PXR-dependent induction of human CYP3A4 gene expression by organochlorine pesticides

OCP are xenobiotics which display various toxic effects on animal and human health. One of their effects is to bind and activate estrogen receptor alpha (ERalpha). We have previously studied the down-regulation of induced CYP1A1 (cytochrome P450) expression by this class of molecules in mammary carcinoma cells and shown the importance of ERalpha in this process. However, an alternative mechanism was suggested by those experiments in hepatoma cells. In this study, we have performed Northern blot and transient transfection assays in various cell lines and shown that OCP activate human pregnane X receptor (PXR) and subsequent CYP3A4 mRNA expression. This effect is mediated by the distal xenobiotic responsive element modulator of the promoter. The induction of CYP3A4 by OCP was dose-dependent within the 1-10 microM range. The data suggest that chronic exposure to OCP could alter a major metabolite pathway in human liver and putatively modify the pharmacokinetics of drugs and pollutants.     

Modulation of mitochondrial metabolic function by phorbol 12-myristate 13-acetate through increased mitochondrial translocation of protein kinase Calpha in C2C12 myocytes

Protein kinase C (PKC) agonists including phorbol 12-myristate 13-acetate (PMA) not only induce the redistribution of cytosolic PKC to various subcellular compartments but also activate the kinase domain of the protein. In the present study we have investigated the nature of mitochondrial PKC pool and its effects on mitochondrial function in cells treated with PMA. Treatment of C2C12 myoblasts, C6 glioma and COS7 cells with PMA resulted in a dramatic redistribution of intracellular PKCalpha pool, with large fraction of the protein pool sequestered in the mitochondrial compartment. We also observed mitochondrial PKCdelta accumulation in a cell restricted manner. The intramitochondrial localization was ascertained by using a combination of protection against protease treatment of isolated mitochondria and immunofluorescence microscopy. PMA-induced mitochondrial localization of PKCalpha was accompanied by increased mitochondrial PKC activity, altered cell morphology, disruption of mitochondrial membrane potential, decreased complex I and pyruvate dehydrogenase activities, and increased mitochondrial ROS production. All of these changes could be retarded by treatment with PKC inhibitors. These results show a direct role for PMA-mediated PKCalpha translocation to mitochondria in inducing mitochondrial toxicity.     

Alterations of insulin secretion following long-term manipulation of ATP-sensitive potassium channels by diazoxide and nateglinide

Previous studies have shown that prolonged exposure to drugs, which act via blocking KATP channels, can desensitize the insulinotropic effects of drugs and nutrients acting via KATP channels. In this study, effects of prolonged exposure to diazoxide, a KATP channel opener, on beta cell function were examined using clonal BRIN-BD11 cells. The findings were compared to the long-term effects of KATP channel blockers nateglinide and tolbutamide. Following 18 h exposure to 200 microM diazoxide, the amounts of insulin secreted in response to glucose, amino acids and insulinotropic drugs were increased. Secretory responsiveness to a variety of agents acting via KATP channels was retained following prolonged diazoxide exposure. In contrast, 18 h exposure to 100 microM nateglinide significantly attenuated the insulin secretory responses to tolbutamide, nateglinide and BTS 67 582. Glucose- and L-alanine-stimulated insulin release were unaffected by prolonged nateglinide exposure, however responsiveness to L-leucine and L-arginine was diminished. Prolonged exposure to nateglinide had no effect on forskolin- and PMA-stimulated insulin release, and the overall pattern of desensitization was similar to that induced by 100 microM tolbutamide. We conclude that in contrast to chronic long-term KATP channel blockade, long-term diazoxide treatment is not harmful to KATP channel mediated insulin secretion and may have beneficial protective effects on beta cell function.     

Cell cycle- and protein kinase C-specific effects of resiniferatoxin and resiniferonol 9,13,14-ortho-phenylacetate in intestinal epithelial cells

We have previously reported that protein kinase C (PKC) signaling can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells, including downregulation of cyclin D1, induction of p21(Waf1/Cip1), and activation of the growth suppressor function of pocket proteins. In the current study, we compared the cell cycle- and PKC-specific effects of the vanilloid resiniferatoxin (RTX), its parent diterpene resiniferonol 9,13,14-ortho-phenylacetate (ROPA), and the PKC agonist PMA in the IEC-18 non-transformed intestinal crypt cell line. ROPA and PMA were found to produce strikingly similar alterations in cell cycle progression and PKC activity in IEC-18 cells, although PMA was approximately 1000-fold more potent in producing these effects. Both agents induced a transient PKC-dependent blockade in G1---> S progression associated with transient downregulation of cyclin D1 and induction of p21(Waf1/Cip1). In contrast, RTX produced a prolonged PKC-independent cell cycle arrest in G(0)/G(1) phase which was maintained for longer than 24h. This arrest was vanilloid receptor-independent and associated with prolonged downregulation of cyclin D1 mRNA and protein, with little effect on levels of p21(Waf1/Cip1). Combined exposure to RTX and ROPA produced a sustained and complete cell cycle blockade in IEC-18 cells, associated with depletion of cyclin D1 and sustained enhancement of p21(Waf1/Cip1) levels. PMA, ROPA, RTX and the RTX/ROPA combination were capable of activating ERK1/2 signaling in IEC-18 cells, albeit with different kinetics. In contrast, only PMA and ROPA activated JNK1/2 and p38 in this system. Notably, some preparations of commercially obtained RTX produced effects indistinguishable from those of the RTX/ROPA combination, suggesting that certain batches of the compound may contain significant amounts of ROPA (or another PKC agonist activity). Together, these data demonstrate that structurally related compounds can produce similar cell cycle-specific effects but through distinct mechanisms. In addition, they add to a growing body of evidence that vanilloids can have antiproliferative effects in a variety of cell types.     

Involvement of the muscarinic acetylcholine receptor in inhibition of cell migration

Activation of G protein-coupled receptors is known to stimulate cell migration, but receptor-mediated signals inhibiting cell migration have not been identified. We investigated the ability of transfected human M(3) muscarinic acetylcholine receptors (mAChR) to regulate the migration of Chinese hamster ovary (CHO) cells. Single cells migrated on colloidal gold applied to fibronectin-coated plates, and videomicroscopy was used to measure cell spreading and migration. Activation of M(3) mAChR with the agonist carbachol was found to inhibit cell migration, whereas direct activation of protein kinase C (PKC) with PMA was found to stimulate migration. The amount of cell adhesion and spreading was found to be equivalent for carbachol- and PMA-treated cells. Selective inactivation of conventional PKC isoforms with Go6976 (C(24)H(18)N(4)O) abolished the PMA-mediated increase in cell migration. In contrast, the mAChR-mediated decrease in migration was not altered by Go6976, but was abolished when both novel and conventional PKC isoforms were inactivated by calphostin C or chelerythrine. These findings suggest involvement of conventional PKC isoforms in the stimulation of migration and of novel PKC isoforms in the inhibition of migration. Carbachol- but not PMA-treated cells exhibited an elongated morphology reminiscent of migrating cells that cannot detach their trailing edges from the substratum. Similarly, carbachol-treated cells detached less readily from fibronectin than control or PMA-treated cells when integrin activity was diminished by the chelation of Ca(2+) and Mg(2+). Finally, the carbachol-induced diminution of cell detachment was preserved after inhibition of the conventional PKC isoforms with Go6976, but was abrogated by treatment with either calphostin C or chelerythrine. These findings suggest that mAChR activation diminishes the ability of cells to detach from the substratum, resulting in diminished migration. This is in contrast to the direct activation of PKC with PMA, which stimulates migration.     

Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1

Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotic detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet–drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes, intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that caution should be taken in PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies.     

Application and interpretation of hPXR screening data: Validation of reporter signal requirements for prediction of clinically relevant CYP3A4 inducers

A human pregnane X receptor (PXR) reporter-gene assay was established and validated using 19 therapeutic agents known to be clinical CYP3A4 inducers, 5 clinical non-inducers, and 6 known inducers in human hepatocytes. The extent of CYP3A4 induction (measured as RIF ratio in comparison to rifampicin) and EC50 was obtained from the dose-response curve. All of the clinical inducers (19/19) and human hepatocyte inducers (6/6) showed positive responses in the PXR assay. One out of five clinical non-inducers, pioglitazone, also showed a positive response. An additional series of 18 commonly used drugs with no reports of clinical induction was also evaluated as putative negative controls. Sixteen of these were negative (89%), whereas two of these, flutamide and haloperidol showed 16-fold (RIF ratio 0.79) and 10-fold (RIF ratio 0.48) maximal induction, respectively in the reporter-gene system. Flutamide and haloperidol were further demonstrated to cause CYP3A4 induction in human cryopreserved hepatocytes based on testosterone 6beta-hydroxylation activity. The induction potential index calculated based on the maximum RIF ratio, EC50, and in vivo maximum plasma concentration was used to predict the likelihood of CYP3A4 induction in humans. When the induction potential index is greater than 0.08, the compound is likely to cause induction in humans. A high-throughput screening strategy was developed based on the validation results at 1microM and 10microM for the same set of drugs. A RIF ratio of 0.4 was set as more practical screening cut-off to minimize the possibility of generating false positives. Thus, a tiered approach was implemented to use the human PXR reporter-gene assay from early lead optimization to late lead characterization in drug discovery.     

Induction by staurosporine of hepatocyte growth factor production in human skin fibroblasts independent of protein kinase inhibition

Staurosporine is one of the most potent and well known inhibitors of protein kinases, and it is often used to study the involvement of protein kinases in signal transduction pathways. We now report that staurosporine can induce the production of hepatocyte growth factor (HGF) independently of protein kinase inhibition. Staurosporine markedly stimulated the production of HGF in various cell types, including human skin fibroblasts. Its effect was accompanied by up-regulation of HGF gene expression. The inhibition of protein kinases appears not to be involved in staurosporine-induced HGF production, because other protein kinase inhibitors, K-252a, H-7, GF 109203X and genistein, had no HGF-inducing activity. UCN-01, 7-hydroxystaurosporine, which differs from staurosporine only in its aglycone moiety, also showed HGF-inducing activity, and inactive K-252a differs from staurosporine only in its sugar moiety. These results indicate that the sugar moiety, a six-atom ring structure, is important in the HGF-inducing activity of staurosporine. Experiments were then carried out to determine whether the characteristics of staurosporine-induced HGF production have similarities to those of HGF production stimulated by other HGF inducers. The effect of staurosporine like that of 8-bromo-cAMP and that of cholera toxin was marked in human skin fibroblasts from all four different sources, whereas the effects of epidermal growth factor and phorbol 12-myristate 13-acetate were variable depending on cells. The net increase in HGF production induced by staurosporine was not reduced in protein kinase C-depleted human skin fibroblasts. Moreover, synergistic induction of HGF was detected between staurosporine and interferon-gamma as well as between 8-bromo-cAMP and interferon-gamma. Staurosporine, however, did not increase intracellular cAMP levels in human skin fibroblasts. These results indicate that staurosporine induced HGF in different cell types via a signaling pathway similar to the cAMP-mediated pathway without increasing cAMP levels.     

Phorbol 12-myristate 13-acetate (PMA)-induced migration of glioblastoma cells is mediated via p38MAPK/Hsp27 pathway

We investigated the mechanism of phorbol 12-myristate 13-acetate (PMA)-induced migration of glioblastoma cells focusing on the p38 mitogen-activated protein kinase (MAPK)/heat shock protein 27 (Hsp27) pathway. PMA-induced cell migration and activation of p38MAPK in A172 glioblastoma cells. PMA-induced formation of lamellipodia and focal complexes was blocked by inhibiting p38MAPK with SB203580 or small interfering RNA (siRNA). Furthermore, activation of p38MAPK resulted in phosphorylation of an F-actin polymerization regulator, Hsp27. Immunohistochemical analysis showed that upon PMA stimulation, both unphosphorylated and phosphorylated Hsp27 were translocated to the lamellipodia. SB203580 or p38MAPK siRNA blocked these phenomena, indicating that Hsp27 phosphorylation and translocation from cytosol to membrane were mediated by p38MAPK. To address the question of whether endogenous Hsp27 participates in PMA-induced migration, we inhibited the expression of Hsp27 using Hsp27 siRNA. Although knockdown of Hsp27 by siRNA had little effect on p38MAPK activation, lamellipodia and focal complex formation was markedly inhibited. Migration was also abolished in Hsp27 siRNA-transfected cells. In conclusion, p38MAPK activation followed by Hsp27 phosphorylation was required for PMA-induced migration. Furthermore, Hsp27 itself played critical roles in PMA-induced migration. Our data provide substantial evidence for a model elucidating the molecular mechanisms of regulation of actin dynamics and migration by PMA-activated protein kinase C in glioblastoma cells.     

Inhibitory effects of wogonin on the invasion of human breast carcinoma cells by downregulating the expression and activity of matrix metalloproteinase-9

Wogonin, a naturally occurring monoflavonoid extracted from Scutellariae radix, has been shown to possess tumor therapeutic potential in vitro and in vivo. However, the effects of wogonin on tumor cells invasion remains poorly understood. In this study, we performed in vitro experiments to investigate the anti-invasive and anti-metastatic activity of wogonin in MDA-MB-231 human breast carcinoma cells. Wogonin caused a concentration-dependent suppression of cell migration, adhesion and invasion. The mechanism revealed that wogonin significantly inhibited the expression and activity of both endogenous and phorbol-12-myristate-13-acetate (PMA)-induced matrix metalloproteinase-9 (MMP-9) potentially associating with the suppression of translocation of protein kinase C (PKC) δ and phosphorylation of extracellular signal-regulated kinase (ERK1/2). These results suggested that wogonin could inhibit the invasion of tumor cells by downregulating the expression and activity of MMP-9, the possible targets may be PKCδ and ERK1/2.     

Differential regulation of NPR-B/GC-B by protein kinase c and calcium

C-type natriuretic peptide (CNP) activation of the guanylyl cyclase-linked natriuretic peptide receptor-B (NPR-B) stimulates vasorelaxation and bone growth. Hormones and phorbol esters (PMA) inhibit NPR-B in calcium and protein kinase c-dependent manners, respectively. Here, we characterize the kinetic properties of NPR-B in membranes from cells exposed to PMA, the calcium ionophore, ionomycin, or sphingosine-1-phosphate (S1P). PMA and ionomycin primarily increased the K(m) and decreased the V(max) of NPR-B for GTP, respectively, whereas S1P caused modest changes in both parameters. PMA and S1P treatment increased the EC50 for CNP activation by eight- and three-fold, whereas ionomycin was ineffective. All three agents caused NPR-B dephosphorylation, but the basis for the loss of phosphate differed between treatments. In vitro phosphorylation of NPR-B in membranes was markedly diminished by prior whole cell PMA or S1P exposure, whereas ionomycin pretreatment had no effect. The involvement of the known phosphorylated residues in each process was tested with a mutant receptor containing glutamates substituted for these sites. While the effect of PMA was lost on this receptor, the effects of S1P and ionomycin were only partially blocked. Our data suggest that the molecular bases for PMA- and calcium-dependent inhibition of NPR-B are unique. The former results from reduced phosphorylation of a known site and primarily affects the affinity of NPR-B for CNP and GTP. The latter is associated with reductions in maximal velocities by a mechanism that does not involve inhibition of NPR-B phosphorylation and requires a process in addition to the dephosphorylation of the known sites.     

The role of protein kinase C in regulation of TCDD-mediated CYP1A1 gene expression.

Cytochrome P450 1A1 (CYP1A1) is induced by halogenated and polycyclic aromatic hydrocarbons following activation of the aryl hydrocarbon receptor (AhR). Protein kinase C (PKC) has been implicated in the regulation of this response. In tissue culture, induction of PKC activity with phorbol esters synergizes the actions of TCDD-induced CYP1A1, while PKC inhibitors block induction of CYP1A1 by TCDD. Here, the actions of specific PKC inhibitors on CYP1A1 induction were examined using a HepG2 human cell line (TV101L) that carries a stably integrated firefly luciferase gene under control of the human CYP1A1 promoter (-1612/+293). TV101 cells were treated with TCDD and either the kinase inhibitor staurosporine or one of the PKC inhibitors GF109203X, G?6983, or G?6976. Aryl hydrocarbon receptor-dependent activation of CYP1A1-luciferase and cellular PKC activity were measured. TCDD treatment induced CYP1A1-luciferase activity in an AhR-dependent manner, as determined by binding of nuclear AhR to xenobiotic response elements (XREs). Dose-dependent inhibition of PKC activity by staurosporine was concordant with inhibition of TCDD-induced CYP1A1-luciferase activity. However, the PKC inhibitors GF109203X, G?6983, and G?6976 blocked PKC activity at concentrations independent of those necessary to block TCDD induction of CYP1A1-luciferase activity. For all inhibitors, reduction in CYP1A1-luciferase activity was independent of AhR activation, as determined by electrophoretic mobility shift analysis of TCDD-activated nuclear AhR. The specific PKC inhibitors did not significantly alter cytosolic or nuclear levels of AhR protein, whether alone or in combination with TCDD. These results suggested that PKC was not the sole factor responsible for regulation of CYP1A1.     

Role of protein kinase C in BSA-AGE-mediated inducible nitric oxide synthase expression in RAW 264.7 macrophages

In the present study, the roles of protein kinase C (PKC) in BSA-derived advanced glycosylation end products (BSA-AGEs)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression were investigated. Treatment of RAW 264.7 cells with BSA-AGEs caused dose- and time-dependent increases in NO release and iNOS expression in RAW 264.7 cells, whereas BSA alone had no effect on iNOS induction. The tyrosine kinase inhibitor (genistein), the phosphatidylinositol-specific phospholipase C inhibitor (U-73122), the phosphatidylcholine-specific phospholipase C inhibitor (D-609), and the PKC inhibitors (staurosporine, Ro 31-8220, and Go 6976) all inhibited BSA-AGE-induced NO release and iNOS expression in RAW 264.7 cells. Stimulation of RAW 264.7 cells with BSA-AGEs resulted in the formation of inositol monophosphate; the response was attenuated by U-73122 and genistein. BSA-AGEs stimulated PKC-alpha, -betaI, -delta, and -eta but not -zeta translocation from the cytosol to the membrane. However, incubation of RAW 264.7 cells with BSA-AGEs increased phosphorylation of PKC-zeta at threonine-410, which reflects activation of PKC-zeta, indicating the possible involvement of these PKC isoforms in AGE-mediated effects. Pretreatment of RAW 264.7 cells with U-73122, D-609, and genistein reduced the AGE-stimulated translocation of PKC-alpha, -betaI, -delta, and -eta and activation of PKC-zeta. Taken together, these data suggest that BSA-AGEs might activate PKC and subsequently induce iNOS expression and NO release.     

蛋白激酶C在孕酮激发的豚鼠精子顶体反应中的作用(英文)

目的:研究蛋白激酶C(PKC)在孕酮激发豚鼠精子顶体反应中的作用.方法:豚鼠精子在Ca~(2 )缺乏MCM培养液中,38.5℃5％CO_2/空气获能培养(6-6.5)h然后经Percoll密度梯度离心,再混悬于MCM中,调数至5×10~9个/升.加入PKC激活剂或抑制剂以及相关试剂15分钟后,用相差显微镜评价AR(以AR％表示).结果:在钙离子存在下,PDB或OAG可激发豚鼠精子AR,并可显著提高孕酮激发豚鼠精子AR的作用.然而,STA不仅可抑制AR,也可阻止孕酮激发豚鼠精子AR.同时,依他酸和硝苯地平均可阻断P_4以及P_4和PDB激发豚鼠精子AR.此外,新霉素可抑制P_4激发豚鼠精子AR,而且PDB可部分逆转这种作用.结论:激活或抑制PKC可明显增加或降低P_4激发的豚鼠精子AR,表明PKC在生理性AR的信号转导通路中起重要作用.     

Prevention of macrophage adhesion molecule-1 (Mac-1)-dependent neutrophil firm adhesion by taxifolin through impairment of protein kinase-dependent NADPH oxidase activation and antagonism of G protein-mediated calcium influx

Taxifolin has been reported to down-regulate the expression of intercellular adhesion molecule-1 (ICAM-1), a receptor-mediating firm adhesion with beta2 integrin (e.g., Mac-1) expressed on leukocytes. To evaluate whether taxifolin could modulate Mac-1-dependent firm adhesion by neutrophils, and the possible mechanism(s) underlying its anti-inflammatory action, its effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol-12-myristate-13-acetate (PMA)-activated peripheral human neutrophils were studied. Pretreatment with taxifolin (1-100 microM) concentration-dependently diminished fMLP- or (PMA)-induced Mac-1-dependent firm adhesion and upexpression of surface Mac-1. Mobilisation of intracellular calcium and production of reactive oxygen species (ROS) signal the upexpression of Mac-1 and firm adhesion by neutrophils. Taxifolin impeded the calcium influx induced by fMLP (a receptor-mediated activator) or AlF(4)(-) (a G protein-mediated activator). Taxifolin also effectively inhibited the fMLP- or PMA-induced ROS production with 50% inhibitory concentration (IC(50)) less than 10microM, possibly through impairing the activation of NADPH oxidase, a major ROS-generating enzyme in neutrophils, by restricting the activation of p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase C (PKC). In conclusion, we propose that impairment of ROS production by NADPH oxidase through interfering with p38 MAPK- and/or PKC-dependent signals, and antagonism of G protein-mediated calcium influx may account for the inhibition of Mac-1-dependent neutrophil firm adhesion that confers taxifolin the anti-inflammatory activity.     

Activation of protein kinase Cα signaling prevents cytotoxicity and mutagenicity following lead acetate in CL3 human lung cancer cells

Protein kinase C (PKC) family of serine/threonine protein kinases is sensitive signaling transducers in response to lead acetate (Pb) that could transmit phosphorylation cascade for proliferation and de-differentiation of neural cells. However, little is known as to the impact of PKC on Pb genotoxicity. Here we investigate whether Pb activates the conventional/classical subfamily of PKC (cPKC) signaling to affect cytotoxicity and mutagenicity in CL3 human non-small-cell lung adenocarcinoma cells. Pb specifically promoted membrane localization of the α isoform of PKC in CL3 cells. Pb also elicited Raf-1 activation as measured by the induction of phospho-Raf-1^S338 and the dissociation from the Raf-1 kinase inhibitor protein. Inhibition of cPKC activity using Gö6976 or depletion of PKCα by introducing specific small interfering RNA blocked the induction of phospho-Raf-1^S338, phospho-MKK1/2 and phospho-ERK1/2 in cells exposed to Pb. Intriguingly, declining PKCα enhanced the Pb cytotoxicity and revealed the Pb mutagenicity at the hprt gene. The results suggest that PKCα is obligatory for activation of the Raf-1–MKK1/2–ERK1/2 signaling module and plays a defensive role against cytotoxicity and mutagenicity following Pb exposure. Results obtained in this study also support our previous report showing that ERK1/2 activity is involved in preventing Pb genotoxicity.