Inhibitor GF109203X Research Articles

Alteration of placental type 2 11β-hydroxysteroid dehydrogenase expression and activity by lead

Alteration of placental type 2 11β-hydroxysteroid dehydrogenase expression and activity by lead

PKCδ is required for porcine reproductive and respiratory syndrome virus replication

Protein kinase C (PKC) that transduces signals to modulate a wide range of cellular functions has been shown to regulate a number of viral infections. Herein, we showed that inhibition of PKC with the PKC inhibitor GF109203X significantly impaired porcine reproductive and respiratory syndrome virus (PRRSV) replication. Inhibition of PKC led to virus yield reduction, which was associated with decreased viral RNA synthesis and lowered virus protein expression. And this inhibitory effect by PKC inhibitor was shown to occur at the early stage of PRRSV infection. Subsequently, we found that PRRSV infection activated PKCδ in PAMs and knockdown of PKCδ by small interfering RNA (siRNA) suppressed PRRSV replication, suggesting that novel PKCδ may play an important factor in PRRSV replication. Taken together, these data imply that PKC is involved in PRRSV infection and beneficial to PRRSV replication, extending our understanding of PRRSV replication.

The mechanism of honokiol-induced intracellular Ca2+ rises and apoptosis in human glioblastoma cells

Honokiol, an active constituent of oriental medicinal herb Magnolia officinalis, caused Ca2+ mobilization and apoptosis in different cancer cells. In vivo, honokiol crossed the blood–brain or –cerebrospinal fluid barrier, suggesting that it may be an effective drug for the treatment of brain tumors, including glioblastoma. This study examined the effect of honokiol on intracellular Ca2+ concentration ([Ca2+]i) and apoptosis in DBTRG-05MG human glioblastoma cells. Honokiol concentration-dependently induced a [Ca2+]i rise. The signal was decreased partially by removal of extracellular Ca2+. Honokiol-triggered [Ca2+]i rise was not suppressed by store-operated Ca2+ channel blockers (nifedipine, econazole, SK&F96365) and the protein kinase C (PKC) activator phorbol 12-myristate 13 acetate (PMA), but was inhibited by the PKC inhibitor GF109203X. GF109203X-induced inhibition was not altered by removal of extracellular Ca2+. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished honokiol-induced [Ca2+]i rise. Conversely, incubation with honokiol abolished TG or BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C (PLC) with U73122 abolished honokiol-induced [Ca2+]i rise. Honokiol (20–80μM) reduced the cell viability, which was not reversed by prechelating cytosolic Ca2+ with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester). Honokiol (20–60μM) enhanced reactive oxygen species (ROS) production, decreased mitochondrial membrane potential, released cytochrome c, and activated caspase-9/caspase-3. Together, honokiol induced a [Ca2+]i rise by inducing PLC-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via PKC-dependent, non store-operated Ca2+ channels. Moreover, honokiol activated the mitochondrial pathway of apoptosis in DBTRG-05MG human glioblastoma cells.

Dying tumor cells stimulate proliferation of living tumor cells via caspase-dependent protein kinase Cδ activation in pancreatic ductal adenocarcinoma

Pancreatic cancer is one of the most lethal human cancers, and radiotherapy is often implemented for locally advanced pancreatic ductal adenocarcinoma. Tumor cell repopulation is a major challenge in treating cancers after radiotherapy. In order to address the problem of tumor repopulation, our previous studies have demonstrated that dying cells stimulate the proliferation of living tumor cells after radiotherapy. In particular, dying cells undergoing apoptosis also activate survival or proliferation signals and release growth factors to surrounding living cells. In the present study, we used an in vitro model to examine the possible mechanisms for dying cell stimulated tumor repopulation in pancreatic cancer. In this model, a small number of living, luciferase-labeled pancreatic cancer cells (reporter) were seeded onto a layer of a much larger number of irradiated, unlabeled pancreatic cancer cells and the growth of the living cells was measured over time as a gage of tumor repopulation. Our results indicate that irradiated, dying Panc1 feeder cells significantly stimulated the proliferation of living Panc1 reporter cells. Importantly, we identified that the percentage of apoptotic cells and the cleavage of caspases 3 and 7 and protein kinase Cδ (PKCδ) were increased in irradiated Panc1 cells. We presumed that caspases 3 and 7 and PKCδ as integral mediators in the process of dying pancreatic cancer cell stimulation of living tumor cell growth. In order to demonstrate the importance of caspases 3, 7 and PKCδ, we introduced dominant-negative mutants of caspase 3 (DN_C3), caspase 7 (DN_C7), or PKCδ (DN_PKCδ) into Panc1 cells using lentiviral vectors. The stably transduced Panc1 cells were irradiated and used as feeders and we found a significant decrease in the growth of living Panc1 reporter cells when compared with irradiated wild-type Panc1 cells as feeders. Moreover, the role of PKCδ in the growth stimulation of living tumor cells was further confirmed using a pan PKC inhibitor GF109203x and a specific PKCδ inhibitor, rottlerin. Additionally, we found significantly increased phosphorylation of Akt, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK1/2) in the irradiated Panc1 cells. Mechanistically, PKCδ cleavage was attenuated in both DN_C3 and DN_C7 transduced Panc1 cells, and both Akt and p38 MAPK phosphorylation were attenuated in DN_PKCδ transduced Panc1 cells following radiation. Thus, this report suggests a novel finding that cellular signaling caspase 3/7-PKCδ-Akt/p38 MAPK is crucial to the repopulation in Panc1 cells after radiotherapy.

MG132, a proteasome inhibitor, enhances LDL uptake in HepG2 cells in vitro by regulating LDLR and PCSK9 expression.

Expression of liver low-density lipoprotein receptor (LDLR), a determinant regulator in cholesterol homeostasis, is tightly controlled at multiple levels. The aim of this study was to examine whether proteasome inhibition could affect LDLR expression and LDL uptake in liver cells in vitro. HepG2 cells were examined. Real-time PCR and Western blot analysis were used to determine the mRNA and protein levels, respectively. DiI-LDL uptake assay was used to quantify the LDLR function. Luciferase assay system was used to detect the activity of proprotein convertase subtilisin/kexin type 9 (PCSK9, a major protein mediating LDLR degradation) promoter. Specific siRNAs were used to verify the involvement of PCSK9. Treatment of HepG2 cells with the specific proteasome inhibitor MG132 (0.03-3 μmol/L) dose-dependently increased LDLR mRNA and protein levels, as well as LDL uptake. Short-term treatment with MG132 (0.3 μmol/L, up to 8 h) significantly increased both LDLR mRNA and protein levels in HepG2 cells, which was blocked by the specific PKC inhibitors GF 109203X, Gö 6983 or staurosporine. In contrast, a longer treatment with MG132 (0.3 μmol/L, 24 h) did not change LDLR mRNA, but markedly increased LDLR protein by reducing PCSK9-mediated lysosome LDLR degradation. Furthermore, MG132 time-dependently suppressed PCSK9 expression in the HepG2 cells through a SREBP-1c related pathway. Combined treatment with MG132 (0.3 μmol/L) and pravastatin (5 μmol/L) strongly promoted LDLR expression and LDL uptake in HepG2 cells, and blocked the upregulation of PCSK9 caused by pravastatin alone. Inhibition of proteasome by MG132 in HepG2 cells plays dual roles in LDLR and PCSK9 expression, and exerts a beneficial effect on cholesterol homeostasis.

Mechanisms of resveratrol-induced changes in cytosolic free calcium ion concentrations and cell viability in OC2 human oral cancer cells.

Resveratrol is a natural compound that affects cellular calcium (Ca(2+)) homeostasis and viability in different cells. This study examined the effect of resveratrol on cytosolic free Ca(2+) concentrations ([Ca(2+)]i) and viability in OC2 human oral cancer cells. The Ca(2+)-sensitive fluorescent dye fura-2 was used to measure [Ca(2+)]i, and water-soluble tetrazolium-1 was used to measure viability. Resveratrol evoked concentration-dependent increase in [Ca(2+)]i. The response was reduced by removing extracellular Ca(2+). Resveratrol also caused manganese-induced fura-2 fluorescence quench. Resveratrol-evoked Ca(2+) entry was inhibited by nifedipine and the protein kinase C (PKC) inhibitor GF109203X but was not altered by econazole, SKF96365, and the PKC activator phorbol 12-myristate 13 acetate. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished resveratrol-evoked [Ca(2+)]i rise. Conversely, treatment with resveratrol inhibited BHQ-evoked [Ca(2+)]i rise. Inhibition of phospholipase C (PLC) with U73122 abolished resveratrol-evoked [Ca(2+)]i rise. At 20-100 μM, resveratrol decreased cell viability, which was not affected by chelating cytosolic Ca(2+)with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. Annexin V-fluorescein isothiocyanate staining data suggest that resveratrol at 20-40 μM induced apoptosis in a concentration-dependent manner. Collectively, in OC2 cells, resveratrol induced [Ca(2+)]i rise by evoking PLC-dependent Ca(2+) release from the endoplasmic reticulum and by causing Ca(2+) entry via nifedipine-sensitive, PKC-regulated mechanisms. Resveratrol also caused Ca(2+)-independent apoptosis.

High-density lipoprotein induces cyclooxygenase-2 expression and prostaglandin I-2 release in endothelial cells through sphingosine kinase-2

High-density lipoprotein (HDL) has a significant cardioprotective effects. HDL induces cyclooxygenase-2 (COX-2) expression and prostacyclin I-2 (PGI-2) release in vascular endothelial cells, which contributes to its anti-atherogenic effects. However, the underlying mechanisms are not fully understood. In the present study, we observed that HDL-stimulated COX-2 expression and PGI-2 production in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent manner. These effects triggered by HDL were inhibited by pertussis toxin (PTX), protein kinase C (PKC) inhibitor GF109203X, and ERK inhibitor PD98059, suggesting that Gαi/Gαo-coupled GPCR, PKC, and ERK pathways are involved in HDL-induced COX-2/PGI-2 activation. More importantly, we found that silencing of sphingosine kinase 2 (SphK-2) also blocked HDL-induced COX-2/PGI-2 activation. In addition, HDL-activated SphK-2 phosphorylation accompanied by increased S1P level in the nucleus. Our ChIP data demonstrated that SphK-2 is associated with CREB at the COX-2 promoter region. Collectively, these results indicate that HDL induces COX-2 expression and PGI-2 release in endothelial cells through activation of PKC, ERK1/2, and SphK-2 pathways. These findings implicate a novel mechanism underlying anti-atherothrombotic effects of HDL.

Aortic Superoxide Production at the Early Hyperglycemic Stage in a Rat Type 2 Diabetes Model and the Effects of Pravastatin

Endothelium-derived superoxide induces vascular dysfunctions. The aim of this study was to examine the activity of protein kinase C (PKC) isoforms and endothelial nitric oxide synthase (eNOS), which leads to vascular superoxide production in type 2 diabetes, in addition to the effects of pravastatin. We studied these mechanisms in Otsuka Long-Evans Tokushima Fatty (OLETF) rats (type 2 diabetes model) at the early hyperglycemic stage (vs. non-diabetic Long-Evans Tokushima Otsuka [LETO] rats). Superoxide production and catalase activity were measured in aortas, as were the protein expressions of PKCδ and phospho-Ser(1177) eNOS. Superoxide production was increased in OLETF rats, and this increase was inhibited by the selective conventional PKC (cPKC) inhibitor Gö6976 and by the non-selective cPKC and novel PKC inhibitor GF109203X. Phospho-Ser(1177) eNOS was significantly increased in OLETF rats, whereas the protein expressions of PKCδ and phosopho-Thr(505) PKCδ and catalase activity were all greatly reduced. Pravastatin administration to OLETF rats in vivo had normalizing effects on all of these variables. The increment in superoxide production seen in OLETF rats (but not the production in pravastatin-treated OLETF rats) was abolished by high concentration of N(ω)-nitro-L-arginine methyl ester (electron transport inhibitor of eNOS), by sepiapterin (precursor of tetrahydrobiopterin), and by LY294002 (phosphatidylinositol 3-kinase [PI3-kinase] inhibitor). In OLETF rats at the early hyperglycemic stage, aortic superoxide production is increased owing to activation of uncoupled eNOS through phosphorylation by PI3-kinase/Akt. This may be related to the observed reduction in PKCδ/catalase activities. Pravastatin inhibited endothelial superoxide production via normalization of PKCδ/catalase activities.

Mepivacaine-induced contraction involves increased calcium sensitization mediated via Rho kinase and protein kinase C in endothelium-denuded rat aorta

Mepivacaine is an aminoamide local anesthetic that produces vasoconstriction in vivo and in vitro. The goals of this in vitro study were to determine whether mepivacaine-induced contraction involves calcium sensitization in isolated endothelium-denuded aortas, and to investigate the specific protein kinases involved. The effects of mepivacaine and potassium chloride on intracellular calcium concentrations ([Ca2+]i) and tension in the presence or absence of Y-27632 or GF 109203X were measured simultaneously using the acetoxymethyl ester of fura-2-loaded aortic strips. Cumulative mepivacaine concentration–response curves were generated in the presence or absence of the following inhibitors: Rho kinase inhibitor Y-27632, protein kinase C (PKC) inhibitor GF 109203X, extracellular signal-regulated kinase (ERK) inhibitor PD 98059, c-Jun NH2-terminal kinase (JNK) inhibitor SP600125, and p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580. Phosphorylation of PKC and MAPK, and membrane translocation of Rho kinase were detected in vascular smooth muscle cells by Western blotting. The slope of the mepivacaine-induced [Ca2+]i-tension curve was higher than that of the KCl-induced [Ca2+]i-tension curve. Pretreatment with Y-27632 or GF 109203X shifted the mepivacaine-induced [Ca2+]i-tension curve to the lower right. Pretreatment with Y-27632, GF 109203X, PD 98059, or SP600125 attenuated mepivacaine-induced contraction in a concentration-dependent manner. Y-27632 and GF 109203X attenuated mepivacaine-induced Rho kinase membrane translocation and PKC phosphorylation, respectively. PD 98059 and SP600125 attenuated mepivacaine-induced ERK and JNK phosphorylation, respectively. Taken together, these results indicate that mepivacaine-induced contraction involves increased calcium sensitization mediated by Rho kinase and PKC. Such contraction mainly involves activation of ERK- and JNK-mediated pathways.

Transient activation of protein kinase C contributes to fluoride-induced apoptosis of rat erythrocytes

Role of PKC in fluoride-induced apoptosis of rat erythrocytes was studied in vitro and in vivo. Treatment of erythrocytes with 5mM NaF for 1–24h caused progressive accumulation of cytosolic Ca2+ and PS exposure at outer membrane surface. After 1h, these processes were suppressed by PKC inhibitors staurosporine, GF 109203X and chelerythrine, but increased by PKC activator PMA. Following 24h, NaF-induced Ca2+ uptake and PS externalization were partly prevented by PMA or staurosporine, but not by GF 109293X and chelerythrine. Application of PP inhibitor OA augmented NaF-induced cell responses within 1h, but not after 24h. Incubation of erythrocytes with 0.1–10mM NaF for 1h produced a dose-dependent PKCα translocation from cytosol to membranes with appearance of active PKM fragment. 24h NaF exposure led to complete loss of cytosolic PKCα and proteolysis of membrane PKCα. Besides, NaF weakly stimulated membrane PKCζ, although its subcellular distribution was not altered. Thus, transient PKCα activation/translocation positively contributes to NaF-induced apoptosis in vitro. Consumption of 2–20ppm fluoride by the rats for 12months also induced dose-dependent PKCα translocation to membranes and activation of membrane PKCζ, what indicates that PKC stimulation is an important physiological mechanism of fluoride toxicity.

Role of PKC-β in chemical allergen-induced CD86 expression and IL-8 release in THP-1 cells

We previously demonstrated an age-related decrease in receptor for activated C-kinase (RACK-1) expression and functional deficit in Langerhans cells' responsiveness. This defect specifically involves the translocation of protein kinase C (PKC)-β. The purpose of this study was to investigate the role of RACK-1 and PKC-β in chemical allergen-induced CD86 expression and IL-8 release in the human promyelocytic cell line THP-1 and primary human dendritic cells (DC). Dinitrochlorobenzene, p-phenylenediamine and diethyl maleate were used as contact allergens. The selective cell-permeable inhibitor of PKC-β and the broad PKC inhibitor GF109203X completely prevented chemical allergen- or lipopolysaccharide (LPS)-induced CD86 expression and significantly modulated IL-8 release (50 % reduction). The selective cell-permeable inhibitor of PKC-ε (also known to bind to RACK-1) failed to modulate allergen- or LPS-induced CD86 expression or allergen-induced IL-8 release, while modulating LPS-induced IL-8 release. The use of a RACK-1 pseudosubstrate, which directly activates PKC-β, resulted in dose-related increase in CD86 expression and IL-8 release. Similar results were obtained with human DC, confirming the relevance of results obtained in THP-1 cells. Overall, our findings demonstrate the role of PKC-β and RACK-1 in allergen-induced CD86 expression and IL-8 production, supporting a central role of PKC-β in the initiation of chemical allergen-induced DC activation.

Tyrosine Phosphorylation on Spleen Tyrosine Kinase (Syk) Is Differentially Regulated in Human and Murine Platelets by Protein Kinase C Isoforms

Protein kinase C (PKC) isoforms differentially regulate platelet functional responses downstream of glycoprotein VI (GPVI) signaling, but the role of PKCs regulating upstream effectors such as Syk is not known. We investigated the role of PKC on Syk tyrosine phosphorylation using the pan-PKC inhibitor GF109203X (GFX). GPVI-mediated phosphorylation on Syk Tyr-323, Tyr-352, and Tyr-525/526 was rapidly dephosphorylated, but GFX treatment inhibited this dephosphorylation on Tyr-525/526 in human platelets but not in wild type murine platelets. GFX treatment did not affect tyrosine phosphorylation on FcRγ chain or Src family kinases. Phosphorylation of Lat Tyr-191 and PLCγ2 Tyr-759 was also increased upon treatment with GFX. We evaluated whether secreted ADP is required for such dephosphorylation. Exogenous addition of ADP to GFX-treated platelets did not affect tyrosine phosphorylation on Syk. FcγRIIA- or CLEC-2-mediated Syk tyrosine phosphorylation was also potentiated with GFX in human platelets. Because potentiation of Syk phosphorylation is not observed in murine platelets, PKC-deficient mice cannot be used to identify the PKC isoform regulating Syk phosphorylation. We therefore used selective inhibitors of PKC isoforms. Only PKCβ inhibition resulted in Syk hyperphosphorylation similar to that in platelets treated with GFX. This result indicates that PKCβ is the isoform responsible for Syk negative regulation in human platelets. In conclusion, we have elucidated a novel pathway of Syk regulation by PKCβ in human platelets.

Phosphorylation of Noxo1 at threonine 341 regulates its interaction with Noxa1 and the superoxide‐producing activity of Nox1

Superoxide production by Nox1, a member of the Nox family NAPDH oxidases, requires expression of its regulatory soluble proteins Noxo1 (Nox organizer 1) and Noxa1 (Nox activator 1) and is markedly enhanced upon cell stimulation with phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC). The mechanism underlying PMA-induced enhancement of Nox1 activity, however, remains to be elucidated. Here we show that, in response to PMA, Noxo1 undergoes phosphorylation at multiple sites, which is inhibited by the PKC inhibitor GF109203X. Among them, Thr341 in Noxo1 is directly phosphorylated by PKC invitro, and alanine substitution for this residue reduces not only PMA-induced Noxo1 phosphorylation but also PMA-dependent enhancement of Nox1-catalyzed superoxide production. Phosphorylation of Thr341 allows Noxo1 to sufficiently interact with Noxa1, an interaction that participates in Nox1 activation. Thus phosphorylation of Noxo1 at Thr341 appears to play a crucial role in PMA-elicited activation of Nox1, providing a molecular link between PKC-mediated signal transduction and Nox1-catalyzed superoxide production. Furthermore, Ser154 in Noxo1 is phosphorylated in both resting and PMA-stimulated cells, and the phosphorylation probably participates in a PMA-independent constitutive activity of Nox1. Ser154 may also be involved in protein kinase A (PKA) mediated regulation of Nox1; this serine is the major residue that is phosphorylated by PKA invitro. Thus phosphorylation of Noxo1 at Thr341 and at Ser154 appears to regulate Nox1 activity in different manners. Noxo1 binds to p22phox by pull down (1, 2, 3) Noxo1 binds to Noxo1 by pull down (View interaction) Noxa1 binds to Noxo1 by pull down (1, 2, 3, 4, 5).

Effects of PTHrP on expression of MMP9 and MMP13 in sika deer antler chondrocytes

Deer antlers are the only mammalian appendages to display an annual cycle of full regeneration. However, little is known about the molecular mechanisms of antler regeneration. Our previous study has demonstrated that parathyroid hormone-related peptide (PTHrP) can promote proliferation of antler chondrocytes and inhibit its differentiation, but the mechanism underlying such regulation is not fully understood. We have determined the role of PTHrP on the mRNA expression of matrix metalloproteinase-9 (MMP9) and MMP13 in the antler chondrocytes. The possible pathways that transduce PTHrP effects were examined. In situ hybridization showed that MMP9 and MMP13 were mainly localized in the dermal fibroblasts, perichondrium, and cartilage in the sika deer antler, of which MMP9 and MMP13 were highly expressed in the chondrocytes. Exogenous PTHrP could inhibit the expression of MMP9 and MMP13 in the antler chondrocytes. The inhibitory effect of PTHrP on MMP9 was abolished by JNK inhibitor, SP600125, while P38MAPK inhibitor SB203850 and PKC inhibitor GF109203X could rescue the inhibitory effect of PTHrP on MMP13. The results suggest that PTHrP can inhibit MMP9 expression by JNK signaling pathway and MMP13 expression by p38MAPK and PKC signaling pathways in the antler chondrocytes. Thus PTHrP is involved in the control of antler chondrocytes maturation and cartilage matrix degradation.

Endothelial Nitric Oxide Synthase Phosphorylation at Threonine 495 and Mitochondrial Reactive Oxygen Species Formation in Response to a High H2O2 Concentration

Background: Hydrogen peroxide (H₂O₂) is produced in vessels during ischemia/reperfusion and during inflammation, both leading to vascular dysfunction. We investigated cellular pathways involved in endothelial nitric oxide synthase (eNOS) phosphorylation at Threonine 495 (Thr⁴⁹⁵) in human umbilical vein endothelial cells (HUVECs) exposed to H₂O₂. Methods: HUVECs were exposed to 400 μ<smlcap>M</smlcap> H₂O₂ for 30 min. Phosphorylation at Thr⁴⁹⁵ was assessed by Western blotting and reactive oxygen species (ROS) monitored by flow cytometry. Protein kinase C (PKC) pathways were investigated by pretreatment with PKC-β inhibitor ruboxistaurin or pan-PKC inhibitor GF109203X. In addition, we investigated ROCK and ERK pathways by MEKK1/2 inhibitor U0126 and ROCK inhibitor Y27632. Results: H₂O₂ increased eNOS phosphorylation at Thr⁴⁹⁵ (to 176% vs. control (100%), p < 0.001) along with increased mitochondrial ROS formation (from 19.7 to 45.3%, p < 0.01). This rise in phosphorylation could be prevented by U0126 and Y27632 in a dose-dependent manner, but did not result in lowered mitochondrial ROS formation. Conversely, addition of the antioxidant N-acetyl-<smlcap>L</smlcap>-cysteine only prevented mitochondrial ROS formation but did not prevent phosphorylation of eNOS Thr⁴⁹⁵. Conclusion: H₂O₂-mediated phosphorylation of eNOS Thr⁴⁹⁵ is mediated by ROCK and ERK activity, but not by PKC, and is uncoupled from mitochondrial ROS signaling. Furthermore, ERK inhibition increased mitochondrial ROS formation.

Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H2O2 in HL-1 mouse cardiac muscle cells

Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H2O2), but not angiotensin II, stimulated MIF expression in HL-1 cells. H2O2-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H2O2-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.

Oleic Acid Inhibits the KATP Channel Subunit Kir6.1 and the KATP Current in Human Umbilical Artery Smooth Muscle Cells

BackgroundThe objective of the present study was to determine the effect of various concentrations of oleic acid (OA) on KATP channel expression and the potential relationship to exogenous nitrogen monoxide and protein kinase C levels. MethodsHuman umbilical artery smooth muscle cells (HUASMCs), between the 7th and 10th passages, were divided into control group, OA group (final OA concentration of 0, 50, 100 or 200 μmol/L), nitric oxide (NO) intervention group, protein kinase C inhibitor group or GF-109203X (GFX) intervention group. Western immunoblotting was used to detect the protein expression of the KATP channel subunit Kir6.1. Also, quantitative real-time polymerase chain reaction analysis to determine Kir6.1 messenger RNA levels and whole-cell patch clamping to measure KATP currents were performed. ResultsThe results suggested that OA inhibited Kir6.1 protein and messenger RNA expression in HUASMCs. Under a high concentration of potassium (140 mmol/L), 100 μmol/L OA significantly reduced ATP-sensitive potassium current density, whereas a low extracellular concentration of potassium (5.4 mmol/L) did not influence KATP density. Pretreatment with either exogenous NO or GFX weakened the OA-induced inhibition of KATP in HUASMCs. ConclusionsThe study demonstrated that OA inhibited Kir6.1, a KATP channel subunit, in HUASMCs, and indirectly inhibited the KATP current. In addition, the results indicated that NO and/or GFX partially reversed OA inhibition in HUASMCs.

Mechanisms of resveratrol-induced changes in [Ca2+]i and cell viability in PC3 human prostate cancer cells

Resveratrol is a natural compound that affects cellular Ca2+ homeostasis and viability in different cells. This study examined the effect of resveratrol on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability in PC3 human prostate cancer cells. The Ca2+-sensitive fluorescent dye fura-2 was used to measure [Ca2+]i and WST-1 was used to measure viability. Resveratrol-evoked [Ca2+]i rises concentration-dependently. The response was reduced by removing extracellular Ca2+. Resveratrol-evoked Ca2+ entry was not inhibited by nifedipine, econazole, SKF96365 and the protein kinase C inhibitor GF109203X, but was nearly abolished by the protein kinase C activator phorbol 12-myristate 13 acetate. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-tert-butylhydroquinone decreased resveratrol-evoked rise in [Ca2+]i. Conversely, treatment with resveratrol inhibited BHQ-evoked rise in [Ca2+]i. Inhibition of phospholipase C with U73122 did not alter resveratrol-evoked rise in [Ca2+]i. Previous studies showed that resveratrol between 10 and 100 µM induced cell death in various cancer cell types including PC3 cells. However, in this study, resveratrol (1–10 μM) increased cell viability, which was abolished by chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid-acetoxymethyl ester (BAPTA/AM). Therefore, it is suggested that in PC3 cells, resveratrol had a dual effect on viability: at low concentrations (1–10 µM) it induced proliferation, whereas at higher concentrations it caused cell death. Collectively, our data suggest that in PC3 cells, resveratrol-induced rise in [Ca2+]i by evoking phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ entry, via protein kinase C-regulated mechanisms. Resveratrol at 1–10 µM also caused Ca2+-dependent cell proliferation.

Protein Kinase C Modulates Aurora-kinase Inhibition Induced by CCT129202 in HMC-1560,816 Cell Line

The human mast cell line HMC-1⁵⁶⁰,⁸¹⁶ carries activating mutations in the proto-oncogene of c-kit that cause autophosphorylation and permanent c-kit receptor activation. The compound CCT129202 is a new and selective inhibitor of Aurora kinase A and B that decreases the viability of a variety of human tumor cell lines. The effect of Aurora kinase inhibition was assessed in the HMC-1⁵⁶⁰,⁸¹⁶ line in order to find a suitable tool for mastocytosis treatment. CCT129202 treatment induces a significant decrease in cell viability in HMC-1⁵⁶⁰,⁸¹⁶ cells after 48 hours of treatment. Moreover, caspase-3 and caspase-8 activation was induced after incubation of HMC-1⁵⁶⁰,⁸¹⁶ cells in the presence of CCT129202. It has been demonstrated that Protein Kinase C (PKC) plays a crucial role in mast cell activation as well as cell migration, adhesion and apoptotic cell death. Co-treatment of Ca²⁺-independent PKCs (δ ε and θ) inhibitor GF109203X with CCT129202, reduces caspase-3 activation which controls cell levels. In contrast, Go6976, an inhibitor of Ca²⁺-dependent PKCs, increases caspase-3 activation. Oppositely, GF109203X does not modify CCT129202-induced apoptosis through the caspase-8 pathway whereas Go6976 treatment abolishes the increase on caspase-8 activity due to CCT129202. This implies that Ca²⁺-independent PKC isoforms seems to be related with CCT129202-induced apoptosis through the caspase- 3 pathway, whereas Ca²⁺-dependent PKC isoforms are related with the CCT129202 effect on the caspase-8 pathway. Interestingly, CCT129202 cytotoxic effect remains even though Ca²⁺-dependent PKCs are inhibited, which shows that the Aurora kinase inhibitor effect is acting through the caspase-3 pathway. On the other hand, Ca²⁺-independent PKCs inhibition does not affect the final apoptotic CCT129202 effect because this seems to be mediated by the caspase-8 pathway. Moreover, CCT129202 does not affect PKCδ and Ca²⁺-dependent PKC translocation, which indicates that PKC translocation pivots on its activation. This demonstrates that Aurora kinase inhibition is not related to this process. Finally, when PKC is silenced in HMC-1⁵⁶⁰,⁸¹⁶ cells, the effect of CCT129202 on the caspase-3 pathway disappears, which indicates that the CCT129202 effect is clearly PKC-dependent.

Effects of rivastigmine on secreted amyloid precursor protein and beta-amyloid secretion in neuroblastoma SK-N-SH cells

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized clinically by progressive impairment of memory and cognition. The currently available pharmacological treatment of AD consists mainly of cholinesterase inhibitors. Rivastigmine is one of the cholinesterase inhibitors clinically used to treat this disease, and many clinical trials have indicated that it did alleviate some AD symptoms without causing apparent side effects. Since the amyloid precursor protein (APP) processing imbalance plays a crucial role in AD pathogenesis, the effects of rivastigmine on APP processing were investigated. In neuroblastoma SK-N-SH cells, rivastigmine significantly increased the secretion of sAPPα and decreased the release of Aβ40 and Aβ42 as compared with control group, but it has no effect on cellular full length APP expression. Rivastigmine significantly increased α-secretase activity and decreased β-secretase activity as compared with control group. The increased sAPPα can be partially blocked by muscarinic receptor inhibitor scopolamine but not by nicotinic receptor antagonist α-Bungarotoxin. The effect of rivastigmine on sAPPα can be partially reversed by PKC inhibitor GF109203X, ERK inhibitor PD98059 and JNK inhibitor SP600125. The data present here indicated that rivastigmine can regulate APP processing in vitro by increasing sAPPα secretion and decreasing Aβ release and this pharmacological property may underlie the clinical effect of the drug in the treatment of AD patients.