bluuebunny/crossref_metadata_2025 huggingface dataset:
Nephroblastoma Overexpressed (Nov) Is a Novel Bone Morphogenetic Protein Antagonist
Authors: CANALIS ERNESTO
Published: December 2007

Abstract:  Nephroblastoma overexpressed (Nov), a member of the CCN family of proteins, is expressed by osteoblasts and its transcription is regulated by transforming growth factor (TGF)‐β and bone morphogenetic proteins (BMP). CCN proteins can interact with TGF‐β, BMPs, and Wnt. We explored the function of Nov in skeletal cells, in vitro and in vivo. Constitutive overexpression of Nov in cells of the osteoblastic lineage impaired osteoblastic differentiation, opposed the biological effects of BMP‐2 and Wnt 3 and impaired BMP‐2 and Wnt signaling, indicating that Nov has BMP‐2 antagonistic activity. Transgenic mice overexpressing Nov under the control of the osteocalcin promoter exhibited osteopenia secondary to decreased bone formation, confirming the effects in vitro. GST pulldown experiments demonstrated direct Nov–BMP interactions. In conclusion, Nov has BMP antagonistic properties and inhibits osteoblastogenesis and osteoblastic function.
Proresorptive Actions of FSH and Bone Loss
Authors: ZAIDI MONE , BLAIR HARRY C. , IQBAL JAMEEL , ZHU LING LING , KUMAR T. RAJENDRA , ZALLONE ALBERTA , SUN LI
Published: December 2007

Abstract:  We review studies that propose follicle‐stimulating hormone (FSH) as a physiologic stimulator of osteoclastic bone resorption. We hypothesize that, in addition to low estrogen, a rising FSH contributes to the increased bone resorption and bone loss in hypergonadism. This is of particular relevance to the perimenopausal transition, wherein profound bone loss is accompanied by trabecular perforations in the face of high FSH and normal estrogen levels. Potential therapeutic implications include the development of antagonists to both circulating FSH and its osteoclastic receptor.
Glucocorticoids and the Osteoclast
Authors: KIM HYUN‐JU , ZHAO HAIBO , KITAURA HIDEKI , BHATTACHARYYA SANDIP , BREWER JUDSON A. , MUGLIA LOUIS J. , PATRICK ROSS F. , TEITELBAUM STEVEN L.
Published: December 2007

Abstract:  Glucocorticoid (GC)‐induced bone loss is the most common cause of secondary osteoporosis but its pathogenesis is controversial. GCs clearly suppress bone formation in vivo but the means by which they impact osteoblasts is unclear. Because bone remodeling is characterized by tethering of the activities of the two cells, the osteoclast is a potential modulator of the effect of GCs on osteoblasts. To address this issue we compared the effects of dexamethasone on wild‐type (WT) osteoclasts with those derived from mice with disruption of the GC receptor in osteoclast lineage cells and found that the bone‐degrading capacity of GC‐treated WT cells is suppressed. The inhibitory effect of dexamethasone on bone resorption reflects failure of osteoclasts to organize their cytoskeleton in response to M‐CSF. Dexamethasone specifically arrests M‐CSF activation of RhoA, Rac, and Vav3, each of which regulate the osteoclast cytoskeleton. In all circumstances, mice lacking the GC receptor in osteoclast lineage cells are spared the impact of dexamethasone on osteoclasts and their precursors. Consistent with osteoclasts modulating the osteoblast‐suppressive effect of dexamethasone, GC receptor‐deficient mice are protected from the steroid's inhibition of bone formation.
Mechanisms of Osteoclastic Secretion
Authors: ZHAO HAIBO , PATRICK ROSS F.
Published: December 2007

Abstract:  A tight balance between bone resorption by osteoclasts and bone formation by osteoblasts is required for the maintenance of bone mass and integrity. A net increase in bone resorption over formation results in osteoporosis, a disease associated with significantly morbidity and mortality. Following attachment via the integrin αvβ3, osteoclasts degrade bone by generation of the ruffled border, the unique resorptive organelle of the cell. The adherent cell then secretes into the subcellular space protons and acidic proteases. We review here the concepts relating to the mechanisms of regulated secretion and provide preliminary data on the role of one protein important for secretion by osteoclasts.
Bone Loss in Thyroid Disease
Authors: ABE ETSUKO , SUN LI , MECHANICK JEFFREY , IQBAL JAMEEL , YAMOAH KOSJ , BALIRAM RAMKUMARIE , ARABI ARIO , MOONGA BALJIT S. , DAVIES TERRY F. , ZAIDI MONE
Published: December 2007

Abstract:  More than 10% of postmenopausal women in the United States receive thyroid hormone replacement therapy and up to 20% of these women are over‐replaced inducing subclinical hyperthyroidism. Because hyperthyroidism and post menopausal osteoporosis overlap in women of advancing age, it is urgent to understand the effect of thyroid hormone excess on bone. We can now provide results that not thyroid hormones but also TSH itself has an equally important role to play in bone remodeling.
Similarities and Contrasts in Ryanodine Receptor Localization and Function in Osteoclasts and Striated Muscle Cells
Authors: HUANG CHRISTOPHER L.‐H. , SUN LI , FRASER JAMES A. , GRACE ANDREW A. , ZAIDI MONE
Published: December 2007

Abstract:  This review compares ryanodine receptor (RyR)‐mediated Ca2+ signaling processes in muscle and osteoclast cells. In muscle, RyR‐mediated release of an intracellularly stored, sarcoplasmic reticular (SR), Ca2+ is triggered by voltage‐sensitive dihydropyridine receptor (DHPR)‐L‐type Ca2+ channels either through an allosteric coupling with the RyR in skeletal muscle or a Ca2+‐induced Ca2+ release initiated by extracellular Ca2+ entry in cardiac muscle. Both cell subtypes are nevertheless capable of Ca2+‐induced SR Ca2+ release with cardiac muscle additionally showing a store overload‐induced Ca2+ release (SOICR) driven by SR luminal Ca2+ under some pathological conditions. Osteoclasts similarly show cytosolic Ca2+ elevations driven by release of intracellular Ca2+ stores that culminate in motile activity in turn modifying bone resorptive activity. However, such triggering is controlled by ambient Ca2+ rather than membrane potential with features strongly suggestive of control by a surface membrane Ca2+ receptor. Yet common actions of the RyR‐specific agents perchlorate, dantrolene Na, ryanodine, caffeine, adenosine 3′,5′‐cyclic diphosphate ribose (cADPr) and ruthenium red implicate RyR in signaling in all these cell types. These findings were reconciled by reports confirming and uniquely localizing a cell surface rather than microsomal osteoclastic RyR that might itself detect ambient Ca2+ possibly through its otherwise intraluminal positioned low‐affinity Ca2+‐binding site in parallel with the SOICR mechanism in cardiac muscle. Such a mechanism could interact with other osteoclast processes transferring Ca2+ between cytosol, intracellular stores and extracellular space and be integrated with systemic processes regulating Ca2+ homeostasis.
T Cells: Unexpected Players in the Bone Loss Induced by Estrogen Deficiency and in Basal Bone Homeostasis
Authors: WEITZMANN M. NEALE , PACIFICI ROBERTO
Published: December 2007

Abstract:  The bone–immune interface has become a subject of intense interest in recent years. It has long been recognized that infection, inflammation, and autoimmune disorders are associated with systemic and local bone loss. Yet, it is only recently that T lymphocytes and their products have been recognized as key regulators of osteoclast formation, life span, and activity. Similarly, sex steroids and aging have been known to regulate the immune system and T cells for decades. In spite of the abundance of clinical and physiological clues, it is only in the last few years that investigators have linked immune cells to the etiology of postmenopausal and senile osteoporosis, as well as to the bone loss caused by a variety of endocrine conditions. As surprising is new evidence showing that in contrast to their bone destructive effects under certain pathological conditions, T cells are highly protective of basal bone homeostasis, through complex regulatory effects on osteoprotegerin (OPG) production by B cells, involving CD40 to CD40 Ligand (CD40L) costimulation. This article examines the experimental evidence suggesting that estrogen prevents bone loss by regulating T cell function, and that T cell costimulation with B cells is critical for OPG production and maintenance of basal bone homeostasis.
Vitamin D
Authors: CHRISTAKOS SYLVIA , DHAWAN PUNEET , BENN BRYAN , PORTA ANGELA , HEDIGER MATTHIAS , OH GOO T. , JEUNG EUI‐BAE , ZHONG YAN , AJIBADE DARE , DHAWAN KOPAL , JOSHI SNEHA
Published: December 2007

Abstract:  Vitamin D maintains calcium homeostasis and is required for bone development and maintenance. Recent evidence has indicated an interrelationship between vitamin D and health beyond bone, including effects on cell proliferation and on the immune system. New developments in our lab related to the function and regulation of target proteins have provided novel insights into the mechanisms of vitamin D action. Studies in our lab have shown that the calcium‐binding protein, calbindin, which has been reported to be a facilitator of calcium diffusion, also has an important role in protecting against apoptotic cell death in different tissues including protection against cytokine destruction of osteoblastic and pancreatic β cells. These findings have important implications for the therapeutic intervention of many disorders including diabetes and osteoporosis. Recent studies in our laboratory of intestinal calcium absorption using calbindin‐D9k null mutant mice as well as mice lacking the 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) inducible epithelial calcium channel, TRPV6, provide evidence for the first time of calbindin‐D9k and TRPV6 independent regulation of active calcium absorption. Besides calbindin, the other major target of 1,25(OH)2D3 in intestine and kidney is 25(OH)D3 24 hydroxylase (24(OH)ase), which is involved in the catabolism of 1,25(OH)2D3. In our laboratory we have identified various factors that cooperate with the vitamin D receptor in regulating 24(OH)ase expression including C/EBP β, SWI/SNF (complexes that remodel chromatin using the energy of ATP hydrolysis) and the methyltransferases, CARM1 and G9a. Evidence is also presented for C/EBP β as a nuclear coupling factor that coordinates regulation of osteopontin by 1,25(OH)2D3 and PTH. Our findings define novel mechanisms that may be of fundamental importance in understanding how 1,25(OH)2D3 mediates its multiple biological effects.
Intracellular Protein Degradation From a Vague Idea through the Lysosome and the Ubiquitin‐Proteasome System and on to Human Diseases and Drug Targeting
Authors: CIECHANOVER AARON
Published: December 2007

Abstract:  Between the 1950s and 1980s, scientists were focusing mostly on how the genetic code is transcribed to RNA and translated to proteins, but how proteins are degraded has remained a neglected research area. With the discovery of the lysosome by Christian de Duve, it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental evidence strongly suggested that intracellular proteolysis is largely nonlysosomal, but the mechanisms involved had remained obscure. The discovery of the ubiquitin–proteasome system resolved this enigma. We now recognize that ubiquitin‐ and proteasome‐mediated degradation of intracellular proteins is involved in the regulation of a broad array of cellular processes, such as cell cycle and division, regulation of transcription factors, and assurance of the cellular quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of many human diseases, malignancies, and neurodegenerative disorders among them, which led subsequently to an increasing effort to develop mechanism‐based drugs; one is already in use.
Evolution, Aging, and Osteoporosis
Authors: TUNG STEPHANIE , IQBAL JAMEEL
Published: December 2007

Abstract:  Aging is a process whereby organisms lose to the capacity to effectively handle life's stresses. Associated with aging are pathophysiological processes, such as decreases in bone mass, which in the extreme form lead to significant morbidity. Evolutionary theory suggests that these pathophysiological processes are permitted to occur because an organism need only preserve its body against stress only for the amount of time needed for effective reproduction. In this review, an understanding of osteoporosis and bone loss is discussed within the context of aging theory. Specific topics covered include: (1) bone loss as an antagonistically pleiotropic physiological process, (2) age‐associated stress accumulation and its negative impact on bone turnover, and (3) the mechanisms whereby gonadal failure, increases in inflammatory cytokines, and cellular bone marrow changes lead to bone loss. This review concludes by examining purported hypotheses in the context of Werner's syndrome, a disease characterized by premature aging. We suggest that future osteoporosis therapy will likely focus on prevention of aging in general as a means to prevent the development of osteoporosis.
Transcriptional Regulation of Osteoblasts
Authors: FRANCESCHI RENNY T. , GE CHUNXI , XIAO GUOZHI , ROCA HERNAN , JIANG DI
Published: December 2007

Abstract:  The differentiation of osteoblasts from mesenchymal precursors requires a series of cell fate decisions controlled by a hierarchy of transcription factors. Among these are RUNX2, Osterix (OSX), ATF4, and a large number of nuclear coregulators. During bone development, initial RUNX2 expression coincides with the formation of mesenchymal condensations well before the branching of chondrogenic and osteogenic lineages. Given that RUNX2 is expressed so early and participates in several stages of bone formation, it is not surprising that it is subject to a variety of controls. These include regulation by nuclear accessory factors and posttranslational modification, especially phosphorylation. Specific examples of RUNX2 regulation include interactions with DLX proteins and ATF4 and phosphorylation by the ERK/MAP kinase pathway. RUNX2 is regulated via phosphorylation of critical serine residues in the P/S/T domain. MAPK activation of RUNX2 was also found to occur in vivo. Transgenic expression of constitutively active MEK1 in osteoblasts accelerated skeletal development while a dominant‐negative MEK1 retarded development in a RUNX2‐dependent manner. These studies allow us to begin understanding the complex mechanisms necessary to fine‐tune bone formation in response to extracellular stimuli including ECM interactions, mechanical loads, and hormonal stimulation.
New Roles for Osteoclasts in Bone
Authors: BOYCE BRENDAN F. , YAO ZHENQIANG , ZHANG QIAN , GUO ROULIN , LU YAN , SCHWARZ EDWARD M. , XING LIANPING
Published: December 2007

Abstract:  Osteoclasts have long been recognized as the cells that resorb bone in normal bone remodeling and in pathologic conditions in which bone resorption is increased. They are derived from precursors in the mononuclear phagocyte lineage, which arise in the bone marrow and fuse with one another to form the multinucleated cells that resorb calcified matrixes under the influence of osteoblastic cells in bone marrow. There is growing evidence that osteoclast precursors (OCPs) and osteoclasts have functions in and around bone other than bone resorption. For example, they modulate the differentiation of other cells, including osteoblastic cells; they regulate hematopoietic stem cell movement from the bone marrow to the bloodstream; and they are secretory cells that participate in immune responses. In this article, we review these findings, which support new roles for osteoclasts and OCPs in the growing field of osteoimmunology and in common pathologic conditions affecting bones and joints.
Substrate-Bound Insulin-Like Growth Factor (IGF)-I-IGF Binding Protein-Vitronectin-Stimulated Breast Cell Migration Is Enhanced by Coactivation of the Phosphatidylinositide 3-Kinase/AKT Pathway by αv-Integrins and the IGF-I Receptor
Authors: Hollier Brett G. , Kricker Jennifer A. , Van Lonkhuyzen Derek R. , Leavesley David I. , Upton Zee
Published: December 2007

IGF-I can bind to the extracellular matrix protein vitronectin (VN) through the involvement of IGF-binding proteins-2, -3, -4, and -5. Because IGF-I and VN have established roles in tumor cell dissemination, we were keen to investigate the functional consequences of the interaction of IGF-I, IGF binding proteins (IGFBPs), and VN in tumor cell biology. Hence, functional responses of MCF-7 breast carcinoma cells and normal nontumorgenic MCF-10A mammary epithelial cells were investigated to allow side-by-side comparisons of these complexes in both cancerous and normal breast cells. We demonstrate that substrate-bound IGF-I-IGFBP-VN complexes stimulate synergistic increases in cellular migration in both cell types. Studies using IGF-I analogs determined this stimulation to be dependent on both heterotrimeric IGF-I-IGFBP-VN complex formation and the involvement of the IGF-I receptor (IGF-IR). Furthermore, the enhanced cellular migration was abolished on incubation of MCF-7 and MCF-10A cells with function blocking antibodies directed at VN-binding integrins and the IGF-IR. Analysis of the signal transduction pathways underlying the enhanced cell migration revealed that the complexes stimulate a transient activation of the ERK/MAPK signaling pathway while simultaneously producing a sustained activation of the phosphatidylinositide 3-kinase/AKT pathway. Experiments using pharmacological inhibitors of these pathways determined a requirement for phosphatidylinositide 3-kinase/AKT activation in the observed response. Overexpression of wild type and activated AKT further increases substrate-bound IGF-I-IGFBP-VN-stimulated migration. This study provides the first mechanistic insights into the action of IGF-I-IGFBP-VN complexes and adds further evidence to support the involvement of VN-binding integrins and their cooperativity with the IGF-IR in the promotion of tumor cell migration.
RFamide-Related Peptide Gene Is a Melatonin-Driven Photoperiodic Gene
Authors: Revel Florent G. , Saboureau Michel , Pévet Paul , Simonneaux Valérie , Mikkelsen Jens D.
Published: December 2007

In seasonal species, various physiological processes including reproduction are organized by photoperiod via melatonin, but the mechanisms of melatonin action are still unknown. In birds, the peptide gonadotropin-inhibiting hormone (GnIH) has been shown to have inhibitory effects on reproductive activity and displays seasonal changes of expression. Here we present evidence in mammals that the gene orthologous to GnIH, the RFamide-related peptide (RFRP) gene, expressed in the mediobasal hypothalamus, is strongly regulated by the length of the photoperiod, via melatonin. The level of RFRP mRNA and the number of RFRP-immunoreactive cell bodies were reduced in sexually quiescent Syrian and Siberian hamsters acclimated to short-day photoperiod (SD) compared with sexually active animals maintained under long-day photoperiod (LD). This was contrasted in the laboratory Wistar rat, a non-photoperiodic breeder, in which no evidence for RFRP photoperiodic modulation was seen. In Syrian hamsters, the reduction of RFRP expression in SD was independent from secondary changes in gonadal steroids. By contrast, the photoperiodic variation of RFRP expression was abolished in pinealectomized hamsters, and injections of LD hamsters with melatonin for 60 d provoked inhibition of RFRP expression down to SD levels, indicating that the regulation is dependent on melatonin. Altogether, these results demonstrate that in these hamster species, the RFRP neurons are photoperiodically modulated via a melatonin-dependent process. These observations raise questions on the role of RFRP as a general inhibitor of reproduction and evoke new perspectives for understanding how melatonin controls seasonal processes via hypothalamic targets.
Aldosterone Induces Superoxide Generation via Rac1 Activation in Endothelial Cells
Authors: Iwashima Fumiko , Yoshimoto Takanobu , Minami Isao , Sakurada Maya , Hirono Yuki , Hirata Yukio
Published: December 2007

Currently, aldosterone is believed to be involved in the development of cardiovascular injury as a potential cardiovascular risk hormone. However, its exact cellular mechanisms remain obscure. This study was undertaken to examine the effect of aldosterone on superoxide production in cultured rat aortic endothelial cells with possible involvement of the small GTP-binding (G) protein Rac1. The aldosterone levels showed a time-dependent (6–24 h) and dose-dependent (10−8 to 10−6m) increase in superoxide generation, whose effect was abolished by mineralocorticoid receptor antagonist (eplerenone), Src inhibitor (PP2), and reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase inhibitor (apocynin). Aldosterone activated NADP(H) oxidase and Rac1, whose effects were abolished by eplerenone. The aldosterone-induced superoxide generation was abolished either by nonselective small G protein inhibitor (Clostridium difficile toxin A) or dominant-negative Rac1. Dominant-negative Rac1 also inhibited aldosterone-induced ACE gene expression. Thus, the present study is the first to demonstrate that aldosterone induces superoxide generation via mineralocorticoid receptor-mediated activation of NAD(P)H-oxidase and Rac1 in endothelial cells, thereby contributing to the development of aldosterone-induced vascular injury.
Resistance to the Nitric Oxide/Cyclic Guanosine 5′-Monophosphate/Protein Kinase G Pathway in Vascular Smooth Muscle Cells from the Obese Zucker Rat, a Classical Animal Model of Insulin Resistance: Role of Oxidative Stress
Authors: Russo I. , Del Mese P. , Doronzo G. , Mattiello L. , Viretto M. , Bosia A. , Anfossi G. , Trovati M.
Published: December 2007

Some in vivo and ex vivo studies demonstrated a resistance to the vasodilating effects of nitric oxide (NO) in insulin-resistant states and, in particular, obese Zucker rats (OZR). To evaluate the biochemical basis of this phenomenon, we aimed to identify defects of the NO/cGMP/cGMP-dependent protein kinase (PKG) pathway in cultured vascular smooth muscle cells (VSMCs) from OZR and lean Zucker rats (LZR) by measuring: 1) NO donor ability to increase cGMP in the absence and presence of inhibitors of soluble guanylate cyclase (sGC) and phosphodiesterases (PDEs); 2) NO and cGMP ability to induce, via PKG, vasodilator-stimulated phosphoprotein (VASP) phosphorylation at serine 239 and PDE5 activity; 3) protein expression of sGC, PKG, total VASP, and PDE5; 4) superoxide anion concentrations and ability of antioxidants (superoxide dismutase+catalase and amifostine) to influence the NO/cGMP/PKG pathway activation; and 5) hydrogen peroxide influence on PDE5 activity and VASP phosphorylation. VSMCs from OZR vs. LZR showed: 1) baseline cGMP concentrations higher, at least in part owing to reduced catabolism by PDEs; 2) impairment of NO donor ability to increase cGMP, even in the presence of PDE inhibitors, suggesting a defect in the NO-induced sGC activation; 3) reduction of NO and cGMP ability to activate PKG, indicated by the impaired ability to phosphorylate VASP at serine 239 and to increase PDE5 activity via PKG; 4) similar baseline protein expression of sGC, PKG, total VASP, and PDE5; and 5) higher levels of superoxide anion. Antioxidants partially prevented the defects of the NO/cGMP/PKG pathway observed in VSMCs from OZR, which were reproduced by hydrogen peroxide in VSMCs from LZR, suggesting the pivotal role of oxidative stress.
Dehydroepiandrosterone Stimulates Endothelial Proliferation and Angiogenesis through Extracellular Signal-Regulated Kinase 1/2-Mediated Mechanisms
Authors: Liu Dongmin , Iruthayanathan Mary , Homan Laurie L. , Wang Yiqiang , Yang Lingling , Wang Yao , Dillon Joseph S.
Published: December 2007

Dehydroepiandrosterone (DHEA) activates a plasma membrane receptor on vascular endothelial cells and phosphorylates ERK 1/2. We hypothesize that ERK1/2-dependent vascular endothelial proliferation underlies part of the beneficial vascular effect of DHEA. DHEA (0.1–10 nm) activated ERK1/2 in bovine aortic endothelial cells (BAECs) by 15 min, causing nuclear translocation of phosphorylated ERK1/2 and phosphorylation of nuclear p90 ribosomal S6 kinase. ERK1/2 phosphorylation was dependent on plasma membrane-initiated activation of Gi/o proteins and the upstream MAPK kinase because the effect was seen with albumin-conjugated DHEA and was blocked by pertussis toxin or PD098059. A 15-min incubation of BAECs with 1 nm DHEA (or albumin-conjugated DHEA) increased endothelial proliferation by 30% at 24 h. This effect was not altered by inhibition of estrogen or androgen receptors or nitric oxide production. There was a similar effect of DHEA to increase endothelial migration. DHEA also increased the formation of primitive capillary tubes of BAECs in vitro in solubilized basement membrane. These rapid DHEA-induced effects were reversed by the inhibition of either Gi/o-proteins or ERK1/2. Additionally, DHEA enhanced angiogenesis in vivo in a chick embryo chorioallantoic membrane assay. These findings indicate that exposure to DHEA, at concentrations found in human blood, causes vascular endothelial proliferation by a plasma membrane-initiated activity that is Gi/o and ERK1/2 dependent. These data, along with previous findings, define an important vascular endothelial cell signaling pathway that is activated by DHEA and suggest that this steroid may play a role in vascular function.
Roles of Rho Guanosine 5′-Triphosphatase A, Rho Kinases, and Extracellular Signal Regulated Kinase (1/2) in Prostaglandin E2-Mediated Migration of First-Trimester Human Extravillous Trophoblast
Authors: Nicola Catalin , Chirpac Andrei , Lala Peeyush K. , Chakraborty Chandan
Published: December 2007

Prostaglandin (PG) E2 may regulate invasiveness of human placenta because we previously reported stimulation of migration of placental trophoblasts by PGE2 acting through PGE receptor (EP)-1 and activating calpain. RhoA GTPase and its important effector Rho kinase (ROCK) have also been previously shown to regulate trophoblast migration. Using immortalized HTR-8/SVneo trophoblast cells and first-trimester human chorionic villus explant cultures on matrigel, we further examined the role of RhoA/ROCK and MAPK (ERK1/2) pathways on PGE2-mediated stimulation of trophoblast migration. Migration of cytotrophoblasts was shown to be inhibited by treatment of the trophoblast cell line and chorionic villus explants with either cell-permeable C3 transferase or selective RhoA small interfering RNA. These inhibitions were significantly mitigated by the addition of PGE2, an EP1/EP3 agonist or an EP3/EP4 agonist, suggesting that RhoA plays an important role in trophoblast migration but may not be obligatory for PGE2 action. Treatment of HTR-8/SVneo cells with nonselective ROCK inhibitor Y27632 or ROCK small interfering RNAs inhibited migration of these cells, which could not be rescued with PGE2 or the other two EP agonists, suggesting the obligatory role of ROCK in PGE2-induced migratory response. Furthermore, U0126, an inhibitor of MAPK kinases MEK1 and MEK2, abrogated PGE2-induced migration of trophoblasts, and PGE2 or the other two EP agonists stimulated ERK1/2 activation in trophoblasts, which was not abrogated by pretreatment with C3 transferase, indicating that ERK signaling pathway is an efficient alternate pathway for RhoA in PGE2-mediated migration of trophoblasts. These results suggest that ROCK and ERK1/2 play more important roles than RhoA in PGE2-mediated migration stimulation of first-trimester trophoblasts.
Norepinephrine Suppresses Gonadotropin-Releasing Hormone Neuron Excitability in the Adult Mouse
Authors: Han Seong-Kyu , Herbison Allan E.
Published: December 2007

Norepinephrine (NE) is considered to exert an important modulatory influence upon the activity of GnRH neurons. In the present study, we used a transgenic GnRH-green fluorescent protein mouse model to examine the effects of NE on the electrical excitability of GnRH neurons in male and female mice. Gramicidin-perforated patch recordings demonstrated that NE (10–100 μm) exerted a robust membrane hyperpolarization, with associated suppression of firing, in more than 85% of male prepubertal and adult GnRH neurons (n = 25). The same hyperpolarizing action was observed in female GnRH neurons from diestrous (91%, n = 11), proestrous (50%, n = 14), estrous (77%, n = 13), and ovariectomized (82%, n = 11) mice. A subpopulation (<10%) of silent GnRH neurons in all groups responded to NE with hyperpolarization followed by the initiation of firing upon NE washout. The hyperpolarizing actions of NE were mimicked by α1-adrenergic (phenylephrine) and β-adrenergic (isoproterenol) receptor agonists, but α2 receptor activation (guanabenz) had no effect. Approximately 75% of the NE-evoked hyperpolarization was blocked by the α1 receptor antagonist prazosin, and 75% of GnRH neurons responded to both phenylephrine and isoproterenol. These findings indicate that NE acts through both α1- and β-adrenergic receptors located on the soma/dendrites of GnRH neurons to directly suppress their excitability throughout the estrous cycle and after ovariectomy. These data force a reanalysis of existing models explaining the effects of NE on gonadotropin secretion.
Cloning, Expression, and Functional Characterization of Relaxin Receptor (Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 7) Splice Variants from Human Fetal Membranes
Authors: Kern András , Hubbard Daniela , Amano Aaron , Bryant-Greenwood Gillian D.
Published: December 2007

The relaxin receptor [leucine-rich repeat-containing G protein-coupled receptor 7 (LGR7)] belongs to the leucine-rich repeat containing G protein-coupled receptors subgroup C. Three new LGR7 splice variants have been cloned from the human fetal membranes and shown to be truncated versions of the full-length receptor, encoded by different lengths of the extracellular domain. The expression of their mRNAs has been confirmed by both qualitative and quantitative PCR and shown to be higher in the chorion and decidua before, compared with after, spontaneous labor. When HEK293 cells were transfected with each LGR7 splice variant, their proteins were retained within the endoplasmic reticulum. However, the protein for the shortest variant was also secreted into the medium. We have characterized the intracellular functions and effects of these LGR7 variants on the function of the wild-type (WT)-LGR7. In coexpression studies, each splice variant interacted directly with the WT-LGR7 and exerted a dominant-negative effect on cAMP accumulation by the WT-LGR7 after relaxin treatment. This interaction resulted in the sequestration of the WT-LGR7 inside the cells by down-regulation of its maturation and cell surface delivery. The constitutive homodimerization of WT-LGR7 has been shown here to take place in the endoplasmic reticulum, and the presence of any one of the splice variants decreased this by the formation of heterodimers with the WT-LGR7, supporting the view that homodimerization is a prerequisite for receptor trafficking to the cell surface. These data suggest that the dominant-negative effects of the LGR7 splice variants expressed in the chorion and decidua could be functionally significant in the peripartal period by inhibiting the function of WT-LGR7 and dampening the responsiveness of these tissues to endogenous relaxin.
Disruption of Growth Hormone Signaling Retards Prostate Carcinogenesis in the Probasin/TAg Rat
Authors: Wang Zhuohua , Luque Raul M. , Kineman Rhonda D. , Ray Vera H. , Christov Konstantin T. , Lantvit Daniel D. , Shirai Tomoyuki , Hedayat Samad , Unterman Terry G. , Bosland Maarten C. , Prins Gail S. , Swanson Steven M.
Published: December 2007

We asked whether down-regulation of GH signaling could block carcinogenesis in the Probasin/TAg rat, a model of aggressive prostate cancer. The Spontaneous Dwarf rat, which lacks GH due to a mutation (dr) in its GH gene, was crossed with the Probasin/TAg rat, which develops prostate carcinomas at 100% incidence by 15 wk of age. Progeny were heterozygous for the TAg oncogene and homozygous for either the wild-type GH gene (TAg/Gh+/+) or the dr mutation (TAg/Ghdr/dr). Prostate tumor incidence and burden were significantly reduced, and tumor latency was delayed in TAg/Ghdr/dr rats relative to TAg/Gh+/+ controls. At 25 wk of age, loss of GH resulted in a 20 and 80% decrease in the area of microinvasive carcinoma in the dorsal and lateral lobes, respectively. By 52 wk of age, invasive prostate adenocarcinomas were observed in all TAg/Gh+/+ rats, whereas the majority of TAg/Ghdr/dr did not develop invasive tumors. Suppression of carcinogenesis could not be attributed to alterations in prostate expression of TAg or androgen receptor or changes in serum testosterone levels. As carcinogenesis progressed in TAg/Gh+/+ rats, prostate GHR mRNA and protein expression increased significantly, but prostate IGF-I receptor mRNA and protein levels dropped. Furthermore, serum IGF-I and prostate IGF-I levels did not change significantly over the course of carcinogenesis. These findings suggest that GH plays a dominant role in progression from latent to malignant prostate cancer driven by the powerful probasin/TAg fusion gene in rats and suggest that GH antagonists may be effective at treating human prostate cancer.
Forkhead Box Protein O1 Negatively Regulates Skeletal Myocyte Differentiation through Degradation of Mammalian Target of Rapamycin Pathway Components
Authors: Wu Ai-Luen , Kim Jeong-Ho , Zhang Chongben , Unterman Terry G. , Chen Jie
Published: December 2007

The forkhead transcription factor forkhead box protein O1 (FoxO1), a downstream target of phosphatidylinositol 3-kinase/Akt signaling, has been reported to suppress skeletal myocyte differentiation, but the mechanism by which FoxO1 regulates myogenesis is not fully understood. We have previously demonstrated that a nutrient-sensing mammalian target of rapamycin (mTOR) pathway controls the autocrine production of IGF-II and the subsequent phosphatidylinositol 3-kinase/Akt signaling downstream of IGF-II in myogenesis. Here we report a regulatory loop connecting FoxO1 to the mTOR pathway. Inducible activation of a FoxO1 active mutant in the C2C12 mouse myoblasts blocks myogenic differentiation at an early stage and meanwhile leads to proteasome-dependent degradation of a specific subset of components in the mTOR signaling network, including mTOR, raptor, tuberous sclerosis complex 2, and S6 protein kinase 1. This function of FoxO1 requires new protein synthesis, consistent with the idea that a transcriptional target of FoxO1 may be responsible for the degradation of mTOR. We further show that active FoxO1 inhibits IGF-II expression at the transcriptional activation level, through the modulation of mTOR protein levels. Moreover, the addition of exogenous IGF-II fully rescues myocyte differentiation from FoxO inhibition. Taken together, we propose that the mTOR-IGF-II pathway is a major mediator of FoxO’s inhibitory function in skeletal myogenesis.
Ligand-Independent Activation of Androgen Receptors by Rho GTPase Signaling in Prostate Cancer
Authors: Lyons Leah S. , Rao Shuyun , Balkan Wayne , Faysal Joanne , Maiorino Carol A. , Burnstein Kerry L.
Published: December 2007

Prostate cancer invariably recurs after androgen deprivation therapy. Growth of this recurrent/androgen-independent form of prostate cancer may be due to increased androgen receptor (AR) transcriptional activity in the absence of androgen. This ligand-independent AR activation is promoted by some growth factors but the mechanism is not well understood. Vav3, a Rho guanosine triphosphatase guanine nucleotide exchange factor, which is activated by growth factors, is up-regulated in human prostate cancer. We show here that Vav3 levels increase during in vivo progression of prostate cancer to androgen independence. Vav3 strikingly enhanced growth factor activation of AR in the absence of androgen. Because Vav3 may be chronically activated in prostate cancer by growth factor receptors, we examined the effects of a constitutively active (Ca) form of Vav3 on AR transcriptional activity. Ca Vav3 caused nuclear localization and ligand-independent activation of AR via the Rho guanosine triphosphatase, Rac1. Ca Rac1 activation of AR occurred, in part, through MAPK/ERK signaling. Expression of active Rac1 conferred androgen-independent growth of prostate cancer cells in culture, soft agar, and mice. These findings suggest that Vav3/Rac 1 signaling is an important modulator of ligand-independent AR transcriptional activity in prostate cancer progression.
Modulation of in Situ Estrogen Synthesis by Proline-, Glutamic Acid-, and Leucine-Rich Protein-1: Potential Estrogen Receptor Autocrine Signaling Loop in Breast Cancer Cells
Authors: Rajhans Rajib , Nair Hareesh B. , Nair Sujit S. , Cortez Valerie , Ikuko Kijima , Kirma Nameer B. , Zhou Dujin , Holden Alan E. , Brann Darrell W , Chen Shiuan , Tekmal Rajeshwar Rao , Vadlamudi Ratna K.
Published: December 2007

In situ estrogen synthesis is implicated in tumor cell proliferation through autocrine or paracrine mechanisms especially in postmenopausal women. Several recent studies demonstrated activity of aromatase, an enzyme that plays a critical role in estrogen synthesis in breast tumors. Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1/MNAR) is an estrogen receptor (ER) coregulator, and its expression is deregulated in breast tumors. In this study, we examined whether PELP1 promotes tumor growth by promoting local estrogen synthesis using breast cancer cells (MCF7) that stably overexpress PELP1. Immunohistochemistry revealed increased aromatase expression in MCF7-PELP1-induced xenograft tumors. Real-time PCR analysis showed enhanced activation of the aromatase promoter in MCF7-PELP1 clones compared with MCF7 cells. Using a tritiated-water release assay, we demonstrated that MCF7-PELP1 clones exhibit increased aromatase activity compared with control MCF-7 cells. PELP1 deregulation uniquely up-regulated aromatase expression via activation of aromatase promoter I.3/II, and growth factor signaling enhanced PELP1 activation of aromatase. PELP1-mediated induction of aromatase requires functional Src and phosphatidylinositol-3-kinase pathways. Mechanistic studies revealed that PELP1 interactions with ER-related receptor-α and proline-rich nuclear receptor coregulatory protein 2 lead to activation of aromatase. Immunohistochemistry analysis of breast tumor array showed increased expression of aromatase in ductal carcinoma in situ and node-positive tumors compared with no or weak expression in normal breast tissue. Fifty-four percent (n = 79) of PELP1-overexpressing tumors also overexpressed aromatase compared with 36% (n = 47) in PELP1 low-expressing tumors. Our results suggest that PELP1 regulation of aromatase represents a novel mechanism for in situ estrogen synthesis leading to tumor proliferation by autocrine loop and open a new avenue for ablating local aromatase activity in breast tumors.
Glucocorticoid/Glucocorticoid Receptor Inhibition of Surfactant Protein-A (SP-A) Gene Expression in Lung Type II Cells Is Mediated by Repressive Changes in Histone Modification at the SP-A Promoter
Authors: Islam Kazi Nazrul , Mendelson Carole R.
Published: December 2007

Surfactant protein-A (SP-A) gene expression in human fetal lung type II cells is stimulated by cAMP and IL-1 and is inhibited by glucocorticoids. cAMP/IL-1 stimulation of SP-A expression is mediated by increased binding of thyroid transcription factor-1 and nuclear factor (NF)-κB to the TTF-1-binding element (TBE) in the SP-A promoter. This is associated with decreased expression of histone deacetylases (HDACs), increased recruitment of coactivators, and enhanced acetylation of histone H3 (K9,14) at the TBE. In the present study, endogenous glucocorticoid receptor (GR) was found to interact with thyroid transcription factor-1 and NF-κB p65 at the TBE. GR knockdown enhanced SP-A expression in type II cells cultured in serum-free medium, suggesting a ligand-independent inhibitory role of endogenous GR. Furthermore, use of chromatin immunoprecipitation revealed that dexamethasone (Dex) treatment of fetal lung type II cells increased recruitment of endogenous GR and HDACs-1 and -2 and blocked cAMP-induced binding of inhibitor of κB kinase-α (IKKα) to the TBE region. Accordingly, Dex reduced basal and blocked cAMP-stimulated levels of acetylated (K9,14) and phosphorylated (S10) histone H3 at the TBE. Dex also increased TBE binding of dimethylated histone H3 (K9) and of heterochromatin protein 1α. Thus, Dex increases interaction of GR with the complex of proteins at the TBE. This facilitates recruitment of HDACs and causes a local decline in basal and cAMP-induced histone H3 phosphorylation and acetylation and an associated increase in H3-K9 dimethylation and binding of heterochromatin protein 1α. Collectively, these events may culminate in the closing of chromatin structure surrounding the SP-A gene and inhibition of its transcription.
Energy saving processes in huddling emperor penguins: from experiments to theory
Authors: Gilbert Caroline , Blanc Stéphane , Le Maho Yvon , Ancel André
Published: December 2007

This paper investigates the energy savings of male emperor penguins Aptenodytes forsteri linked to their huddling behaviour, the key factor that allows them to assume their incubating task while undergoing a long fast. Drawing on new studies by our team, this review examines the energetic benefits accrued from huddling and estimates the respective contributions of wind protection, exposure to mild ambient temperatures,reduction in cold-exposed body surfaces and body temperature adjustments in these energy savings. The metabolic rate of `loosely grouped' birds(restrained in small groups of 5–10 individuals, which are unable to huddle effectively) is reduced by 39% compared to metabolic rate of `isolated'birds, with 32% of these energetic benefits due to wind protection. In addition, metabolic rate of `free-ranging' emperors, i.e. able to move freely and to huddle, is on average 21% lower than that of `loosely grouped' birds. Exposure to mild ambient temperatures within the groups and reduction in cold-exposed body surfaces while huddling, though overestimated, would represent a 38% metabolic reduction. About two thirds of metabolic lowering is attributable to the reduction in cold-exposed body surfaces and one third to the mild microclimate created within the groups. Moreover, body temperature adjustments contribute to these energetic benefits: maintaining body temperatures 1°C lower would represent a 7–17% reduction in energy expenditure. These processes, linked together, explain how huddling emperors save energy and maintain a constant body temperature, which ensures a successful incubation in the midst of the austral winter.
Impaired escape flight ability in butterflies due to low flight muscle ratio prior to hibernation
Authors: Almbro Maria , Kullberg Cecilia
Published: December 2007

For an animal to escape an attacking predator, speed and manoeuvrability are likely to be crucial factors. Previous studies on reptiles and birds have revealed that gaining weight, due to for instance egg load or lipid accumulation, leads to impaired escape ability and possibly increases the risk of being caught by predators. Here we tested whether the flight performance of an insect, the small tortoiseshell butterfly (Aglais urticae), is affected by variations in body mass due to feeding by comparing flight performance parameters between individuals that (i) took flight spontaneously or after being subjected to a simulated predator attack and (ii) varied in flight muscle ratio (FMR: thorax mass/total body mass). The results show that butterflies that were subjected to a simulated predator attack flew at higher velocities and straighter than butterflies that were allowed to take off spontaneously. Furthermore, our study suggests, for the first time, that butterflies may experience impaired escape ability after feeding, which directly leads to a decrease in FMR; a reduction from 0.5 to 0.3 reduced escape flight velocity by about 37% at 0.5 m from the start. Finally, this study shows that FMR affects take-off angle and sinuosity, with steeper angles and more turning in butterflies with low FMR.
A cumulative feeding threshold required for vitellogenesis can be obviated with juvenile hormone treatment in lubber grasshoppers
Authors: Fronstin R. B. , Hatle J. D.
Published: December 2007

Developmental thresholds can ensure that an adequate condition has been attained to proceed through major transitions (e.g. initiation of reproduction, metamorphosis). Nutrition is critical to attaining most thresholds, because it is needed for both growth and storage. Attaining a threshold typically stimulates the release of hormones that commit the animal to the developmental transition, yet the relationships between the nutrition needed for developmental thresholds and these endocrine signals are poorly understood. Lubber grasshoppers require a cumulative feeding threshold to initiate vitellogenesis and potentially commit to oviposition. We tested the relative roles of the nutritional threshold and the major gonadotropin(juvenile hormone; JH) in initiating vitellogenesis and committing to oviposition. The source of JH was removed from all females, and then JH analog was applied after different amounts of feeding. Threshold feeding was not required to initiate vitellogenesis, suggesting that sub-threshold grasshoppers are competent to respond to JH. Further, sub-threshold grasshoppers went on to oviposit earlier than supra-threshold grasshoppers treated with JH at the same time. Hence, threshold feeding is required only to cause the production and release of JH. At the same time, we also found that individuals that were restored with JH late in life tended to favor current reproduction, at the expense of future reproduction. Both time to oviposition and vitellogenin profiles were consistent with this developmental allocation. Taken together, our results suggest that lubber grasshoppers adjust reproductive tactics primarily in response to nutrition (which only serves to release JH) and secondarily in response to age.
Jamming avoidance response of big brown bats in target detection
Authors: Bates Mary E. , Stamper Sarah A. , Simmons James A.
Published: December 2007

When searching for prey, big brown bats (Eptesicus fuscus) enhance the range of their sonar by concentrating more energy in the nearly constant-frequency (CF) tail portion of their frequency-modulated (FM) sweeps. We hypothesize that this portion of their signals may be vulnerable to interference from conspecifics using the same frequencies in their own emissions. To determine how bats modify their signals when confronted with an interfering stimulus, we compared the echolocation calls of bats when a CF jamming tone was on and off. The bats performed a two-alternative forced-choice detection task in the laboratory that required the use of echolocation. All three bats shifted the tail-end CF component of their emitted frequency bidirectionally away from the CF jamming stimulus only when the jamming frequency was within 2–3 kHz of the preferred baseline frequency of the bat. The duration of their emissions did not differ between the jamming and no-jamming trials. The jamming avoidance response of bats may serve to avoid masking or interference in a narrow range of frequencies important for target detection.