bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of the Leptin Receptor during Germ Cell Development in the Mouse Testis*
Authors: El-Hefnawy Talal , Ioffe Sergey , Dym Martin
Published: January 2014

Leptin, a recently identified hormonal product of the ob gene, is known to regulate appetite, body metabolism, and reproductive functions. We investigated the expression of the leptin receptor (Ob-R) in testes from different age groups. The messenger RNA for Ob-R was found in testes from all age groups using RT-PCR. Using immunohistochemistry, we observed age- and stage-dependent distribution of the Ob-R in mouse testis. In testis of 5-day-old mice, its expression was mainly in type A spermatogonia. In the 20- and 30-day-old testis, Ob-R expression was in the spermatocytes; in the adult testis, it was specific to spermatocytes in stages IX and X of the cycle of the seminiferous epithelium. Five main immunoreactive proteins were detected using Western blot (220, 120, 90, 66, and 46 kDa). The 120-kDa protein was evident only in 20-day-old and older testes, whereas the 90-kDa band was present only in the 5- and 10-day-old testis. Leptin treatment induced phosphorylation of signal transducer and activator of transcription-3 in cultured seminiferous tubules from adult and 5-day-old testes. Our results show for the first time age- and stage-specific localization of a functional Ob-R in testicular germ cells. We hypothesize a direct role for leptin, through phosphorylation of signal transducer and activator of transcription-3, in proliferation and differentiation of germ cells, which may partially explain the infertility observed in leptin-deficient mice.
Deficient Mineralization of Intramembranous Bone in Vitamin D-24-Hydroxylase-Ablated Mice Is Due to Elevated 1,25-Dihydroxyvitamin D and Not to the Absence of 24,25-Dihydroxyvitamin D*
Authors: St-Arnaud René , Arabian Alice , Travers Rose , Barletta Frank , Raval-Pandya Mihali , Chapin Kelli , Depovere Jos , Mathieu Chantal , Christakos Sylvia , Demay Marie B. , Glorieux Francis H.
Published: January 2014

The 25-hydroxyvitamin D-24-hydroxylase enzyme (24-OHase) is responsible for the catabolic breakdown of 1,25-dihydroxyvitamin D[ 1,25(OH)2D], the active form of vitamin D. The 24-OHase enzyme can also act on the 25-hydroxyvitamin D substrate to generate 24,25-dihydroxyvitamin D, a metabolite whose physiological importance remains unclear. We report that mice with a targeted inactivating mutation of the 24-OHase gene had impaired 1,25(OH)2D catabolism. Surprisingly, complete absence of 24-OHase activity during development leads to impaired intramembranous bone mineralization. This phenotype was rescued by crossing the 24-OHase mutant mice to mice harboring a targeted mutation in the vitamin D receptor gene, confirming that the elevated 1,25(OH)2D levels, acting through the vitamin D receptor, were responsible for the observed accumulation of osteoid. Our results confirm the physiological importance of the 24-OHase enzyme for maintaining vitamin D homeostasis, and they reveal that 24,25-dihydroxyvitamin D is a dispensable metabolite during bone development.
Sp1 and SF-1 Interact and Cooperate in the Regulation of Human Steroidogenic Acute Regulatory Protein Gene Expression*
Authors: Sugawara Teruo , Saito Masaki , Fujimoto Seiichiro
Published: January 2014

Steroidogenic acute regulatory (StAR) protein plays a critical role in the movement of cholesterol from the outer to the inner mitochondrial membrane. Steroidogenic factor 1 (SF-1) controls basal and cAMP-stimulated transcription of the StAR gene. The 1.3-kb StAR promoter has three SF-1 binding sites, and two consensus transcription factor Sp1 binding sequences near the two most distal SF-1 binding sites. Sp1 mediates cAMP-dependent transcription of steroidogenic P450 enzyme genes, raising the possibility of Sp1 involvement in cAMP regulation of the StAR gene. However, the mechanism of Sp1-mediated, cAMP-stimulated responsiveness is not known. In this study, we elucidated the roles of Sp1 and SF-1 in the regulation of the human StAR gene promoter. We found that there was negligible promoter activity in a pGL2 StAR construct (−235 to +39) in which Sp1 and SF-1 binding sites were mutated in Y-1 adrenal tumor cells. An Sp1 binding site mutation (pGL2Sp1M) did not support promoter activity, suggesting that Sp1 cooperates with SF-1 in regulating StAR promoter function. In gel shift assays, the SF-1 binding site formed a complex with an SF-1-GST fusion protein and Sp1. Coimmunoprecipitation cross-linking experiments indicated that SF-1 physically interacts with Sp1 in vitro. Finally, a mammalian two-hybrid system was employed to demonstrate that Sp1 and SF-1 associate in vivo. In conclusion, our data indicate that Sp1 and SF-1 physically interact and cooperate in the regulation of human StAR promoter activity.
Improved Glucose and Lipid Metabolism in Genetically Obese Mice Lacking aP2
Authors: Uysal K. Teoman , Scheja Ludger , Wiesbrock Sarah M. , Bonner-Weir Susan , Hotamisligil Gökhan S.
Published: January 2014

Adipocyte fatty acid-binding protein, aP2, is a member of the intracellular fatty acid binding protein family. Previously, studies have shown increased insulin sensitivity in aP2-deficient mice with dietary obesity. Here, we asked whether aP2-related alterations in lipolytic response and insulin production are features of obesity-induced insulin resistance and investigated the effects of aP2-deficiency on glucose homeostasis and lipid metabolism in ob/ob mice, a model of extreme obesity. ob/ob mice homozygous for the aP2 null allele (ob/ob-aP2−/−) became more obese than ob/ob mice as indicated by significantly increased body weight and fat pad size but unaltered body length. However, despite their extreme adiposity, ob/ob-aP2−/− animals were more insulin-sensitive compared with ob/ob controls, as demonstrated by significantly lower plasma glucose and insulin levels and better performance in both insulin and glucose tolerance tests. These animals also showed improvements in dyslipidemia and had lower plasma triglyceride and cholesterol levels. Lipolytic response toβ -adrenergic stimulation and lipolysis-associated insulin secretion was significantly reduced in ob/ob-aP2−/− mice. Interestingly, glucose-stimulated insulin secretion, while virtually abolished in ob/ob controls, was significantly improved in ob/ob-aP2−/− animals. There were no apparent morphological differences in the structure or size of the pancreatic islets between genotypes. Taken together, the data indicate that in obesity, aP2-deficiency not only improves peripheral insulin resistance but also preserves pancreatic β cell function and has beneficial effects on lipid metabolism.
The Peroxisome Proliferator Perfluorodecanoic Acid Inhibits the Peripheral-Type Benzodiazepine Receptor (PBR) Expression and Hormone-Stimulated Mitochondrial Cholesterol Transport and Steroid Formation in Leydig Cells*
Authors: Boujrad Noureddine , Vidic Branislav , Gazouli Maria , Culty Martine , Papadopoulos Vassilios
Published: January 2014

The peroxisome proliferator perfluordecanoic acid (PFDA) has been shown to exert an antiandrogenic effect in vivo by acting directly on the interstitial Leydig cells of the testis. The objective of this study was to examine the in vitro effects of PFDA and identify its site of action in steroidogenesis using as model systems the mouse tumor MA-10 and isolated rat Leydig cells. PFDA inhibited in a time- and dose-dependent manner the hCG-stimulated Leydig cell steroidogenesis. This effect was localized at the level of cholesterol transport into the mitochondria. PFDA did not affect either the total cell protein synthesis or the mitochondrial integrity. Moreover, it did not induce any DNA damage. Morphological studies indicated that PFDA induced lipid accumulation in the cells, probably due to the fact that cholesterol mobilized by hCG did not enter the mitochondria to be used for steroidogenesis. In search of the target of PFDA, we examined its effect on key regulatory mechanisms of steroidogenesis. PFDA did not affect the hCG-induced steroidogenic acute regulatory protein (StAR) levels. However, it was found to inhibit the mitochondrial peripheral-type benzodiazepine receptor (PBR) ligand binding capacity, 18-kDa protein, and messenger RNA (mRNA) levels. Further studies indicated that PFDA did not affect PBR transcription, but it rather accelerated PBR mRNA decay. Taken together, these data suggest that PFDA inhibits the Leydig cell steroidogenesis by affecting PBR mRNA stability, thus inhibiting PBR expression, cholesterol transport into the mitochondria, and the subsequent steroid formation. Moreover, this action of PFDA on PBR mRNA stability indicates a new mechanism of action of peroxisome proliferators distinct from the classic transcription-mediated regulation of target genes.
Regulation of the GABAA Receptor by Nitric Oxide in Frog Pituitary Melanotrophs*
Authors: Castel Hélène , Jégou Sylvie , Tonon Marie-Christine , Vaudry Hubert
Published: January 2014

Nitric oxide (NO) is implicated in the regulation of various endocrine functions, but the effect of NO on GABAA receptor transmission has never been reported in endocrine cells. In the present study, we have investigated the effects of various agents acting on the NO transduction pathway on GABAA receptor function in frog pituitary melanotrophs. Histochemical studies using the NADPH-diaphorase reaction and immunohistochemical labeling with antibodies against neuronal NO synthase (nNOS) revealed that nNOS is expressed in the intermediate lobe of the pituitary and in cultured melanotrophs. Whole-cell patch-clamp recordings showed that the specific substrate of NOS l-arginine (l-Arg, 10−4m) or the NO donor sodium nitroprusside (10−5m) provoked a long-lasting inhibition of the current evoked by GABA (5 × 10−6m). The NOS inhibitor l-nitroarginine (10−5m) produced a biphasic effect, i.e. a transient decrease followed by a delayed increase of the GABA-evoked current amplitude. Similarly, the specific nNOS inhibitor 7-nitroindazole and the specific inducible NOS (iNOS) inhibitor aminoguanidine (10−5m each) provoked a transient depression of the current followed by a sustained potentiation. Formation of cGMP in neurointermediate lobes was enhanced by l-Arg (10−4m) and by the calcium-releasing agent caffeine (10−4m), and inhibited by the calmodulin (CaM)/Ca2+ complex blocker W7 (10−5m). The GABA-evoked current was potentiated by the guanylyl cyclase inhibitor ODQ (10−8–10−7m) and inhibited by the protein kinase G (PKG) activator 8pCPT-cGMP (3 × 10−7–3 × 10−5m). The present data indicate that NO, produced by a CaM/Ca2+-dependent NOS in frog melanotrophs, exerts an autocrine inhibitory effect on the GABA-evoked current. The action of NO on the GABAA receptor function is mediated through activation of the cGMP/PKG pathway.
Stimulation of Intracellular Free Calcium in GH3 Cells byγ 3-Melanocyte-Stimulating Hormone. Involvement of a Novel Melanocortin Receptor?
Authors: Langouche Lies , Roudbaraki Morad , Pals Katrien , Denef Carl
Published: January 2014

The melanocortin (MC) γ3MSH is a peptide that can be generated from the N-terminal domain of POMC and is believed to signal through the MC3 receptor. We recently showed that it induces a sustained rise in intracellular free calcium levels ([Ca2+]i) in a subpopulation of pituitary cells, particularly in the lactosomatotroph lineage. In the present study we report that γ3MSH and some analogs increase [Ca2+]i in the GH- and PRL-secreting GH3 cell line and evaluate on the basis of pharmacological experiments and gene expression studies which MC receptor may be involved.

A dose as low as 1 pm γ3MSH induced an oscillating[ Ca2+]i increase in a significant percentage of GH3 cells. Increasing the dose recruited an increasing number of responding cells; a maximum was reached at 0.1 nm. γ2MSH,α MSH, and NDP-αMSH displayed a similar effect. SHU9119, an MC3 and MC4 receptor antagonist, and an MC5 receptor agonist, did not affect the number of cells showing a [Ca2+]i rise in response to γ3MSH. SHU9119 had also no effect when added alone. MTII, a potent synthetic agonist of the MC3, MC4, and MC5 receptor as well as an N-terminally extended recombinant analog of γ3MSH showed low potency in increasing [Ca2+]i in GH3 cells, but high potency in stimulating cAMP accumulation in HEK 293 cells stably transfected with the MC3 receptor. In contrast, a peptide corresponding to the γ2MSH sequence of POMC-A of Acipenser transmontanus increased [Ca2+]i in GH3 cells, but was about 50 times less potent than γ2- or γ3MSH in stimulating cAMP accumulation in the MC3 receptor expressing HEK 293 cells. By means of RT-PCR performed on a RNA extract from GH3 cells, the messenger RNA of the MC2, MC3, and MC4 receptor was undetectable, but messenger RNA of the MC5 receptor was clearly present.

These data suggest that the GH3 cell line does not mediate the effect of γ3MSH through the MC3 receptor. The involvement of the MC5 receptor is unlikely, but cannot definitely be excluded. The findings animate the hypothesis that there exists a second, hitherto unidentified, MC receptor that displays high affinity for γ3MSH.
Novel Long-Acting Somatostatin Analog with Endocrine Selectivity:
Potent Suppression of Growth Hormone But Not of Insulin
Authors: Afargan Michel , Janson Eva Tiensuu , Gelerman Garry , Rosenfeld Rakefet , Ziv Offer , Karpov Olga , Wolf Amnon , Bracha Moshe , Shohat Dvira , Liapakis George , Gilon Chaim , Hoffman Amnon , Stephensky David , Oberg Kjell
Published: January 2014

Somatostatin, also known as somatotropin release-inhibiting factor (SRIF), is a natural cyclic peptide inhibitor of pituitary, pancreatic, and gastrointestinal secretion. Its long-acting analogs are in clinical use for treatment of various endocrine syndromes and gastrointestinal anomalies. These analogs are more potent inhibitors of the endocrine release of GH, glucagon, and insulin than the native SRIF; hence, they do not display considerable physiological selectivity. Our goal was to design effective and physiologically selective SRIF analogs with potential therapeutic value. We employed an integrated approach consisting of screening of backbone cyclic peptide libraries constructed on the basis of molecular modeling of known SRIF agonists and of high throughput receptor binding assays with each of the five cloned human SRIF receptors (hsst1–5). By using this approach, we identified a novel, high affinity, enzymatically stable, and long-acting SRIF analog, PTR-3173, which binds with nanomolar affinity to human SRIF receptors hsst2, hsst4, and hsst5. The hsst5 and the rat sst5 (rsst5) forms have the same nanomolar affinity for this analog. In the human carcinoid-derived cell line BON-1, PTR-3173 inhibits forskolin-stimulated cAMP accumulation as efficiently as the drug octreotide, indicating its agonistic effect in this human cell system. In hormone secretion studies with rats, we found that PTR-3173 is 1000-fold and more than 10,000-fold more potent in inhibiting GH release than glucagon and insulin release, respectively. These results suggest that PTR-3173 is the first highly selective somatostatinergic analog for the in vivo inhibition of GH secretion, with minimal or no effect on glucagon and insulin release, respectively.
Somatostatin Inhibits Akt Phosphorylation and Cell Cycle
Entry, But Not p42/p44 Mitogen-Activated Protein (MAP) Kinase
Activation in Normal and Tumoral Pancreatic Acinar Cells**This research was supported by Grant 970384 (to N.R.) from the Natural Sciences and Engineering Research Council of Canada.
Authors: Charland Stéphanie , Boucher Marie-Josée , Houde Mathieu , Rivard Nathalie
Published: January 2014

Somatostatin, or its structural analog SMS 201–995 (SMS), is recognized to exert a growth-inhibitory action in rat pancreas, but the cellular mechanisms are not completely understood. This study was undertaken to evaluate the effect of SMS on p42/p44 MAP kinases and phosphatidylinositol 3-kinase activation and to analyze expression of some cell cycle regulatory proteins in relation to pancreatic acinar cell proliferation in vivo (rat pancreas), as well as in the well-established tumoral cell line AR4–2J. We herein report that: 1) SMS inhibits caerulein-induced pancreatic weight and DNA content and abolishes epidermal growth factor (EGF)-stimulated AR4–2J proliferation; 2) SMS only moderately reduces the stimulatory effect of caerulein on p42/p44 MAP kinase activities in pancreas and has no effect on EGF-stimulated MAP kinase activities in AR4–2J cells; 3) SMS repressed caerulein-induced Akt activity in normal pancreas; 4) SMS has a strong inhibitory action on cyclin E expression induced by caerulein in pancreas and EGF in AR4–2J cells and as expected, the resulting cyclin E-associated cyclin-dependent kinase (cdk)2 activity, as well as pRb phosphorylation, are blunted by SMS treatment in both models; and 5) SMS suppresses mitogen-induced p27Kip1 down-regulation, as well as marginally induces p21Cip expression. Thus, our data suggest that somatostatin-induced growth arrest is mediated by inhibition of phosphatidylinositol 3-kinase pathway and by enhanced expression of p21Cip and p27Kip1, leading to repression of pRb phosphorylation and cyclin E-cdk2 complex activity.
Up-Regulation of p27Kip1 by Progestins Is Involved in the Growth Suppression of the Normal and Malignant Human Endometrial Glandular Cells
Authors: Shiozawa Tanri , Horiuchi Akiko , Kato Kiyoshi , Obinata Miyuki , Konishi Ikuo , Fujii Shingo , Nikaido Toshio
Published: January 2014

Progestins are known to suppress the growth of normal human endometrial glands and endometrial carcinomas possessing PRs. To elucidate the molecular mechanisms of progestin-induced growth inhibition, the expression and functional involvement of p27Kip1 (p27), a cyclin-dependent-kinase inhibitor, was investigated using cultured normal endometrial glandular cells and endometrial carcinoma cell lines (Ishikawa; PR-positive, KLE; PR-negative). Growth of the normal endometrial glandular cells and Ishikawa cells was suppressed by treatment with progesterone and medroxyprogesterone acetate, respectively, in association with an increase in p27 protein expression. Immunoprecipitation revealed that progestins accelerated the complex formation of p27 and cdk2 in both types of cells. However, treatment with progestins did not show any marked alterations in the mRNA expression of p27 in either normal glandular cells or Ishikawa cells. On the other hand, p27 protein degradation experiments indicated that treatment with progesterone and medroxyprogesterone acetate prolonged the degradation time of the normal endometrial glandular cells and Ishikawa cells, respectively. Forced expression of the p27 protein using a p27 expression plasmid reduced the growth activity of normal endometrial glandular cells. These findings suggest that p27 is functionally involved in progestin-induced growth suppression of normal and malignant endometrial epithelial cells and that up-regulation of the p27 protein by progestins possibly occurs via posttranslational mechanisms.
Experimental IGF-I Receptor Deficiency Generates a Sexually Dimorphic Pattern of Organ-Specific Growth Deficits in Mice, Affecting Fat Tissue in Particular
Authors: Holzenberger Martin , Hamard Ghislaine , Zaoui Randa , Leneuve Patricia , Ducos Bertrand , Beccavin Caroline , Périn Laurence , Le Bouc Yves
Published: January 2014

Reduced IGF type I receptor levels diminish postnatal growth rate and adult body weight in mice. Here, we studied the impact of experimental IGF receptor deficiency on tissue-specific growth by Cre-lox-mediated dosage of a floxed IGF-IR gene. We generated mice with a wide spectrum of receptor deficiency (5–82%), and separated them into two groups with either strong (≥50%) IGF-IR deficiency (XS mice) or moderate deficiency (<50%, M mice). The growth of XS mice was significantly retarded from 3 wk after birth onward, with respect to M littermates. This effect was twice as strong in males as in females. Growth deficits persisted throughout adult life, and at 10–12 months, most organs and tissues showed specific weight defects. Skin, bone and connective tissue, muscle, spleen, heart, lung, and brain were the most severely affected organs in the XS males. With the exception of muscle and spleen, the same tissues were also significantly reduced in size in females, although to a lesser extent. The most severe growth defect, however, concerned adipose tissue. Fat pad size in XS males was only 29% (females, 44%) of M mice. The estimated number of adipocytes in XS male fat pads was only 21% that of M males (XS female, 27%). Lipid content per cell was significantly higher in XS adipocytes, whereas plasma glucose and insulin levels were low in XS males. Thus, IGF type I receptor deficiency produced mice with disproportionate postnatal organ growth, and these effects depended strongly on sex. A marked reduction in IGF-IR levels resulted in a major defect in adipose tissue.
α1-Adrenergic Receptors Mediate LH-Releasing Hormone Secretion through Phospholipases C and A2 in Immortalized Hypothalamic Neurons
Authors: Kreda Silvia M. , Sumner Martina , Fillo Silvia , Ribeiro Carla M. , Luo Guo X. , Xie Weihua , Daniel Kiefer W. , Shears Stephen , Collins Sheila , Wetsel William C.
Published: January 2014

Norepinephrine has long been known to stimulate the pulsatile and preovulatory release of LH-releasing hormone (LHRH). In vivo and in vitro studies indicate that these effects are mediated primarily through α1-adrenergic receptors (α1-ARs). With the immortalized hypothalamic LHRH neurons, we have found that α1-adrenergic agents directly stimulate the secretion of LHRH in a dose-dependent manner. Ligand binding and RNA studies demonstrate that the GT1 cells contain both α1A- and α1B-ARs. Competition binding experiments show that approximately 75% of the binding is due toα 1B-ARs; the remainder is made up ofα 1A-ARs. Receptor activation leads to stimulation of PLC. PLCβ1 and PLCβ3 are expressed in GT1 neurons, and these PLCs are probably responsible for the release of diacylglycerol and IP as well as the increase in intracellular calcium. The mobilization of cytoplasmic calcium is sufficient to stimulate cytosolic PLA2 (cPLA2) and release arachidonic acid. A dissection of the contributions of the phospholipases to LHRH secretion suggests that cPLA2 acts downstream of PLC and that it significantly augments the PLC-stimulated LHRH secretory response. Inasmuch as the α1-ARs are known to play a critical role in LHRH physiology, we propose that both PLC and cPLA2 are critical in regulating and amplifying LHRH release.
Estrogen Reduces the Depth of Resorption Pits by Disturbing the Organic Bone Matrix Degradation Activity of Mature Osteoclasts
Authors: Parikka Vilhelmiina , Lehenkari Petri , Sassi Mirja-Liisa , Halleen Jussi , Risteli Juha , Härkönen Pirkko , Väänänen H. Kalervo
Published: January 2014

Decreased E2 levels after menopause cause bone loss through increased penetrative resorption. The reversal effect of E2 substitution therapy is well documented in vivo, although the detailed mechanism of action is not fully understood. To study the effects of E2 on bone resorption, we developed a novel in vitro bone resorption assay in which degradation of inorganic and organic matrix could be measured separately. E2 treatment significantly decreased the depth of resorption pits, although the area resorbed was not changed. Electron microscopy further revealed that the resorption pits were filled with nondegraded collagen, suggesting that E2 disturbed the organic matrix degradation. Two major groups of proteinases, matrix metalloproteinases (MMPs) and cysteine proteinases, have been suggested to participate in organic matrix degradation by osteoclasts. We show here that MMP-9 released a cross-linked carboxyl-terminal telopeptide of type I collagen from bone collagen, and cathepsin K released another C-terminal fragment, the C-terminal cross-linked peptide of type I collagen. E2 significantly inhibited the release of the C-terminal cross-linked peptide of type I collagen into the culture medium without affecting the release of cross-linked carboxyl-terminal telopeptide of type I collagen in osteoclast cultures. These results suggest that organic matrix degradation is initiated by MMPs and continued by cysteine proteases; the latter event is regulated by E2.
Peptones Stimulate Intestinal Cholecystokinin Gene Transcription via Cyclic Adenosine Monophosphate Response Element-Binding Factors
Authors: Bernard Christine , Sutter Anne , Vinson Charles , Ratineau Christelle , Chayvialle Jean-Alain , Cordier-Bussat Martine
Published: January 2014

Cholecystokinin (CCK) is a potent intestinal hormone that regulates several digestive functions. Despite the physiological importance of CCK, the cellular and molecular mechanisms that govern its synthesis and secretion are not completely identified. Peptones, which are fair counterparts of the protein fraction in the intestinal lumen, are good stimulants of CCK secretion. We have previously shown that peptones activate CCK gene transcription in STC-1 enteroendocrine cells. The DNA element(s) necessary to induce the transcriptional stimulation was preliminary, localized in the first 800 bp of the CCK gene promoter. In the present study, we identify a DNA element [peptone-response element (PepRE)] essential to confer peptone-responsiveness to the CCK promoter, and we characterize the transcription factors implicated. Localization of the PepRE between −93 and −70 bp of the promoter was established using serial 5′-3′deletions. Systematic site-directed mutagenesis demonstrated that the core PepRE sequence, spanning from nucleotide −72 to −83, overlapped with the putative AP-1/CRE site. Mutations in the core sequence dramatically decreased peptone-responsiveness of CCK promoter fragments. The PepRE functioned as a low-affinity CRE consensus site, binding only transcription factors of the CREB family. Overexpression, in STC-1 cells, of a dominant-negative protein (A-CREB), that prevented the binding of CREB factors to DNA, completely abolished the peptone-induced transcriptional stimulation. Peptone treatment did not modify the nature and the abundance of proteins bound to the PepRE but led to increased phosphorylation of the CREB factors. In conclusion, the present study first demonstrates that CCK gene expression is under the control of protein-derived nutrients in the STC-1 enteroendocrine cell line.
Hedgehog Signaling Regulation of Homeodomain Protein Islet Duodenum Homeobox-1 Expression in Pancreatic β-Cells*
Authors: Thomas Melissa K. , Lee Jee H. , Rastalsky Naina , Habener Joel F.
Published: January 2014

Insulin gene expression in pancreatic β-cells is regulated by signals from developmental morphogen proteins known as hedgehogs (Hhs). By analyzing 5′-deletion insulin promoter-reporter constructs in transient transfections of clonal INS-1 β-cells, we located activating Hh-responsive regions within the rat insulin I promoter that include the glucose-response elements Far (E2) and Flat (A2/A3). Activation of Hh signaling in INS-1 cells by ectopic Hh expression increased (and inhibition of Hh signaling with the Hh-specific inhibitor cyclopamine decreased) transcriptional activation of a multimerized FarFlat enhancer-reporter construct. In DNA-binding studies, nuclear extracts from INS-1 cells activated by ectopic Hh expression increased (and extracts from INS-1 cells treated with cyclopamine decreased) protein binding to a radiolabeled FarFlat oligonucleotide probe. An antiserum directed against the transcription factor islet duodenum homeobox-1 (IDX-1), a regulator of pancreas development and activator of the insulin gene promoter, attenuated the binding activity of Hh-responsive protein complexes. Nuclear IDX-1 protein levels on Western blots were increased by ectopic Hh expression, thereby providing a mechanism for Hh-mediated regulation of the insulin promoter. Addition of cyclopamine to INS-1 cells decreased IDX-1 messenger RNA expression. In transient transfections of a− 4.5-kb mouse IDX-1 promoter-reporter construct, ectopic Hh expression increased (and cyclopamine administration decreased) transcriptional activation of the IDX-1 promoter in a dose-dependent manner. Thus, the IDX-1 gene is a direct regulatory target of Hh signaling in insulin-producing pancreatic β-cells. We propose that Hh signaling activates the insulin gene promoter indirectly via the direct activation of IDX-1 expression. Because IDX-1 gene expression is essential for insulin gene expression, pancreatic β-cell development, and normal glucose homeostasis, our findings that Hh signaling regulates IDX-1 expression in the endocrine pancreas suggest possible novel therapeutic approaches for diabetes mellitus.
Immediate and Prolonged Patterns of Agouti-Related
Peptide-(83–132)-Induced c-Fos Activation in Hypothalamic and
Extrahypothalamic Sites*
Authors: Hagan Mary M. , Benoit Stephen C. , Rushing Paul A. , Pritchard Laurel M. , Woods Stephen C. , Seeley Randy J.
Published: January 2014

Several lines of evidence substantiate the important role of the central nervous system melanocortin 3- and 4-receptor (MC3/4-R) system in the control of food intake and energy balance. Agouti-related peptide (AgRP), an endogenous antagonist of these receptors, produces a robust and unique pattern of increased food intake that lasts up to 7 days after a single injection. Little is known about brain regions that may mediate this powerful effect of AgRP on food intake. To this end we compared c-Fos-like immunoreactivity (c-FLI) in several brain sites of rats injected intracerebroventricularly with 1 nmol AgRP-(83–132) 2 and 24 h before death and compared c-FLI patterns to those induced by another potent orexigenic peptide, neuropeptide Y (NPY). Although both NPY and AgRP induced c-FLI in hypothalamic areas, AgRP also produced increased c-FLI in the accumbens shell and lateral septum. Although NPY elicited no changes in c-FLI 24 h after administration, AgRP induced c-FLI in the accumbens shell, nucleus of the solitary tract, central amygdala, and lateral hypothalamus. These results indicate that an NPY-like hypothalamic circuit mediates the short-term effects of AgRP, but that the unique sustained effect of AgRP on food intake involves a complex circuit of key extrahypothalamic reward and feeding regulatory nuclei.
Inability of Overexpressed des(1–3)Human Insulin-Like Growth Factor I (IGF-I) to Inhibit Forced Mammary Gland Involution Is Associated with Decreased Expression of IGF Signaling Molecules*
Authors: Hadsell Darryl L. , Alexeenko Tatiana , Klemintidis Yann , Torres Daniel , Lee Adrian V.
Published: January 2014

Overexpression of des(1–3) human insulin-like growth factor I (IGF-I) in the mammary glands of transgenic mice (WAP-DES) inhibits apoptosis during natural, but not forced, mammary involution. We hypothesized that this differential response would correlate with the expression of IGF signal transducers. Forced and natural involution were analyzed in nontransgenic and WAP-DES mice beginning on day 16 postpartum. During natural involution, mammary gland wet weight was higher and apoptosis was lower in WAP-DES than in nontransgenic mice. The WAP-DES transgene had no effect on these parameters during forced involution. Mammary tissue concentrations of the transgene protein were 2- to 10-fold higher than those of endogenous IGF-I. Western blot analysis of pooled mammary tissue extracts demonstrated only slightly higher phosphorylation of the IGF signal transducers insulin receptor substrate-1 (IRS-1) and Akt in the WAP-DES than in nontransgenic mice. Dramatic early reductions in phospho-IRS-1, phospho-Akt, IRS-1, IRS-2, and Akt proteins occurred during forced, but not natural, involution. The abundance of the IGF-I receptor and the messenger RNAs for the IGF-I receptors, IRS-1 and -2, were not affected by either genotype or involution. These findings support the conclusions that mammary cells lose their responsiveness to insulin-like signals during forced involution, and that posttranscriptional or posttranslational regulation of IRS-1 and IRS-2 may play a role in this loss.
Receptor-Operated Osteoclast Calcium Sensing*
Authors: Bennett Brian D. , Alvarez Ulises , Hruska Keith A.
Published: January 2014

Osteoclasts “sense” elevated extracellular calcium, which leads to cytoskeletal changes that may be linked to phospholipase C (PLC) activation and the associated rise in intracellular calcium ([Ca2+]i). Since PLC is linked to transient receptor potential channels (trp), we hypothesized that receptor activated calcium influx due to this channel type would be activated by osteoclasts sensing [Ca2+]e. We found that high [Ca2+]e induced similar intracellular Ca2+ rises in chicken osteoclasts with or without intracellular Ca2+ store depletion by either TPEN or thapsigargin, thus defining store-insensitive Ca2+ influx. This store-insensitive calcium sensing component was blocked by the PLC antagonist U73122. Also, the calcium channel inhibitor SKF 96365, a blocker of store-independent trp-like channels, was effective in inhibiting calcium sensing in the presence of thapsigargin. Thus, a store-independent component of calcium sensing was associated with ion channels linked to PLC. Since receptor activated transient receptor potential (trp) family cation channels open in a PLC-dependent and store-independent manner, we suggest that receptor operated channels are activated in osteoclasts stimulated by high extracellular Ca2+.
The Insulin-Like Growth Factor I Receptor-Induced Interaction of Insulin Receptor Substrate-4 and Crk-II
Authors: Karas M. , Koval A. P. , Zick Y. , LeRoith D.
Published: January 2014

Stimulation of the insulin or insulin-like growth factor (IGF)-I receptor results in activation of several signaling pathways. Proteins of the insulin receptor substrate (IRS) family play important roles in mediating these signaling cascades. To date, four members of the IRS family of docking proteins have been characterized. Recently, we have reported that stimulation of the IGF-I receptor in 293 HEK cells regulates interaction of the newly discovered IRS-4 molecule with the Crk family of proteins. In the present study, we characterize the molecular basis of these interactions. C- and N termini truncation analysis of IRS-4 demonstrated that the region between amino acids 678 and 800 of the IRS-4 molecule is involved in this interaction. This region contains a cluster of four tyrosines (Y700, Y717, Y743, and Y779). We hypothesize that one or more of these tyrosines are involved in the interaction between the SH2 domain of the Crk-II molecule when IRS-4 is phosphorylated upon IGF-I receptor activation. Additional mutational analyses confirmed this hypothesis. Interestingly, none of these four tyrosines was individually critical for the interaction between Crk-II and IRS-4, but when all four tyrosines were simultaneously mutated to phenylalanine, the IGF-I induced interaction between these molecules was abolished. Taken together, these results suggest a novel mechanism of Crk-II binding to tyrosine phosphorylated proteins.
Estrogen Receptor (ER)α and ERβ Exhibit Unique Pharmacologic Properties When Coupled to Activation of the Mitogen-Activated Protein Kinase Pathway*
Authors: Wade Christian B. , Robinson Siobhan , Shapiro Robert A. , Dorsa Daniel M.
Published: January 2014

The rapid, nongenomic effects of estrogen are increasingly recognized as playing an important role in several aspects of estrogen action. Rapid activation of the mitogen-activated protein kinase (MAPK) signaling pathway by estrogen is among the more recently identified of these effects. To explore the role of estrogen receptors (ERs) in mediating these effects, we have transfected ER-negative Rat-2 fibroblasts with complementary DNA clones encoding either human ERα or rat ERβ and examined their ability to couple to activation of MAPK in response to 17β-estradiol (17β-E2) and other ligands. For both receptors, addition of E2 resulted in a rapid phosphorylation of MAPK. Activation of MAPK in ERα-transfected cells was partially and completely blocked by the antiestrogens tamoxifen and ICI 182,780, respectively. In ERβ-transfected cells, MAPK activation was less sensitive to inhibition by tamoxifen and ICI 182,780. We have also observed that, in this model system, a membrane-impermeable estrogen (BSA-E2) and 17α-E2 were both able to activate MAPK in a manner similar to E2 alone. Here also, ICI 182,780 blocked the ability of BSA-E2 to activate MAPK through ERα, but failed to block ERβ-mediated effects. BSA-E2 treatment, however, failed to activate nuclear estrogen-response-element-mediated gene transcription. These data show that these nuclear ERs are necessary for estrogen’s effects at the membrane. This model system will be useful in identifying molecular interactions involved in the rapid effects mediated by the ERs.
Analysis of Ovarian Gene Expression in Follicle-Stimulating Hormone β Knockout Mice*
Authors: Burns Kathleen H. , Yan Changning , Kumar T. Rajendra , Matzuk Martin M.
Published: January 2014

FSH is a heterodimeric glycoprotein hormone that is produced in the gonadotroph cells of the anterior pituitary. It acts on Sertoli cells of the testis and granulosa cells of the ovary. We previously demonstrated that FSHβ knockout female mice are infertile due to a block in folliculogenesis preceding antral stage development. To investigate aberrations of ovarian gene regulation in the absence of FSH, we analyzed the expression of several important marker genes using Northern blot and in situ hybridization techniques. Key findings are as follows: 1) Follicles of FSHβ knockout mice develop a well organized thecal layer, which is positive for P450 17α-hydroxylase and LH receptor messenger RNAs (mRNAs). This indicates that theca recruitment is completed autonomously with respect to FSH. 2) Granulosa cells in FSH-deficient mice demonstrate an increase in FSH receptor mRNA, and decreases in P450 aromatase, serum/glucocorticoid-induced kinase, and inhibin/activin subunit mRNAs. These data support studies that implicate FSH signaling cascades in the expression of these genes. 3) In contrast to the thecal layer, granulosa cell populations in FSHβ knockout mice do not accumulate LH receptor mRNA. This suggests that although the granulosa cells have a block in proliferation at the antral follicle stage in the absence of FSH, they do not initiate programs of terminal differentiation as seen in luteinizing cells of wild-type ovaries. 4) Ovaries of FSH-deficient mice demonstrate a modest decrease in cyclin D2 mRNA, without up-regulation of cell cycle inhibitor mRNAs associated with luteinization (i.e. p15, p27, and p21). Although components of the FSH null phenotype may be caused by partial cyclin D2 loss of function, these findings indicate that the mechanisms of granulosa cell cycle arrest in FSHβ knockout mice are distinct from those of cycle withdrawal at luteinization. Underscoring the usefulness of the FSH-deficient mouse model, this study clarifies aspects of gonadotropin-dependent folliculogenesis, thecal layer development, cycle control in granulosa cells, and luteinization.
Characterization of Signal Transduction Pathway in Neurotropic Action of Angiotensin II in Brain Neurons
Authors: Yang Hong , Wang Xiangyu , Raizada Mohan K.
Published: January 2014

Interaction of angiotensin II with the neuronal angiotensin type 1 receptor stimulates the PI3K signaling pathway. Our objective in this study was to investigate the hypothesis that the PI3K cascade regulates the neurotropic actions of angiotensin II in rat brain neurons. We followed growth associated protein-43 expression and neurite extension as markers of neurotropic activity. Angiotensin II, through its interaction with the angiotensin type 1 receptor, increased growth associated protein-43 expression and neurite extension. These effects were abolished by pretreatment of neurons with wortmannin and rapamycin, but not by PD 98059. Antisense oligonucleotides specific for p70S6 kinase also inhibited angiotensin II-stimulated neurotropic activity. These data confirm the involvement of PI3K and p70S6 kinase in angiotensin II-mediated neurotropic action. Further support for this was provided by the observation that angiotensin II caused a time-dependent stimulation of p70S6 kinase by an angiotensin type 1 receptor-mediated process. We also found that the neurotropic actions of angiotensin II are mediated by plasminogen activator inhibitor-1. Evidence for this includes 1) angiotensin II-stimulated neuronal plasminogen activator inhibitor-1 gene expression, 2) potent neurotropic action of exogenous plasminogen activator inhibitor-1, and 3) inhibitory neurotropic effect of angiotensin II by antisense oligonucleotide-mediated depletion of plasminogen activator inhibitor-1. Finally, we found that the neurotropic action of plasminogen activator inhibitor-1 is not blocked by either angiotensin type 1 receptor antagonist or inhibitors of PI3K or p70S6 kinase, indicating that the plasminogen activator inhibitor-1 step is downstream from the p70S6 kinase. These observations demonstrate that angiotensin II is a neurotropic hormone that engages a distinct PI3K-p70S6 kinase-plasminogen activator inhibitor-1 signaling pathway for this action.
Knockout Mice Lacking Steroidogenic Factor 1 Are a Novel Genetic Model of Hypothalamic Obesity
Authors: Majdic Gregor , Young Morag , Gomez-Sanchez Elise , Anderson Paul , Szczepaniak Lidia S. , Dobbins Robert L. , McGarry J. Denis , Parker Keith L.
Published: January 2014

Knockout (KO) mice lacking steroidogenic factor 1 (SF-1) exhibit a phenotype that includes adrenal and gonadal agenesis, impaired gonadotropin expression, and abnormalities of the ventromedial hypothalamic nucleus (VMH). Studies in rodents with lesions of the ventromedial hypothalamus have implicated the VMH in body weight regulation, suggesting that SF-1 KO mice may provide a genetic model of obesity. To prevent death, SF-1 KO mice were rescued with corticosteroid injections, followed by syngeneic adrenal transplants from wild-type (WT) littermates. Corticosterone and ACTH levels in WT and SF-1 KO mice were indistinguishable, documenting restoration of hypothalamic-pituitary-adrenal function. Although weights at earlier ages did not differ significantly from WT littermates, SF-1 KO mice were significantly heavier by 8 wk of age and eventually weighed almost twice as much as WT controls. Obesity in SF-1 KO mice predominantly resulted from decreased activity rather than increased food intake. Leptin was increased markedly, insulin was modestly elevated, and glucose was indistinguishable from WT mice. Although sex steroids in rodents affect weight, ovariectomy did not abolish the weight difference between WT and SF-1 KO mice. These SF-1 KO mice are a genetic model of late-onset obesity that may help elucidate the role of the VMH in weight regulation.
Molecular Regulation of the IGF-Binding Protein-4 Protease System in Human Fibroblasts: Identification of a Novel Inducible Inhibitor
Authors: Chen Bing-Kun , Overgaard Michael T. , Bale Laurie K. , Resch Zachary T. , Christiansen Michael , Oxvig Claus , Conover Cheryl A.
Published: January 2014

The IGF-binding protein-4 (IGFBP-4) protease system is an important regulator of local IGF bioavailability and cell growth. Recently, the IGF-dependent IGFBP-4 protease secreted by cultured human fibroblasts was identified as pregnancy-associated plasma protein A (PAPP-A). In pregnancy serum, PAPP-A circulates as a disulfide-bound complex with the precursor form of major basic protein (pro-MBP), and in this complex PAPP-A’s proteolytic activity is not evident. In this study we analyzed the IGFBP-4 protease system in normal human fibroblasts to determine regulation outside of pregnancy. Treatment with the phorbol ester tumor promoter, β-phorbol 12,13-didecanoate (β-PDD), resulted in time-dependent inhibition of the IGF-dependent IGFBP-4 protease activity in cell-conditioned medium, which was evident at 6 h and complete by 24 h. PAPP-A mRNA was constitutively expressed in control cells, and levels were decreased only after 24 h of β-PDD treatment. Secretion of PAPP-A protein into conditioned medium did not change with β-PDD treatment. On the other hand, pro-MBP mRNA was undetectable in control human fibroblasts, and treatment with β-PDD induced pro-MBP mRNA and protein expression within 6 h. β-PDD-induced pro-MBP mRNA expression and protease inhibition were blocked with an inhibitor of RNA synthesis, actinomycin D. Actinomycin D had no effect on PAPP-A mRNA levels in the absence or presence of β-PDD. Similarly, transformation of human fibroblasts with simian virus 40 large T antigen resulted in the synthesis of pro-MBP mRNA and protein and inhibition of IGFBP-4 protease activity. Coculture of fibroblasts with cells transfected with pro-MBP cDNA resulted in inhibition of IGFBP-4 proteolytic activity without having any effect on PAPP-A synthesis. In summary, phorbol ester tumor promoters and simian virus 40 transformation regulate IGFBP-4 proteolysis in human fibroblasts through induction of a novel inhibitor of PAPP-A, pro-MBP. These findings expand our understanding of the IGFBP-4 protease system and suggest an additional level of local cell growth control.
Transcriptional Activation of the Ovine Follicle-Stimulating Hormone-β Gene by Gonadotropin- Releasing Hormone Involves Multiple Signal Transduction Pathways
Authors: Vasilyev Vyacheslav V. , Pernasetti Flavia , Rosenberg Suzanne B. , Barsoum Mark J. , Austin Darrell A. , Webster Nicholas J. G. , Mellon Pamela L.
Published: January 2014

GnRH regulates gonadotrope cells through GnRH receptor activation of the PKC-, MAPK-, and calcium-activated signaling cascades. Due to the paucity of homologous model systems expressing FSHβ, little is known about the specific mechanisms involved in transcriptional regulation of this gene by GnRH. Previous studies from our laboratory demonstrated that the gonadotrope-derived LβT2 cell line expresses FSHβ mRNA. In the present study we characterized the mechanisms involved in GnRH regulation of the FSHβ promoter using this cell model. Using transfection assays, we show that GnRH regulation of the ovine FSHβ promoter involves at least two elements, present between −4152/−2878 and −2550/−1089 bp, in association with one or several elements within the proximal region of the promoter. Surprisingly, the two activating protein-1 sites previously shown to be involved in the FSHβ response to GnRH in heterologous cells do not play a role in GnRH responsiveness in the gonadotrope cell model. Here we demonstrate that calcium influx itself is not sufficient to confer the response, but it is necessary for both 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and GnRH induction of the FSHβ gene. Moreover, we show that GnRH regulation of FSHβ gene expression is mediated by PKC and establish the presence of multiple PKC isozymes in LβT2 cells. Interestingly, GnRH and TPA induce activity of the FSHβ promoter through different, although possibly overlapping, pools of PKC isoforms. This is further supported by the use of a MAPK inhibitor, which abolishes the induction of FSHβ by GnRH, but not by TPA. In conclusion, we have demonstrated that calcium, PKC, and MAPK signaling systems are all involved in the induction of FSHβ gene expression by GnRH in the LβT2 mouse gonadotrope cell model.