Phospholipid-dependent Protein Kinase C Research Articles

Nailing down PKC isoform specificity in diabetic nephropathy two's company, three's a crowd

Diabetic nephropathy is characterized by early vascu-lar dysfunction and increasing matrix accumulation inthe kidney, eventually leading to proteinuria, glomer-ulosclerosis and interstitial fibrosis [1]. Over the lastdecade, our understanding of the molecular mecha-nisms and the pathogenesis of diabetic nephropathy(and other diabetic microvascular complications) hasbeen greatly enhanced [2]. However, we still do notcompletely understand how metabolic disturbances inthe diabetic state, i.e. hyperglycaemia, induce sucha vast array of distinct cellular events leading toprogressive renal failure [2]. Several hypotheses linkinghyperglycaemia and altered cellular biology have beenproposed [3]. One of these hypotheses postulates thathigh glucose concentration leads to the activation ofthe calcium- and phospholipid-dependent proteinkinase C (PKC) signalling pathway which subse-quently mediates cellular response, e.g. with alteredgene expression [4]. It is generally believed thatintracellular PKC activation is achieved by thediabetes-induced accumulation of one of its co-factors,diacylglycerol (DAG), inside the cell [2,5]. Diabetesmellitus causes elevated DAG levels in vascular tissuesassociated with diabetic complications, includingretina, heart, aorta and renal glomeruli, and in non-vascular tissues such as liver and skeletal muscle [2].However, PKC may also be activated by othermechanisms [6,7]. Oxidative stress has been reportedto induce prolonged activation of PKC within cells [8]through reactive oxygen species (ROS), producedby hyperglycaemia or through ‘advanced glycationend products’ (AGEs) which have been shown todirectly activate PKC [9,10]. Since PKC is animportant intracellular messenger system and plays acentral role in cell proliferation, matrix expression,apoptosis and regulation of gene transcription [4],PKC activation as one of the important underlyingmolecular mechanism of diabetic complications isan attractive hypothesis and a multitude of reportssuggest such a mechanism [4]. Further support comesfrom experimental and human studies, where inhibi-tion of PKC by specific drugs has been shown toprevent early changes in the diabetic retina andkidney [11–16].However, PKC is not a single entity but consists of afamily of at least 12 serine-threonine kinases withdistinct co-factor activation, expression patterns andcellular functions [17]. These isoforms were first clonedin 1986 and have been divided on the basis of theirregulatory domains into three larger subgroups: theclassical (conventional) PKC isoforms a, bI/II and g(regulated by calcium and DAG), the novel PKCisoforms , and (regulated by DAG) and theatypical (non-calcium-/non-DAG-regulated) PKCisoforms and / (Figure 1) [4]. Multiple studiesover the last decade have clearly demonstrated that thevarious PKC isoforms have distinct cellular functionsin different cell types, cellular compartments andsignalling pathways [4,18].

Phase I trial of UCN-01 in combination with topotecan in patients with advanced solid cancers: a Princess Margaret Hospital Phase II Consortium study

Background: 7-Hydroxystaurosporine (UCN-01) inhibits serine–threonine kinases including the Ca2+ and phospholipid-dependent protein kinase C (PKC), CDKs 2, 4, 6, Chk-1 and PDK1. UCN-01 mediates distinct effects in vitro/in vivo: cell cycle arrest in G1, abrogation of G2 arrest by inhibiting chk1, induction of apoptosis and potentiation of cytotoxicity of S-phase-active chemotherapeutics including the topoisomerase 1 inhibitor topotecan (T). This phase I study was designed to determine the maximal tolerated dose (MTD), recommended phase 2 dose (RPTD), toxicity profile, pharmacokinetics and antitumor activity of T and UCN-01 in patients with refractory solid tumors.Design: Both agents were administered every 21 days intravenously through central venous access in escalating doses to eligible patients. On day 1, following antiemetic prophylaxis with dexamethasone and a serotonin type 3(A) receptor (5HT3) inhibitor, UCN-01 was infused over 3h, followed by T infused over 30min. On days 2–5, patients received T only. UCN-01 doses were reduced by 50% in cycles 2 and beyond because of its prolonged half-life.Results: Thirty-three patients were entered in three cohorts: Dose Level (DL) 1 (UCN-01 70mg/m2, T 0.75mg/m2), three patients; DL 2 (UCN-01 70mg/m2, T 1.0mg/m2), 24 patients; DL 3 (UCN-01 90mg/m2, T 1.0mg/m2), six patients. All but three patients were PS 0 or 1, median age was 54 years (range, 29–72), 91% were female. Primary tumor types: ovary/peritoneal (23 patients), colon (three patients), salivary gland (two patients), others (five patients). All patients were eligible for adverse event (AE) analysis and 22 patients were eligible for survival and tumor response analysis. Two of six patients had dose limiting toxicity (DLT) at DL 3 (grade 3 N/V; grade 4 neutropenia with infection). One DLT was seen in one patient at DL 2, consisting of grade 4 leukopenia. This cohort was expanded and no further DLTs were observed. Most common drug-related AEs were mild (grade 1–2). Non-hematological grade 3–4 AEs consisted of transient hyperglycemia (4), infection (3), coagulation, fatigue, hypotension, nausea (2), hypomagnesemia, vomiting, headache (1). Hematologic toxicities occurred in 100% of patients. Grade 3–4 hematologic abnormalities included neutropenia (16, including three with infection), leukopenia (11), lymphopenia (7), thrombocytopenia (5). Best response for 22 evaluable patients was PD (8), SD for at least six cycles (12), PR (1: carcinoma of ovary, dose level 2) and one not assessable. Pharmacokinetic analysis confirmed the prolonged half-life of UCN-01 of ∼15 days.Conclusions: DLT was observed at DL 3 and RPTD was determined to be DL 2. To date, this combination has been relatively well tolerated with some preliminary evidence of efficacy. A phase II study of this combination in patients with ovarian cancer is underway.

Lead (Pb +2) impairs long-term memory and blocks learning-induced increases in hippocampal protein kinase C activity

The long-term storage of information in the brain known as long-term memory (LTM) depends on a variety of intracellular signaling cascades utilizing calcium (Ca 2+) and cyclic adenosine monophosphate as second messengers. In particular, Ca +2/phospholipid-dependent protein kinase C (PKC) activity has been proposed to be necessary for the transition from short-term memory to LTM. Because the neurobehavioral toxicity of lead (Pb +2) has been associated to its interference with normal Ca +2 signaling in neurons, we studied its effects on spatial learning and memory using a hippocampal-dependent discrimination task. Adult rats received microinfusions of either Na + or Pb +2 acetate in the CA1 hippocampal subregion before each one of four training sessions. A retention test was given 7 days later to examine LTM. Results suggest that intrahippocampal Pb +2 did not affect learning of the task, but significantly impaired retention. The effects of Pb +2 selectively impaired reference memory measured in the retention test, but had no effect on the general performance because it did not affect the latency to complete the task during the test. Finally, we examined the effects of Pb +2 on the induction of hippocampal Ca +2/phospholipid-dependent PKC activity during acquisition training. The results showed that Pb +2 interfered with the learning-induced activation of Ca +2/phospholipid-dependent PKC on day 3 of acquisition. Overall, our results indicate that Pb +2 causes cognitive impairments in adult rats and that such effects might be subserved by interference with Ca +2-related signaling mechanisms required for normal LTM.

Extracellular Regulated Kinase, but Not Protein Kinase C, Is an Antiapoptotic Signal of Insulin-like Growth Factor-1 on Cultured Cardiac Myocytes

This study aims to elucidate the signaling pathway for insulin-like growth factor-1 (IGF-1) in cultured neonatal rat cardiomyocytes and particularly the role of IGF-1 in cardiac apoptosis. IGF-1 stimulated polyphosphoinositide turnover, translocation of protein kinase C (PKC) isoforms (α, ε, and δ) from the soluble to the particulate fraction, activation of phospholipid-dependent and Ca2+-, phospholipid-dependent PKC, and activation of the extracellular-regulated kinase (ERK). IGF-1 attenuated sorbitol-induced cardiomyocyte viability and nuclear DNA fragmentation. These antiapoptotic effects of IGF-1 were blocked by PD-098059 (an MEK inhibitor) but not by bisindolylmaleimide I (BIM, a specific PKC inhibitor). The ERK pathway may therefore be an important component in the mechanism whereby IGF-1 exerts its antiapoptotic effect on the cardiomyocyte.

Different hippocampal activity profiles for PKA and PKC in spatial discrimination learning.

Protein kinases are considered essential for the processing and storage of information in the brain. However, the dynamics of protein kinase activation in the hippocampus during spatial learning are poorly understood. In this study, rats were trained to learn a holeboard spatial discrinmination task and the activity profiles for cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and Ca2+/ phospholipid-dependent protein kinase C (PKC) in the hippocampus were examined. Hippocampal PKA activity increased rapidly on Day 1 of spatial learning and remained moderately high at later stages of acquisition. In contrast, PKC activity increased in particulate fractions compared with cytosolic fractions after habituation training and was maximal at Day 3 of spatial acquisition. The results establish a temporal dissociation between PKA and PKC during acquisition of spatial discrimination learning.

Prostaglandin E2 up-regulates insulin-like growth factor binding protein-3 expression and synthesis in human articular chondrocytes by a c-AMP-independent pathway: role of calcium and protein kinase A and C.

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism and its bioactivity and bioavailability is regulated, in part, by IGF-1 binding protein 3 (IGFBP-3). Prostaglandin E2 (PGE2) stimulates IGF-1 production by articular chondrocytes and we determined whether the eicosanoid could regulate IGFBP-3 and, as such, act as a modifier of IGF-1 action at a different level. Using human articular chondrocytes in high density primary culture, Western and Western ligand blotting to measure secreted IGFBP-3 protein, and Northern analysis to monitor IGFBP-3 mRNA levels, we demonstrated that PGE2 provoked a 3.9 +/- 1.1 (n = 3) fold increase in IGFBP-3 mRNA and protein. This effect was reversed by the Ca++ channel blockers, verapamil and nifedipine, and the Ca++/calmodulin inhibitor, W-7. The Ca+2 ionophore, ionomycin, mimicked the effects of PGE2 as did the phorbol ester PMA, which activates Ca++/-phospholipid-dependent protein kinase C (PKC). Cyclic AMP mimetics, such as forskolin, IBMX, Ro-20-1724, and Sp-cAMP, inhibited the expression and synthesis of the binding protein. PGE2 did not increase the levels of cAMP or protein kinase A (PKA) activity in chondrocytes. The PGE2 secretagogue, IL-1 beta, down-regulated control levels of IGFBP-3 which could be completely abrogated by pre-incubation with the tyrosine kinase inhibitor, erbstatin, and partially reversed (50 +/- 8%) by KT-5720, a PKA inhibitor. These observations suggested that PGE2 does not mediate the effect of its secretagogue and that IL-1 beta signalling in chondrocytes may involve multiple kinases of diverse substrate specificities. Dexamethasone down-regulated control, constitutive levels of IGFBP-3 mRNA and protein eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE2 receptor signalling pathways. Taken together, our results suggest that PGE2 modulates IGFBP-3 expression, protein synthesis, and secretion, and that such regulation may modify human chondrocyte responsiveness to IGF-1 and influence cartilage metabolism.

Inhibition of eukaryote protein kinases and of a cyclic nucleotide-binding phosphatase by prenylated xanthones

A series of prenylated xanthones are variously potent inhibitors of the catalytic subunit (cAK) of rat liver cyclic AMP-dependent protein kinase (PKA), rat brain Ca 2+and phospholipid-dependent protein kinase C (PKC), chicken gizzard myosin light chain kinase (MLCK), wheat embryo Ca 2+-dependent protein kinase (CDPK) and potato tuber cyclic nucleotide-binding phosphatase (Pase). The prenylated xanthones examined are mostly derivatives of α-mangostin in which the 3-hydroxyl and 6-hydroxyl are variously substituted with groups R or R′, respectively, or derivatives of 3-isomangostin (mangostanol) in which the 9-hydroxyl is substituted with groups R′ or the prenyl side chain is modified. The most potent inhibitors of cAK have non-protonatable and relatively small R′ and R groups. Conversely, the most potent inhibitors of PKC and MLCK have bulkier and basic R′ groups. Some prenylated xanthones are also potent inhibitors of CDPK. PKC and cAK are competitively inhibited by particular prenylated xanthones whereas the compounds that are the most potent inhibitors of MLCK and CDPK are non-competitive inhibitors. Prenylated xanthones having relatively small and non-protonatable R′ and R groups inhibit a high-affinity cyclic nucleotide binding Pase in a non-competitive fashion.

Localization of a molecular form of interferon-regulated RNase L in the cytoskeleton.

RNase L (also termed 2-5A-dependent RNase) is a crucial enzyme involved in the molecular mechanism of interferon (IFN) action. Activated by 2',5'-oligoadenylate oligomers (2-5A), this enzyme controls the regulation of RNA stability in IFN-treated or virus-infected mammalian cells. Knowledge of RNase location within cells may provide additional information about its function. Previous work located RNase as a detergent-soluble molecule in nuclei and cytoplasm. In this study, we demonstrate that this enzyme was also present in a detergent-insoluble fraction associated with proteins of the cytoskeleton. A cellular fractionation procedure was used to prepare the cytoskeleton, which was shown to contain 2-5A binding activity not due to cytoplasmic contaminants. In contrast to the cytoplasmic fraction, which contained RNase L with a 2-5A-accessible site, the insoluble RNase molecular form of the cytoskeleton could not be assayed by the classic radiobinding method or the covalent UV cross-linking procedure, which only detects the 2-5A binding site in an open position, that is, free of 2-5A or with an unmasked 2-5A site. The 2-5A binding site present in the cytoskeleton was completely masked and not directly accessible to its 2-5A activator. This particular molecular form of RNase can be detected after a specific denaturing-renaturing treatment of the cytoskeleton, which separates the RNase from cytoskeletal proteins, unmasking the 2-5A site. The cytoskeletal RNase was no longer present at this site when cells were stimulated for a short time with 12-O-tetradecanoylphorbol-13-acetate (TPA). Our data suggest the existence of a pathway that targets the RNase to another subcellular location. To explore the issue further, we examined in vitro the ability of calcium and phospholipid-dependent protein kinase C (PKC) to catalyze significant phosphorylation of the RNase.

Inhibition of eukaryote serine/threonine-specific protein kinases by piceatannol.

The protein tyrosine kinase (PTK) inhibitor piceatannol is also an inhibitor of the rat liver cyclic AMP-dependent protein kinase (PKA) catalytic subunit (cAK), rat brain Ca(2+)- and phospholipid-dependent protein kinase C (PKC), avian gizzard Ca(2+)-calmodulin-dependent myosin light chain kinae (MLCK), and of wheat embryo Ca(2+)-dependent protein kinase (CDPK) (IC50 values 3, 8, 12, and 19 microM, respectively). However, a number of piceatannol-related compounds with fewer or no phenolic hydroxy substituents are inactive or very poor inhibitors of these serine/threonine protein kinases. Similarly, the PTK inhibitor ellagic acid is a potent inhibitor of cAK and of PKC (IC50 values 2 and 8 microM, respectively), whereas the non-phenolic perylene is ineffective as a protein kinase inhibitor. Ellagic acid is a competitive inhibitor of both cAK and of PKC but piceatannol inhibits these enzymes in a fashion that is competitive and non-competitive, respectively. Interaction with calmodulin may contribute to the inhibition of MLCK and CDPK by piceatannol.

Ginsenoside Rh 2 and Rh 3 induce differentiation of HL-60 cells into granulocytes: modulation of protein kinase C isoforms during differentiation by ginsenoside Rh 2

Ginsenoside Rh 1 or Rh 2 differentiated B16 melanoma or F9 teratocarnoma to phenotypic normal melanocyte-like cells or parietal endoderm-like cells. Ginsenoside Rh 3 and Rh 4 were recently isolated from Panax ginseng, but their biochemical and pharmacological effects remain unidentified. The present study investigated whether the ginsenoside Rh group (G-Rh 1, -Rh 2, -Rh 3 and -Rh 4) having similar structures induce differentiation of HL-60 cells and whether protein kinase C (PKC) is involved in differentiation by ginsenoside. Differentiation was assessed by Wright–Giemsa stain and nitroblue tetrazolium reduction. G-Rh 2 and G-Rh 3 induced differentiation of HL-60 cells into morphologically and functionally granulocytes but G-Rh 1 and G-Rh 4 did not. G-Rh 2 and G-Rh 3 arrested the cell cycle at the G1/S phase, consistent with the ability to induce differentiation in a decreasing order of retinoic acid>G-Rh 2>G-Rh 3. During differentiation by G-Rh 2, Ca 2+/phospholipid-dependent PKC activity was increased in both the cytosol and total cell extract and Ca 2+/phospholipid-dependent phosphorylation of 38 and 200 kDa endogenous proteins increased, while phosphorylation of 60, 64, 66 and 97 kDa proteins was Ca 2+/phospholipid-independent. When cytosolic PKC isoforms were analyzed by immunoblotting, no significant change was observed in the α level, however, the immunoreactive 60 kDa band of a similar mass to the PKC catalytic fragment appeared following treatment with G-Rh 2. The β isoform was gradually increased with prolonged treatment. The γ isoform was not detected in the cytosol of untreated cells, whereas a small amount was detected 5 days after treatment. It is concluded that G-Rh 2 and G-Rh 3 can induce differentiation of HL-60 cells into granulocytes and modulation of PKC isoform levels may contribute to differentiation of HL-60 cells by G-Rh 2.

Inhibition of eukaryote protein kinases by isoquinoline and oxazine alkaloids.

The aporphine isoquinoline alkaloid apomorphine is a potent inhibitor of the catalytic subunit (cAK) of rat liver cyclic AMP-dependent protein kinase (PKA), myosin light chain kinase (MLCK), and Ca(2+)- and phospholipid-dependent protein kinase C (PKC) (IC50 values 1, 11, and 8 microM, respectively). However, a number of O-methylated analogues of apomorphine are inactive or poor inhibitors of cAK. The benzophenanthridine isoquinoline alkaloid sanguinarine is a potent inhibitor of cAK but is a relatively poor inhibitor of PKC (IC50 values 6 and 217 microM respectively). However a number of methylated analogues of sanguinarine are inactive as cAK inhibitors. The aporphine isoquinoline alkaloids (+)-boldine and bulbocapnine are non-competitive inhibitors of MLCK with respect to both peptide substrate and ATP. The inhibition of cAK, MLCK, and PKC by apomorphine and sanguinarine is competitive with respect to ATP as substrate. The oxazine alkaloids darrow red, nile blue A, and oxazine 170 are variously effective as inhibitors of cAK, MLCK, PKC, and CDPK (IC50 values 4-65 microM). Ca2+ binds to apomorphine and (+)-boldine which, together with nile blue A and oxazine 170, are potent inhibitors of calmodulin (CaM)-dependent MLCK (IC50 values 11, 12, 4, and 7 microM, respectively), and interact with dansyl-CaM.

Protein kinase C activity and protein levels in Alzheimer's disease

Patients with Alzheimer's disease (AD) have been reported to have abnormalities in the levels and activities of protein kinase C (PKC) in brain and other tissues. We have measured Ca 2+-activated, phospholipid-dependent PKC activities and levels in cerebral cortex from frontal, motor, temporal and parietal regions, as well as in leukocytes and platelets from AD patients and controls. No significant differences in PKC histone H1 phosphotransferase activity were seen in frontal, motor, temporal or parietal cortex, or in leukocytes and platelets from AD patients and controls. Elevated PKC protein was present in cytosolic fractions from frontal cortex, but not in other brain regions, or in leukocytes and platelets. These data suggest that abnormalities of PKC phosphorylating activity are absent in AD.

Prostaglandin E2 stimulates insulin-like growth factor binding protein-4 expression and synthesis in cultured human articular chondrocytes: Possible mediation by Ca++ -calmodulin regulated processes

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism whose action is mediated by high affinity cell surface receptors and bioactivity and bioavailability regulated, in part, by IGF-1 binding proteins (IGFBPs). Prostaglandin E2 (PGE2) stimulates collagen and proteoglycan synthesis in cartilage via an autocrine feedback loop involving IGF-1. We determined whether the eicosanoid could regulate IGFBP-4, a major form expressed by chondrocytes and, as such, act as a modifier of IGF-1 action at another level. Using human articular chondrocytes in high-density primary culture, Western and Western ligand blotting to measure secreted IGFBP-4 protein, and Northern analysis to monitor IGFBP-4 mRNA levels, we demonstrated that PGE2 provoked a 2.7 +/- 0.3- and 3.8 +/- 0.5- (n = 3) fold increase in IGFBP-4 mRNA and protein, respectively. This effect was reversed by the Ca(++) channel blocker, verapamil, and the Ca(++)/calmodulin inhibitor, W-7. The Ca(++)ionophore, ionomycin, mimicked the effects of PGE2. The phorbol ester, PMA, which activated phospholipid-dependent protein kinase C (PKC) in chondrocytes, had no effect on IGFBP-4 production. Cyclic AMP mimetics and PKA activators, IBMX, and Sp-cAMP, inhibited the expression of the binding protein as did the PGE2 secretagogue, interleukin-1 beta (IL-beta). The inhibitory effect of the latter cytokine was mediated by a erbstatin/genistein (tyrosine) sensitive kinase. Dexamethasone, an inhibitor of cyclooxygenase (COX-2) expression and PGE2 synthesis, down-regulated control, constitutive levels of IGFBP-4 mRNA and protein, eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE2-receptor signalling pathways. The results suggest that extracellular signals control IGFBP-4 production by a number of different transducing networks with changes in Ca(++) and calmodulin activity exerting a strong positive influence, possibly maintaining the constitutivity of IGFBP-4 synthesis under basal conditions. PGE2 activation of the IGF-1/IGFBP axis may play a pivotal role in the metabolism of cartilage and possibly connective tissues in general. Eicosanoid biosynthesis may be a rate-limiting step in cartilage repair processes.

Lead may affect glucocorticoid signal transduction in cultured hepatoma cells through inhibition of protein kinase C

Specific cellular sites of lead action have not been completely defined. To elucidate the effects of lead exposure on glucocorticoid-mediated signal transduction in hepatic hormonal target tissues, the induction of tyrosine aminotransferase (TAT) specific activity in the H4-IIE-C3 hepatoma cell culture model system was employed. It had been found that lead acetate (3–10 μM) exposure of HTC cells significantly reduced TAT specific activity in a concentration- and time-dependent manner. Two possible molecular targets of the lead-induced effect were investigated: interference with calcium-mediated cellular processes and calcium- and phospholipid-dependent protein kinase C (PKC) activity and isoform-type interactions. Lead acetate treatment (5 μM) reduced TAT specific activity below sodium acetate treated controls by 31%. One-half of the TAT specific activity was recovered by co-treatment with 5 μM lead acetate and 10 mM calcium chloride. As the concentration of lead acetate was increased to 10 μM, interference in calcium-mediated events also increased. Potentiation of glucocorticoid induction by phorbol myristate acetate (PMA) (300 nM) in control cells was 34%, but was abolished by exposure of cells to 10 μM lead acetate (48 h). Treatment with the kinase inhibitor genistein decreased TAT specific activity by 55% and 45% in control and lead acetate exposed cells, respectively. Following treatment with dexamethasone (100 nM), significant increases in both cytosolic and particulate PKC were noted in control cells but not lead acetate exposed cells. Western blot results indicated that lead exposure may increase PKC β and decrease PKC α translocation from cytosolic to particulate fractions, respectively. Taken together, these results suggest that glucocorticoid signal transduction pathways in HTC cells involve calcium-mediated cellular events and PKC isoforms. Exposure of cells to lead results in interference with calcium-mediated events and aberrant modulation of PKC activities. Within hormonal target cells, these may be toxic molecular sites of action of the heavy metal lead and may contribute to the overall toxicity of lead exposure.

Specific inhibition of cyclic amp-dependent protein kinase by warangalone and robustic acid

The prenylated isoflavone warangalone from the insecticidal plant Derris scandens is a selective and potent inhibitor of rat liver cyclic AMP-dependent protein kinase catalytic subunit (cAK) (IC 50 3.5 μM). The inhibition of rat liver cAK by warangalone is non-competitive with respect to both ATP and the synthetic peptide substrate (LRRASLG) employed in this study. Warangalone is a poor inhibitor of avian calmodulin-dependent myosin light chain kinase (MLCK), rat brain Ca 2+- and phospholipid-dependent protein kinase C (PKC) and wheat embryo Ca 2+-dependent protein kinase (CDPK). The related plant derived prenylisoflavones are also potent cAK inhibitors. Thus, 8-γ-γ-dimethylallylwighteone, 3′-γ-γ-dimethlallylwighteone and nallanin are inhibitors of cAK with IC 50 values in the range 20–33 μM. The prenyl-substituted isoflavones tested in this study are ineffective or poor as inhibitors of PKC. Thus nallanin is a poor PKC inhibitor (IC 50 value of 120 μM). The related isoflavones biochanin A and genistein are poor inhibitors of cAK (IC 50 values 100 μM and 126 μM, respectively). Genistein inhibits MLCK (IC 50 value 14 μM) but biochanin A is a poor MLCK inhibitor (IC 50 value 300 μM). The D. scandens prenyl-isoflavones and related isoflavones are ineffective inhibitors of wheat embryo Ca 2+-dependent protein kinase (CDPK). The 4-methoxy-3-phenyl-coumarin robustic acid is a potent inhibitor of rat liver cAK (IC 50 value 10 μM) but is a poor inhibitor of rat brain PKC, avian MLCK and wheat embryo CDPK. The coumarins 5-methoxypsoralen and 4,4′-di- O-methyl scandenin are poor cAK inhibitors (IC 50 values 240 and 248 μM, respectively). All of the non-prenylated coumarins examined are ineffective as inhibitors of the eukaryote signal-regulated protein kinases cAK, MLCK, PKC and CDPK. The selective, high affinity interaction of warangalone and robustic acid with cAK may contribute to their biological effects in vivo and to the insecticidal activity of the plant D. scandens.

Involvement of calcium-dependent protein kinase C in arsenite-induced genotoxicity in chinese hamster ovary cells

Arsenic is the first metal to be identified as a human carcinogen. Arsenite, one inorganic form of arsenic, has been found to induce sister chromatid exchange, chromosome aberrations, and gene amplification in a variety of in vitro systems. In this study of arsenite-induced genotoxicity represented a micronuclei production in Chinese hamster ovary cells (CHO-K1), we found that the calcium channel blocker, verapamil, can potentiate arsenite-induced micronuclei. And after arsenite treatment, the elevation of intracellular calcium was observed. When extracellular calcium was depleted during arsenite treatment, the arsenite-induced micronuclei formation was significantly suppressed. These data indicated that a calcium ion plays an essential role in arsenite-induced genotoxicity. Further, it was found that the cotreatment of arsenite and a calcium ionophore, A23187, can increase the micronuclei induction. In contrast, pretreatment of the intracellular calcium chelator, quin 2, significantly inhibited micronuclei production of arsenite administration. In addition, we measured the activity of calcium- and phospholipid-dependent protein kinase C (PKC) and found that arsenite can activate PKC activity in a dose-dependent manner. Subsequently, some PKC activators and inhibitors were applied to investigate the involvement of PKC on arsenite-induced micronuclei formation. It was found that H7, a PKC inhibitor, can depress but TPA, a PKC activator, can enhance arsenite-induced micronuclei significantly. These data indicated that arsenite exposure perturbs intracellular calcium homeostasis and activates PKC activity. As a result, the activation of PKC activity may play an important role in arsenite-induced genotoxicity.

Prostaglandin E2 up‐regulates insulin‐like growth factor binding protein‐3 expression and synthesis in human articular chondrocytes by a c‐AMP‐independent pathway: Role of calcium and protein kinase A and C

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism and its bioactivity and bioavailability is regulated, in part, by IGF-1 binding protein 3 (IGFBP-3). Prostaglandin E2 (PGE2) stimulates IGF-1 production by articular chondrocytes and we determined whether the eicosanoid could regulate IGFBP-3 and, as such, act as a modifier of IGF-1 action at a different level. Using human articular chondrocytes in high density primary culture, Western and Western ligand blotting to measure secreted IGFBP-3 protein, and Northern analysis to monitor IGFBP-3 mRNA levels, we demonstrated that PGE2 provoked a 3.9 ± 1.1 (n = 3) fold increase in IGFBP-3 mRNA and protein. This effect was reversed by the Ca++ channel blockers, verapamil and nifedipine, and the Ca++/calmodulin inhibitor, W-7. The Ca++ ionophore, ionomycin, mimicked the effects of PGE2 as did the phorbol ester PMA, which activates Ca++-phospholipid-dependent protein kinase C (PKC). Cyclic AMP mimetics, such as forskolin, IBMX, Ro-20-1724, and Sp-cAMP, inhibited the expression and synthesis of the binding protein. PGE2 did not increase the levels of cAMP or protein kinase A (PKA) activity in chondrocytes. The PGE2 secretagogue, IL-1β, down-regulated control levels of IGFBP-3 which could be completely abrogated by pre-incubation with the tyrosine kinase inhibitor, erbstatin, and partially reversed (50 ± 8%) by KT-5720, a PKA inhibitor. These observations suggested that PGE2 does not mediate the effect of its secretagogue and that IL-1β signalling in chondrocytes may involve multiple kinases of diverse substrate specificities. Dexamethasone down-regulated control, constitutive levels of IGFBP-3 mRNA and protein eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE2 receptor signalling pathways. Taken together, our results suggest that PGE2 modulates IGFBP-3 expression, protein synthesis, and secretion, and that such regulation may modify human chondrocyte responsiveness to IGF-1 and influence cartilage metabolism. © 1996 Wiley-Liss, Inc.

Prostaglandin E2 up-regulates insulin-like growth factor binding protein-3 expression and synthesis in human articular chondrocytes by a c-AMP-independent pathway: Role of calcium and protein kinase A and C

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism and its bioactivity and bioavailability is regulated, in part, by IGF-1 binding protein 3 (IGFBP-3). Prostaglandin E2 (PGE2) stimulates IGF-1 production by articular chondrocytes and we determined whether the eicosanoid could regulate IGFBP-3 and, as such, act as a modifier of IGF-1 action at a different level. Using human articular chondrocytes in high density primary culture, Western and Western ligand blotting to measure secreted IGFBP-3 protein, and Northern analysis to monitor IGFBP-3 mRNA levels, we demonstrated that PGE2 provoked a 3.9 ± 1.1 (n = 3) fold increase in IGFBP-3 mRNA and protein. This effect was reversed by the Ca++ channel blockers, verapamil and nifedipine, and the Ca++/calmodulin inhibitor, W-7. The Ca++ ionophore, ionomycin, mimicked the effects of PGE2 as did the phorbol ester PMA, which activates Ca++-phospholipid-dependent protein kinase C (PKC). Cyclic AMP mimetics, such as forskolin, IBMX, Ro-20-1724, and Sp-cAMP, inhibited the expression and synthesis of the binding protein. PGE2 did not increase the levels of cAMP or protein kinase A (PKA) activity in chondrocytes. The PGE2 secretagogue, IL-1β, down-regulated control levels of IGFBP-3 which could be completely abrogated by pre-incubation with the tyrosine kinase inhibitor, erbstatin, and partially reversed (50 ± 8%) by KT-5720, a PKA inhibitor. These observations suggested that PGE2 does not mediate the effect of its secretagogue and that IL-1β signalling in chondrocytes may involve multiple kinases of diverse substrate specificities. Dexamethasone down-regulated control, constitutive levels of IGFBP-3 mRNA and protein eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE2 receptor signalling pathways. Taken together, our results suggest that PGE2 modulates IGFBP-3 expression, protein synthesis, and secretion, and that such regulation may modify human chondrocyte responsiveness to IGF-1 and influence cartilage metabolism. © 1996 Wiley-Liss, Inc.

Down regulation of protein kinase C during growth enhancement induced by interleukin-6 on a human myeloma cell line, KMS-11.

Interleukin-6 (IL-6) is one of the most important growth factors for myeloma cells. We examined the effect of recombinant IL-6 on the proliferation of five human myeloma cell lines, which were independently established AT Kawasaki Medical School. Only the KMS-11 cell line among these five lines showed growth enhancement induced by IL-6. Based on the results, a possible contribution of Ca(2+)-phospholipid-dependent protein kinase C (PKC) to the signal transduction in KMS-11 cells during growth enhancement was studied, since PKC may play an important role in malignant transformation or cell proliferation induced by some growth factors, such as IL-6. When exogenous IL-6 was added to KMS-11 culture, we observed (1) reduction of total PKC activity, and (2) translocation of PKC activity from its cytosol fraction to the membrane fraction. These findings may indicate that down regulation of PKC occurred during the myeloma cell proliferation induced by IL-6. However, IL-6 does not appear to be involved in cell proliferation and differentiation in the other cell lines studied.

Differential inhibition of eukaryote protein kinases by condensed tannins

Condensed tannins, isolated from a variety of plant sources, were characterized according to the constituent flavans, being based on procyanidin and/or prodelphinidin and having a cis or trans stereochemistry at positions 2 and 3. All the tannin preparations are potent inhibitors of rat liver cyclic AMP-dependent protein kinase catalytic subunit (cAK) with IC 50 values (concentrations for 50% inhibition) ranging from 0.009 to 0.2 μM. The tannin preparations are very good inhibitors of rat brain Ca 2+- and phospholipid-dependent protein kinase C (PKC) (IC 50 values in the range 0.3–7 μM), wheat embryo Ca 2+-dependent protein kinase (CDPK) (IC 50 values in the range 0.8–7 μM) and of calmodulin (CaM)-dependent myosin light chain kinase (MLCK) (IC 50 values in the range 7–24 μM). One of the most effective preparations, that from the leaves of Ribes nigrum, has IC 50 values with respect to cAK, PKC, CDPK and MLCK of 0.009, 0.6, 2.0 and 16 μM, respectively. In general, the order with respect to sensitivity to inhibition by these condensed tannins is cAK > PKC > CDPK > MLCK. The Ribes nigrum preparation is a competitive inhibitor of cAK with respect to both ATP and synthetic peptide substrate. These condensed tannin preparations are the most potent plant-derived inhibitors of cAK yet found.