Characterization of free radical defense system in high glucose cultured HeLa-tat cells: consequences for glucose-induced cytotoxicity

HeLa cell line stably transfected with the tat gene from human immunodeficiency virus type 1 has a decreased antioxidant potential. In this work, we used this model to investigate the effect of a high glucose level (20 mM) on the glucose induced cytotoxicity and on the antioxidant system. In comparison to cell culture under control medium, HeLa-wild cell cultured under 20 mM glucose did not exhibit necrosis or apoptosis, contrary to HeLa-tat cell presenting a significant increase in necrotic or apoptotic state. Moreover after 48 h culture under high glucose level the HeLa-tat proliferation rate was not higher than the one of HeLa-wild cells. In HeLa-wild cell high glucose level resulted in an induction of glutathione reductase activity in opposition to HeLa-tat cells where no change was observed. High glucose level resulted in 20% increase in GSSG/GSH ratio in HeLa-wild cells and 38% increase in HeLa-tat cells. Moreover, high glucose level resulted in a dramatic cytosolic thiol decrease and an important lipid peroxidation in HeLa-tat cells. No significant change of these two parameters was observed in HeLa-wild cells. In both cell lines, high glucose resulted in an increase of total SOD activity, as a consequence of the increase in Cu,Zn-SOD activity. High glucose did not result in an increase of Mn-SOD activity in both cell lines. As a consequence of tat tranfection Mn-SOD activity was 50% lower in HeLa-tat cells in comparison to HeLa-wild cells. This work emphasizes the importance of the antioxidant system in the glucose induced cytotoxicity.     

Structural distinction between soluble and particulate protein kinase C species

A number of peripheral membrane proteins functioning as regulatory enzymes are distributed between soluble and particulate fractions upon homogenization and subcellular fractionation. One such enzyme, the Ca²⁺/phospholipid-dependent protein kinase, protein kinase C, was analyzed in order to examine this characteristic of differential localization. The soluble and particulate forms of this enzyme were purified to relative homogeneity, and their biochemical and biophysical properties were analyzed and compared. Based on biochemical activities, the particulate form required lower phospholipid concentrations for maximal activation than for the soluble species. The particulate species had a more hydrophobic structure as demonstrated by a hydrophobic fluorescence probe, and had almost 50% more -helical structures according to secondary structure estimation, determined from far ultra-violet-circular dichroism spectra (200–250 nm). Using Fourier transform infrared spectroscopy, specific lipid spectra were detected associated with the soluble protein kinase C species. Further analyses with a fluorescent neutral membrane probe suggested that there was more lipid associated with the purified particulate form, which was of a less mobile nature than those associated with the soluble species. These structural differences provide an explanation for the preferential localization of the enzyme and may prove to be the basis for distribution of other membrane-active peripheral membrane regulatory enzymes.     

Effects of glucose, tetrapyrroles and protein kinase C activators on cell proliferation in cultures of Saccharomyces cerevisiae

Abstract Saccharomyces cerevisiae was inoculated into a yeast nitrogen base with either glycerol or glucose as carbon source. Cell proliferation was followed by colony counts on agar medium. Cells in the glycerol-supplemented medium divided less than once in 10 days. When glucose, 6-deoxy-glucose or protoporphyrin IX was added, the cells had doubling times of about 24 h and increased in number to about 0.5 × 10⁶ cells ml⁻¹ Addition of either of the protein kinase C activators oleoyl-acetylglycerol or phorbol-12-myristate-13-acetate did not activate cell proliferation in the glycerol medium. However, when (i) glucose was combined with either protoporphyrin IX or chlorophyllin, or (ii) either protoporphyrin IX or chlorophyllin was combined with either of the protein kinase C activators, the cells had doubling times of about 12 h. Hence, (i) glucose can act as both a carbon source and a signalling molecule for proliferation, and (ii) two systems are involved in activating cell proliferation in S. cerevisiae : one operating through a protein kinase C system and another through a guanylate cyclase system.     

Activation of protein kinase C by a tumor-promoting phorbol ester in pancreatic islets

Rat pancreatic islet homogenates display protein kinase C activity. This phospholipid-dependent and calcium-sensitive enzyme is activated by diacylglycerol or the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In the presence of TPA, the Ka for Ca2+ is close to 5 microM. TPA does not affect phosphoinositide turnover but stimulates [32P]- and [3H]choline-labelling of phosphatidylcholine in intact islets. Exogenous phospholipase C stimulates insulin release, in a sustained and glucose-independent fashion. The secretory response to phospholipase C persists in media deprived of CaCl2. It is proposed that protein kinase C participates in the coupling of stimulus recognition to insulin release evoked by TPA, phospholipase C and, possibly, those secretatogues causing phosphoinositide breakdown in pancreatic islets.     

Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages

This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca²⁺-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-κB (NF-κB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca²⁺-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca²⁺-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC- and -β activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-κB-specific DNA–protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-κB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-κB activation. These data suggest that persistent inhibition of ER Ca²⁺-ATPase by TG would influence calcium release from ER Ca²⁺ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-κB activation that induces iNOS expression and NO production.     

Structure and function of the protein kinase C gene family

Protein kinase C is a serine/threonine protein kinase which is activated in the cell in response to production of diacylglycerol. Gene cloning has revealed the presence of a highly related family of enzymes, which can be sub-divided into groups on the basis of sequence conservation. Differences are seen in both isoform distribution and associated biochemical activity, for example in substrate specificity and activator requirements. Comparison of the protein sequences andin vitro activities of the protein kinase C isoforms has identified regions important for particular aspects of kinase function. Some of these regions are also found associated with other proteins, allowing confirmation of the assigned activity. Site-directed mutagenesis has confirmed the presence of an autoinhibitory sequence involved in protein kinase C regulation and generated constitutively activated proteins which can be used to study differential isoform function. These same sequences have been shown to play a role in substrate selection, perhaps by competition for binding to the active site. Protein kinase C is known to be a phosphoprotein and the identification of regulatory sites phosphorylated by a ‘PKC-kinase’ suggest a possible alternative route for regulation of protein kinase C activity.     

Parallel regulation of arginine transport and nitric oxide synthesis by angiotensin II in vascular smooth muscle cells role of protein kinase C

Summary Experiments were performed to characterize arginine transport in vascular smooth muscle cells (SMCs) and the effect of angiotensin II (Ang II) on this process. In addition, the role of arginine transport in the cytokineinduced nitric oxide (NO) production was assessed. Arginine transport takes place through Na⁺-independent (60%) and Na⁺-dependent pathways (40%). The Na⁺-independent arginine uptake appears to be mediated by system y⁺ because of its sensitivity to cationic amino acids such as lysine, ornithine and homoarginine. The transport system was relatively insensitive to acidification of the extracellular medium. By contrast, the Na⁺-dependent pathway is consistent with system B^0,+ since it was inhibited by both cationic and neutral amino acids (i.e., glutamine, phenylalanine, and asparagine), and did not accept Li⁺ as a Na⁺ replacement. Treatment of SMCs with 100nM Ang II significantly inhibited the Na⁺-dependent arginine transport without affecting systems y⁺, A, and L. This effect occurred in a dose-dependent manner (IC₅₀ of 8.9 ± 0.9nM) and is mediated by the AT-1 receptor subtype because it was blocked by DUP 753, a non-peptide antagonist of this receptor. The inhibition of system B^0,+ by Ang II is mediated by protein kinase C (PKC) because it was mimicked by phorbol esters (phorbol 12-myristate 13-acetate) and was inhibited by staurosporine. Ang II also inhibited the IL-1 induced nitrite accumulation by SMCs. This action was also inhibited by staurosporine and reproduced with phorbol esters, suggesting a coupling between arginine uptake and NO synthesis through a PKC-dependent mechanism. However, arginine supplementation in the medium (10mM) failed to prevent the inhibitory action of Ang II on NO synthesis. These findings suggest that although Ang II inhibits concomitantly arginine transport and NO synthesis in SMCs, the reduction of NO synthesis is not associated with alterations in the cellular transport of arginine.Abbreviations Arg arginine - Orn ornithine - HmR homoarginine - Lys lysine - Gln glutamine - Asn asparagine - His histidine - Phe phenylalanine - Leu leucine - Cys Cysteine - Ala alanine - Ser serine - Thr threonine - Glu glutamate - mAIB -methyl-aminoisobutyric acid - BCH bicycloaminoheptane     

Angiotensin II-induced decrease in expression of inducible nitric oxide synthase in rat astroglial cultures: role of protein kinase C

Inducible nitric oxide synthase (iNOS) has been implicated as a mediator of cellular toxicity in a variety of neurodegenerative disorders. Nitric oxide, which is generated in high quantities following induction of iNOS, combines with other oxygen radicals to form highly reactive, death-inducing compounds. Given the frequency of neuronal death due to neurodegenerative diseases, cerebral trauma, and stroke, it is important to study the mechanisms of regulation of iNOS in the brain. We demonstrated previously that angiotensin II (Ang II) decreases the expression of iNOS produced by bacterial endotoxin or cytokines in cultured astroglia prepared from adult rat brain. Here, we have addressed the mechanisms by which Ang II negatively modulates iNOS. The inhibitory effects of Ang II on lipopolysaccharide-induced expression of iNOS mRNA and protein and nitrite accumulation were mimicked by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate. Down-regulation of PKC produced by long-term treatment of astroglia with phorbol 12-myristate 13-acetate abolished the inhibitory effect of Ang II on lipopolysaccharide-stimulated expression of iNOS mRNA and nitrite accumulation. Finally, the reduction of lipopolysaccharide-induced nitrite accumulation by Ang II was attenuated by the selective PKC inhibitor chelerythrine. Collectively, these data indicate a role for PKC in the inhibitory actions of Ang II on iNOS expression in cultured astroglia.     

Mutual regulation of conventional protein kinase C and a ubiquitin ligase complex

Several isoforms of protein kinase C (PKC) are degraded by the ubiquitin-proteasome pathway after phorbol ester-mediated activation. However, little is known about the ubiquitin ligase (E3) that targets activated PKCs. We recently showed that an E3 complex composed of HOIL-1L and HOIP (LUBAC) generates linear polyubiquitin chains and induces the proteasomal degradation of a model substrate. HOIL-1L has also been characterized as a PKC-binding protein. Here we show that LUBAC preferentially binds activated conventional PKCs and their constitutively active mutants. LUBAC efficiently ubiquitinated activated PKC in vitro, and degradation of activated PKCalpha was delayed in HOIL-1L-deficient cells. Conversely, PKC activation induced cleavage of HOIL-1L and led to downregulation of the ligase activity of LUBAC. These results indicate that LUBAC is an E3 for activated conventional PKC, and that PKC and LUBAC regulate each other for proper PKC signaling.     

Phosphorylation of liver gap junction protein by protein kinase C

The 27 kDa protein, a major component of rat liver gap junctions, was shown to be phosphorylated in vitro by protein kinase C. The stoichiometry of the phosphorylation indicated that approx. 0.33 mol phosphate was incorporated per mol 27 kDa protein. Phosphorylation was entirely dependent on the presence of calcium and was virtually specific for serine residues. For comparison, the gap junction protein was also examined for its phosphorylation by cAMP-dependent protein kinase, the extent of phosphorylation being one-tenth that exerted by protein kinase C.     

蛋白激酶C对胰岛素抵抗骨骼肌细胞的影响

目的探讨蛋白激酶C(Protein Kinase C,PKC)在棕榈酸(Palmitic Acid,PA)诱导的骨骼肌细胞胰岛素抵抗(Isulin Resistance,IR)中的作用。方法免疫荧光鉴定原代大鼠骨骼肌细胞,氧化酶-过氧化物酶偶联法(GOD-POD法)检测培养液中葡萄糖浓度。设立对照组、棕榈酸组(PA组)、罗格列酮组(Rosiglitazone,Ros组),每组一分为二,分别加PKC抑制剂白屈莱红碱(Chelerythrine Chloride,CC)与正常培养液作用1h,Western Blot检测PKB及P-Ser473 PKB表达水平。结果 90%以上的细胞-αsarcometric actin免疫荧光染色呈阳性反应,表明培养的细胞为骨骼肌细胞;0.6mmol/L的PA作用24h可诱导骨骼肌细胞产生胰岛素抵抗;PA组与对照组相比P-Ser473 PKB水平显著降低,与本组未加CC相比显著升高。同时,罗格列酮组及本组加CC中P-Ser473PKB水平均高于PA组。结论在PA诱导的骨骼肌细胞IR方面PKC起重要作用,罗格列酮与PKC抑制剂CC均能改善PA引起的IR。     

NADPH oxidase NOX1 is involved in activation of protein kinase C and premature senescence in early stage diabetic kidney

Increased oxidative stress and activation of protein kinase C (PKC) under hyperglycemia have been implicated in the development of diabetic nephropathy. Because reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, NOX1 accelerate the translocation of PKC isoforms, NOX1 is postulated to play a causative role in the development of diabetic nephropathy. Hyperglycemia was induced in wild-type and Nox1-deficient mice (KO) by two doses of streptozotocin injection. At 3 weeks after the induction of hyperglycemia, glomeruli and cortical tubules were isolated from kidneys. The mRNA level of Nox1 was significantly upregulated in the renal cortex at 3 weeks of hyperglycemia. Urinary albumin and expression of inflammatory or fibrotic mediators were similarly elevated in diabetic wild-type and KO; however, increases in glomerular volume and mesangial matrix area were attenuated in diabetic KO. Nox1 deficiency significantly reduced the levels of renal thiobarbituric acid-reacting substances and 8-hydroxydeoxyguanosine, membranous translocation of PKCα/β, activity of PKC, and phosphorylation of p38 mitogen-activated protein kinase in the diabetic kidney. Furthermore, increased staining of senescence-associated β-galactosidase in glomeruli and cortical tubules of diabetic mice was significantly suppressed in KO. Whereas the levels of cyclin-dependent kinase inhibitors, p16^INK4A and p21^Cip1, were equivalent between the genotypes, increased levels of p27^Kip1 and γ-H2AX, a biomarker for DNA double-strand breaks, were significantly attenuated in isolated glomeruli and cortical tubules of diabetic KO. Taken together, NOX1 modulates the p38/p27^Kip1 signaling pathway by activating PKC and promotes premature senescence in early stage diabetic nephropathy.     

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

Rises in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca²⁺]_cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca²⁺]_cyt (Ca²⁺ buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca²⁺ or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca²⁺ signalling, we here monitor Ca²⁺ flux around the platelet by measuring net Ca²⁺ fluxes to or from the extracellular space and the intracellular Ca²⁺ stores, which act as the major sources and sinks for Ca²⁺ influx into and efflux from the cytosol, as well as monitoring the cytosolic Na⁺ concentration ([Na⁺]_cyt), which influences platelet Ca²⁺ fluxes via Na⁺/Ca²⁺ exchange. The intracellular store Ca²⁺ concentration ([Ca²⁺]_st) was monitored using Fluo-5N, the extracellular Ca²⁺ concentration ([Ca²⁺]_ext) was monitored using Fluo-4 whilst [Ca²⁺]_cyt and [Na⁺]_cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca²⁺]_cyt in the absence of extracellular Ca²⁺. PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca²⁺ release and Ca²⁺ removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca²⁺]_cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na⁺]_cyt which would be expected to reduce Ca²⁺ removal via the Na⁺/Ca²⁺ exchanger (NCX). Thrombin-evoked rises in [Na⁺]_cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non-selective cation permeability of the plasma membrane as assessed by Mn²⁺ quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca²⁺]_cyt following SERCA inhibition and either removal of extracellular Na⁺ or inhibition of Na⁺/K⁺-ATPase activity by removal of extracellular K⁺ or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca²⁺]_cyt by acceleration of SERCA activity, whilst rises in [Ca²⁺]_cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na⁺/K⁺-ATPase activity, with the latter limiting the effect of thrombin on rises in [Na⁺]_cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca²⁺ signalling.     

Activation by phorbol esters of protein kinase C in MCF-7 human breast cancer cells

Exposure of MCF-7 human breast cancer cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to the inhibition of cell proliferation. We investigate here the short-term effects of TPA on subcellular distribution of protein kinase C, and on protein phosphorylation in cultured MCF-7 cells. We report a rapid and dramatic decrease in cytosolic protein kinase C activity after TPA treatment. Only 30% of the enzymatic activity lost in the cytosol was recovered in the particulate fraction. These data suggest that subcellular translocation of protein kinase C is accompanied by a rapid down-regulation of the enzyme (70%). Furthermore, TPA and other protein kinase C activators rapidly induce the phosphorylation of a 28 kDa protein in intact MCF-7 cells. Phorbol esters devoid of tumor-promoting activity are ineffective both for inducing these early biochemical events and for inhibiting cell proliferation.     

Actinomycin D decreases protein kinase C content and induces neuritogenesis in neuroblastoma cells

We have previously reported that inhibition of protein kinase C induces differentiation of neuroblastoma cells in culture. It is shown now that actinomycin D, a well known inhibitor of DNA synthesis, reduces selectively the content of protein kinase C and induces neuritogenesis in Neuro 2a cells in culture.     

Palmitoylcarnitine modulates interaction protein kinase C delta-GAP-43

Palmitoylcarnitine, reported previously to promote neuronal differentiation, was observed to affect distribution of protein kinase C (PKC) isoforms in neuroblastoma NB-2a cells, leading to retardation in cytoplasm of high molecular weight species of PKCbeta and delta. Growth cone protein-GAP-43, a PKC substrate, was co-immunoprecipitated with all the conventional and novel PKCs: palmitoylcarnitine, however, decreased its amount exclusively in the complex with PKCdelta. Administration of palmitoylcarnitine, although did not change the subcellular distribution of GAP-43, decreased its phosphorylation, which could regulate other signal transduction pathways (calmodulin and G(0)-dependent).