Overexpression of Par‐4 sensitizes TRAIL‐induced apoptosis via inactivation of NF‐κB and Akt signaling pathways in renal cancer cells

The prostate‐apoptosis‐response‐gene‐4 (Par‐4) is up‐regulated in prostate cells undergoing programmed cell death. Furthermore, Par‐4 protein has been shown to function as an effector of cell death in response to various apoptotic stimuli that trigger mitochondria and membrane receptor‐mediated cell death pathways. In this study, we investigated how Par‐4 modulates TRAIL‐mediated apoptosis in TRAIL‐resistant Caki cells. Par‐4 overexpressing cells were strikingly sensitive to apoptosis induced by TRAIL compared with control cells. Par‐4 overexpressing Caki cells treated with TRAIL showed an increased activation of the initiator caspase‐8 and the effector caspase‐3, together with an enforced cleavage of XIAP and c‐FLIP. TRAIL‐induced reduction of XIAP and c‐FLIP protein levels in Par‐4 overexpressing cells was prevented by z‐VAD pretreatment. In addition, the surface DR5 protein level was increased in TRAIL‐treated Par‐4 overexpressing cells. Interestingly, even though a deletion of leucine zipper domain in Par‐4 recovered Bcl‐2 level to basal level induced by wild type Par‐4, it partly decreased sensitivity to TRAIL in Caki cells. In addition, exposure of Caki/Par‐4 cells to TRAIL led to reduction of phosphorylated Akt levels, but deletion of leucine zipper domain of Par‐4 did not affect these phosphorylated Akt levels. In conclusion, we here provide evidence that ectopic expression of Par‐4 sensitizes Caki cells to TRAIL via modulation of multiple targets, including DR5, Bcl‐2, Akt, and NF‐κB. J. Cell. Biochem. 109: 885–895, 2010. © 2010 Wiley‐Liss, Inc.     

Regulation of cadmium-induced apoptosis by PKCδ in U937 human promonocytic cells

Pulse treatment with cadmium chloride followed by recovery caused apoptosis in U937 human promonocytic cells. In addition, the treatment-induced PKCδ translocation from cytosol to membrane fraction, which was already detected at 30 min of treatment; and also caused PKCδ cleavage to give a 41-kDa fragment, which was detected at 3–6 h of recovery, concomitantly with the execution of apoptosis. All these effects were reduced by the PKCδ-specific inhibitor rottlerin. By contrast, rottlerin did not prevent the cadmium-provoked stimulation of the stress response (as measured by HSP70 expression), nor inhibited the generation of apoptosis by heat-shock, which failed to cause PKCδ translocation. Cadmium chloride rapidly induced p38^MAPK activation, which was not affected by rottlerin. By contrast, the p38^MAPK inhibitor SB203580 reduced PKCδ translocation and cleavage, indicating that p38^MAPK activation precedes and regulates PKCδ activation. It is concluded that PKCδ mediates apoptosis induction by cadmium ions via early membrane translocation, and also possibly through late kinase proteolytic cleavage and phosphorylation on tyrosine residues.     

Rottlerin inhibits migration of follicular thyroid carcinoma cells by PKCδ‐independent destabilization of the focal adhesion complex

This study examined the effect of rottlerin on the focal adhesion‐mediated cell migration of CGTH W‐2 human follicular thyroid carcinoma cells. Rottlerin (10 µM) resulted in decreased adhesion of CGTH W‐2 cells to matrix substance, which was correlated with metastatic potential. Rottlerin treatment also resulted in a marked reduction in the migration of CGTH W‐2 cells. Protein levels of integrin β1, FAK, and paxillin were decreased by rottlerin. Consistent with this, immunostaining of FAK, vinculin, and paxillin revealed disassembly of the focal adhesions. Disruption of actin stress fibers was noted, which was compatible with reduced expression levels and activities of Rac‐1 and Rho. The effect of rottlerin on cell migration was not attributable to inhibition of PKCδ activity since siRNA knockdown of PKCδ did not recapitulate the effects of rottlerin on cell adhesion and migration. Furthermore, activation of PKCδ by phorbol esters failed to restore the rottlerin‐inhibited migratory ability. The mitochondrial uncoupler, carbonylcyanide‐4‐(trifluoromethoxy)‐phenylhydrazone, was able to mimic several rottlerin's effects. In summary, we demonstrated that rottlerin inhibits the migration of CGTH W‐2 cells by disassembly of focal adhesion complexes in a PKCδ‐independent manner, and might play as a mitochondrial uncoupler role in these events. J. Cell. Biochem. 110: 428–437, 2010. © 2010 Wiley‐Liss, Inc.     

Mitochondrial PKC‐ε deficiency promotes I/R‐mediated myocardial injury via GSK3β‐dependent mitochondrial permeability transition pore opening

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)‐induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase‐2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross‐clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2^?/?) and wild‐type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin‐related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N‐acetylcysteine (NAC) or PKC‐δ shRNA treatment on glycogen synthase kinase‐3β (GSK‐3β) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2^?/? mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC‐ε translocation was lower in ALDH2^?/? mice than in WT mice, and PKC‐δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre‐treatment under I/R injury. In addition, PKC‐ε inhibition caused activation of caspase9, caspase3 and Drp1Ser⁶¹⁶ in response to I/R stress. Importantly, expression of phosphorylated GSK‐3β (inactive form) was lower in ALDH2^?/? mice than in WT mice, and both were increased by NAC pre‐treatment. I/R‐induced mitochondrial translocation of GSK‐3β was inhibited by PKC‐δ shRNA or NAC pre‐treatment. In addition, mitochondrial membrane potential (?Ψ_m) was reduced in ALDH2^?/? mice after I/R, which was partly reversed by the GSK‐3β inhibitor (SB216763) or PKC‐δ shRNA. Collectively, our data provide the evidence that abnormal PKC‐ε/PKC‐δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK‐3β‐dependent mPTP opening, which results in mitochondrial injury‐triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2^?/? mice following I/R stress.     

Heterogeneity in apoptotic responses of microvascular endothelial cells to oxidative stress

Oxidative stress contributes to disease and can alter endothelial cell (EC) function. EC from different vascular beds are heterogeneous in structure and function, thus we assessed the apoptotic responses of EC from lung and heart to oxidative stress. Since protein kinase Cδ (PKCδ) is activated by oxidative stress and is an important modulator of apoptosis, experiments assessed the level of apoptosis in fixed lung and heart sections of PKCδ wild-type (PKCδ(+/+)) and null (PKCδ(-/-)) mice housed under normoxia (21% O(2)) or hyperoxia (~95% O(2)). We noted a significantly greater number of TUNEL-positive cells in lungs of hyperoxic PKCδ(+/+) mice, compared to matched hearts or normoxic organs. We found that 33% of apoptotic cells identified in hyperoxic lungs of PKCδ(+/+) mice were EC, compared to 7% EC in hyperoxic hearts. We further noted that EC apoptosis was significantly reduced in lungs of PKCδ(-/-) hyperoxic mice, compared to lungs of PKCδ(+/+) hyperoxic mice. In vitro, both hyperoxia and H(2)O(2) promoted apoptosis in EC isolated from microvasculature of lung (LMVEC), but not from the heart (HMVEC). H(2)O(2) treatment significantly increased p38 activity in LMVEC, but not in HMVEC. Inhibition of p38 attenuated H(2)O(2)-induced LMVEC apoptosis. Baseline expression of total PKCδ protein, as well as the caspase-mediated, catalytically active PKCδ cleavage fragment, was higher in LMVEC, compared to HMVEC. PKCδ inhibition significantly attenuated H(2)O(2)-induced LMVEC p38 activation. Conversely, overexpression of wild-type PKCδ or the catalytically active PKCδ cleavage product greatly increased H(2)O(2)-induced HMVEC caspase and p38 activation. We propose that enhanced susceptibility of lung EC to oxidant-induced apoptosis is due to increased PKCδ→p38 signaling, and we describe a PKCδ-centric pathway which dictates the differential response of EC from distinct vascular beds to oxidative stress.     

Activation of PKCδ and ERK1/2 in the sensitivity to UV-induced apoptosis of human cells harboring 4977 bp deletion of mitochondrial DNA

The 4977 bp deletion of mitochondrial DNA (mtDNA), often found in patients with chronic progressive external ophthalmoplegia (CPEO), has been demonstrated to increase the susceptibility to apoptosis of human cells. We investigated the mechanism underlying the apoptotic susceptibility of the Δ4977 cybrid harboring about 80% 4977 bp-deleted mtDNA. The production of hydrogen peroxide (H₂O₂) and phosphorylation of PKCδ and ERK1/2 were increased in the Δ4977 cybrid, which was more susceptible to UV-induced apoptosis. Moreover, treatment with N-acetyl-l-cysteine (NAC) or blocking of activation of PKCδ by rottlerin or PKCδ-siRNA, and inhibition of ERK1/2 by PD98059 or ERK1/2-siRNA significantly attenuated the susceptibility of the Δ4977 cybrid to apoptosis. Furthermore, the increase of PKCδ expression in the Δ4977 cybrid also amplified the apoptotic signal through caspase 3-mediated proteolytic activation of PKCδ. In addition, PKCδ and ERK1/2 were hyperphosphorylated in skin fibroblasts of CPEO patients harboring 4977 bp-deleted mtDNA. We suggest that the activation of PKCδ and ERK1/2 elicited by 4977 bp-deleted mtDNA-induced oxidative stress plays a role in the susceptibility of the mutant cells to apoptosis. This may explain, at least in part, the degenerative manifestation of brain and muscle in patients with mitochondrial encephalomyopathies such as CPEO syndrome.     

NDRG1 is down‐regulated in the early apoptotic event induced by camptothecin analogs: The potential role in proteolytic activation of PKCδ and apoptosis

We previously reported that NSC606985, a new camptothecin analog, induces apoptosis of acute myeloid leukemic cells, which is triggered by proteolytic activation of protein kinase C delta (PKCδ). Here, we performed quantitative proteomic analysis of NSC606985‐treated and untreated leukemic U937 cells with two‐dimensional fluorescence difference gel electrophoresis (2‐D DIGE) in combination with matrix‐assisted laser desorption/ionization time‐of‐flight/time‐of‐flight tandem mass spectrometry. Thirty‐three proteins were found to be deregulated. Then, we focused on N‐myc downstream regulated gene 1 (NDRG1) down‐regulated during apoptosis induction. The results demonstrated that the down‐regulation of NDRG1 protein but not its mRNA was an early event prior to proteolytic activation of PKCδ in U937 cells under treatments of NSC606985 as well as other camptothecin analogs. With the ectopic expression of NDRG1, the proteolytic activation of PKCδ in NSC606985‐treated leukemic cells was delayed and the cells were less sensitive to apoptosis. On the contrary, the suppression of NDRG1 expression by specific small interfering RNA significantly enhanced NSC606985‐induced activation of PKCδ and apoptosis of U937 cells. In summary, our study suggests that the down‐regulation of NDRG1 is involved in proteolytic activation of PKCδ during apoptosis induction, which would shed new light on the understanding the apoptotic process initiated by camptothecin.     

Protein kinase Cδ but not PKCα is involved in insulin-induced glucose metabolism in hepatocytes

The liver is a major insulin‐responsive tissue responsible for glucose regulation. One important mechanism in this phenomenon is insulin‐induced glycogen synthesis. Studies in our laboratory have shown that protein kinase Cs delta (PKCδ) and alpha (α) have important roles in insulin‐induced glucose transport in skeletal muscle, and that their expression and activity are regulated by insulin. Their importance in glucose regulation in liver cells is unclear. In this study we investigated the possibility that these isoforms are involved in the mediation of insulin‐induced glycogen synthesis in hepatocytes. Studies were done on rat hepatocytes in primary culture and on the AML‐12 (alpha mouse liver) cell line. Insulin increased activity and tyrosine phosphorylation of PKCδ within 5 min. In contrast, activity and tyrosine phosphorylation of PKCα were not increased by insulin. PKCδ was constitutively associated with IR, and this was increased by insulin stimulation. Suppression of PKCδ expression by transfection with RNAi, or overexpression of kinase dead (dominant negative) PKCδ reduced both the insulin‐induced activation of PKB/Akt and the phosphorylation of glycogen synthase kinase 3 (GSK3) and reduced significantly insulin‐induced glucose uptake. In addition, treatment of primary rat hepatocytes with rottlerin abrogated insulin‐induced increase in glycogen synthesis. Neither overexpression nor inhibition of PKCα appeared to alter activation of PKB, phosphorylation of GSK3 or glucose uptake in response to insulin. We conclude that PKCδ, but not PKCα, plays an essential role in insulin‐induced glucose uptake and glycogenesis in hepatocytes. J. Cell. Biochem. 113: 2064–2076, 2012. © 2012 Wiley Periodicals, Inc.     

Caspase‐3‐mediated cyclic stretch‐induced myoblast apoptosis via a Fas/FasL‐independent signaling pathway during myogenesis

Skeletal muscle cells are exposed to mechanical stretch during embryogenesis. Increased stretch may contribute to cell death, and the molecular regulation by stretch remains incompletely understood. The aim of this study was to investigate the effects of cyclic stretch on cell death and apoptosis in myoblast using a Flexercell Strain Unit. Apoptosis was studied by annexin V binding and PI staining, DNA size analysis, electron microphotograph, and caspase assays. Fas/FasL expression was determined by Western blot. When myoblasts were cultured on a flexible membrane and subjected to cyclic strain stress, apoptosis was observed in a time‐dependent manner. We also determined that stretch induced cleavage of caspase‐3 and increased caspase‐3 activity. Caspase‐3 inhibition reduced stretch‐induced apoptosis. Protein levels of Fas and FasL remained unchanged. Our findings implicated that stretch‐induced cell death is an apoptotic event, and that the activation of caspase cascades is required in stretch‐induced cell apoptosis. Furthermore, we had provided evidence that caspase‐3 mediated cyclic stretch‐induced myoblast apoptosis. Mechanical forces induced activation of caspase‐3 via signaling pathways independent of Fas/FasL system. J. Cell. Biochem. 107: 834–844, 2009. © 2009 Wiley‐Liss, Inc.     

Role of calpain-9 and PKC-δ in the apoptotic mechanism of lumen formation in CEACAM1 transfected breast epithelial cells

CEACAM1-4S (carcinoembryonic antigen-related cell adhesion molecule 1) is a type I membrane protein with a short (12-amino acid) cytoplasmic tail. Wild type CEACAM1-4S-transfected MCF7 cells form glands with lumena when grown in 3D culture, while null mutations of two putative phosphorylation sites (T457A and S459A) in the cytoplasmic domain fail to undergo lumen formation. When gene chip analysis was performed on mRNA isolated from both wild type and T457A,S459A mutated CEACAM1-4S-transfected MCF7 cells grown in 3D culture, calpain-9 (CAPN9) was identified out of over 400 genes with a > 2 log 2 difference as a potential inducer of lumen formation. Inhibition of CAPN9 expression in MCF7/CEACAM1-4S cells by RNAi or by calpeptin or PD150606 inhibited lumen formation. Transfection of CAPN9 into wild type MCF7 cells restores lumen formation demonstrating that calpain-9 may play a critical role in lumen formation. Additionally, we demonstrate that the apoptosis related kinase, PKC-δ, is activated by proteolytic cleavage during lumen formation exclusively in wild type CEACAM1-4S-transfected MCF7 cells grown in 3D culture and that lumen formation is inhibited by either RNAi to PKC-δ or by the PKC-δ inhibitor rottlerin.     

PKCδ knockdown inhibits free fatty acid induction of endothelial cell apoptosis

The mechanisms whereby free fatty acids induce endothelial cell apoptosis are not yet understood. The present study aimed to investigate the role of PKCδ in free fatty acid–induced endothelial cell apoptosis. In addition, we looked for evidence of apoptosis‐related interactions between PKCδ and Fas signal pathway. Human umbilical vein endothelial cells were treated with various concentrations of free fatty acids and transiently transfected with PKCδ siRNA or Fas siRNA to inhibit PKCδ or Fas expression. Cell proliferation was determined through colorimetric assays, and apoptosis was quantified using flow cytometry. Protein expression was determined from cell lysates using Western blots with antibodies against p‐PKCδTyr512, PKCδ, and Fas. Statistical analyses were performed. Free fatty acids had multiple effects on human umbilical vein endothelial cells, including concentration‐dependent inhibition of cell proliferation, induction of apoptosis, increased Fas expression, and increased PKCδ expression and phosphorylation. Inhibition of PKCδ mRNA expression by PKCδ siRNA led to a reduction in both free fatty acid–induced apoptosis and Fas expression. However, Fas siRNA treatment inhibited Fas, but not PKCδ, expression in human umbilical vein endothelial cells. The free fatty acid–induced apoptosis in endothelial cells are possibly mediated by PKCδ and may involve upregulation of its downstream Fas. Copyright © 2012 John Wiley & Sons, Ltd.     

Involvement of protein kinase Cδ in induction of apoptosis by cationic liposomes in macrophage-like RAW264.7 cells

We have recently demonstrated that reactive oxygen species (ROS) play an important role in RAW264.7 cell apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes). In this study, we investigated whether protein kinase Cδ PKCδ) is involved in apoptosis induced by cationic liposomes. Tyrosine phosphorylation, nuclear localization, and cleavage of PKCδ were observed following the treatment of cells with SA-liposomes, suggesting that SA-liposomes activate PKCδ. Rottlerin, a specific inhibitor of PKCδ, inhibited ROS generation and also suppressed apoptosis. Cell surface proteoglycans may contribute to PKCδ activation by SA-liposomes. These findings suggest that PKCδ is strongly associated with apoptosis induced by SA-liposomes.     

Elevated AKT activity protects the prostate cancer cell line LNCaP from TRAIL-induced apoptosis

We find that the prostate cancer cell lines ALVA-31, PC-3, and DU 145 are highly sensitive to apoptosis induced by TRAIL (tumor-necrosis factor-related apoptosis-inducing ligand), while the cell lines TSU-Pr1 and JCA-1 are moderately sensitive, and the LNCaP cell line is resistant. LNCaP cells lack active lipid phosphatase PTEN, a negative regulator of the phosphatidylinositol (PI) 3-kinase/Akt pathway, and demonstrate a high constitutive Akt activity. Inhibition of PI 3-kinase using wortmannin and LY-294002 suppressed constitutive Akt activity and sensitized LNCaP cells to TRAIL. Treatment of LNCaP cells with TRAIL alone induced cleavage of the caspase 8 and XIAP proteins. However, processing of BID, mitochondrial release of cytochrome c, activation of caspases 7 and 9, and apoptosis did not occur unless TRAIL was combined with either wortmannin, LY-294002, or cycloheximide. Blocking cytochrome c release by Bcl-2 overexpression rendered LNCaP cells resistant to TRAIL plus wortmannin treatment but did not affect caspase 8 or BID processing. This indicates that in these cells mitochondria are required for the propagation rather than the initiation of the apoptotic cascade. Infection of LNCaP cells with an adenovirus expressing a constitutively active Akt reversed the ability of wortmannin to potentiate TRAIL-induced BID cleavage. Thus, the PI 3-kinase-dependent blockage of TRAIL-induced apoptosis in LNCaP cells appears to be mediated by Akt through the inhibition of BID cleavage.     

Imiquimod‐induced apoptosis of melanoma cells is mediated by ER stress‐dependent Noxa induction and enhanced by NF‐κB inhibition

Melanoma is characterized by dysregulated intracellular signalling pathways including an impairment of the cell death machinery, ultimately resulting in melanoma resistance, survival and progression. This explains the tumour's extraordinary resistance to the standard treatment. Imiquimod is a topical immune response modifier (imidazoquinoline) with both antiviral and antitumour activities. The mechanism by which imiquimod triggers the apoptosis of melanoma cells has now been carefully elucidated. Imiquimod‐induced apoptosis is associated with the activation of apoptosis signalling regulating kinase1/c‐Jun‐N‐terminal kinase/p38 pathways and the induction of endoplasmic stress characterized by the activation of the protein kinase RNA‐like endoplasmic reticulum kinase signalling pathway, increase in intracellular Ca²⁺ release, degradation of calpain and subsequent cleavage of caspase‐4. Moreover, imiquimod triggers the activation of NF‐κB and the expression of the inhibitor of apoptosis proteins (IAPs) such as, X‐linked IAP (XIAP) together with the accumulation of reactive oxygen species (ROS). Also, imiquimod triggers mitochondrial dysregulation characterized by the loss of mitochondrial membrane potential (Δψm), the increase in cytochrome c release, and cleavage of caspase‐9, caspase‐3 and poly(ADP‐ribose) polymerase (PARP). Inhibitors of specific pathways, permit the elucidation of possible mechanisms of imiquimod‐induced apoptosis. They demonstrate that inhibition of NF‐kB by the inhibitor of nuclear factor kappa‐B kinase (IKK) inhibitor Bay 11‐782 or knockdown of XIAP induces melanoma apoptosis in cells exposed to imiquimod. These findings support the use of either IKK inhibitors or IAP antagonists as adjuvant therapies to improve the effectiveness topical imiquimod in the treatment of melanoma.     

Overexpression of thioredoxin reductase 1 inhibits migration of HEK-293 cells

Background information. TrxR (thioredoxin reductase), in addition to protecting against oxidative stress, plays a role in the redox regulation of intracellular signalling pathways controlling, among others, cell proliferation and apoptosis. The aim of the present study was to determine whether TrxR1 is involved in the regulation of cell migration. Results. Stably transfected HEK‐293 (human embryonic kidney) cells which overexpress cytosolic TrxR1 (HEK‐TrxR15 and HEK‐TrxR11 cells) were used in the present study. We found that the stimulation of cell motility induced by PKC (protein kinase C) activators, PMA and DPhT (diphenyltin), was inhibited significantly in the HEK‐TrxR15 and HEK‐TrxR11 cells compared with control cells. The overexpression of TrxR1 also inhibited characteristic morphological changes and reorganization of the F‐actin cytoskeleton induced by PMA and DPhT. In addition, the selective activation of PKCδ by DPhT was inhibited in cells that overexpressed cytosolic TrxR1. Furthermore, rottlerin, a selective inhibitor of PKCδ, and PKCδ siRNA (small interfering RNA), suppressed the morphological changes induced by DPhT in the control cells. Conclusions. The overexpression of TrxR1 inhibits migration of HEK‐293 cells stimulated with PMA and DPhT. Moreover, our observations suggest that this effect is mediated by the inhibition of PKCδ activation.     

Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: purification and characterization of the inhibitory complex

Protein kinase C (PKC) δ plays an important role in cellular proliferation and apoptosis. The catalytic fragment of PKCδ generated by caspase-dependent cleavage is essential for the initiation of etoposide-induced apoptosis. In this study, we identified a novel mouse PKCδ isoform named PKCδIX (Genebank Accession No. HQ840432). PKCδIX is generated by alternative splicing and is ubiquitously expressed, as seen in its full-length PKCδ. PKCδIX lacks the C1 domain, the caspase 3 cleavage site, and the ATP binding site but preserves an almost intact c-terminal catalytic domain and a nuclear localization signal (NLS). The structural characteristics of PKCδIX provided a possibility that this PKCδ isozyme functions as a novel dominant-negative form for PKCδ due to its lack of the ATP-binding domain that is required for the kinase activity of PKCδ. Indeed, overexpression of PKCδIX significantly inhibited etoposide-induced apoptosis in NIH3T3 cells. In addition, an in vitro kinase assay showed that recombinant PKCδIX protein could competitively inhibit the kinase activity of PKCδ. We conclude that PKCδIX can function as a natural dominant-negative inhibitor of PKCδ in vivo.     

PKCδ mediates Nrf2-dependent protection of neuronal cells from NO-induced apoptosis

A chemical inhibitor library of 84 compounds was screened to investigate the signaling pathway(s) leading to activation of Nrf2 in response to nitric oxide (NO). We identified the protein kinase C delta (PKCδ) inhibitor rottlerin as the only compound that reduced NO-induced ARE-luciferase reporter activity and diminished NO-induced up-regulation of two Nrf2/ARE-regulated proteins - NAD(P)H:quinone oxidoreductase-1 (NQO1) and hemeoxygenase-1 (HO-1) in SH-Sy5y cells. Rottlerin also sensitized neuroblastoma cells and mouse primary cortical neurons to NO-induced apoptosis. Stable over-expression of PKCδ augmented NO-induced, ARE-dependent gene expression of HO-1 in SH-Sy5y cells, which were more protected from NO killing. Conversely, NO-induced ARE-dependent gene expression was reduced in PKCδ-knockdown SH-EP cells, which displayed greater sensitivity to apoptosis. PKCδ^−/− cortical neurons exhibited increased NO-induced apoptosis and less HO-1 mRNA and protein induction compared with wild type neurons. Hence, PKCδ is an important positive modulator of NO-induced Nrf2/ARE-dependent signaling that counteracts NO-mediated apoptosis in neuronal cells.     

Chlamydia trachomatis‐infected host cells resist dsRNA‐induced apoptosis

Human pathogenic Chlamydia trachomatis have evolved sophisticated mechanisms to manipulate host cell signalling pathways in order to prevent apoptosis. We show here that host cells infected with C. trachomatis resist apoptosis induced by polyI:C, a synthetic double‐stranded RNA that mimics viral infections. Infected cells displayed significantly reduced levels of PARP cleavage, caspase‐3 activation and a decrease in the TUNEL positive population in the presence of polyI:C. Interestingly, the chlamydial block of apoptosis was upstream of the initiator caspase‐8. Processing of caspase‐8 was reduced in infected cells and coincided with a decrease in Bid truncation and downstream caspase‐9 cleavage. Moreover, the enzymatic activity of caspase‐8, measured by a luminescent substrate, was significantly reduced in infected cells. Caspase‐8 inhibition by Chlamydia was dependent on cFlip as knock‐down of cFlip decreased the chlamydial block of caspase‐8 activation and consequently reduced apoptosis inhibition. Our data implicate that chlamydial infection interferes with the host cell's response to viral infections and thereby influences the fate of the cell.     

Apoptosis signal-regulating kinase1 is inducible by protein kinase Cδ and contributes to phorbol ester-mediated G1 phase arrest through persistent JNK activation

Although protein kinase Cδ (PKCδ) has been suggested in the negative control of the cell cycle machinery in many types of cancer cells, its underlying mechanisms are partly understood. Here we report that the expression of apoptosis signal-regulating kinase1 (ASK1) is inducible in a PKCδ-dependent manner, and contributes to phorbol ester-induced cell cycle arrest through persistent JNK activation in breast cancer epithelial cells. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) gradually up-regulated the expression of ASK1 mRNA and protein, and subsequently enhanced its catalytic activity in MCF-7 cells. Importantly, such PMA-induced ASK1 expression was completely abolished by pretreatment of rottlerin, a specific PKCδ inhibitor or by knocking down the expression of PKCδ, while ectopic expression of a constitutively active form of PKCδ strongly up-regulated ASK1 expression. We also found that the persistent activation of mitogen-activated protein kinase, JNK in response to PMA was greatly attenuated by RNA interference-mediated knockdown of ASK1. Taken together, these results suggest that inducible expression of ASK1 by PKCδ contributes to the G1 arrest by enhancing persistent JNK signaling activation which represents a novel alternative mechanism of PKCδ-dependent cell cycle arrest and limiting proliferation of breast cancer epithelial cells.     

Osteoprotegerin production by breast cancer cells is suppressed by dexamethasone and confers resistance against TRAIL‐induced apoptosis

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of NF‐κB ligand (RANKL) and TNF‐related apoptosis‐inducing ligand (TRAIL). While RANKL is essential for osteoclastogenesis and facilitates breast cancer migration into bone, TRAIL promotes breast cancer apoptosis. We analyzed the expression of OPG and TRAIL and its modulation in estrogen receptor‐positive MCF‐7 cells and receptor‐negative MDA‐MB‐231 cells. In both cells, OPG mRNA levels and protein secretion were dose‐ and time‐dependently enhanced by interleukin (IL)‐1β and suppressed by dexamethasone. In contrast to MCF‐7 cells, MDA‐MB‐231 abundantly expressed TRAIL mRNA, which was enhanced by IL‐1β and inhibited by dexamethasone. TRAIL activated pro‐apoptotic caspase‐3, ‐7, and poly‐ADP‐ribose polymerase and decreased cell numbers of MDA‐MB‐231, but had no effect on MCF‐7 cells. Gene silencing siRNA directed against OPG resulted in a 31% higher apoptotic rate compared to non‐target siRNA‐treated MDA‐MB‐231 cells. Furthermore, TRAIL induced significantly less apoptosis in cells cultured in conditioned media (containing OPG) compared to cells exposed to TRAIL in fresh medium lacking OPG (P < 0.01) and these protective effects were reversed by blocking OPG with its specific ligand RANKL (P < 0.05). The association between cancer cell survival and OPG production by MDA‐MB‐231 cells was further supported by the finding, that modulation of OPG secretion using IL‐1β or dexamethasone prior to TRAIL exposure resulted in decreased and increased rate of apoptosis, respectively (P < 0.05). Thus, OPG secretion by breast cancer cells is modulated by cytokines and dexamethasone, and may represent a critical resistance mechanism that protects against TRAIL‐induced apoptosis. J. Cell. Biochem. 108: 106–116, 2009. © 2009 Wiley‐Liss, Inc.