Phosphorylation Of PKCδ Research Articles

Longifolioside A inhibits TLR4-mediated inflammatory responses by blocking PKCδ activation in LPS-stimulated THP-1 macrophages

Longifolioside A is an iridoid glucoside compound isolated from Pseudolysimachion rotundum var. subintegrum, which has been used in traditional herbal medicines to treat respiratory inflammatory diseases. Logifolioside A is a potent antioxidant; however, its underlying pharmacological mechanisms of action in inflammatory diseases are unknown.Here, we investigated the inhibitory effects of longifolioside A in lipopolysaccharide (LPS)-stimulated toll-like receptor 4 (TLR4) signal transduction systems using human THP-1 macrophages and HEK293 cells stably expressing human TLR4 protein (293/HA-hTLR4).Longifolioside A significantly reduced the release of inflammatory cytokines such as interleukin (IL)-6, -8, and tumor necrosis factor (TNF)-α in LPS-stimulated THP-1 macrophages. Furthermore, longifolioside A inhibited the expression of inflammatory mediator genes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 that produce nitric oxide (NO) and prostaglandin E2 (PGE2), respectively. Longifolioside A suppressed the phosphorylation of PKCδ, IRAK4, IKKα/β, IκBα, and mitogen-activated protein (MAP) kinases (ERK 1/2 and JNK, but not p38), thereby inactivating the nuclear localization of NF-κB and AP-1, and thus decreasing the expression of inflammatory response genes.Notably, longifolioside A disrupted the interaction between human TLR4 and the TIR domain-containing adaptor protein (TIRAP), an early step during TLR4 activation, thereby reducing IL-8 secretion in 293/HA-hTLR4 cells. This inhibitory effect was comparable to that of TAK-242 (a TLR4 inhibitor, or resatorvid). Our results indicate that longifolioside A prevents inflammatory response by suppressing TLR4 activation required for NF-κB and AP-1 activation.

Acetylation‐phosphorylation cross‐talk: A role for HDACs in the regulation of PKCdelta/theta phosphorylation

Post‐translational modifications (PTMs) such as acetylation and phosphorylation have been implicated in contractile dysfunction and muscle wasting. Moreover, mounting evidence demonstrates that histone deacetylase (HDAC) inhibitors attenuate muscle wasting in animal models of disuse and muscular dystrophy. Historically, lysine acetylation was studied in the regulation of gene transcription, where for instance HDACs remove acetyl groups on lysine residues of histone proteins to suppress gene expression. However, recent evidence has shown that non‐histone proteins are also regulated by acetylation; functioning in protein stability, intracellular signaling, mitochondrial function and muscle contraction. Despite our current understanding for lysine acetylation and protein phosphorylation in muscle, how these PTMs cross‐talk to regulate muscle physiology remains unclear. In our attempt to understand acetylation‐phosphorylation cross‐talk, we examined the role for HDAC inhibitors on protein kinase C delta (PKCδ) and delta/theta (PKCδ/θ) phosphorylation. PKC is composed of multiple isoforms and its activation has been linked to muscle disease and dysfunction. Myotubes were treated with or without a pan‐HDAC inhibitor (trichostatin A; TSA) in the absence or presence of a pathological agonist (i.e. PMA). Phorbol 12‐myristate 13‐acetate (PMA) increased phosphorylation of PKCδ and PKCδ/θ. Interestingly, PMA‐induced PKCδ and PKCδ/θ phosphorylation was amplified in myotubes treated with the pan‐HDAC inhibitor TSA. Class selective HDAC inhibitors confirmed these findings and further demonstrated that inhibition of class I HDACs (HDAC1, 2, 3) was sufficient to increase PKCδ and PKCδ/θ phosphorylation. It should be noted that total PKCδ and PKCδ/θ protein expression increased with class I HDAC inhibition concomitant to the increase in protein phosphorylation. These data suggest that HDAC inhibition increases total PKC gene/protein expression that can then be phosphorylated. Finally, we report that the HDAC 1/2 selective inhibitor Romidepsin also increased total and phosphorylated PKCδ and PKCδ/θ in response to PMA. Genetic knockdown studies are currently underway to elucidate the specific HDAC that regulates PKC gene/protein expression, while chromatin immunoprecipitation (ChIP) is being used to determine HDAC binding to the PKC promoter. Lastly, elucidating a functional consequence for HDAC inhibitor‐mediated regulation of PKCδ and PKCδ/θ protein expression and phosphorylation in muscle could yield significant insight into muscle wasting and function.Support or Funding InformationThis work is supported by the USDA NIFA (Hatch‐NEV00727, Hatch‐NEV00767), the Dennis Meiss & Janet Ralston Fund for Nutri‐epigenetic Research, the National Institute for General Medical Sciences (NIGMS) of the NIH (P20 GM130459) and the National Heart, Lung, and Blood Institute of the NIH (R15 HL143496) and NSF EPSCOR Track II (OIA‐1826801) to B.S.F.

Melatonin restores Muc2 depletion induced by V. vulnificus VvpM via melatonin receptor 2 coupling with G\u03b1q

BackgroundMelatonin (5-methoxy-N-acetyltryptamine), a hormone produced in the pineal gland, has a variety of biological functions as an antioxidant, but a functional role of melatonin in the regulation of intestinal mucin (Muc) production during bacterial infection has yet to be described in detail. In this study, we investigate the effects of melatonin during Muc2 repression elicited by the Gram-negative bacterium V. vulnificus.MethodsMucus-secreting human HT29-MTX cells were used to study the functional role of melatonin during Muc2 depletion induced by the recombinant protein (r) VvpM produced by V. vulnificus. The regulatory effects of melatonin coupling with melatonin receptor 2 (MT2) on the production of reactive oxygen species (ROS), the activation of PKCδ and ERK, and the hypermethylation of the Muc2 promoter as induced by rVvpM were examined. Experimental mouse models of V. vulnificus infection were used to study the role of melatonin and how it neutralizes the bacterial toxin activity related to Muc2 repression.ResultsRecombinant protein (r) VvpM significantly reduced the level of Muc2 in HT29-MTX cells. The repression of Muc2 induced by rVvpM was significantly restored upon a treatment with melatonin (1 μM), which had been inhibited by the knockdown of MT2 coupling with Gαq and the NADPH oxidase subunit p47 phox. Melatonin inhibited the ROS-mediated phosphorylation of PKCδ and ERK responsible for region-specific hypermethylation in the Muc2 promoter in rVvpM-treated HT29-MTX cells. In the mouse models of V. vulnificus infection, treatment with melatonin maintained the level of Muc2 expression in the intestine. In addition, the mutation of the VvpM gene from V. vulnificus exhibited an effect similar to that of melatonin.ConclusionsThese results demonstrate that melatonin acting on MT2 inhibits the hypermethylation of the Muc2 promoter to restore the level of Muc2 production in intestinal epithelial cells infected with V. vulnificus.

5-Caffeoylquinic acid ameliorates oxidative stress-mediated cell death via Nrf2 activation in hepatocytes

Context 5-Caffeoylquinic acid (5-CQA) is one of the most abundant compounds found in natural foods including coffee. Objective We investigated whether 5-CQA had a cytoprotective effect through the NF-E2-related factor 2 (Nrf2)-antioxidant response element (ARE) signalling pathway. Materials and methods Nrf2 activation in response to 5-CQA treatment at the concentration of 10–100 μM is evaluated by Western blotting of Nrf2 and ARE reporter gene assay as well as its target gene expression in HepG2 cells. Intracellular reactive oxygen species (ROS) and glutathione (GSH) levels were measured in the tert-butyl hydroperoxide-induced hepatocytes to examined cytoprotective effect of 5-CQA (10–100 μM). The specific role of 5-CQA on Nrf2 activation was examined using Nrf2 knockout cells or Nrf2 specific inhibitor, ML-385. Results Nuclear translocation of Nrf2 is increased by 5-CQA in HepG2 cells which peaked at 6 h. Consequently, 5-CQA significantly increases the ARE reporter gene activity and downstream antioxidant proteins, including glutamate cysteine ligase (GCL), hemeoxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1, and Sestrin2. Nrf2 deficiency or inhibition completely antagonized ability of 5-CQA to induce HO-1 and GCL expression. Cells pre-treated with 5-CQA were rescued from tert-butyl hydroperoxide-induced ROS production and GSH depletion. Nrf2 activation by 5-CQA was due to increased phosphorylation of MAPKs, AMPK and PKCδ. Discussion and conclusions Taken together, our results demonstrate that as a novel Nrf2 activator, 5-CQA, may be a promising candidate against oxidative stress-mediated liver injury. Additional efforts are needed to assess 5-CQA, as a potential therapeutic in liver diseases in vivo and in humans.

Src Kinase Is Biphosphorylated at Y416/Y527 and Activates the CUB-Domain Containing Protein 1/Protein Kinase C δ Pathway in a Subset of Triple-Negative Breast Cancers

Targeted therapeutics are needed for triple-negative breast cancer (TNBC). In this study, we investigated the activation of Src family of cytoplasmic tyrosine kinases (SFKs) and two SFK substrates-CUB-domain containing protein 1 (CDCP1) and protein kinase C δ (PKCδ)-in 56 formalin-fixed, paraffin-embedded (FFPE) TNBCs. Expression of SFK phosphorylated at Y416 (SFK_pY416+) in tumor cells was strongly associated with phosphorylation of CDCP1 and PKCδ (CDCP1_ pY743+ and PKCδ_pY311+), as assessed by immunohistochemistry, indicating increased SFK activity in situ. To enable biochemical analysis, protein extraction from FFPE tissue was optimized. Cleaved CDCP1 isoform (70 kDa) was expressed to a varying degree in all samples but only phosphorylated in TNBC tumor cells that expressed SFK_pY416. Interestingly, active SFK was found to be biphosphorylated (SFK_pY416+/pY527+). Biphosphorylated active SFK was observed more frequently in forkhead box protein A1 (FOXA1)- TNBCs. In addition, in SFK_pY416- samples, FOXA1+ TNBC tended to be SFK_pY527+ (classic inactive SFK), and FOXA1- TNBC tended to be SFK_pY527- (SFK poised for activation). Strong SFK_pY416 staining was also observed in tumor-infiltrating lymphocytes in a subset of TNBCs with high tumor-infiltrating lymphocyte content. This report will facilitate protein biochemical analysis of FFPE tumor samples and justifies the development oftherapies targeting the SFK/CDCP1/PKCδ pathway for TNBC treatment.

The effects of 6-hydroxydopamine on iron metabolish in astrocytes are mediated by hypoxia-inducible factor- 2Α

Disrupted iron homeostasis in the substantia nigra (SN) is involved in Parkinson's disease (PD). In astrocytes, 6-OHDA increased both DMT1 and FPN1 expression, increased iron traffic. However, the mechanisms remain elusive. Hypoxia-inducible factors (HIFs) are known to be important in regulating iron homeostasis. To explore that the mechanism of iron metabolism in astrocytes treated with 6-OHDA, using primary cultured rat astrocytes, we observed that protein levels of HIF-1α and HIF-2α in astrocytes were significantly increased by treatment with 6-OHDA, whereas expression did not change in ventral mesencephalic (VM) neurons. Furthermore using inhibitors of HIF-1α and HIF-2α, we observed that HIF-2α inhibitor markedly reduced the up-regulation of DMT1 and FPN1 by 6-OHDA and decreased ferrous iron influx and efflux. However, there was no effect following treatment of astrocytes with HIF-1α inhibitor. Activation of HIF-2α by Phorbol 12-myristate 13-acetate (PMA) and 6-OHDA was blocked by bisindolylmaleimide I hydrochloride (Bisl) and N-acetyl-l-cysteine (NAC) or Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) preventing downstream activation of DMT1 and FPN1. NAC and L-NAME both block the activation of PKCδ phosphorylation by 6-OHDA in primary cultured astrocytes. Our data indicate that HIF-2α, but not HIF-1α, regulates expression of DMT1 and FPN1 in astrocytes, and PKC pathway is involved in 6-OHDA activation of HIF-2α in astrocytes, which may be associated with ROS and NO activation of PKCδ.

High Glucose Environments Interfere with Bone Marrow-Derived Macrophage Inflammatory Mediator Release, the TLR4 Pathway and Glucose Metabolism

Macrophages may be a crucial aspect of diabetic complications associated with the inflammatory response. In this study, we examined how hyperglycaemia, a common aspect of diabetes, modulates bone marrow-derived macrophages (BMDMs) under an inflammatory stimulus. To perform this study, BMDMs from non-diabetic and diabetic (60 mg/kg alloxan, i.v.) male C57BL/6 mice (CEUA/FCF/USP-488) were cultured under normal (5.5 mM) and high glucose (HG, 25 or 40 mM) conditions and stimulated or not stimulated with lipopolysaccharide (LPS, 100 ng/mL). Compared to the BMDMs from the normoglycaemic mice, the LPS-stimulated BMDMs from the diabetic mice presented reduced TLR4 expression on the cell surface, lower phagocytic capacity, and reduced secretion of NO and lactate but greater oxygen consumption and greater phosphorylation of p46 SAPK/JNK, p42 ERK MAPK, pAKT and pPKC-δ. When the BMDMs from the non-diabetic mice were cultured under high-glucose conditions and stimulated with LPS, TLR4 expression was reduced on the cell surface and NO and H2O2 levels were reduced. In contrast, the diabetic BMDMs cultured under high glucose conditions presented increased levels of lactate and reduced phosphorylation of AKT, PKC-δ and p46 SAPK/JNK but enhanced phosphorylation of the p46 subunit of SAPK/JNK after LPS stimulation. High glucose levels appear to modify macrophage behaviour, affecting different aspects of diabetic and healthy BMDMs under the same LPS stimulus. Thus, hyperglycaemia leaves a glucose legacy, altering the basal steady state of macrophages.

Asprosin attenuates insulin signaling pathway through PKCδ-activated ER stress and inflammation in skeletal muscle.

It has been reported that asprosin is a novel adipokine which is augmented in mice and humans with type 2 diabetes (T2DM). Asprosin stimulates hepatic gluconeogenesis under fasting conditions. However, the roles of asprosin in inflammation, endoplasmic reticulum (ER) stress, and insulin resistance in skeletal muscle has not been studied. In the currents study, elevated levels of asprosin expression were observed in adipocytes under hyperlipidemic conditions. Treatment of C2C12 myocytes with asprosin-induced ER stress markers (phosphorylated inositol-requiring enzyme 1 and eukaryotic initiation factor 2, and CHOP expression) as well as inflammation markers (interleukin-6 expression, phosphorylated IκB, and nuclear translocated nuclear factor-κβ). Finally, asprosin treatment promoted exacerbation of insulin sensitivity as determined by levels of insulin receptor substrate 1 and Akt phosphorylation as well as glucose uptake. Moreover, treatment of asprosin augmented protein kinase C-δ (PKCδ) phosphorylation and nuclear translocation, but suppressed messenger RNA expression of sarcoplasmic reticulum Ca2+ ATPase 2b in both C2C12 myocytes and in mouse soleus skeletal muscle. These asprosin-induced effects were markedly decreased in small interfering (si) RNA-mediated PKCδ-knockdown in C2C12 myocytes. These results suggest that asprosin results in impairment of insulin sensitivity in skeletal muscle through PKCδ-associated ER stress/inflammation pathways and may be a valuable strategy for management of insulin resistance and T2DM.

Phosphorylation of TSC2 by PKC-δ reveals a novel signaling pathway that couples protein synthesis to mTORC1 activity.

Downstream of insulin-like growth factor receptor, the TSC1/2/ TCB1D7 (tuberous sclerosis complex) and mTOR (mechanistic target of rapamycin) pathways are implicated in many human diseases, including cancer and diabetes. Targeting this pathway is currently an important approach for palliating or eradicating cancer. Downstream of mTOR, translational machinery targeting holds great promise for anticancer drug development. Therefore, we investigated whether the protein synthesis machinery that is regulated by mTORC1 (mTOR complex 1) signaling can in turn regulate mTORC1 activity. We found that inhibition of protein synthesis results in rapid activation of mTORC1 signaling, thereby uncovering a feedback loop between mTOR and the translation machinery. This mTORC1 activation requires tuberous sclerosis complex (TSC) but is independent of AKT. In addition, by using a PKC-δ (protein kinase c delta)-specific inhibitor and PKC-δ siRNA knockdown, we found that PKC-δ kinase activity is required for mTORC1 activation in response to translation inhibitors. Furthermore, translation inhibition activates PKC-δ. Subsequently, we investigated whether PKC-δ can phosphorylate and inactivate TSC1/2, leading to mTORC1 activation. In vitro kinase assays showed direct phosphorylation of TSC2 (S932 and S939) by PKC-δ, which was confirmed by mass spectrometry. In vivo kinase analysis further indicated that both S932 and S939 are phosphorylated in response to translation inhibitors. Finally, phosphorylation defective TSC2 mutants (S932A and S939A single mutants and a S932A/S939A double mutant) failed to upregulate mTORC1 activity in the presence of translation inhibitors, suggesting that activation of mTORC1 by translation inhibitors is mediated by PKC-δ phosphorylation of TSC2 at S932/S939, which inactivates TSC.

Anti-inflammatory property of quercetin through downregulation of ICAM-1 and MMP-9 in TNF-α-activated retinal pigment epithelial cells

Quercetin is a flavonoid polyphenolic compound present in fruits and vegetables that has proven anti-inflammatory activity. The goal of the present investigation was to investigate the effects of quercetin on tumor necrosis factor-α (TNF-α)-induced inflammatory responses via the expression of ICAM-1 and MMP-9 in human retinal pigment epithelial cells (ARPE-19 cells). Real-time PCR, gelatin zymography, and Western blot analysis showed that TNF-α induced the expression of ICAM-1 and MMP-9 protein and mRNA in a time-dependent manner. These effects were attenuated by pretreatment of ARPE-19 cells with quercetin. Quercetin inhibited the TNF-α-induced phosphorylation of PKCδ, JNK1/2, ERK1/2. Quercetin, rottlerin, SP600125 and U0126 attenuated TNF-α-stimulated c-Jun phosphorylation and AP-1–Luc activity. Pretreatment with quercetin, rottlerin, SP600125, or Bay 11-7082 attenuated TNF-α-induced NF-κB (p65) phosphorylation, translocation and RelA/p65–Luc activity. TNF-α significantly increased MMP-9 promoter activity and THP-1 cell adherence, and these effects were attenuated by pretreatment with quercetin, rottlerin, SP600125, U0126, tanshinone IIA or Bay 11-7082. These results suggest that quercetin attenuates TNF-α-induced ICAM-1 and MMP-9 expression in ARPE-19 cells via the MEK1/2–ERK1/2 and PKCδ–JNK1/2–c-Jun or NF-κB pathways.

C-Src regulates Nrf2 activity through PKCδ after oxidant stimulus

Nrf2 is the main transcription factor involved in expression of cell defense enzymes, which is altered in several oxidant-related disorders. Cytosolic Nrf2 activation is modulated through phosphorylation by PKCδ, an enzyme controlled by Src tyrosine kinases. Of relevance, Src family members are involved in numerous cellular processes and regulated by hydrogen peroxide (H2O2). In this study we analysed the activation of cell survival-related signaling proteins, c-Src and Nrf2, and the influence of c-Src kinase on Nrf2 regulation after exposure to H2O2. Acute exposure of HT22 mouse hippocampal neural cells to H2O2 increased c-Src and Nrf2 phosphorylation/activation at Tyr416 and Ser40, respectively. Nrf2 phosphorylation at Ser40, its nuclear accumulation and transcriptional activity involving heme oxygenase-1 (HO-1) expression were dependent on c-Src kinase activation. Moreover, modulation of Nrf2 activity by c-Src occurred through PKCδ phosphorylation at Tyr311. We demonstrate, for the first time, c-Src-mediated regulation of Nrf2 transcriptional activity, via PKCδ activation, following an acute H2O2 stimulus. This work supports that the c-Src/PKCδ/Nrf2 pathway may constitute a novel signaling pathway stimulated by H2O2 and a potential target for the treatment of diseases involving redox deregulation.

Pro-Inflammatory Stimuli Influence Expression of Intercellular Adhesion Molecule 1 in Human Anulus Fibrosus Cells through FAK/ERK/GSK3 and PKCδ Signaling Pathways.

Objective: Intervertebral disc (IVD) degeneration and disc herniation are major causes of lower back pain, which involve the presence of inflammatory mediators and tissue invasion by immune cells. Intercellular adhesion molecule 1 (ICAM1, also termed CD54) is an adhesion molecule that mediates cell-cell interactions, particularly between immune cells and target tissue. The aim of this study was to examine the intracellular signaling pathways involved in inflammatory stimuli-induced ICAM1 expression in human anulus fibrosus (AF) cells. Methods: Quantitative reverse transcription-polymerase chain reaction (qPCR), western blotting, and flow cytometry were performed to dissect the roles of different signaling pathways in inflammatory stimuli-mediated ICAM1 expression. Results: Using qPCR and western blot analyses, a significant increase in ICAM1 expression was observed in AF cells after stimulation of lipopolysaccharide (LPS) plus interferon-gamma (IFNγ) in a time-dependent manner. Flow cytometry revealed ICAM1 upregulation on the surface of AF cells. Importantly, LPS plus IFNγ treatment also significantly promoted Chemokine ligand (CCL)2 expression, but not CCL3. The enhanced ICAM1 expression was abolished after incubation with antibody against CCL2. In AF cells, treatment with LPS plus IFNγ activated the FAK/ERK/GSK3 signaling pathways, promoted a time-dependent increase in PKCδ phosphorylation, and promoted PKCδ translocation to the nucleus. Treatment with the pharmacological PKCδ inhibitor; rottlerin, effectively blocked the enhanced productions of ICAM1 and CCL2. Conclusions: Inflammatory stimuli in AF cells are part of a specific pathophysiology in IVD degeneration and disc herniation that modulates CCL2/ICAM1 activation through the FAK/ERK/GSK3 and PKCδ signaling pathways in AF cells.

Inhibitory mechanism of ginsenoside Rh3 on granulocyte–macrophage colony-stimulating factor expression in UV-B–irradiated murine SP-1 keratinocytes

BackgroundUltraviolet (UV) goes through the epidermis and promotes release of inflammatory cytokines in keratinocytes. Granulocyte–macrophage colony-stimulating factor (GM-CSF), one of the keratinocyte-derived cytokines, regulates proliferation and differentiation of melanocytes. Extracellular signal–regulated kinase (ERK1/2) and protein kinase C (PKC) signaling pathways regulate expression of GM-CSF. Based on these results, we found that ginsenoside Rh3 prevented GM-CSF production and release in UV-B–exposed SP-1 keratinocytes and that this inhibitory effect resulted from the reduction of PKCδ and ERK phosphorylation. MethodsWe investigated the mechanism by which ginsenoside Rh3 from Panax ginseng inhibited GM-CSF release from UV-B–irradiated keratinocytes. ResultsTreatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or UV-B induced release of GM-CSF in the SP-1 keratinocytes. To elucidate whether the change in GM-CSF expression could be related to PKC signaling, the cells were pretreated with H7, an inhibitor of PKC, and irradiated with UV-B. GM-CSF was decreased by H7 in a dose-dependent manner. When we analyzed which ginsenosides repressed GM-CSF expression among 15 ginsenosides, ginsenoside Rh3 showed the largest decline to 40% of GM-CSF expression in enzyme-linked immunosorbent assay. Western blot analysis showed that TPA enhanced the phosphorylation of PKCδ and ERK in the keratinocytes. When we examined the effect of ginsenoside Rh3, we identified that ginsenoside Rh3 inhibited the TPA-induced phosphorylation levels of PKCδ and ERK. ConclusionIn summary, we found that ginsenoside Rh3 impeded UV-B–induced GM-CSF production through repression of PKCδ and ERK phosphorylation in SP-1 keratinocytes.

Effect of Royal Jelly and Brazilian Green Propolis on the Signaling for Histamine H1 Receptor and Interleukin-9 Gene Expressions Responsible for the Pathogenesis of the Allergic Rhinitis.

The significant correlation between nasal symptom scores and level of histamine H1 receptor (H1R) mRNA in nasal mucosa was observed in patients with pollinosis, suggesting that H1R gene is an allergic disease sensitive gene. We demonstrated that H1R and interleukin (IL)-9 gene are the allergic rhinitis (AR)-sensitive genes and protein kinase Cδ (PKCδ) signaling and nuclear factor of activated T-cells (NFAT) signaling are involved in their expressions, respectively. Honey bee products have been used to treat allergic diseases. However, their pathological mechanism remains to be elucidated. In the present study, we investigated the mechanism of the anti-allergic effect of royal jelly (RJ) and Brazilian green propolis (BGPP). Treatment with RJ and BGPP decreased in the number of sneezing on toluene 2,4-diissocyanate (TDI)-stimulated rats. The remarkable suppression of H1R mRNA in nasal mucosa was observed. RJ and BGPP also suppressed the expression of IL-9 gene. RJ and BGPP suppressed phorbol-12-myristate-13-acetate-induced Tyr311 phosphorylation of PKCδ in HeLa cells. In RBL-2H3 cells, RJ and BGPP also suppressed NFAT-mediated IL-9 gene expression. These results suggest that RJ and BGPP improve allergic symptoms by suppressing PKCδ and NFAT signaling pathways, two important signal pathways for the AR pathogenesis, and suggest that RJ and BGPP could be good therapeutics against AR.

Chemotactic effect of S100A8 and S100A9 on human eosinophilic leukemia cells, EoL-1 through TLR4

S100A8 and S100A9 function as key factors in inflammatory responses including cell survival, differentiation and chemotactic activity. Chronic eosinophilic leukemia (CEL) is a rare hematological malignancy with eosinophilia. In this study, we investigated the contribution of S100A8 and S100A9 to chemotaxis of the human eosinophilic leukemia cell line, EoL-1. A chemotaxis assay, western blot analysis and a NF-κB transcription factor assay were conducted to investigate the chemotactic mechanism. S100A8 and S100A9 induced the migration of EoL-1 cells via the phosphorylation of PKCδ, AKT, and MAPKs and the translocation of NF-κB; however, they had no effect on eosinophils from normal peripheral blood. PKCδ, AKT, and MAPKs such as ERK, p38 MAPK, and JNK are upstream regulator molecules of NF-κB activation. Iκ-Bα degradation is needed for NF-κB activation. These findings suggest that S100A8 and S100A9 induce the cell movement and contribute to an understanding of S100A8 and S100A9 in eosinophil biology and the pathogenic mechanism of hematological malignancy.

Exposure to far-infrared rays attenuates methamphetamine-induced recognition memory impairment via modulation of the muscarinic M1 receptor, Nrf2, and PKC

We demonstrated that activation of protein kinase Cδ (PKCδ) and inactivation of the glutathione peroxidase-1 (GPx-1)-dependent systems are critical for methamphetamine (MA)-induced recognition memory impairment. We also demonstrated that exposure to far-infrared rays (FIR) causes induction of the glutathione (GSH)-dependent system, including induction of the GPx-1 gene. Here, we investigated whether exposure to FIR rays affects MA-induced recognition memory impairment and whether it modulates PKC, cholinergic receptors, and the GSH-dependent system. Because the PKC activator bryostatin-1 mainly induces PKCα, PKCε, and PKCδ, we assessed expression of these proteins after MA treatment. MA treatment selectively increased PKCδ expression and its phosphorylation. Exposure to FIR rays significantly attenuated MA-induced increases in PKCδ phosphorylation. Importantly, bryostatin-1 potentiated MA-induced phosphorylation of PKCδ. MA treatment significantly decreased M1, M3, and M4 muscarinic acetylcholine receptors (mAChRs) and β2 nicotinic acetylcholine receptor expression. Of these, the decrease was most pronounced in M1 mAChR. Exposure to FIR significantly attenuated MA-induced decreases in the M1 mAChR and phospho-ERK1/2, while it facilitated Nrf2-dependent GSH induction. Dicyclomine, an M1 mAChR antagonist, and l-buthionine-(S, R)-sulfoximine (BSO), an inhibitor of GSH synthesis, counteracted against the protective potentials mediated by FIR. More importantly, the memory-enhancing potential of FIR rays was significantly counteracted by bryostatin-1, dicyclomine, and BSO. Our results suggest that exposure to FIR rays attenuates MA-induced impairment in recognition memory via up-regulation of M1 mAChR, Nrf2-dependent GSH induction, and ERK1/2 phosphorylation by inhibiting PKCδ phosphorylation by bryostatin-1.

Isoniazid induces apoptosis: Role of oxidative stress and inhibition of nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)

AimsLong-term treatment of Isoniazid (INH) in tuberculosis (TB) patients can lead to anti-tuberculosis drug-induced hepatotoxicity. To understand the mechanism of hepatotoxicity, an attempt has been made to elucidate the role of Nrf2, a transcription factor induced by oxidative stress, in INH induced apoptosis liver cancer cell lines. Materials and methodsCytotoxicity was evaluated by MTT assay. Apoptosis and reactive oxygen species (ROS) generation was performed by flow cytometry. mRNA and protein expression of various genes involves in INH induced toxicity was evaluated via Real-time PCR and western blot analysis respectively. Differential protein expression was performed by two-dimensional gel electrophoresis followed by identification using MALDI TOF/TOF. Key findingsINH induced ROS and apoptosis in HepG2 as well as THLE-2 cells. Nuclear damage was also observed by INH treatment in HepG2 cells. Expression of apoptotic (Cytochrome C and Caspase 9) and antioxidative (Keap1 and Nrf2) genes were observed to increase. INH induced PKCδ phosphorylation and released Nrf2 from its inhibitor Keap1 in the cytoplasm of HepG2 cells. However, over-expression of Nrf2 did not affect nuclear Nrf2 protein level as well as its downstream target NQO1. Nrf2 importer, Karyopherin β1 level was observed to decrease in HepG2 as well as THLE-2 cells following INH treatment. SignificanceThese findings suggest that INH prevented Nrf2 translocation into the nucleus by inhibiting its importer Karyopherin β1. Therefore Nrf2 might not able to bind ARE sequences from inducing antioxidative response for protecting the cells undergoing apoptosis.

Intracellular zinc increase affects phosphorylation state and subcellular localization of protein kinase C delta (δ)

Protein kinase C delta (PKCδ) is a Ser/Thr-specific kinase involved in many fundamental cellular processes including growth, differentiation and apoptosis. PKCδ is expressed ubiquitously in all known cell types, and can be activated by diacylglycerol, phorbol esters and other kinases. Multiple lines of evidence have indicated that the mode of activation greatly influences the role PKCδ plays in cellular function. Divalent metal ions, such as zinc are released as a response to cellular stress and injury, often resulting in oxidative damage and cell death. In this study, we evaluate the effect increased concentrations of intracellular zinc has on the phosphorylation state and subcellular localization of PKCδ. More specifically, we demonstrate that intracellular zinc inhibits the phosphorylation of PKCδ at Thr505 in a concentration-dependent manner and facilitates the translocation of PKCδ from the cytosol to the Golgi complex. Analysis of a PKCδ structural model revealed a potential His-Cys3 zinc-binding domain adjacent to residue Thr505 and suggests that interaction with a Zn2+ ion may preclude phosphorylation at this site. This study establishes zinc as a potent modulator of PKCδ function and suggests a novel mechanism by which PKCδ is able to “sense” changes in the concentration of intracellular zinc. These findings illuminate a new paradigm of metal ion-protein interaction that may have significant implications on a broad spectrum of cellular processes.

Tyrosine Kinase Inhibitors Protect the Salivary Gland from Radiation Damage by Inhibiting Activation of Protein Kinase C-δ.

In patients undergoing irradiation (IR) therapy, injury to nontumor tissues can result in debilitating, and sometimes permanent, side effects. We have defined protein kinase C-δ (PKCδ) as a regulator of DNA damage-induced apoptosis and have shown that phosphorylation of PKCδ by c-Abl and c-Src activates its proapoptotic function. Here, we have explored the use of tyrosine kinase inhibitors (TKI) of c-Src and c-Abl to block activation of PKCδ for radioprotection of the salivary gland. Dasatinib, imatinib, and bosutinib all suppressed tyrosine phosphorylation of PKCδ and inhibited IR-induced apoptosis in vitro To determine whether TKIs can provide radioprotection of salivary gland function in vivo, mice were treated with TKIs and a single or fractionated doses of irradiation. Delivery of dasatinib or imatinib within 3 hours of a single or fractionated dose of irradiation resulted in >75% protection of salivary gland function at 60 days. Continuous dosing with dasatinib extended protection to at least 5 months and correlated with histologic evidence of salivary gland acinar cell regeneration. Pretreatment with TKIs had no impact on clonogenic survival of head and neck squamous cell carcinoma (HNSCC) cells, and in mice harboring HNSCC cell-derived xenografts, combining dasatinib or imatinib with fractionated irradiation did not enhance tumor growth. Our studies indicate that TKIs may be useful clinically to protect nontumor tissue in HNC patients undergoing radiotherapy, without negatively impacting cancer treatment. Mol Cancer Ther; 16(9); 1989-98. ©2017 AACR.

N-Methyl-D-Aspartate Receptor-Driven Calcium Influx Potentiates the Adverse Effects of Myocardial Ischemia-Reperfusion Injury Ex Vivo.

Background:Despite the adverse effects of N-methyl-d-aspartate receptor (NMDAR) activity in cardiomyocytes, no study has yet examined the effects of NMDAR activity under ex vivo ischemic-reperfusion (I/R) conditions. Therefore, our aim was to comprehensively evaluate the effects of NMDAR activity through an ex vivo myocardial I/R rat model.Methods:Isolated rat hearts were randomly segregated into 6 groups (n = 20 in each group): (1) an untreated control group; (2) a NMDA-treated control group; (3) an untreated I/R group; (4) an I/R+NMDA group treated with NMDA; (5) an I/R+NMDA+MK-801 group treated with NMDA and the NMDAR inhibitor MK-801; and (6) an I/R+NMDA+[Ca2+]-free group treated with NMDA and [Ca2+]-free buffer. The 4 I/R groups underwent 30 minutes of ischemia followed by 50 minutes of reperfusion. Left ventricular pressure signals were analyzed to assess cardiac performance. Myocardial intracellular calcium levels ([Ca2+]i) were assessed in isolated ventricular cardiomyocytes. Creatine kinase, creatine kinase isoenzyme MB, lactate dehydrogenase, cardiac troponin I, and cardiac troponin T were assayed from coronary effluents. TTC and TUNEL staining were used to measure generalized myocardial necrosis and apoptosis levels, respectively. Western blotting was applied to assess the phosphorylation of PKC-δ, PKC-ε, Akt, and extracellular signal-regulated kinase.Results:Enhanced NMDAR activity under control conditions had no significant effects on the foregoing variables. In contrast, enhanced NMDAR activity under I/R conditions produced significant increases in [Ca2+]i levels (∼1.2% increase), significant losses in left ventricular function (∼5.4% decrease), significant multi-fold increases in creatine kinase, creatine kinase isoenzyme MB, lactate dehydrogenase, cardiac troponin I, and cardiac troponin T, significant increases in generalized myocardial necrosis (∼36% increase) and apoptosis (∼150% increase), and significant multi-fold increases in PKC-δ, PKC-ε, Akt, and extracellular signal-regulated kinase phosphorylation (all P < 0.05). These adverse effects were rescued by the NMDAR inhibitor MK-801 or [Ca2+]-free buffer (all P < 0.05).Conclusions:NMDAR-driven calcium influx potentiates the adverse effects of myocardial I/R injury ex vivo.