Agonist-induced internalization and recycling of the glucagon-like peptide-1 receptor in transfected fibroblasts and in insulinomas.

Glucagon-like peptide-1 (GLP-1) is the most potent stimulator of glucose-induced insulin secretion and its pancreatic beta-cell receptor is a member of a new subfamily of G-protein-coupled receptors which includes the receptors for vasoactive intestinal polypeptide, secretin and glucagon. Here we studied agonist-induced GLP-1 receptor internalization in receptor-transfected Chinese hamster lung fibroblasts using three different approaches. First, iodinated GLP-1 bound at 4 degrees C to transfected cells was internalized with a t 1/2 of 2-3 min following warming up of the cells to 37 degrees C. Secondly, exposure to GLP-1 induced a shift in the distribution of the receptors from plasma membrane-enriched to endosomes-enriched membrane fractions, as assessed by Western blot detection of the receptors using specific antibodies. Thirdly, continuous exposure of GLP-1 receptor-expressing cells to iodinated GLP-1 led to a linear accumulation of peptide degradation products in the medium following a lag time of 20-30 min, indicating a continuous cycling of the receptor between the plasma membrane and endosomal compartments. Potassium depletion and hypertonicity inhibited transferrin endocytosis, a process known to occur via coated pit formation, as well as GLP-1 receptor endocytosis. In contrast to GLP-1, the antagonist exendin-(9-39) did not lead to receptor endocytosis. Surface re-expression following one round of GLP-1 receptor endocytosis occurred with a half-time of about 15 min. The difference in internalization and surface re-expression rates led to a progressive redistribution of the receptor in intracellular compartments upon continuous exposure to GLP-1. Finally, endogenous GLP-1 receptors expressed by insulinoma cells were also found to be internalized upon agonist binding. Together our data demonstrate that the GLP-1 receptor is internalized upon agonist binding by a route similar to that taken by single transmembrane segment receptors. The characterization of the pathway and kinetics of GLP-1-induced receptor endocytosis will be helpful towards understanding the role of internalization and recycling in the control of signal transduction by this receptor.

Level of accumulation of epoxy fatty acid in Arabidopsis thaliana expressing a linoleic acid delta12-epoxygenase is influenced by the availability of the substrate linoleic acid.

Arabidopsis thaliana (L.) Heynh. expressing the Crepis palaestina (L.) linoleic acid delta12-epoxygenase in its developing seeds typically accumulates low levels of vernolic acid (12,13-epoxy-octadec-cis-9-enoic acid) in comparison to levels found in seeds of the native C. palaestina. In order to determine some of the factors limiting the accumulation of this unusual fatty acid, we have examined the effects of increasing the availability of linoleic acid (9cis, 12cis-octadecadienoic acid), the substrate of the delta12-epoxygenase, on the quantity of epoxy fatty acids accumulating in transgenic A. thaliana. The addition of linoleic acid to liquid cultures of transgenic plants expressing the delta12-epoxygenase under the control of the cauliflower mosaic virus 35S promoter increased the amount of vernolic acid in vegetative tissues by 2.8-fold. In contrast, the addition to these cultures of linoelaidic acid (9trans, 12trans-octadecadienoic acid), which is not a substrate of the delta12-epoxygenase, resulted in a slight decrease in vernolic acid accumulation. Expression of the delta12-epoxygenase under the control of the napin promoter in the A. thaliana triple mutant fad3/fad7-1/fad9, which is deficient in the synthesis of tri-unsaturated fatty acids and has a 60% higher level of linoleic acid than the wild type, was found to increase the average vernolic acid content of the seeds by 55% compared to the expression of the delta12-epoxygenase in a wild-type background. Together, these results reveal that the availability of linoleic acid is an important factor affecting the synthesis of epoxy fatty acid in transgenic plants.

Acid-sensing channels in human bladder: expression, function and alterations during bladder pain syndrome.

Purpose: To examine the possible role of H+-activated acid-sensing ion channels (ASICs) in pain perception we characterized their expression in bladder dome biopsies of Bladder Pain Syndrome (BPS) patients and controls, in cultured human urothelium and in urothelial TEU-2 cells.Materials and Methods: Cold cut biopsies from the bladder dome were obtained in 8 asymptomatic controls and 28 patients with symptoms of BPS. ASIC expression was analyzed by QPCR and immunofluorescence. The channel function was measured by electrophysiology.Results: ASIC1a, ASIC2a and ASIC3 mRNAs were detected in human bladder. Similar amounts of ASIC1a and -3 were detected in detrusor smooth muscle, whereas in urothelium ASIC3 levels were higher than -1a. ASIC2a mRNA levels were lower than either -1a or -3 in both layers. ASIC currents were measured in TEU-2 cells and in primary cultures of human urothelium, and ASIC expression was confirmed by QPCR. Differentiation of TEU-2 cells caused an up-regulation of ASIC2a and ASIC3, and a down-regulation of ASIC1a mRNAs. BPS patients showed an up-regulation of ASIC2a and -3 mRNA, whereas ASIC1a remained unchanged. In contrast, the mRNA levels of TRPV1 were down-regulated during BPS. All differences were statistically significant (p<0.05)Conclusions: Several different ASIC subunits are expressed in human bladder and TEU-2 cells, where their levels are regulated during urothelial differentiation. An up-regulation of ASIC2a and -3 in BPS suggests their involvement in increased pain and hyperalgesia. A down-regulation of TRPV1 mRNA levels might indicate a different regulatory mechanism, controlling its expression in human bladder.

Macrophage migration inhibitory factor deficiency leads to age-dependent impairment of glucose homeostasis in mice.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by many cells and tissues including pancreatic beta-cells, liver, skeletal muscle, and adipocytes. This study investigates the potential role of MIF in carbohydrate homeostasis in a physiological setting outside of severe inflammation, utilizing Mif knockout (MIF-/-) mice. Compared with wild-type (WT) mice, MIF-/- mice had a lower body weight, from birth until 4 months of age, but subsequently gained weight faster, resulting in a higher body weight at 12 months of age. The lower weight in young mice was related to a higher energy expenditure, and the higher weight in older mice was related to an increased food intake and a higher fat mass. Fasting blood insulin level was higher in MIF-/- mice compared with WT mice at any age. After i.p. glucose injection, the elevation of blood insulin level was higher in MIF-/- mice compared with WT mice, at 2 months of age, but was lower in 12-month-old MIF-/- mice. As a result, the glucose clearance during intraperitoneal glucose tolerance tests was higher in MIF-/- mice compared with WT mice until 4 months of age, and was lower in 12-month-old MIF-/- mice. Insulin resistance was estimated (euglycemic-hyperinsulinemic clamp tests), and the phosphorylation activity of AKT was similar in MIF-/- mice and WT mice. In conclusion, this mouse model provides evidence for the role of MIF in the control of glucose homeostasis.

Stem cell populations derived from heart and pancreas show a with a unique phenotype and give rise to functional cardiomyocytes and insulin-producing cells, respectively

Introduction: Recently, mesenchymal stem cells (MSC) of perivascular origin have been identified in several organs not including the heart. Using a novel cell isolation protocol, we have isolated cells sharing common characteristics from mouse hearts and pancreas. The aim of the present study was to characterize these cells in vitro.Methods: Cells were isolated from neonatal and adult mouse hearts and pancreas and cultured for more than 6 months. Surface marker expression was analyzed by flow cytometry and immunocytochemistry. Cell differentiation was tested using multiple differentiation media. Insulin production by pancreas-derived cells was tested by dithizone staining.Results: Cells showing a similar, distinctive morphology were obtained from the heart and pancreas after 4-8 weeks of culture. Cells from the two organs also showed a very similar immunophenotype, characterized by expression of c-kit (stem cell factor receptor), CD44, the common leukocyte marker CD45, and the monocytic markers CD11b and CD14. A significant proportion of cardiac and pancreatic cells expressed NG2, a marker for pericytes and other vascular cells. A significant proportion of cardiac, but not of pancreatic cells expressed stem cell antigen-1 (Sca-1). However, cells did not express T, B or dendritic cell markers. Cells of both cardiac and pancreatic origin spontaneously formed "spheres" (spherical cell aggregates similar to "neurospheres" formed by neural stem cells) in vitro. Cardiosphere formation was enhanced by TNF-alpha. Several cardiospheres (but no "pancreatospheres") derived from neonatal (but not adult) cells showed spontaneous rhythmic contractions, thus demonstrating cardiac differentiation (this was confirmed by immunostaining for alpha-sarcomeric actinin). Beating activity was enhanced by low serum conditions. Cells from both organs formed adipocytes, osteocytes and osteocytes under appropriate conditions, the typical differentiation pattern of MSCs. Pancreas-derived cells also formed dithizonepositive insulin-producing cells.Conclusions: We have defined cardiac and pancreatic cell populations that share a common morphology, growth characteristics, and a unique immunophenotype. Expression of perivascular and monocytic markers, along with stem/priogenitor cell markers by these cells suggests a relationship with pericytes-mesoangioblasts and so-called multipotent monocytes. Cells show MSC-typical growth and differentiation patterns, together with tissue-specific differentiation potential: cardiomyocytes for cardiac-derived cells and insulinproducing cells for pancreas-derived cells.

Conditional BDNF release under pathological conditions improves Huntington's disease pathology by delaying neuronal dysfunction

Background Brain-Derived Neurotrophic Factor (BDNF) is the main candidate for neuroprotective therapy for Huntington's disease (HD), but its conditional administration is one of its most challenging problems. Results Here we used transgenic mice that over-express BDNF under the control of the Glial Fibrillary Acidic Protein (GFAP) promoter (pGFAP-BDNF mice) to test whether up-regulation and release of BDNF, dependent on astrogliosis, could be protective in HD. Thus, we cross-mated pGFAP-BDNF mice with R6/2 mice to generate a double-mutant mouse with mutant huntingtin protein and with a conditional over-expression of BDNF, only under pathological conditions. In these R6/2:pGFAP-BDNF animals, the decrease in striatal BDNF levels induced by mutant huntingtin was prevented in comparison to R6/2 animals at 12 weeks of age. The recovery of the neurotrophin levels in R6/2:pGFAP-BDNF mice correlated with an improvement in several motor coordination tasks and with a significant delay in anxiety and clasping alterations. Therefore, we next examined a possible improvement in cortico-striatal connectivity in R62:pGFAP-BDNF mice. Interestingly, we found that the over-expression of BDNF prevented the decrease of cortico-striatal presynaptic (VGLUT1) and postsynaptic (PSD-95) markers in the R6/2:pGFAP-BDNF striatum. Electrophysiological studies also showed that basal synaptic transmission and synaptic fatigue both improved in R6/2:pGAP-BDNF mice. Conclusions These results indicate that the conditional administration of BDNF under the GFAP promoter could become a therapeutic strategy for HD due to its positive effects on synaptic plasticity.

Pro-inflammatory gene expression and neurotoxic effects of activated microglia are attenuated by absence of CCAAT/enhancer binding protein beta

Background. Microglia and astrocytes respond to homeostatic disturbances with profound changes of gene expression. This response, known as glial activation or neuroinflammation, can be detrimental to the surrounding tissue. The transcription factor CCAAT/enhancer binding protein ß (C/EBPß) is an important regulator of gene expression in inflammation but little is known about its involvement in glial activation. To explore the functional role of C/EBPß in glial activation we have analyzed pro-inflammatory gene expression and neurotoxicity in murine wild type and C/EBPß-null glial cultures. Methods. Due to fertility and mortality problems associated with the C/EBPß-null genotype we developed a protocol to prepare mixed glial cultures from cerebral cortex of a single mouse embryo with high yield. Wild-type and C/EBPß-null glial cultures were compared in terms of total cell density by Hoechst-33258 staining; microglial content by CD11b immunocytochemistry; astroglial content by GFAP western blot; gene expression by quantitative real-time PCR, western blot, immunocytochemistry and Griess reaction; and microglial neurotoxicity by estimating MAP2 content in neuronal/microglial cocultures. C/EBPß DNA binding activity was evaluated by electrophoretic mobility shift assay and quantitative chromatin immunoprecipitation. Results. C/EBPß mRNA and protein levels, as well as DNA binding, were increased in glial cultures by treatment with lipopolysaccharide (LPS) or LPS + interferon ¿ (IFN¿). Quantitative chromatin immunoprecipitation showed binding of C/EBPß to pro-inflammatory gene promoters in glial activation in a stimulus- and gene-dependent manner. In agreement with these results, LPS and LPS+IFN¿ induced different transcriptional patterns between pro-inflammatory cytokines and NO synthase-2 genes. Furthermore, the expressions of IL-1ß and NO synthase-2, and consequent NO production, were reduced in the absence of C/EBPß. In addition, neurotoxicity elicited by LPS+IFN¿-treated microglia co-cultured with neurons was completely abolished by the absence of C/EBPß in microglia.

Heat perception and signalling in plants: a tortuous path to thermotolerance.

An accurate assessment of the rising ambient temperature by plant cells is crucial for the timely activation of various molecular defences before the appearance of heat damage. Recent findings have allowed a better understanding of the early cellular events that take place at the beginning of mild temperature rise, to timely express heat-shock proteins (HSPs), which will, in turn, confer thermotolerance to the plant. Here, we discuss the key components of the heat signalling pathway and suggest a model in which a primary sensory role is carried out by the plasma membrane and various secondary messengers, such as Ca(2+) ions, nitric oxide (NO) and hydrogen peroxide (H(2) O(2) ). We also describe the role of downstream components, such as calmodulins, mitogen-activated protein kinases and Hsp90, in the activation of heat-shock transcription factors (HSFs). The data gathered for land plants suggest that, following temperature elevation, the heat signal is probably transduced by several pathways that will, however, coalesce into the final activation of HSFs, the expression of HSPs and the onset of cellular thermotolerance.

Oral administration of the KATP channel opener diazoxide ameliorates disease progression in a murine model of multiple sclerosis

Background Multiple Sclerosis (MS) is an acquired inflammatory demyelinating disorder of the central nervous system (CNS) and is the leading cause of nontraumatic disability among young adults. Activated microglial cells are important effectors of demyelination and neurodegeneration, by secreting cytokines and others neurotoxic agents. Previous studies have demonstrated that microglia expresses ATP-sensitive potassium (KATP) channels and its pharmacological activation can provide neuroprotective and anti-inflammatory effects. In this study, we have examined the effect of oral administration of KATP channel opener diazoxide on induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Methods Anti-inflammatory effects of diazoxide were studied on lipopolysaccharide (LPS) and interferon gamma (IFNy)-activated microglial cells. EAE was induced in C57BL/6J mice by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Mice were orally treated daily with diazoxide or vehicle for 15 days from the day of EAE symptom onset. Treatment starting at the same time as immunization was also assayed. Clinical signs of EAE were monitored and histological studies were performed to analyze tissue damage, demyelination, glial reactivity, axonal loss, neuronal preservation and lymphocyte infiltration. Results Diazoxide inhibited in vitro nitric oxide (NO), tumor necrosis factor alpha (TNF-¿) and interleukin-6 (IL-6) production and inducible nitric oxide synthase (iNOS) expression by activated microglia without affecting cyclooxygenase-2 (COX-2) expression and phagocytosis. Oral treatment of mice with diazoxide ameliorated EAE clinical signs but did not prevent disease. Histological analysis demonstrated that diazoxide elicited a significant reduction in myelin and axonal loss accompanied by a decrease in glial activation and neuronal damage. Diazoxide did not affect the number of infiltrating lymphocytes positive for CD3 and CD20 in the spinal cord. Conclusion Taken together, these results demonstrate novel actions of diazoxide as an anti-inflammatory agent, which might contribute to its beneficial effects on EAE through neuroprotection. Treatment with this widely used and well-tolerated drug may be a useful therapeutic intervention in ameliorating MS disease.

Intralesional regulatory T-cell suppressive function during human acute and chronic cutaneous leishmaniasis due to Leishmania guyanensis.

The levels of regulatory T cells (Treg cells), analyzed by Foxp3 mRNA expression, were determined in lesions from patients with acute cutaneous leishmaniasis (ACL) and chronic cutaneous leishmaniasis (CCL). We demonstrated that Treg cells preferentially accumulate in lesions from ACL patients during the early phase of infection (lesion duration of less than 1 month). In addition, levels of Foxp3 mRNA transcripts were significantly higher in specimens from patients with CCL than in those from patients with ACL, suggesting a critical role of intralesional Treg cells in CCL. Intralesional Treg cells from both ACL and CCL patients were shown to have suppressive functions in vitro, since they inhibited the gamma interferon (IFN-gamma) produced by CD4(+) CD25(-) T cells purified from peripheral blood mononuclear cells from the same patient in response to Leishmania guyanensis stimulation. Intralesional 2,3-indoleamine dioxygenase (IDO) mRNA expression was associated with that of Foxp3, suggesting a role for IDO in the suppressive activity of intralesional Treg cells. In addition, a role, albeit minor, of interleukin-10 (IL-10) was also demonstrated, since neutralization of IL-10 produced by intralesional T cells increased IFN-gamma production by effector cells in an in vitro suppressive assay. These results confirm the role of intralesional Treg cells in the immunopathogenesis of human Leishmania infection, particularly in CCL patients.

Control of thrombin signaling through PI3K is a mechanism underlying plasticity between hair follicle dermal sheath and papilla cells.

In hair follicles, dermal papilla (DP) and dermal sheath (DS) cells exhibit striking levels of plasticity, as each can regenerate both cell types. Here, we show that thrombin induces a phosphoinositide 3-kinase (PI3K)-Akt pathway-dependent acquisition of DS-like properties by DP cells in vitro, involving increased proliferation rate, acquisition of ;myofibroblastic' contractile properties and a decreased capacity to sustain growth and survival of keratinocytes. The thrombin inhibitor protease nexin 1 [PN-1, also known as SERPINE2) regulates all those effects in vitro. Accordingly, the PI3K-Akt pathway is constitutively activated and expression of myofibroblastic marker smooth-muscle actin is enhanced in vivo in hair follicle dermal cells from PN-1(-/-) mice. Furthermore, physiological PN-1 disappearance and upregulation of the thrombin receptor PAR-1 (also known as F2R) during follicular regression in wild-type mice also correlate with such changes in DP cell characteristics. Our results indicate that control of thrombin signaling interferes with hair follicle dermal cells plasticity to regulate their function.

Immunogenicity of the carcinoembryonic antigen derived peptide 694 in HLA-A2 healthy donors and colorectal carcinoma patients.

Carcinoembryonic antigen (CEACAM5) is commonly overexpressed in human colon cancer. Several antigenic peptides recognized by cytolytic CD8+ T-cells have been identified and used in colon cancer phase-I vaccination clinical trials. The HLA-A*0201-binding CEA(694-702) peptide was recently isolated from acid eluted MHC-I associated peptides from a human colon tumor cell line. However, the immunogenicity of this peptide in humans remains unknown. We found that the peptide CEA(694-702) binds weakly to HLA-A*0201 molecules and is ineffective at inducing specific CD8+ T-cell responses in healthy donors. Immunogenic-altered peptide ligands with increased affinity for HLA-A*0201 were identified. Importantly, the elicited cytolytic T lymphocyte (CTL) lines and clones cross-reacted with the wild-type CEA(694-702) peptide. Tumor cells expressing CEA were recognized in a peptide and HLA-A*0201 restricted fashion, but high-CEA expression levels appear to be required for CTL recognition. Finally, CEA-specific T-cell precursors could be readily expanded by in vitro stimulation of peripheral blood mononuclear cell (PBMC) from colon cancer patients with altered CEA peptide. However, the CEA-specific CD8+ T-cell clones derived from cancer patients revealed low-functional avidity and impaired tumor-cell recognition. Together, using T-cells to demonstrate the processing and presentation of the peptide CEA694-702, we were able to corroborate its presentation by tumor cells. However, the low avidity of the specific CTLs generated from cancer patients as well as the high-antigen expression levels required for CTL recognition pose serious concerns for the use of CEA694-702 in cancer immunotherapy.

RANKL expression, function, and therapeutic targeting in multiple myeloma and chronic lymphocytic leukemia.

Bone destruction is a prominent feature of multiple myeloma, but conflicting data exist on the expression and pathophysiologic involvement of the bone remodeling ligand RANKL in this disease and the potential therapeutic benefits of its targeted inhibition. Here, we show that RANKL is expressed by primary multiple myeloma and chronic lymphocytic leukemia (CLL) cells, whereas release of soluble RANKL was observed exclusively with multiple myeloma cells and was strongly influenced by posttranscriptional/posttranslational regulation. Signaling via RANKL into multiple myeloma and CLL cells induced release of cytokines involved in disease pathophysiology. Both the effects of RANKL on osteoclastogenesis and cytokine production by malignant cells could be blocked by disruption of RANK-RANKL interaction with denosumab. As we aimed to combine neutralization of RANKL with induction of antibody-dependent cellular cytotoxicity of natural killer (NK) cells against RANKL-expressing malignant cells and as denosumab does not stimulate NK reactivity, we generated RANK-Fc fusion proteins with modified Fc moieties. The latter displayed similar capacity compared with denosumab to neutralize the effects of RANKL on osteoclastogenesis in vitro, but also potently stimulated NK cell reactivity against primary RANKL-expressing malignant B cells, which was dependent on their engineered affinity to CD16. Our findings introduce Fc-optimized RANK-Ig fusion proteins as attractive tools to neutralize the detrimental function of RANKL while at the same time potently stimulating NK cell antitumor immunity.

Ligand-dependent inhibition of CD1d-restricted NKT cell development in mice transgenic for the activating receptor Ly49D.

In addition to their CD1d-restricted T cell receptor (TCR), natural killer T (NKT) cells express various receptors normally associated with NK cells thought to act, in part, as modulators of TCR signaling. Immunoreceptor-tyrosine activation (ITAM) and inhibition (ITIM) motifs associated with NK receptors may augment or attenuate perceived TCR signals respectively, potentially influencing NKT cell development and function. ITIM-containing Ly49 family receptors expressed by NKT cells are proposed to play a role in their development and function. We have produced mice transgenic for the ITAM-associated Ly49D and ITIM-containing Ly49A receptors and their common ligand H2-Dd to determine the importance of these signaling interplays in NKT cell development. Ly49D/H2-Dd transgenic mice had selectively and severely reduced numbers of thymic and peripheral NKT cells, whereas both ligand and Ly49D transgenics had normal numbers of NKT cells. CD1d tetramer staining revealed a blockade of NKT cell development at an early precursor stage. Coexpression of a Ly49A transgene partially rescued NKT cell development in Ly49D/H2-Dd transgenics, presumably due to attenuation of ITAM signaling. Thus, Ly49D-induced ITAM signaling is incompatible with the early development of cells expressing semi-invariant CD1d-restricted TCRs and appropriately harmonized ITIM-ITAM signaling is likely to play an important role in the developmental program of NKT cells.

Plasmacytoid Dendritic Cells Are Productively Infected and Activated through TLR-7 Early after Arenavirus Infection.

The antiviral response is largely mediated by dendritic cells (DCs), including conventional (c) DCs that function as antigen-presenting cells, and plasmacytoid (p) DCs that produce type I interferons, making them an attractive target for viruses. We find that the Old World arenaviruses lymphocytic choriomeningitis virus clone 13 (LCMV Cl13) and Lassa virus bind pDCs to a greater extent than cDCs. Consistently, LCMV Cl13 targets pDCs early after in vivo infection of its natural murine host and establishes a productive and robust replication cycle. pDCs coproduce type I interferons and proinflammatory cytokines, with the former being induced in both infected and uninfected pDCs, demonstrating a dissociation from intrinsic virus replication. TLR7 globally mediates pDC responses, limits pDC viral load, and promotes rapid innate and adaptive immune cell activation. These early events likely help dictate the outcome of infections with arenaviruses and other DC-replicating viruses and shed light on potential therapeutic targets.