightly different sequence motifs is relatively weak. The biological function of all three AR-binding sites as transcription control elements has been studied. The ARBSd/crp2 element clearly shows androgen-response element characteristics. The contribution of the common upstream element to the androgen-dependent control of reporter gene transcription is less clear. The transcription of a reporter gene construct containing the crp2 footprint fragment crp2F (−273/+88) is hormonally regulated as determined by transfection into the human breast cancer cell line T-47D. Androgens, but also glucocorticoids, efficiently stimulate steroid-dependent transcription of the chloramphenicol acetyltransferase gene. Mutation of the 5′-TGTACA-3′ sequence in ARBSd/crp2 destroys the AR binding and abolishes the androgen-dependent synthesis of chloramphenicol acetyltransferase. A large fragment derived from intron 1 of the crp1 and crp2 gene can also provide the androgen-dependent transcription of chimeric constructs in T-47D cells. However, the induction measured is less than the one observed with crp2F (−273/+88), and this activity seems to reside in several subfragments that each display a low but consistent androgen responsiveness.
Identification of Transcripts Initiated from an Internal Promoter in the c-erbAα Locus That Encode Inhibitors of Retinoic Acid Receptor-α and Triiodothyronine Receptor Activities
Authors: Chassande Olivier , Fraichard Alexandre , Gauthier Karine , Flamant Frédéric , Legrand Claude , Savatier Pierre , Laudet Vincent , Samarut Jacques
Published: January 2014

The thyroid hormone receptor-coding locus, c-erbAα, generates several mRNAs originating from a single primary transcript that undergoes alternative splicing. We have identified for the first time two new transcripts, called TRΔα1 and TRΔα2[ mRNA for isoform α1 and α2 of the T3 receptor (TR), respectively], whose transcription is initiated from an internal promoter located within intron 7 of the c-erbAα gene. These two new transcripts exhibit tissue-specific patterns of expression in the mouse. These two patterns are in sharp contrast with the expression patterns of the full-length transcripts generated from the c-erbAα locus. TRΔα1 and TRΔα2 mRNAs encode N-terminally truncated isoforms of T3Rα1 and T3Rα2, respectively. The protein product of TRΔα1 antagonizes the transcriptional activation elicited by T3 and retinoic acid. This protein inhibits the ligand-induced activating functions of T3Rα1 and 9-cis-retinoic acid receptor-α but does not affect the retinoic acid-dependent activating function of retinoic acid receptor-α. We predict that these truncated proteins may work as down-regulators of transcriptional activity of nuclear hormone receptors in vivo.
Dissociation of Early Folding Events from Assembly of the Human Lutropin β-Subunit
Authors: Muyan Mesut , Ruddon Raymond W. , Norton Sheila E. , Boime Irving , Bedows Elliott
Published: January 2014

The human LH of the anterior pituitary is a member of the glycoprotein hormone family that includes FSH, TSH, and placental CG. All are noncovalently bound heterodimers that share a common α-subunit and β-subunits that confer biological specificity. LHβ and CGβ share more than 80% amino acid sequence identity; however, in transfected Chinese hamster ovary (CHO) cells, LHβ assembles with the α-subunit more slowly than does hCGβ, and only a fraction of the LHβ synthesized is secreted, whereas CGβ is secreted efficiently. To understand why the assembly and secretion of these related β-subunits differ, we studied the folding of LHβ in CHO cells transfected with either the LHβ gene alone, or in cells cotransfected with the gene expressing the common α-subunit, and compared our findings to those previously seen for CG. We found that the rate of conversion of the earliest detectable folding intermediate of LH, pβ1, to the second major folding form, pβ2, did not differ significantly from the pβ1-to-pβ2 conversion of CGβ, suggesting that variations between the intracellular fates of the two β-subunits cannot be explained by differences in the rates of their early folding steps. Rather, we discovered that unlike CGβ, where the folding to pβ2 results in an assembly-competent product, apparently greater than 90% of the LH pβ2 recovered from LHβ-transfected CHO cells was assembly incompetent, accounting for inefficient LHβ assembly with the α-subunit. Using the formation of disulfide (S-S) bonds as an index, we observed that, in contrast to CGβ, all 12 LHβ cysteine residues formed S-S linkages as soon as pβ2 was detected. Attempts to facilitate LH assembly with protein disulfide isomerase in vitro using LH pβ2 and excess urinary α-subunit as substrate were unsuccessful, although protein disulfide isomerase did facilitate CG assembly in this assay. Moreover, unlike CGβ, LHβ homodimers were recovered from transfected CHO cells. Taken together, these data suggest that differences seen in the rate and extent of LH assembly and secretion, as compared to those of CG, reflect conformational differences between the folding intermediates of the respectiveβ -subunits.
Cell-Specific Expression of the Mouse Glycoprotein Hormone α-Subunit Gene Requires Multiple Interacting DNA Elements in Transgenic Mice and Cultured Cells
Authors: Brinkmeier Michelle L. , Gordon David F. , Dowding Janet M. , Saunders Thomas L. , Kendall Susan K. , Sarapura Virginia D. , Wood William M. , Ridgway E. Chester , Camper Sally A.
Published: January 2014

The glycoprotein hormone α-subunit gene is expressed and differentially regulated in pituitary gonadotropes and thyrotropes. Previous gene expression studies suggested that cell specificity may be regulated by distinct DNA elements. We have identified an enhancer region between −4.6 and −3.7 kb that is critical for high level expression in both gonadotrope and thyrotrope cells of transgenic mice. Fusion of the enhancer to −341/+43 mouseα -subunit promoter results in appropriate pituitary cell specificity and transgene expression levels that are similar to levels observed with the intact −4.6 kb/+43 construct. Deletion of sequences between− 341 and −297 resulted in a loss of high level expression and cell specificity, exhibited by ectopic transgene activation in GH-, ACTH-, and PRL-producing pituitary cells as well as in other peripheral tissues. Consistent with these results, transient cell transfection studies demonstrated that the enhancer stimulated activity of a− 341/+43 α-promoter in both αTSH and αT3 cells, but it did not enhance α-promoter activity significantly in CV-1 cells. Removal of sequences between −341 and −297 allowed the enhancer to function in heterologous cells. Loss of high level expression and cell specificity may be due to loss of sequences required for binding of the LIM homeoproteins or the α-basal element 1. These data demonstrate that the enhancer requires participation by both proximal and distal sequences for high level expression and suggests that sequences from− 341 to −297 are critical for restricting expression to the anterior pituitary.
Calcitonin Receptor-Mediated Growth Suppression of HEK-293 Cells Is Accompanied by Induction of p21WAF1/CIP1 and G2/M Arrest
Authors: Evdokiou Andreas , Raggatt Liza-Jane , Atkins Gerald J. , Findlay David M.
Published: January 2014

We investigated the mechanisms by which calcitonin (CT) suppresses cellular proliferation, using HEK-293 cells stably transfected with either the rat C1a CT receptor (CTR) or the insert-negative form of the human CTR. CT treatment of clonal cell lines expressing either receptor type, but not untransfected HEK-293 cells, strongly suppressed cell growth in a concentration-dependent manner. The reduction in cell growth with CT treatment could not be attributed to cellular necrosis or apoptotic cell death, the latter assessed by both DNA fragmentation analysis and caspase 3 (CPP-32) assay. Growth inhibition was associated with an accumulation of cells in the G2 phase of the cell cycle. CT treatment of the human and rat CTR-expressing cell lines resulted in a rapid and sustained induction of mRNA encoding the cyclin-dependent kinase inhibitor, p21WAF1/CIP1, increased levels of which were maintained at least 48 h after initiation of treatment. Western blot analysis showed a rapid corresponding increase in p21WAF1/CIP1 protein, whereas protein levels of another member of the cyclin-dependent kinase inhibitor family, p27kip1, were unchanged. In parallel with the induction of p21, CT treatment reduced levels of p53 mRNA and protein. CT treatment resulted in a specific cell cycle block in G2, which was associated with inhibition of Cdc2/cyclin B kinase activity as measured by histone H1 phosphorylation. There was no evidence for p21 association with this complex despite the inhibition of Cdc2 activity. Evidence that p21 induction was causative of cell growth suppression was obtained from p21 antisense oligonucleotide experiments. Treatment with a p21 antisense oligonucleotide blocked induction of p21 expression and significantly reduced the CT-mediated growth inhibition. These observations suggest that p21 is required for the G2 arrest in response to CT, but argue against a direct role of p21 in the inhibition of Cdc2 activity. These studies suggest a novel regulation of cell cycle progression by CT and will provide a basis for detailed examination of the molecular mechanisms involved.
Signals from the AT2 (Angiotensin Type 2) Receptor of Angiotensin II Inhibit p21ras and Activate MAPK (Mitogen-Activated Protein Kinase) to Induce Morphological Neuronal Differentiation in NG108–15 Cells
Authors: Gendron∗ Louis , Laflamme∗ Liette , Rivard Nathalie , Asselin Claude , Payet Marcel D. , Gallo-Payet Nicole
Published: January 2014

In a previous study, we had shown that activation of the AT2 (angiotensin type 2) receptor of angiotensin II (Ang II) induced morphological differentiation of the neuronal cell line NG108–15. In the present study, we investigated the nature of the possible intracellular mediators involved in the AT2 effect. We found that stimulation of AT2 receptors in NG108–15 cells resulted in time-dependent modulation of tyrosine phosphorylation of a number of cytoplasmic proteins. Stimulation of NG108–15 cells with Ang II induced a decrease in GTP-bound p21ras but a sustained increase in the activity of p42mapk and p44mapk as well as neurite outgrowth. Similarly, neurite elongation, increased polymerized tubulin levels, and increased mitogen-activated protein kinase (MAPK) activity were also observed in a stably transfected NG108–15 cell line expressing the dominant-negative mutant of p21ras, RasN17. These results support the observation that inhibition of p21ras did not impair the effect of Ang II on its ability to stimulate MAPK activity. While 10 μm of the MEK inhibitor, PD98059, only moderately affected elongation, 50 μm PD98059 completely blocked the Ang II- and the RasN17-mediated induction of neurite outgrowth. These results demonstrate that some of the events associated with the AT2 receptor-induced neuronal morphological differentiation of NG108–15 cells not only include inhibition of p21ras but an increase in MAPK activity as well, which is essential for neurite outgrowth.
Cellular Distribution of Constitutively Active Mutant Parathyroid Hormone (PTH)/PTH-Related Protein Receptors and Regulation of Cyclic Adenosine 3′,5′-Monophosphate Signaling by β-Arrestin2
Authors: Ferrari Serge L. , Bisello Alessandro
Published: January 2014

PTH promotes endocytosis of human PTH receptor 1 (PTH1Rc) by activating protein kinase C and recruitingβ -arrestin2. We examined the role of β-arrestin2 in regulating the cellular distribution and cAMP signaling of two constitutively active PTH1Rc mutants, H223R and T410P. Overexpression of aβ -arrestin2-green fluorescent protein (GFP) conjugate in COS-7 cells inhibited constitutive cAMP accumulation by H223R and T410P in a dose-dependent manner, as well as the response to PTH of both mutant and wild-type PTH1Rcs. The cellular distribution of PTH1Rc-GFP conjugates, fluorescent ligands, and βarrestin2-GFP was analyzed by fluorescence microscopy in HEK-293T cells. In cells expressing either receptor mutant, a ligand-independent mobilization ofβ -arrestin2 to the cell membrane was observed. In the absence of ligand, H223R and wild-type PTH1Rcs were mainly localized on the cell membrane, whereas intracellular trafficking of T410P was also observed. While agonists promoted β-arrestin2-mediated endocytosis of both PTH1Rc mutants, antagonists were rapidly internalized only with T410P. The protein kinases inhibitor, staurosporine, significantly decreased internalization of ligand-PTH1Rc mutant complexes, although the recruitment of β-arrestin2 to the cell membrane was unaffected. Moreover, in cells expressing a truncated wild-type PTH1Rc lacking the C-terminal cytoplasmic domain, agonists stimulated translocation ofβ -arrestin2 to the cell membrane followed by ligand-receptor complex internalization without associated β-arrestin2. In conclusion, cAMP signaling by constitutively active mutant and wild-type PTH1Rcs is inhibited by a receptor interaction withβ -arrestin2 on the cell membrane, possibly leading to uncoupling from Gsα. This phenomenon is independent from protein kinases activity and the receptor C-terminal cytoplasmic domain. In addition, there are differences in the cellular localization and internalization features of constitutively active PTH1Rc mutants H223R and T410P.