Decreased Cell Surface Research Articles

Minocycline Inhibits <i>Candida albicans</i> Budded-to-Hyphal-Form Transition and Biofilm Formation

Candida albicans frequently causes bloodstream infections; its budded-to-hyphalform transition (BHT) and biofilm formation are major contributors to virulence. During an analysis of antibacterial compounds that inhibit C. albicans BHT, we found that the tetracycline derivative minocycline inhibited BHT and subsequent biofilm formation. Minocycline decreased expression of hypha-specific genes HWP1 and ECE1, and adhesion factor gene ALS3 of C. albicans. In addition, minocycline decreased cell surface hydrophobicity and the extracellular β-glucan level in biofilms. Minocycline has been widely used for catheter antibiotic lock therapy to prevent bacterial infection; this compound may also be prophylactically effective against Candida infection.

Exposure to High Glucose Concentration Decreases Cell Surface ABCA1 and HDL Biogenesis in Hepatocytes.

Aim: To study atherosclerosis risk in diabetes, we investigated ATP-binding cassette transporter A1 (ABCA1) expression and high-density lipoprotein (HDL) biogenesis in the liver and hepatocytes under hyperglycemic conditions.Methods and Results: In streptozotocin-induced diabetic mice, plasma HDL decreased while ABCA1 protein increased without changing its mRNA in the liver, only in the animals that responded to the treatment to show hypoinsulinemia and fasting hyperglycemia but not in the poor responders not showing those. To study the mechanism for this finding, hepatocytes were isolated from the control and diabetic mice, and they showed no difference in expression of ABCA1 protein, its mRNA, and HDL biogenesis in 1 g/l d-glucose but showed decreased HDL biogenesis in 4.5 g/l d-glucose although ABCA1 protein increased without change in its mRNA. Similar findings were confirmed in HepG2 cells with d-glucose but not with l-glucose. Thus, these cell models reproduced the in vivo findings in hyperglycemia. Labeling of cell surface protein revealed that surface ABCA1 decreased in high concentration of d-glucose in HepG2 cells despite the increase of cellular ABCA1 while not with l-glucose. Immunostaining of ABCA1 in HepG2 cells demonstrated the decrease of surface ABCA1 but increase of intracellular ABCA1 with high d-glucose. Clearance of ABCA1 was retarded both in primary hepatocytes and HepG2 cells exposed to high d-glucose but not to l-glucose, being consistent with the decrease of surface ABCA1.Conclusions: It is suggested that localization of ABCA1 to the cell surface is decreased in hepatocytes in hyperglycemic condition to cause decrease of HDL biogenesis.

Clinical and Genetic Features of Patients With TNFRSF1A Variants in Japan: Findings of a Nationwide Survey.

To elucidate the clinical and genetic features of patients with TNFRSF1A variants in Japan using data obtained from a nationwide survey conducted by the Ministry of Health, Labor, and Welfare of Japan study group for tumor necrosis factor receptor-associated periodic syndrome (TRAPS). Inquiries were sent to 2,900 departments of internal medicine and pediatrics in all hospitals with more than 200 beds in Japan, asking whether they had patients in whom TRAPS was suspected. Genetic tests for TNFRSF1A, MEFV, and MVK were performed on 169 patients. Cell surface expression of TNFRSF1A variants was assessed using 293T cells. Ten patients from 10 independent families were found to have TNFRSF1A variants. We collected clinical and genetic information on 41 additional patients with TNFRSF1A variants and symptoms of inflammation from 23 independent families; 17 of these patients had not been described in the literature. The common clinical features of Japanese patients were fever of >38°C (100% of patients), arthralgia (59%), and rash (55%). The prevalence of abdominal pain (36%), myalgia (43%), and amyloidosis (0%) was significantly lower in Japanese patients than in Caucasian patients. The most common variant was T61I (appearing in 49% of patients), and it was identified in 7 of 363 healthy controls. Defects in cysteine residues and the T50M variant were associated with decreased cell surface expression, while other variants, including T61I, were not. Patients with TNFRSF1A variants are very rare in Japan, as in other countries, but there are a number of clinical and genetic differences between Japanese and Caucasian patients. The pathogenic significance of the T61I variant remains unclear.

Genetic engineering of CHO cells for viral resistance to minute virus of mice.

Contamination by the parvovirus minute virus of mice (MVM) remains a challenge in Chinese hamster ovary (CHO) biopharmaceutical production processes. Although infrequent, infection of a bioreactor can be catastrophic for a manufacturer, can impact patient drug supply and safety, and can have regulatory implications. We evaluated engineering a CHO parental cell line (CHOZN® GS-/- ) to create a new host cell line that is resistant to MVM infection by modifying the major receptors used by the virus to enter cells. Attachment to a cell surface receptor is a key first step in the infection cycle for many viruses. While the exact functional receptor for MVM binding to CHO cell surface is unknown, sialic acid on the cell surface has been implicated. In this work, we used the zinc finger nuclease gene editing technology to validate the role of sialic acid on the cell surface in the binding and internalization of the MVM virus. Our approach was to systematically mutate genes involved in cell surface sialylation and then challenge each cell line for their ability to resist viral entry and propagation. To test the importance of sialylation, the following genes were knocked out: the CMP-sialic acid transporter, solute carrier family 35A1 (Slc35a1), the core 1-β-1,3-galactosyltransferase-1 specific chaperone (Cosmc), and mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase (Mgat1) as well as members of the sialyltransferase family. Slc35a1 is responsible for transporting sialic acid into the Golgi. Knocking out function of this gene in a cell results in asialylated glycan structures, thus eliminating the ability of MVM to bind to and enter the cell. The complete absence of sialic acid on the Slc35a1 knockout cell line led to complete resistance to MVM infection. The Cosmc and Mgat1 knockouts also show significant inhibition of infection likely due to their effect on decreasing cell surface sialic acid. Previously in vitro glycan analysis has been used to elucidate the precise sialic acid structures required for MVM binding and internalization. In this work, we performed the sequential knockout of various sialyltransferases that add terminal sialic acid to glycans with different linkage specificities. Cell lines with modifications of the various genes included in this study resulted in varying effects on MVM infection expanding on the knowledge of MVM receptors. MVM resistant host cell lines were also tested for the production of model recombinant proteins. Our data demonstrate that resistance against the MVM virus can be incorporated into CHO production cell lines, adding another level of defense against the devastating financial consequences of MVM infection without compromising recombinant protein yield or quality. Biotechnol. Bioeng. 2017;114: 576-588. © 2016 Wiley Periodicals, Inc.

The Rho-ROCK pathway as a new pathological mechanism of innate immune subversion in chronic myeloid leukaemia.

CD1d-restricted invariant natural killer T (iNKT) cells are believed to play a key role in cancer immune surveillance, and are functionally deficient in patients with chronic myeloid leukaemia (CML). Herein, we have hypothesized that this defect might originate from BCR-ABL-dependent dysfunctions in myeloid dendritic cells (mDCs). Indeed, flow cytometry and confocal microscopy revealed that cell surface expression of CD1d was downregulated in CML mDCs, relative to healthy donor (HD) controls. The decreased cell surface display of CD1d could not be ascribed to defective mDC differentiation, as attested by normal expression of HLA-DR and the CD86 maturation marker. On the other hand, reduced membrane expression was not associated with decreased intracytoplasmic levels of CD1d or its mRNA transcripts, consistent with intracellular retention. In vitro treatment of CML mDCs with the Rho-associated protein kinase (ROCK) inhibitor Y-27632 partially restored both cell surface CD1d expression and CD1d-mediated antigen presentation, whereas it had no effect on HD mDCs. An inhibitor of BCR-ABL tyrosine kinase (TK), imatinib mesylate (IM), had no such activity. Similar recovery of CD1d expression occurred with fasudil, another ROCK inhibitor that is commonly used in clinical trials. Our data support the conclusion that BCR-ABL-dependent ROCK, but not TK, is involved in CD1d downregulation. We propose that ROCK, which is most likely activated by the DH/PH domain of BCR-ABL, mediates iNKT-cell immune subversion in CML patients by downregulating CD1d expression on CML mDCs. Our study reveals the ROCK-mDC axis as a new potential target to restore immune surveillance in patients with CML, offering new therapeutic perspectives for CML treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Pharmacotherapy of Tobacco Use Disorder

Pharmacotherapy of Tobacco Use Disorder

MARCH1 regulates insulin sensitivity by controlling cell surface insulin receptor levels.

Insulin resistance is a key driver of type 2 diabetes (T2D) and is characterized by defective insulin receptor (INSR) signalling. Although surface INSR downregulation is a well-established contributor to insulin resistance, the underlying molecular mechanisms remain obscure. Here we show that the E3 ubiquitin ligase MARCH1 impairs cellular insulin action by degrading cell surface INSR. Using a large-scale RNA interference screen, we identify MARCH1 as a negative regulator of INSR signalling. March1 loss-of-function enhances, and March1 overexpression impairs, hepatic insulin sensitivity in mice. MARCH1 ubiquitinates INSR to decrease cell surface INSR levels, but unlike other INSR ubiquitin ligases, MARCH1 acts in the basal state rather than after insulin stimulation. Thus, MARCH1 may help set the basal gain of insulin signalling. MARCH1 expression is increased in white adipose tissue of obese humans, suggesting that MARCH1 contributes to the pathophysiology of T2D and could be a new therapeutic target.

Abstract 3243: Neutrophil elastase regulates PD-L1 expression in breast cancer

Abstract Introduction: We have previously reported that breast cancer cells take up the inflammatory mediator neutrophil elastase (NE) from tumor-associated neutrophils (TANs). NE uptake leads to: 1) increased cleavage of cyclin E (CCNE) to its low molecular weight isoforms and generation of the CCNE-derived immunogenic epitope CCNE144-152, 2) cross-presentation of the NE-derived epitope PR1, and 3) increased human leukocyte antigen (HLA) class I expression. NE is therefore a link between innate and adaptive immune responses in breast cancer. The current study was undertaken to investigate the effects of NE uptake on PD-L1 expression as another mechanism impacting adaptive immune responses in breast cancer. Methods: The breast cancer cell line MDA-MB-231 was maintained in standard media or media supplemented with NE. The effect of NE on cell surface expression of PD-L1 was determined using flow cytometry. Total PD-L1 protein expression was determined using western blot analysis performed on whole cell lysates. NE was inhibited with either elafin or alpha-1-antitrypsin to assess the requirement of enzymatic activity for the observed effects. Transcriptional regulation of PD-L1 expression by NE was assessed by qPCR. To evaluate specific transcription factors (TFs) involved in PD-L1 regulation, nuclear protein was isolated and analyzed for the activity of 16 TFs using a commercial plate array. The impact of NE uptake on TFs identified on the array was confirmed by western blot analysis. The functional effects of changes in PD-L1 expression were evaluated using an annexin V assay to assess T-cell apoptosis. Results: Addition of NE to breast cancer cells resulted in a reduction in PD-L1 surface expression as determined by flow cytometry and in total PD-L1 expression as shown by western blot analysis. The effect was reversed after removal of the cells from NE-supplemented media. The addition of elafin to inhibit NE enzymatic activity led to increased NE uptake by MDA-MB-231 cells with no change in PD-L1 cell surface expression. Treatment with alpha-1-antitrypsin prevented NE uptake and also abrogated its effect on PD-L1 expression. Together, these results suggest that uptake of enzymatically active NE is required to decrease PD-L1 expression. qPCR showed a decrease in the PD-L1 transcript suggesting that the effect of NE on PD-L1 is transcriptionally regulated. NE treatment resulted in decreased expression of TFs known to be involved in PD-L1 regulation, including STAT1, STAT3, and JUN. NE-treated breast cancer cells induced less apoptosis of T-cells compared with untreated breast cancer cells. Conclusions: Uptake of enzymatically active NE by breast cancer results in decreased cell surface and total PD-L1 expression, an effect that is in part transcriptionally regulated. These findings suggest another mechanism whereby the innate inflammatory mediator NE may impact adaptive immune responses in breast cancer. Citation Format: Victor Gall, Anne V. Philips, Akhil Chawla, Na Qiao, Lisa S. St. John, Pariya Sukhumalchandra, Mao Zhang, Jeffrey J. Molldrem, Gheath Alatrash, Elizabeth A. Mittendorf. Neutrophil elastase regulates PD-L1 expression in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3243.

Abstract 1547: Circulating tumor cells from a syngeneic mouse model of hepatocellular carcinoma demonstrate epithelial-mesenchymal transition, decreased MHC I expression, and increased CCR7 expression

Abstract Metastasis is the leading cause of death in cancer patients, worldwide. A better understanding of how cancer cells detach from the primary tumor, escape from immunosurveillance while in circulation, and successfully colonize distant parts of the body, is needed. Circulating tumor cells (CTCs) are excellent tools to study this process. Using a syngeneic hepatocellular carcinoma mouse model, we were able to isolate cancer cells from the primary tumor and CTCs from the blood and establish novel primary tumor-derived cell lines and novel CTC lines. Wound healing assays revealed that CTCs have greater migratory capacity than the cancer cells from the primary tumors. However, MTT assays revealed that CTCs had less proliferative capacity than the cancer cells from the primary tumors. Furthermore, molecular characterization of the cancer cells from primary tumors and CTCs using Western blotting showed a decreased expression of E-cadherin and an increased expression of fibronectin by CTCs, suggesting that epithelial-to-mesenchymal transition (EMT) takes place in CTCs in comparison to cancer cells from primary tumors. To address the question of how CTCs may evade immunosurveillance, we investigated the profile of expression of two cell surface proteins involved in immune system function: the major histocompatibility complex class I (MHCI)- required for cytotoxic T cell activation and subsequent cancer cell killing, and chemokine receptor 7 (CCR7)- exploited by cancer cells for immune evasion. Our analysis revealed that there is decreased cell surface expression of MHCI and increased expression of CCR7 in the CTCs in comparison to the cancer cells from primary tumors. These findings indicate that although CTCs are not more proliferative, they have acquired significantly increased migratory capacity than cancer cells in primary tumors. This may be explained by their demonstration of EMT. Furthermore, their loss of MHC I expression and their gain of CCR7 expression may help with evasion of killing by cytotoxic T cells and result in enhanced metastasis. Citation Format: Jeannette A. Huaman, Ubayed Muhith, Dibash K. Das, Michelle Naidoo, Olorunseun O. Ogunwobi. Circulating tumor cells from a syngeneic mouse model of hepatocellular carcinoma demonstrate epithelial-mesenchymal transition, decreased MHC I expression, and increased CCR7 expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1547.

Abstract 4005: Understanding the mechanism of 2FF-induced immune modulation

Abstract Fucosylation is the process of adding a fucose sugar to a glycan chain. The fucose analog, 2-fluorofucose (2FF), has been shown to inhibit cellular fucosylation by depletion of the fucosylation substrate, GDP-fucose, as well as direct inhibition of fucosyltransferases. 2FF has shown promising activity in a number of tumor models. For example 2FF enhanced the protective effect of a lymphoma vaccine (Okeley et al, PNAS 110, 2013), and this protection was determined to be immune dependent since depletion of CD4 and CD8 T-Cells reduced the 2FF/vaccine activity. Due to the complex nature of immune interactions in vivo we established an experimental system using human PBMCs to assess how 2FF-mediated immune changes may contribute to 2FF anti-tumor activity. Human PBMCs were matured in the presence of 2FF for 10 days and phenotypic analysis was performed. Using lectin binding, we found that 2FF dose-dependently decreased cell surface fucosylation which correlated with decreased levels of GDP-fucose and formation of GDP-2FF. 2FF-treatment alone had minimal effect on immune cell phenotype and no significant changes in activation or lineage markers were identified. Recently receptor fucosylation has been shown to be critical for BCR antigen recognition and antibody production (Li et al, J Immunol 194, 2015) as well as TLR recognition and signaling through the scavenger receptor DC-SIGN (Gringhuis et al, Nat Commun 5, 2014). As the TCR is reported to be fucosylated and basal signaling and activation of this receptor is regulated by a galectin-glycoprotein lattice we hypothesized that afucosylation following 2FF treatment may affect TCR functionality. During TCR engagement and activation the galectin-glycoprotein lattice is disrupted. 2FF-treated T cells show decreased galectin-3 levels and when activated have increased and more sustained TCR signaling, shown by increased levels of phosphorylated TCR associated proteins. Additionally, 2FF-treated T cells show increased tetramer specific binding. These data indicate that alteration in surface fucosylation on T cells impacts the regulatory glycoprotein lattice that negatively influences T cell action. Following up on this observation, in co-cultures 2FF-treated T cells activate dendritic cells in a contact dependent and antigen specific manner, shown by upregulation of maturation markers MHCII, CD83, CD86 and CD40. Overall we hypothesize that 2FF-treated T cells are more easily activated due to decreased avidity of galectin-3 binding to glycoproteins which lowers the threshold of lattice disruption by peptide-MHC resulting in easier TCR engagement, increased TCR signaling, and dendritic cell maturation. Citation Format: Jessica J. Field, Nicole M. Okeley, Weiping Zeng, Che-Leung Law, Peter D. Senter, Shyra J. Gardai. Understanding the mechanism of 2FF-induced immune modulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4005.

Identification and pharmacological analyses of eight naturally occurring caprine melanocortin-1 receptor mutations in three different goat breeds

The melanocortin-1 receptor (MC1R) belongs to the family of seven transmembrane G protein-coupled receptors and plays a central role in animal coat color. We have sequenced the full coding region of 954bp of the MC1R gene in 72 goats of three breeds with different coat colors and identified five missense mutations (K226E, F250V, G255D, V265I, and C267W) and one silent mutation (A61A), among which two haplotypes with complete linkage disequilibrium (A61A and F250V, G255D and V265I) were found. We performed detailed functional studies on the six single and two double mutations in transiently transfected HEK293T cells. We found that none of the mutants had decreased cell surface expression. However, all the mutants except A61A had decreased constitutive activities in the cAMP pathway. Five mutations (F250V, G255D, G267W, A61A/F250V, G255D/V265I) exhibited significant defects in ligand binding and consequent agonist-induced cAMP signaling and ERK1/2 activation. Additionally, K226E, with normal ligand binding affinity and cAMP signaling, showed a significant defect in ERK1/2 activation, exhibiting biased signaling. Co-expression studies showed that the five defective mutants did not affect wild-type MC1R signaling, hence they were not dominant negative. In summary, we provided detailed data of these goat MC1R mutations leading to a better understanding of the role of MC1R mutation and coat color in goats.

Unique pattern of PMN-MDSC migration in cancer

Abstract Myeloid-derived suppressor cells (MDSC) are pathologically activated immature myeloid cells that contribute to an immunosuppressive tumor microenvironment. In order to exert their effects at either the primary tumor site or in metastases, PMN-like MDSC (PMN-MDSC) must first migrate to these sites. There is some evidence to suggest that chemokine gradients can mediate their recruitment. We have observed that human PMN-MDSC accumulation in colon and lung tumors strongly correlates with the expression of S100A9, CXCL1 and CXCL8 in those tumors. Moreover, we have found that, in comparison to PMN, patient PMN-MDSC have an increased chemotactic response to CXCL8 in spite of decreased cell surface CXCR2 expression. Similarly, in murine transgenic models of melanoma, pancreatic and prostate cancer, we have found that PMN-MDSC migrate more than PMN when stimulated with CXCL1, in spite of similar levels of cell surface CXCR2 expression. Interestingly, murine PMN-MDSC are more migratory even without stimulation, and have greater speed, mean squared displacement and random motility coefficient than PMN. It is possible that in addition to specific factors secreted in the primary and metastatic tumor sites, PMN-MDSC recruitment to these sites is regulated by an altered migratory behavior of the cells themselves.

TRPM4 Is Expressed in and Carries a Nonselective Cation Current Through the Apical Membrane of Mouse Cortical Collecting Duct Principal Cells

Maintaining daily water, K+‐ and Na+‐balance is an important function of principal cells of the mouse cortical collecting duct (CCD). Under physiological conditions, these cells express a Ca2+ ‐activated nonselective cation channel (NSCCa), the molecular identity of which has been unknown. This study sought to identify whether the nonselective cation channel, TRPM4, functions as a NSCCa in principal cells of mouse cortical collecting duct.Using immunohistochemistry and western blotting, we show the presence of TRPM4 channel protein in the mouse cortical collecting duct cell line mpkCCDc14. Single channel recordings showed a current that is carried by monovalent cations and is Ca2+ sensitive and exhibits equal permeability for Na+ and K+. The corresponding current–voltage relationship of this channel was linear with a slope conductance 23.4 pS, which is the same as that observed from heterologously expressed TRPM4 channels. TRPM4 is inhibited by ATP and stimulated by PIP2. Application of 1 mM ATP to the “cytoplasmic” bath solution of an inside‐out patch almost completely inhibited channel activity but washing out ATP fully restored the channel activity. The channel activity significantly increased after adding 1 μM PI(4,5)P2 to the “cytoplasmic” bath solution. The TRPM4 inhibitor 9‐phenanthrol (100 μM) strongly inhibited the endogenous current. These biophysical properties of NSCCa are identical to those described for TRPM4. Acute application of H2O2 (100 μM and 500 μM) was unable to stimulate TRPM4 activity inside‐out patches; however, using a cell surface biotinylation assay, we showed that treating the mpkCCDc14 with 100 μM H2O2 for 24hrs significantly decreased cell surface levels of TRPM4 protein.Overall, we conclude that TRPM4 is the molecular identity of NSCCa in mpkCCDc14. Further study will be needed to determine the physiological relevance of this channel.Support or Funding InformationNIH R01‐DK100582 to HM

Inhibition of inflammatory signaling pathways in 3T3-L1 adipocytes by apolipoprotein A-I.

Activation of inflammatory signaling pathways links obesity with metabolic disorders. TLR4-mediated activation of MAPKs and NF-κB are 2 such pathways implicated in obesity-induced inflammation. Apolipoprotein A-I (apoA-I) exerts anti-inflammatory effects on adipocytes by effluxing cholesterol from the cells via the ATP binding cassette transporter A1 (ABCA1). It is not known if these effects involve inhibition of inflammatory signaling pathways by apoA-I. This study asks if apoA-I inhibits activation of MAPKs and NF-κB in mouse 3T3-L1 adipocytes and whether this inhibition is ABCA1 dependent. Incubation of differentiated 3T3-L1 adipocytes with apoA-I decreased cell surface expression of TLR4 by 16 ± 2% and synthesis of the TLR4 adaptor protein, myeloid differentiation primary response 88, by 24 ± 4% in an ABCA1-dependent manner. ApoA-I also inhibited downstream activation of MAPKs, such as ERK, p38MAPK, and JNK, as well as expression of proinflammatory adipokines in bacterial LPS-stimulated 3T3-L1 adipocytes in an ABCA1-dependent manner. ApoA-I, by contrast, suppressed nuclear localization of the p65 subunit of NF-κB by 30 ± 3% in LPS-stimulated 3T3-L1 adipocytes in an ABCA1-independent manner. In conclusion, apoA-I inhibits TLR4-mediated inflammatory signaling pathways in adipocytes by preventing MAPK and NF-κB activation.-Sultana, A., Cochran, B. J., Tabet, F., Patel, M., Cuesta Torres, L., Barter, P. J., Rye, K.-A. Inhibition of inflammatory signaling pathways in 3T3-L1 adipocytes by apolipoprotein A-I.

Dysbindin-1 modifies signaling and cellular localization of recombinant, human D₃ and D₂ receptors.

Dystrobrevin binding protein-1 (dysbindin-1), a candidate gene for schizophrenia, modulates cognition, synaptic plasticity and frontocortical circuitry and interacts with glutamatergic and dopaminergic transmission. Loss of dysbindin-1 modifies cellular trafficking of dopamine (DA) D2 receptors to increase cell surface expression, but its influence upon signaling has never been characterized. Further, the effects of dysbindin-1 upon closely related D3 receptors remain unexplored. Hence, we examined the impact of dysbindin-1 (isoform A) co-expression on the localization and coupling of human D2L and D3 receptors stably expressed in Chinese hamster ovary or SH-SY5Y cells lacking endogenous dysbindin-1. Dysbindin-1 co-transfection decreased cell surface expression of both D3 and D2L receptors. Further, while their affinity for DA was unchanged, dysbindin-1 reduced the magnitude and potency of DA-induced adenylate cylase recruitment/cAMP production. Dysbindin-1 also blunted the amplitude of DA-induced phosphorylation of ERK1/2 and Akt at both D2L and D3 receptors without, in contrast to cAMP, affecting the potency of DA. Interference with calveolin/clathrin-mediated processes of internalization prevented the modification by dysbindin-1 of ERK1/2 and adenylyl cyclase stimulation at D2L and D3 receptors. Finally, underpinning the specificity of the influence of dysbindin-1 on D2L and D3 receptors, dysbindin-1 did not modify recruitment of adenylyl cyclase by D1 receptors. These observations demonstrate that dysbindin-1 influences cell surface expression of D3 in addition to D2L receptors, and that it modulates activation of their signaling pathways. Accordingly, both a deficiency and an excess of dysbindin-1 may be disruptive for dopaminergic transmission, supporting its link to schizophrenia and other CNS disorders. Dysbindin-1, a candidate gene for schizophrenia, alters D2 receptors cell surface expression. We demonstrate that dysbindin-1 expression also influences cell surface levels of D3 receptors. Further, Dysbindin-1 reduces DA-induced adenylate cylase recruitment/cAMP production and modifies major signaling pathways (Akt and extracellular signal-regulated kinases1/2 (ERK1/2)) of both D2 and D3 receptors. Dysbindin-1 modulates thus D2 and D3 receptor signaling, supporting a link to schizophrenia.

Thymoquinone strongly inhibits fMLF-induced neutrophil functions and exhibits anti-inflammatory properties in vivo

Polymorphonuclear neutrophils are key players in host defense against pathogens through the robust production of superoxide anion by the NADPH oxidase and the release of antibacterial proteins from granules. However, inappropriate release of these agents in the extracellular environment induces severe tissue injury, thereby contributing to the physiopathology of acute and chronic inflammatory disorders. Many studies have been carried out to identify molecules capable of inhibiting phagocyte functions, in particular superoxide anion production, for therapeutic purposes. In the present study, we show that thymoquinone (TQ), the major component of the volatile oil from Nigella sativa (black cumin) seeds strongly inhibits fMLF-induced superoxide production and granules exocytosis in neutrophils. The inhibition of superoxide anion was not due to a scavenger effect, as TQ did not inhibit superoxide anion produced by the xanthine/xanthine oxidase system. Interestingly, TQ impaired the phosphorylation on Ser-304 and Ser-328 of p47PHOX, a cytosolic subunit of the NADPH oxidase. TQ also attenuated specific and azurophilic granule exocytosis in fMLF-stimulated neutrophils as evidenced by decreased cell surface expression of gp91PHOX and CD11b, and release of myeloperoxidase. Furthermore, both the PKC and MAPK pathways, which are involved in p47PHOX phosphorylation and granules exocytosis, respectively, were inhibited by TQ in fMLF-stimulated neutrophils. Finally, in a model of pleurisy induced by λ-carrageenan in rats, TQ reduced neutrophil accumulation in the pleural space, showing that it not only inhibits PMN functions in vitro, but also exhibits anti-inflammatory properties in vivo. Thus, TQ possesses promising anti-inflammatory therapeutic potential.

Sonic Hedgehog regulates thymic epithelial cell differentiation

Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus.

Glut4 Is Sorted from a Rab10 GTPase-independent Constitutive Recycling Pathway into a Highly Insulin-responsive Rab10 GTPase-dependent Sequestration Pathway after Adipocyte Differentiation

The RabGAP AS160/TBC1D4 controls exocytosis of the insulin-sensitive glucose transporter Glut4 in adipocytes. Glut4 is internalized and recycled through a highly regulated secretory pathway in these cells. Glut4 also cycles through a slow constitutive endosomal pathway distinct from the fast transferrin (Tf) receptor recycling pathway. This slow constitutive pathway is the only Glut4 cycling pathway in undifferentiated fibroblasts. The α2-macroglobulin receptor LRP1 cycles with Glut4 and the Tf receptor through all three exocytic pathways. To further characterize these pathways, the effects of knockdown of AS160 substrates on the trafficking kinetics of Glut4, LRP1, and the Tf receptor were measured in adipocytes and fibroblasts. Rab10 knockdown decreased cell surface Glut4 in insulin-stimulated adipocytes by 65%, but not in basal adipocytes or in fibroblasts. This decrease was due primarily to a 62% decrease in the rate constant of Glut4 exocytosis (kex), although Rab10 knockdown also caused a 1.4-fold increase in the rate constant of Glut4 endocytosis (ken). Rab10 knockdown in adipocytes also decreased cell surface LRP1 by 30% by decreasing kex 30-40%. There was no effect on LRP1 trafficking in fibroblasts or on Tf receptor trafficking in either cell type. These data confirm that Rab10 is an AS160 substrate that limits exocytosis through the highly insulin-responsive specialized secretory pathway in adipocytes. They further show that the slow constitutive endosomal (fibroblast) recycling pathway is Rab10-independent. Thus, Rab10 is a marker for the specialized pathway in adipocytes. Interestingly, mathematical modeling shows that Glut4 traffics predominantly through the specialized Rab10-dependent pathway both before and after insulin stimulation.

Targeting Endothelial Adhesion Molecule Transcription for Treatment of Inflammatory Disease: A Proof-of-Concept Study.

Targeting the endothelial adhesion molecules that control leukocyte trafficking into a tissue has been explored as a biological therapy for inflammatory diseases. However, these molecules also participate in leukocyte migration for immune surveillance, and inhibiting the physiological level of an adhesion molecule might promote infection or malignancy. We explored the concept of targeting endothelial adhesion molecule transcription during inflammation in a human system. Intercellular adhesion molecule 1 (ICAM-1) mediates leukocyte migration across the retinal endothelium in noninfectious posterior uveitis. We observed an increase in the transcription factor, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NF-κB1), in parallel with ICAM-1, in human retinal endothelial cells treated with tumor necrosis factor-alpha (TNF-α), and identified putative binding sites for NF-κB1 within the ICAM-1 regulatory region. We targeted induced NF-κB1 expression in endothelial cells with small interfering (si)RNA. Knockdown of NF-κB1 significantly decreased cell surface expression of ICAM-1 protein induced by TNF-α but did not reduce constitutive ICAM-1 expression. Consistently, NF-κB1 knockdown significantly reduced leukocyte binding to cell monolayers in the presence of TNF-α but did not impact baseline binding. Findings of this proof-of-concept study indicate that induced transcription of endothelial adhesion molecules might be targeted therapeutically for inflammatory disease in humans.

Annexin A6 and Late Endosomal Cholesterol Modulate Integrin Recycling and Cell Migration

Annexins are a family of proteins that bind to phospholipids in a calcium-dependent manner. Earlier studies implicated annexin A6 (AnxA6) to inhibit secretion and participate in the organization of the extracellular matrix. We recently showed that elevated AnxA6 levels significantly reduced secretion of the extracellular matrix protein fibronectin (FN). Because FN is directly linked to the ability of cells to migrate, this prompted us to investigate the role of AnxA6 in cell migration. Up-regulation of AnxA6 in several cell models was associated with reduced cell migration in wound healing, individual cell tracking and three-dimensional migration/invasion assays. The reduced ability of AnxA6-expressing cells to migrate was associated with decreased cell surface expression of αVβ3 and α5β1 integrins, both FN receptors. Mechanistically, we found that elevated AnxA6 levels interfered with syntaxin-6 (Stx6)-dependent recycling of integrins to the cell surface. AnxA6 overexpression caused mislocalization and accumulation of Stx6 and integrins in recycling endosomes, whereas siRNA-mediated AnxA6 knockdown did not modify the trafficking of integrins. Given our recent findings that inhibition of cholesterol export from late endosomes (LEs) inhibits Stx6-dependent integrin recycling and that elevated AnxA6 levels cause LE cholesterol accumulation, we propose that AnxA6 and blockage of LE cholesterol transport are critical for endosomal function required for Stx6-mediated recycling of integrins in cell migration.