PKC Inhibitor Bisindolylmaleimide Research Articles

Aerobic exercise alleviates ventilator-induced lung injury by inhibiting NLRP3 inflammasome activation

BackgroundVentilator-induced lung injury (VILI) is caused by stretch stimulation and other factors related to mechanical ventilation (MV). NOD-like receptor protein 3 (NLRP3), an important innate immune component, is strongly associated with VILI. This study aimed to investigate the effect and mechanisms of aerobic exercise (EX) on VILI.MethodsTo test the effects of the PKC inhibitor bisindolylmaleimide I on PKC and NLRP3, male C57BL/6 mice (7 weeks old, 19 ~ 23 g) were randomly divided into four groups: control group(C), bisindolylmaleimide I-pretreated group(B), MV group, and bisindolylmaleimide I-pretreated + MV (B + MV) group. The mice were pretreated with bisindolylmaleimide I through intraperitoneal injection (0.02 mg/kg) 1 h before MV. MV was performed at a high tidal volume (30 ml/kg). To explore the ameliorative effect of EX on VILI, the mice were randomly divided into C group, MV group, EX group and EX + MV group and subjected to either MV or 5 weeks of EX training. After ventilation, haematoxylin-eosin (HE) staining and wet/dry weight ratio was used to assess lung pathophysiological changes. PKCɑ, P-PKCɑ, ASC, procaspase-1, caspase-1, pro-IL-1β, IL-1β, NLRP3 and occludin (tight junction protein) expression in lung tissues was determined by Western blotting. The level of IL-6 in alveolar lavage fluid was determined by ELISA.ResultsNLRP3, P-PKCɑ, and PKCɑ levels were inceased in MV group, but bisindolylmaleimide I treatment reversed these changes. Inhibition of PKC production prevented NLRP3 activation. Moreover, MV increased ASC, procaspase-1, caspase-1, pro-IL-1β, and IL1β levels and decreased occludin levels, but EX alleviated these changes. HE staining and lung injury scoring confirmed an absence of obvious lung injury in C group and EX group. Lung injury was most severe in MV group but was improved in EX + MV group. Overall, these findings suggest that MV activates the NLRP3 inflammasome by activating PKCɑ and inducing occludin degradation, while Exercise attenuates NLRP3 inflammasome and PKCɑ activation. Besides, exercise improves cyclic stretch-induced degradation of occludin.ConclusionPKC activation can increase the level of NLRP3, which can lead to lung injury. Exercise can reduce lung injury by inhibiting PKCɑ and NLRP3 activation. Exercise maybe a potential measure for clinical prevention of VILI.

Targeting Cutaneous T-Cell Lymphoma Cells by Ingenol Mebutate (PEP005) Correlates with PKCδ Activation, ROS Induction as Well as Downregulation of XIAP and c-FLIP.

New therapeutic strategies are needed for cutaneous T-cell lymphoma (CTCL), and the plant extract ingenol mebutate (PEP005) may be considered. PEP005 has been approved for actinic keratosis, and proapoptotic activities were described in different cancer cells. Here, we aimed to investigate its efficacy in four CTCL cell lines and its mode of action. While HuT-78 and HH responded with induced apoptosis as well as with loss of cell viability and cell proliferation, MyLa and SeAx remained resistant. Interestingly, both sensitive and resistant cells showed caspase-8 activation and enhanced levels of reactive oxygen species (ROS), while final caspase-3 activation was restricted to sensitive cells. Apoptosis induction was prevented by the caspase inhibitor QVD-Oph as well as by the antioxidant vitamin E. Caspase activation by PEP005 may be explained to some extent by the downregulation of the caspase antagonistic proteins c-FLIP and XIAP in sensitive cells, whereas both proteins were strongly expressed in resistant cells. Finally, PEP005 resulted in the activation of proapoptotic PKCδ, and the PKC inhibitor bisindolylmaleimide I reduced apoptosis, caspase-3 processing and ROS production, as well as restored cell viability. In conclusion, PKCδ appeared as a central player in apoptosis regulation in CTCL cells, also suggesting its therapeutic targeting.

Mitochondrial ROS activates PKC alpha translocation to the membrane of vagal sensory neurons.

Hyperexcitability of airway sensory nerves can trigger disease‐associated reflexes resulting in cough and dyspnea. We have observed that mitochondrial ROS production increases hyperexcitability in nociceptive bronchopulmonary C‐fibers. We also found that this increased excitability can be blocked by the PKC inhibitor bisindolylmaleimide (BIM) I, but not the inactive PKC analog BIM V. In previous work we have shown, using a pan‐PKC antibody, that mitochondrial ROS production via Antimycin A (AntA; mitochondrial complex III Qi site inhibitor) causes translocation to the cell membrane. However, there are several PKC isoforms expressed in nociceptive neurons. We wanted to determine which isoforms were present, their distribution in nociceptive cells and whether specific isoforms were activated by AntA. Studies looking at mRNA expression have highlighted PKC alpha, beta1, beta2, delta and epsilon as abundant in nociceptors so our studies have focused on these particular isoforms. Sensory neuron cell bodies were isolated from mouse vagal ganglia. To identify nociceptive cells, we used a mouse strain that co‐expresses a red fluorescent protein (tdTomato) with the Transient Receptor Potential V1 ion channel. Cells were treated for 2 min with: vehicle, 10 μM AntA or 300 nM PMA then immediately fixed. We performed immunocytochemistry to determine cellular location of different isoforms. Of the TRPV1 expressing nociceptive cells, more than 50% were positive for any given isoform of PKC. All isoforms translocated to the membrane in response to PMA treatment. However, only PKC alpha exhibited translocation to the membrane in response to AntA treatment. In the case of PKC delta, AntA appeared to cause translocation of this isoform to the nucleus. Our experiments suggest that nociceptor hyperexcitability, in response to ROS produced via mitochondrial dysfunction, may occur through the translocation and activation of the PKC alpha isoform.Support or Funding InformationThis work was supported by the National Heart Lung and Blood Institute in Bethesda, USA(R01‐HL119802)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

M2-like tumor-associated macrophages drive vasculogenic mimicry through amplification of IL-6 expression in glioma cells.

Vasculogenic mimicry (VM) has offered a new horizon for understanding tumor angiogenesis, but the mechanisms of VM in glioma progression have not been studied explicitly until now. As a significant component of immune infiltration in tumor microenvironment, macrophages have been demonstrated to play an important role in tumor growth and angiogenesis. However, whether macrophages could play a potential key role in glioma VM is still poorly understood. Herein we reported that both VM and CD163+ cells were associated with WHO grade and reduced patient survival, and VM channel counting was correlated to the number of infiltrated CD163+ cells in glioma specimens. In vitro studies of glioma cell lines implicated that M2-like macrophages (M2) promoted glioma VM. We found that conditional medium derived from M2 amplified IL-6 expression in glioma cells. Furthermore, our data indicated that IL-6 could promote glioma VM, as blocking IL-6 with neutralizing antibodies abrogated M2-mediated VM enhancement. In addition, the potent PKC inhibitor bisindolylmaleimide I could prevent M2-induced IL-6 upregulation and further inhibited glioma VM facilitation. Taken together, our results suggested that M2-like macrophages drove glioma VM through amplifying IL-6 secretion in glioma cells via PKC pathway.

121 - Redox Control of the Critical Immune Regulatory Enzyme Indoleamine 2,3-Dioxygenase by NADPH Oxidase 2-Derived Reactive Oxygen Species in Human Monocytes

The heme enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is a critical immune regulatory enzyme capable of signalling for immune suppression in diverse pathological settings including chronic inflammation, infection and cancer. During inflammation, IDO1 is expressed within activated innate immune cells that also produce the reactive oxygen species (ROS) hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) by NADPH oxidase (Nox) and myeloperoxidase (MPO), respectively. We recently reported that H2O2 inhibits recombinant IDO1 dioxygenase activity by engaging the enzyme’s heme peroxidase activity resulting in oxidative self-inactivation (JBC 2013;288:1548). To test if Nox-derived H2O2 or MPO-derived HOCl also control cellular IDO1 activity, we studied IFN-γ-stimulated human monocytes that co-express IDO1, Nox and MPO. Stimulation of monocytes with PMA increased cellular production of H2O2 and HOCl that coincided with the post-translational inhibition of IDO1 activity. The ability of PMA to inhibit IDO1 was abrogated by inhibition of Nox-derived H2O2 production with the PKC inhibitor Bisindolylmaleimide or the flavoprotein oxidase inhibitor diphenyleniodonium, but not by inhibition of MPO-derived HOCl with 4-aminobenzoic acid hydrazide. IFN-γ-stimulated human monocytes not only expressed the phagocytic Nox2 isoform but also Nox4. While selective Nox4 inhibition reduced cellular H2O2 production by ~50%, it did not reverse PMA’s inhibitory action on IDO1. Also, PMA did not inhibit IDO1 activity in IFN-γ-stimulated monocytes isolated from chronic granulomatous disease (CGD) patients expressing defective Nox2. These studies establish that Nox2-derived ROS control IDO1 activity in human innate immune cells and provide important new insights into the redox control of IDO1’s immune regulatory actions.

Pharmacological Profiling of Lysophosphatidylinositol Species at GPR55 Using xCELLigence Cellular Impedance Analysis

L-α-lysophosphatidylinositol (LPI) is the endogenous ligand for GPR55, a Gα13-coupled GPCR. The most abundant endogenous LPI species are stearoyl-LPI (16:0 LPI), palmitoyl-LPI (18:0 LPI) and arachidonoyl-LPI (20:4 LPI). The pharmacology of ligands at GPR55 is contentious therefore further studies are required to elucidate the pharmacology of GPR55 in native cells. This study aimed to elucidate the signaling profiles of LPI species in stably expressing GPR55-HEK293 cells and human osteoclasts using xCELLigence cellular impedance in order to validate this approach as a screening tool for GPR55 ligands. In GPR55-HEK293 cells, all LPI species produced a dose dependent decrease in cellular impedance that was absent in HEK293 cells and antagonised by GPR55 antagonists CID1261822 and CID16020046. Immunocytochemical staining confirmed that the decrease in impedance was attributable to cellular retraction. GPR55 mediated signaling via Gαi, Gαs, Gαq/11 or Gβγ subunits was excluded due to lack of inhibition by pre-treatment with pertussis toxin, SQ22536, U73122 and gallein respectively. Responses to all species were completely inhibited by Y27632 and H89 suggesting that the main pathways contributing to the decrease in impedance are Rho and PKA. Interestingly, the response to 20:4 LPI was also significantly attenuated by the PKC inhibitor Bisindolylmaleimide II, indicative of biased agonism. Treatment with all LPI species in human osteoclasts produced a dose dependant decrease in cellular impedance attributed to Rho, ERK and PKC as well as Gi and Gβγ, reflective of more diverse signaling. These experiments validate use of xCELLigence technology to screen novel GPR55 ligands in native cells.

Map kinase and PKC signaling pathways modulate NGF-mediated apoE transcription

The present study assessed the mechanisms by which nerve growth factor (NGF) increased the level of apolipoprotein E (apoE) in PC12 cells. NGF (50ng/mL) significantly increased apoE protein levels following 72h of treatment. Similarly NGF increased luciferase activity in cells transfected with a luciferase reporter construct containing a 500bp fragment of the apoE promoter, indicating NGF-induced apoE expression is regulated, at least in part, at the level of transcription. The non-selective nitric oxide synthase (NOS) inhibitor Nɷ-nitro-L-arginine methylester (L-NAME; 20mM) did not attenuate the NGF-mediated increase in luciferase activity, while the inducible NOS inhibitor s-methylisothiourea (S-MIU; 2mM) partially attenuated this action of NGF. Inhibition of MAP kinase activation with 50μM U0126 or pre-treatment with the PKC inhibitor bisindolylmaleimide 1 (BIS-1; 10μM) prevented the NGF-mediated activation of the apoE promoter. Pre-treatment with the phospholipase C (PLC) inhibitor U73122 (5μM) partially inhibited the NGF-induced increase in luciferase activity while the Akt inhibitor LY294002 (10μM) had no effect. These data suggest NGF-induced apoE transcription requires MAP kinase and PKC activation and that these TrkA signaling pathways may be modulated by NO.

Mitochondrial reactive oxygen species induce hyperexcitability in nociceptive airway sensory nerves via PKC activation (663.5)

Activation of airway nociceptive C‐fibers evokes cough and parasympathetic reflexes. Nociceptor excitability is increased by inflammation, worsening symptoms in asthma. Airway C‐fiber terminals are packed with mitochondria. Inflammatory signaling modulates mitochondrial function, causing reactive oxygen species (ROS) production. We hypothesize that these ROS increase nociceptor excitability. We recorded action potentials in single C‐fibers using an ex vivo mouse lung‐ganglia preparation. “Nociceptors” are slow fibers, sensitive to capsaicin (via TRPV1). Antimycin A, a mitochondrial complex III inhibitor that evokes ROS, had no effect on the excitability of non‐nociceptive nerves. Ten minutes after antimycin A, nociceptive nerves developed hyperexcitability to chemical and mechanical stimuli. Antimycin‐induced hyperexcitability was blocked by N‐acetyl cysteine and dithiothreitol but not by TRPV1 knockout. ROS can activate protein kinase C independently of Ca2+. Here, the PKC inhibitor bisindolylmaleimide I (BIM I), but not the inactive analogue BIM V, abolished antimycin‐induced hyperexcitability. Antimycin A also caused PKC translocation via ROS. Lastly, exogenous H2O2 induced nociceptor hyperexcitability, which was blocked by BIM I. We conclude that acute modulation of nerve terminal mitochondria can induce airway nociceptor hyperexcitability via ROS‐activated PKC pathways.Grant Funding Source: Supported by R01HL119802‐01

Nerve Growth Factor Enhances Tau Isoform Expression and Transcription in IMR32 Cells

The present study characterized the nerve growth factor (NGF)-mediated regulation of tau protein expression and transcription in IMR32 human neuroblastoma cells. Treatment of IMR32 cells with 50 ng/mL NGF resulted in increased levels of specific tau protein isoforms. A 550 bp fragment of the tau promoter was cloned and treatment of transfected IMR32 and PC12 cells with NGF also resulted in increased promoter activation, suggesting that the NGF-mediated increase in tau isoforms is regulated, at least in part, at the level of transcription. Pretreatment with the MAP kinase inhibitor U0126 or the PKC inhibitor bisindolylmaleimide 1 (BIS-1) attenuated the NGF-mediated increase in tau transcription, indicating that the NGF-mediated activation of the MAP kinase and PKC signaling pathways modulate tau transcription. Pre-treatment of cells with the Akt inhibitor, LY294002 or with NOS inhibitors Nω-nitro-L-arginine methylester (L-NAME) or s-methylisothiourea (S-MIU) had no effect on the NGF-mediated increase in tau promoter activation, suggesting that NO and the NGF-Akt signaling pathway do not modulate tau transcription. Taken together, these data demonstrate that NGF increases the levels of multiple human tau isoforms in IMR32 cells which may result, at least in part, from NGF-mediated PKC and MAP kinase-induced tau transcription.

Investigation of signalling cascades induced by neurotrophic synaptolepis factor K7 reveals a critical role for novel PKCε

This study elucidates signalling cascades involved in the neurotrophic effects induced by an active compound of Synaptolepis kirkii, a plant that is used against snakebites and for treatment of epilepsy. The active compound of this plant, synaptolepis factor K7 (K7), is suggested to exert anti-tumoral and neurotrophic actions via modulation of PKC. In SH-SY5Y cells synthesis of the neuronal marker growth-associated protein 43 was increased upon 48h treatment with K7. Immunofluorescent staining of neurites revealed an increased neurite formation by synaptolepis factor K7. Short-term signal transduction events were followed at the level of extracellular-regulated kinase phosphorylation. Extracellular-regulated kinase (ERK) phosphorylation was transiently increased upon stimulation with synaptolepis factor K7 (300nM) with a maximal effect at 30min. Use of the general PKC inhibitor bisindolylmaleimide I blocked the K7-induced ERK phosphorylation suggesting involvement of PKC. Conversely, inhibition of conventional PKCs, α, β and γ by treatment with Go6976 did not inhibit ERK phosphorylation up to 1μM. Use of a specific-PKCε translocation inhibitor peptide or RNAi-mediated knockdown of PKC-epsilon (ε) abolished the K7-induced ERK phosphorylation implicating PKCε in K7 function. This was confirmed by the observed increase in PKCε translocation and autophosphorylation induced by the compound. These data show that synaptolepis factor K7 induces neuronal differentiation of SH-SY5Y cells concomitant with a transient increase in ERK phosphorylation that is mediated by activation of PKCε.

Potentiation of Sulfonylurea Action by an EPAC-selective cAMP Analog in INS-1 Cells: Comparison of Tolbutamide and Gliclazide and a Potential Role for EPAC Activation of a 2-APB-sensitive Ca2+Influx

Tolbutamide and gliclazide block the K(ATP) channel K(ir)6.2/Sur1, causing membrane depolarization and stimulating insulin secretion in pancreatic beta cells. We examined the ability of the EPAC-selective cAMP analog 8-pCPT-2'-O-Me-cAMP-AM to potentiate the action of these drugs and the mechanism that might account for it. Insulin secretion stimulated by both 200 μM tolbutamide and 20 μM gliclazide, concentrations that had equivalent effects on membrane potential, was inhibited by thapsigargin (1 μM) or the L-type Ca(2+) channel blocker nicardipine (2 μM) and was potentiated by 8-pCPT-2'-O-Me-cAMP-AM at concentrations ≥2 μM in INS-1 cells. Ca(2+) transients stimulated by either tolbutamide or gliclazide were inhibited by thapsigargin or nicardipine and were significantly potentiated by 8-pCPT-2'-O-Me-cAMP-AM at 5 μM but not 1 μM. Both tolbutamide and gliclazide stimulated phospholipase C activity; however, only gliclazide did so independently of its activity at K(ATP) channels, and this activity was partially inhibited by pertussis toxin. 8-pCPT-2'-O-Me-cAMP-AM alone (5 μM) did not stimulate insulin secretion, but did increase intracellular Ca(2+) concentration significantly, and this activity was inhibited by 25 μM 2-aminoethoxydiphenylborate (2-APB) or the removal of extracellular Ca(2+). 8-pCPT-2'-O-Me-cAMP-AM potentiation of insulin secretion stimulated by tolbutamide was markedly inhibited by 2-APB (25 μM) and enhanced by the PKC inhibitor bisindolylmaleimide I (1 μM). Our data demonstrate that the actions of both tolbutamide and gliclazide are strongly potentiated by 8-pCPT-2'-O-Me-cAMP-AM, that gliclazide can stimulate phospholipase C activity via a partially pertussis toxin-sensitive mechanism, and that 8-pCPT-2'-O-Me-cAMP-AM potentiation of tolbutamide action may involve activation of a 2-APB-sensitive Ca(2+) influx.

PKC‐dependent activation of human K2P18.1 K+ channels

Two-pore-domain K(+) channels (K(2P) ) mediate K(+) background currents that modulate the membrane potential of excitable cells. K(2P) 18.1 (TWIK-related spinal cord K(+) channel) provides hyperpolarizing background currents in neurons. Recently, a dominant-negative loss-of-function mutation in K(2P) 18.1 has been implicated in migraine, and activation of K(2P) 18.1 channels was proposed as a therapeutic strategy. Here we elucidated the molecular mechanisms underlying PKC-dependent activation of K(2P) 18.1 currents. Human K(2P) 18.1 channels were heterologously expressed in Xenopus laevis oocytes, and currents were recorded with the two-electrode voltage clamp technique. Stimulation of PKC using phorbol 12-myristate-13-acetate (PMA) activated the hK(2P) 18.1 current by 3.1-fold in a concentration-dependent fashion. The inactive analogue 4α-PMA had no effect on channel activity. The specific PKC inhibitors bisindolylmaleimide I, Ro-32-0432 and chelerythrine reduced PMA-induced channel activation indicating that PKC is involved in this effect of PMA. Selective activation of conventional PKC isoforms with thymeleatoxin (100 nM) did not reproduce K(2P) 18.1 channel activation. Current activation by PMA was not affected by pretreatment with CsA (calcineurin inhibitor) or KT 5720 (PKA inhibitor), ruling out a significant contribution of calcineurin or cross-talk with PKA to the PKC-dependent hK(2P) 18.1 activation. Finally, mutation of putative PKC phosphorylation sites did not prevent PMA-induced K(2P) 18.1 channel activation. We demonstrated that activation of hK(2P) 18.1 (TRESK) by PMA is mediated by PKC stimulation. Hence, PKC-mediated activation of K(2P) 18.1 background currents may serve as a novel molecular target for migraine treatment.

OSU-2S, A Novel FTY720 derivative, Mediates Cytotoxicity In Chronic Lymphocytic Leukemia Through PKC Dependent Modulation of SHP1 Tyrosine Phosphatase

Abstract 2499Despite development of numerous chemotherapeutic agents against Chronic Lymphocytic Leukemia (CLL), drug resistance remains impediment in the successful treatment of CLL. Currently several kinases including SYK, BTK, Pim and PI3K are being targeted with inhibitors for therapy in CLL. However, no phosphatase activation directed therapeutics has been described for CLL. We have recently reported FTY720, a potent immunosuppressive agent derived from fungal sphingosine analog ISP-1 to exhibit potent in-vitro and in-vivo preclinical activity against CLL and Mantle cell Lymphoma through protein phosphatase 2A (PP2A) activation dependent mechanisms. However, the immunosuppressive nature of this drug hinders its further development for clinical therapy in cancer. To overcome this limitation we have developed several derivatives of FTY720 by structure-activity relationship analysis. Here we demonstrate OSU-2S as an agent that modulates SHP1 and PP2A protein phosphatases in CLL with potential therapeutic options. OSU-2S is a novel FTY720 derivative that does not interact with sphingosine 1 phosphate receptor 1, thus lacking immunosuppressive property without compromising the cytotoxic potential. Here we have shown preclinical activity of OSU-2S in cell lines representing CLL (MEC-1), ALL (697), lymphoblastic lymphoma cell (Raji, Ramos), Mantle cell Lymphoma (Mino, Jeko), T-cells (Jurkat) and primary CLL and T cell leukemia cells. In primary CLL B-cells, OSU-2S induces activation of major caspases including caspase 3, 8, and 9 resulting in Poly (ADP-ribose) polymerase cleavage. It also induces reactive oxygen species in CLL primary cells with no modulation of Bcl-2 or Mcl-1 levels. Interestingly, OSU-2S induces phosphorylation of Ser 591 of the SHP1 phosphatase in time dependent manner. Consistent with a previously described role for pSer-591 to serve as the nuclear localization signal for SHP1, OSU-2S induced accumulation of pSer-591 SHP1 in the nuclear fraction. Interestingly, OSU-2S induced phosphorylation of Ser-591 SHP1 appears to be mediated through PKC dependent mechanism as activation of PKC with PMA induced phosphorylation of Ser-591 SHP1 and PKC inhibitor Bisindolylmaleimide inhibited OSU-2S induced Serine 591 phosphorylation of SHP1. Moreover, concentrations of Bisindolylmaleimide that prevented the SHP1 Ser-591 phosphorylation also partially rescued OSU-2S induced apoptosis of primary CLL B cells. Co-immunoprecipitation experiments revealed association of PP2A with pSer-591 SHP1 in CLL cells treated with OSU-2S indicating a potential crosstalk between these two phosphatases. Consistent with this hypothesis OSU-2S treatment resulted in activation of PP2A in primary CLL B cells. Further, administration of OSU-2S into Eμ-Tcl-1 transgenic mice with high circulating peripheral blood leukemic cells resulted in significant reduction in circulating CD19+CD5+ leukemic B cells indicating therapeutic efficacy. Ongoing studies on the biochemical basis of cross-talk between the Serine threonine (PP2A) and protein tyrosine (SHP1) phosphatases in response to OSU-2S treatment in CLL will be presented.This work was supported by LLS-Specialized Center of Research in Leukemia and NCI-Leukemia SPORE Grants Disclosures:No relevant conflicts of interest to declare.

The podosome marker protein Tks5 regulates macrophage invasive behavior.

Tks5 is a Src substrate and adaptor protein previously recognized for its regulation of cancer cell invasion through modulation of specialized adhesion structures called podosomes/invadopodia. Here we show for the first time that Tks5 localizes to the podosomes of primary macrophages, and that Tks5 protein levels increase concurrently with podosome deposition during the differentiation of monocytes into macrophages. Similar results are reported for model THP-1 cells, which differentiate into macrophages and form proteolytically active podosomes in response to a PKC signaling agonist (PMA) and with sensitivity to a PKC inhibitor (bisindolylmaleimide). Genetic manipulation of Tks5 expression (silencing and overexpression) in stable THP-1 cell lines does not independently alter this macrophage differentiation process. Nor do these cells lose the ability to focalize F-actin and its accessory proteins into podosome-like structures following PMA treatment. However, Tks5 directly controls podosome-associated gelatin degradation and invasion through collective changes in adhesion, chemotaxis, and the expression/proteolytic activity of MMP9. The Src family kinase-dependent phosphorylation of Tks5 is also implicated in the regulation of THP-1 macrophage invasive behavior. These results therefore define a previously unappreciated function of Tks5 signaling specific to the functional attributes of the macrophage podosome in adhesion, motility, and extracellular matrix-remodeling.

Klotho Regulates ETB Receptor Expression via the PKC/c‐Jun Pathway

Background Klotho is a newly-discovered anti-aging gene. We recently reported that klotho gene delivery prevented progression of spontaneous hypertension and renal damage. In vivo expression of klotho upregulated renal ETB receptors in spontaneous hypertensive rats. The purpose of this study was to determine the molecular mechanism of the regulation of ETB receptors by klotho in HEK 293 cells. Methods & Results Overexpression of klotho was achieved using AAV plasmid carrying the mouse klotho full-length cDNA (mKL). Silencing of klotho gene was achieved using small interfering RNA (siRNA). Protein expression of ETA and ETB receptors and c-Jun was measured by western blot. Nitric Oxide (NO) production was evaluated by flowcytometry using the DAF probe. Overexpression of klotho dose-dependently increased ETB protein expression and NO production in HEK 293 cells. RNAi knockdown of klotho resulted in a selective decrease in mRNA and protein expression of ETB. On the other hand, klotho gene delivery increased PKC activity and enhanced c-Jun expression. Blockade of PKC activation by a PKC inhibitor bisindolylmaleimide abolished the klotho-induced increases in ETB and c-Jun expression in HEK 293 cells. These results suggest that the upregulation of ETB by klotho may be PKC/c-Jun-dependent. Conclusions Klotho may regulate ETB expression via the PKC/c-Jun pathway in HEK 293 cells. This finding may offer new insights into the molecular mechanism of klotho-mediated renal protection.

Epac stimulation induces rapid increases in connexin43 phosphorylation and function without preconditioning effect

It has been recently shown that beta-adrenergic receptors are able to activate phospholipase C via the cyclic adenosine monophosphate-binding protein Epac. This new interconnection may participate in isoproterenol (Iso)-induced preconditioning. We evaluated here whether Epac could induce PKCepsilon activation and could play a role in ischemic preconditioning through the phosphorylation of connexin43 (Cx43) and changes in gap junctional intercellular communication (GJIC). In cultured rat neonatal cardiomyocytes, we showed that in response to Iso and 8-CPT, a specific Epac activator, PKCepsilon content was increased in particulate fractions of cell lysates independently of protein kinase A (PKA). This was associated with an increased Cx43 phosphorylation. Both Iso and 8-CPT induced an increase in GJIC that was blocked by the PKC inhibitor bisindolylmaleimide. Interestingly, inhibition of PKA partly suppressed both Iso-induced increases in Cx43 phosphorylation and in GJIC. The same PKCepsilon-dependent Cx43 phosphorylation by beta-adrenergic stimulation via Epac was found in adult rat hearts. However, in contrast with Iso that induced a preconditioning effect, perfusion of isolated hearts with 8-CPT prior to ischemia failed to improve the post-ischemia functional recovery. In conclusion, Epac stimulation induces PKCepsilon activation and Cx43 phosphorylation with an increase in GJIC, but Epac activation does not induce preconditioning to ischemia in contrast with beta-adrenergic stimulation.

Phenolic fraction of tobacco smoke condensate potentiates benzo[a]pyerene diol epoxide-induced cell transformation: Role of protein kinase C

In this study we separated weakly acidic phenolic components from other neutral, acidic and basic components of tobacco smoke condensate (TSC) and observed that phenolic fraction of TSC significantly increased the number of colonies of promotion-sensitive JB6 Cl41 cells that showed anchorage-independent growth on soft agar in response to BPDE (an ultimate carcinogen produced by metabolic activation of the PAH benzo[a]pyrene). Anchorage-independent cell growth is indicative of cell transformation resulting in acquisition of tumorigenic potential. In order to understand the underlying mechanism by which TSC phenolic fraction potentiates BPDE-induced tumorigenicity, we examined its effect on the activation of two transcription factors AP-1 and NF-κB which are known to be influenced by established tumor promoter TPA. BPDE treatment caused induction of both AP-1 and NF-κB activity as determined by luciferase reporter assay and only NF-κB induction in response to BPDE was significantly attenuated by TSC phenolic fraction whereas AP-1 induction remains unaltered. Attenuation of NF-κB activation by TSC phenolic fraction was associated with significant decrease of intracellular PKC substrate phosphorylation in BPDE treated cells. Non-specific PKC inhibitors staurosporine and bisindolylmaleimide II as well as inhibitors specific to conventional PKCs (Go6976) and PKC-δ (rottlerin) attenuated NF-κB activation in BPDE treated cells to a varying degree indicating a possible link between PKC down-regulation and the attenuation of NF-κB activity by TSC phenolic fraction. Treatment of cells with PKC inhibitors also potentiated anchorage-independent growth of BPDE treated cells on soft agar. Our data suggest a possible role of PKC down-regulation in potentiation of BPDE-induced tumorogenicity by TSC phenolic fraction.

Neurokinins inhibit low threshold inactivating K + currents in capsaicin responsive DRG neurons

Neurokinins (NK) released from terminals of dorsal root ganglion (DRG) neurons may control firing of these neurons by an autofeedback mechanism. In this study we used patch clamp recording techniques to determine if NKs alter excitability of rat L4-S3 DRG neurons by modulating K + currents. In capsaicin (CAPS)-responsive phasic neurons substance P (SP) lowered action potential (AP) threshold and increased the number of APs elicited by depolarizing current pulses. SP and a selective NK 2 agonist, [βAla 8]-neurokinin A (4–10) also inhibited low threshold inactivating K + currents isolated by blocking non-inactivating currents with a combination of high TEA, (−) verapamil and nifedipine. Currents recorded under these conditions were heteropodatoxin-sensitive (Kv4 blocker) and α-dendrotoxin-insensitive (Kv1.1 and Kv1.2 blocker). SP and NKA elicited a > 10 mV positive shift of the voltage dependence of activation of the low threshold currents. This effect was absent in CAPS-unresponsive neurons. The effect of SP or NKA on K + currents in CAPS-responsive phasic neurons was fully reversed by an NK 2 receptor antagonist (MEN10376) but only partially reversed by a PKC inhibitor (bisindolylmaleimide). An NK 1 selective agonist ([Sar 9, Met 11]-substance P) or direct activation of PKC with phorbol 12,13-dibutyrate, did not change firing in CAPS-responsive neurons, but did inhibit various types of K + currents that activated over a wide range of voltages. These data suggest that the excitability of CAPS-responsive phasic afferent neurons is increased by activation of NK 2 receptors and that this is due in part to inhibition and a positive voltage shift in the activation of heteropodatoxin-sensitive Kv4 channels.

Identification of bisindolylmaleimides and indolocarbazoles as inhibitors of HCV replication by tube-capture-RT-PCR

We devised a screening method for hepatitis C virus (HCV) inhibitors by exploiting the JFH1 viral culture system. The viral RNA released in the medium was adsorbed onto PCR plates, and real-time RT-PCR was performed by directly adding the one-step RT-PCR reaction mixture to the wells. The “tube-capture-RT-PCR” method obviates the need for labor-intensive RNA isolation and should allow high-throughput screening of HCV inhibitors. To substantiate the validity of the assay for drug screening, a pilot screen of an inhibitor library composed of 95 compounds was performed. In addition to the known inhibitors of HCV replication included in the library, the assay identified the PKC inhibitor bisindolylmaleimide I (BIM I) as an HCV replication inhibitor. BIM I was also effective in reducing the viral protein level in genotype 1b and 2a subgenomic replicon cells, indicating inhibition of HCV replication. Further assays revealed that a broad range of bisindolylmaleimides and indolocarbazoles inhibit HCV, but no correlation was found between the PKC inhibition pattern and anti-HCV activity. These series of compounds represent new classes of inhibitors that may warrant further development.

Parathyroid hormone synergizes with non‐cyclic AMP pathways to activate the cyclic AMP response element

Parathyroid hormone (PTH) activates multiple signaling pathways following binding to the PTH1 receptor in osteoblasts. Previous work revealed a discrepancy between cAMP stimulation and CRE reporter activation of truncated PTH peptides, suggesting that additional signaling pathways contribute to activation of the CRE. Using a CRE-Luciferase reporter containing multiple copies of the CRE stably transfected into the osteoblastic cell line Saos-2, we tested the ability of modulators of alternative pathways to activate the CRE or block the PTH-induced activation of the CRE. Activators of non-cyclic AMP pathways, that is, EGF (Akt, MAPK, JAK/STAT pathways); thapsigargin (intracellular calcium pathway); phorbol myristate acetate (protein kinase C, PKC pathway) induced minor increases in CRE-luciferase activity alone but induced dramatic synergistic effects in combination with PTH. The protein kinase A (PKA) inhibitor H-89 (10 microM) almost completely blocked PTH-induced activation of the CRE-reporter. Adenylate cyclase inhibitors SQ 22536 and DDA had profound and time-dependent biphasic effects on the CRE response. The MAPK inhibitor PD 98059 partially inhibited basal and PTH-induced CRE activity to the same degree, while the PKC inhibitor bisindolylmaleimide (BIS) had variable effects. The calmodulin kinase II inhibitor KN-93 had no significant effect on the response to PTH. We conclude that non-cAMP pathways (EGF pathway, calcium pathway, PKC pathway) converge on, and have synergistic effects on, the response of a CRE reporter to PTH.