bluuebunny/crossref_metadata_2025 huggingface dataset:
Blockade of Growth Hormone Receptor Shedding by a Metalloprotease Inhibitor*
Authors: Alele Jimmy , Jiang Jing , Goldsmith Jeffrey F. , Yang Xiaoyong , Maheshwari Hiralal G. , Black Roy A. , Baumann Gerhard , Frank Stuart J.
Published: January 2014

GH, an important growth-promoting and metabolic hormone, exerts its biological effects by interacting with cell surface GH receptors (GHRs). The GHR is a single membrane-spanning protein that binds GH via its extracellular domain. The high affinity GH-binding protein (GHBP), which corresponds to a soluble form of the GHR extracellular domain, carries a substantial fraction of the GH in the circulation of various species and probably has a role in modulation of the hormone’s bioavailability. Although in rodents, it is believed that the GHBP is largely derived by translation of an alternatively spliced GHR messenger RNA, in humans and rabbits, proteolytic cleavage of the membrane-anchored receptor releases the GHR extracellular domain, which is believed to thereby become the GHBP. In this study, we used human IM-9 lymphocytes and GHR antibodies to study this proteolytic shedding of the GHBP. As determined by immunoblotting with anti-GHR cytoplasmic domain serum, addition of phorbol 12-myristate 13-acetate (PMA; 1μ g/ml) to serum-starved cells led to rapid loss (roughly 60% decline after 1 h; t1/2 = ∼5 min) of mature GHRs (115–140 kDa) from either total cell or detergent-soluble extracts. Loss of full-length GHRs was accompanied by accumulation of four proteins (65–68 kDa), each reactive with the cytoplasmically directed antiserum. The pattern of appearance of these GHR ctyoplasmic domain proteins, the electrophoretic and immunological characteristics of which are similar to those of a recombinant rabbit GHR mutant that lacks the extracellular domain, was such that progressively faster migrating forms were evident between 5–60 min of PMA exposure. Treatment with N-ethylmaleimide (NEM; 5 mm), an agent known to cause GHBP shedding from IM-9 cells, promoted a similar rapid loss of full-length GHRs and an accumulation of GHR cytoplasmic domain remnant proteins. PMA-induced, but not NEM-induced, GHR proteolysis was blocked by the protein kinase C inhibitor, GF109203X. Both PMA- and NEM-induced receptor proteolysis were, however, inhibited by the metalloprotease inhibitor, Immunex Compound 3 (minimum effective concentration, 10 μm). Notably, PMA and NEM also promoted shedding of GHBP into the conditioned medium of the cells, as determined by a chromatographic [125I]human GH binding assay; this GHBP shedding was also inhibited by Immunex Compound 3. These results strongly implicate a member(s) of the metalloprotease family as a potential GHBP-generating enzyme.
Penetration of Dexamethasone into Brain Glucocorticoid Targets Is Enhanced in mdr1A P-Glycoprotein Knockout Mice*
Authors: Meijer O. C. , de Lange E. C. M. , Breimer D. D. , de Boer A. G. , Workel J. O. , de Kloet E. R.
Published: January 2014

Mice with a genetic disruption of the multiple drug resistance (mdr1a) gene were used to examine the effect of the absence of its drug-transporting P-glycoprotein product from the blood-brain barrier on the distribution and cell nuclear uptake of[ 3H]-dexamethasone in the brain.[ 3H]-dexamethasone (4 μg/kg mouse) was administered sc to adrenalectomized mdr1a (−/−) and mdr1a (+/+) mice. One hour later, the mice were decapitated, and the radioactivity was measured in homogenates of cerebellum, blood, and liver following extraction of the radioactive steroid. The frontal brain was cut in sections for autoradiography.

In the cerebellum of the mdr1a mutants, the amount of[ 3H]-dexamethasone relative to blood was about 5-fold higher than observed in the controls, whereas the ratio in blood vs. liver was not different. Using autoradiography, it was found that brain areas expressing the glucocorticoid receptor (GR) in high abundance, such as the hippocampal cell fields and the paraventricular nucleus (PVN), showed a 10-fold increase in cell nuclear uptake of radiolabeled steroid. The amount of retained steroid increased toward levels observed in the pituitary, which contains a similar density of GRs. The [3H]-dexamethasone concentration in pituitary was not affected by mdr1a gene disruption. The GR messenger RNA expression pattern in hippocampus was not different between the wild types and mdr1a mutants, which rules out altered receptor expression as a cause of the enhanced dexamethasone uptake.

In conclusion, the present study demonstrates that the brain is resistant to penetration by dexamethasone because of mdr1a activity at the level of the blood-brain barrier. The data support the concept of a pituitary site of action of dexamethasone in blockade of stress-induced ACTH release. Dexamethasone poorly substitutes for depletion of the endogenous glucocorticoid from the brain and therefore, in this tissue, may cause a condition resembling that of adrenalectomy.
Insulin-Like Growth Factor Binding Protein -2 and -4 Messenger Ribonucleic Acid Expression in Bovine Ovarian Follicles: Effect of Gonadotropins and Developmental Status*
Authors: Armstrong D. G. , Baxter G. , Gutierrez C. G. , Hogg C. O. , Glazyrin A. L. , Campbell B. K. , Bramley T. A. , Webb R.
Published: January 2014

This work is concerned with the role of insulin-like growth factor binding protein (IGFBP)-2 and -4 in the regulation of IGF bioactivity in bovine follicles during the development of dominance. We measured the expression of IGFBP-2 and -4 messenger RNA (mRNA) in small (1–4 mm) gonadotropin-sensitive follicles and medium (4–8 mm) and large (>8 mm) gonadotropin-dependent follicles using in situ hybridization. In healthy nonatretic bovine follicles, IGFBP-2 and -4 mRNA expression was confined to granulosa and theca tissue, respectively. Moreover, during the development of follicular atresia, there were distinct changes in the temporal and spatial expression of these genes. IGFBP-2 immunoactivity was localized in granulosa tissue and the basement membrane of healthy preantral follicles, whereas IGFBP-4 immunoactivity was localized in both theca and granulosa tissue. Of particular interest was the lack of IGFBP-2 mRNA expression in large (>8 mm) gonadotropin-dependent follicles, an observation that was confirmed by the lack of immunoreactive IGFBP-2 in these follicles. The regulation of IGFBP-2 and -4 mRNA expression in granulosa and theca cells was analyzed using a serum-free cell culture system. FSH inhibited the expression of IGFBP-2 mRNA in granulosa cells, whereas LH stimulated IGFBP-4 mRNA expression in theca cells. Our results provide evidence for the existence of different roles for IGFBP-2 and -4 in the developing follicle.
Effect of Twenty-Four-Week Treatment with the Antiestrogen EM-800 on Estrogen-Sensitive Parameters in Intact and Ovariectomized Mice
Authors: Luo Shouqi , Sourla Antigone , Labrie Claude , Gauthier Sylvain , Merand Yves , Belanger Alain , Labrie Fernand
Published: January 2014

Treatment with the antiestrogen EM-800, at the daily oral dose of 3μ g, 10 μg, 30 μg, or 100 μg for 24 weeks, caused a marked inhibition of uterine and vaginal weight in both intact and ovariectomized mice. Maximal 64% and 41% inhibitions of uterine weight were achieved in intact and ovariectomized animals, respectively. Similar inhibitory effects of EM-800 were observed on vaginal weight with maximal inhibitions of 71% and 35%, in intact and ovariectomized animals, respectively. The pure antiestrogenic activity of EM-800 on the hypothalamo-pituitary-ovarian axis is illustrated by the 76–91% increases in ovarian weight observed in intact animals treated with the 10–100 μg doses of the antiestrogen. Serum 17β-estradiol was 93% increased at the 100 μg daily dose of EM-800, whereas serum androstenedione, testosterone, and dihydrotestosterone were 141–713% increased over control at the same dose of the antiestrogen. Serum LH was increased by treatment with EM-800 in intact animals, whereas no effect was observed on the elevated gonadotropin levels in ovariectomized animals. At all doses used in intact animals, the antiestrogen caused a complete disappearance of the glandular elements of the mammary gland, the atrophy being comparable with that observed in ovariectomized mice. The mammary gland of EM-800-treated animals was exclusively composed of an atrophied ductal system lined by atrophied epithelial cells with an absence of lobulo-glandular elements. No effect of the compound was observed on the histology of the mammary gland in ovariectomized animals, thus showing the pure antiestrogenic effect of EM-800 on the mammary gland, as shown also for the uterus, vagina, and hypothalamo-pituitary axis. At histopathology, all doses of EM-800 in intact animals led to a moderate to severe uterine and vaginal atrophy. The uterine atrophy affected both the myometrium and the endometrium. Interestingly, the uterine atrophy achieved in intact animals treated with EM-800 was greater than that observed after ovariectomy alone, thus clearly demonstrating the pure antiestrogenic activity of EM-800. The present data show the highly potent and pure antiestrogenic activity of EM-800 on all parameters measured after 6 months of treatment in both intact and ovariectomized mice, a maximal effect being reached at the daily 10 μg dose of the antiestrogen in intact animals.
Glucose Metabolism in Goto-Kakizaki Rat Islets*
Authors: Zong-Chao Ling , Efendic Suad , Wibom Rolf , Abdel-Halim Samy M. , Östenson Claes-Göran , Landau Bernard R. , Khan Akhtar
Published: January 2014

Islets from Goto-Kakizaki (GK) rats from our colony, despite marked impairment of glucose-induced insulin release, used glucose and produced CO2 at a rate 3 times that of islets from control Wistar rats. Almost all glucose used was accounted for in CO2 and lactate production. The percentages of glucose carbon used collected in CO2 and lactate were similar for control and GK islets. GK islets also oxidized 40% more acetate and leucine to CO2 than did control islets. The fraction of carbon leaving the Krebs cycle relative to CO2 production was the same in GK and control islets. The capacities of mitochondria from GK islets to generate ATP from glutamate and malate were similar and that to generate ATP from succinate and rotenone was somewhat less from GK islets. The reason for the enhanced utilization of substrates by islets of the GK rat is not apparent. In conclusion, there is no decrease in islet glucose utilization, glucose oxidation, Krebs cycle function, or the electron transport system evident from these measurements to explain the impaired insulin release in islets from GK rats.
Obesity and Insulin Resistance in Human Growth Hormone Transgenic Rats*
Authors: Ikeda Akihiro , Chang Kyu-Tae , Matsumoto Yoshiki , Furuhata Yasufumi , Nishihara Masugi , Sasaki Fumihiko , Takahashi Michio
Published: January 2014

A line of transgenic rats (heterozygotes) carrying a chimeric gene comprising a regulatory portion of murine whey acidic protein and a structural portion of human GH (hGH) genes developed severe obesity with age. To characterize physiological mechanisms that lead to fat accumulation, an array of parameters related to obesity were studied. Blood hGH levels were continuously low, endogenous rat GH secretion was suppressed, and the pulsatility in peripheral GH levels was absent. Plasma glucose, insulin, triglyceride, and FFA levels in the male transgenic rats significantly exceeded those in nontransgenic littermates at 12 and 17 weeks, but not at 7 weeks, of age. All symptoms except hyperlipidemia were restored to normal by treatment with an antidiabetic agent, thiazolidinedione (troglitazone), for 1 week from 17 weeks of age. As phenotypic expression of obesity was already evident before aberration of physiological parameters, it was assumed that animals had a condition in which obesity or hyperlipidemia caused hyperinsulinemia. Gene expression and enzymatic activity of lipoprotein lipase in the adipose tissue in the transgenic rats were not different from those in normal rats. In contrast, the gene expression level of glycerol-3-phosphodehydrogenase was markedly elevated, suggesting that glycerol synthesis was much enhanced in the adipocytes of the transgenic rats. In an ip glucose tolerance test, the transgenic rats were not hyperglycemic at 7 weeks of age; however, the animal became hyperglycemic at 15–17 weeks of age. Finally, treatment with recombinant hGH for 1 week to produce pulsatile secretion reduced the size of epididymal and kidney fat pads and restored normal weight gain. These observations suggest that continuously low peripheral GH levels with the lack of pulsatile secretion resulted in obesity and noninsulin-dependent diabetes mellitus.
Dehydroepiandrosterone Decreases Serum Tumor Necrosis Factor-α and Restores Insulin Sensitivity: Independent Effect from Secondary Weight Reduction in Genetically Obese Zucker Fatty Rats*
Authors: Kimura Mari , Tanaka Shun-ichi , Yamada Yoshihiko , Kiuchi Yoshihiro , Yamakawa Tadashi , Sekihara Hisahiko
Published: January 2014

Dehydroepiandrosterone (DHEA) and its sulfate ester are the most abundant circulating adrenal steroids in humans. Administration of DHEA has been reported to have beneficial effects on obesity, hyperlipidemia, diabetes, and atherosclerosis in obese rodents, although its effects on insulin resistance have not been fully elucidated.

In this study, the effects of DHEA treatment on insulin sensitivity were investigated in genetically obese Zucker rats, an animal model of insulin resistance, using the euglycemic clamp technique. After 0.4% DHEA was administered for 10 days to female obese Zucker rats aged 16 weeks, body weight and plasma insulin decreased and glucose disposal rate (GDR), which was normally reduced in obese rats, rose significantly compared with age- and sex-matched control obese rats. On the other hand, although the pair-fed obese rats also showed levels of weight reduction similar to those of DHEA-treated rats, the increase in GDR of DHEA-treated rats was significantly greater than in pair-fed rats, suggesting a direct ameliorating effect of DHEA on insulin sensitivity of obese rats. Serum concentration of tumor necrosis factor (TNF)-α, one of cytokines causing insulin resistance, was also reduced significantly in DHEA-treated, but not in pair-fed obese rats. In conclusion, our results suggest that DHEA treatment reduces body weight and serum TNF-α independently, and that both may ameliorate insulin resistance in obese Zucker fatty rats.
Immunoexpression of Aquaporin-1 in the Efferent Ducts of the Rat and Marmoset Monkey during Development, Its Modulation by Estrogens, and Its Possible Role in Fluid Resorption*
Authors: Fisher Jane S. , Turner Katie J. , Fraser Hamish M. , Saunders Philippa T. K. , Brown Dennis , Sharpe Richard M.
Published: January 2014

Recent data suggest that estrogens play a role in regulating fluid resorption from the efferent ducts, though the biochemical mechanisms involved are unknown. The present study has used immunocytochemistry to localize a water channel protein, Aquaporin-1 (AQP-1), to the efferent ducts of male rats and marmoset monkeys from perinatal life through to adulthood and has then investigated its potential hormonal regulation in neonatal/peripubertal life, via administration of a GnRH antagonist (GnRHa) or diethylstilbestrol (DES) to rats. AQP-1 was immunoexpressed intensely in the apical brush border of the epithelium lining the efferent ducts at all ages studied, from late fetal life through puberty to adulthood. In the marmoset, but not the rat, AQP-1 was also expressed in the epithelium of the rete testis. Once the cell types within the efferent duct epithelium had differentiated, it was clear that only nonciliated cells of the rat localized AQP-1. When gonadotropin secretion was suppressed in rats by neonatal administration of GnRHa, immunoexpression of AQP-1 at age 18 and 25 days was virtually unchanged in intensity, though the efferent ducts were reduced in size. In contrast, when DES was administered neonatally to rats (up to day 12), immunoexpression of AQP-1 was reduced at day 10, virtually abolished at day 18, reduced markedly at day 25 and to a small extent at day 35; these findings were confirmed by Western blot analysis at day 18. The DES-induced decrease in immunoexpression of AQP-1 was accompanied by pronounced distension of the efferent ducts and rete, consistent with reduced fluid resorption. The epithelial cells of the efferent ducts in DES-treated rats were cuboidal rather than columnar in shape as in controls and were reduced significantly in height compared with controls at all ages through to adulthood. These findings suggest that estrogens may play a role in regulating fluid resorption from the efferent ducts during fetal/neonatal development and/or a role in the gross and functional development of the efferent ducts and rete testis. The present data also suggest that AQP-1 is one of the elements involved in the regulation of fluid resorption in the efferent ducts. The importance of fluid flow in fetal/neonatal development of the excurrent duct system of the male is also suggested by these observations.
Coordinate and Divergent Regulation of Corticotropin-Releasing Factor (CRF) and CRF-Binding Protein Expression in an Immortalized Amygdalar Neuronal Cell Line**Portions of this report were presented in abstract form at the 1997 and 1998 annual meetings of The Endocrine Society.
Authors: Mulchahey J. J. , Regmi A. , Sheriff S. , Balasubramaniam A. , Kasckow J. W.
Published: January 2014

CRF is a 41-amino acid neuropeptide best known for its hypophysiotropic actions. CRF is widely distributed in the central nervous system in areas beyond the hypothalamus. CRF-binding protein (CRF-BP) regulates the bioavailability of CRF, and knowledge of the regulation of CRF-BP synthesis is an integral component of understanding the actions of CRF. To better study the regulation of CRF and CRF-BP, primary amygdalar cultures were immortalized by transfection with the SV 40 large T antigen. A clonal line that expresses CRF immunoreactivity and messenger RNA was selected. The production of CRF peptide and message by this line is regulated in a manner indistinguishable from primary cultures. We also observed that the immortalized cells express CRF-BP immunoreactivity and messenger RNA. The expression of both CRF and CRF-BP is positively regulated by forskolin and interleukin-6. Unlike CRF, the expression of CRF-BP message and peptide was increased by phorbol 12-myristate 13-acetate or dexamethasone. These results demonstrate that the synthesis of CRF and CRF-BP in this clonal cell line may be regulated in parallel by some agents but not by others. These data also suggest that dexamethasone may decrease the biological availability of CRF in the amygdala by increasing the expression of CRF-BP, rather than by decreasing CRF expression.
Quantitative Analysis of Oxytocin and Vasopressin Messenger Ribonucleic Acids in Single Magnocellular Neurons Isolated from Supraoptic Nucleus of Rat Hypothalamus
Authors: Xi Dan , Kusano Kiyoshi , Gainer Harold
Published: January 2014

Oxytocin (OT) and vasopressin (VP) are peptide hormones that are derived from genes predominantly expressed in distinct magnocellular neurons in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Recent evidence suggests that some magnocellular neurons coexpress both peptides. Our qualitative RT-PCR experiments on single cells show that the majority of magnocellular neurons coexpress both peptide messenger RNAs (mRNAs) in varying amounts. Using a competitive RT-PCR method combined with a standard calibration curve, we quantitatively determined OT and VP mRNA in single magnocellular neurons from the normal female rat SON, with a detection sensitivity of less than 30 mRNA molecules/cell. We defined the phenotypes of the single magnocellular neurons according to their ratios of these two peptide mRNAs. Using this approach, we identified three major phenotypes: oxytocin neurons, where the average OT to VP mRNA ratio is about 256; vasopressin neurons, where the average VP to OT mRNA ratio is about 182; and one oxytocin/vasopressin coexisting neuron, where the OT/VP mRNA ratio is 2. Thus, there is some OT and VP mRNA coexpression in virtually all of the magnocellular neurons in supraoptic nuclei of hypothalamus. However, clear phenotypes are identifiable by considering quantitative as opposed to qualitative differences.
Amphiregulin Is Coordinately Expressed with Heparin-Binding Epidermal Growth Factor-Like Growth Factor in the Interstitial Smooth Muscle of the Human Prostate1
Authors: Adam Rosalyn M. , Borer Joseph G. , Williams B. Jill , Eastham James A. , Loughlin Kevin R. , Freeman Michael R.
Published: January 2014

Peptide growth factors have been proposed as mediators of smooth muscle-epithelial cell interactions in the human prostate; however, the identity of these molecules has not been established. In this study, we compared expression levels of messenger RNAs (mRNAs) encoding the epidermal growth factor (EGF) receptor-related receptor tyrosine kinases (ErbB1 through 4), the six EGF receptor ligands, EGF, transforming growth factor (TGF)-α, amphiregulin (ARG), HB-EGF, betacellulin, and epiregulin, and the related molecule heregulin-α, in a series of 10 prostate tissue specimens. Only EGF showed a disease-specific association, with increased mRNA levels in four of five PCa specimens in comparison to matched normal tissue from the same subject. In contrast, ARG and HB-EGF mRNAs showed a coordinate pattern of expression in 7/10 specimens that was distinct from all other growth factor or receptor genes examined and from mRNAs for prostate specific antigen, the androgen receptor and GAPDH, a housekeeping enzyme. Analysis of an additional series of benign prostatic hyperplasia and prostate cancer specimens from 60 individuals confirmed that ARG and HB-EGF mRNA levels varied in a highly coordinate manner (r = 0.93; P < 0.0001) but showed no association with disease. ARG was immunolocalized largely to interstitial smooth muscle cells (SMC), previously identified as the site of synthesis of HB-EGF in the prostate, while the cognate ARG and HB-EGF receptor, ErbB1, was localized exclusively to ductal epithelial cells and carcinoma cells. Although ARG was a relatively poor mitogen for Balb/c3T3 cells in comparison to HB-EGF, it was similar in potency to HB-EGF in stimulating human prostate epithelial cell growth, suggesting that prostate epithelia may be a physiologic target for ARG in vivo. Expression of both ARG and HB-EGF mRNAs was induced in cultured prostate SMC by fibroblast growth factor-2, a human prostate SMC mitogen linked to prostate disease. These findings indicate that ARG and HB-EGF are likely to be key mediators of directional signaling between SMC and epithelial cells in the human prostate and appear to be coordinately regulated.
Orexin Receptors Are Expressed in the Adrenal Medulla of the Rat
Authors: López M. , Señaris R. , Gallego R. , García-Caballero T. , Lago F. , Seoane L. , Casanueva F. , Diéguez C.
Published: January 2014

Two recently discovered hypothalamic peptides, orexin-A and orexin-B, play a role as mediators in the central mechanisms that regulate feeding behavior and sleep control. These peptides bind and activate two orexin receptors that belong to the G-protein coupled receptor superfamily. Morphological studies have detected mRNA expression of orexin receptors exclusively in the rat central nervous system. In this paper we demonstrate a strong level of expression of orexin receptor 1 and 2 in the adrenal medulla of the rat by RT-PCR immunohistochemistry. The results of the present study provide the first evidence showing that the adrenal medulla expresses orexin receptors, and thus appears to be a target tissue for orexins. This could open a new loop in which the central and autonomous nervous system may be involved in body weight homeostasis and sleep control.
Induction of Rat Aldose Reductase Gene Transcription Is Mediated through the cis-Element, Osmotic Response Element (ORE): Increased Synthesis and/or Activation by Phosphorylation of ORE-Binding Protein Is a Key Step
Authors: Aida Kaoru , Tawata Masato , Ikegishi Yukinobu , Onaya Toshimasa
Published: January 2014

We isolated the rat aldose reductase gene and examined the 5′-flanking sequence for the presence of transcription regulatory element responsive to hyperosmolarity. Deletion of aldose reductase gene up to− 1047 bp abolished the transcriptional activation in response to osmotic stimuli in transient transfection experiments. A 17-bp sequence[ rat osmotic response element (rORE)], which is located in bp− 1073/−1057 and contains the TGGAAAATCAC sequence, confers osmotic response on a heterologous promoter. Electrophoretic mobility shift assays using the 17-bp fragment demonstrated that distinct DNA-protein complexes (I and II) were formed predominantly with nuclear extracts from the cells exposed to hyperosmolarity. When the nuclear extracts were preincubated with calf intestinal alkaline phosphatase or protein phosphatase 1, formation of complexes I and II was reduced to the control level. However, incubation with protein tyrosine phosphatase and addition of antiphosphotyrosine antibody had no effect on the complexes. When the nuclear extracts were preincubated with diamide to oxidize the thiols, complexes I and II were not affected. Pretreatment of the cells with cycloheximide abolished the complexes. All of these data indicate that activation by phosphorylation and/or increased synthesis of rORE-binding protein(s) are the key steps in induction of transcription of the rat aldose reductase gene by hyperosmolarity. Furthermore, we showed that glucose was more effective than NaCl in induction of aldose reductase both in transient transfection experiments and by Northern blot analysis. The results suggest the presence of a glucose-specific mechanism of induction in addition to that by NaCl.
Role of Signal Transduction in Internalization of the G Protein-Coupled Receptor for Parathyroid Hormone (PTH) and PTH-Related Protein*
Authors: Huang Zhengmin , Bambino Tom , Chen Ying , Lameh Jelveh , Nissenson Robert A.
Published: January 2014

For G protein-coupled receptors, limited information is available on the role of agonist binding or of the second-messenger products of receptor signaling on receptor endocytosis. We explored this problem using the opossum PTH/PTH-related protein (PTHrP) receptor, a prototypical Class II G protein-coupled receptor, as a model. In one approach, we evaluated the endocytic properties of mutated forms of the opossum PTH/PTHrP receptor that we had previously shown to be impaired in their ability to initiate agonist-induced signaling when expressed in COS-7 cells. A point mutation in the third cytoplasmic loop (K382A) that severely impairs PTH/PTHrP receptor signaling significantly reduced internalization, whereas two mutant receptors that displayed only partial defects in signaling were internalized normally. To explore more directly the role of second-messenger pathways, we used a cleavable biotinylation method to assess endocytosis of the wild-type receptor stably expressed in human embryonic kidney (HEK) 293 cells. A low rate of constitutive internalization was detected (<5% over a 30-min incubation at 37 C); the rate of receptor internalization was enhanced about 10-fold by the receptor agonists PTH(1–34) or PTHrP(1–34), whereas the receptor antagonist PTH(7–34) had no effect. Forskolin treatment produced a minimal increase in constitutive receptor endocytosis, and the protein kinase (PK)-A inhibitor H-89 failed to block agonist-stimulated endocytosis. Similarly, activation of PK-C, by treatment with phorbol 12-myristate 13-acetate, elicited only a minimal increase in constitutive receptor endocytosis; and blockade of the PK-C pathway, by treatment with a bisindolylmaleimide, failed to inhibit agonist-induced receptor endocytosis. Immunofluorescence confocal microscopic studies of PTH/PTHrP receptor internalization confirmed the results using receptor biotinylation. These findings suggest that: 1) agonist binding is required for the efficient endocytosis of the PTH/PTHrP receptor; 2) receptor activation (agonist-induced receptor conformational change) and/or coupling to G proteins plays a critical role in receptor internalization; and 3) activation of PK-A and PK-C is neither necessary nor sufficient for agonist-stimulated receptor internalization.
The Gonadotropin-Releasing Hormone Receptor Gene Promoter Directs Pituitary-Specific Oncogene Expression in Transgenic Mice*
Authors: Albarracin Constance T. , Frosch Matthew P. , Chin William W.
Published: January 2014

Our previous work has shown that 1.2 kb of the 5′ flanking region of the mouse GnRH receptor (mGnRH-R) gene is sufficient to direct tissue-specific expression in vitro. In this study, we have used the cell-specific regulatory sequences of the mGnRH-R gene promoter to target the expression of the simian virus 40 virus T antigen (TAg) to the pituitary gland of transgenic mice. A hybrid transgene, GnRH-R/TAg, was prepared using the −1164/+52 region of the mGnRH-R gene and +2533/+5234 sequences encoding the large T antigen of the simian virus 40. Two founders developed tumors of apparent pituitary origin at 44 (M28, female) and 50 (M25, male) days of age. M28 and M25 mice were about 50% underweight, and their gonads were grossly underdeveloped compared with wild-type litter mates. A third male founder, M29, developed a tumor at a later time (109 days). M29 was able to breed successfully and stably transmit the GnRH-R/TAg transgene. Mice of the M29 transgene line developed tumors at 4–5 months of age. Gross examination showed that the tumors extend from the sella and infiltrate into the inferior surface of the brain. In small tumors collected from young transgenic animals, normal pituitary cells as well as transition areas of increasing cellular atypia are evident. Frankly malignant cells are seen in all tumors. The pituitary tumors express the α-, FSHβ-, and LHβ-subunits and the GnRH-R messenger RNA, all markers of a gonadotrope but not of other anterior pituitary cell lineages. In summary, our studies indicate that 1.2 kb of the 5′-flanking region of the mGnRH-R gene can be used to target expression specifically to the gonadotropes of the pituitary gland in transgenic mice. The GnRH-R gene promoter-directed expression appears to be cell-specific and results in the formation of tumors that are primarily of gonadotropic origin.
The Oct-2 POU Domain Gene in the Neuroendocrine Brain: A Transcriptional Regulator of Mammalian Puberty*
Authors: Ojeda Sergio R. , Hill Jennifer , Hill Diane F. , Costa Maria E. , Tapia Veronica , Cornea Anda , Ma Ying J.
Published: January 2014

POU homeodomain genes are transcriptional regulators that control development of the mammalian forebrain. Although they are mostly active during embryonic life, some of them remain expressed in the postnatal hypothalamus, suggesting their involvement in regulating differentiated functions of the neuroendocrine brain. We show here that Oct-2, a POU domain gene originally described in cells of the immune system, is one of the controlling components of the cell-cell signaling process underlying the hypothalamic regulation of female puberty. Lesions of the anterior hypothalamus cause sexual precocity and recapitulate some of the events leading to the normal initiation of puberty. Prominent among these events is an increased astrocytic expression of the gene encoding transforming growth factor-α (TGFα), a tropic polypeptide involved in the stimulatory control of LHRH secretion. The present study shows that such lesions result in the rapid and selective increase in Oct-2 transcripts in TGFα-containing astrocytes surrounding the lesion site. In both lesion-induced and normal puberty, there is a preferential increase in hypothalamic expression of the Oct-2a and Oct-2c alternatively spliced messenger RNA forms of the Oct-2 gene, with an increase in 2a messenger RNA levels preceding that in 2c and antedating the peripubertal activation of gonadal steroid secretion. Both Oct-2a and 2c trans-activate the TGFα gene via recognition motifs contained in the TGFα gene promoter. Inhibition of Oct-2 synthesis reduces TGFα expression in astroglial cells and delays the initiation of puberty. These results suggest that the Oct-2 gene is one of the upstream components of the glia to neuron signaling process that controls the onset of female puberty in mammals.
The Pancreatic Homeodomain Transcription Factor IDX1/IPF1 Is Expressed in Neural Cells during Brain Development
Authors: Perez-Villamil Beatriz , Schwartz Petra T. , Vallejo Mario
Published: January 2014

Expression of the homeodomain transcription factor IDX1/IPF1 has been shown to be restricted to cells in the developing foregut that form the pancreatic primordium. In the adult, IDX1/IPF1 is expressed in the duodenum and pancreatic islets. The IDX1/IPF1 gene is required for pancreatic development, and in the human, heterozygous mutations have been linked to diabetes mellitus. In the present communication, we report that IDX1/IPF1 is expressed in discrete cells of the rat central nervous system during embryonic development. Using RT-PCR, IDX1/IPF1 mRNA was detected in neural precursor RC2.E10 cells, as well as in both forebrain and hindbrain of developing rats at embryonic day 15 (E15). The presence of IDX1/IPF1 protein was confirmed by Western immunoblotting. Immunohistochemical analyses of sagittal sections of E15 rats demonstrated the presence of scattered IDX1/IPF1-immunopositive cells in the forebrain. Finally, electrophoretic mobility shift assays using nuclear extracts from neural cells revealed the presence of IDX1/IPF1 bound to a putative homeodomain protein DNA-binding site present in the promoter of the glial fibrillary acidic protein gene. Our results suggest that IDX1/IPF1 may have previously unsuspected extrapancreatic functions during development of neural cells in the central nervous system.
Expression and Localization of Serum/Glucocorticoid-Induced Kinase in the Rat Ovary: Relation to Follicular Growth and Differentiation1
Authors: Alliston Tamara N. , Gonzalez-Robayna Ignacio J. , Buse Patricia , Firestone Gary L. , Richards JoAnne S.
Published: January 2014

Expression of serum/glucocorticoid-inducible kinase (Sgk), one member of an inducible serine/threonine kinase family, is induced by FSH/cAMP in rat granulosa cells cultured in defined medium. The FSH-stimulated pattern of sgk expression is biphasic, and transcriptional activation of the sgk gene depends on an intact Sp1/Sp3 binding site within the proximal promoter. To determine whether sgk was expressed in a hormone-dependent and physiologically relevant manner in vivo, the cellular levels of sgk messenger RNA (mRNA) and protein as well as the subcellular localization of this kinase were analyzed in ovaries containing follicles and corpora lutea at specific stages of differentiation. To stimulate follicular development and luteinization, hypophysectomized (H) rats were treated with estradiol (E; HE) and FSH (FSH; HEF) followed by hCG (hCG; HEF/hCG). To analyze Sgk in functional corpora lutea, PRL was administered to HEF/hCG rats, or ovaries of pregnant rats were obtained on day 7, 15, or 22 of gestation. In situ hybridization indicated that sgk mRNA was low/undetectable in granulosa cells of H and HE rats. An acute injection (iv) of FSH to HE rats rapidly increased sgk mRNA at 2 and 8 h. Sgk mRNA was also elevated in granulosa cells of preovulatory follicles of HEF rats and in luteal cells of HEF/hCG and pregnant rats. Northern blots and Western blots confirmed the in situ hybridization data, indicating that the amount and cellular localization Sgk protein were related to that of sgk mRNA. When the subcellular localization of this kinase was analyzed by immunohistochemistry, Sgk protein was nuclear in granulosa cells and some thecal cells of large preovulatory follicles. In contrast, Sgk protein was cytoplasmic in luteal cells as well as some cells within the stromal compartment. Intense immunostaining was also observed in oocytes present in primordial follicles, but not in growing follicles. Collectively, these results show that FSH and LH stimulate marked increases in the cellular content of Sgk, as well as dramatic changes in the subcellular distribution of this kinase. The specific nuclear vs. cytoplasmic compartmentalization of Sgk in granulosa cells and luteal cells, respectively, indicates that Sgk controls distinct functions in proliferative vs. terminally differentiated granulosa cells.
Cholestenoic Acid Is a Naturally Occurring Ligand for Liver X Receptor α**This work was supported by NIH grants.
Authors: Song Ching , Liao Shutsung
Published: January 2014

Excessive cholesterol is eliminated from extrahepatic cells by reverse cholesterol transport, a process by which neutral sterols are transferred to extracellular acceptor lipoproteins for further transport to the liver. Another process independent of lipoproteins involves excretion of 3β-hydroxy-5-cholesten-25(R)-26-carboxylic (cholestenoic) acid, a metabolite of 27-hydroxycholesterol. Physiological concentrations of cholestenoic acid activated the nuclear receptor liver X receptor α (LXRα; NR1H3), but not other oxysterol receptors. As a ligand, cholestenoic acid modulated interaction of LXRα with the nuclear receptor coactivator Grip-1. Cholestenoic acid, therefore, may function as a signaling molecule for regulation of lipid metabolism via LXRα.
Breeding Stock-Specific Variation in Peptidylglycineα-Amidating Monooxygenase Messenger Ribonucleic Acid Splicing in Rat Pituitary1
Authors: Ciccotosto Giuseppe D. , Hand Tracey A. , Mains Richard E. , Eipper Betty A.
Published: January 2014

Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the carboxyl-terminal amidation of glycine-extended peptides in a two-step reaction involving a monooxygenase and a lyase. Several forms of PAM messenger RNA result from alternative splicing of the single copy PAM gene. The presence of alternately spliced exon A between the two enzymatic domains allows endoproteolytic cleavage to occur in selected tissues, generating soluble monooxygenase and membrane lyase from integral membrane PAM. While using an exon A antiserum, we made the unexpected observation that Charles River Sprague Dawley rats expressed forms of PAM containing exon A in their pituitaries, whereas Harlan Sprague Dawley rats did not. Forms of PAM containing exon A were expressed in the atrium and hypothalamus of both types of Sprague Dawley rat, although in different proportions. PAM transmembrane domain splicing also differed between rat breeders, and full-length PAM-1 was not prevalent in the anterior pituitary of either type of rat. Despite striking differences in PAM splicing, no differences in levels of monooxygenase or lyase activity were observed in tissue or serum samples. The splicing patterns of other alternatively spliced genes, pituitary adenylate cyclase-activating polypeptide receptor type 1 and cardiac troponin T, did not vary with rat breeder. Strain-specific variations in the splicing of transcripts such as PAM must be taken into account in analyzing the resultant proteins, and knowledge of these differences should identify variations with functional significance.
The Role of the Dorsal Vagal Complex and the Vagus Nerve in Feeding Effects of Melanocortin-3/4 Receptor Stimulation*
Authors: Williams Diana L. , Kaplan Joel M. , Grill Harvey J.
Published: January 2014

Fourth intracerebroventricular (4th-icv) administration of the melanocortin-3/4 receptor (MC3/4-R) agonist, MTII, reduces food intake; the antagonist, SHU9119, increases feeding. The dorsal motor nucleus of the vagus nerve (DMX) contains the highest density of MC4-R messenger RNA in the brain. To explore the possibility that the DMX contributes to 4th-icv MC4-R effects, we delivered doses of MTII and SHU9119 that are subthreshold for ventricular response unilaterally through a cannula centered above the DMX. MTII markedly suppressed 2-h (50%), 4-h (50%), and 24-h (33%) intake. Feeding was significantly increased 4 h (50%) and 24 h (20%) after SHU9119 injections. These results suggest that receptors in the DMX, or the dorsal vagal complex more generally, underlie effects obtained with 4th-icv administration of these ligands. We investigated possible vagal mediation of 4th-icv MTII effects by giving the agonist to rats with subdiaphragmatic vagotomy. MTII suppressed 2-, 4-, and 24-h liquid diet intake (∼80%) to the same extent in vagotomized and surgical control rats. We conclude that stimulation or antagonism of MC3/4-Rs in the dorsal vagal complex yields effects on food intake that do not require an intact vagus nerve.
The gp130 Cytokines Interleukin-11 and Ciliary Neurotropic Factor Regulate through Specific Receptors the Function and Growth of Lactosomatotropic and Folliculostellate Pituitary Cell Lines*
Authors: Castro Carolina Perez , Nagashima Alberto Carbia , Pereda Marcelo Páez , Goldberg Victoria , Chervin Alberto , Largen Penny , Renner Ulrich , Stalla Günter K. , Arzt Eduardo
Published: January 2014

Two of the most potent cytokines regulating anterior pituitary cell function are leukemia inhibitory factor and interleukin-6 (IL-6), which belong to the cytokine receptor family using the common gp130 signal transducer. We studied the actions of two other members of this family, IL-11 and ciliary neurotropic factor (CNTF), on folliculostellate (FS) cells (TtT/GF cell line) and lactosomatotropic cells (GH3 cell line). The messenger RNA (mRNA) for the α-chain specific for the IL-11 receptor (1.7 kb) and CNTF receptor (2 kb) are expressed on both cell types. In addition, we detected CNTF receptor mRNA in normal rat anterior pituitary cells. IL-11 (1.25–5 nm) dose dependently stimulated the proliferation of FS cells. CNTF, at doses from 0.4–2 nm, also significantly stimulated the growth of these cells. In addition, both cytokines significantly stimulated proliferation of lactosomatotropic GH3 cells, and CNTF stimulated hormone production (GH and PRL) at 24 h by these cells. At 16–72 h, IL-11 stimulates the secretion of the angiogenic factor vascular endothelial growth factor by FS cells. In addition, both GH3 and FS cells express CNTF mRNA. These data suggest that IL-11 and CNTF may act as growth and regulatory factors in anterior pituitary cells.
Regulation of Corticotropin-Releasing Factor Receptor Type 2β Messenger Ribonucleic Acid in the Rat Cardiovascular System by Urocortin, Glucocorticoids, and Cytokines*
Authors: Kageyama Kazunori , Gaudriault Georges E. , Bradbury Margaret J. , Vale Wylie W.
Published: January 2014

CRF receptor type 2 (CRF R2) messenger RNA (mRNA) expression in the rodent heart is modulated by exposure to both the bacterial endotoxin lipopolysaccharide (LPS) and glucocorticoids. In this study we examined the roles of glucocorticoids, cytokines, and CRF R2β ligands in the regulation of CRF R2β expression in the cardiovascular system both in vivo and in vitro. Using ribonuclease protection assays, we found that, in addition to the injection of LPS or corticosterone, physical restraint caused a decrease in CRF R2β mRNA levels in the rat heart and aorta. Adrenalectomy with corticosterone replacement at constant levels partially blocked LPS-induced decreases in CRF R2β mRNA expression in the heart. Thus, elevations of endogenous circulating corticosterone could contribute to the down-regulation of CRF R2β mRNA expression in heart. To identify other putative modulating factors, we examined CRF R2β expression in the aorta- derived A7R5 cell line. Incubation with CRF R2 ligands or dexamethasone reduced CRF R2β mRNA levels. In addition, incubation with a variety of cytokines, proteins released during immune challenge, also reduced CRF R2β mRNA expression. The multifactorial regulation of CRF R2β mRNA expression in the cardiovascular system may serve to limit the inotropic and chronotropic effects of CRF R2 agonists such as urocortin during prolonged physical or immune challenge.
Production of Immunoreactive Thyroglobulin C-Terminal Fragments during Thyroid Hormone Synthesis
Authors: Duthoit Christine , Estienne Valérie , Delom Frédéric , Durand-Gorde Josée-Martine , Mallet Bernard , Carayon Pierre , Ruf Jean
Published: January 2014

Here, we studied the fragmentation of the prothyroid hormone, thyroglobulin (Tg), which occurs during thyroid hormone synthesis, a process which involves iodide, thyroperoxidase, and the H2O2-generating system, consisting of glucose and glucose oxidase. Various peptides were found to be immunoreactive to autoantibodies to Tg from patients and monoclonal antibodies directed against the immunodominant region of Tg. The smallest peptide (40 kDa) bore thyroid hormones and was identified at the C-terminal end of the Tg molecule, which shows homologies with acetylcholinesterase. Similar peptides were obtained by performing metal-mediated oxidation of Tg via a Fenton reaction. It was concluded that the oxidative stress induced during hormone synthesis generates free radicals, which, in turn, cleave Tg into immunoreactive peptides.
The Distribution of Cells Containing Estrogen Receptor-α (ERα) and ERβ Messenger Ribonucleic Acid in the Preoptic Area and Hypothalamus of the Sheep: Comparison of Males and Females
Authors: Scott Christopher J. , Tilbrook Alan J. , Simmons Donna M. , Rawson John A. , Chu Simon , Fuller Peter J. , Ing Nancy H. , Clarke Iain J.
Published: January 2014

We have used in situ hybridization to compare the distributions of estrogen receptor α (ERα) and ERβ messenger RNA (mRNA)-containing cells in the preoptic area and hypothalamus of ewes and rams. Perfusion-fixed brain tissue was collected from luteal phase ewes and intact rams (n = 4) during the breeding season. Matched pairs of sections were hybridized with sheep-specific, 35S-labeled riboprobes, and semiquantitative image analysis was performed on emulsion-dipped slides.

A number of sex differences were observed, with females having a greater density of labeled cells than males (P < 0.001) and a greater number of silver grains per cell (P < 0.01) in the ventromedial nucleus for both ER subtypes. In addition, in the retrochiasmatic area, males had a greater (P < 0.05) cell density for ERα mRNA-containing cells than females, whereas in the paraventricular nucleus, females had a greater density (P < 0.05) of ERα mRNA-containing cells than males. There was a trend (P = 0.068) in the arcuate nucleus for males to have a greater number of silver grains per cell labeled for ERα mRNA.

In both sexes, there was considerable overlap in the distributions of ERα and ERβ mRNA-containing cells, but the density of labeled cells within each nucleus differed in a number of instances. Nuclei that contained a higher (P < 0.001) density of ERα than ERβ mRNA-containing cells included the preoptic area, bed nucleus of the stria terminalis, and ventromedial nucleus, whereas the subfornical organ (P < 0.001), paraventricular nucleus (males only, P < 0.05), and retrochiasmatic nucleus (females only, P < 0.05) had a greater density of ERα than ERβ mRNA-containing cells. The anterior hypothalamic area and supraoptic nucleus had similar densities of cells containing both ER subtypes. The lateral septum and arcuate nucleus contained only ERα, whereas only ERβ mRNA-containing cells were seen in the zona incerta.

The sex differences in the populations of ER mRNA-containing cells in the ventromedial and arcuate nuclei may explain in part the sex differences in the neuroendocrine and behavioral responses to localized estrogen treatment in these nuclei. Within sexes, the differences between the distributions of ERα and ERβ mRNA-containing cells may reflect differential regulation of the actions of estrogen in the sheep hypothalamus. Low levels of ERβ mRNA in the preoptic area and ventromedial and arcuate nuclei, regions known to be important for the regulation of reproduction, suggest that ERβ may not be involved in these functions.