Activation of post-synaptic dopamine D₁ receptors promotes the release of tissue plasminogen activator in the nucleus accumbens via PKA signaling

We have previously demonstrated that tissue plasminogen activator (tPA) plays an important role through the conversion of plasminogen to plasmin in the release of dopamine in the nucleus accumbens (NAc) evoked by depolarization or the systemic administration of drugs of abuse such as morphine and nicotine. In the present study, we examined the mechanisms by which drugs of abuse increase extracellular tPA activity in the NAc in vivo using in situ zymography. The dopamine D₁ receptor (D₁R) agonist SKF38393, but not D₂ receptor agonist quinpirole, significantly increased extracellular tPA activity in the NAc. The effect of SKF38393 was blocked by pre-treatment with the dopamine D₁R antagonist SCH23390. Microinjection of Rp-cAMPs, a protein kinase A inhibitor, into the NAc completely blocked the effect of SKF38393. Systemic administration of morphine and methamphetamine increased extracellular tPA activity in the NAc, and these effects were completely blocked by pre-treatment with SCH23390 and raclopride. The results suggest that activation of post-synaptic dopamine D₁Rs in the NAc leads to an increase in extracellular tPA activity via protein kinase A signaling. Furthermore, dopamine D₂ receptors are also involved in the release of tPA induced by morphine and methamphetamine.     

Barbituric acid derivative BAS 02104951 inhibits PKCε, PKCη, PKCε/RACK2 interaction, Elk-1 phosphorylation in HeLa and PKCε and η translocation in PC3 cells following TPA-induction

Protein kinase C (PKC) is a family of at least 10 isozymes involved in the activation of different signal transduction pathways. The exact function of these isozymes is not known at present. Isozyme-selective inhibitors would be important to explain the function of the different PKCs and are anticipated to have pharmaceutical potential. Here we report that the small organic molecule BAS 02104951 [5-(1,3-benzodioxol-5-ylmethylene)-1-(phenylmethyl)-2,4,6(1H,3H,5H)-pyrimidinetrion], a barbituric acid derivative, inhibited PKCη and PKCε in vitro (IC(50) 18 and 36 μM, respectively). BAS 02104951 also inhibited the interaction of PKCε with its adaptor protein receptor for activated C-kinase 2 (RACK2) (IC(50) 28.5 μM). BAS 02104951 also inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Elk-1 phosphorylation in HeLa cells, translocation of PKCε and PKCη to the membrane following treatment of PC3 cells with TPA. The compound did not inhibit the proliferation of PC3 and HeLa cells. BAS 02104951 can be used as selective inhibitor of PKCε in cells not expressing PKCη and may serve as a basis for the rational development of a selective inhibitor of PKCε or PKCη, or for an inhibitor of the PKCε/RACK2 interaction.     

Rituximab inhibits Kv1.3 channels in human B lymphoma cells via activation of FcγRIIB receptors

Kv1.3 channels play an important role in modulating lymphocyte proliferation and apoptosis. We hypothesized that Kv1.3 channels in B lymphocytes might be regulated by rituximab, an antibody to CD20, a drug for treatments of B-cell lymphomas and autoimmune diseases. Using both whole-cell and cell-attached patch-clamp techniques, we found that rituximab inhibited Kv1.3 channels in Daudi human B lymphoma cells by promoting the channel inactivation at a concentration which was much greater than that required for activation of CD20. The effect of rituximab on Kv1.3 channels was abolished after selective blockade of FcγRIIB receptors with anti-FcγRIIB antibody. Western blot experiments showed that Daudi B cells expressed both Kv1.3 channel and the low affinity Fc receptor, FcγRIIB, which could be activated by the Fc region of rituximab. In contrast, normal lymphocytes expressed less Kv1.3 channels with faster inactivation. Confocal microscopy and flow cytometry data showed that rituximab induced apoptosis of Daudi B cells and that the effect was attenuated by blockade of FcγRIIB receptors and partially mimicked by inhibition of Kv1.3 channels. These results suggest that in addition to previously described complement-dependent cytotoxicity, rituximab also induces apoptosis of malignant B lymphocyte by stimulating FcγRIIB receptors and inhibiting Kv1.3 channels.     

Seahorse-derived peptide suppresses invasive migration of HT1080 fibrosarcoma cells by competing with intracellular α-enolase for plasminogen binding and inhibiting uPA-mediated activation of plasminogen

α-Enolase is a glycolytic enzyme and a surface receptor for plasminogen. α-Enolase-bound plasminogen promotes tumor cell invasion and cancer metastasis by activating plasmin and consequently degrading the extracellular matrix degradation. Therefore, α-enolase and plasminogen are novel targets for cancer therapy. We found that the amino acid sequence of a peptide purified from enzymatic hydrolysates of seahorse has striking similarities to that of α-enolase. In this study, we report that this peptide competes with cellular α-enolase for plasminogen binding and suppresses urokinase plasminogen activator (uPA)-mediated activation of plasminogen, which results in decreased invasive migration of HT1080 fibrosarcoma cells. In addition, the peptide treatment decreased the expression levels of uPA compared to that of untreated controls. These results provide new insight into the mechanism by which the seahorse-derived peptide suppresses invasive properties of human cancer cells. Our findings suggest that this peptide could emerge as a potential therapeutic agent for cancer. [BMB Reports 2014; 47(12): 691-696]     

Nitric oxide increases the invasion of pancreatic cancer cells via activation of the PI3K–AKT and RhoA pathways after carbon ion irradiation

Previous studies have shown that serine proteases and Rho-associated kinase contribute to carbon ion radiation-enhanced invasion of the human pancreatic cancer cell line PANC-1. The results presented here show that nitric oxide synthase (NOS) also plays a critical role in this process. Irradiation of PANC-1 cells promoted invasion and production of nitric oxide (NO), which activated the PI3K–AKT signaling pathway, while independently activating RhoA. Inhibition of PI3K, Rho-associated kinase, and serine protease alone or in conjunction with NOS suppressed the radiation-enhanced invasion of PANC-1 cells, suggesting that they could serve as possible targets for the management of tumor metastasis.     

Proteasome inhibitors sensitize glioma cells and glioma stem cells to TRAIL-induced apoptosis by PKCε-dependent downregulation of AKT and XIAP expressions

In this study we examined the effects of proteasome inhibitors on cell apoptosis in TRAIL-resistant glioma cells and glioma stem cells (GSCs). Treatment with proteasome inhibitors and TRAIL induced apoptosis in all the resistant glioma cells and GSCs, but not in astrocytes and neural progenitor cells. Since PKCε has been implicated in the resistance of glioma cells to TRAIL, we examined its role in TRAIL and proteasome inhibitor-induced apoptosis. We found that TRAIL did not induce significant changes in the expression of PKCε, whereas a partial decrease in PKCε expression was obtained by proteasome inhibitors. A combined treatment of TRAIL and proteasome inhibitors induced accumulation of the catalytic fragment of PKCε and significantly and selectively decreased its protein and mRNA levels in the cancer but not in normal cells. Overexpression of PKCε partially inhibited the apoptotic effect of the proteasome inhibitors and TRAIL, and the caspase-resistant PKCεD383A mutant exerted a stronger inhibitory effect. Silencing of PKCε induced cell apoptosis in both glioma cells and GSCs, further supporting its role in cell survival. TRAIL and the proteasome inhibitors decreased the expression of AKT and XIAP in a PKCε-dependent manner and overexpression of these proteins abolished the apoptotic effect of this treatment. Moreover, silencing of XIAP sensitized glioma cells to TRAIL. Our results indicate that proteasome inhibitors sensitize glioma cells and GSCs to TRAIL by decreasing the expression of PKCε, AKT and XIAP. Combining proteasome inhibitors with TRAIL may be useful therapeutically in the treatment of gliomas and the eradication of GSCs.     

Inhibitory effect of 30-kDa phytoglycoprotein on expression of TNF-α and COX-2 via activation of PKCα and ERK 1/2 in LPS-stimulated RAW 264.7 cells

Endothelial cells may play a potential role in cholesterol efflux from peripheral tissues to liver. Cholesterol efflux from cells is essential for activation of the reverse cholesterol transport pathway and cardiovascular health. One of the cholesterol transporters is steroidogenic acute regulatory protein (StAR) which promotes intramitochondrial delivery of cholesterol to the cholesterol side-chain cleavage system. The aim of the present study was to determine the effects of a niacin–chromium complex on aortas of hyperlipidemic rats and on the cholesterol efflux from aorta endothelial cells by examination under light and transmission electron microscopes and evaluating the StAR immunoreactivity, respectively. Aorta lipid peroxidation (LPO) and glutathione (GSH) levels were determined by spectrophotometric methods. After treating hyperlipidemic animals with the complex, the StAR immunoreactivity in endothelial cells increased to achieve cholesterol homeostasis and efflux. Combined treatment with niacin and chromium resulted in an inhibition in the mast cell secretion and a decrease in lipid vacuole size in unilocular adipose tissue surrounding aorta, as well as in a decrease in morphological degenerations observing in aorta of hyperlipidemic rats. Aorta LPO levels increased and GSH levels decreased in the hyperlipidemic group, whereas treatment with niacin and chromium reversed these effects. In conclusion, this study reveals that combined treatment with niacin and chromium prevents the morphological and biochemical changes observed in thoracic aorta of hyperlipidemic rats, and may regulate effectively cardiovascular diseases inducing an increase in StAR levels on endothelial cells.     

Estrogen deficiency inhibits the odonto/osteogenic differentiation of dental pulp stem cells via activation of the NF-κB pathway

Various factors can affect the functions of dental pulp stem cells (DPSCs). However, little knowledge is available about the effects of estrogen deficiency on the differentiation of DPSCs. In this study, an estrogen-deficient rat model was constructed and multi-colony-derived DPSCs were obtained from the incisors of ovariectomized (OVX) or sham-operated rats. Odonto/osteogenic differentiation and the possible involvement of the nuclear factor kappa B (NF-κB) pathway in the OVX-DPSCs/Sham-DPSCs of these rats were then investigated. OVX-DPSCs presented decreased odonto/osteogenic capacity and an activated NF-κB pathway, as compared with Sham-DPSCs. When the cellular NF-κB pathway was specifically inhibited by BMS345541, the odonto/osteogenic potential in OVX-DPSCs was significantly upregulated. Thus, estrogen deficiency down-regulated the odonto/osteogenic differentiation of DPSCs by activating NF-κB signaling and inhibition of the NF-κB pathway effectively rescued the decreased differentiation potential of DPSCs.     

Piezoelectric effect stimulates the rearrangement of chondrogenic cells and alters ciliary orientation via atypical PKCζ

Therapeutic ultrasound was administered to patients suffering from bone fracture with FDA approval. Bone and cartilage are piezoelectric materials. To investigate the effects of piezoelectricity on the cells of chondrogenic lineage, we applied ultrasound stimulation on an AT-cut quartz coverslip to generate electric field fluctuations. The bone-marrow-derived mesenchymal stem cells (BMMSC) and primary chondrocytes were cultured on either glass or quartz coverslips for ultrasound stimulation. The cells were immunofluorescent-labeled for the assessment of cell arrangement and ciliary orientation. Ultrasound and piezoelectricity both stimulate cell migration and disrupt ciliary orientation induced by directional migration. In particular, piezoelectric effects on cell rearrangement can be abolished by the inhibitor specifically targeting atypical Protein kinase C zeta (PKCζ). Our findings shed light on the possibility of cellular modulation by using piezoelectric manipulation.     

Exosomes released from M. tuberculosis infected cells can suppress IFN-γ mediated activation of naïve macrophages

Background

Macrophages infected with Mycobacterium tuberculosis (M.tb) are known to be refractory to IFN-γ stimulation. Previous studies have shown that M.tb express components such as the 19-kDa lipoprotein and peptidoglycan that can bind to macrophage receptors including the Toll-like receptor 2 resulting in the loss in IFN-γresponsiveness. However, it is unclear whether this effect is limited to infected macrophages. We have previously shown that M.tb-infected macrophages release exosomes which are 30–100 nm membrane bound vesicles of endosomal origin that function in intercellular communication. These exosomes contain mycobacterial components including the 19-kDa lipoprotein and therefore we hypothesized that macrophages exposed to exosomes may show limited response to IFN-γ stimulation.

Methodology/Principal Findings

Exosomes were isolated from resting as well as M.tb-infected RAW264.7 macrophages. Mouse bone marrow-derived macrophages (BMMØ) were treated with exosomes +/− IFN-γ. Cells were harvested and analyzed for suppression of IFN-γ responsive genes by flow cytometry and real time PCR. We found that exosomes derived from M.tb H37Rv-infected but not from uninfected macrophages inhibited IFN-γ induced MHC class II and CD64 expression on BMMØ. This inhibition was only partially dependent on the presence of lipoproteins but completely dependent on TLR2 and MyD88. The exosomes isolated from infected cells did not inhibit STAT1 Tyrosine phosphorylation but down-regulated IFN-γ induced expression of the class II major histocompatibity complex transactivator; a key regulator of class II MHC expression. Microarray studies showed that subsets of genes induced by IFN-γ were inhibited by exosomes from H37Rv-infeced cells including genes involved in antigen presentation. Moreover, this set of genes partially overlapped with the IFN-γ-induced genes inhibited by H37Rv infection.

Conclusions

Our study suggests that exosomes, as carriers of M.tb pathogen associated molecular patterns (PAMPs), may provide a mechanism by which M.tb may exert its suppression of a host immune response beyond the infected cell.     

Cytokine-mediated induction of human immunodeficiency virus (HIV) expression and cell death in chronically infected U1 cells: do tumor necrosis factor alpha and gamma interferon selectively kill HIV-infected cells?

Infection with several DNA or RNA viruses induces a state of increased sensitivity to cell lysis mediated by tumor necrosis factor (TNF), particularly in the presence of gamma interferon (IFN-gamma). Infection of human cells with the human immunodeficiency virus (HIV) may induce a similar phenomenon. However, TNF and IFN-gamma are known upregulators of HIV replication, raising the question of the potential role of these cytokines in the selective elimination of cells infected with this virus. The present study demonstrates that chronically infected U1 cells were killed with much greater efficiency by costimulation with TNF-alpha and IFN-gamma than their uninfected parental cell line U937. However, synergistic induction of viral expression also occurred in U1 cells as a consequence of treatment with the two cytokines. Cell death in U1 cells was not caused by the massive production of virions, in that costimulation with glucocorticoid hormones and TNF-alpha or IFN-gamma resulted in high levels of virion production without cytopathicity. To investigate the nature of the selective cytotoxic effect observed in U1 cells costimulated with TNF-alpha plus IFN-gamma, a panel of uninfected cell clones was generated by limiting dilution of U937 cells and tested for response to TNF-alpha and/or IFN-gamma. In contrast to the uncloned bulk parental U937 cell line, most uninfected cell clones showed a very high susceptibility to being killed by TNF-alpha and IFN-gamma. Similar findings were obtained when both infected U1 cells and several uninfected U937 cell clones were costimulated with an anti-Fas monoclonal antibody in the presence of IFN-gamma, although, unlike cells stimulated with TNF-alpha, cells treated with anti-Fas antibody did not express virus. Therefore, the increased susceptibility to cytokine-mediated lysis observed in cell lines infected with HIV is likely due to the selection of preexisting cell clones rather than viral infection.     

Sanguinarine inhibits osteoclast formation and bone resorption via suppressing RANKL-induced activation of NF-κB and ERK signaling pathways

Sanguinarine is a natural plant extract that has been supplemented in a number of gingival health products to suppress the growth of dental plaque. However, whether sanguinarine has any effect on teeth and alveolar bone health is still unclear. In this study, we demonstrated for the first time that sanguinarine could suppress osteoclastic bone resorption and osteoclast formation in a dose-dependent manner. Sanguinarine diminished the expression of osteoclast marker genes, including TRAP, cathepsin K, calcitonin receptor, DC-STAMP, V-ATPase d2, NFATc1 and c-fos. Further investigation revealed that sanguinarine attenuated RANKL-mediated IκBα phosphorylation and degradation, leading to the impairment of NF-κB signaling pathway during osteoclast differentiation. In addition, sanguinarine also affected the ERK signaling pathway by inhibiting RANKL-induced ERK phosphorylation. Collectively, this study suggested that sanguinarine has protective effects on teeth and alveolar bone health.     

Ca2+-dependent and -independent release of neurotransmitters from PC12 cells: a role for protein kinase C activation?

The intracellular mechanisms regulating the process of evoked neurotransmitter release were studied in the cloned neurosecretory cell line PC12. Various agents were employed that were known, from previous studies in other systems, to stimulate release in a manner either strictly dependent or independent of the concentration of extracellular Ca2+, [Ca2+]o. Three parameters were investigated in cells suspended in either Ca2+-containing or Ca2+-free Krebs-Ringer media: release of previously accumulated [3H]dopamine; average free cytoplasmic Ca2+ concentration, [Ca2+]i (measured by the quin2 technique); and cell ultrastructure, with special reference to the number and structure of secretion granules. The release induced by the ionophores transporting monovalent cations, X537A and monensin, occurred concomitantly with profound alterations of secretory granule structure (swelling and dissolution of the dense core). These results suggest that the effect of these drugs is due primarily to leakage of dopamine from granules to the cytoplasm and extracellular space. In contrast, the changes induced by other stimulatory drugs used concerned not the structure but the number of secretory granules, indicating that with these drugs stimulation of exocytosis is the phenomenon underlying the increased transmitter release. The release response induced by the Ca2+-ionophore ionomycin was dependent on [Ca2+]o, occurred rapidly, was concomitant with a marked rise of [Ca2+]i, and ceased after 1-2 min even though [Ca2+]i remained elevated for many minutes. 12-O-tetradecanoylphorbol, 13-acetate and diacylglycerol (both of which are known as activators of protein kinase C) induced slow responses almost completely independent of [Ca2+]o and not accompanied by changes of [Ca2+]i. Combination of an activator of protein kinase C with a low concentration of ionomycin failed to modify the [Ca2+]i rise induced by the ionophore, but elicited a marked potentiation of the release response, which was two- to fourfold larger than the sum of the responses elicited separately by either drugs. Thus, activation of protein kinase C seems to play an important role in the regulation of exocytosis in neurosecretory cells, possibly by increasing and maintaining the sensitivity to Ca2+ of the intracellular apparatus regulating granule discharge by exocytosis.     

Phagocytosis of cells dying through autophagy induces inflammasome activation and IL-1β release in human macrophages

Phagocytosis of naturally dying cells usually blocks inflammatory reactions in host cells. We have recently observed that clearance of cells dying through autophagy leads to a pro-inflammatory response in human macrophages. Investigating this response further, we found that during engulfment of MCF-7 or 293T cells undergoing autophagic death, but not apoptotic or anoikic ones, caspase-1 was activated and IL-1β was processed, then secreted in a MyD88-independent manner. Autophagic dying cells were capable of preventing some LPS-induced pro-inflammatory responses, such as TNFα, IL-6 and IL-8 induction, but synergized with LPS for IL-1β production. Caspase-1 inhibition prevented macrophage IL-1β release triggered by the dying cells and also other pro-inflammatory cytokines which were not formed in the presence of IL-1 receptor antagonist anakinra either. IL-1β secretion was also observed using calreticulin knock down or necrostatin treated autophagic MCF-7 cells and it required phagocytosis of the dying cells which led to ATP secretion from macrophages. Blocking K (+) efflux during phagocytosis, the presence of apyrase, adding an antagonist of the P2X7 receptor or silencing the NOD-like receptor protein NALP3 inhibited IL-1β secretion. These data suggest that during phagocytosis of autophagic dying cells ATP, acting through its receptor, initiates K (+) efflux, inflammasome activation and secretion of IL-1β, which initiates further pro-inflammatory events. Thus, autophagic death of malignant cells and their clearance may lead to immunogenic response.     

Binding of anti-SSA antibodies to apoptotic fetal cardiocytes stimulates urokinase plasminogen activator (uPA)/uPA receptor-dependent activation of TGF-β and potentiates fibrosis

In congenital heart block (CHB), binding of maternal anti-SSA/Ro Abs to fetal apoptotic cardiocytes impairs their removal by healthy cardiocytes and increases urokinase plasminogen activator (uPA)/uPA receptor (uPAR)-dependent plasmin activation. Because the uPA/uPAR system plays a role in TGF-β activation, we evaluated whether anti-Ro binding to apoptotic cardiocytes enhances plasmin-mediated activation of TGF-β, thereby promoting a profibrosing phenotype. Supernatants from cocultures of healthy cardiocytes and apoptotic cardiocytes bound by IgG from a mother whose child had CHB (apoptotic-CHB-IgG [apo-CHB-IgG]) exhibited significantly increased levels of active TGF-β compared with supernatants from cocultures of healthy cardiocytes and apoptotic cardiocytes preincubated with IgG from a healthy donor. Treatment of the culture medium with anti-TGF-β Ab or TGF-β inhibitor (SB431542) abrogated the luciferase response, thereby confirming TGF-β dependency. Increased uPA levels and activity were present in supernatants generated from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes compared with healthy cardiocytes and apoptotic cardiocytes preincubated with IgG from a healthy donor, respectively. Treatment of apo-CHB-IgG cardiocytes with anti-uPAR or anti-uPA Abs or plasmin inhibitor aprotinin prior to coculturing with healthy cardiocytes attenuated TGF-β activation. Supernatants derived from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes promoted Smad2 phosphorylation and fibroblast transdifferentiation, as evidenced by increased smooth muscle actin and collagen expression, which decreased when fibroblasts were treated with supernatants from cocultures pretreated with uPAR Abs. These data suggested that binding of anti-Ro Abs to apoptotic cardiocytes triggers TGF-β activation, by virtue of increasing uPAR-dependent uPA activity, thus initiating and amplifying a cascade of events that promotes myofibroblast transdifferentiation and scar.     

GEF-H1/RhoA signalling pathway mediates lipopolysaccharide-induced intercellular adhesion molecular-1 expression in endothelial cells via activation of p38 and NF-κB

The purpose of study is to investigate the effects of GEF-H1/RhoA pathway in regulating intercellular adhesion molecule-1 (ICAM-1) expression in lipopolysaccharide (LPS)-activated endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) to LPS induced GEF-H1 and ICAM-1 expression in dose- and time-dependent up-regulating manners. Pretreatment with Clostridium difficile toxin B-10463 (TcdB-10463), an inhibitor of Rho activity, reduced LPS-related phosphorylation of p65 at Ser 536 in a dose-dependent manner. Inhibition of TLR4 expression significantly blocked LPS-induced RhoA activity, NF-κB transactivation, GEF-H1 and ICAM-1 expression. Coimmunoprecipitation assay indicated that LPS-activated TLR4 and GEF-H1 formed a signalling complex, suggesting that LPS, acting through TLR4, stimulates GEF-H1 expression and RhoA activity, and thereby induces NF-κB transactivation and ICAM-1 gene expression. However, GEF-H1/RhoA regulates LPS-induced NF-κB transactivation and ICAM-1 expression in a MyD88-independent pathway because inhibition of MyD88 expression could not block LPS-induced RhoA activity. Furthermore, pretreatment with Y-27632, an inhibitor of ROCK, significantly reduced LPS-induced p38, ERK1/2 and p65 phosphorylation, indicating that ROCK acts as an upstream effector of p38 and ERK1/2 to promote LPS-induced NF-κB transactivation and ICAM-1 expression. What is more, the p38 inhibitor (SB203580) but not ERK1/2 inhibitor (PD98059) blocked LPS-induce NF-κB transactivation and ICAM-1 expression, which demonstrates that RhoA mediates LPS-induced NF-κB transactivation and ICAM-1 expression dominantly through p38 but not ERK1/2 activation. In summary, our data suggest that LPS-induced ICAM-1 synthesis in HUVECs is regulated by GEF-H1/RhoA-dependent signaling pathway via activation of p38 and NF-κB.