The CCCH-Type Zinc Finger Antiviral Protein Relieves Immunosuppression of T Cells Induced by Avian Leukosis Virus Subgroup J via the NLP-PKC-δ-NFAT Pathway.

The CCCH-type zinc finger antiviral protein (ZAP) can recognize and induce the degradation of mRNAs and proteins of certain viruses, as well as exerting its antiviral activity by activating T cells. However, the mechanism of ZAP that mediates T cell activation during virus infection remains unclear. Here, we found a potential function of ZAP that relieves immunosuppression of T cell induced by avian leukosis virus subgroup J (ALV-J) via a novel signaling pathway that involves norbin-like protein (NLP), protein kinase C delta (PKC-δ), and nuclear factor of activated T cell (NFAT). Specifically, ZAP expression activated T cells by promoting the dephosphorylation and nuclear translocation of NFAT. Furthermore, knockdown of ZAP weakened the reactivity and antiviral response of T cells. Mechanistically, ZAP reduced PKC-δ activity by upregulating and reactivating NLP by competitively binding with viral protein. Knockdown of NLP decreased the dephosphorylation of PKC-δ by ZAP expression. Moreover, we show that knockdown of PKC-δ reduced the phosphorylation levels of NFAT and enhanced its nuclear translocation. Taken together, these data revealed that ZAP relieves immunosuppression caused by ALV-J and mediates T cell activation through the NLP-PKC-δ-NFAT pathway. IMPORTANCE The evolution of the host defense system is driven synchronously in the process of resisting virus invasion. Accordingly, host innate defense factors effectively work to suppress virus replication. However, it remains unclear whether the host innate defense factors are involved in antiviral immune responses against the invasion of immunosuppressive viruses. Here, we found that CCCH-type zinc finger antiviral protein (ZAP) effectively worked in resistance to immunosuppression caused by avian leukosis virus subgroup J (ALV-J), a classic immunosuppressive virus. Evidence showed that ZAP released the phosphatase activity of NLP inhibited by ALV-J and further activated NFAT by inactivating PKC-δ. This novel molecular mechanism, i.e., ZAP regulation of the antiviral immune response by mediating the NLP-PKC-δ-NFAT pathway, has greatly enriched the understanding of the functions of host innate defense factors and provided important scientific ideas and a theoretical basis for research on immunosuppressive viruses and antiviral immunity.

PKC-δ-dependent mitochondrial ROS attenuation is involved as 9-OAHSA combats lipoapotosis in rat hepatocytes induced by palmitic acid and in Syrian hamsters induced by high-fat high-cholesterol high-fructose diet.

Metabolic-associated fatty liver disease (MAFLD) is a global concern, often undetected until reaching an advanced stage. Palmitic acid (PA) is a type of fatty acid that increases and leads to liver apoptosis in MAFLD. However, there is currently no approved therapy or compound for MAFLD. Recently, branched fatty acid esters of hydroxy fatty acids (FAHFAs), a group of bioactive lipids, have emerged as promising agents to treat associated metabolic diseases. This study utilizes one type of FAHFA, oleic acid ester of 9-hydroxystearic acid (9-OAHSA), to treat PA-induced lipoapoptosis in an in vitro MAFLD model using rat hepatocytes and a high-fat high-cholesterol high-fructose (HFHCHFruc) diet in Syrian hamsters. The results indicate that 9-OAHSA rescues hepatocytes from PA-induced apoptosis and attenuates lipoapoptosis and dyslipidemia in Syrian hamsters. Additionally, 9-OAHSA decreases the generation of mitochondrial reactive oxygen species (mito-ROS) and stabilizes the mitochondrial membrane potential in hepatocytes. The study also demonstrates that the effect of 9-OAHSA on mito-ROS generation is at least partially mediated by PKC-δ signaling. These findings suggest that 9-OAHSA shows promise as a therapy for MAFLD.

Non-synonymous SNPs variants of PRKCG and its association with oncogenes predispose to hepatocellular carcinoma.

BACKGROUND: PRKCG encodes PKC γ, which is categorized under the classical protein kinase C family. No studies have specifically established the relationship between PRKCG nsSNPs with structural and functional variations in PKC γ in the context of hepatocellular carcinoma (HCC). The present study aims to uncover this link through in-silico and experimental studies. METHODS: The 3D structure of PKC γ was predicted. Molecular Dynamic (MD) Simulations were run and estimates were made for interactions, stability, conservation and post-translational alterations between wild and mutant structures. The association of PRKCG levels with HCC survival rate was determined. Genotyping analyses were conducted to investigate the deleterious PRKCG nsSNP association with HCC. mRNA expression of PKC γ, HIF-1 alpha, AKT, SOCS3 and VEGF in the blood of controls and HCC patients was analyzed and a genetic cascade was constructed depicting these interactions. RESULTS: The expression level of studied oncogenes was compared to tumour suppressor genes. Through Alphafold, the 3D structure of PKC γ was explored. Fifteen SNPs were narrowed down for in-silico analyses that were identified in exons 5, 10 and 18 and the regulatory and kinase domain of PKC γ. Root mean square deviation and fluctuation along with the radius of gyration unveiled potential changes between the wild and mutated variant structures. Mutant genotype AA (homozygous) corresponding to nsSNP, rs386134171 had more frequency in patients with OR (2.446), RR (1.564) and P-values (< 0.0029) that highlights its significant association with HCC compared to controls in which the wild genotype GG was found more prevalent. CONCLUSION: nsSNP rs386134171 can be a genetic marker for HCC diagnosis and therapeutic studies. This study has laid down a road map for future studies to be conducted on HCC.

Inhibition of PKC-δ reduce rhabdomyolysis-induced acute kidney injury.

Despite extensive research, the mechanisms underlying rhabdomyolysis-induced acute kidney injury (AKI) remain largely elusive. In this study, we established both cell and murine models of rhabdomyolysis-induced AKI by using myoglobin and glycerin, respectively, and provided evidence that protein kinase Cδ (PKC-δ) was activated in both models and subsequently promoted cell apoptosis. Moreover, we found that this detrimental effect of PKC-δ activation can be reversed by its pharmaceutical inhibitor rottlerin. Furthermore, we detected and confirmed the existence of PKC-δ-mediated myoglobin-induced cell apoptosis and the expression of TNF-α and IL1-ß via regulation of the p38MAPK and ERK1/2 signalling pathways. In summary, our research revealed the role of PKC-δ in renal cell apoptosis and suggests that PKC-δ is a viable therapeutic target for rhabdomyolysis-induced AKI.

aPKCi triggers basal extrusion of luminal mammary epithelial cells by tuning contractility and vinculin localization at cell junctions.

Metastasis is the main cause of cancer-related deaths. How a single oncogenic cell evolves within highly organized epithelium is still unknown. Here, we found that the overexpression of the protein kinase atypical protein kinase C ι (aPKCi), an oncogene, triggers basally oriented epithelial cell extrusion in vivo as a potential mechanism for early breast tumor cell invasion. We found that cell segregation is the first step required for basal extrusion of luminal cells and identify aPKCi and vinculin as regulators of cell segregation. We propose that asymmetric vinculin levels at the junction between normal and aPKCi+ cells trigger an increase in tension at these cell junctions. Moreover, we show that aPKCi+ cells acquire promigratory features, including increased vinculin levels and vinculin dynamics at the cell-substratum contacts. Overall, this study shows that a balance between cell contractility and cell-cell adhesion is crucial for promoting basally oriented cell extrusion, a mechanism for early breast cancer cell invasion.

CGRP-dependent sensitization of PKC-δ positive neurons in central amygdala mediates chronic migraine.

BACKGROUND: To investigate specific brain regions and neural circuits that are responsible for migraine chronification. METHODS: We established a mouse model of chronic migraine with intermittent injections of clinically-relevant dose of nitroglycerin (0.1 mg/kg for 9 days) and validated the model with cephalic and extracephalic mechanical sensitivity, calcitonin gene-related peptide (CGRP) expression in trigeminal ganglion, and responsiveness to sumatriptan or central CGRP blockade. We explored the neurons that were sensitized along with migraine chronification and investigated their roles on migraine phenotypes with chemogenetics. RESULTS: After repetitive nitroglycerin injections, mice displayed sustained supraorbital and hind paw mechanical hyperalgesia, which lasted beyond discontinuation of nitroglycerin infusion and could be transiently reversed by sumatriptan. The CGRP expression in trigeminal ganglion was also upregulated. We found the pERK positive cells were significantly increased in the central nucleus of the amygdala (CeA), and these sensitized cells in the CeA were predominantly protein kinase C-delta (PKC-δ) positive neurons co-expressing CGRP receptors. Remarkably, blockade of the parabrachial nucleus (PBN)-CeA CGRP neurotransmission by CGRP8-37 microinjection to the CeA attenuated the sustained cephalic and extracephalic mechanical hyperalgesia. Furthermore, chemogenetic silencing of the sensitized CeA PKC-δ positive neurons reversed the mechanical hyperalgesia and CGRP expression in the trigeminal ganglion. In contrast, repetitive chemogenetic activation of the CeA PKC-δ positive neurons recapitulated chronic migraine-like phenotypes in naïve mice. CONCLUSIONS: Our data suggest that CeA PKC-δ positive neurons innervated by PBN CGRP positive neurons might contribute to the chronification of migraine, which may serve as future therapeutic targets for chronic migraine.

The roles of PKC-δ and PKC-ε in myocardial ischemia/reperfusion injury.

Ischemia and reperfusion (I/R) cause a reduction in arterial blood supply to tissues, followed by the restoration of perfusion and consequent reoxygenation. The reestablishment of blood flow triggers further damage to ischemic tissue through reactive oxygen species (ROS) accumulation, interference with cellular ion homeostasis, opening of mitochondrial permeability transition pores (mPTPs) and promotion of cell death (apoptosis or necrosis). PKC-δ and PKC-ε, belonging to a family of serine/threonine kinases, have been demonstrated to play important roles during I/R injury in cardiovascular diseases. However, the cardioprotective mechanisms of PKC-δ and PKC-ε in I/R injury have not been elaborated until now. This article discusses the roles of PKC-δ and PKC-ε during myocardial I/R in redox regulation (redox signaling and oxidative stress), cell death (apoptosis and necrosis), Ca2+ overload, and mitochondrial dysfunction.

Moracin D induces apoptosis in prostate cancer cells via activation of PPAR gamma/PKC delta and inhibition of PKC alpha.

Herein, apoptotic mechanism of Moracin D was explored in prostate cancer cells in association with peroxisome proliferator-activated receptor gamma (PPAR-γ)-related signaling involved in lipid metabolism. Moracin D augmented cytotoxicity and sub G1 population in PC3 and DU145 prostate cancer cells, while DU145 cells were more susceptible to Moracin D than PC3 cells. Moracin D attenuated the expression of caspase-3, poly (ADP-ribose) polymerase (PARP), B-cell lymphoma 2 (Bcl-2), and B-cell lymphoma-extra-large (Bcl-xL) in DU145 cells. Consistently, Moracin D significantly augmented the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in DU145 cells. Interestingly, Moracin D activated PPAR-γ and phospho-protein kinase C delta (p-PKC-δ) and inhibited phospho-protein kinase C alpha (p-PKC-α) in DU145 cells. Furthermore, STRING bioinformatic analysis reveals that PPAR-γ interacts with nuclear factor-κB (NF-κB) that binds to PKC-α/PKC-δ or protein kinase B (AKT) or extracellular signal-regulated kinase (ERK). Indeed, Moracin D decreased phosphorylation of NF-κB, ERK, and AKT in DU145 cells. Conversely, PPAR-γ inhibitor GW9662 reduced the apoptotic ability of Moracin D to activate caspase 3 and PARP in DU145 cells. Taken together, these findings provide a novel insight that activation of PPAR-γ/p-PKC-δ and inhibition of p-PKC-α are critically involved in Moracin D-induced apoptosis in DU145 prostate cancer cells.

Deficiency in the phosphatase PHLPP1 suppresses osteoclast-mediated bone resorption and enhances bone formation in mice.

Enhanced osteoclast-mediated bone resorption and diminished formation may promote bone loss. Pleckstrin homology (PH) domain and leucine-rich repeat protein phosphatase 1 (Phlpp1) regulates protein kinase C (PKC) and other proteins in the control of bone mass. Germline Phlpp1 deficiency reduces bone volume, but the mechanisms remain unknown. Here, we found that conditional Phlpp1 deletion in murine osteoclasts increases their numbers, but also enhances bone mass. Despite elevating osteoclasts, Phlpp1 deficiency did not increase serum markers of bone resorption, but elevated serum markers of bone formation. These results suggest that Phlpp1 suppresses osteoclast formation and production of paracrine factors controlling osteoblast activity. Phlpp1 deficiency elevated osteoclast numbers and size in ex vivo osteoclastogenesis assays, accompanied by enhanced expression of proto-oncogene C-Fms (C-Fms) and hyper-responsiveness to macrophage colony-stimulating factor (M-CSF) in bone marrow macrophages. Although Phlpp1 deficiency increased TRAP+ cell numbers, it suppressed actin-ring formation and bone resorption in these assays. We observed that Phlpp1 deficiency increases activity of PKCζ, a PKC isoform controlling cell polarity, and that addition of a PKCζ pseudosubstrate restores osteoclastogenesis and bone resorption of Phlpp1-deficient osteoclasts. Moreover, Phlpp1 deficiency increased expression of the bone-coupling factor collagen triple helix repeat-containing 1 (Cthrc1). Conditioned growth medium derived from Phlpp1-deficient osteoclasts enhanced mineralization of ex vivo osteoblast cultures, an effect that was abrogated by Cthrc1 knockdown. In summary, Phlpp1 critically regulates osteoclast numbers, and Phlpp1 deficiency enhances bone mass despite higher osteoclast numbers because it apparently disrupts PKCζ activity, cell polarity, and bone resorption and increases secretion of bone-forming Cthrc1.

Insulin directly stimulates mitochondrial glucose oxidation in the heart.

BACKGROUND: Glucose oxidation is a major contributor to myocardial energy production and its contribution is orchestrated by insulin. While insulin can increase glucose oxidation indirectly by enhancing glucose uptake and glycolysis, it also directly stimulates mitochondrial glucose oxidation, independent of increasing glucose uptake or glycolysis, through activating mitochondrial pyruvate dehydrogenase (PDH), the rate-limiting enzyme of glucose oxidation. However, how insulin directly stimulates PDH is not known. To determine this, we characterized the impacts of modifying mitochondrial insulin signaling kinases, namely protein kinase B (Akt), protein kinase C-delta (PKC-δ) and glycogen synthase kinase-3 beta (GSK-3ß), on the direct insulin stimulation of glucose oxidation. METHODS: We employed an isolated working mouse heart model to measure the effect of insulin on cardiac glycolysis, glucose oxidation and fatty acid oxidation and how that could be affected when mitochondrial Akt, PKC-δ or GSK-3ß is disturbed using pharmacological modulators. We also used differential centrifugation to isolate mitochondrial and cytosol fraction to examine the activity of Akt, PKC-δ and GSK-3ß between these fractions. Data were analyzed using unpaired t-test and two-way ANOVA. RESULTS: Here we show that insulin-stimulated phosphorylation of mitochondrial Akt is a prerequisite for transducing insulin's direct stimulation of glucose oxidation. Inhibition of mitochondrial Akt completely abolishes insulin-stimulated glucose oxidation, independent of glucose uptake or glycolysis. We also show a novel role of mitochondrial PKC-δ in modulating mitochondrial glucose oxidation. Inhibition of mitochondrial PKC-δ mimics insulin stimulation of glucose oxidation and mitochondrial Akt. We also demonstrate that inhibition of mitochondrial GSK3ß phosphorylation does not influence insulin-stimulated glucose oxidation. CONCLUSION: We identify, for the first time, insulin-stimulated mitochondrial Akt as a prerequisite transmitter of the insulin signal that directly stimulates cardiac glucose oxidation. These novel findings suggest that targeting mitochondrial Akt is a potential therapeutic approach to enhance cardiac insulin sensitivity in condition such as heart failure, diabetes and obesity.

12-Deoxyphorbol Esters Induce Growth Arrest and Apoptosis in Human Lung Cancer A549 Cells Via Activation of PKC-δ/PKD/ERK Signaling Pathway.

Prostratin, a non-tumor promoting 12-deoxyphorbol ester, has been reported as a protein kinase C (PKC) activator and is shown to have anti-proliferative activity in certain cancer cell types. Here we show that GRC-2, a prostratin analogue isolated from Euphorbia grandicornis, is ten-fold more potent than prostratin for inhibiting the growth of human non-small cell lung cancer (NSCLC) A549 cells. Flow cytometry assay revealed that GRC-2 and prostratin inhibited cell cycle progression at the G2/M phase and induced apoptosis. The cytotoxic effect of GRC-2 and prostratin was accompanied by activation and nuclear translocation of PKC-δ and PKD as well as hyperactivation of extracellular signal-related kinase (ERK). Knockdown of either PKC-δ, PKD or ERK significantly protected A549 cancer cells from GRC-2- and prostratin-induced growth arrest as well as apoptosis. Taken together, our results have shown that prostratin and a more potent analogue GRC-2 reduce cell viability in NSCLC A549 cells, at least in part, through activation of the PKC-δ/PKD/ERK pathway, suggesting the potential of prostratin and GRC-2 as anticancer agents.

Protein Kinase C-ζ stimulates colorectal cancer cell carcinogenesis via PKC-ζ/Rac1/Pak1/ß-Catenin signaling cascade.

Colorectal cancer (CRC) is the second most common cancer in the world and death from CRC accounts for 8% of all cancer deaths both in men and women in the United States. CRC is life-threatening disease due to therapy resistant cancerous cells. The exact mechanisms of cell growth, survival, metastasis and inter & intracellular signaling pathways involved in CRC is still a significant challenge. Hence, investigating the signaling pathways that lead to colon carcinogenesis may give insight into the therapeutic target. In this study, the role of atypical Protein Kinase C (aPKC) on CRC was investigated by using two inhibitors of that protein class: 1) ζ-Stat (8-hydroxynaphthalene-1,3,6-trisulfonic acid) is a specific inhibitor of PKC-ζ and 2) ICA-I 5-amino-1-(2,3-dihydroxy-4-hydroxymethyl)cyclopentyl)-1H-imidazole-4-carboxamide) is a specific inhibitor of PKC-ι. The cell lines tested were CCD18CO normal colon epithelial and LOVO metastatic CRC cells. The inhibition of aPKCs did not bring any significant toxicity on CCD18CO normal colon cell line. Although PKC-ι is an oncogene in many cancers, we found the overexpression of PKC-ζ and its direct association with Rac1. Our findings suggest that the PKC-ζ may be responsible for the abnormal growth, proliferation, and migration of metastatic LOVO colon cancer cells via PKC-ζ/Rac1/Pak1/ß-Catenin pathway. These results suggest the possibility of utilizing PKC-ζ inhibitor to block CRC cells growth, proliferation, and metastasis.

Loss-of-function phenotype of a PKCθT219A knockin mouse strain.

BACKGROUND: Protein kinase C θ has been established as an important signaling intermediate in T-effector-cell activation and survival pathways by controlling activity of the key transcription factors NF-κB and NFAT. Previous studies identified an activation-induced auto-phosphorylation site at Thr-219, located between the tandem C1 domains of the regulatory fragment in PKCθ, as a structural requirement for its correct membrane translocation and the subsequent transactivation of downstream signals leading to IL-2 production in a human T cell line. METHODS: The present work aimed to define the role of this phosphorylation switch on PKCθ in a physiological context through a homozygous T219A knockin mouse strain. T cell activation was analyzed by H3-thymidine uptake (proliferative response), qRT-PCR and luminex measurements (cytokine production). NFAT and NF-κB transactivation responses were estimated by Gel mobility shift and Alpha Screen assays. Frequencies of T cell subsets were analyzed by flow cytometry. RESULTS: Despite a normal T cell development, in vitro activated effector T cells clearly revealed a requirement of Thr-219 phosphorylation site on PKCθ for a transactivation of NF-κB and NFAT transcription factors and, subsequently, robust IL-2 and IFN-γ expression. CONCLUSION: This phenotype is reminiscent of the PKCθ knockout T cells, physiologically validating that this (p) Thr-219 auto-phosphorylation site indeed critically regulates PKCθ function in primary mouse T cells.

Characterisation of amphioxus protein kinase C-δ/θ reveals a unique proto-V3 domain suggesting an evolutionary mechanism for PKC-θ unique V3.

A primitive adaptive immune system has recently been suggested to be present in a basal chordate amphioxus (Branchiostoma belcheri, Bb), making it an ideal model for studying the origin of adaptive immune. The novel protein kinase C isoform PKC-θ, but not its closest isoform PKC-δ, plays a critical role for mammalian T-cell activation via translocation to immunological synapse (IS) mediated by a unique PKC-θ V3 domain containing one PxxP motif. To understand the evolution of this unique PKC-θ V3 domain and the primitive adaptive immune system in amphioxus, we comparatively studied the orthologs of PKC-δ and -θ from amphioxus and other species. Phylogenetic analysis showed BbPKC-δ/θ to be the common ancestor of vertebrate PKC-δ and PKC-θ, with a V3 domain containing two PxxP motifs. One motif is conserved in both zebrafish and mammalian PKC-θ but is absent in PKC-δ V3 domain of these species, and has already emerged in drosophila PKC-δ. The other non-conserved motif emerged in BbPKC-δ/θ, and only retained in Danio rerio PKC-δ (DrPKC-δ) but lost in mammalian PKC-δ and -θ. Comparative analyses of the sequence and function of BbPKC-δ/θ, DrPKC-δ, DrPKC-θ and Homo sapiens PKC-θ (HsPKC-θ) in IS translocation and T-cell receptor (TCR)-induced NF-κB activation revealed that retention of the conserved PxxP motif and loss of the non-conserved PxxP motif in mammalian PKC-θ and loss of both PxxP motifs in mammalian PKC-δ accomplish the unique function of PKC-θ in T cells. Together, this study suggests an evolutionary mechanism for PKC-θ unique V3 and reveals BbPKC-δ/θ is the common ancestor of PKC-δ and -θ with a functional proto-V3 domain, supplying new evidence for the existence of primitive adaptive immune system in amphioxus.

Bioactive staurosporine derivatives from the Streptomyces sp. NB-A13.

Six new (1-6) and nine known (7-15) staurosporine derivatives were isolated from the rice solid fermentation of the marine-derived Streptomyces sp. NB-A13. The structures of the new staurosporine derivatives were established by extensive spectroscopic data interpretation. The absolute configurations of 1 and 2 were assigned by quantum chemical calculations of the electronic circular dichroism (ECD) spectra. All of these compounds were screened for their cytotoxic activities against PC-3 and SW-620 cell lines. Compound 7 exhibited stronger inhibitory activity against SW-620 cell lines than the positive control staurosporine (25.10 nM), with IC50 values of 9.99 nM. Moreover, compounds 1-5, 8-13 and 15 also showed significant cytotoxicities with IC50 values ranging from 0.02 to 16.60 µM, while 6 exhibited no cytotoxic potency. Additionally, compounds 1-7 were also tested for enzyme inhibition activities of Protein kinase C theta (PKC-θ), and showed activity with IC50 values ranging from 0.06 to 9.43 µM except for compound 6, which has no inhibition activity.

Determination of the Role and Active Sites of PKC-Delta-Like from Lamprey in Innate Immunity.

Protein kinase C-δ (PKC-δ) is an important protein in the immune system of higher vertebrates. Lampreys, as the most primitive vertebrates, have a uniquevariable lymphocyte receptor (VLR) immune system. PKC-δ-like is a crucial functional gene in lampreys and is highly expressed in their immune organs. In this study, lampreys were stimulated with different immunogens, and lipopolysaccharide (LPS) was found to increase the expression of PKC-δ-like. Overexpression of PKC-δ-like could also effectively activate the innate immune response. We further demonstrated that PKC-δ-like-CF, a catalytic fragment of PKC-δ-like, is responsible for activating the innate immune response, and Thr-211, which is Thr-419 of PKC-δ-like, was confirmed to be the key site affecting PKC-δ-like-CF activity. These results indicated that PKC-δ-like from lamprey may have an important role in the innate immune response.

Inhibition of ZL55 cell proliferation by ADP via PKC-dependent signalling pathway.

Extracellular nucleotides can regulate cell proliferation in both normal and tumorigenic tissues. Here, we studied how extracellular nucleotides regulate the proliferation of ZL55 cells, a mesothelioma-derived cell line obtained from bioptic samples of asbestos-exposed patients. ADP and 2-MeS-ADP inhibited ZL55 cell proliferation, whereas ATP, UTP, and UDP were inactive. The nucleotide potency profile and the blockade of the ADP-mediated inhibitory effect by the phospholipase C inhibitor U-73122 suggest that P2Y1 receptor controls ZL55 cell proliferation. The activation of P2Y1 receptor by ADP leads to activation of intracellular transduction pathways involving [Ca2+ ]i , PKC-δ/PKC-α, and MAPKs, ERK1/2 and JNK1/2. Cell treatment with ADP or 2-MeS-ADP also provokes the activation of p53, causing an accumulation of the G1 cyclin-dependent kinase inhibitors p21WAF1 and p27Kip . Inhibition of ZL55 cell proliferation by ADP was completely reversed by inhibiting MEK1/2, or JNK1/2, or PKC-δ, and PKC-α. Through the inhibition of ADP-activated transductional kinases it was found that PKC-δ was responsible for JNK1/2 activation. JNK1/2 has a role in transcriptional up-regulation of p53, p21WAF1/CIP1 , and p27kip1 . Conversely, the ADP-activated PKC-α provoked ERK1/2 phosphorylation. ERK1/2 increased p53 stabilization, required to G1 arrest of ZL55 cells. Concluding, the importance of the study is twofold: first, results shed light on the mechanism of cell cycle inhibition by ADP; second, results suggest that extracellular ADP may inhibit mesothelioma progression.

Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation.

Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation. This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood. By genome-wide ChIP-sequencing ex vivo human CD4(+) T cells, here, we show that the signaling enzyme, protein kinase C theta (PKC-θ) directly relays stimulatory signals to chromatin by binding to transcriptional-memory-responsive genes to induce transcriptional activation. Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4(+) T cells. Within the nucleus, PKC-θ catalytic activity maintains the Ser536 phosphorylation on the p65 subunit of NF-κB (also known as RelA) and can directly influence chromatin accessibility at transcriptional memory genes by regulating H2B deposition through Ser32 phosphorylation. Furthermore, using a cytoplasm-restricted PKC-θ mutant, we highlight that chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit transcriptional memory responses in human memory T cells.

Measuring the lactate-to-creatine ratio via 1H NMR spectroscopy can be used to noninvasively evaluate apoptosis in glioma cells after X-ray irradiation.

BACKGROUND: Radiotherapy is among the commonly applied treatment options for glioma, which is one of the most common types of primary brain tumor. To evaluate the effect of radiotherapy noninvasively, it is vital for oncologists to monitor the effects of X-ray irradiation on glioma cells. Preliminary research had showed that PKC-ι expression correlates with tumor cell apoptosis induced by X-ray irradiation. It is also believed that the lactate-to-creatine (Lac/Cr) ratio can be used as a biomarker to evaluate apoptosis in glioma cells after X-ray irradiation. In this study, we evaluated the relationships between the Lac/Cr ratio, apoptotic rate, and protein kinase C iota (PKC-ι) expression in glioma cells. METHODS: Cells of the glioma cell lines C6 and U251 were randomly divided into 4 groups, with every group exposed to X-ray irradiation at 0, 1, 5, 10 and 15 Gy. Single cell gel electrophoresis (SCGE) was conducted to evaluate the DNA damage. Flow cytometry was performed to measure the cell cycle blockage and apoptotic rates. Western blot analysis was used to detect the phosphorylated PKC-ι (p-PKC-ι) level. 1H NMR spectroscopy was employed to determine the Lac/Cr ratio. RESULTS: The DNA damage increased in a radiation dose-dependent manner (p < 0.05). With the increase in X-ray irradiation, the apoptotic rate also increased (C6, p < 0.01; U251, p < 0.05), and the p-PKC-ι level decreased (C6, p < 0.01; U251, p < 0.05). The p-PKC-ι level negatively correlated with apoptosis, whereas the Lac/Cr ratio positively correlated with the p-PKC-ι level. CONCLUSION: The Lac/Cr ratio decreases with an increase in X-ray irradiation and thus can be used as a biomarker to reflect the effects of X-ray irradiation in glioma cells.

Low Level Pro-inflammatory Cytokines Decrease Connexin36 Gap Junction Coupling in Mouse and Human Islets through Nitric Oxide-mediated Protein Kinase Cδ.

Pro-inflammatory cytokines contribute to the decline in islet function during the development of diabetes. Cytokines can disrupt insulin secretion and calcium dynamics; however, the mechanisms underlying this are poorly understood. Connexin36 gap junctions coordinate glucose-induced calcium oscillations and pulsatile insulin secretion across the islet. Loss of gap junction coupling disrupts these dynamics, similar to that observed during the development of diabetes. This study investigates the mechanisms by which pro-inflammatory cytokines mediate gap junction coupling. Specifically, as cytokine-induced NO can activate PKCδ, we aimed to understand the role of PKCδ in modulating cytokine-induced changes in gap junction coupling. Isolated mouse and human islets were treated with varying levels of a cytokine mixture containing TNF-α, IL-1ß, and IFN-γ. Islet dysfunction was measured by insulin secretion, calcium dynamics, and gap junction coupling. Modulators of PKCδ and NO were applied to determine their respective roles in modulating gap junction coupling. High levels of cytokines caused cell death and decreased insulin secretion. Low levels of cytokine treatment disrupted calcium dynamics and decreased gap junction coupling, in the absence of disruptions to insulin secretion. Decreases in gap junction coupling were dependent on NO-regulated PKCδ, and altered membrane organization of connexin36. This study defines several mechanisms underlying the disruption to gap junction coupling under conditions associated with the development of diabetes. These mechanisms will allow for greater understanding of islet dysfunction and suggest ways to ameliorate this dysfunction during the development of diabetes.