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Recently we have shown that PKC beta1 and beta2 are able to inhibit the tyrosine kinase activity of the human insulin receptor (HIR). Now we have investigated whether a distinct PKC isoform might be involved in the inhibitory effect of TNF alpha on insulin signaling in HEK293 cells. TNF alpha induces a rapid translocation of the PKC isoform epsilon (TNF alpha 10(-9) M, maximal effect within 5-10 min) in rat-1 fibroblasts, while no effect occurred on other isoforms. Cotransfection of HIR with PKC epsilon did not significantly reduce the insulin stimulated receptor kinase activity; however, when cells were incubated with TNF alpha for 10 min (10(-9) M) a 62 +/- 17% (n = 5) inhibition of the insulin receptor kinase activity was observed which was significantly (P<0.01) higher than that observed in cells which were not transfected with PKC (32 +/- 11.5%, n = 5). The data suggest that translocation of PKC epsilon induced by TNF alpha enables this PKC isoform to interact with insulin signaling and to inhibit the insulin receptor kinase activity.

A large soluble N-terminal fragment of Alzheimer's disease amyloid precursor protein (secreted form of APP: APPs) is produced by constitutive processing in the middle of the amyloid beta-protein portion of APP. Recent studies indicate that the activation of endogenous protein kinase C (PKC) with phorbol ester raises the rate of secretion of APPs. We constructed rat fibroblast 3Y1 cells that stably overexpress PKC isoenzymes alpha, delta, or epsilon, and analyzed the amount of APPs released from these PKC transfectants. The levels of APPs released from 3Y1 cells overexpressing PKC alpha and -epsilon were higher than those from PKC delta-transfected and control cells expressing vector only. These results suggest that specific isoforms of PKC regulate the secretion of APPs through a signaling pathway.

R59022, a diacylglycerol kinase inhibitor, enhances the prostaglandin F2 alpha (PGF2 alpha)-induced diacylglycerol (DAG) synthesis in Swiss 3T3 cells. It also potentiates the PGF2 alpha-mediated protein kinase C (PKC)-dependent 80 kDa protein (80K) phosphorylation and initiation of DNA replication. R59022 enhances the PGF2 alpha mitogenic response by increasing the rate of entry into the S phase. Insulin does not cause 80K phosphorylation, and does not enhance its induction but it potentiates the PGF2 alpha mitogenic response. These results suggest that mitogenically triggered fluctuations in DAG content and PKC activity play a pivotal role in controlling the PGF2 alpha-induced DNA synthesis while insulin acts via a different mechanism.

The depolarisation-induced influx of 45Ca2+ into anterior pituitary tissue and GH3 cells through 'L'-type, nimodipine-sensitive channels was investigated. In anterior pituitary prisms, phorbol esters, activators of protein kinase C, caused an enhancement of K(+)-induced 45Ca2+ influx. However, in the GH3 anterior pituitary cell line, phorbol esters inhibited K(+)-induced 45Ca2+ influx. The modulation by phorbol esters in both tissues was stereo-specific and time- and concentration-dependent. The diacylglycerol analogue, 1,2-dioctanoyl sn-glycerol was able to mimic the phorbol ester-induced enhancement of calcium influx into anterior pituitary pieces, but was ineffective in GH3 cells. 1,2-Dioctanoyl sn-glycerol may selectively activate an isoform of protein kinase C which is responsible for enhanced 'L'-type Ca(2+)-channel activity.

Abstract: We isolated and characterized four new PKCδ isoforms, PKCδIV, δV, δVI, and δVII, specifically expressed in the mouse testis. These isoforms possess neither V1 nor C2-like domains. Moreover, PKCδVI and δVII have a different last exon as their V5 domain. The transcription of PKCδIV, δV, δVI, and δVII is initiated from the same site in the upstream region of exon4 of the PKCδ gene. They are expressed exclusively in the testis in an age-dependent manner. PKCδIV and δV are expressed in spermatids with sperm maturation stage-specific manner, and that PKCδVI and δVII are expressed in spermatogonia and spermatocytes. [Copyright &y& Elsevier]

Semaphorins (Semas) are a family of secreted and transmembrane proteins that play critical roles in development. Interestingly, several vertebrate transmembrane Sema classes are capable of producing functional soluble ectodomains. However, little is known of soluble Sema6 ectodomains in the nervous system. Herein, we show that the soluble Sema6A ectodomain, sSema6A, exhibits natural and protein kinase C (PKC)‐induced release. We show that PKC mediates Sema6A phosphorylation at specific sites and while this phosphorylation is not the primary mechanism regulating sSema6A production, we found that the intracellular domain confers resistance to ectodomain release. Finally, sSema6A is functional as it promotes the cohesion of zebrafish early eye field explants. This suggests that in addition to its canonical contact‐mediated functions, Sema6A may have regulated, long‐range, forward‐signaling capacity. [ABSTRACT FROM AUTHOR]

Abstract: Protein kinase C (PKC) is a regulator of apoptosis with both pro- and anti-apoptotic effects. The mechanistic basis for the discrepant effects is not completely understood. Here we show that Smac interacts with PKC. The interaction depends on the N-terminus of Smac and is disrupted upon treatment with paclitaxel. This is associated with release of Smac into the cytosol. Activation of PKC rescues the interaction during paclitaxel exposure and suppresses the paclitaxel-mediated cell death. However, under these conditions the complex is mainly found in the cytosol suggesting that cytosolic Smac can be bound by PKC when PKC is activated. The data unravel a previously unrecognized interaction and suggest that PKC by associating with Smac may prevent its apoptotic effects. Structured summary of protein interactions: PKC delta physically interacts with SMAC by anti bait coimmunoprecipitation (View Interaction: 1 , 2 , 3 , 4 ) XIAP physically interacts with SMAC by anti tag coimmunoprecipitation (View interaction) [Copyright &y& Elsevier]

Abstract: An acidic microenvironment induces disruption of adherens junctions (AJs) of hepatoma cells. This study investigated the impact of an acidic extracellular pH (pHe) on p120-catenin (p120-ctn) serine phosphorylation. pH 6.6 treatment increased intracellular calcium levels, activated protein kinase C (PKC)α and PKCδ, and decreased serine phosphorylation of p120-ctn. Further knockdown of PKCα and δ by small interference RNA (siRNA) prevented the pH 6.6-induced downregulation of p120-ctn at AJ and the serine dephosphorylation of p120-ctn. Moreover, PP2 pretreatment and siRNA of c-Src abrogated the pH 6.6-induced PKCδ activation. Together, the c-Src-PKCδ cascade and PKCα regulate the acidic pHe-induced AJ disruption. [Copyright &y& Elsevier]

Abstract: In this study, we investigated the role of protein kinase C (PKC) and mitochondrial permeability transition pore (mPTP) on the effect of ceramide in an in vitro model of ischemia in SH-SY5Y neuroblastoma cells. In ischemic cell viability studies, a dual effect of ceramide was observed, depending on ceramide concentration. PKC isoforms are involved in the protective effect of low concentrations of ceramide. During ischemia, ceramide treatment leads to an increase in the formation of reactive oxygen species (ROS), which induces a controlled opening of mPTP. This fact prevents mitochondrial Ca2+ overload, which is clearly protective. [ABSTRACT FROM AUTHOR]

Abstract: Adenosine (Ado), an endogenous nucleoside, can stimulate corticosterone synthesis in adrenal cells via the A2A/A2B adenosine receptors (ARs). This study evaluated the contribution of protein kinase C (PKC) isoforms in Ado-induced steroidogenesis. The PKC inhibitor calphostin c blocked Ado-induced steroidogenesis, the mitogen-activated protein kinase (MEK)-extracellular signal-related regulated kinase (ERK)-cyclic AMP responsive element-binding protein cascade, and the mRNA expression of steroidogenic acute regulatory protein and CYP11B1. Further analyses revealed that PKCμ was indeed activated by Ado. Moreover, downregulation of PKCμ by small interfering RNA (siRNA) inhibited Ado-stimulated steroidogenesis and ERK phosphorylation. Finally, inhibition of either A2AAR or A2BAR led to the suppression of PKCμ phosphorylation. Together, these findings suggest that A2AR-PKCμ-MEK signaling mediates Ado-stimulated adrenal steroidogenesis. [ABSTRACT FROM AUTHOR]

Abstract: The molecular mechanism of the anti-adipogenic effect of evodiamine (which has several capsaicin-like pharmacological actions) was investigated. The evodiamine effect was not blocked by the specific TRPV1 antagonist capsazepine in 3T3-L1 preadipocytes, whereas its effect was greatly curtailed by inhibitors of protein kinase C (PKC) and epidermal growth factor receptor (EGFR). Signal analyses showed that evodiamine stimulated the phosphorylation of EGFR, PKCα, and ERK, all of which were reduced by an EGFR inhibitor. Silencing experiments of EGFR mRNA supported the involvement of these signaling molecules in the inhibitory effect of evodiamine. An unidentified mechanism whereby evodiamine inhibits adipogenesis via the EGFR–PKCα–ERK signaling pathway was revealed. [Copyright &y& Elsevier]

Abstract: The complexity of the mammalian p53 pathway and protein kinase C (PKC) family has hampered the discrimination of the effect of PKC isoforms on p53 activity. Using yeasts co-expressing the human wild-type p53 and a mammalian PKC-α, -δ, -ε or -ζ, we showed a differential regulation of p53 activity and phosphorylation state by PKC isoforms. Whereas PKC-α reduced the p53-induced yeast growth inhibition and cell cycle arrest, PKC-δ and -ε enhanced the p53 activity through p53 phosphorylation, and PKC-ζ had no effect on p53. This work identified positive and negative p53 regulators which represent promising pharmacological targets in anti-cancer therapy. [Copyright &y& Elsevier]

Abstract: We recently reported that diacylglycerol kinase (DGK) α enhanced tumor necrosis factor-α (TNF-α)-induced activation of nuclear factor-κB (NF-κB). However, the signaling pathway between DGKα and NF-κB remains unclear. Here, we found that small interfering RNA-mediated knockdown of DGKα strongly attenuated protein kinase C (PKC) ζ-dependent phosphorylation of a large subunit of NF-κB, p65/RelA, at Ser311 but not PKCζ-independent phosphorylation at Ser468 or Ser536. Moreover, knockdown and overexpression of PKCζ suppressed and synergistically enhanced DGKα-mediated NF-κB activation, respectively. These results strongly suggest that DGKα positively regulates TNF-α-dependent NF-κB activation via the PKCζ-mediated Ser311 phosphorylation of p65/RelA. [Copyright &y& Elsevier]

Abstract: Integrin avidity is regulated by changes in the conformation of the heterodimer and cluster formation. We measured cell adhesion by integrin α2β1 (CHO-α2) to collagen at short contact times (0.5–60s) by single cell force spectroscopy (SCFS). The adhesion increased rapidly with contact time and was further strengthened by the addition of 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) and integrin activator. TPA also improved the strength of adhesive units. Furthermore, changes in membrane nanotube properties indicated better coupling of integrins to the cell cytoskeleton. We conclude that in addition to increasing integrin avidity TPA strengthens integrin–cytoskeletal linkage. [Copyright &y& Elsevier]

Abstract: Adiponectin, one of the adipokines secreted by adipocytes, possesses insulin sensitizing and anti-atherosclerotic properties. Tumor necrosis factor-alpha (TNF-α) is known to suppress the expression and secretion of adiponectin in adipocytes; however, the underlying mechanism remains poorly understood. Here, we demonstrate that GO6976 (a selective inhibitor of conventional protein kinase C (PKC)) prevents TNF-α-induced suppression of adiponectin secretion and expression in fully differentiated 3T3-L1 adipocytes, accompanied by attenuation of c-Jun N-terminal kinase (JNK) activation. Additionally, the transcriptional activity of peroxisome proliferator-activated receptor-gamma (PPARg) (a strong inducer of adiponectin) on the adiponectin promoter was inhibited in a PKC isoform-specific manner. These results raise the possibility that PKC is involved in TNF-α-induced suppression of adiponectin in 3T3-L1 adipocytes. [Copyright &y& Elsevier]

Abstract: Ras association domain family 1A (RASSF1A) is a tumour suppressor that contains an amino-terminal cysteine-rich region, similar to the diacylglycerol (DAG)-binding domain (C1 domain) found in the protein kinase C (PKC) family of proteins, and a carboxy-terminal Ras-association (RA) domain. In the present study, RASSF1A was identified as a substrate for PKC. Using classical biochemical approaches, it was established that S197 and S203 within the RA domain of RASSF1A are phosphorylated by PKC in vitro and in vivo. Unlike the WT protein, the S197, 203D double mutant of RASSF1A failed to modulate microtubule organization and perinuclear vimentin collapse. By contrast, the equivalent AA mutant of RASSF1A phenocopied the WT protein. These findings indicate that PKC phosphorylation of RASSF1A regulates its ability to reorganize the microtubule network. [Copyright &y& Elsevier]

Abstract: The goal of the current study, conducted in freshly isolated thymocytes was (1) to investigate the possibility that the activation of poly(ADP-ribose) polymerase-1 (PARP-1) in an intact cell can be regulated by protein kinase C (PKC) mediated phosphorylation and (2) to examine the consequence of this regulatory mechanism in the context of cell death induced by the genotoxic agent. In cells stimulated by the PKC activating phorbol esters, DNA breakage was unaffected, PARP-1 was phosphorylated, 1-methyl-3-nitro-1-nitrosoguanidine-induced PARP activation and cell necrosis were suppressed, with all these effects attenuated by the PKC inhibitors GF109203X or Gö6976. Inhibition of cellular PARP activity by PKC-mediated phosphorylation may provide a plausible mechanism for the previously observed cytoprotective effects of PKC activators. [Copyright &y& Elsevier]

Abstract: Phosphoinoisitide dependent kinase l (PDK1) is proposed to phosphorylate a key threonine residue within the catalytic domain of the protein kinase C (PKC) superfamily that controls the stability and catalytic competence of these kinases. Hence, in PDK1-null embryonic stem cells intracellular levels of PKCα, PKCβ1, PKCγ, and PKCε are strikingly reduced. Although PDK1-null cells have reduced endogenous PKC levels they are not completely devoid of PKCs and the integrity of downstream PKC effector pathways in the absence of PDK1 has not been determined. In the present report, the PDK1 requirement for controlling the phosphorylation and activity of a well characterised substrate for PKCs, the serine kinase protein kinase D, has been examined. The data show that in embryonic stem cells and thymocytes loss of PDK1 does not prevent PKC-mediated phosphorylation and activation of protein kinase D. These results reveal that loss of PDK1 does not functionally inactivate all PKC-mediated signal transduction. [Copyright &y& Elsevier]

Abstract: Doxorubicin (DOX) transport activity of Ral-interacting protein (RLIP76) in non-small cell lung cancer (NSCLC) is approximately twice that of in small cell lung cancer (SCLC). Since protein-kinase-C (PKC)α mediated phosphorylation of RLIP76 causes doubling of the specific activity of RLIP76, and NSCLC cells are known to have greater PKCα activity, we examined the contribution of PKC mediated phosphorylation of RLIP76 towards intrinsic DOX-resistance in human NSCLC. Expression of a deletion mutant RLIP76delPKCα-sites followed by depletion of the wild-type RLIP76 using a siRNA targeted at one of the deleted regions resulted in generation of cells expressing only the mutant protein, which could not be phosphorylated by PKCα. DOX-transport activity of the mutant RLIP76 purified from NSCLC and SCLC was similar and comparable to that of RLIP76 purified from the wild-type SCLC. However, this activity was significantly lower than that of RLIP76 purified from the wild-type NSCLC. After siRNA mediated depletion of PKCα, DOX-transport activities of RLIP76 purified from SCLC and NSCLC were indistinguishable. Depletion of PKCα inhibited the growth of NSCLC more than SCLC cells (70±3% vs. 43±5%, respectively). PKCα-depletion lowered the IC50 of NSCLC cell lines for DOX to the same level as that observed for SCLC. RLIP76−/− mouse embryonic fibroblasts (MEFs) were significantly more sensitive to DOX as compared with RLIP76+/+ MEFs (IC50 25 vs. 125nM, respectively). However, PKCα-depletion did not affect DOX-cytotoxicity towards RLIP76−/− MEFs, as opposed to RLIP76+/+ MEFs which were sensitized by 2.2-fold. These results demonstrate that RLIP76 is a primary determinant of DOX-resistance, and that PKCα mediated accumulation defect and DOX-resistance in NSCLC is primarily due to differential phosphorylation of RLIP76 in SCLC and NSCLC. [Copyright &y& Elsevier]

Abstract: The cysteine in the M/IXXCW motif is conserved in all but one (threonine in place of cysteine) of the human protein tyrosine kinases (PTKs). We showed that all RET-PTC-1 mutants in which the C in this motif (C376) was replaced with glycine, lysine, threonine or serine lost their activity in vitro. However, the C376T/S mutants showed normal tyrosine phosphorylation in vivo (in cells). Further analyses reveled that protein kinase C (PKC) initiated the activities of the C376T/S mutants in cells. We conclude that the M/IXXCW motif-mediated mechanisms which initiate PTK activities are partially replaced by a PKC-mediated mechanism. [Copyright &y& Elsevier]

Abstract: Osteoblastic differentiation is an essential part of bone formation. Dimethyl sulfoxide (DMSO) is a water miscible solvent that is used extensively for receptor ligands in osteoblast studies. However, little is known about its effects on osteoblastogenic precursor cells. In this study, we have used a murine preosteoblast cell line MC3T3-E1 cells to demonstrate that DMSO effectively induces osteoblastic differentiation of MC3T3-E1 cells via the activation of Runx2 and osterix and is dependent upon the protein kinase C (PKC) pathways. We further demonstrated that prolonged activation of PKC pathways is sufficient to induce osteoblastic differentiation, possibly via the activation of PKD/PKCμ. [Copyright &y& Elsevier]

In this study, we investigated the role of calcium/calmodulin‐dependent protein kinase II (CaMKII) in contraction‐stimulated glucose uptake in skeletal muscle. C2C12 myotubes were contracted by electrical pulse stimulation (EPS), and treadmill running was used to exercise mice. The activities of CaMKII, the small G protein Rac1, and the Rac1 effector kinase PAK1 were elevated in muscle by running exercise or EPS, while they were lowered by the CaMKII inhibitor KN‐93 and/or small interfering RNA (siRNA)‐mediated knockdown. EPS induced the mRNA and protein expression of the Rac1‐GEF Kalirin in a CaMKII‐dependent manner. EPS‐induced Rac1 activation was lowered by the Kalirin inhibitor ITX3 or siRNA‐mediated Kalirin knockdown. KN‐93, ITX3, and siRNA‐mediated Kalirin knockdown reduced EPS‐induced glucose uptake. These findings define a CaMKII‐Kalirin‐Rac1 signaling pathway that contributes to contraction‐stimulated glucose uptake in skeletal muscle myotubes and tissue. [ABSTRACT FROM AUTHOR]

GPCR kinase 2 (GRK2)/β-arrestins and protein kinase A (PKA)/protein kinase C (PKC) mediate homologous and heterologous regulations of GPCRs, respectively. Conventional protein kinase C enzymes (PKCs), as exemplified by PKCβII, selectively inhibit internalization of dopamine D 2 receptor and β 2 adrenoceptor in a β-arrestin- but not GRK2-dependent manner. PKCβII interacts with β-arrestin2 upon autophosphorylation at T250, and inhibits the receptor internalization by decreasing the ubiquitination of β-arrestin2. PKCβII interferes with the interaction between β-arrestin2 and MDM2 in the cytosol, resulting in the redistribution of MDM2 to the nucleus. Subsequently, deubiquitination of β-arrestin2 and inhibition of agonist-induced receptor internalization follow. Thus, our study suggests that the extent of β-arrestin ubiquitination and the autophosphorylation status of PKCs determine PKCβII-mediated inhibition of homologous regulatory processes of GPCRs. [ABSTRACT FROM AUTHOR]

Previous studies have shown that amyloid‐β oligomers (AβO) bind with high affinity to cellular prion protein (PrPC). The AβO‐PrPC complex binds to cell‐surface co‐receptors, including the laminin receptor (67LR). Our current studies revealed that in Neuroscreen‐1 cells, 67LR is the major co‐receptor involved in the cellular uptake of AβO and AβΟ‐induced cell death. Both pharmacological (dibutyryl‐cAMP, forskolin and rolipram) and physiological (pituitary adenylate cyclase‐activating polypeptide) cAMP‐elevating agents decreased cell‐surface PrPC and 67LR, thereby attenuating the uptake of AβO and the resultant neuronal cell death. These cAMP protective effects are dependent on protein kinase A, but not dependent on the exchange protein directly activated by cAMP. Conceivably, cAMP protects neuronal cells from AβO‐induced cytotoxicity by decreasing cell‐surface‐associated PrPC and 67LR. [ABSTRACT FROM AUTHOR]

Resurgent sodium currents likely play a role in modulating neuronal excitability. Here we studied whether protein kinase C (PKC) activation can increase resurgent currents produced by the human sodium channel hNav1.7. We found that a PKC agonist significantly enhanced hNav1.7-mediated resurgent currents and this was prevented by PKC antagonists. The enhancing effects were replicated by two phosphorylation-mimicking mutations and were prevented by a phosphorylation-deficient mutation at a conserved PKC phosphorylation site (Serine 1479). Our results suggest that PKC can increase sodium resurgent currents through phosphorylation of a conserved Serine residue located in the domain III–IV linker of sodium channels. [ABSTRACT FROM AUTHOR]

Hypertrophic cardiomyopathy (HCM), a disease characterized by cardiac muscle hypertrophy and hypercontractility, is the most frequently inherited disorder of the heart. HCM is mainly caused by variants in genes encoding proteins of the sarcomere, the basic contractile unit of cardiomyocytes. The most frequently mutated among them is MYBPC3, which encodes cardiac myosin-binding protein C (cMyBP-C), a key regulator of sarcomere contraction. In this review, we summarize clinical and genetic aspects of HCM and provide updated information on the function of the healthy and HCM sarcomere, as well as on emerging therapeutic options targeting sarcomere mechanical activity. Building on what is known about cMyBP-C activity, we examine different pathogenicity drivers by which MYBPC3 variants can cause disease, focussing on protein haploinsufficiency as a common pathomechanism also in nontruncating variants. Finally, we discuss recent evidence correlating altered cMyBP-C mechanical properties with HCM development. [ABSTRACT FROM AUTHOR]

Abstract: Members of the protein kinase C (PKC) family of serine/threonine kinases have been implicated in several physiological processes regulating the activation response of platelets. They are involved in processes leading to granule secretion, integrin activation, platelet aggregation and spreading, and procoagulation. The protein kinase C θ (PKCθ) isoform, which was originally identified in T lymphocytes, is also expressed at relatively high levels in platelets, wherein it is involved in the regulation of hemostasis and thrombosis. Recent studies suggest a role for PKCθ in protease-activated receptor (PAR)-, glycoprotein VI (GPVI) receptor- and glycoprotein αIIbβ3 integrin receptor-linked signal transduction pathways. The present review focuses on the latest observations relevant to the role of PKCθ in platelet activation. [Copyright &y& Elsevier]

Abstract: Platelets tightly regulate haemostasis and arterial thrombosis. Protein kinase C (PKC) is involved in most platelet responses implicated in thrombus formation. Recent pharmacological and mouse gene knockout approaches show that the conventional PKC isoforms and the novel PKC isoforms contribute in distinct ways to these platelet responses. We hypothesize that, in platelets and other cells, the characteristic functions of PKC isoforms are established through unique activation mechanisms and unique interacting protein partners, which result in isoform-specific patterns of substrate phosphorylation. For identifying the substrate proteins in a living cell, new methodology is available and discussed. [Copyright &y& Elsevier]

Abstract: We tested the hypothesis that activation of the polyol pathway and protein kinase C (PKC) during diabetes is due to loss of NO. Our results show that after 4 weeks of streptozotocin-induced diabetes, treatment with l-arginine restored NO levels and prevented tissue accumulation of sorbitol in mice, which was accompanied by an increase in glutathiolation of aldose reductase. l-Arginine treatment decreased superoxide generation in the aorta, total PKC activity and PKC-βII phosphorylation in the heart, and the plasma levels of triglycerides and soluble ICAM. These data suggest that increasing NO bioavailability by l-arginine corrects the major biochemical abnormalities of diabetes. [Copyright &y& Elsevier]

Abstract: Many studies have indicated that differentiated cells inhibit drug-induced cytotoxicity but undifferentiated cells do not, though the mechanisms are unclear. Currently, HL-60 cells are induced to differentiate into macrophage-like cells with Phorbol-12-myristate-13-acetate (TPA) treatment (TPA-differentiated cells). Our study shows that caspase-9/-3-mediated cytotoxicity can be induced in undifferentiated HL-60 cells but not in TPA-differentiated HL-60 cells. However, caspase-9/-3-mediated cytotoxicity can be induced in TPA-differentiated cells if they are pretreated with a protein kinase C (PKC) or a mitogen activated protein kinase (MEK) inhibitor. Taken together, this study demonstrates that TPA-differentiated HL-60 cells inhibit caspases-9/-3-mediated cytotoxicity through the PKC and MEK signaling pathways. [Copyright &y& Elsevier]

Abstract: We previously showed that endothelin-1 (ET-1) stimulates the synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells, and that protein kinase C (PKC)-dependent p44/p42 mitogen-activated protein (MAP) kinase plays a part in the IL-6 synthesis. In the present study, we investigated the effect of (−)-epigallocatechin gallate (EGCG), one of the major flavonoids containing in green tea, on ET-1-induced IL-6 synthesis in osteoblasts and the underlying mechanism. EGCG significantly reduced the synthesis of IL-6 stimulated by ET-1 in MC3T3-E1 cells as well primary cultured mouse osteoblasts. SB203580, a specific inhibitor of p38 MAP kinase, but not SP600125, a specific SAPK/JNK inhibitor, suppressed ET-1-stimulated IL-6 synthesis. ET-1-induced phosphorylation of p38 MAP kinase was not affected by EGCG. On the other hand, EGCG suppressed the phosphorylation of p44/p42 MAP kinase induced by ET-1. Both the IL-6 synthesis and the phosphorylation of p44/p42 MAP kinase stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA), a direct activator of PKC, were markedly suppressed by EGCG. The phosphorylation of MEK1/2 and Raf-1 induced by ET-1 or TPA were also inhibited by EGCG. These results strongly suggest that EGCG inhibits ET-1-stimulated synthesis of IL-6 via suppression of p44/p42 MAP kinase pathway in osteoblasts, and the inhibitory effect is exerted at a point between PKC and Raf-1 in the ET-1 signaling cascade. [Copyright &y& Elsevier]

Abstract: Expression of the telomerase catalytic subunit (TERT) is the rate-limiting determinant of telomerase activity in most human cells. In this work, we examined the participation of protein kinase C (PKC) in the regulation of hTERT expression in human T lymphocytes. Transient expression assays using luciferase reporter plasmids containing hTERT promoter showed that overexpression of PKC θ, but not the other PKC isoforms, could activate the promoter activity of hTERT in resting T lymphocytes. Among the PKC θ-activated signalings, we presented evidence that the expression of hTERT is mediated through NFκB but not through MEK or c-Jun N-terminal kinase pathways. Analysis of the hTERT promoter occupancy in vivo using chromatin immunoprecipitation assays, however, did not detect an increased binding of NFκB to the hTERT promoter in the activated T cells, although an increased binding of cMyc and Sp1 was detected. Together with the observation that inhibition of NFκB eliminated the induction of cMyc in activated T cells, these results suggest that PKC θ-activated NFκB signaling regulates the expression of hTERT via cMyc in human T lymphocytes. [Copyright &y& Elsevier]

Abstract: Small cell lung cancer (SCLC) cells migration across human brain microvascular endothelial cells (HBMECs) is an essential step of brain metastases. Here we investigated signalling pathways in HBMECs contributing to the process. Inhibition of endothelial Rho kinase (ROCK) with Y27632 and overexpression of ROCK dominant-negative mutant prevented SCLC cells, NCI-H209, transendothelial migration and the concomitant changes of tight junction. Conversely, inhibition of phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) had no effects. Furthermore, endothelial RhoA protein was activated during NCI-H209 cells transendothelial migration. Rho/ROCK participated in NCI-H209 cells transendothelial migration through regulating actin cytoskeleton reorganization. These results suggested that Rho/ROCK was required for SCLC cells transendothelial migration. [Copyright &y& Elsevier]

Abstract: Vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are key angiogenic stimulators during normal development and wound healing, as well as in a variety of pathological conditions. Recent studies have demonstrated a synergistic effect of VEGF and PlGF in pathological angiogenesis and suggest a role for PlGF in amplifying VEGF action in endothelial cells. We show here in the mouse model of oxygen-induced retinopathy that VEGF is significantly increased (P &#60;0.01) in the retina at both the mRNA and protein levels. In this mouse model, PlGF was significantly upregulated in the retina at the protein level (P &#60;0.01) without a corresponding change in mRNA levels. In cultured human retinal and umbilical vein endothelial cells, VEGF induced the production of PlGF protein by over 10-fold (P &#60;0.01) in a dose-dependent manner through a post-transcriptional mechanism. The increased PlGF expression upon VEGF treatment was significantly reduced by inhibition of the protein kinase C (PKC) and MEK signaling pathways, as well as by treatment with the calcium ionophore A23187. Taken together, our findings demonstrate that VEGF can amplify its effects on endothelial cells by inducing the production of PlGF via a post-transcriptional mechanism in a PKC-dependent manner, and provide a potential link between PKC inhibition and amelioration of vascular complications in the development of angiogenic diseases. [Copyright &y& Elsevier]