A receptors at post-synaptic sites. The molecular events involved in gephyrin-dependent GABAA receptor clustering are still unclear. Evidence has been recently provided that gephyrin phosphorylation plays a key role in these processes. Gephyrin post-translational modifications have been shown to influence the structural remodeling of GABAergic synapses and synaptic plasticity by acting on post-synaptic scaffolding properties as well as stability. In addition, gephyrin phosphorylation and the subsequent phosphorylation-dependent recruitment of the chaperone molecule Pin1 provide a mechanism for the regulation of GABAergic signaling. Extensively characterized as pivotal enzyme controlling cell proliferation and differentiation, the prolyl-isomerase activity of Pin1 has been shown to regulate protein synthesis necessary to sustain the late phase of long-term potentiation at excitatory synapses, which suggests its involvement at synaptic sites. In this review we summarize the current state of knowledge of the signaling pathways responsible for gephyrin post-translational modifications. We will also outline future lines of research that might contribute to a better understanding of molecular mechanisms by which gephyrin regulates synaptic plasticity at GABAergic synapses.Gephyrin is a multifunctional scaffold protein essential for accumulation of inhibitory glycine and GABA A receptors  family Südhof,  and the ARs at synaptic sites at any given time, and thereby may influence the strength of synaptic transmission: gephyrin-gephyrin interaction, gephyrin-receptor  binding affinities, gephyrin turnover and synaptic transport. Recently new mechanistic insights on the regulation of gephyrin oligomerization, stability and receptor binding capability have been provided. They suggest that phosphorylation, (a versatile mechanism for regulating protein activity in a specific and controlled manner), already involved in the functional modulation of receptors at synapses, is determinant for all aspect of gephyrin dynamics. Interestingly, the signaling pathways altering the phosphorylation status of gephyrin have been previously identified as modulator of glutamatergic signaling. The functional cross-talk between excitatory and inhibitory transmission may have important implications for the long-term stability of neuronal networks.In summary, several gephyrin-dependent mechanisms affect the number of GABAviade novo regulation of protein synthesis but also via trafficking of pre-existing synaptic proteins. Therefore, it is not surprising that these signaling pathways contribute to regulate gephyrin transport at synapses  signaling; in particular the tropomyosin-related kinase B (Trk-B) and its ligand the brain-derived neurotrophic factor  and Dual specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1A), two protein kinases implicated in the inhibitory phosphorylation of sprouty proteins, in particular sprouty2, that specifically inhibit the Ras-Raf-MAPK pathway triggered by BDNF   site that lies in the C-terminal E-domain Figure . The C-dIn this region, conformational changes induced by phosphorylation could affect the folding of the C-domain itself and of the neighboring G- and E-domains, thus altering gephyrin clustering properties. A recent study , glutamic acid (E), serine (S) and threonine (T) , can target the same site, making this residue at the cross-road of different signaling pathways  by altering NMDA receptor channel conductance upon Cdk5-depenent phosphorylation of certain receptor subunits  pathway has been implicated in many forms of synaptic plasticity at glutamatergic synapses, including NMDA-dependent and independent forms of LTP. ERK1/2 activity enhances AMPA receptor functional properties by affecting their trafficking, by promoting the structural remodeling of activated spines as well as local protein synthesis  or rotamase  has been shown to regulate cell signaling through conformational changes that are not simply due to the phosphorylation event tau, amyloid-β-protein precursor, α-synuclein, and neurofilaments since aberrant interactions with these have implications for the development of neurodegenerative disorders such as Alzheimer disease , pointing to an exclusive role for Pin1 in mitosis (Lu et al., Gephyrin was identified as a novel target of post-phosphorylation prolyl-isomerization long before its identification as target of Ras/MAPK and PI3K/Akt signaling cascades (Zita et al., AR dynamics requires the coordinated activity of several other associated proteins whose identification and functional characterization has just started to be addressed. At least two key molecules have emerged to play an essential role in regulating gephyrin accumulation at postsynapses, namely NL2 and collybistin (Poulopoulos et al., in vivo (Tyagarajan et al., ARs α1, α2, and α3 subunits, namely Asp327 and Phe330 (Kim et al., ARs. Pin1 come into play once proline-directed phophorylation has occurred. This molecular switch is therefore positioned downstream the signaling cascades that orchestrate the precise phosphorylation patterns on their corresponding target molecules, thus being able to tune GABAergic transmission.Is Pin1 also involved in GABAergic synaptic signaling? As already mentioned, gephyrin contribution to GABAARs being juxtaposed to pre-synaptic releasing sites. Despite its overall stability, the gephyrin scaffold must ensure rapid changes in its composition to sustain several forms of synaptic plasticity. One mechanism promoting dynamic changes at inhibitory PSD is represented by post-translational modifications, and in particular by reversible phosphorylation of several key components of the PSDs. The fact that phosphorylation plays a key role in regulating synapse re-arrangement is not new, being extensively characterized at the level of neurotransmitter receptors. The novelty consists in having identified new signaling pathways able to affect synaptic strength by acting on the scaffolding molecule itself via alterations of its clustering properties. We are still at the beginning of this new challenge but the data obtained so far disclose a complex scenario. Several serine and threonine residues were found phosphorylated on gephyrin isolated from mouse and rat brains, thus indicating that multiple pathways converge on gephyrin, modifying residues that are very close to each other and possibly functionally interconnected. Interestingly some of the phosphorylated sites were also found acetylated in vivo, raising the possibility that acetylation exerts and additional level of control by directly modulating gephyrin protein-protein interaction or by competing with specific phosphorylation targets.The different roles played by the scaffolding molecule gephyrin at GABAergic synapses are still not completely understood. Gephyrin builds a stable scaffold underneath the synaptic plasma membrane to guarantee, over time, the appropriate number of GABAARs trafficking and synaptic localization.Unveiling the hierarchy of each phosphorylation event, their cross-talks and their respective contribution to the functional organization of GABAergic synapses will require not only the identification of all kinases and phosphatases involved, but also an accurate analysis of their impact on various gephyrin activities, and in particular on GABAThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background. Allergen-specific immunotherapy (SIT) is one of the important regimens for the treatment of allergic diseases. Predictive tests for the clinical response to SIT are limited. In this study we aimed to evaluate whether specific IgE/total IgE levels can predict clinical improvement in monosensitized patients to house dust mite treated with immunotherapy. Patients and Methods. We analyzed 32 patients who had undergone 2 years of SIT. Serum t-IgE and s-IgE levels, and serum s-IgE/t-IgE ratios were calculated and tested for correlation with clinical response to SIT. Asthma symptom score (ASS), rhinitis symptom score (RSS), pulmonary functions and visual analogue scales (VAS) were evaluated at the beginning and after 2 years. Results. There were 17 boys and 15 girls with the mean age of 10.78 ± 3.03 years. The mean serum house dust mite s-IgE level was 128.62 ± 142.61 kU/L, t-IgE 608.90 ± 529.98 IU/mL, and s-IgE/t-IgE ratio 33.83 ± 53.18. Before immunotherapy, ASS was 6.23 ± 1.63, RSS; 8.20 ± 1.88, VAS; 7.38 ± 2.01, FEV1 (%); 89.14 ± 8.48, PEF (%); 88.93 ± 13.57, and after 2 years, these values were determined as 1.90 ± 1.10, 3.05 ± 1.39, 1.35 ± 1.24, 97.6 ± 11.26, and 97.0 ± 11.55, respectively. s-IgE/t-IgE ratio was correlated with change in RSS  and VAS . Conclusion. Although SIT is very effective treatment, all patients do not benefit from treatment. We assumed that s-IgE/t-IgE ratio would be useful to predict the clinical response to SIT. Allergen-specific immunotherapy (SIT) has been used to treat IgE-mediated rhinoconjunctivitis, asthma, and venom hypersensitivity for almost a century. The clinical efficacy of SIT has been shown in several randomized, prospective, double blind placebo-controlled studies –5. A numβ decrease IgE production and enhance IgG4 and IgA production, respectively [Many complex immunologic changes occur during the course of SIT. Immunological changes induced by SIT are encompassing modulation of allergen-specific antibody responses, reduction in recruitment and activation of proinflammatory cells, and changes in the pattern of allergen-specific T-cell responses –9. It haectively , 10–12. ectively , 14. Morectively , 15–17. ectively –19. It iectively .In this study, we aimed to evaluate whether specific IgE/total IgE levels can predict clinical improvement in monosensitized patients to house dust mite treated with immunotherapy. We analyzed 32 children monosensitized to house dust mite with the diagnosis of asthma and/or allergic rhinitis that were on followup in Cukurova University, Faculty of Medicine, Division of Pediatric Allergy-Immunology, Adana, Turkey between January 2007 and September 2010. All patients underwent SIT by means of subcutaneous route as part of the therapy.All patients presented with a clinical diagnosis of asthma and/or allergic rhinitis. Diagnosis of asthma and allergic rhinitis was defined according to GINA and ARIA criteria , 22. SkiImmunotherapy was administered by means of subcutaneous route, and each patient received the maximum tolerated dose, as per the manufacturer's recommendations. The maintenance dose was 0.8 mL which corresponds to 3.84 mg of the major allergens of D pteronyssinus and D farinae.Patients were divided into two groups according to the s-IgE/t-IgE ratio. We set up 16.2 as a cut-off point for s-IgE/t-IgE ratio, and Group 1 included patients who had s-IgE/t-IgE ratio <16.2, while Group 2 s-IgE/t-IgE ratio >16.2 .D. pteronyssinus, D. farinae, grass mix, tree mix, mold mix, and Alternaria species) , and an induration with a diameter of ≥3 mm was accepted as a positive reaction.A routine skin prick test was performed for all patients using kits containing common inhalant allergens .A blood sample was processed at the time of diagnosis and after 2 years of SIT. s-IgE levels were determined by using the fluoroimmunoassay technique  according to the manufacturer's instructions. Serum s-IgE levels were determined with a detection limit of 0.35 kAU/L. In all patients, the s-IgE level was measured for the same allergens used in the skin prick tests.spIgE/t-IgE ratio was calculated asβ2-agonist  was recorded daily as the number of puffs. Daytime asthma symptoms and nocturnal awakenings were scored subjectively, as follows: 0, no symptoms during the day/night; 1, symptoms did not affect daily activities or nighttime sleep; 2, symptoms affected at least 1 daily activity or disturbed nighttime sleep; 3, symptoms affected 2 or more daily activities or disturbed sleep all night or most of the night. Use of β2-agonists was scored as follows: 0, none; 1, once a day; 2, between 2 and 3 times a day; 3, more than 3 times a day. The minimum score for each day was 0 , and the maximum score was 9  [Asthma symptom scores were determined based on the daytime symptoms and nocturnal awakening. In addition, the amount of as-needed gonists) .Each nasal symptoms including itching, congestion, sneezing, and rhinorrhea were scored subjectively as follows: 0, no symptoms; 1, symptom positive however did not affect daily activities or nighttime sleep; 2, symptoms were bothering but did not affect daily activities or nighttime sleep; 3, symptoms affected daily activities or disturbed sleep. Scores for each symptoms recorded and sum of the scores were calculated (0–12) .Visual analogue scale was evaluated on 10 cm card .PFTs were performed according to the American Thoracic Society (ATS) Criteria by using ZAN 100 Spiromed , and the results were expressed as percentages of the predicted values for age and height .Changes in ASS, RSS, VAS, and pulmonary functions throughout 2 years were calculated by the following formula:Decreases in RSS, FEV1, PEF, and VAS were calculated with same method.P < 0.05 value was considered as statistical significant.All statistical analysis was carried out using a computer software . Nonparametric Wilcoxon test was utilized to compare the groups. Spearman's correlation test was used to determine the statistical relationships between s-IgE/t-IgE ratio and changes in ASS, RSS, and VAS. The There were 17 boys and 15 girls with the mean age of 10.78 ± 3.03 years . Sixteen patients had asthma, 4 patients had allergic rhinitis, and 12 patients had both asthma and allergic rhinitis. At beginning of the immunotherapy, the mean total IgE value was found to be 608.9 ± 529.987 IU/mL, spIgE and spIgE/t-IgE ratio were 128.62 ± 142.61 kUA/L (median: 69.05) and 33.83 ± 53.18 (median: 17.68), respectively. The characteristics of the patients were given in We found statistically significant improvement in asthma symptom score, rhinitis symptom score, pulmonary functions, and VAS at the end of the 2 years of SIT .r = −0.392,  P = 0.08). On the other hand, significant inverse correlation was determined between the s-IgE/t-IgE ratio and change VAS . There was no significant correlation between the s-IgE/t-IgE ratio and changes in ASS and pulmonary functions  and VAS . However, there were no significant correlations in Group 1 regarding these parameters. In addition, no statistically significant relation was found between the s-IgE/t-IgE ratio and changes in ASS and pulmonary functions in both groups. In addition, patients were divided into, two groups according to the s-IgE/t-IgE ratio. Previously Di Lorenzo et al. reported that serum s-IgE/t-IgE ratio of greater than 16.2 correlated with an effective clinical response to SIT . Based oAllergen-specific immunotherapy has the potential to modify the natural course of allergic diseases , 27, 28.β + Treg cells, and increases in “protective” noninflammatory blocking antibodies, particularly IgG4 and IgA2 subclasses with inhibitory activity. This suppression occurs within weeks or months as a consequence of the appearance of a population of regulatory T cells that exert their effects by mechanisms involving cell-cell contact, but also by the release of cytokines such as IL-10 (increases IgG4) and TGF-β (increases specific IgA). Mast cell and basophil desensitization are very early effects. Intermediate effects are related to changes in allergen-specific T cells, and late affects are related to B cells and IgE as well as mast cells, basophils, and eosinophils [Parietaria judaica [Successful immunotherapy is accompanied by the suppression of numbers of T-helper 2 (Th2) effector cells, eosinophils, basophils, mast cells and neutrophils infiltration in target organs, induction of IL-10 and/or TGF-inophils . Severalinophils , 27, 28.inophils –5. There judaica . To suppThe continuous hyperreactivity in skin prick test might have negative impact on motivation of both physicians and patients. Therefore, different objective and practical parameters are needed to evaluate the clinical response to SIT. Nasal and/or bronchial provocation tests, diluted skin prick tests, SpIG4 levels, or other detailed tests are usually used in clinical studies or studies aimed to investigate the effectiveness of SIT. However, it is generally difficult to perform all these tests in routine practice because of ethical and economic issues. In this study, we evaluate whether this simple s-IgE/t-IgE ratio can predict clinical improvement in such patients routinely and we assumed that this ratio would be helpful to predict the efficacy of SIT in clinical practice.Allergen-specific immunotherapy is of long-duration therapy, and at least 3–5-year treatment was recommended for the best clinical benefits , 30. Bec


Pueraria thomsonii Benth. Although its anti-inflammatory and anti-hyperglycosemia effects have been well documented, the effect of tectorigenin on endothelial dysfunction insulin resistance involved has not yet been reported. Herein, this study aims to investigate the action of tectorigenin on amelioration of insulin resistance in the endothelium. Palmitic acid (PA) was chosen as a stimulant to induce ROS production in endothelial cells and successfully established insulin resistance evidenced by the specific impairment of insulin PI3K signaling. Tectorigenin effectively inhibited the ability of PA to induce the production of reactive oxygen species and collapse of mitochondrial membrane potential. Moreover, tectorigenin presented strong inhibition effect on ROS-associated inflammation, as TNF-α and IL-6 production in endothelial cells was greatly reduced with suppression of IKKβ/NF-κB phosphorylation and JNK activation. Tectorigenin also can inhibit inflammation-stimulated IRS-1 serine phosphorylation and restore the impaired insulin PI3K signaling, leading to a decreased NO production. These results demonstrated its positive regulation of insulin action in the endothelium. Meanwhile, tectorigenin down-regulated endothelin-1 and vascular cell adhesion molecule-1 overexpression, and restored the loss of insulin-mediated vasodilation in rat aorta. These findings suggested that tectorigenin could inhibit ROS-associated inflammation and ameliorated endothelial dysfunction implicated in insulin resistance through regulating IRS-1 function. Tectorigenin might have potential to be applied for the management of cardiovascular diseases involved in diabetes and insulin resistance.Tectorigenin is a plant isoflavonoid originally isolated from the dried flower of  Diabetes mellitus is a devastating metabolic disorder and continues to be a major risk factor of morbidity and mortality throughout the world Insulin plays a key role in the regulation of glucose and lipid homeostasis in adipose tissue, skeletal muscle and liver. In addition to crucial metabolic actions, insulin exerts desirable effects on the maintenance of physiological endothelial function through its ability to stimulate NO release. This action was mainly mediated by a cascade of signaling that involves activation of the PI3K-Akt axis and the downstream activation of NO synthase (eNOS). Except NO-dependent vasodilatory actions, insulin also regulates proliferation, ET-1 and adhesion molecule expression in endothelial cells and this action is confirmed by increased insulin vasodilatory effects in humans under ET-1 receptor blockade Pueraria thomsonii Benth is well known as a Chinese traditional medicine for the management of diabetes and cardiovascular diseases, and the flowers of this plant (Fen-ge-hua in Chinese) have been traditionally used to counteract the overconsumption of alcohol P. thomsonii Benth included preventive effect on hangovers P. thomsonii Benth has been reported to lower serum lipid and blood glucose levels P. thomsonii Benth might be efficacious against metabolic disorders, including diabetes mellitus.In oriental herbal medicine, the roots of P. thomsonii Benth. This compound has been shown to possess hypoglycemic effect in streptozotocin-induced diabetic rats, and potently inhibited aldose reductase, suggesting tectorigenin is a promising compound for the prevention and/or treatment of diabetic complications Tectorigenin is a major isoflavonoid originally isolated from the dried flower of Male Sprague-Dawley rats (200–250 g), supplied by the Laboratory Animal Center of Nanjing Qinglongshan (Certification number: SCXK 2009-0002). The care and treatment of these rats was maintained in accordance with the Provisions and General Recommendation of Chinese Experimental Animals Administration Legislation. Experimenters have License of Staff Engaged in Laboratory Animal (No. 2102044).Puerariae thomsonii flos, following the previously reported methods of Wang et al. 1H and 13C NMR, and MS. The purity was determined to be higher than 98% by HPLC. In the present study, it was dissolved in DMSO before use. PA was obtained from Sinopharm Chemical Reagent Co., Ltd.  and dissolved in absolute ethanol at 200 mM as stock solution and then was further diluted with medium containing 10% FFA-free and low endotoxin BSA to obtain a concentration of 5 mM before use.Tectorigenin (98.5% in purity) was isolated from the 4-hydroxypheny-L-glycine from Sigma.The following items were purchased from the cited commercial sources: RPMI Medium 1640 from Gibco ; FBS from Sijiqing Biological Engineering Materials Co. Ltd. ; insulin from Wanbang Biochemical Pharmaceutical Company ; sodium salicylate from Tianjin Kemiou Chemical Agent Center ; human TNF-α and IL-6 ELISA kits from R&D (USA), PI3K ELISA kits from Echelon Biosciences Inc. . All primers from Sangon Biotec Co., Ltd. ; TRNzol Plus, BU-Taq DNA polymerase, DNA Marker and BU-SuperScript RT kit from Biouniquer Technology Co., Ltd. . Anti-phospho-IRS-1 (Ser307), anti-IRS-1(R301), anti-phospho-Akt (T308), anti-Akt (A444), anti-phospho-IKKβ (Y199) and anti-IKKβ (F182) from Bioworld Technology ; horseradish-peroxidase conjugated anti-rabbit and anti-mouse IgG antibodys, phospho-NF-κB p65 (Ser536) antibody, NF-κB p65 antibody, anti-SAPK/JNK, anti-phospho-SAPK/JNK (Thr183/Tyr185), phospho-eNOS (Ser 1177) antibody and eNOS (49G3) antibody from Cell Signaling Technology, Inc. ; PY99antibody from Santa Cruz biotechnology, inc.; glutathione reduced (GSH), Wortmannin and ECL Plus Western blotting detection reagent and Mitoquinone mesylate (MitoQ) were purchased from Beyotime Institute of Biotechnology , v/v) FBS, streptomycin (100 U/ml) and penicillin (100 U/ml) under an atmosphere of 5% CO2 and 95% humidified air at 37°C. The medium was renewed every 2 days until the cells were grown to confluence.Human umbilical vein endothelial cells (HUVECs) were purchased from the Type Culture Collection of the Chinese Academy of Sciences  and were examined morphologically and identified as endothelial cells by positive immunocytochemistry staining for Factor VIII. HUVECs were grown in RPMI Medium 1640 supplemented with 10% , and then were starved in serum-free medium for 12 h. After that, HUVECs were incubated with tectorigenin , salicylate (5 mM), glutathione reduced , Mitoquinone mesylate  for 30 min, then PA (100 μM) was added to the culture system for another 30 min. After washed with PBS and loaded with 10 μM DCFH-DA at 37°C for 30 min, cells were rinsed thrice with PBS and fixed in 2% paraformaldehyde (v/v) at 4°C for 5 min. Fixed cells were examined using an Olympus IX81 inverted microscope with attached charge-coupled device camera  using appropriate filters with a peak excitation wavelength of 488 nm and a peak emission wavelength of 525 nm.Intracellular ROS generation was assessed using the ROS-specific fluorescent dye 2′, 7′-dichlorofluorescein, diacetate . When HUVECs were grown to conuence in 48-well plates, cells were washed three times with phosphate buffered solution (PBS) . When HUVECs cultured in 48-well plates reached conuence, cells were washed three times with PBS, then starved in serum-free medium for 12 h. Pretreated with tectorigenin , salicylate (5 mM), glutathione reduced , Mitoquinone mesylate  for 30 min, cells were stimulated without or with PA (100 μM) for 30 min and then washed thrice with PBS. For another experiment, cells were pretreated with tectorigenin (10 μM) for 30 min. After loading with 5 μM DAF-FM DA at 37°C for 30 min and kept in the dark, the HUVECs were rinsed three times with PBS. After that, cells were incubated with insulin (100 nM) at 37°C for 5 min, and fixed in 2% paraformaldehyde (vol/vol) at 4°C for another 5 min. Fixed cells were examined using an Olympus IX81 inverted microscope with attached charge-coupled device camera  using appropriate filters with a peak excitation wavelength of 495 nm and a peak emission wavelength of 515 nm.Mitochondrial membrane potential production was assessed using the fluorescent dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylimidacarbocyanineiodide . When HUVECs grown to conuence in 48-well plates, cells were washed three times with PBS, then starved in serum-free medium for 12 h. Pretreated with tectorigenin , glutathione reduced  or Mitoquinone mesylate  for 30 min, cells were stimulated with PA (100 μM) for additional 30 min and then washed with PBS. After loading with 25 μg/ml JC-1 at 37°C for 30 min and kept in the dark, the HUVECs were rinsed three times with PBS and fixed in 2% paraformaldehyde (vol/vol) at 4°C for 5 min. In addition, a positive control which will induce potent collapse of mitochondrial membrane potential was designed. In this group, HUVECs were treated with 10 μM carbonylcyanide-m-chlorophenylhydrazone (CCCP) for 20 min without PA stimulation before loading with JC-1. Fixed cells were examined using an Olympus IX81 inverted microscope with attached charge-coupled device camera  using appropriate filters with a peak excitation wavelength of 585 nm and a peak emission wavelength of 590 nm.HUVECs were serum-starved for 12 h and incubated with tectorigenin , salicylate (5 mM) or GSH (1 mM) for 30 min, then stimulated with 100 μM PA for 30 min. After incubated in the absence or presence of insulin (100 nM) for 20 min, treated cells were washed with ice-cold PBS and lysed on ice in cell lysis buffer for western  supplemented with 1 mM PMSF. Proteins were quantified using the Bicinchoninic Acid (BCA) Protein Assay kit  in accordance with the manufacturer's instructions. Equal amounts of protein samples were separated by 10% SDS-PAGE and transferred to PVDF membranes  in a Mini Trans-Blot Electrophoretic Transfer Cell . The membranes were blocked with 5% non-fat milk in TBST  for 2 h at room temperature, and probed with primary antibodies (1∶800 dilution in TBST) overnight at 4°C. Then incubated with the secondary antibody at 37°C for 1 h. HRP-labeled anti-rabbit or anti-mouse IgG was used as the secondary antibody (1∶2000 dilution in TBST). Signals were detected with an ECL western detection reagent and quantized by densitometry with Image-Pro Plus 6.0 (IPP 6.0) software.HUVECs grown to confluence in 24-well plates were pretreated with tectorigenin , salicylate (5 mM) or GSH (1 mM) for 30 min, then stimulated with PA (100 μM) for further 12 h in serum-free medium, and the medium was then collected on ice. The levels of TNF-α and IL-6 in the supernatant were assayed with commercial ELISA Kits.5′-ACATCTGCTGGA AGGTGGAC-3′, Reverse primer: 5′-GGTACCACCATGTACCCAGG-3′; ET-1 (436 bp), forward primer: 5′-CGTTGTTCCGTATGGACTT G-3′; reverse primer: 5′-AGGCTATGGCTTCAGACA GG-3′; VCAM-1 (540 bp), forward primer: 5′-AGCCACATCAGTATCCAAGAGGAG-3′; reverse primer: 5′-CAATAACCAACTCTATGTTCCTTTTC-3′. Comparison to β-actin values was carried out for normalization. Relative expression levels in each sample were determined by comparison with the standard. The PCR products were electrophoresed in 1.5% agarose gel with 0.5 mg/mL ethidium bromide and visualized under UV light, then the signal intensity was detected by Bio-Rad image system and analyzed with computer program Quantity One (Bio-Rad).For endothelin-1 (ET-1) and vascular cell adhesion molecule-1 (VCAM-1) gene determination, HUVECs were grown to confluence in six-well plates and then incubated with tectorigenin , salicylate, wortmannin or PD98059 for 0.5 h, respectively, and then stimulated with PA for 2 h. After incubation, cells were treated with insulin for 20 min in the continued presence of the inhibitors or tectorigenin. Total RNA was extracted from the cells using TRNzol Plus and its concentration was calculated from spectrophotometric measurements at 260 nm. Total RNA (500 ng) from each RNA sample was reverse-transcribed into cDNA with the RT kit . Amplification of individual target cDNA was carried out with specific primers as followings: β-actin (161 bp), Forward primer: 4 1.2, KH2PO4 1.2, CaCl2 2.5, NaHCO3 25, Calcium Disodium Edetate (EDTA) 0.026, glucose 5.0, mM, pH 7.4) and gassed with 95% O2 –5% CO2. The dissected aortae were cleaned of connective tissue and cut into rings (5 mm long). Take care to avoid abrading the intimal surface to maintain the integrity of the endothelial layer. For measurement of vascular responses, aortic rings were suspended in an organ bath containing 30 mL of K-H solution maintained at 37°C, pH 7.4, and continuously aerated with 95% O2 and 5% CO2. A resting tension of 2.0 g was applied to the aortic ring and changes in tension were measured with a force-displacement transducer connected to a polygraph. After an hour equilibration period, segments were exposed to 60 mM of KCl to assess the constriction function. The functionality of vascular endothelium was confirmed by the ability of relaxation after exposed to 10−5 M acetylcholine (Ach) to relax segments contracted with 10−6 M phenylephrine (the relaxation was over 80%). After confirmed the integrity of the endothelium, the aortic ring was pre-contracted with phenylephrine (10−6 M) and then insulin (10−9–10−6 M) was added cumulatively to evoke an endothelium-dependent relaxation. The relaxation induced by insulin was expressed as a percentage of the phenylephrine-induced contraction. After washout, the aortic ring was incubated with tectorigenin  or salicylate (5 mM) for 30 min followed by the addition of PA (100 μM) for another 30 min. After the washout of tectorigenin and PA, the aortic ring was pre-contracted with phenylephrine (10−6 M). When the contraction was stable, increasing concentrations of insulin (10−9–10−6 M) were added to produce endothelium-dependent relaxation.Rats were killed by cervical dislocation. The thoracic aorta was immediately removed and placed in 4°C Krebs-Henseleit (K-H) solution  is tightly associated with endothelial dysfunction, we first investigated the effect of tectorigenin on PA-induced ROS generation in HUVECs. After a 30 min treatment with PA (100 μM), intracellular oxidative stress was measured using a DCFH-DA fluorescent probe. Compared with the control, the fluorescence intensity in PA exposed cells was significantly higher, indicating ROS production was enhanced in endothelial cells. However, PA-stimulated ROS production was abolished by treatment with tectorigenin in a dose-dependent manner . The inhibitory potency of tectorigenin at concentration of 10 μM was similar to that of 1 mM GSH Figure 1The effect of tectorigenin on mitochondrial membrane potential of cells was determined by a JC-1 mitochondrial membrane potential assay kit. JC-1 is a fluorescent dye that is concentrated by respiring mitochondria. JC-1 exists as a monomer that fluoresces green and forms J-aggregates with red fluorescence at low and high Δψm, respectively. In this study, Δψm was measured in terms of the relative intensity of JC-1 after HUVECs were stimulated with PA and tectorigenin. IKKβ/NF-κB signaling is a key regulator of inflammation. As evidenced in IKKβ/NF-κB and JNK regulate the expression of a wide range of proinflammatory cytokines, including TNF-α and IL-6. As presented in IRS-1 serine phosphorylation was a key molecular event linking inflammation to the impairment of insulin signaling PA stimulation attenuated insulin-meidated IRS-1 tyrosine phosphorylaiton, leading to the impairment of downstream insulin signaling evidenced by reduced Akt and eNOS phosphorylation in response to insulin  in endothelial cells through MAPK (ERK) pathways. The balance between PI3K-dependent and MAPK-dependent functions of insulin plays a key role in the maintenance of the endothelial homeostasis. In this study, insulin increased the basal expression of ET-1 and VCAM-1, and this action was enhanced by wortmannin treatment. When endothelial cells were exposed to PA, ET-1 and VCAM-1 expression in response to insulin was further increased. But tectorigenin effectively inhibited ET-1 and VCAM-1 overexpression in a concentration-dependent manner. PD98059, a specific inhibitor of ERK, also downregulated ET-1 and VCAM-1 expression as tectorigenin did, indicating the involvement of ERK activation. The results were shown in −9 to 10−6 M. Whereas the stimulation of rat aorta with PA impaired insulin action, resulting significant decrease of relaxation. The percentages of relaxation induced by 0.1 μM and 1 μM of insulin were reduced from 67.28% to 15.35% (0.1 μM of insulin) and from 86.26% to 20.74% (1 μM of insulin), respectively. Tectorigenin treatment significantly resotored insulin-meidated vessel relaxation evidenced by largest relaxation (1 μM of insulin) were restored to 72.75%, 83.34% and 90.44% by tectorigenin at concentrations of 0.1, 1 and 10 μM. The result demonstrated that tectorigenin protected endothelium-dependent relaxation against inflammatory insult.Endothelial dysfunction is characterized by the loss of endothelium-dependent vessel relaxation. Data presented in Oxidative stress, which may be precipitated by hyperglycemia and vascular inflammation, plays a pivotal role in the development of diabetes complications, both microvascular and cardiovascular. Hyperglycemic conditions of cells cause ROS overproduction and the deregulation of ROS signaling pathways, leading to impaired endothelial function and consequently to vascular dysfunction. Consequently, reducing oxidative stress by lowering ROS production is crucial in the management of diabetes complications. It has been established that excessive circulating free fatty acids (FFAs) stimulates ROS formation in endothelial cells through PKC activation via IKK/NF-κB and JNK/SAPK signaling pathways Oxidative stress is tightly concerned with inflammation, and ROS evoke inflammatory response It has been well established that inflammation is a key feature of insulin resistance. And serine phosphorylation of insulin receptor substrate-1 (IRS-1) is a key event linking inflammation and insulin resistance. Inflammatory molecules such as TNF-α, IL-6 and IKKβ can affect IRS-1 function and impaired insulin signaling Insulin resistance is a characteristic feature of the impairment of PI3K signaling. Binding of insulin to its receptor on endothelial cells leads to phosphorylation of IRS-1, which subsequent binds and activates PI3K.The activated PI3K phosphorylates and activates downstream Akt and eNOS, leading to NO production. PA inhibited insulin-mediated IRS-1 tyrosine phosphorylation, and subsequently impaired insulin signaling along the PI3K/Akt/eNOS pathway. As tectorigenin attenuated PA-induced IRS-1 serine phosphorylation and enhanced insulin mediated IRS-1 tyrosine phosphorylation, we reasoned that the phosphorylating modification of IRS-1 should contribute to the regulation of insulin PI3K signaling. As expected, tectorigenin restored insulin-mediated Akt and eNOS phosphorylation impaired by PA  and the vasoconstrictor (ET-1) effects The present study aimed to investigate the actions of tectorigenin on PA-induced endothelial dysfunction, and provide insight into the possible mechanisms. Tectorigenin inhibited ROS-associated inflammation and restored insulin signaling transduction along the IRS-1/PI3K /Akt/eNOS pathways. Meanwhile, tectorigenin suppressed enhanced mitogenic action of insulin by inhibition of ET-1 and VCAM-1 expression, well demonstrating its beneficial modulation of insulin actions in endothelium. The resulting increase in insulin-mediated NO should be responsible for actions of tectorigenin in amelioration of endothelial dysfunction. Taken together, the action of tectorigenin on attenuating endothelial dysfunction implicated in insulin resistance provides us new insights to its possible application for the prevention or treatment of cardiovascular disorders involved in insulin resistance and diabetes.


To evaluate the value of different MRI derived parameters to differentiate left ventricular noncompaction of the myocardium (LVNC) in patients with known familial LVNC from other cardiomyopathies.LVNC is a rare disease characterized by numerous, excessively prominent ventricular trabeculations and deep intertrabecular recesses communicating with the ventricular cavity. Cardiac MRI (cMRI) has been reported as an ideal imaging modality to characterize patients with LVNC. Nevertheless, qualitative parameters to differentiate normal compaction of the myocardium in healthy subjects from LVNC or from other cardiomyopathies like dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM) may fail due to highly variable left ventricular trabeculations. Therefore, absolute quantification should be performed.comp), MM-Index=MMI [g/m2], MMnon-comp, percental MMnon-comp, ventricular volumes and function were calculated using the CAAS MRV  with known familial LVNC carrying a gene mutation, 11 DCM (32.6 years±17) and 10 HCM patients compared with 24 healthy volunteers (20.2 years±6.3) using a 1.5 T scanner and a standard steady state free precession sequence in standard orientations. Total left ventricular muscle mass (MM), the compacted muscle mass , but only a poor specificity (27%). None of the LVNC patients demonstrated with intramyocardial late gadolinium enhancement (LGE), but HCM and DCM patients did. Furthermore, increased trabeculations in the basal and septal segments, especially in the segments 4-6, are additional strong indicators for LVNC.The mean EF was significantly reduced (p<0.05) in LVNC patients compared to all cardiomyopathies and healthy subjects, total MM was increased, but not significantly different from HCM patients. The total MMInon-comp or the percental MMnon-comp together with an absence of LGE and increased trabeculations in basal segments allows to reliably diagnose familial LVNC.Absolute cMRI quantification of the MMI


The cystatin α is modified by glutathione or make their dimmer, and they are inactive. Those modifications are regulated by the redox-potential by the glutathione.Cathepsins are an essential protease family in all living cells. The cathepsins play an essential roles such as protein catabolism and protein synthesis. To targeting to various organella and to regulate their activity, the post translational-processing and modification play an important role Cathepsins are translated in polysome as the pre-pro-mature forms. The pre-peptide is removed cotranslationally and then translocated to Golgi-apparatus and the pro-part is removed and the mature-part is glycosylated, and the mature-part is targeted into the lysosome mediated by mannose-6-phosphate signal and the mature-part is bound with their coenzymes. The degradation of the mature-part is started by the limited proteolysis of the ordered nicked bonds to make hydrophobic peptides. The peptides are incorporated into phagosome or proteasome after ubiquitinated and are degrade into amino-acids. Cystatins are endogenous inhibitors of cathepsins. Cystatin  We will introduce recent advances on the study of post-translational processing, modification, and targeting of cathepsins and cystatins. Almost all the intracellular proteins are passed through principally similar processes from the synthesis to their degradation in general. Therefore, I would like to introduce the general fate of intracellular proteins, from the post-translational processing, modification, and targeting to the ordered particles. As Biological merit of post-translational processing  and modiThe glycosylated cathepsins are targeted into lysosomes mediated by mannose-6 phosphate receptors which are located on the lysosomal.As Figures Post-translational processing and maturation are summarized in Cathepsin L is translated as 17 amino acids of prepart, 96 amino acids of pro-part and 221 amino acids of mature-part . The preCathepsin B  is transThen the mannose-6-phosphate-moities play a role as the targeting marker to the lysosomes. The pro-part was cleaved off to make mature cathepsins in the lysosome. The cleavage ordered nicking at 47th bond was starting to be degraded.Cathepsin H is translated as 21 amino acids of prepart , 114 amiIntracellular protein degradation (= autophagosome formation) is regulated by nutritional and hormonal conditions. Fasting or insulin enrichment caused stimulation of the phagosome formation and cathepsin L amount are increased; on the contrary, refeeding and glucagon enrichment resulted in the suppression of phagosome formation and decreasing of cathepsin L amount.t1/2) of cathepsins and the contents in lysosomes clearly increased by treatment by E-64 .As you see in The initiations of degradation of cathepsins are started from the ordered limited proteolysis by cysteine protease in the lysosomes, as Procathepsins or mature cathepsins are secreted from the various cells and play individual physiological, or pathological roles.As 3, E-64, CA-148 eta. Therefore, bone metastasis of cancers is inhibited by cathepsin inhibitors, such as by E-64 or CLIK-148 [Bone collagen is degraded mainly by cathepsin L and K, therefore they are called collagenolytic cathepsins. The secretion of these cathepsins from osteoclasts is stimulated by PHT (Parathyroid Hormone) and suppressed by vitamin DCLIK-148 .As Pit formation test in α (A) [β (B) [γ-trace is secreted from cerebrospinal fluid. Another secretary group having high-molecular weight (repeated low molecular cystatins) is kininogen family in serum [Endogenous low-molecular weight cathepsin inhibitors are consisted of two big groups , one is α (A)  is only ) [β (B)  is locat) [β (B) . The secin serum .α does not inhibit cathepsin B activity but strongly inhibit cathepsin L activity and cathepsin H activities [Each cystatin shows different inhibitory specificity for individual cathepsins, for example, cystatin tivities , 19. ThiThe amino-acid sequences of low-molecular weight cystatin family have strong homology, however, their post-translational processing are quite different. Because their localizations and biological functions is quite different.α antibody, only the cornified-envelope of skin was stained [α into the cornified envelope. The sphingosine is a powerful inhibitor for protein kinase C. As shown in Figures α is phosphorylated by protein kinase C, and the phosphorglated cystatin α is incorporated into cornified envelope. The participation of protein kinase C in the phosphorylation of cystatin α was also confirmed using specific inhibitor of protein kinase C, (Hidaka H-7) inhibited the incorporation of P32 into cystatin α.When skin was stained by immunohistochemistry using anticystatin  stained . The sphα was incorporated into skin cornified envelop. In the envelop, the phosphorylated cystatin α was conjugated with filaggrin-linker sediment peptide which is rich in glutamine residues, mediated by epidermal transglutaminase in the presence of calcium, and yield a high-molecular weight protein (skin fiber), as shown in The phosphorylated cystatin α showed strong inhibition to cathepsin L, but not to the cathepsin B and H, it also showed strong inhibition to the bacterial cysteine proteases and virus cathepsins. Therefore, cystatin α and phosphorylated cystatin α  showed strong protection from infections of bacteria or viruses in skin.The phosphorylated cystatin β  and the changing of their inhibitory activities. Cystatin β is located ubiquitously in lysosomes of various organs. The third position of the N-termius-cysteine residue of cystatin β reacts with glutathione to form a mixed disulfate complex and also is able to make dimmer by the disulfate bond. Those are inactive forms. As . The coefficient of oxidized or reduced forms of glutathiones regulates the inhibitory activity of cystatin β. Therefore, the activities of cathepsins are regulated by the intracellular redox potentials in spite of the changes of the inhibitory activities of cystatin β.Post-translational covalent modification of cystatin β-cell of pancreas, the cystatin β is located in insulin secretion particles, therefore, cystatin β is secreted with insulin in the pancreas. As


Furthermore, administration of LY294002, an inhibitor of PI3K, compromised the protective effect of CO and decreased the level of phospho-GSK3β after I/R injury. These results suggest that CO protects against liver damage by maintaining GSK3β phosphorylation, which may be mediated by the PI3K/Akt signaling pathway. Our study provides additional support for the therapeutic potential of CO in organ injury and identifies GSK3β as a therapeutic target for CO in the amelioration of hepatic injury.Carbon monoxide (CO) may exert important roles in physiological and pathophysiological states through the regulation of cellular signaling pathways. CO can protect organ tissues from ischemia/reperfusion (I/R) injury by modulating intracellular redox status and by inhibiting inflammatory, apoptotic, and proliferative responses. However, the cellular mechanisms underlying the protective effects of CO in organ I/R injury remain incompletely understood. In this study, a murine model of hepatic warm I/R injury was employed to assess the role of glycogen synthase kinase-3 (GSK3) and phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathways in the protective effects of CO against inflammation and injury. Inhibition of GSK3 through the PI3K/Akt pathway played a crucial role in CO-mediated protection. CO treatment increased the phosphorylation of Akt and GSK3-beta (GSK3 Hepatic ischemia/reperfusion (I/R) injury is a cause of significant morbidity and mortality after liver transplantation, hemorrhagic shock, and extended liver resection for cancer. The pathophysiology of liver I/R injury includes both initial cellular damage due to ischemia as well as delayed liver dysfunction following reperfusion-initiated and inflammation-induced hepatocellular damage . During ex vivo isolated liver perfusion model [TLR4 activation by lipopolysaccharide (LPS) can be suppressed by the cytoprotective heme oxygenase-1/carbon monoxide (HO-1/CO) system . CO, a ron model . Althougon model –9, the mβ (GSK3β) by Ser9 phosphorylation can confer cardioprotective effects during myocardial infarction [β activity has recently been identified in a number of studies as crucial in the regulation of the inflammatory response. Phosphatidylinositol-3-kinase (PI3 K)/Akt-dependent inhibition of GSK3β activity in monocytes can regulate TLR-dependent activation [β, the molecular mechanisms by which GSK3β confers cytoprotective effects in hepatic I/R injury through TRL4 modulation have not been well studied.Recent studies have shown that inhibition of glycogen synthase kinase 3farction , 11, andfarction . GSK3β ativation , 14. Altβ activity through a PI3 K/Akt-mediated pathway, leading to the downregulation of TLR4-dependent proinflammatory cytokines, and the upregulation of IL-10. Our results further validate the use of CO as a pharmacological cytoprotective agent against hepatic I/R injury and identify GSK3β as a major therapeutic target of CO action in the liver.In this study, we establish a signaling pathway by which CO can confer anti-inflammatory protection for liver homeostasis. We demonstrate that CO inhibits GSK3Male C57BL/6 wild type (WT) mice at 8–10 weeks of age were purchased from the Orient Bio . Animals were maintained in a specific pathogen-free facility. Animal studies were approved by the University of Ulsan Animal Care and Use Committee.The human hepatocarcinoma cell line (HepG2) and the murine macrophage cell line, RAW 264.7, were cultured in DMEM . All media was supplemented with 10% fetal bovine serum and a 100 units/mL penicillin-streptomycin mixture (Gibco).To evaluate the protective effect of inhaled CO, animals were randomly assigned to receive preconditioning with room air or room air supplemented with 250 parts per million (ppm) CO, for 12 hours in a sealed exposure chamber prior to the experiment. Mice were exposed to CO 250 ppm for 1 hour and 6 hours after reperfusion.i.p.) or vehicle (10% DMSO in PBS i.p.) was given 30 min prior to the ischemic insult.We used a well-established mouse model of warm hepatic ischemia followed by reperfusion . An atraTo detect serum alanine aminotransferase (sALT), serum was collected from peripheral blood. ALT activity, an indicator of hepatocellular injury, was measured using the EnzyChrom Alanine Transaminase Assay Kit .μm thick sections. Tissue sections were mounted on regular glass slides, deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol, and stained with hematoxylin and eosin (H&E). Overall pathological changes, including immune cell infiltration and hepatic cell necrosis, were diagnosed according to previously described methods [For histopathological observations, portions of liver were fixed in 10% neutral-buffered formalin solution and then dehydrated in graded alcohol. The fixed tissue was embedded in paraffin and sliced into 4  methods .β (S9) by immunohistochemistry, the tyramide signal amplification (TSA) biotin system  was used according to the protocols recommended by the manufacturer. Briefly, after blocking, the sections were incubated first with either anti-p-GS (S641) or anti-p-GSK3β (S9) antibody . After overnight incubation, the sections were washed and then incubated with biotinylated anti-rabbit IgG antibody, next with streptavidin-horseradish peroxidase (HRP), and then with the biotinyl tyramide amplification reagent. Deposition of the biotin-tyramide on tissue sections was visualized with streptavidin-HRP and the substrate diaminobenzidine ; and then the sections were then counterstained with hematoxylin.For the detection of p-GS (S641) and p-GSK3κB p65 and anti-phospho-CREB antibodies .Preparation of nuclear extracts was carried out using the Nuclear/Cytosol Fractionation Kit . Proteins in the cell lysates were immunoprecipitated with anti-CBP antibodies for 3 h at 4°C, followed by incubation with Dynabeads protein G overnight at 4°C. Proteins in the immunoprecipitates were resolved using SDS-PAGE, followed by Western blotting with anti-phospho-NF-β-mercaptoethanol for 5 min. Proteins were then subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes . After blocking with 5% skim milk in PBS, membranes were incubated with appropriate dilutions of antibodies at 4°C overnight as follows: polyclonal rabbit anti-phospho glycogen synthase kinase (Ser9), rabbit anti-phospho glycogen synthase (Ser641), mouse anti-glycogen synthase kinase, rabbit anti-glycogen synthase, rabbit anti-phospho CREB (Ser133), rabbit anti-phospho Akt (Ser473), rabbit anti-HMGB1 , rabbit anti-CBP, rabbit anti-phospho-NF-κB-p65 (Ser276), and β-actin  were used. Membranes were then washed with 0.05% PBS-Tween 20 and incubated with a 1/5000 dilution of HRP-conjugated secondary Abs at room temperature for 1 h. Immunoreactivity was detected using the ECL detection system . Films were exposed at multiple time points to ensure that the images were not saturated.Harvested liver tissues and cells were lysed with mammalian lysis buffer containing phosphatase and protease inhibitors. Equal amounts of cell lysates were measured with the BCA protein assay reagent . Lysates were boiled in sample buffer containing Total RNA was prepared using Trizol reagent . Three microgram of total RNA was used to synthesize the first-strand cDNA by using oligo-dT primers  and M-MLV reverse transcriptase  according to the manufacturer's instructions. The synthesized cDNA was subjected to the PCR-based amplification. Semiquantitative RT-PCR was performed using Taq polymerase . Real-time PCR was performed using SYBR Green PCR Master Mix  on an ABI 7500 Fast Real-Time PCR System . PCR primer pairs were as follows: TNF: 5′-AGA CCC TCA CAC TCA GAT CAT CTT C-3′, 5′-TTG CTA CGA CGT GGG CTA CA-3′, IL-6: 5′-CGA TGA TGC ACT TGC AGA AA-3′, 5′-TGG AAA TTG GGG TAG GAA GG-3′ and IL-10: 5′-CAG TAC AGC CGG GAA GAC AA-3′, 5′-CAG CTT CTC ACC CAG GGA AT-3′.Neutrophil sequestration in liver was quantified by measuring tissue MPO activity. Tissue samples for MPO analysis were frozen in liquid nitrogen immediately after removal from the animal and were thawed and homogenized and centrifuged to remove insoluble materials. MPO activities were measured using a mouse myeloperoxidase DuoSet ELISA kit  according to the manufacturer's instruction. The supernatants were analyzed for MPO levels by sandwich ELISA. The levels of MPO in organ extracts were expressed as ng/mg of protein.t-test.All data were expressed as mean ± SD. Differences between experimental groups were compared using the Student's two-tailed unpaired versus  1091 ± 230 U/L, respectively, P < 0.01). panels (A)–(C)) and immune cell infiltration (panel (D)–(F)) observed in the air-treated I/R group were markedly reduced with CO inhalation. Furthermore, myeloperoxidase (MPO) activity, reflecting liver neutrophil activity, was decreased in the CO inhalation group, compared with the air-treated group after hepatic I/R  (Carbon monoxide (CO) has been shown to exert protective effects in various tissue models of I/R injury , 9, 17.  < 0.01) . Since H < 0.01) .β, which is negatively regulated by Akt. As shown in β in HepG2 cells. Conversely, CO decreased the phosphorylation of GS, the substrate of GSK3β.Previous reports have shown that HO-1 derived CO can activate the PI3 K-Akt pathway  and therβ was modulated by ROS generation. Treatment with CO dramatically increased LPS-induced phosphorylation of Akt and GSK3β. Inhibition of ROS generation with NAC attenuated LPS/CO-induced phosphorylation of Akt and GSK3β. These results suggest that CO stimulates LPS-dependent PI3 K/Akt-GSK3β phosphorylation via enhanced ROS production.A previous study has shown that both endogenous and exogenous CO can increase cellular ROS generation . ROS canβ axis may be involved in CO-mediated protection in the liver I/R model.Therefore, to examine whether CO activates PI3 K/Akt signaling during hepatic I/R injury, we examined the expression of p-Akt in mice subjected to hepatic I/R. There was a significant increase in p-Akt in the CO-treated mice subjected to hepatic I/R injury at 1 hour of reperfusion relative to air-treated controls, which was sustained until 6 hours of reperfusion . These fβ phosphorylation in mouse liver as a self-regulatory mechanism [β is involved in amelioration of liver pathology, we hypothesized that GSK3β inhibition may represent the underlying mechanism for CO-mediated liver protection. To evaluate the potential role of GSK3β in CO-mediated liver protection, we analyzed the effects of CO on GSK3β phosphorylation at Ser-9 and glycogen synthase (GS) phosphorylation at Ser-641, the substrate of GSK3β. As shown in β in the ischemic livers, compared with the sham control and air-inhaled groups. Conversely, the level of GS phosphorylation was decreased in ischemic mice given CO inhalation, compared with the air inhalation group. According to a previous report (26), the decrease of GS phosphorylation that occurs in response to I/R stimulation alone is due to GS phosphatase activation by I/R. Thus, transient ischemia induces GS activation and glycogen synthesis in vivo. These observations suggest that this occurs through a different mechanism than through the increase of pGSK3-β, as observed with CO treatment.I/R stimulation has been previously shown to trigger GSK3echanism . Given tPrevious studies have demonstrated that the cytoprotective effects of CO involve p38 MAPK signaling . Consistβ phosphorylation in ischemic liver and reduced GS phosphorylation, as compared with the air-inhalation group. Consistent with immunohistochemistry data, decreased mRNA levels of TNF-α, IL-6, and CXCL10 were consistently found in the livers of mice subjected to CO-inhalation, compared with those of the air-inhalation group  (panel (D)), mice given the PI3 K inhibitor revealed significant hepatocellular necrosis, cytoplasmic vacuolization, and sinusoidal congestion (panel (C)). Livers of animals treated with the PI3 K inhibitor after CO inhalation showed moderate to severe hepatocellular changes (panel (E)). As shown in P < 0.01). In contrast, livers from the CO inhalation group showed decreased MPO activity (3.738 ± 1.203 ng/mg), compared with the group subjected to PI3 K inhibitor treatment after CO inhalation . Thus, PI3 K/Akt-dependent GSK3β phosphorylation, a therapeutic target of CO, serves as a self-regulatory mechanism of liver homeostasis to limit the excessive I/R-induced tissue damage.The PI3 K-Akt pathway has been shown  of TLR4 . We usedfter I/R . Increas < 0.01) . Unlike β in a PI3 K/Akt-dependent pathway [in vivo findings, we analyzed whether CO-dependent activation of the PI3 K/Akt pathway may regulate TLR4-dependent inflammatory responses through GSK3β inhibition in vitro. LPS-stimulated macrophages were first pretreated with the PI3 K inhibitor (LY294002) and then treated with a CO releasing molecule (CORM2). Western blot analysis showed that treatment with CORM2 dramatically increased LPS-induced phosphorylation of Akt, GSK3β, and its downstream target, the cAMP response element-binding protein (CREB). However, pretreatment with PI3 K inhibitor (LY294002) inhibited phosphorylation of GSK3β, Akt, and CREB in LPS-stimulated macrophages treated with CORM2 , leading to proinflammatory gene activation, which competes against the binding of CREB to CBP, the latter regulating IL-10 expression [We next sought to validate the cellular mechanism by which CORM2-induced phosphorylation of GSK3onocytes . GSK3β c of CREB . GSK3β ipression .β inactivation by CORM2 influenced the ability of CREB and NF-κB p65 to associate with CBP. As shown in Figures β inhibition.We sought to determine whether GSK3β pathway as a novel therapeutic target of the anti-inflammatory effects of CO in a hepatic I/R injury model. The GSK3β pathway has previously been identified as an important target of inflammatory regulation. GSK3β is a proline-directed Ser/Thr kinase that phosphorylates a number of substrates including glycogen synthase (GS) as well as constituents of numerous intracellular signaling pathways including SMAD3, β-catenin, NOTCH2, CREB, and others [β can negatively regulate the inflammatory response and protect mice from endotoxin shock [β inhibition was associated with increased cAMP-response element binding (CREB) protein DNA binding activity, resulting in the increased production of the anti-inflammatory cytokine IL-10 and the decreased NF-κB-dependent production of pro-inflammatory genes [β ameliorates liver I/R injury through an IL-10 mediated immune regulatory mechanism [In the current study, we identify the GSK3d others –31. Recein shock . In thisry genes . Recent echanism . During via downregulation of the MEK/ERK1/2 signaling pathway [Carbon monoxide (CO), which can be applied by inhalation or by pharmacological delivery with CORMs, continues to show promise as an anti-inflammatory therapeutics in several models of organ I/R injury. For example, inhaled CO conferred tissue protection in rodents subjected to lung I/R injury, as evidenced by reduced markers of apoptosis, which depended on activation of the MKK3/p38 MAPK pathway . Additio pathway .α challenge [ex vivo in an isolated perfused liver model subjected to cold ischemia injury, in part by upregulating the p38 MAPK pathway [α and iNOS expression [In rodent models, CO has been shown to protect against acute liver injury caused by TNFhallenge  and can hallenge , 17, 34.via inhibition of cytochrome c oxidase leading to the generation of low levels of reactive oxygen species (ROS) that mediate adaptive signaling pathways [β through a mechanism that involves increased ROS-induced Akt phosphorylation. These results provide evidence that the protective effects of CO are mediated through redox mechanisms that can lead to the activation of adaptive pathways.ROS have been shown to be involved in the counter-regulation of inflammation in response to LPS treatment through modulation of macrophage production of IL-10 . Additiopathways . Our datin vivo has also been shown to be dependent on the activation of the p38 MAPK and PI3 K/Akt pathways.The phosphatidylinositol-3-kinase (PI3 K)/Akt pathway represents another multifunctional signaling pathway that promotes cell survival under adverse conditions. Previous studies have implicated the PI3 K/Akt pathway in the cytoprotective effects of CO. For example, during anoxia/reoxygenation of pulmonary endothelial cells, CO treatment protected against apoptosis by upregulating the p38 MAPK and PI3 K/Akt-dependent STAT3 pathway. The protection afforded by CO in a cardiac I/R injury model β activity in monocytes regulates TLR-dependent activation of inflammatory responses [β signaling pathway and inhibition of NF-κB nuclear translocation were shown to contribute to cardioprotection during myocardial I/R injury [in vivo data demonstrate that CO activates PI3 K/Akt signaling to promote GSK3β inhibition through phosphorylation at Ser9 during hepatic I/R injury. Furthermore, we have shown that activation of this pathway by CO therapy is a crucial mediator of the protection afforded by CO against hepatic injury and inflammation during I/R injury in mice.The PI3 K/Akt pathway has been identified as an important regulator of GSK3 signaling. PI3 K/Akt-dependent inhibition of GSK3esponses , 14. ActR injury . Consistβ can differentially regulate TLR4-dependent signaling leading to modulation of anti-/proinflammatory cytokine balance. LPS stimulation in monocytes has been shown to result in Ser9 phosphorylation of GSK3β in a PI3 K/Akt-dependent pathway [β inhibition was associated with hepatoprotection through the augmentation of the expression of the anti-inflammatory cytokine IL-10. Consistently, our results demonstrate that GSK3β inactivation by CO results in IL-10 upregulation in macrophages through a mechanism involving the augmentation of the binding of CREB to the nuclear co-activator CBP, leading and the suppression of the binding of NF-κB p65 to the nuclear coactivator CBP.GSK3 pathway , which d pathway . GSK3β iβ through Ser9 phosphorylation, leading to the downregulation of TLR4-dependent proinflammatory cytokines, and the upregulation of IL-10. The latter effect is mediated through activation of CREB and disruption of the p65/CBP interaction. Our results further validate the use of CO as a pharmacological cytoprotective agent against hepatic I/R injury and identify GSK3β as a major therapeutic target of CO action in the liver.In summary, our results establish a signaling pathway by which CO can confer anti-inflammatory protection in the liver: CO activates PI3 K/Akt which results in inhibition of GSK3


The development of melanocytes is regulated by the tyrosine kinase receptor c-KIT and the basic-helix-loop-helix-leucine zipper transcription factor Mitf. These essential melanocyte survival regulators are also well known oncogenic factors in malignant melanoma. Despite their importance, not much is known about the regulatory mechanisms and signaling pathways involved. In this study, we therefore sought to identify the signaling pathways and mechanisms involved in c-KIT mediated regulation of Mitf. We report that c-KIT stimulation leads to the activation of Mitf specifically through the c-KIT phosphorylation sites Y721 (PI3 kinase binding site), Y568 and Y570 (Src binding site). Our study not only confirms the involvement of Ras-Erk signaling pathway in the activation of Mitf, but also establishes that Src kinase binding to Y568 and Y570 of c-KIT is required. Using specific inhibitors we observe and verify that c-KIT induced activation of Mitf is dependent on PI3-, Akt-, Src-, p38- or Mek kinases. Moreover, the proliferative effect of c-KIT is dependent on Mitf in HEK293T cells. In contrast, c-KIT Y568F and Y721F mutants are less effective in driving cell proliferation, compared to wild type c-KIT. Our results reveal novel mechanisms by which c-KIT signaling regulates Mitf, with implications for understanding both melanocyte development and melanoma. Cell signaling plays an important role in the fine tuning of cellular function and behavior. Signaling cascades generated by the cell surface tyrosine kinase receptor c-KIT through the binding of its ligand stem cell factor (SCF) are involved in the regulation of many cell types including melanocytes Mitf give rise to phenotypic defects in mice similar to that found in c-KIT and SCF mutant mice in that coat color is lacking due to absence of melanocytes and mutations in all three genes also affect mast cells Mitf mutations on the one hand and c-KIT/SCF mutations on the other hand in that Mitf affects eye and bone development whereas c-KIT and SCF do not; c-KIT and SCF have severe hematopoietic deficiencies which Mitf mutations do not exhibit, at least not to the same degree.Melanocytes are derived from the neural crest during embryogenesis. In order for these cells to fully differentiate into functional pigment producing melanocytes, these cells first have to migrate and colonize target tissues, including the skin and hair follicle. The program that enables such behavior is orchestrated by the c-KIT tyrosine kinase receptor and its target, the melanocyte master regulator Microphthalmia associated transcription factor (Mitf) CDK2 is maintained by Mitf in melanocytes and melanoma and the BCL2 gene was found to be transcribed and upregulated by Mitf under SCF stimulation CDKN1A and CDKN2A expression to inhibit cell growth and initiate differentiation Mitf is a basic helix-loop-helix-leucine-zipper transcription factor that is not only essential for melanogenesis and melanocyte function, but is also involved in bone and mast cell development To date, two mechanisms are known by which c-KIT regulates the activity of Mitf. First, c-KIT phosphorylation activates the Ras-Erk pathway where Erk2 directly phosphorylates S73 of Mitf. As a result, the transactivation activity of Mitf is increased and the half-life of the protein is decreased as a consequence of ubiquitin-dependent degradation In this report we have used a series of c-KIT mutants and pharmacological inhibitors to identify the signaling pathways radiating from c-KIT to Mitf in human embryonic kidney (HEK) 293T cells and in Melan-A melanocytes. We show that the tyrosine-to- phenylalanine mutations Y568F, Y570F and Y721F in c-KIT prevent activation of Mitf during SCF stimulation. In contrast, the Y703F/Y936F c-KIT double mutant does not block SCF-induced activation of Mitf. In addition, by using selective inhibitors against Src, Mek 1 and 2, p38, PI3 kinase and Akt, we show that the corresponding downstream signaling pathways are involved in the activation of Mitf. Finally, our transfection model revealed that the different c-KIT mutants together with wild type (wt) Mitf yielded a lower degree of SCF-induced proliferation compared to wild type c-KIT expressing HEK293T cells.http://www.sgul.ac.uk/depts/anatomy/pages/dcbm&m.htm). The Melan-A cell line was a kind gift from Dr. Bennett.HEK293T cells (Thermo Scientific Open Biosystems) were cultured in Dulbecco's modified Eagle's medium and supplemented with 10% fetal bovine serum and 100 units/ml penicillin- streptomycin. Mouse melanocyte Melan-A cells were cultured according to recommended protocol  was used to generate tyrosine-to-phenylalanine substitution mutants of c-KIT contained in pcDNA3 vector. P3XFLAG-CMV-14 (Sigma Aldrich) was used as a vector backbone for the Mitf construct.Turbofect transfection reagent (Fermentas) was used for all the experiments in this study according to manufacturer's protocol.2, 37°C prior to SCF stimulation.SCF (ORF Genetics) was used for cell culture stimulation in a final concentration of 100 ng/ml. In the inhibitor experiments, 10 µM of SU6656 , U0126 (Promega), LY294002 (Sigma Aldrich) or 3 µM of Akt IV  was pre-incubated in cell culture medium for 30 minutes in 5% CO2 cell culture flasks (Nunc). One day after plating, cells were transfected with both c-KIT and/or Mitf plasmids and incubated for 8 hours in a cell culture incubator, after which the cells were washed 3 times with PBS and cultured in serum starved medium. Cells that were detached from culture flasks during the washing steps were collected and resuspended. SCF was added the day after the initial starvation procedure. Cells were trypsinized, resuspended in culture medium, stained with trypan blue and analyzed with Countess automated cell counter (Invitrogen) after 48 hours of SCF stimulation.HEK293T (4 million) cells were plated in 75 cm3VO4, 1% Trasylol, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA and 25mM beta-glycerophosphate. The lysates were centrifuged at 17,000 X g for 20 minutes at 4°C. The FLAG-tagged Mitf protein was immunoprecipitated with monoclonal anti-FLAG M2 antibody (Sigma Aldrich). Human c-KIT was immunoprecipitated with the KitC1 antibody Cells were lysed for 15 minutes in cold lysis buffer containing 1% Triton X-100, 25 mM Tris, pH 7.5, 1 mM Nahttp://rsbweb.nih.gov/ij/docs/menus/analyze.html. The intensity of the low molecular weight band was subtracted from the intensity of the heavy molecular weight band. According to this method a protein mobility shift seen as increased intensity of the heavy molecular weight protein band, produces a positive value. A resulting negative value indicates inactive state of Mitf. Each bar in the mobility shift quantitation charts in this study represents the average from three independent experiments. Error bars are reported as standard error of the mean.Mitf protein intensity was measured with ImageJ as outlined at the NIH website: p-value of <0.05 was considered significant. A 2-tail non parametric sign test was performed on Mitf mobility shift assay data, where the criterion for significance was set as p<0.05.Each data set from Mitf mobility shift assays and cell proliferation experiments represents the mean for three and nine separate experiments, respectively. One way analysis of variance was used to analyze significant differences in cell proliferation assay. Significant results were further examined by a Newman- Keuls multiple comparison test. A Ligand-stimulation of c-KIT post transcriptionally modifies Mitf through Erk-dependent phosphorylation of Mitf at S73 and S409. Addition of these phospho-groups can easily be detected by Western blotting where the modifications generate a mobility shift of the Mitf protein SCF binding to c-KIT causes auto-phosphorylation of various tyrosine residues in the intracellular domain of c-KIT. To pinpoint the involvement of the specific phospho-tyrosine residues involved in the SCF-induced activation of Mitf, we generated tyrosine-to-phenylalanine c-KIT mutants. In this experiment we used the HEK293T and mouse melanocyte Melan-A cell lines and transfected both c-KIT and Mitf into the cells. Because the Melan-A cells have endogenous expression of both wt Mitf and wt c-KIT, we needed to bypass the signals generated from wt mouse c-KIT in order to avoid interference with the transfected human c-KIT mutants. The activation of c-KIT can be detected by a general anti-phosphotyrosine monoclonal antibody (4G10 from Sigma Aldrich) after immunoprecipitation of the c-KIT protein. In the Melan-A cells, endogenous mouse c-KIT was phosphorylated after 5–15 min of mouse SCF (mSCF) stimulation . FurtherPhosphorylation of c-KIT sites Y568 and Y570 (with Y568 being the primary activation site) allows docking and activation of Src family kinases leading to the activation of the Ras-Raf-Mek-Erk signaling cascade The PI3 kinase survival pathway involving the serine/ threonine kinase Akt has been shown to protect melanocytes from apoptosis In all, these observations illustrate that the activation of Mitf by c-KIT is dependent on functional direct docking sites for the PI3 kinase subunit p85 and Src kinase on c-KIT Y721 and Y568, respectively. Also, the data show that c-KIT phosphorylation sites Y703/936 are obsolete.The role of Src kinase in the activation of Mitf has not been investigated previously nor has the PI3 kinase-Akt pathway been studied in the context of c-KIT- induced Mitf activation. To investigate this, we treated cells with either Src family kinase inhibitor (SU6656), Mek inhibitor (U0126), PI3 kinase inhibitor (LY294002) or Akt inhibitor (Akt IV) for 30 minutes prior to SCF stimulation. The addition of SCF to untreated samples caused a mobility shift or activation of Mitf. However, pre-treatment with SU6656, U0126, LY294002 or Akt IV inhibited the activation of Mitf in all cases . These oThe stress activated protein kinase p38 acts downstream of c-KIT and is known to be involved in the activation of Mitf gene expression in response to UV radiation Our results not only confirm that c-KIT activates Mitf through the Ras-Erk pathway as previously reported p<0.01, p<0.01 and p<0.001, respectively) augmented cell growth compared to respective 0 hour negative control. In addition, control cells transfected with only c-KIT or Mitf did not produce any proliferative effects upon SCF stimulation (Mitf activates transcription of a wide array of genes that regulate both cell survival and cell death mulation .Our results show that SCF-dependent cell proliferation is only achieved in the HEK293T cells when wt c-KIT and wt Mitf are present. Although, c-KIT mutants that lack functional Src and p85 binding sites can mediate ligand-induced cell proliferation, the response is severely impaired compared to the wild-type receptor.The mechanisms behind c-KIT activation are well studied. When c-KIT is bound to its ligand, SCF, the receptor dimerizes, leading to auto-activation of the cytoplasmic tyrosine kinase domain in vitro phosphatase treatment of SCF-stimulated Mitf only reversed SCF-induced mobility shift of the protein, leaving the upper band intact. Thus, it seems mobility shift of Mitf is the consequence of S73 and S409 phosphorylation and subsequent post translational modification that directly dictates the occurrence of the upper band. Since, in vivo inhibition of Mek did not completely abrogate the shift, this suggests the involvement of additional signaling pathways that might regulate the modification of Mitf.In melanoma, Src family of kinases has the ability to suppress differentiation and favor proliferation The importance of the Ras-Erk pathway in the activation of Mitf is highlighted in this experiment, in agreement with previous studies. But, more importantly, we show that the interaction between Src kinase and c-KIT phosphorylation site Y568 is required for the activation of Mitf. The Ras-Erk pathway is known to be critical for cell proliferation and both c-KIT One of the functions of Mitf is to indirectly protect the skin cells from UVB-induced DNA damage by transcribing genes that controls the production and transfer of melanin to adjacent keratinocytes. The p38 stress response pathway is triggered by UVB exposure in human melanocytes. As a result, the level of Mitf is increased and pigment producing genes are transcribed During progression of melanoma, expression of the scaffolding and adaptor protein Gab2 is often found to be amplified. The presence of Gab2 aggravates the invasiveness and mestastatic capabilities of melanoma The PI3 kinase-Akt survival pathway protects both melanocytes and melanoma from programmed cell death Our cell proliferation results point out another level of signaling complexity. As expected, the highest level of cell proliferation was achieved through the stimulation of wt c-KIT in the presence of Mitf. Even though SCF stimulation of the different mutants of c-KIT results in reduced Mitf- dependent cell growth, the mutants are still quite capable of promoting proliferation. This phenomenon can be partly explained by the involvement of other regulators in c-KIT signaling. The c-KIT phosphorylation sites Y568 and Y570 in the juxtamembrane domain serve as multifunctional docking sites. Mutation of one or the other leads to a loss of signalling through multiple pathways. The phosphorylated Y568 residue does not only act as binding site for Src family kinases Tyrosine 703 and 936 are consensus Grb2 binding sites in that they have an asparagine residue located two amino acids downstream of the phosphorylated tyrosine residue. However, they do not seem to be directly involved in recruitment of Grb2-Sos to the receptor. Rather, Grb2 acts as an adapter recruiting either the negative regulator Cbl Y721 is a consensus binding site for the p85 subunit of PI3-kinase. In cells not expressing GAB2, Y721 is essential for activation of PI3 kinase by c-KIT To summarize, the signalling income of ligand-stimulation of c-KIT is very complex, with several pathways initiated by the receptor feeding into each other and influencing each other. Thus, when all these components come into play, an apparent loss of Mitf mobility shift caused by c-KIT mutants Y568F and Y721F does not necessarily translate into a phenotypic response such as complete lack of cell proliferation.In summary, our data show that c-KIT regulates Mitf through the c-KIT phosphorylation sites Y568, Y570 and Y721 while Y703 and Y936 are dispensable . We have


Inhibitors of dipeptidyl peptidase-IV (DPP-IV), which decrease the degradation of glucose-lowering GLP-1(7-36) to the metabolically inactive GLP-1(9-36), are current new treatment options for patients with type 2 diabetes mellitus, a high-risk population for cardiovascular disease. However, the effects of the metabolite GLP-1(9-36) on atherosclerosis are unknown. Thus, the present study examined the effect of GLP-1(9-36) on chemokine-induced CD4-positive lymphocyte migration as one of the early and critical steps in atherogenesis.Stimulation of isolated human CD4-positive lymphocytes with SDF-1 led to a 3.4 fold  increase in cell migration. Pretreatment of cells with GLP-1(9-36) reduced this effect in a concentration-dependent manner by 41% to a 2.0 fold induction at 10 nmol/L GLP-1(9-36) . Similar effects were obtained when RANTES was used as a chemokine to induce cell migration. The action of GLP-1(9-36) on CD4-positive lymphocyte migration was mediated through an early inhibition of chemokine-induced PI-3 kinase activity. Downstream in the PI-3 kinase signaling pathway, GLP-1(9-36) inhibited SDF-1-induced phosphorylation of MLC and cofilin and decreased f-actin formation as well as ICAM3 translocation as shown by Western blotting, flow cytometry and immunohistochemistry, respectively. However, the effect of GLP-1(9-36) on PI-3 kinase signaling was not associated with increased intracellular levels of cAMP. Furthermore, experiments with siRNA demonstrated that the inhibitory effect of GLP-1(9-36) on SDF-1-induced ICAM3-translocation was preserved in human CD4-positive lymphocytes lacking the GLP-1 receptor, suggesting signaling independent of the known GLP-1 receptor.Thus, GLP-1(9-36) inhibits chemokine-induced CD4-positive lymphocyte migration by inhibition of the PI3-kinase pathway independent of cAMP and GLP-1 receptor signaling. Further studies are needed to assess whether such effects may be clinically relevant for patients with type 2 diabetes treated with DPP-IV inhibitors. Glucagon-like peptide-1 (GLP-1) analoga and inhibitors of dipeptidyl peptidase-IV (DPP-IV) are current new treatment options for patients with type 2 diabetes mellitus, a high-risk population for atherosclerosis Whereas various effects of metabolically active GLP-1(7-36) on multiple organs have been suggested, only limited evidence exists about the actions of GLP-1(9-36) on the cardiovascular system  on mechanisms involved in atherogenesis. Thus, we investigated the effects of GLP-1(9-36) on the chemokine-induced migration of human CD4-positive lymphocytes as an early and critical step in atherogenesis.Buffy coats were obtained completely anonymized from the Department of Transfusion Medicine, University Hospital of Aachen. As the buffy coats were obtained completely anonymized, no ethical approval was necessary for this study in Germany. Human CD4-positive lymphocytes were isolated from buffy coats as has been previously described For in vitro cell migration assays, human CD4-positive lymphocytes were cultured in serum-free media for 16 h. A 48-well microchemotaxis chamber (Neuroprobe) was used to investigate T-cell chemotaxis Standard western blot analysis was performed using specific antibodies against p-cofilin, p-MLC  and human GLP-1 receptor (Upstate). Western blot images were analyzed by Carestream molecular imaging software (Carestream). For determination of protein levels, bands were normalized to ß-actin bands.RNA was extracted using the RNeasy extraction kit (Quiagen). cDNA was generated by reverse transcription . Quantitative RT-PCR was performed using a commercially available Taqman Gene Expression Assay for the full length GLP-1 receptor (Applied Biosystems). Samples were normalized to a control gene .6 cells were treated with the same conditions as for the migration experiments and stained with trypan blue (Sigma Aldrich). Vital cells were counted in a Neubauer chamber.Standard flow cytometry was performed using a CANTO II (BD) and a PE-labeled antibody against human CXCR4 (BD Pharmigen). For the determination of vitality, 1×10A PI-3 kinase activity assay was performed as has been previously described 6 cells/mL) were treated with 100 ng/mL SDF-1 for times indicated. Cell stimulation was stopped by fixation using 3.7% (wt/vol) paraformaldehyde in PBS, pH 7.4. Cells were then washed at room temperature in TBS . Cells were permeabilized with 0.1% Triton X-100 for intracellular staining of f-actin for 10 min. Cell suspensions were incubated for 30 min in PBS with FITC-conjugated phalloidin (Sigma). Flow cytometry analysis was performed in a FACScan cytofluorometer (Guave) and induction of f-actin was measured as previously described For f-actin staining CD4-positive lymphocytes were pre-treated with 10 nmol/L GLP-1(9-36) for 30 min. Then, CD4-positive lymphocytes (3×105 CD4 positive lymphocytes were incubated in 4-well plates (Costar Corp.) in a final volume of 1 ml complete medium on coverslips coated with collagen. Prior to treatment with SDF-1 , cells were pre-incubated with GLP-1(9-36) for 30 min. CD4-positive lymphocytes were then fixated with 3.7% (wt/vol) paraformaldehyde in PBS and rinsed in TBS. Cells were incubated with a specific mouse anti-human ICAM3 antibody (BD Pharmingen) and after washing with PBS, an carboxymethylindocyanine 3-Cy3-coupled goat anti-mouse antibody (Jackson) was added for 30 min. After washing slides were counterstained with DAPI. Images were recorded with a fluorescence microscope (Leica) and cells in 5 randomly selected fields (∼ 100 cells) were analysed. If ICAM-3 was located evenly on the outer side of the plasma membrane, T-cells were considered to be resting. When a clear clustering of ICAM3 at the uropod was visible, ICAM3 translocation was considered to be present.To determine ICAM3 translocation, immunofluorescence staining for ICAM3 was performed on human CD4-positive lymphocytes as has been previously described 6 isolated, human CD4-positive lymphocytes were treated with 10 nmol/L GLP-1(9-36) (Phoenix) or 100 ng/ml SDF-1 (Upstate) for times indicated. 100 ng/ml Forskolin  was used as a positive control. The cAMP competitive ELISA (Thermo Scientific) was performed according to the manufactureŕs protocol. Briefly, cAMP was extracted by adding 0.1 mol/l HCl with 0.5% triton X-100 to the cells. After centrifugation at 600 g for 10 min, the supernatant was used for the determination of cAMP levels by competitive cAMP ELISA.For determination of intracellular cAMP concentrations 1×10Transfection of human CD4-positive lymphocytes with siRNA of the GLP-1 receptor has been performed as has been previously described The data are reported as mean±standard error of the mean (SEM). Differences were analyzed by one- or two-way-ANOVA followed by the appropriate post-hoc test where applicable. A p-value <0.05 was regarded as statistically significant.To investigate the effect of GLP-1(9-36) on CD4-positive lymphocyte migration, experiments in the modified Boyden chamber were performed with isolated human CD4-positive lymphocytes. SDF-1 treatment increased cell migration by 3.4 fold  and pretreatment of cells with GLP-1(9-36) reduced this effect in a concentration-dependent manner by 41% to a 2.0 fold induction at 10 nmol/L GLP-1(9-36)  . SimilarFurther migration experiments were performed to exclude that the phenomenon observed is caused by unspecific effects of a 28 amino acid peptide. In these experiments a scrambled GLP-1(9-36) was used, which consists of the same amino acids as GLP-1(9-36) but in a random order. Scrambled GLP-1(9-36) did not inhibit SDF-1-induced human CD4-positive lymphocyte migration . NeitherGLP-1(9-36) did not affect the expression of the chemokine receptor CXCR4 and did not influence cell viability as shown by flow cytometry and trypan blue staining, respectively .Downstream of the chemokine receptor, the activation of PI-3 kinase is a crucial step in chemokine-induced CD4-positive lymphocyte migration Downstream in the PI3-kinase signaling cascade, chemokine stimulation of CD4-positive lymphocytes leads to serine phosphorylation of the enzymes cofilin and MLC. GLP-1(9-36) inhibited both SDF-1-induced phosphorylation of cofilin  and MLC As ICAM3 translocation to the uropod of migrating CD4-positive lymphocytes is a crucial step in cell adhesion during migration Given that f-actin formation in isolated human CD4-positive lymphocytes is an important mechanism in cell polarisation during cell migration As cAMP has been suggested to be involved in GLP-1(9-36) signalling As binding to the GLP-1 receptor protein has been shown to reduce CD4-positive lymphocyte migration This study demonstrates that the degradation product GLP-1(9-36) inhibits chemokine-induced migration of human CD4-positive lymphocytes, which is mediated through an early inhibition of PI-3 kinase activity with subsequent inhibition of MLC and cofilin phosphorylation as well as f-actin formation and ICAM3 translocation. The action of GLP-1(9-36) on CD4-positive lymphocyte migration and the PI3-kinase signaling cascade is not mediated by cAMP signaling or the GLP-1 receptor protein.Current new treatment options for patients with type 2 diabetes include DPP-IV inhibitors, which decrease the rapid degradation of GLP-1(7-36) to the metabolite GLP-1(9-36). Given that patients with type 2 diabetes are a high-risk population for cardiovascular events The effect of GLP-1(9-36) on human CD4-positive lymphocyte migration is mediated by an early inhibition of PI-3 kinase activity, an important step in chemokine-induced human CD4-positive lymphocyte migration. PI-3 kinase activity results in the activation of small Rho GTPases. Downstream of these GTPases, the phosphorylation of cofilin is important to allow for actin polymerization of migrating cells, while the phosphorylation of MLC contributes to cell contraction at the uropod of migrating T-cells. In this study, GLP-1(9-36) reduces chemokine-induced phosphorylation of cofilin with subsequent f-actin formation. Moreover, GLP-1(9-36) limits the phosphorylation of MLC and inhibits SDF-1-mediated ICAM3 translocation to the uropod of migrating cells, thus inhibiting two crucial steps in T-cell migration.GLP-1(9-36) diminishes both SDF-1- as well as RANTES-induced lymphocyte migration and thus the effect of GLP-1(9-36) on cell migration does not appear to be depended on the chemotactic stimulus employed. However, the concentrations of GLP-1(9-36) necessary to inhibit T-cell migration were physiological for RANTES, but supraphysiological for SDF. This may be caused by the fact that SDF was observed to be a much stronger inducer for T-cell migration (3.43-fold for SDF vs 1.98-fold for RANTES), with higher GLP-1(9-36) concentrations necessary to inhibit SDF compared to RANTES - induced T-cell migration.Moreover, the GLP-1(9-36) concentrations employed in our study correspond to the concentrations used in previous experiments Potential mechanisms by which GLP-1(9-36) may inhibit PI3-kinase activity may be at the level of the chemokine receptor via interaction with a so far unknown GLP-1(9-36) receptor, mediated by G-proteins, caused by dimerization of and with PI3-kinase or via modification of the regulatory p85 or catalytic p110 subunit.Thus, the present study shows that GLP-1(9-36) inhibits chemokine-induced CD4-positive lymphocyte migration by inhibition of the PI3-kinase pathway independent of cAMP and GLP-1 receptor signaling. Further studies are needed to assess whether such effects may be clinically relevant for type 2 diabetes patients treated with DPP-IV inhibitors.