Anti-inflammatory effects of N-cyclooctyl-5-methylthiazol-2-amine hydrobromide on lipopolysaccharide-induced inflammatory response through attenuation of NLRP3 activation in microglial cells

Microglial activation is closely associated with neuroinflammatory pathologies. The nucleotide-binding and oligomerization domain-like receptor containing a pyrin domain 3 (NLRP3) inflammasomes are highly organized intracellular sensors of neuronal alarm signaling. NLRP3 inflammasomes activate nuclear factor kappa-B (NF-κB) and reactive oxygen species (ROS), which induce inflammatory responses. Moreover, NLRP3 dysfunction is a common feature of chronic inflammatory diseases. The present study investigated the effect of a novel thiazol derivative, N-cyclooctyl-5-methylthiazol-2-amine hydrobromide ({"type":"entrez-protein","attrs":{"text":"KHG26700","term_id":"728847257"}}KHG26700), on inflammatory responses in lipopolysaccharide (LPS)-treated BV-2 microglial cells. {"type":"entrez-protein","attrs":{"text":"KHG26700","term_id":"728847257"}}KHG26700 significantly attenuated the expression of several pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin-1β, and interleukin-6, in these cells, as well as the LPS-induced increases in NLRP3, NF-κB, and phospho-IkBα levels. {"type":"entrez-protein","attrs":{"text":"KHG26700","term_id":"728847257"}}KHG26700 also suppressed the LPS-induced increases in protein levels of autophagy protein 5 (ATG5), microtubule-associated protein 1 light chain 3 (LC3), and beclin-1, as well as downregulating the LPS-enhanced levels of ROS, lipid peroxidation, and nitric oxide. These results suggest that the anti-inflammatory effects of {"type":"entrez-protein","attrs":{"text":"KHG26700","term_id":"728847257"}}KHG26700 may be due, at least in part, to the regulation of the NLRP3-mediated signaling pathway during microglial activation.     

NIMA-related kinase 7 amplifies NLRP3 inflammasome pro-inflammatory signaling in microglia/macrophages and mice models of spinal cord injury

BackgroundNIMA-related kinase-7 (NEK7) is a serine/threonine kinase that drives cell-cycle dynamics by modulating mitotic spindle formation and cytokinesis. It is also a crucial modulator of the pro-inflammatory effects of NOD-like receptor 3 (NLRP3) inflammasome. However, the role of NEK7 in microglia/macrophages post-spinal cord injury (SCI) is not well defined.MethodsIn this study, we performed both in vivo and in vitro experiments. Using an in vivo mouse SCI model, NEK7 siRNAs were administered intraspinally. For in vitro analysis, BV-2 microglia cells with NEK7-siRNA were stimulated with 1 μg/ml lipopolysaccharide (LPS) and 2 mM Adenosine triphosphate (ATP).ResultsHere, we found that the mRNA and protein levels of NEK7 and NLRP3 inflammasomes were upregulated in spinal cord tissues of injured mice and BV-2 microglia cells exposed to Lipopolysaccharide (LPS) and Adenosine triphosphate (ATP). Further experiments established that NEK7 and NLRP3 interacted in BV-2 microglia cells, an effect that was eliminated following NEK7 ablation. Moreover, NEK7 ablation suppressed the activation of NLRP3 inflammasomes. Although NEK7 inhibition did not significantly improve motor function post-SCI in mice, it was found to attenuate local inflammatory response and inhibit the activation of NLRP3 inflammasome in microglia/macrophages of the injured spinal cord.ConclusionNEK7 amplifies NLRP3 inflammasome pro-inflammatory signaling in BV-2 microglia cells and mice models of SCI. Therefore, agents targeting the NEK7/NLRP3 signaling offers great promise in the treatment of inflammatory response post-SCI.     

NLRP3 inflammasome inhibition attenuates silica-induced epithelial to mesenchymal transition (EMT) in human bronchial epithelial cells

Silicosis is an incurable and progressive lung disease characterized by chronic inflammation and fibroblasts accumulation. Studies have indicated a vital role for epithelial-mesenchymal transition (EMT) in fibroblasts accumulation. NLRP3 inflammasome is a critical mediator of inflammation in response to a wide range of stimuli (including silica particles), and plays an important role in many respiratory diseases. However, whether NLRP3 inflammasome regulates silica-induced EMT remains unknown. Our results showed that silica induced EMT in human bronchial epithelial cells (16HBE cells) in a dose- and time-dependent manner. Meanwhile, silica persistently activated NLRP3 inflammasome as indicated by continuously elevated extracellular levels of interleukin-1β (IL-1β) and IL-18. NLRP3 inflammasome inhibition by short hairpin RNA (shRNA)-mediated knockdown of NLRP3, selective inhibitor MCC950, and caspase-1 inhibitor Z-YVAD-FMK attenuated silica-induced EMT. Western blot analysis indicated that TAK1-MAPK-Snail/NF-κB pathway involved NLRP3 inflammasome-mediated EMT. Moreover, pirfenidone, a commercially and clinically available drug approved for treating idiopathic pulmonary fibrosis (IPF), effectively suppressed silica-induced EMT of 16HBE cells in line with NLRP3 inflammasome inhibition. Collectively, our results indicate that NLRP3 inflammasome is a promising target for blocking or retarding EMT-mediated fibrosis in pulmonary silicosis. On basis of this mechanism, pirfenidone might be a potential drug for the treatment of silicosis.     

Vascular endothelial cells senescence is associated with NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation via reactive oxygen species (ROS)/thioredoxin-interacting protein (TXNIP) pathway

Endothelial dysfunction caused by endothelial cells senescence and chronic inflammation is tightly linked to the development of cardiovascular diseases. NLRP3 (NOD-like receptor family pyrin domain-containing3) inflammasome plays a central role in inflammatory response that is associated with diverse inflammatory diseases. This study explores the effects and possible mechanisms of NLRP3 inflammasome in endothelial cells senescence. Results show an increment of pro-inflammatory cytokine interleukin (IL) −1β secretion and caspase-1 activation during the senescence of endothelial cells induced by bleomycin. Moreover, secreted IL-1β promoted endothelial cells senescence through up-regulation of p53/p21 protein expression. NLRP3 inflammasome was found to mediate IL-1β secretion through the production of ROS (reactive oxygen species) during the senescence of endothelial cells. Furthermore, the association of TXNIP (thioredoxin-interacting protein) with NLRP3 induced by ROS promoted NLRP3 inflammasome activation in senescent endothelial cells. In addition, the expressions of NLRP3 inflammasome related genes, ASC (apoptosis associated speck-like protein containing a CARD), TXNIP, cleaved caspase-1 and IL-1β, were also increased in vitro and in vivo studies. These findings indicate that endothelial senescence could be mediated through ROS and NLRP3 inflammasome signaling pathways, suggesting a potential target for the prevention of endothelial senescence-related cardiovascular diseases.     

2-[3-(2-Thioxopyrrolidin-3-ylidene)methyl]-tryptophan,a Novel Yellow Pigment in Salted Radish Roots

The structure of the yellow pigment found in salted radish roots was studied. It was found that 1-(2-thioxopyrrolidin-3-yl)-1,2,3,4-tetrahydro-β-carboline-3- carboxylic acid (TPCC) was unstable under neutral pH, and was easily converted into the yellow pigment. The yellow pigment was isolated and identified as 2-[3-(2-thioxopyrrolidin-3-ylidene)methyl]-tryptophan (TPMT) by IR, MS, ¹H-, and ¹³C-NMR spectroscopy. In addition, we proved that this compound was the main yellow pigment in salted radish roots. This compound induced no mutagenicity in Salmonella typhimurium TA98 and TA100, either with or without prior activation.     

Design and synthesis of novel 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-thiadiazol- 2-yl)urea derivatives with potent anti-CML activity throughout PI3K/AKT signaling pathway

In this investigation, a series of 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4- thiadiazol-2-yl)urea receptor tyrosine kinase inhibitors were synthesized by a simple and efficient structure-based design. Structure-activity relationship (SAR) analysis of these compounds based on cellular assays led to the discovery of a number of compounds that showed potent activity against human chronic myeloid leukemia (CML) cell line K562, but very weak or no cellular toxicity through monitoring the growth kinetics of K562 cell during a period of 72 h using the real-time live-cell imaging. Among these compounds, 1-(5-((6-((3-morpholinopropyl) amino)pyrimidin-4-yl)thio)-1,3,4-thiadiazol-2-yl)-3-(4-(trifluoromethyl)phenyl)urea (7) exhibited the least cellular toxicity and better biological activity in cellular assays (K562, IC₅₀: 0.038 μM). Compound 7 also displayed very good induced-apoptosis effect for human CML cell line K562 and exerted its effect via a significantly reduced protein phosphorylation of PI3K/Akt signal pathway by Human phospho-kinase array analysis. In vitro results indicate that 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4- thiadiazol-2-yl)urea derivatives are lead molecules for further development as treatment of chronic myeloid leukemia and cancer.     

Insulinomimetic Zn complex (Zn(opt)2) enhances insulin signaling pathway in 3T3-L1 adipocytes

Zinc (Zn) is an essential trace element with multiple regulatory functions, involving insulin synthesis, secretion, signaling and glucose transport. Since 2000, we have proposed that Zn complexes with different coordination environments exhibit high insulinomimetic and antidiabetic activities in type 2 diabetic animals. However, the molecular mechanism for the activities is still unsolved. The purpose of this study was to reveal the molecular mechanism of several types of Zn complexes in 3T3-L1 adipocytes, with respect to insulin signaling pathway. Obtained results shows that bis(1-oxy-2-pyridine-thiolato)Zn(II), Zn(opt)2, with S(2)O(2) coordination environment induced most strongly Akt/protein kinase B (Akt/PKB) phosphorylation, in which the optimal phosphorylation was achieved at a concentration of 25 microM, and this Zn(opt)2-induced Akt/PKB phosphorylation was inhibited by wortmannin at 100 nM. Further, the phosphorylation was maximal at 5-10 min stimulation, in agreement with the Zn uptake which was also maximal at 5-10 min stimulation. The Akt/PKB phosphorylation was in concentration- and time-dependent manners. Zn(opt)2 was also capable to translocate GLUT4 protein to the plasma membrane. We conclude that Zn(opt)2 was revealed to exhibit both insulinomimetic and antidiabetic activities by activating insulin signaling cascade through Akt/PKB phosphorylation, which in turn caused the GLUT4 translocation from the cytosol to the plasma membrane.     

Inhibition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) endocytosis by ouabain in human endothelial cells

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake and reduction is widely used to evaluate cell proliferation and viability. MTT is taken up by the cells through endocytosis. We find that ouabain (1-200 nM) inhibits MTT reduction in human umbilical vein endothelial cells (HUVEC) without affecting cell viability. Ouabain does not inhibit MTT reduction when cell lysates substituted for the intact cells. Disruption of caveolae by cholesterol depletion, completely prevents the effect of ouabain. Treatment of HUVEC with Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine partially abrogates the inhibitory effect of ouabain. The data suggest that ouabain interaction with caveolar Na/K-ATPase inhibits MTT endocytosis through the activation of signaling proteins such as Src kinase.     

The effect of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline on cell growth, cell cycle, induction of DNA fragmentation, and activity of caspase 3 in murine leukemia L1210 cells and fibroblast NIH-3T3 cells

Quinazolines are multitarget agents, which have broad spectrum of biological activity, and some of them are now in cancer clinical testing. 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline is a new synthetically prepared derivative, which in our previous study showed cytotoxic effects on cancer cell lines HeLa and B16. Quinazoline, at micromolar concentrations, induced morphological changes and necrosis of B16 cells, and at nanomolar concentrations it produced changes of F-actin cytoskeleton. It did not cause changes in the cell cycle, did not induce apoptotic cell death in B16 cells, did not have a mutagenic effect, and did not even behave as a typical intercalating agent. Little significant reduction of tumor volume in intramuscular transplanted B16 cells was observed. The aim of the present study was to examine the cytotoxic effect of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline on murine leukemia L1210 cells and fibroblast NIH-3T3 cells. Induction of cell morphology and cell cycle changes, induction of apoptosis and caspase 3 activity were studied. Quinazoline acted cytotoxically on both cell lines. The sensitivity of leukemia L1210 cells to the quinazoline was higher than that of fibroblast NIH-3T3. The IC(100) was 12 microM for L1210 cells and 24 microM for NIH-3T3 cells. No effect of quinazoline on the cell cycle profile of L1210 and NIH-3T3 was detected, however, quinazoline induced an increase of the sub-G(0) cell fraction, apoptotic DNA fragmentation, and apoptotic morphological changes at a concentration of 12 microM. This quinazoline concentration induced caspase 3 activity. Our results demonstrated that induction of apoptotic cell death via activation of caspase 3 contributed to the cytotoxic effects of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline in murine leukemia L1210 cells.     

Synthesis,Biological Evaluation and in Silico Studies of 3-Hydroxy-N-(2-(substituted phenyl)-4-oxothiazolidin-3-yl)-2-napthamide Derivatives

In the present study, a series of 3-hydroxy-N-(2-(substituted phenyl)-4-oxothiazolidin-3-yl)-2-napthamide derivatives were synthesized, characterized and evaluated for theirin vitroactivity, i. e., antimicrobial, antioxidant and anti-inflammatory. The target compounds were synthesized by condensation reaction of 3-hydroxy-2-naphthoic acid hydrazide with substituted benzaldehydes which were subjected to cyclization reaction with thioglycolic acid and ZnCl₂ to get target compounds. The synthesized 3-hydroxy-N-(2-(substituted phenyl)-4-oxothiazolidin-3-yl)-2-napthamide derivatives were examined for their antimicrobial activity and 3-hydroxy-N-(4-oxo-2-(3,4,5-trimethoxyphenyl)thiazolidin-3-yl)-2-naphthamide ( S20 ) exhibited the highest antimicrobial potential. The N′-(2,3-dichlorobenzylidene)-3-hydroxy-2-naphthohydrazide ( S5 ) displayed good antifungal potential against Rhizopus oryzae, whereas N′-(2,3-dichlorobenzylidene)-3-hydroxy-2-naphthohydrazide ( S20 ) showed the highest antioxidant potential and N-(2-(2,6-dichlorophenyl)-4-oxothiazolidin-3-yl)-3-hydroxy-2-naphthamide ( S16 ) displayed the highest anti-inflammatory activity. The results of molecular docking studies revealed that existence of hydrogen bonding and hydrophobic interactions with their respective proteins. In silico ADMET studies were carried out by Molinspiration, Pre-ADMET and OSIRIS property explorer to predict the pharmacokinetic behaviour of synthesized 3-hydroxy-N-(2-(substituted phenyl)-4-oxothiazolidin-3-yl)-2-napthamide derivatives.     

Syntheses and pharmacokinetic evaluations of four metabolites of 2-(4-(2-((1H-benzo[d]imidazol-2-yl)thio)ethyl)piperazin-1-yl)-N-(6-methyl-2,4-bis-(methylthio)pyridin-3-yl)acetamide hydrochloride [K-604], an acyl-CoA:cholesterol O-acyltransferase-1 inhibitor

We synthesized and identified four metabolites of acyl-coenzyme A:cholesterol O-acyltransferase (ACAT)-1 inhibitor, K-604 (1). Two of the metabolites M1 and M2, were prepared from 1 using a combination reagent of hydrogen peroxide and sodium tungstate with either phosphoric acid or trifluoroethanol as the solvent to control the regioselectivity. Upon exposure of 4b to tert-butyl hypochlorite at −78 °C, the monosulfoxidation afforded synthetic intermediate of M3 in excellent yield. The efficient synthesis of M4 was established. The in vitro metabolic study exhibited a high clearance value (720 μL/min/mg protein) of 1 using human liver microsomes. We orally administered a single dose of 10 mg/kg of 1 to monkeys because the in vitro metabolic patterns are quite similar. Fortunately, the drug concentration of 1 was much higher than those of M1, M2, M3 and M4.     

A Pitfall of the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) Assay due to Evaporation in wells on the Edge of a 96 well Plate

The 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) calorimetric assay is replacing the traditional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as a fast, one-step assay of cell viability. We have observed that evaporation of the outer wells of a 96 well plate increases the absorbancy by 52% compared to the inner wells. Filling the outer 2 rows of wells with media and replacement of the media prior to addition of the MTS reagent will, however, correct this inaccuracy.     

(2E)-1-(2,4,6-Trimethoxyphenyl)-3-(4-chlorophenyl)prop-2-en-1-one,a Chalcone Derivative,Promotes Apoptosis by Suppressing RAS-ERK and AKT/FOXO3a Pathways in Hepatocellular Carcinoma Cells

Background : Liver cancer is an extremely common cancer with the highest mortality rate and poor prognosis. Owing to their low systemic toxicity and few side effects, natural compounds may provide better therapeutic effects for patients. (2E)-1-(2,4,6-trimethoxyphenyl)-3-(4-chlorophenyl)prop-2-en-1-one (TMOCC), a chalcone derivative, exhibits cytotoxicity towards many tumor cells. However, the anticancer mechanism of TMOCC has not been elucidated in human hepatocellular carcinoma (HCC). Methods : Cell Counting Kit-8 and colony formation assays were used to evaluate the effects of TMOCC on viability and proliferation. Mitochondrial transmembrane potential and flow cytometry assays were used to detect apoptosis. The expression levels of proteins related to apoptosis, the RAS-ERK and AKT/FOXO3a signaling pathways were assessed using western blot. Potential targets of TMOCC were detected using molecular docking analysis. Results : TMOCC inhibited viability and proliferation, and induced the loss of mitochondrial transmembrane potential, apoptosis and DNA double-strand breaks in both HCC cells. The RAS-ERK and AKT/FOXO3a signaling pathways were suppressed by TMOCC. Finally, ERK1, PARP-1, and BAX were identified as potential targets of TMOCC. Conclusion : Taken together, our results show that TMOCC promotes apoptosis by suppressing the RAS-ERK and AKT/FOXO3a signaling pathways. TMOCC may be a potential multi-target compound that is effective against liver cancer.     

Inhibition of anti-inflammatory pathway through suppressors of cytokine signalling (Socs2/Socs3) in the initiation of hepatocellular carcinoma

Hepatocellular carcinoma (HCC), a leading cause of cancer related deaths is predominantly driven by chronic inflammatory responses. Due to asymptomatic nature and lack of early patient biopsies, precise involvement of inflammation in hepatic injury initiation remains unidentified. Aim of the study was to elucidate the regulation patterns of inflammatory signalling from initiation of hepatic injury to development of HCC. HCC mice model was established using DEN followed by repeated doses of CCl₄ and sacrificed at three different stages of disease comprising 7, 14 and 21 weeks. Serum biochemical tests, hepatic lipids quantification, histopathology and qPCR analyses were conducted to characterize the initiation and progression of liver injury and inflammatory signalling. Notably, at 7 weeks, we observed hepatocyte damage and periportal necrotic bodies coupled with induction of Socs2/Socs3 and anti-inflammatory cytokine Il-10. At 14 weeks, mice liver showed advancement of liver injury with micro-vesicular steatosis and moderate collagen deposition around portal zone. With progression of injury, the expression of Socs3 was declined with further reduction of Il-10 and Tgf-β indicating the disturbance of anti-inflammatory mechanism. In contrast, pro-inflammatory cytokines Il1-β, Il6 and Tnf-α were upregulated contributing inflammation. Subsequently, at 21 weeks severe liver damage was estimated as characterized by macro-vesicular steatosis, perisinusoidal collagen bridging, immune cell recruitment and significant upregulation of Col-1α and α-Sma. In parallel, there was significant upregulation of pro/anti-inflammatory cytokines highlighting the commencement of chronic inflammation.Findings of the study suggest that differential regulation of cytokine suppressors and inflammatory cytokines might play role in the initiation and progression of hepatic injury leading towards HCC.     

Membrane receptor-initiated signaling in 1,25(OH)2D3-stimulated calcium uptake in intestinal epithelial cells

Demonstrating 1,25(OH)2D3-stimulated calcium uptake in isolated chick intestinal epithelial cells has been complicated by simultaneous enhancement of both uptake and efflux. We now report that in intestinal cells of adult birds, or those of young birds cultured for 72 h, 1,25(OH)2D3-stimulates 45Ca uptake to greater than 140% of corresponding controls within 3 min of addition. Such cells have lost hormone-stimulated protein kinase C (PKC) activity, believed to mediate calcium efflux. To further test this hypothesis, freshly isolated cells were preincubated with calphostin C, and calcium uptake monitored in the presence or absence of steroid. Only cells treated with the PKC inhibitor demonstrated a significant increase in 45Ca uptake in response to 1,25(OH)2D3, relative to corresponding controls. In addition, phorbol ester was shown to stimulate efflux, while forskolin stimulated uptake. To further investigate the mechanisms involved in calcium uptake, we assessed the role of TRPV6 and its activation by beta-glucuronidase. beta-Glucuronidase secretion from isolated intestinal epithelial cells was significantly increased by treatment with 1,25(OH)2D3, PTH, or forskolin, but not by phorbol ester. Treatment of cells with beta-glucuronidase, in turn, stimulated 45Ca uptake. Finally, transfection of cells with siRNA to either beta-glucuronidase or TRPV6 abolished 1,25(OH)2D3-enhanced calcium uptake relative to controls transfected with scrambled siRNA. Confocal microscopy further indicated rapid redistribution of enzyme and calcium channel after steroid. 1,25(OH)2D3 and PTH increase calcium uptake by stimulating the PKA pathway to release beta-glucuronidase, which in turn activates TRPV6. 1,25(OH)2D3-enhanced calcium efflux is mediated by the PKC pathway.     

3-[2-Amino-2-imidazolin-4(5)-yl]alanine (enduracididine) and 2-[2-amino-2-imidazolin-4(5)-yl] acetic acid in seeds of Lonchocarpus sericeus

3-[2-Amino-2-imidazolin-4(5)-yl]alanine (enduracididine) and 2-[2-amino-2-imidazolin-4(5)-yl] acetic acid have been isolated from seeds of Lonchocarpus sericeus. The concentration of each compound was ca 0.5 % of the fresh seed weight.     

Biosynthesis and biological activity of β-(5-methylisoxazolin-3-on-2-yl)alanine in higher plants

Enzyme preparations from Leucaena seedlings catalysed the formation of β-(5-methylisoxazolin-3-on-2-yl)alanine (MIA) by using 3-hydroxy-5-methylisoxazole (HMI) and O-acetyl-L-serine. Some properties of this enzyme are described. The β-substituted alanine synthases from Pisum and Citrullus seedlings could not catalyse the synthesis of MIA. The phytotoxic effect of HMI on rice seedlings is reduced by alanylation.     

Src homology domain 2-containing protein-tyrosine phosphatase-1 (SHP-1) binds and dephosphorylates G(alpha)-interacting, vesicle-associated protein (GIV)/Girdin and attenuates the GIV-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway

GIV (Gα-interacting vesicle-associated protein, also known as Girdin) is a bona fide enhancer of PI3K-Akt signals during a diverse set of biological processes, e.g. wound healing, macrophage chemotaxis, tumor angiogenesis, and cancer invasion/metastasis. We recently demonstrated that tyrosine phosphorylation of GIV by receptor and non-receptor-tyrosine kinases is a key step that is required for GIV to directly bind and enhance PI3K activity. Here we report the discovery that Src homology 2-containing phosphatase-1 (SHP-1) is the major protein-tyrosine phosphatase that targets two critical phosphotyrosines within GIV and antagonizes phospho-GIV-dependent PI3K enhancement in mammalian cells. Using phosphorylation-dephosphorylation assays, we demonstrate that SHP-1 is the major and specific protein-tyrosine phosphatase that catalyzes the dephosphorylation of tyrosine-phosphorylated GIV in vitro and inhibits ligand-dependent tyrosine phosphorylation of GIV downstream of both growth factor receptors and GPCRs in cells. In vitro binding and co-immunoprecipitation assays demonstrate that SHP-1 and GIV interact directly and constitutively and that this interaction occurs between the SH2 domain of SHP-1 and the C terminus of GIV. Overexpression of SHP-1 inhibits tyrosine phosphorylation of GIV and formation of phospho-GIV-PI3K complexes, and specifically suppresses GIV-dependent activation of Akt. Consistently, depletion of SHP-1 enhances peak tyrosine phosphorylation of GIV, which coincides with an increase in peak Akt activity. We conclude that SHP-1 antagonizes the action of receptor and non-receptor-tyrosine kinases on GIV and down-regulates the phospho-GIV-PI3K-Akt axis of signaling.     

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) causes Akt phosphorylation and morphological changes in intracellular organellae in cultured rat astrocytes

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is widely used for cell viability and cytotoxicity assays, but cell biological effects of MTT itself have not been investigated. In this paper we show that MTT induces a morphological change in an intracellular membranous compartment labeled with anti-Rab5 antibody, dissociation of early endosomal auto-antigen (EEA1) from the membrane fraction, and phosphorylation of Akt probably through a phosphatidylinositol-3-OH kinase [PI(3)K] pathway in cultured rat astrocytes. These findings suggest that MTT affects cellular functions and conditions to some extent, and such effects of MTT may cause some discrepancies of measurement of cell viability using MTT assay and other assays. That is, the effects of MTT on cells could influence the results of cell viability assay. Moreover, MTT or other tetrazolium salts could be used as interesting activators of Akt to investigate the mechanism by which Akt or PI(3)K is activated.