Importance of Extranuclear Estrogen Receptor-�� and Membrane G Protein�CCoupled Estrogen Receptor in Pancreatic Islet Survival

OBJECTIVE

We showed that 17β-estradiol (E₂) favors pancreatic β-cell survival via the estrogen receptor-α (ERα) in mice. E₂ activates nuclear estrogen receptors via an estrogen response element (ERE). E₂ also activates nongenomic signals via an extranuclear form of ERα and the G protein–coupled estrogen receptor (GPER). We studied the contribution of estrogen receptors to islet survival.

RESEARCH DESIGN AND METHODS

We used mice and islets deficient in estrogen receptor-α (αERKO^−/−), estrogen receptor-β (βERKO^−/−), estrogen receptor-α and estrogen receptor-β (αβERKO^−/−), and GPER (GPERKO^−/−); a mouse lacking ERα binding to the ERE; and human islets. These mice and islets were studied in combination with receptor-specific pharmacological probes.

RESULTS

We show that ERα protection of islet survival is ERE independent and that E₂ favors islet survival through extranuclear and membrane estrogen receptor signaling. We show that ERβ plays a minor cytoprotective role compared to ERα. Accordingly, βERKO^−/− mice are mildly predisposed to streptozotocin-induced islet apoptosis. However, combined elimination of ERα and ERβ in mice does not synergize to provoke islet apoptosis. In αβERKO^−/− mice and their islets, E₂ partially prevents apoptosis suggesting that an alternative pathway compensates for ERα/ERβ deficiency. We find that E₂ protection of islet survival is reproduced by a membrane-impermeant E₂ formulation and a selective GPER agonist. Accordingly, GPERKO^−/− mice are susceptible to streptozotocin-induced insulin deficiency.

CONCLUSIONS

E₂ protects β-cell survival through ERα and ERβ via ERE-independent, extra-nuclear mechanisms, as well as GPER-dependent mechanisms. The present study adds a novel dimension to estrogen biology in β-cells and identifies GPER as a target to protect islet survival.Preserving insulin secretion by the pancreatic β-cells is critical in both type 1 and the late stages of type 2 diabetes. In type 1 diabetes, the death of insulin-producing β-cells of the pancreas by apoptosis leads to insulin dependence. Insulin replacement therapy by pancreatic islet transplantation is a treatment that most closely replicates normal physiological conditions for treatment of type 1 diabetes (1), but its effectiveness is reduced by the loss of functional islet mass from apoptosis, impairing the survival of islet grafts. Similarly, in the late stages of type 2 diabetes, evidence of β-cell apoptosis is documented in animal models (2,3) and in humans (4). Thus, in the absence of novel immunotherapy and antiapoptotic drugs, novel strategies to protect insulin-producing cells in vivo represent a major opportunity for therapeutic intervention. One promising approach to protect β-cells from apoptosis involves the cytoprotective actions of estrogens. In addition to its reproductive functions, the female sex steroid 17β-estradiol (E₂) is a neuroprotective hormone against multiple oxidative and proapoptotic insults in vivo and in vitro, acting via classic estrogen receptors (rev. in 5). Recently, we reported that E₂ protects β-cells from streptozotocin (STZ)-induced apoptosis in mice of both sexes via the estrogen receptor (ER)-α (6). In cultured mouse and human islets, E₂ has potent antiapoptotic properties against proinflammatory cytokines and reactive oxygen species (6,7). E₂ acts via classic estrogen receptors, ERα and ERβ (8). In ERα-deficient female mice, E₂ still partially protects β-cell survival via an alternative pathway (6), suggesting that ERβ may mediate the effects of E₂ in the absence of ERα.The G protein–coupled estrogen receptor (GPER), also known as GPR30, has been recognized as a membrane receptor for estrogens that mediates nongenomic signals (9). GPER is expressed in islets and has recently been suggested to mediate the estrogenic effect on islet insulin release (10). We analyzed the contribution of ERα, ERβ, and GPER to islet survival. We used mice individually deficient in ERα, ERβ, ERα and ERβ, and GPER; a mouse lacking ERα binding to the ERE; and human islets. These mutant mice and islets were exposed to oxidative stress using STZ or hydrogen peroxide, respectively, in combination with the use of specific pharmacological probes.     

Lung and respiratory muscle function in facioscapulohumeral muscular dystrophy

Pulmonary dysfunction is not a well‐recognized feature of facioscapulohumeral muscular dystrophy (FSHD). The aim of this study was to establish the prevalence and type of pulmonary and respiratory muscle dysfunction in FSHD. Sixteen patients with moderately advanced FSHD and 16 healthy controls were evaluated. Standard lung and respiratory muscle function tests were performed. Diaphragm muscle inspiratory action was evaluated with transdiaphragmatic pressure measurements. Lung function tests showed an increased residual volume in five patients. There was a significant difference in global respiratory muscle function in patients versus controls; weakness was mild, and it affected expiratory more than inspiratory muscles. There was no significant difference in the diaphragm inspiratory action of patients versus controls. The dystrophic process that underlies FSHD did not significantly involve the muscles of the diaphragm, but it caused mild global respiratory muscle weakness that affected expiratory more than inspiratory muscles. It is probably not necessary to routinely monitor respiratory muscle function in ambulant FSHD patients who lack symptoms or signs of respiratory impairment. Muscle Nerve, 2009     

Identification of plasma glucocorticoids in pallid sturgeon in response to stress

Compared to teleosts, little is known about the stress response in chondrosteans, and the glucocorticoid(s) most responsive to stress have never been definitively determined in sturgeon. In terms of cortisol production, pallid sturgeon (Scaphirhynchus albus) have a low physiological response to stress compared to other sturgeons (Acipenser s.p.). Because of this, our null hypothesis was that cortisol is not the predominant glucocorticoid secreted in response to stress in pallid sturgeon. Our objective was to identify the putative glucocorticoids present in the plasma of pallid sturgeon during the stress response. Pallid sturgeon were subjected to a severe confinement stress (12 h) with an additional handling stressor for the first 6 h. Control fish were not subjected to confinement but were handled only to collect blood. Blood plasma was collected at time 0, 6, and 12 h. Gas chromatography/mass spectrometry was used to screen the plasma for the spectrum of glucocorticoids and determine the putative steroid secreted during the stress response. Cortisol was the primary glucocorticoid detected in stressed pallid sturgeon. In addition, the cortisol metabolites cortisone, alloTHE (5alpha-pregnane-3alpha,17alpha,21-triol-11,20-dione), allo-alpha-cortolone (3alpha,17alpha,20alpha,21-tetrahydro-5alpha-pregnan-11-one), and allo-beta-cortolone (3alpha,17alpha,20beta,21-tetrahydro-5alpha-pregnan-11-one) were detected. Plasma cortisol increased from a resting concentration of 0.67 ng/ml to 10.66 ng/ml at 6h followed by a decrease to 6.78 ng/ml by 12 h. Plasma glucose increased significantly by time 6 and 12 h in both stressed and unstressed groups and remained elevated at time 12h, while resting lactate concentrations were low to non-detectable and did not increase significantly with the stressor over time. Cortisol was the primary glucocorticoid synthesized and secreted in response to a stressor in pallid sturgeon. Though the proportional increase in plasma cortisol in stressed pallid sturgeon was lower than many other species of sturgeon, the concentration was high enough to elicit a secondary stress response as seen by changes in plasma glucose.     

Developing an interstitial ultrasound applicator for thermal ablation in liver: results of animal experiments

BACKGROUND: In this project, an interstitial ultrasound applicator was developed for the treatment of primary and secondary cancers of the liver. Experiments on animals were used to check the destructive capabilities of this probe within the hepatic parenchyma of the pig in vivo, with a study of the physical parameters of the ultrasound treatment. In parallel, the possibility of visualizing the lesions induced by means of ultrasound imaging was also studied. MATERIALS AND METHODS: Thirteen pigs were used in this project, which had received the prior approval of the ethics committee of Lyon Veterinary School. Ultrasound lesions were performed by varying the physical parameters of the treatment (acoustic intensity and shot time) with the aim of obtaining larger and larger areas of destruction. An operative device was developed to ensure precision in treatments. Two types of lesions were performed: elementary lesions corresponding to single shots at 40 degrees to 50 degrees rotation intervals, and cylindrical lesions obtained by a continuous rotary deployment of the probe. The effect of hepatic pedicle clamping on the size of ultrasound lesions was studied. The aspect and dimension of the lesions were analyzed by means of operative ultrasound imaging and macroscopic examination. Histological analysis showed the impact of the treatment on the hepatic parenchyma. RESULTS: This work made it possible to study the elementary ultrasound lesions produced by our probe. Seventy elementary ultrasound lesions were analyzed. Treatments could be performed on all pigs without any difficulty. There were no operative incidents. The ultrasound-induced elementary lesions showed complete necrosis, with lesion length of up to 37 mm obtained without resort to pedicle clamping; this must be considered as a radius of the final lesion obtained over a complete rotary deployment (360 degrees ), then a diameter of 7 cm of thermal ablation can theoretically be obtained. The effect of pedicle clamping was studied and showed improvement of the lesion length. Results of continuous rotary deployment of the probe were encouraging. Operative ultrasound imaging proved to be a simple tool for directing and positioning the applicator in the target zone on the one hand and which, on the other hand, enabled accurate, real-time visualization of the ultrasound lesions. On histological analysis, the ultrasound-induced necrosis was complete and well defined. CONCLUSION: This work shows that it is feasible to treat cancers of the liver using interstitial ultrasound probe. Thermal damage obtained on the hepatic parenchyma of pigs in vivo is complete and can be monitored using simple diagnostic ultrasound. The ultrasound parameters can be adapted to obtain destruction of variable size.     

Up-Regulation of Soluble Axl and Mer Receptor Tyrosine Kinases Negatively Correlates with Gas6 in Established Multiple Sclerosis Lesions

Multiple sclerosis is a disease that is characterized by inflammation, demyelination, and axonal damage; it ultimately forms gliotic scars and lesions that severely compromise the function of the central nervous system. Evidence has shown previously that altered growth factor receptor signaling contributes to lesion formation, impedes recovery, and plays a role in disease progression. Growth arrest-specific protein 6 (Gas6), the ligand for the TAM receptor tyrosine kinase family, consisting of Tyro3, Axl, and Mer, is important for cell growth, survival, and clearance of debris. In this study, we show that levels of membrane-bound Mer (205 kd), soluble Mer (∼150 kd), and soluble Axl (80 kd) were all significantly elevated in homogenates from established multiple sclerosis lesions comprised of both chronic active and chronic silent lesions. Whereas in normal tissue Gas6 positively correlated with soluble Axl and Mer, there was a negative correlation between Gas6 and soluble Axl and Mer in established multiple sclerosis lesions. In addition, increased levels of soluble Axl and Mer were associated with increased levels of mature ADAM17, mature ADAM10, and Furin, proteins that are associated with Axl and Mer solubilization. Soluble Axl and Mer are both known to act as decoy receptors and block Gas6 binding to membrane-bound receptors. These data suggest that in multiple sclerosis lesions, dysregulation of protective Gas6 receptor signaling may prolong lesion activity.Multiple sclerosis (MS) is a debilitating white matter disease of the central nervous system (CNS). Although much of the evidence from animal models and MS suggests it to be an autoimmune disorder mediated by TH-1 type T cells,¹ other possible causes include genetic and environmental factors, antibody-dependent cytotoxicity, and bacterial and viral infections that may mediate altered protein expression resulting in inflammation, axonal and oligodendrocyte damage, demyelination and CNS scarring.² Growth and survival factors that protect against axonal and oligodendrocyte damage or loss, and dampen the inflammatory response are actively being pursued for MS therapy.^2,3,4,5,6 One growth factor associated with oligodendrocyte maturation, survival and dampening the immune response is growth-arrest specific protein 6 (Gas6). Gas6 is a secreted protein that is widely expressed in the central and peripheral nervous systems by endothelial cells and neurons, and is involved in numerous physiological and pathological functions including cell growth, survival and apoptosis.^{7,8,9,10,11,12} Gas6 binds and activates the TAM family of receptor tyrosine kinases consisting of Tyro3 (Rse/Dtk/Sky), Axl (Ufo), and Mer (Eyk).^{8,11,13,14,15} Many cell types express all three receptors and receptor activation can result from homophilic and heterophilic interactions.^16,17 Axl contains the major and minor Gas6 binding groove. Only the minor groove is conserved in Tyro3 and Mer and as a result, response to Gas6 is mediated in a concentration-dependent manner; Gas6 binding affinity is Axl>Tyro3>Mer.¹⁸We previously reported mRNA expression of Axl, Tyro3, and Mer receptors on human fetal oligodendrocytes and the ability of Gas6 to promote oligodendrocyte survival in vitro by activating Axl, resulting in Axl directly and indirectly recruiting phosphatidylinositol 3 kinase and activating the Akt pathway.^19,20 Moreover, we have shown that Gas6/Axl signaling through the Akt pathway can protect oligodendrocytes from tumor necrosis factor α (TNFα)-induced apoptosis.²¹ Down-regulation or deletion of Axl, even in the presence of Gas6, results in loss of protection against TNFα.²²During the relapse phase of relapsing-remitting MS, serum TNFα levels and TNFα mRNA are elevated.^23,24 TNFα is one of the major cytokines expressed in MS lesions.²⁵ TNFα is cleaved to its mature, soluble, secretable form by the matrix metalloproteinase (MMP) TNFα converting enzyme, also known as ADAM17.^26,27,28 MMPs, including ADAM17 and ADAM10 are involved in normal processes such as wound repair and tissue remodeling and are associated with disease states, including MS.^{29,30,31,32,33,34,35,36} Expression of ADAM17 is observed in acute and chronic active MS lesions, primarily in perivascular cuffs and cells morphologically resembling lymphocytes.³⁷ ADAM17 up-regulation in cerebrospinal fluid of MS patients is associated with inflammation and increased soluble TNFα.^37,38 ADAM10 is constitutively expressed on astrocytes in normal appearing white matter and on astrocytes and perivascular macrophages in MS lesions.^38,39,40,41 ADAM10 cleaves Axl, and ADAM17 cleaves Axl and Mer. Cleaved forms of receptors can result in internalization of the receptor and transport off the membrane for recycling. Cleavage can also result in shortened, soluble forms that act as a decoy to regulate the level of a growth factor at the membrane.^41,42Soluble forms of Axl and Mer can reduce the number of viable receptors for Gas6 binding and act as a decoy by sequestering Gas6 extracellularly; potentially a normal mechanism of receptor activation regulation.^40,41 During inflammation, soluble Mer has been reported to inhibit macrophage clearance of apoptotic cells.^41,43 Also, soluble Axl blocked the protective effect of membrane-bound Axl by inhibiting Gas6 induced tyrosine phosphorylation of Axl.⁴⁴ The binding of Gas6 to TAM receptors acts as an inhibitor of inflammation by inhibiting Toll-like receptor and cytokine receptor cascades.⁴⁴ Up-regulation of Axl and its subsequent interaction with interferon α and β receptors results in the expression of cytokine and Toll-like receptor inhibitors.^44,45 Thus, loss of Gas6 signaling, along with dysregulation of the balance between Gas6, Axl and Mer by increased extracellular levels of soluble Axl and Mer, might detrimentally impact the nervous system, especially in established (chronic active and chronic silent) lesions associated with MS. Chronic active MS lesions are characterized by ongoing demyelination, astrogliosis, macrophage and lymphocyte infiltration, astroglial hypertrophy, and oligodendrocyte hyperplasia.⁴⁶ Chronic silent MS lesions are characterized by the absence of actively infiltrating and inflammatory cells, oligodendrocyte loss and no evidence of ongoing demyelination.^46,47,48In this study, we investigated in chronic active and chronic silent MS lesions whether increased expression of soluble Axl and Mer was associated with increased expression of the MMPs ADAM17 and ADAM10, similar to previous studies that showed an association between increased ADAM17 and ADAM10 with TNFα in the CNS of MS patients.^37,38 We also investigated whether in lesions increased soluble Axl and Mer was associated with decreased Gas6, resulting in loss of the beneficial effects from activating membrane-bound Axl and Mer receptors.     

Caveolin-1 Expression Determines the Route of Neutrophil Extravasation through Skin Microvasculature

Interleukin-8 plays a key role in the acute inflammatory response by mediating recruitment of neutrophils through vessel walls into affected tissues. During this process, molecular signals guide circulating blood neutrophils to target specific vessels for extravasation and to migrate through such vessels via particular routes. Our results show that levels of endothelial caveolin-1, the protein responsible for the induction of the membrane domains known as caveolae, are critical to each of these processes. We demonstrate that, in response to the intradermal injection of interleukin-8, neutrophils are preferentially recruited to a unique subset of venules that express high levels of intercellular adhesion molecule-1 and low levels of caveolin-1. Our results show that neutrophils traverse human dermal microvascular endothelial cells using one of two pathways: a transcellular route directly through the cell or a paracellular route through cellular junctions. Caveolin-1 expression appears to favor the transcellular path while down-regulation of caveolin-1 promotes the paracellular route.Wounding of the epithelium and entry of a foreign body elicit a series of responses from the innate immune system. One of the main hallmarks of acute inflammation is neutrophil infiltration at the affected site.^1,2 In response to injury or infection, resident phagocytic cells become activated and release inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-8. TNF-α activates the vascular endothelium causing vasodilation and cellular infiltration.³ IL-8 functions as a critical chemotactic factor attracting neutrophils from the blood to the affected area.^1,4It is currently thought that leukocyte recruitment and migration through the vasculature is an active process not only for migrating blood cells but also for endothelial cells lining the vessels. Initially, inflammatory cytokines or bacterial endotoxins induce expression of P- and E-selectin on the surface of microvascular endothelial cells.^5,6 These molecules recognize carbohydrate counterligands on the surface of circulating leukocytes and mediate the tethering and rolling of these cells along vessel walls.^5,6,7 Firm adhesion is then initiated through the upregulation of endothelial adhesion molecules such as intercellular adhesion molecule (ICAM)-1 and vascular adhesion molecule (VCAM)-1, which bind to integrins expressed on the leukocyte surface.^5,7,8 Finally, the leukocyte is induced to migrate through the vessel in a process known as diapedesis.^5,6,7Among the many proteins implicated in the process of diapedesis, the adhesion molecule ICAM-1, which is up-regulated on activated endothelium, and caveolin-1, which is expressed on most terminally differentiated cell types but is largely undetectable in white blood cells, have been most closely associated with the route of transendothelial migration in in vitro systems.^7,9,10 A recent study by Millan et al clearly demonstrates that ICAM-1 and caveolin-1 are involved in directing the path of T lymphoblast migration through human umbilical vein endothelial cells (HUVECs).⁷Although both caveolin-1 and ICAM-1 have been associated with leukocyte transendothelial migration in vitro, the distribution of these proteins in vessels used by migrating leukocytes in vivo remain unclear. While all endothelial cells (ECs) share common features, the vascular tree is known to be extremely heterogeneous. As a result, the precise molecular profile of selectins and adhesion molecules defining vessels targeted for extravasation by circulating leukocytes is unknown. Furthermore, since the phenotype of vessel ECs is determined in large part by their unique in vivo microenvironment, site specific and regional differences in the expression of molecules contributing to the regulation of leukocyte transmigration have yet to be thoroughly characterized.^11,12 In this study, we have examined the in vivo molecular profile of vessels targeted by circulating neutrophils in response to IL-8 in the skin and have determined the effect of the expression of these factors on the route of neutrophil transmigration in vitro.     

Stem cells ameliorate EAE via an indoleamine 2,3-dioxygenase (IDO) mechanism

Syngeneic, pluripotent Lin(-)Sca1(+) bone marrow stem cells (SC), transferred to mice with experimental autoimmune encephalomyelitis, a model of multiple sclerosis, enhanced recovery, prevented relapses and promoted myelin repair. SC-treated mice showed elevated interferon-gamma production and induction of indoleamine 2,3-dioxygenase (IDO) in CD11c(+) dendritic cells (DC). IDO induction was specific since in the presence of IDO-producing CD11c(+) DC, PLP stimulated T cell proliferation was inhibited and the IDO-inhibitor, 1-MT, abrogated the SC effect. Relapse prevention during chronic disease correlated with decreased responsiveness to PLP(178-191) and MBP(85-99). Thus, pluripotent SC induce IDO in DC leading to inhibition of antigen reactivity and spreading in EAE.     

Relationship between low ultraviolet B irradiance and higher breast cancer risk in 107 countries

Abstract:  Epidemiological data show an inverse relationship between vitamin D levels and breast cancer incidence. This study investigates the relationship of modeled and measured serum 25-hydroxyvitamin D [25(OH)D] levels with age-standardized incidence rates of breast cancer in 107 countries. The hypothesis being tested is that breast cancer incidence is inversely related to geographically-dependent cutaneous sunlight exposure. A multiple regression approach was used to examine the contributions of ultraviolet B (UVB) irradiance to age-standardized incidence rates of breast cancer in the 107 countries with data on these covariates—total column ozone thickness, per capita intake of alcohol and energy from animal and vegetable sources, cigarettes, proportion of female population overweight, and total fertility. Age-standardized incidence rates were substantially higher at latitudes distant from the equator ( R ²  = 0.43, p   <   0.0001). The dose–response gradient between modeled serum 25(OH)D levels and incidence rates of breast cancer followed a standard inverse dose–response curve. Increasing increments in serum 25(OH)D in the range above 22 ng/mL were associated with incrementally lower incidence rates of breast cancer. According to multiple regression, UVB irradiance adjusted for cloud cover was inversely associated with incidence rates (p   =   0.04) after controlling for covariates. Intake of energy from animal sources was also positively associated with incidence rates (p   <   0.01). The overall coefficient of determination, R ², was 0.81 (p   <   0.0001). There was a protective effect of UVB irradiance on risk of breast cancer that was independent of fertility rate, proportion of the population overweight, alcohol intake, animal energy intake, and other covariates.