Independent PKC-delta Research Articles

Antiepileptic drugs reduce efficacy of methotrexate chemotherapy by downregulation of Reduced folate carrier transport activity

Concurrent treatment with methotrexate (MTX) and antiepileptic drugs, such as phenobarbital (PB), reduces the efficacy of MTX chemotherapy in childhood acute lymphoblastic leukemia (ALL). This can result from defective Reduced folate carrier (Rfc1)-dependent cellular uptake of MTX. Indeed, we have shown that functional Rfc1 activity is significantly reduced by clinically relevant concentrations of the anticonvulsant drugs PB or carbamazepine in an adequate in vitro model. As PB is known to regulate carrier-associated transport by the nuclear receptor constitutive androstane receptor (CAR), we investigated the involvement of the CAR signaling cascade and the mode of PB-induced downregulation of Rfc1 activity. CAR activation by PB or the CAR agonist 1,4-bis[2-(3,5-dichloro- pyridyloxy)]-benzene resulted in translocation of Ca(2+)-dependent protein kinase Calpha (cPKCalpha) to the plasma membrane related to significantly elevated PKC activities. In contrast, subcellular localization of Ca(2+)-independent PKCdelta was only marginally altered. Studies on intracellular distribution of the Rfc1 protein indicated that PB-induced activation of cPKCalpha was associated with carrier internalization from the plasma membrane into the cytosol independent of the Rfc1 phosphorylation status. In conclusion, we identified for the first time the molecular mechanism of this clinically relevant drug resistance in patients with ALL concurrently receiving MTX chemotherapy and antiepileptic drugs.

Alterations in the protein kinase C signaling activated by a parthenogenetic agent in oocytes from reproductively old mice

To investigate the effect of female age on oocyte developmental competence, we focused on protein kinase C (PKC), a major component of the signalling pathway involved in oocyte activation, and put forward the hypothesis that, as it occurs in many organs and tissues, aging affects PKC function in mouse oocytes. Biochemical activity of PKC along with the expression and subcellular distribution of some PKC isoforms were monitored in young and old mouse oocytes parthenogenetically activated by SrCl(2). We found that PKC activity increased reaching a level that was lower in old compared to young oocytes in association with an incomplete translocation of PKCbetaI to the plasma membrane. Moreover, old oocytes exhibited a reduced expression of PKCbeta1 and PKCalpha at the protein level, without significant effects on the expression of the Ca(2+)-independent PKCdelta. Detectable amounts of PKCbeta1 mRNA were observed in young and old oocytes at GV stage with no difference between the two groups of age. When meiotic progression to anaphase II up to first cleavage were analyzed, a delay in meiosis resumption and significantly lower rates of pronuclei formation and first cleavage were observed in old compared to young oocytes. Moreover, we found that, in contrast to SrCl(2), PMA (12-O-tetradecanoyl phorbol-13-acetate), a PKC agonist, was ineffective in activating old oocytes. Present findings provide evidence that aging affects the correct storage and activation of some PKCs, functional components of the machinery involved in oocyte activation, and suggest that these changes may negatively influence the activation competence of old oocytes.

Mechanism of the negative inotropic effects of alpha 1-adrenoceptor agonists on mouse myocardium.

This study was done to identify the mechanism of the alpha1-adrenoceptor (AR) mediated negative inotropic effects of phenylephrine (PE) on adult mouse myocardium. As reported by others, we also found that the nonselective alpha1AR agonist PE produced a negative inotropic effect on ventricular strips from adult mice that was inhibited by the alpha1AAR antagonist 5-methylurapidil (5MU) but not by the alpha1BAR antagonist chloroethylclonidine (CEC) or the alpha1DAR antagonist BMY 7378. The selective alpha1AAR agonist A61603 also produced a negative inotropic effect, which was antagonized by 5MU. Phorbol 12,13-dibutyrate (activator of all PKC isoforms) mimicked the negative inotropic responses to PE and A61603. The negative inotropic effects of PE were inhibited by bisindolylmaleimide (inhibitor of all PKC isoforms) but not by Gö 6976 (inhibitor of Ca2+-dependent PKC). Rottlerin, an inhibitor of Ca2+-independent PKCdelta, antagonized the negative inotropic effects of PE and A61603. PE and A61603 increased the translocation of PKCdelta, which was prevented by rottlerin. These data suggest that the alpha1AR-mediated negative inotropy on adult mouse myocardium is signaled by Ca2+-independent PKCdelta.

Protein kinase C-epsilon mediates bradykinin-induced cyclooxygenase-2 expression in human airway smooth muscle cells.

We previously reported that proinflammatory mediator bradykinin (BK) induces cyclooxygenase (COX)-2 expression in human airway smooth muscle (HASM), but the mechanism is unknown in any biological system. Here, we studied the role of specific protein kinase C (PKC) isozyme(s) in COX-2 expression. Among the eight PKC isozymes present in HASM cells, the Ca2+-independent PKC-delta and -epsilon and the Ca2+-dependent PKC-alpha and -betaI were translocated to the nucleus upon BK stimulation. BK-induced COX-2 expression and prostaglandin E2 (PGE2) accumulation were mimicked by the direct PKC activator phorbol 12-myristate 13-acetate (PMA) and inhibited by the broad spectrum PKC inhibitor bisindolylmaleimide I. However, the selective Ca2+-dependent PKC isozyme inhibitor Go 6976 had no effect. Furthermore, the membrane-permeable calcium chelator BAPTA-AM had no effect on BK-induced COX-2 expression and COX activity despite its inhibition of PGE2 accumulation, suggesting the involvement of Ca2+-independent PKC isozymes. Rottlerin, a PKC-delta inhibitor, also had no effect, likely implicating PKC-epsilon. BK-stimulated transcriptional activation of a COX-2 promoter reporter construct was enhanced by overexpression of wild-type PKC-epsilon and abolished by a dominant negative PKC-epsilon, but it was not affected by wild-type or dominant negative PKC-alpha or -delta. Collectively, our results demonstrate that PKC-e mediates BK-induced COX-2 expression in HASM cells.

The C1 domain of protein kinase C as a lipid bilayer surface sensing module.

The activity of membrane-associated protein kinase C (PKC) is tightly controlled by the physical properties of the membrane lipid bilayer, in particular, curvature stress, which is induced by bilayer-destabilizing lipid components. An important example of this is the weakened lipid headgroup interactions induced by phosphatidylethanolamine (PE) and cholesterol. In this work our previous observation with a mixed isoform PKC showing a biphasic dependence of activity as a function of membrane curvature stress [Slater et al. (1994) J. Biol. Chem. 269, 4866-4871] was here extended to individual isoforms. The Ca(2+)-dependent PKCalpha, PKCbeta, and PKCgamma, along with Ca(2+)-independent PKCdelta, but not PKCepsilon or PKCzeta, displayed a biphasic activity as a function of membrane PE content. The fluorescence anisotropy of N-(5-dimethylaminonaphthalene-1-sulfonyl)dioleoylphosphatidylserine (dansyl-PS), which probes the lipid environment of PKC, also followed a biphasic profile as a function of PE content for full-length PKCalpha, PKCbetaIotaIota, and PKCgamma as did the isolated C1 domain of PKCalpha. In addition, the rotational correlation time of both PKCalpha and PKCdelta C1-domain-associated sapintoxin D, a fluorescent phorbol ester, was also a biphasic function of membrane lipid PE content. These results indicate that the C1 domain acts as a sensor of the bilayer surface properties and that its conformational response to these effects may directly underlie the resultant effects on enzyme activity.

Effects of PKC isozyme inhibitors on constrictor responses in the feline pulmonary vascular bed.

The effects of Gö-6976, a Ca(2+)-dependent protein kinase C (PKC) isozyme inhibitor, and rottlerin, a PKC-delta isozyme/calmodulin (CaM)-dependent kinase III inhibitor, on responses to vasopressor agents were investigated in the feline pulmonary vascular bed. Injections of angiotensin II, norepinephrine (NE), serotonin, BAY K 8644, and U-46619 into the lobar arterial constant blood flow perfusion circuit caused increases in pressure. Gö-6976 reduced responses to angiotensin II; however, it did not alter responses to serotonin, NE, or U-46619, whereas Gö-6976 enhanced BAY K 8644 responses. Rottlerin reduced responses to angiotensin II and NE, did not alter responses to serotonin or U-46619, and enhanced responses to BAY K 8644. Immunohistochemistry of feline pulmonary arterial smooth muscle cells demonstrated localization of PKC-alpha and -delta isozymes in response to phorbol 12-myristate 13-acetate and angiotensin II. Localization of PKC-alpha and -delta isozymes decreased with administration of Gö-6976 and rottlerin, respectively. These data suggest that activation of Ca(2+)-dependent PKC isozymes and Ca(2+)-independent PKC-delta isozyme/CaM-dependent kinase III mediate angiotensin II responses. These data further suggest that Ca(2+)-independent PKC-delta isozyme/CaM-dependent kinase III mediate responses to NE. A rottlerin- or Gö-6976-sensitive mechanism is not involved in mediating responses to serotonin and U-46619, but these PKC isozyme inhibitors enhanced BAY K 8644 responses in the feline pulmonary vascular bed.

Identification of protein kinase C isoforms in marine mussels.

In this paper we have examined the distribution of some isoforms of protein kinase C in different tissues from the sea mussel Mytilus galloprovincialis Lmk.. By immunoblot analysis, we have detected the presence of at least three PKC isoforms, all preferably associated with the cellular cytosolic fraction. The Ca(2+)-independent form PKC delta was separated from the Ca(2+)-dependent forms (PKC alpha and beta) by means of an ionic change chromatography on DE-52. A comparative study was carried out on the phosphorylatable non-artificial substrates. The M.B.P. protein proved to be the best substrate, while the worst was HIIIS histone which, however, is frequently used in evaluation assays of the activity.

Ca2+‐Dependent and Ca2+‐Independent Protein Kinase C Changes in the Brains of Patients with Alzheimer's Disease

We examined protein kinase C (PKC) activity in Ca2+-dependent PKC (Ca2+-dependent PKC activities) and Ca2+-independent PKC (Ca2+-independent PKC activities) assay conditions in brains from Alzheimer's disease (AD) patients and age-matched controls. In cytosolic and membranous fractions, Ca2+-dependent and Ca2+-independent PKC activities were significantly lower in AD brain than in control brain. In particular, reduction of Ca2+-independent PKC activity in the membranous fraction of AD brain was most enhanced when cardiolipin, the optimal stimulator of PKC-epsilon, was used in the assay; whereas Ca2+-independent PKC activity stimulated by phosphatidylinositol, the optimal stimulator of PKC-delta, was not significantly reduced in AD. Further studies on the protein levels of Ca2+-independent PKC-delta, PKC-epsilon and PKC-zeta in AD brain revealed reduction of the PKC-epsilon level in both cytosolic and membranous fractions, although PKC-delta and PKC-zeta levels were not changed. These findings indicated that Ca2+-dependent and Ca2+-independent PKC are changed in AD, and that among Ca2+-independent PKC isozymes, the alteration of PKC-epsilon is a specific event in AD brain, suggesting its crucial role in AD pathophysiology.

Developmental expression of protein kinase C isozymes in oligodendrocytes and their differential modulation by 4 beta-phorbol-12,13-dibutyrate.

Myelin gene expression in normal oligodendrocytes (OLG) depends on developmentally regulated protein kinase C (PKC) enzyme activity (Asotra and Macklin: J Neurosci Res 34:571-588, 1993). We studied the developmental expression of the Ca(++)-dependent PKC-alpha, -beta 1, -beta II and -gamma isozymes, and the Ca(++)-independent PKC-delta, -epsilon, -zeta and -eta isozymes in enriched rat brain OLG cultures. In A2B5+ O-2A progenitors, only PKC-delta, PKC-epsilon and PKC-zeta were detected immunocytochemically. In 04+ proligondendrocytes, PKC-beta I, -delta and -zeta were expressed moderately and low levels of PKC-alpha and -epsilon were detected. GD3+ OLG, GC+ OLG and MBP+ OLG showed increased levels of PKC-alpha, -beta I, -delta and -zeta isozymes. PKC-beta II, -gamma and -eta were poorly expressed in OLG. On immunoblots, PKC-alpha was present early and increased continually up to 18 days but PKC-beta I increased until 12 days in cultured OLG. High levels of PKC-delta, PKC-epsilon and PKC-zeta, the most abundant PKC isozymes in OLG, were maintained up to 12 days and were then slightly reduced. Interestingly, relatively high levels of PKC-alpha, PKC-beta I, PKC-beta II, PKC-gamma and PKC-epsilon isozymes were detected in purified myelin membrane although greater levels of PKC-delta were found in OLG than in purified myelin. Thus, most of the PKC isozymes found in cultured OLG were also present in myelin, although at different levels. Treatment with 50 nM 4 beta-phorbol-12,13-dibutyrate (PDB) caused a delayed downregulation of PKC-delta levels after 8 hr without modulating the expression of other PKC isozymes in 1-day OLG; in the 3-day-old and 6-day-old OLG, PDB downmodulated PKC-beta I, -delta and epsilon isozymes with only a minor effect on PKC-alpha and no reduction in PKC-zeta. Induction or downmodulation of individual PKC isozymes by phorbol esters appears to depend on the differentiation state of OLG. These data suggest that PKC-beta I, -delta and -epsilon isozymes have an important function in different cellular events of OLG differentiation. We conclude that the PKC-dependent modulation of myelin gene expression in OLG results predominantly from the Ca(++)-dependent PKC-beta I isozyme activity and the CA(++)-independent PKC-delta and PKC-epsilon activitives in a cell differentiation state-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

EFFECT OF 12-DEOXYPHORBOL 13-PHENYLACETATE ON DAUNORUBICIN RESISTANCE AND CALCIUM-INDEPENDENT PROTEIN-KINASE-C ISOZYMES IN DRUG-SENSITIVE MURINE LEUKEMIA P388 CELLS

Because of a suggested role of protein kinase C (PKC) in multidrug resistance (MDR) and recent. demonstration of molecular and biochemical heterogeneity of PKC, we examined the effect of 12-deoxyphorbol 13-phenylacetate (dPP) on intracellular accumulation and drug sensitivity of daunorubicin (DNR) in drug sensitive P388 murine leukemia cell line. In addition, effect of dPP on Ca++-independent PKC (delta, epsilon and zeta) isozymes was examined with Western blot, using laser densitometry. dPP induced significant resistance to DNR in P388 cells, however had no significant effect on DNR accumulation. In addition, dPP increased the levels of plasma membrane associated PKCdelta and epsilon isozymes in P388 cells and but had no effect on plasma membrane-associated PKCzeta isozyme. This study demonstrates a role of Ca++-independent PKCdelta and epsilon isozymes in drug resistance.

EXPRESSION OF PROTEIN-KINASE-C ISOZYMES IN MULTIDRUG-RESISTANT MURINE LEUKEMIA P388/ADR CELLS

Because of a suggested role of protein kinase C (PKC) in multidrug resistance (MDR) and recent molecular cloning of PKC isozymes, we compared the expression of Ca++-dependent PKCalpha. beta and gamma, and Ca++-independent PKCdelta, epsilon, and zeta isozymes between drug sensitive murine leukemia P388 and its MDR subline P388/ADR, using qualitative and quantitative polymerase chain reaction (PCR) and Western blot techniques. The expression of PKCalpha and PKCbeta mRNA and their proteins was higher in P388/ADR as compared to P388 cells. In contrast, PKCdelta, epsilon, zeta mRNA were decreased in P388/ADR cells as compared to P388 cells. However, at the protein level the expression of PKCdelta, epsilon, and zeta was also increased in P388/ADR cells as compared to P388 cells, suggesting an increased rate of translation of PKCdelta, epsilon, and zeta isozymes in P388/ADR cells. No PKCgamma isozyme was detected by PCR and Western blot analyses. Confocal microscopic examination revealed a distinct pattern of subcellular distribution of PKCbeta isozymes in P388/ADR when compared with P388 cells. This study demonstrates the presence of altered levels of PKC isozymes in P388/ADR cells that may suggest a role of certain PKC isozymes in MDR.