bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Posttranscriptional Regulation of Androgen Receptor Gene Expression by Androgen in Prostate and Breast Cancer Cells*
Authors: Yeap Bu B. , Krueger Romano G. , Leedman Peter J.
Published: January 2014

Androgens, via the androgen receptor (AR), modulate the growth and proliferation of prostate and breast cancer cells. However, the molecular mechanisms underlying the regulation of AR gene expression by androgen in these cells remain to be fully elucidated. To explore differences in AR gene expression between these hormone-responsive tumor cell types, we studied androgen-responsive LNCaP prostate cancer and AR positive MDA453 breast cancer cells. Dihydrotestosterone (DHT) 10 nm increased LNCaP cell proliferation and the proportion of LNCaP cells in S-phase of the cell cycle but inhibited MDA453 cell proliferation and reduced the proportion of MDA453 cells in S-phase of cell cycle. In both these cell lines, DHT decreased total AR messenger RNA (mRNA) but increased AR protein. In LNCaP cells, DHT down-regulated AR mRNA transcription but stabilized AR mRNA. In contrast, in MDA453 cells, DHT had no effect on AR mRNA transcription but destabilized AR mRNA. In summary, transcriptional down-regulation induced by androgens in LNCaP cells results in down-regulation of steady-state AR mRNA despite an androgen-induced increase in AR mRNA stability. However, in MDA453 cells, posttranscriptional destabilization of AR mRNA appears to be the predominant mechanism resulting in down-regulation of AR mRNA by androgen. These results demonstrate cell-specific and divergent regulation of AR mRNA turnover by androgen and identify a novel pathway of androgen-induced posttranscriptional destabilization and down-regulation of AR mRNA in human breast cancer cells. Furthermore, these data establish an important role for posttranscriptional pathways in the regulation of AR gene expression by androgen in human prostate and breast cancer cells.
Histaminergic and Catecholaminergic Interactions in the Central Regulation of Vasopressin and Oxytocin Secretion*
Authors: Knigge Ulrich , Willems Edwin , Kjær Andreas , Jørgensen Henrik , Warberg Jørgen
Published: January 2014

Activation of histaminergic and noradrenergic/adrenergic neurons in the brain stimulates the release of the neurohypophysial hormones arginine vasopressin (AVP) and oxytocin (OT) and are involved the mediation of the hormone responses to physiological stimuli such as dehydration and suckling. We therefore investigated whether the two neuronal systems interact in their regulation of AVP and OT secretion in conscious male rats. When administered intracerebroventricularly (icv), histamine (HA) as well as the H1 receptor agonist 2-thiazolylethylamine or the H2 receptor agonist 4-methylHA stimulated AVP and OT secretion. Prior icv infusion of antagonists specific to α or β adrenergic receptors or their subtypes did not significantly affect the hormone response to HA or the histaminergic agonists. Infused icv norepinephrine (NE) or epinephrine (E) increased AVP and OT secretion. Prior icv infusion of the H1 receptor antagonist mepyramine or the H2 receptor antagonist cimetidine significantly inhibited the AVP and OT responses to NE and the AVP response to E, whereas only cimetidine inhibited the OT response to E significantly. Systemic pretreatment with imetit, which by activation of presynaptic H3 receptors inhibits neuronal synthesis and release of HA, decreased the AVP and OT responses to NE and E significantly. In the doses used, HA and E had no significant effect on mean arterial blood pressure. NE increased mean arterial blood pressure 10% at 1 and 2.5 min, whereafter the blood pressure returned to basal level within 10 min. The results indicate that noradrenergic and adrenergic neurons stimulate AVP and OT secretion via an involvement of histaminergic neurons, which may occur at magnocellular neurons in the supraoptic and paraventricular nuclei of the hypothalamus. The stimulatory effect of the amines on neurohypophysial hormone secretion seems to be independent of a central action on blood pressure. In contrast, a functionally intact noradrenergic and adrenergic neuronal system seems not to be a prerequisite for a HA-induced release of AVP and OT. The present findings further substantiate the role of histaminergic neurons in the central regulation of neurohypophysial hormone secretion.
Parathyroid Hormone-Related Peptide Is a Potent Tumor Angiogenic Factor
Authors: Akino Kozo , Ohtsuru Akira , Kanda Kazuaki , Yasuda Akiko , Yamamoto Toshinori , Akino Yuki , Naito Shinji , Kurokawa Mamoru , Iwahori Nobuharu , Yamashita Shunichi
Published: January 2014

Rat pituitary malignant tumor cells; mGH3, show hypervascularization in in vivo xenografts and overexpress parathyroid hormone-related peptide (PTHrP) compared to original GH3 cells. To elucidate whether PTHrP is involved in tumor-derived angiogenesis, we examined the effect of PTHrP on vascular endothelial cells both in vitro and in vivo. Results of in vivodiffusion chamber assay showed a clear hypervascularization on the outer surface of diffusion chambers containing mGH3 tumor cell implants but not in those containing GH3 cells. Co-incubation with antisense PTHrP oligonucleotide (10 μM), but not sense or mismatched PTHrP oligonucleotide, suppressed hypervascularization in diffusion chambers. To further examine the role of PTHrP on endothelial cell function, PTHrP(1–34) was added at various concentrations to cultured bovine endothelial cells (BAECs) harvested from the aorta. PTHrP(1–34) did not alter the proliferation or migration of endothelial cells, but rather dose-dependently increased capillary formation by endothelial cells on the collagen gel matrix. Furthermore, 0.1 mM of 8-bromo-cAMP caused a similar increase in tube formation, which was dose-dependently inhibited by H89, a protein kinase A inhibitor. Our results indicate for the first time that PTHrP is a potential paracrine factor acting via the PKA pathway to enhance angiogenesis through capillary tube formation by endothelial cells in malignant pituitary tumors.
The Acute and Chronic Stimulatory Effects of Endothelin-1 on Glucose Transport Are Mediated by Distinct Pathways in 3T3-L1 Adipocytes**This work was supported by NIH Grant DK-33651.
Authors: Ishibashi Ken-ichi , Imamura Takeshi , Sharma Prem M. , Ugi Satoshi , Olefsky Jerrold M.
Published: January 2014

We have recently shown that pretreatment with endothelin-1 (ET-1) for 20 min stimulates GLUT4 translocation in a PI3-kinase-dependent manner in 3T3-L1 adipocytes (Imamura, T. et al., J Biol Chem 274:33691–33695). This study presents another pathway by which ET-1 potentiates glucose transport in 3T3-L1 adipocytes. ET-1 treatment (10 nm) leads to approximately 2.5-fold stimulation of 2-deoxyglucose (2-DOG) uptake within 20 min, reaching a maximal effect of ∼4-fold at ∼6 h, and recovering almost to basal levels after 24 h. Insulin treatment (3 ng/ml) results in an approximately 5-fold increase in 2-DOG uptake at 1 h, and recovering to basal levels after 24 h. The ETA receptor antagonist, BQ 610, inhibited ET-1 induced glucose uptake both at 20 min and 6 h, whereas the ETB receptor antagonist, BQ 788, was without effect. Interestingly, ET-1 stimulated 2-DOG uptake at 6 h, not at 20 min, was almost completely blocked by the protein-synthesis inhibitor, cycloheximide and the RNA-synthesis inhibitor, actinomycin D, suggesting that the short-term (20 min) and long-term (6 h) effects of ET-1 involve distinct mechanisms. GLUT4 translocation assay showed that 20 min, but not 6 h, exposure to ET-1 led to GLUT4 translocation to the plasma membrane. In contrast, 6 h, but not 20 min, exposure to ET-1 increased expression of the GLUT1 protein, without affecting expression of GLUT4 protein. ET-1 induced 2-DOG uptake and GLUT1 expression at 6 h were completely inhibited by the MEK inhibitor, PD 98059, and partially inhibited by the PI3-kinase inhibitor, LY 294002, and the Gαi inhibitor, pertussis toxin. The PLC inhibitor, U 73122, was without effect. These findings suggest that ET-1 induced GLUT1 protein expression is primarily mediated via MAPK, and partially via PI3K in 3T3-L1 adipocytes.
Molecular Cloning and Expression of a Functionally Different Alternative Splice Variant of Prointerleukin-1α from the Rat Testis*
Authors: Sultana Taranum , Svechnikov Konstantin , Weber Günther , Söder Olle
Published: January 2014

We report here the characterization of an alternative splice variant of prointerleukin-1α (proIL-1α), constitutively expressed by the normal adult rat testis. In addition to the classical 32K proIL-1α (32proIL-1α) messenger RNA, the testis produced a shorter variant encoding a putative protein of 24K (24proIL-1α). In situ hybridization demonstrated constitutive expression of the splice transcript in the seminiferous tubules. This alternative complementary DNA lacked the fifth exon, harboring the calpain cleavage site essential for generation of mature 17K IL-1α. This was verified by calpain treatment, producing the expected cleavage products of recombinant 32proIL-1α, but not of 24proIL-1α. Similarly, expression in COS-7 cells demonstrated processing of 32proIL-1α to the mature 17K form and secretion, whereas 24proIL-1α remained unprocessed. Both 32proIL-1α and 24proIL-1α showed a dose-dependent stimulatory effect in a thymocyte proliferation assay, although at lower potency than mature 17K IL-1α. In contrast, when tested on hCG-stimulated Leydig cells in vitro, a dose-dependent inhibition of testosterone production was obtained with mature 17K IL-1α and at a lower potency with 32proIL-1α, whereas 24proIL-1α was inactive. In conclusion, the three IL-1 bioactive proteins described here contribute to IL-1 protein heterogeneity and may serve as constitutive paracrine mediators in the testis.
Neuroendocrine Aging in the Female Rat: The Changing Relationship of Hypothalamic Gonadotropin-Releasing Hormone Neurons and N-Methyl-d-Aspartate Receptors**Preliminary versions of this work were presented at the 28th and 29th Annual Meetings of the Society for Neuroscience (Abstracts 110.11 and 777.10). This work was supported by the Brookdale Foundation (to A.C.G.), NIH Grant 1-PO1-AG16765–01 (to A.C.G. and J.H.M.).
Authors: Gore Andrea C. , Yeung Glendy , Morrison John H. , Oung Twethida
Published: January 2014

The reproductive axis undergoes alterations during aging, resulting in acyclicity and the loss of reproductive function. In the hypothalamus, changes intrinsic to GnRH neurons may play a critical role in this process, as may changes in inputs to GnRH neurons from neurotransmitters such as glutamate. We investigated the effects of age and reproductive status on neuroendocrine glutamatergic NMDA receptors (NRs), their regulation of GnRH neurons, and their expression on GnRH neurons, in female rats. First, we quantified NR subunit messenger RNAs (mRNAs) in preoptic area-anterior hypothalamus (POA-AH) and medial basal hypothalamus (MBH), the sites of GnRH perikarya and neuroterminals, respectively. In POA-AH, NR1 mRNA levels varied little with age or reproductive status. NR2a and NR2b mRNA levels decreased significantly between cycling and acyclic rats. In MBH, NR mRNAs all increased with aging, particularly in acyclic animals. Second, we tested the effects of N-methyl-d,l-aspartate (NMA) on GnRH mRNA levels in POA-AH of aging rats. NMA elevated GnRH mRNA levels in young rats, but decreased them in middle-aged rats. Third, we quantified expression of the NR1 subunit on GnRH perikarya in aging rats using double label immunocytochemistry. NR1 expression on GnRH cell bodies varied with age and reproductive status, with 30%, 19%, and 46% of GnRH somata double labeled with NR1 in young proestrous, middle-aged proestrous, and middle-aged persistent estrous rats, respectively. Thus, 1) the expression of hypothalamic NR subunit mRNAs correlates with reproductive status; 2) changes in NR subunit mRNA levels, if reflected by changes in protein levels, may result in alterations in the stoichiometry of the NR during aging, with possible physiological consequences; 3) the effects of NR activation on GnRH mRNA switches from stimulatory to inhibitory during reproductive aging; and 4) expression of the NR1 subunit on GnRH perikarya changes with reproductive status. These molecular, physiological, and cellular neuroendocrine changes are proposed to be involved in the transition to acyclicity in aging female rats.
p38 Mitogen-Activated Protein Kinase Mediates Tumor Necrosis Factor-α-Induced Apoptosis in Rat Fetal Brown Adipocytes*
Authors: Valladares Amparo , Álvarez Alberto M. , Ventura Juan José , Roncero Cesar , Benito Manuel , Porras Almudena
Published: January 2014

Tumor necrosis factor-α (TNFα) induces apoptosis and cell growth inhibition in primary rat fetal brown adipocytes. Here, we examine the role played by some members of the mitogen-activated protein kinase (MAPK) superfamily. TNFα activates extracellular regulated kinase-1/2 (ERK1/2) and p38MAPK. Inhibition of p38MAPK by either SB203580 or SB202190 highly reduces apoptosis induced by TNFα, whereas ERK inhibition potentiates it. Moreover, cotransfection of an active MKK3 mutant and p38MAPK induces apoptosis. p38MAPK inhibition also prevents TNFα-induced cell cycle arrest, whereas MEK1 inhibition enhances this effect, which correlates with changes in proliferating cell nuclear antigen expression, but not in cyclin D1.

c-Jun and activating transcription factor-1 are potential downstream effectors of p38MAPK and ERKs upon TNFα treatment. Thus, TNFα-induced c-Jun messenger RNA expression requires ERKs activation, whereas p38MAPK inhibition enhances its expression. In addition, TNFα-induced activating transcription factor-1 phosphorylation is extensively decreased by SB203580. However, TNFα- induced NF-κB DNA-binding activity is independent of p38MAPK and ERK activation. On the other hand, C/EBP homology protein does not appear to mediate the actions of TNFα, because its expression is almost undetectable and even reduced by TNFα.

Finally, although TNFα induces c-Jun N-terminal kinase (JNK) activation, transfection of a dominant negative of either JNK1 or JNK2 had no effect on TNFα-induced apoptosis. These results suggest that p38MAPK mediates TNFα-induced apoptosis and cell cycle arrest, whereas ERKs do the opposite, and JNKs play no role in this process of apoptosis.
Opposite Effects of Androgens and Estrogens on Adipogenesis in Rat Preadipocytes: Evidence for Sex and Site-Related Specificities and Possible Involvement of Insulin-Like Growth Factor 1 Receptor and Peroxisome Proliferator-Activated Receptorγ 21
Authors: Dieudonne M. N. , Pecquery R. , Leneveu M. C. , Giudicelli Y.
Published: January 2014

To investigate the role of sex steroid hormones in adipose tissue development and distribution, we have studied the effect of various sex steroids (testosterone, dihydrotestosterone (DHT), and 17β-estradiol) in vitro, on the proliferation and differentiation processes in rat preadipocytes from deep (epididymal and parametrial) and superficial (femoral sc) fat deposits. All added steroids failed to affect the growth rate of preadipocytes from male rats when determined from day 1 to day 4 after plating, whether FCS was present or not in the culture medium. In contrast, in preadipocytes from female rats, we observed a positive effect (×2) of 17β-estradiol (0.01μ m) on the proliferative capacities of sc but not parametrial preadipocytes. When preadipocytes were exposed to testosterone or DHT (0.1 μm) during the differentiation process, the glycerol 3-phosphate dehydrogenase activity was significantly decreased in epididymal preadipocytes only. When preadipocytes from male rats were exposed to 17β-estradiol (0.01μ m), the differentiation capacities of preadipocytes were not modified. However, in parametrial preadipocytes from ovariectomized female rats, 17β-estradiol significantly increased (×1.34) the glycerol 3-phosphate dehydrogenase activity. In differentiated preadipocytes that had been exposed to sex steroids, expression of peroxisome proliferator-activated receptor γ2 was up-regulated by 17β-estradiol but not by androgens. As described in other cell types, sex steroids modulate insulin growth factor 1 receptor (IGF1R) expression in preadipocytes. Indeed, IGF1R levels were either enhanced by 17 β-estradiol (0.01 μm) in sc preadipocytes from female ovariectomized rats or decreased by DHT (0.01 μm) in epididymal preadipocytes. These effects were reversed by simultaneous exposure to androgen or estrogen receptor antagonists. In conclusion, this study demonstrates that, in rat preadipocytes kept in primary culture and chronically exposed to sex hormones, androgens elicit an antiadipogenic effect, whereas estrogens behave as proadipogenic hormones. Moreover, our results suggest that these opposite effects could be related to changes in IGF1R (androgens and estrogens) and peroxisome proliferator-activated receptor γ2 expression (estrogens).
Immortalized Luteinizing Hormone-Releasing Hormone Neurons Show a Different Migratory Activity in Vitro*
Authors: Maggi Roberto , Pimpinelli Federica , Molteni Laura , Milani Mario , Martini Luciano , Piva Flavio
Published: January 2014

The development of two cell lines (GT1 and GN) of immortalized LHRH neurons has allowed an accurate study of the mechanisms controlling the synthesis and the secretion of LHRH. These cell lines, obtained in mice by genetic targeted tumorigenesis, retain many of the phenotypic characteristics of LHRH neurons. Of interest, GT1 cells derive from an hypothalamic tumor, whereas GN cells were obtained from a tumor localized in the olfactory bulb. The different origin of these cell lines lead to hypothesize that they might represent hypothalamic postmigratory neurons (GT1 cells), or LHRH neurons blocked at an early stage of their migration (GN cells). Using different experimental procedures, we found that the two cell subclones GT1–7 and GN11 express a different morphology and migratory behavior in vitro. In particular, we found that GN11 cells, but not GT1–7 cells, show the morphological shape of migrating neurons. When analyzing the spontaneous motility we found that only GN11 cells express a high capacity of migrating in a matrix of collagen gel. Moreover, in a chemomigratory assay GN11 cells did show a significant response to the chemotactic stimulus represented by the FBS. On the contrary, GT1–7 cells show very low spontaneous motility and appear insensitive to the FBS stimulus. These results suggest that the simultaneous use of the GT1–7/GN11 cells may represent an experimental tool for screening the factors possibly involved in the control of the migratory processes of LHRH neurons in normal and in pathological conditions, such as those due to their impaired migration, like it happens in Kallmann’s syndrome.
Transforming Growth Factor-β Stimulates Inorganic Phosphate Transport and Expression of the Type III Phosphate Transporter Glvr-1 in Chondrogenic ATDC5 Cells*
Authors: Palmer Gaby , Guicheux Jerôme , Bonjour Jean-Philippe , Caverzasio Joseph
Published: January 2014

Members of the transforming growth factor (TGF)-β family are important regulators of skeletal development. In this study, we investigated the effect of TGF-β1 on inorganic phosphate (Pi) transport and on expression of the type III Pi carriers Glvr-1 and Ram-1 in murine ATDC5 chondrocytes. TGF-β1 induced a selective, dose- and time-dependent increase in sodium-dependent Pi transport in ATDC5 cells. This response was dependent on RNA and protein synthesis and reflected a change in the maximal rate of the transport system, suggesting that TGF-β1 induces the synthesis of new Pi carriers and their insertion into the plasma membrane. Consistently, Northern blotting analysis showed a dose-dependent increase in Glvr-1 messenger RNA expression in response to TGF-β1, which preceded the maximal stimulation of Pi transport by several hours. Glvr-1 thus likely mediates at least part of the increase in Pi uptake induced by TGF-β1. Ram-1 messenger RNA expression was not affected by TGF-β1. TGF-β1 activated the Smad signaling pathway and the mitogen-activated protein kinases ERK and p38 in ATDC5 cells. Unlike the regulation of Pi transport by receptor tyrosine kinase agonists in osteoblasts, the effect of TGF-β1 on Pi uptake in ATDC5 cells did not involve protein kinase C or mitogen-activated protein kinases, suggesting that a specific, possibly Smad-dependent, signal mediates this response. In conclusion, TGF-β1 stimulates Pi transport and Glvr-1 expression in chondrocytes, suggesting that, like proliferation, differentiation, and matrix synthesis, Pi handling is subject to regulation by TGF-β family members in bone-forming cells.
Thyroid Hormone Regulation of Phospholamban Phosphorylation in the Rat Heart*
Authors: Ojamaa Kaie , Kenessey Agnes , Klein Irwin
Published: January 2014

Thyroid hormone exerts predictable effects on the contractile performance of the heart in part by regulating the transcription of genes encoding specific calcium transporter proteins. In a rat model of hypothyroidism, left ventricular (LV) contractile function as measured by ejection fraction was decreased by 22% (P < 0.05), and this was returned to control values with T3 treatment. In confirmation of prior studies, LV phospholamban (PLB) protein content was significantly decreased by 25% and 40% compared with hypothyroid LV when the animals were treated with T3 at two doses, 2.5 and 7.0 μg/day, respectively. The ratio of sarcoplasmic reticulum calcium adenosine triphosphatase (SERCA2) to PLB protein content was thus increased by 171% and 207%, respectively (P < 0.01). Resolution of the phosphorylated PLB pentamers by SDS-PAGE showed that T3 infusion at 2.5 and 7.0 μg/day decreased (P < 0.001) the amount nonphosphorylated pentamers by 82% and 95%, respectively, in a dose-dependent manner. T3 treatment produced an increase in the proportion of highly phosphorylated PLB pentamers (more than five phosphates) when expressed as a fraction of total pentameric molecules (P < 0.05). Site-specific antibodies showed that the T3-induced increase in phosphorylated PLB pentamers was the result of an increase in both serine 16 and threonine 17 phosphorylation. We conclude that thyroid hormone, in addition to regulating the expression of cardiac PLB, is able to alter the degree of PLB phosphorylation, which correlates with enhancement of LV contractile function. These studies suggest that T3 may augment myocyte calcium cycling via changes in both cAMP- and calcium/calmodulin-dependent protein kinase activities.
Cell Type-Specific Metabolism of Peptidylglycineα -Amidating Monooxygenase in Anterior Pituitary*
Authors: El Meskini Rajaa , Mains Richard E. , Eipper Betty A.
Published: January 2014

Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme expressed in each major anterior pituitary cell type. We used primary cultures of adult male rat anterior pituitary to examine PAM expression, processing, and secretion in the different pituitary cell types and to compare these patterns to those observed in transfected AtT-20 corticotrope tumor cells. Immunostaining and subcellular fractionation identified PAM in pituitary secretory granules and additional vesicular compartments; in contrast, in AtT-20 cells, transfected PAM was primarily localized to the trans-Golgi network. PAM expression was highest in gonadotropes, with moderate levels in somatotropes and thyrotropes and lower levels in corticotropes and lactotropes. Under basal conditions, less than 1% of the cell content of monooxygenase activity was secreted per h, a rate comparable to the basal rate of release of individual pituitary hormones. General secretagogues stimulated PAM secretion 3- to 5-fold. Stimulation with specific hypothalamic releasing hormones demonstrated that different pituitary cell types secrete characteristic sets of PAM proteins. Gonadotropes and thyrotropes release primarily monofunctional monooxygenase. Somatotropes secrete primarily bifunctional PAM, whereas corticotropes secrete a mixture of mono- and bifunctional proteins. As observed in transfected AtT-20 cells, pituitary cells rapidly internalize the PAM/PAM-antibody complex from the cell surface. The distinctly different steady-state localizations of endogenous PAM in primary pituitary cells and transfected PAM in AtT-20 cell lines may simply reflect the increased storage capacity of primary pituitary cells.
Establishment and Characterization of a Human Adrenocortical Carcinoma Xenograft Model*
Authors: Logié Armelle , Boudou Philippe , Boccon-Gibod Liliane , Baudin Eric , Vassal Gilles , Schlumberger Martin , Le Bouc Yves , Gicquel Christine
Published: January 2014

Adrenocortical carcinomas are rare malignant tumors. They have a poor prognosis, as they are often diagnosed late and are usually resistant to chemotherapy. The lack of a suitable animal model for these tumors has been a major obstacle to the evaluation of new therapeutic agents. The aim of this study was to establish and characterize xenografts of the human adrenocortical carcinoma NCI H295R cell line as a model of adrenocortical carcinoma for future therapeutic trials. This cell line was sc injected (6 × 106 cells) into nude mice (n = 20). Solid tumors were locally measurable after 45 days at 90% of the inoculation sites. The xenografts were similar histologically to the original adrenocortical carcinoma from which the cell line was derived. The xenografts precisely reproduced the dysregulation of the insulin-like growth factor (IGF) system[ overexpression of the IGF-II and IGF-binding protein-2 (IGFBP-2) genes] typical of adrenocortical carcinoma. Similarly to adrenocortical carcinomas, human IGFBP-2 (but not IGF-II) was secreted in mouse plasma. We analyzed steroid production (cortisol, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone,Δ 4-androstenedione, 11-deoxycortisol, corticosterone, and testosterone). Xenografts produced all three class of steroids, with the preferential production of androgens of the Δ4 pathway.

The H295R xenograft model is a good model of human adrenocortical carcinoma, as it mimics dysregulation of the IGF system usually found in these tumors. It also produces IGFBP-2 and steroids that can be used as tumor markers. This model may therefore be useful for evaluating therapeutic agents.
Anti-Müllerian Hormone Attenuates the Effects of FSH on Follicle Development in the Mouse Ovary
Authors: Durlinger Alexandra L. L. , Gruijters Maria J. G. , Kramer Piet , Karels Bas , Kumar T. Rajendra , Matzuk Martin M. , Rose Ursula M. , de Jong Frank H. , Uilenbroek Jan Th. J. , Grootegoed J. Anton , Themmen Axel P. N.
Published: January 2014

Although ovarian follicle growth is under the influence of many growth factors and hormones of which FSH remains one of the most prominent regulators. Therefore, factors affecting the sensitivity of ovarian follicles to FSH are also important for follicle growth. The aim of the present study was to investigate whether anti-Müllerian hormone (AMH) has an inhibitory effect on follicle growth by decreasing the sensitivity of ovarian follicles to FSH. Furthermore, the combined action of AMH and FSH on ovarian follicle development was examined. Three different experiments were performed. Using an in vitro follicle culture system it was shown that FSH-stimulated preantral follicle growth is attenuated in the presence of AMH. This observation was confirmed by an in vivo experiment showing that in immature AMH-deficient females, more follicles start to grow under the influence of exogenous FSH than in their wild-type littermates. In a third experiment, examination of the follicle population of 4-month-old wild-type, FSHβ-, AMH-, and AMH-/FSHβ-deficient females revealed that loss of FSH expression has no impact on the number of primordial and preantral follicles, but the loss of inhibitory action of AMH on the recruitment of primordial follicles in AMH-deficient mice is increased in the absence of FSH. In conclusion, these studies show that AMH inhibits FSH-stimulated follicle growth in the mouse, suggesting that AMH is one of the factors determining the sensitivity of ovarian follicles for FSH and that AMH is a dominant regulator of early follicle growth.
Impaired Interaction of Mutant Thyroid Hormone Receptors Associated with Human Hepatocellular Carcinoma with Transcriptional Coregulators**This work was supported by grants from Chang-Gung University (CMRP 737, CMRP893, NMRP 407) and the National Science Council of the Republic of China (NSC 87-2316-B-182002).
Authors: Lin Kwang-huei , Wu Yi-hsin , chen shen-liang
Published: January 2014

Thyroid hormone (T3) exerts its many biological activities through interaction with specific nuclear receptors (TRs) that function as ligand-dependent transcription factors at genes that contain a thyroid hormone response element (TRE). Mutant TRs have been detected in human hepatocellular carcinoma cell lines and tissue, but their contribution to carcinogenesis has remained unclear. The interaction of four such mutant TRs (J7-TRα1, J7-TRβ1, H-TRα1, and l-TRα1) with transcriptional coregulators has now been investigated. With the exception of J7-TRα1, which in the absence of T3 exhibited transcriptional silencing activity with a TRE-reporter gene construct in transfected cells, the mutant TRs had little effect (compared with that of wild-type receptors) on transcriptional activity of the reporter gene in the absence or presence of T3, of the transcriptional corepressors SMRT, NCoR or of the transcriptional coactivator SRC. Electrophoretic mobility-shift assays revealed that, in the presence of T3, the J7-TRβ1 mutant did not interact with SRC, whereas J7-TRα1 and H-TRα1 exhibited reduced abilities to associate with this coactivator and l-TRα1 showed an ability to interact with SRC similar to that of wild-type TRα1. The dominant negative activity of the mutant TRs in transfected cells appeared inversely related to the ability of the receptors to interact with SRC. Whereas J7-TRβ1, H-TRα1, and l-TRα1 did not interact with SMRT, and NCoR. J7-TRα1 bind to corepressors but failed to dissociate from them in the presence of T3. These aberrant interactions between the mutant TRs and transcriptional coregulators may contribute to the highly variable clinical characteristics of human hepatocellular carcinoma.
The Expression of Thyrotropin Receptor in the Brain**This work was supported in part by a grant from the Faculté de Médecine de Bicêtre (Université Paris XI, Orsay, France) and Program Tournesol (Ministère des Affaires Etrangères, Paris, France).
Authors: Crisanti Patricia , Omri Boubaker , Hughes Eleanor J. , Meduri Geri , Hery Christiane , Clauser Eric , Jacquemin Claude , Saunier Bertrand
Published: January 2014

The regulation of the thyroid gland by TSH is mediated by a heterotrimeric G protein-coupled receptor. Nonthyroid effects of TSH have been reported, and expression of its receptor has been described in adipocytes and lymphocytes. We have previously reported the existence of specific and saturable binding sites of TSH and specific TSH effects in primary cultured rat brain astroglial cells. We now report expression of the TSH receptor gene in these cells; the coding sequence of the corresponding complementary DNA is identical to that previously established in thyroid. Using specific antisense RNA probe, expression of this gene was detected in some isolated or clustered glial fibrillary acidic protein-positive primary cultured cells by in situ hybridization. With this technique, we further detected TSH receptor messenger RNA (mRNA) expression in rat brain cryoslices in both neuronal cells and astrocytes. Its presence predominated in neuron-rich areas (pyriform and postcingulate cortex, hippocampus, and hypothalamic nuclei) and was mostly colocalized with neuron-specific enolase. In astrocytes, this mRNA was detected in the ependymal cell layer and the subependymal zone, and several isolated cells were also found in the brain parenchyma. We also detected TSH receptor mRNA and protein in primary cultured human astrocytes. The protein was detected as well in both rat and human brain cryoslices. Together, these findings clearly demonstrate the expression of the TSH receptor gene in the brain in both neuronal cells and astrocytes.
Pregnancy and Murine Thyroiditis: Thyroglobulin Immunization Leads to Fetal Loss in Specific Allogeneic Pregnancies**This work was supported in part by NIH Grants DK-52464, DK-35764, and DK-45011 (to T.F.D.), Cellular and Molecular Endocrinology Training Grant DK-07645 (to L.A.) and the David Owen Segal Endowment (to M.I. and M.K.).
Authors: Imaizumi M. , Pritsker A. , Kita M. , Ahmad L. , Unger P. , Davies T. F.
Published: January 2014

Thyroid autoantibodies are risk factors in human pregnancy. To investigate the influence of autoimmune thyroiditis on pregnancy, we have studied the impact of murine experimental autoimmune thyroiditis (EAT) on pregnancy outcome by using thyroglobulin (Tg) immunized CBA/J (H2k) female mice. When Tg immunized mice were mated with BALB/c (H2d) males, only 57% (47/83) of pregnant mice maintained their conceptions compared with >85% of other strain combinations (P < 0.05). We also found that MHC class II antigens were expressed on placental cells from Tg immunized pregnant mice but not in control normal pregnancies. Furthermore, the frequency and severity of thyroiditis, assessed by histological analyses, was also increased in Tg immunized mice mated with the BALB/c strain compared with syngeneic pregnancies (P < 0.05). In these pregnant mice mated with BALB/c, interleukin-4 secretion by mitogen-stimulated spleen cells was significantly suppressed and interferon-γ secretion by mixed lymphocyte reactions with BALB/c cells was significantly increased. These data demonstrated enhanced Th1 cell proliferation and fetal loss in CBA/J X BALB/c pregnancies. We concluded, therefore, that pregnancy loss was increased in experimental autoimmune thyroiditis in a manner that was dependent on paternal antigens. These observations have broad implications for understanding the immunology of pregnancy.
Targeted Protein Kinase A and PP-2B Regulate Insulin Secretion through Reversible Phosphorylation*
Authors: Lester Linda B. , Faux Maree C. , Nauert J. Brian , Scott John D.
Published: January 2014

Protein kinases and phosphatases play key roles in integrating signals from various insulin secretagogues. In this study, we show that the activities of the cAMP-dependent protein kinase (PKA) and the calcium/calmodulin-dependent phosphatase, PP-2B are coordinated resulting in the regulation of insulin secretion. Transient inhibition of PP-2B, using the immunosuppressant FK506, increased forskolin stimulated insulin secretion by 2.5-fold ± 0.3 (n = 6) in rat islets and RINm5F cells. Surprisingly, forskolin treatment resulted in the dephosphorylation of the vesicle-associated protein synapsin 1 and increased PP-2B activity by 2.98 ± 0.97-fold (n = 4). One potential explanation for the observed coordination of PKA and PP-2B activity is their colocalization through a mutual anchoring protein, AKAP79/150. Accordingly, RINm5F cells expressing AKAP79 exhibited decreased insulin secretion, reduced PP-2B activity and were insensitive to FK506. This suggests that AKAP targeting of PKA and PP-2B maintains a signal transduction complex that may regulate reversible phosphorylation events involved in insulin secretion.
Selective Delivery of Estradiol to Bone by Aspartic Acid Oligopeptide and Its Effects on Ovariectomized Mice
Authors: Yokogawa Koichi , Miya Kazuhiro , Sekido Tohru , Higashi Yasuhiko , Nomura Masaaki , Fujisawa Ryuichi , Morito Keiko , Masamune Yukito , Waki Yoshihiro , Kasugai Shohei , Miyamoto Ken-ichi
Published: January 2014

We have developed a novel osteotropic prodrug of estradiol (E2) conjugated with l-Asp-hexapeptide (E2·3D6), which has very low affinity for estrogen receptors, and in this study, we examined its pharmacokinetic behavior and pharmacological potential. After a single iv injection of E2·3D6 to mice, the half-time for elimination from plasma was about 100 min; however, E2 was selectively delivered to the bone and eliminated very slowly, declining to the endogenous level at about 7 days. After a single iv injection of E2, the half-time in plasma was about 70 min, whereas E2 was highly distributed to the uterus, and the bone concentration of E2 was only slightly increased at 6 h. When E2 (0.37 μmol/kg, sc, every third day) or E2·3D6 (0.11 to 1.1 μmol/kg, sc, every seventh day) was administered to OVX mice for 4 weeks, E2 increased the bone mineral density (BMD) together with weights of liver and uterus, whereas E2·3D6 increased only the BMD, in a dose-dependent manner. E2·3D6 enhanced the expression of messenger RNAs of bone matrix proteins (osteopontin, bone sialoprotein, type I collagen α) of OVX mice at 4 h after administration, but E2 did very slightly. These results indicate that the E2 prodrug was delivered to the bone, where it gradually released E2, thereby ameliorating bone loss. This acidic oligopeptide appears to be a good candidate for selective drug delivery to bone.
Comprehensive Messenger Ribonucleic Acid Profiling Reveals That Peroxisome Proliferator-Activated Receptor γ Activation Has Coordinate Effects on Gene Expression in Multiple Insulin-Sensitive Tissues
Authors: Way James M. , Harrington W. Wallace , Brown Kathleen K. , Gottschalk William K. , Sundseth Scott S. , Mansfield Traci A. , Ramachandran Ravi K. , Willson Timothy M. , Kliewer Steven A.
Published: January 2014

Peroxisome proliferator-activated receptor γ (PPARγ) agonists, including the glitazone class of drugs, are insulin sensitizers that reduce glucose and lipid levels in patients with type 2 diabetes mellitus. To more fully understand the molecular mechanisms underlying their therapeutic actions, we have characterized the effects of the potent, tyrosine-based PPARγ ligand GW1929 on serum glucose and lipid parameters and gene expression in Zucker diabetic fatty rats. In time-course studies, GW1929 treatment decreased circulating FFA levels before reducing glucose and triglyceride levels. We used a comprehensive and unbiased messenger RNA profiling technique to identify genes regulated either directly or indirectly by PPARγ in epididymal white adipose tissue, interscapular brown adipose tissue, liver, and soleus skeletal muscle. PPARγ activation stimulated the expression of a large number of genes involved in lipogenesis and fatty acid metabolism in both white adipose tissue and brown adipose tissue. In muscle, PPARγ agonist treatment decreased the expression of pyruvate dehydrogenase kinase 4, which represses oxidative glucose metabolism, and also decreased the expression of genes involved in fatty acid transport and oxidation. These changes suggest a molecular basis for PPARγ-mediated increases in glucose utilization in muscle. In liver, PPARγ activation coordinately decreased the expression of genes involved in gluconeogenesis. We conclude from these studies that the antidiabetic actions of PPARγ agonists are probably the consequence of 1) their effects on FFA levels, and 2), their coordinate effects on gene expression in multiple insulin-sensitive tissues.
Adenosine Triphosphate Activates Mitogen-Activated Protein Kinase in Human Granulosa-Luteal Cells*
Authors: Tai Chen-Jei , Kang Sung Keun , Tzeng Chii-Ruey , Leung Peter C. K.
Published: January 2014

ATP has been shown to activate the phospholipase C/diacylglycerol/protein kinase C (PKC) pathway. However, little is known about the downstream signaling events. The present study was designed to examine the effect of ATP on activation of the mitogen-activated protein kinase (MAPK) signaling pathway and its physiological role in human granulosa-luteal cells. Western blot analysis, using a monoclonal antibody that detected the phosphorylated forms of extracellular signal-regulated kinase-1 and -2 (p42mapk and p44 mapk, respectively), demonstrated that ATP activated MAPK in a dose- and time-dependent manner. Treatment of the cells with suramin (a P2 purinoceptor antagonist), neomycin (a phospholipase C inhibitor), staurosporin (a PKC inhibitor), or PD98059 (an MAPK/ERK kinase inhibitor) significantly attenuated the ATP-induced activation of MAPK. In contrast, ATP-induced MAPK activation was not significantly affected by pertussis toxin (a Gi inhibitor). To examine the role of Gs protein, the intracellular cAMP level was determined after treatment with ATP or hCG. No significant elevation of intracellular cAMP was noted after ATP treatment. To determine the role of MAPK in steroidogenesis, human granulosa-luteal cells were treated with ATP, hCG, or ATP plus hCG in the presence or absence of PD98059. RIA revealed that ATP alone did not significantly affect the basal progesterone concentration. However, hCG-induced progesterone production was reduced by ATP treatment. PD98059 reversed the inhibitory effect of ATP on hCG-induced progesterone production. To our knowledge, this is the first demonstration of ATP-induced activation of the MAPK signaling pathway in the human ovary. These results support the idea that the MAPK signaling pathway is involved in mediating ATP actions in the human ovary.
Cytokine-Activated Endothelium Recruits Osteoclast Precursors
Authors: McGowan Neil W. A. , Walker Emily J. , Macpherson Heather , Ralston Stuart H. , Helfrich Miep H.
Published: January 2014

Osteoclast precursors reach sites of osteoclast formation and remodelling via the vasculature and are therefore destined to encounter endothelium before migrating to the bone surface. Here we investigated the hypothesis that endothelium may be involved in the regulation of osteoclast precursor recruitment to sites of bone resorption. Osteoclast precursors in human peripheral blood were identified by their ability to form mature osteoclasts in 21-day cultures supplemented with RANKLigand, M-CSF, 1,25(OH)2-vitamin D3, dexamethasone and prostaglandin E2. Under control conditions few osteoclast precursors adhered to endothelial cells (the human bone marrow-derived endothelial cell line BMEC-1). However, BMEC-1 cells treated with the resorption stimulating cytokines IL-1β and TNFα depleted the PBMC population of all osteoclast precursors. These results provide the first evidence that osteoclast precursors can adhere to endothelium and suggest that endothelium could play an important role in the recruitment of osteoclast precursors to sites of bone resorption.
Is Cadmium Chloride-Induced Inter-Sertoli Tight Junction Permeability Barrier Disruption a Suitable in Vitro Model to Study the Events of Junction Disassembly during Spermatogenesis in the Rat Testis?*
Authors: Chung Nancy P. Y. , Cheng C. Yan
Published: January 2014

The events of germ cell movement during spermatogenesis are composed of intermittent phases of junction disassembly and reassembly. Although primary Sertoli cells cultured in vitro can be used to study junction reassembly, an in vitro model to study the events of junction disassembly is still lacking. We have assessed whether the CdCl2-induced inter-Sertoli tight junction (TJ) permeability barrier disruption in vitro can fill this gap. When Sertoli cells (1.2 × 106 cells/cm2) were cultured on Matrigel-coated bicameral units to allow the assembly of inter-Sertoli TJs, it was manifested by a steady rise in transepithelial electrical resistance across the Sertoli cell epithelia. Exposure of these cells on day 1 (i.e. 24 h after their isolation) to CdCl2 at 5–10μ m for 8 h could perturb the inter-Sertoli TJ assembly dose dependently without any apparent cytotoxicity. Likewise, when cells were exposed to CdCl2 (0.1–5 μm) on day 4 for 8 h after inter-Sertoli TJs were already assembled, CdCl2 also perturbed the maintenance of inter-Sertoli TJ permeability barrier dose dependently without signs of cell cytotoxicity. Although the perturbed inter-Sertoli TJs were not capable of resealing even after the removal of CdCl2, the presence of testosterone (T) at 1 × 10−9m allowed resealing of the inter-Sertoli TJ barrier after CdCl2 was removed, whereas the presence of 2 × 10−7m testosterone even protected Sertoli cells from CdCl2-induced damage. More important, the reassembly of inter-Sertoli TJs after CdCl2-induced TJ disruption was accompanied by changes in cellular gene expression of occludin and urokinase plasminogen activator, which mimicked their patterns during inter- Sertoli TJ assembly in vitro without CdCl2 treatment. Based on these results, it is apparent that CdCl2-induced inter-Sertoli TJ disassembly is a potential in vitro model to study the events of junction disassembly.
Phenotypic Manifestations of Insulin-Like Growth Factor-Binding Protein-3 Overexpression in Transgenic Mice*
Authors: Modric Tomislav , Silha Josef V. , Shi Zengdun , Gui Yaoting , Suwanichkul Adisak , Durham Susan K. , Powell David R. , Murphy Liam J.
Published: January 2014

In cell culture systems insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) can both enhance and inhibit IGF-I action. To investigate the biological role of IGFBP-3 in vivo, transgenic (Tg) mice that constitutively overexpress the human IGFBP-3 complementary DNA (cDNA) driven by the mouse phosphoglycerate kinase I (PGK) and the cytomegalovirus (CMV) promoters were examined. Serum levels of human IGFBP-3 in CMVBP-3 and PGKBP-3 Tg mice were 4.7 and 5.8μ g/ml, respectively and total IGFBP-3 was increased 4.9- and 7.7-fold compared with that in wild-type (Wt) mice. In PGKBP-3 Tg mice the levels of transgene expression were similar in all tissues. Although CMVBP-3 mice demonstrated similar levels of expression of the transgene as PGKBP-3 mice in most tissues, markedly elevated expression was apparent in the kidney and heart. The transgene-derived IGFBP-3 circulated as a 150-kDa ternary complex, and serum IGF-I levels were elevated 1.9- to 2.8-fold in Tg mice compared with Wt mice. A significant reduction in birth weight of approximately 10% and a modest reduction in litter size were apparent in both Tg strains. Early postnatal growth, as assessed by both body weight and length, was significantly reduced in Tg mice compared with Wt mice. This was more marked in PGKBP-3 than in CMVBP-3 mice, who demonstrated a propensity to adiposity after weaning. The relative organ weights of brain and kidney were reduced in both Tg strains, whereas liver size and epididymal fat were significantly increased in CMVBP-3, but not PGKBP-3, mice. Our data indicate that overexpression of IGFBP-3 is associated with modest intrauterine and postnatal growth retardation despite elevated circulating IGF-I levels.
Isolation and Characterization of a Novel Member of the Relaxin/Insulin Family from the Testis of the Frog Rana esculenta*
Authors: de Rienzo Gianluca , Aniello Francesco , Branno Margherita , Minucci Sergio
Published: January 2014

A complementary DNA (cDNA) encoding a frog relaxin/insulin member family (fRLX) from testis cDNA library was isolated and characterized. The fRLX cDNA predicted a 155-amino acid protein with a low homology to mammalian RLF and relaxin. Northern blot analysis revealed a single transcript expressed in the interstitial compartment, RT-PCR, evidenced that fRLX is expressed at low levels in the oviduct and ovary too. The predicted mature fRLX protein, composed of the signal peptide, B, C, and A domains, has conserved amino acid sequences in the characteristic functional domains. A different expression of the transcript was found during the frog reproductive cycle, with a peak in Spring. After administration of ethane dimethane sulfonate, by in situ hybridization, fRLX messenger RNA disappeared from the interstitial compartment and reappeared again at the time of generating of a new population of Leydig cells (LC), strongly indicating that LC are the interstitial cell type expressing fRLX. Preliminary results obtained by in situ hybridization, performed on testis of hypophysectomized frogs evidenced a pituitary control of fRLX expression. This study is the first cloning of a relaxin/insulin family member in a nonmammalian vertebrate. In addition, because fRLX expression changes during the annual cycle suggesting its involvement in spermatogenesis, fRLX may be considered a new marker for the study of spermatogenesis in the Rana esculenta.