PURPOSE: Our goal was to investigate the effects of inhibition of transglutaminase 2 (TGase 2) on endotoxin-induced uveitis (EIU) METHODS: EIU was induced in female Lewis rats by single footpad injections of 200 microgram of lipopolysaccharide (LPS). TGase 2 inhibitors were administered intraperitoneally 30 minutes before and at the time of LPS administration. Rats were sacrificed 24 hours after injection, and the effects of the TGase 2 inhibitors were evaluated by the number of intraocular inflammatory cells present on histologic sections and by measuring the TGase 2 activity and TGase products in the aqueous humor (AqH). TGase 2 substrates were also assayed in AqH from uveitis patients. RESULTS: Clinical indications of EIU, the number of cells present on histologic sections, and TGase 2 activity in AqH increased in a time-dependent manner, peaking 24 hours after LPS injection. Inflammation in EIU was significantly reversed by treatment with TGase inhibitors. A 23-kDa cross-linked TGase substrate was identified in the AqH from EIU rats and uveitis patients. MALDI-TOF analysis showed that this substrate in uveitis patients was human Ig kappa chain C region. CONCLUSIONS: TGase 2 activity and its catalytic product were increased in the AqH of EIU rats. TGase 2 inhibition attenuated the degree of inflammation in EIU. Safe and stable TGase inhibitors may have great potential for the treatment of inflammatory uveitis.
Animals ; Disease Progression ; Enzyme Inhibitors/*therapeutic use ; Female ; GTP-Binding Proteins/*antagonists & inhibitors/*metabolism ; Lipopolysaccharides ; Rats ; Rats, Inbred Lew ; Transglutaminases/*antagonists & inhibitors/*metabolism ; Uveitis/chemically induced/*enzymology/pathology

OBJECTIVETo investigate the effect of exenatide on chemotactic migration of adipose-derived stem cells (ADSCs) and confirm that Rho GTPase is the downstream effector protein of SDF-1/CXCR-4 migration pathway.

METHODSADSCs were isolated, cultured, identified by flow cytometry, and induced to differentiate in vitro. RTCA xCELLigence system was used to analyze the effect of exenatide on ADSC proliferation. The effects of exenatide at different concentrations, AMD3100 (CXCR-4 antagonist), and CCG-1423 (Rho GTPase antagonist) on chemotactic migration of ADSCs were tested using Transwell assay. The expression of CXCR-4 in exenatide-treated ADSCs was measured by flow cytometry and Western blotting. Active Rho pull-down detection kit was used to detect the expression of Rho GTPase. Laser confocal microscopy was used to observe the formation of stress fibers in ADSCs with different treatments.

RESULTSExenatide treatment for 24 h had no significant effect on ADSC proliferation. Exenatide obviously promoted chemotactic migration of ADSCs in a concentration-dependent manner, and this effect was blocked by either AMD3100 or CCG-1423. Both flow cytometry and Western blotting showed that exenatide dose-dependently up-regulated CXCR-4 expression in ADSCs. Western blotting showed that the expression of Rho GTPase was related to SDF-1/CXCR-4 pathway, and laser confocal microscopy revealed that the formation of stress fibers in ADSCs was related to SDF-1/CXCR-4/ Rho GTPase pathway.

CONCLUSIONExenatide promotes chemotactic migration of ADSCs, and Rho GTPase is the downstream effector protein of SDF-1/CXCR-4 pathway.

Adipose Tissue ; cytology ; Anilides ; pharmacology ; Benzamides ; pharmacology ; Cells, Cultured ; Chemokine CXCL12 ; metabolism ; Chemotaxis ; Heterocyclic Compounds ; pharmacology ; Humans ; Peptides ; pharmacology ; Receptors, CXCR4 ; antagonists & inhibitors ; metabolism ; Signal Transduction ; Stem Cells ; cytology ; Venoms ; pharmacology ; rho GTP-Binding Proteins ; antagonists & inhibitors ; metabolism

Animals ; COS Cells ; Cercopithecus aethiops ; Endoplasmic Reticulum ; GTP Phosphohydrolases ; antagonists & inhibitors ; chemistry ; genetics ; metabolism ; GTP-Binding Proteins ; antagonists & inhibitors ; chemistry ; genetics ; metabolism ; Gene Expression ; Genetic Complementation Test ; HeLa Cells ; Humans ; Kinetics ; Membrane Fusion ; genetics ; Membrane Proteins ; antagonists & inhibitors ; chemistry ; genetics ; metabolism ; Protein Multimerization ; RNA, Small Interfering ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism ; Vesicular Transport Proteins ; genetics ; metabolism

Actins ; chemistry ; Amides ; therapeutic use ; Animals ; Apoptosis ; drug effects ; Cytoskeleton ; drug effects ; Enzyme Inhibitors ; therapeutic use ; Female ; Liver Neoplasms, Experimental ; drug therapy ; pathology ; Mice ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Pyridines ; therapeutic use ; ras Proteins ; analysis ; rho-Associated Kinases ; analysis ; antagonists & inhibitors ; rhoA GTP-Binding Protein ; analysis ; rhoC GTP-Binding Protein

Diabetic nephropathy (DN) is a progressive kidney disease that is caused by injury to kidney glomeruli. Podocytes are glomerular epithelial cells and play critical roles in the glomerular filtration barrier. Recent studies have shown the importance of regulating the podocyte actin cytoskeleton in early DN. The phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, simultaneously regulates Rac1 and Cdc42, which destabilize the podocyte actin cytoskeleton during early DN. In this study, in order to evaluate the reno-protective effects of wortmannin in early DN by regulating Rac1 and Cdc42, streptozotocin (STZ)-induced proteinuric renal disease (SPRD) rats were treated with wortmannin. The albuminuria value of the SPRD group was 3.55 +/- 0.56 mg/day, whereas wortmannin group was 1.77 +/- 0.48 mg/day. Also, the albumin to creatinine ratio (ACR) value of the SPRD group was 53.08 +/- 10.82 mg/g, whereas wortmannin group was 20.27 +/- 6.41 mg/g. Changes in the expression level of nephrin, podocin and Rac1/Cdc42, which is related to actin cytoskeleton in podocytes, by wortmannin administration were confirmed by Western blotting. The expression levels of nephrin (79.66 +/- 0.02), podocin (87.81 +/- 0.03) and Rac1/Cdc42 (86.12 +/- 0.02) in the wortmannin group were higher than the expression levels of nephrin (55.32 +/- 0.03), podocin (53.40 +/- 0.06) and Rac1/Cdc42 (54.05 +/- 0.04) in the SPRD group. In addition, expression and localization of nephrin, podocin and desmin were confirmed by immunofluorescence. In summary, we found for the first time that wortmannin has a reno-protective effect on SPRD rats during the early DN. The beneficial effects of wortmannin in SPRD rats indicate that this compound could be used to delay the progression of the disease during the early DN stage.
Albumins/metabolism ; Androstadienes/*administration & dosage/pharmacology ; Animals ; Creatinine/blood ; Desmin/genetics/metabolism ; Diabetes Mellitus, Experimental/*drug therapy/metabolism/pathology ; Diabetic Nephropathies/*drug therapy/metabolism/pathology ; Disease Models, Animal ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/metabolism ; Kidney/*pathology ; Membrane Proteins/genetics/metabolism ; Phosphatidylinositol 3-Kinases/*antagonists & inhibitors ; Podocytes/*drug effects/metabolism/pathology ; Rats ; Rats, Inbred Strains ; cdc42 GTP-Binding Protein/genetics/metabolism ; rac1 GTP-Binding Protein/genetics/metabolism

Lysophosphatidic acid (LPA) is a bioactive phospholipids and involves in various cellular events, including tumor cell migration. In the present study, we investigated LPA receptor and its transactivation to EGFR for cyclooxygenase-2 (COX-2) expression and cell migration in CAOV-3 ovarian cancer cells. LPA induced COX-2 expression in a dose-dependent manner, and pretreatment of the cells with pharmacological inhibitors of Gi (pertussis toxin), Src (PP2), EGF receptor (EGFR) (AG1478), ERK (PD98059) significantly inhibited LPA- induced COX-2 expression. Consistent to these results, transfection of the cells with selective Src siRNA attenuated COX-2 expression by LPA. LPA stimulated CAOV-3 cell migration that was abrogated by pharmacological inhibitors and antibody of EP2. Higher expression of LPA2 mRNA was observed in CAOV-3 cells, and transfection of the cells with a selective LPA2 siRNA significantly inhibited LPA-induced activation of EGFR and ERK, as well as COX-2 expression. Importantly, LPA2 siRNA also blocked LPA-induced ovarian cancer cell migration. Collectively, our results clearly show the significance of LPA2 and Gi/Src pathway for LPA-induced COX-2 expression and cell migration that could be a promising drug target for ovarian cancer cell metastasis.
Butadienes/pharmacology ; Cell Line, Tumor ; Cell Movement/drug effects/*physiology ; Cyclooxygenase 2/*biosynthesis ; Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors/metabolism ; Female ; Flavonoids/pharmacology ; GTP-Binding Protein alpha Subunits, Gi-Go/antagonists & inhibitors/*metabolism ; Humans ; Lysophospholipids/pharmacology ; Nitriles/pharmacology ; Ovarian Neoplasms/metabolism/*pathology ; Pertussis Toxin/pharmacology ; Protein-Tyrosine Kinases/antagonists & inhibitors/*metabolism ; Proto-Oncogene Proteins/antagonists & inhibitors/*metabolism ; Pyrimidines/pharmacology ; Receptor, Epidermal Growth Factor/antagonists & inhibitors/metabolism ; Receptors, Lysophosphatidic Acid/*metabolism ; Receptors, Prostaglandin E/metabolism ; Signal Transduction ; Transcriptional Activation ; Tyrphostins/pharmacology

This study was performed to examine the role of transglutaminase 2 (TG2) in ventilator-induced lung injury (VILI). C57BL/6 mice were divided into six experimental groups: 1) control group; 2) lipopolysaccharide (LPS) group; 3) lung protective ventilation (LPV) group; 4) VILI group; 5) VILI with cystamine, a TG2 inhibitor, pretreatment (Cyst+VILI) group; and 6) LPV with cystamine pretreatment (Cyst+LPV) group. Acute lung injury (ALI) score, TG2 activity and gene expression, inflammatory cytokines, and nuclear factor-kappaB (NF-kappaB) activity were measured. TG2 activity and gene expression were significantly increased in the VILI group (P < 0.05). Cystamine pretreatment significantly decreased TG2 activity and gene expression in the Cyst+VILI group (P < 0.05). Inflammatory cytokines were higher in the VILI group than in the LPS and LPV groups (P < 0.05), and significantly lower in the Cyst+VILI group than the VILI group (P < 0.05). NF-kappaB activity was increased in the VILI group compared with the LPS and LPV groups (P < 0.05), and significantly decreased in the Cyst+VILI group compared to the VILI group (P = 0.029). The ALI score of the Cyst+VILI group was lower than the VILI group, but the difference was not statistically significant (P = 0.105). These results suggest potential roles of TG2 in the pathogenesis of VILI.
Acute Lung Injury/pathology ; Animals ; Cystamine/therapeutic use ; Cytokines/analysis ; Enzyme Inhibitors/therapeutic use ; Enzyme-Linked Immunosorbent Assay ; GTP-Binding Proteins/*antagonists & inhibitors/genetics/metabolism ; Gene Expression ; Lipopolysaccharides/toxicity ; Male ; Mice ; Mice, Inbred C57BL ; NF-kappa B/metabolism ; Respiration, Artificial ; Transglutaminases/*antagonists & inhibitors/genetics/metabolism ; Ventilator-Induced Lung Injury/*enzymology/pathology/prevention & control

Tumor cells are known to produce larger amounts of reactive oxygen species (ROS) than normal cells. Although numerous reports have indicated the importance of ROS in urokinase plasminogen activator (uPA) production, the precise mechanisms remain controversial. In our study, we investigated the effect of ROS on uPA generation in human hepatoma cells, HepG2 and Hep 3B. We determined the effects of hepatocyte growth factor (HGF) on the regulation of ROS, which resulted in suppression of ROS production, as measured with the fluorescent probe, 2'-7'-dichlorofluorescein diacetate. The role of HGF in modulating ROS production, particularly that regulated by Rac-1, was determined. HGF suppressed the increment in Rac-1-regulated ROS in both cell lines. Treatment with 200 microM of H2O2 showed a 1.6-2.1 fold increment in HGF, but a little increment occurred at 500 microM of H2O2. It looks no dose dependent manner. Combined treatment with H2O2 and HGF, resulted in a slightly increased production of HGF compared to no treatment (control). Also, H2O2 upregulated uPA expression in both hepatoma cell lines. To identify the downstream pathways regulated by ROS, we treated cells with PD 98059, an MEK inhibitor, and SB 203580, a p38 inhibitor, after treatment with H2O2, and showed negative control between ERK and p38 kinase activities for uPA regulation. We found that HGF modulate Rac-1-regulated ROS production through activation of Akt and ROS regulates uPA production via MAP kinase, which provides a novel clue to clarify the mechanism underlying hepatoma progression.
Cell Line, Tumor ; Fluorescent Dyes/chemistry ; Hepatocyte Growth Factor/pharmacology/*physiology ; Humans ; Hydrogen Peroxide/pharmacology ; Imidazoles/pharmacology ; Liver Neoplasms/drug therapy ; Mitogen-Activated Protein Kinases/antagonists & inhibitors/*metabolism ; Pyridines/pharmacology ; Reactive Oxygen Species/*metabolism ; Recombinant Proteins/pharmacology ; Urokinase-Type Plasminogen Activator/*biosynthesis ; rac1 GTP-Binding Protein/metabolism

Our previous studies have shown that subthreshold concentrations of two platelet agonists exert synergistic effects on platelet aggregation. Here we studied the mechanism of synergistic interaction of 5-hydroxytryptamine (5-HT) and epinephrine mediated platelet aggregation. We show that 5-HT had no or little effect on aggregation but it did potentiate the aggregation response of epinephrine. The synergistic interaction of 5-HT (1-5 microM) and epinephrine (0.5-2 microM) was inhibited by alpha2-adrenoceptor blocker (yohimbine; IC50= 0.4 microM), calcium channel blockers (verapamil and diltiazem with IC50 of 10 and 48 mM, respectively), PLC inhibitor (U73122; IC50=6 microM) and nitric oxide (NO) donor, SNAP (IC50=1.6 microM)). The data suggest that synergistic effects of platelet agonists are receptor-mediated and occur through multiple signalling pathways including the activation PLC/Ca2+ signalling cascades.
Blotting, Western ; Calcium Channel Blockers/pharmacology ; Calcium Signaling* ; Drug Synergism ; Enzyme Activation ; Enzyme Inhibitors/pharmacology ; Epinephrine/pharmacology ; G-Protein, Inhibitory Gi/metabolism* ; GTP-Binding Proteins/metabolism* ; Human ; Phospholipase C/metabolism* ; Phospholipase C/antagonists & inhibitors ; Platelet Aggregation/physiology ; Platelet Aggregation/drug effects* ; Serotonin/pharmacology ; Signal Transduction

Transglutaminase 2 (TG2), a cross-linking enzyme, is involved in drug resistance and in the constitutive activation of nuclear factor kappa B (NF-kappaB). We investigated the association of non-small cell lung cancer (NSCLC) treatment efficacy with TG2 and NF-kappaB expression in 120 patients: 102 with adenocarcinoma and 18 with other histologic types. All patients underwent surgery; 88 received adjuvant chemotherapy, with 28 receiving platinum-based doublet chemotherapy as first-line treatment and 29 receiving epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy. Patients' TG2 and NF-kappaB expression values were calculated semiquantitatively. The median TG2 value was 50 (range, 0-300) and the median NF-kappaB value was 20 (range, 0-240). Disease-free survival did not differ between the low- and high-TG2 groups. Among patients who received palliative platinum-based doublet chemotherapy, progression free survival (PFS) was longer in the low-TG2 group than in the high-TG2 group (11.0 vs. 7.0 months; P=0.330). Among those who received EGFR-TKI therapy, PFS was also longer in the low-TG2 group than in the high-TG 2 group (11.0 vs. 2.0 months; P=0.013). Similarly, in EGFR wild-type patients treated with EGFR-TKI, PFS was longer in patients with low TG2 expression (9.0 vs. 2.0 months; P=0.013). TG2 expression levels can predict PFS in patients with NSCLC treated with EGFR-TKI.
Adenocarcinoma/*drug therapy/mortality/surgery ; Adult ; Aged ; Aged, 80 and over ; Antineoplastic Agents/therapeutic use ; Carcinoma, Non-Small-Cell Lung/*drug therapy/mortality/surgery ; Disease-Free Survival ; Female ; GTP-Binding Proteins/*biosynthesis ; Humans ; Lung Neoplasms/*drug therapy/mortality/surgery ; Male ; Middle Aged ; NF-kappa B/biosynthesis ; Protein Kinase Inhibitors/therapeutic use ; Receptor, Epidermal Growth Factor/*antagonists & inhibitors/genetics ; Transglutaminases/*biosynthesis ; Treatment Outcome