Modulation Of Protein Kinase C Activity Research Articles

Roads to Dysfunction

See related article, pages 951–960 Oxidized low-density lipoprotein (oxLDL) is considered to be the strongest proatherogenic lipoprotein. OxLDL is rapidly taken up by endothelial cells and macrophages and via the steps of foam cell formation and cell death, oxLDL eventually accumulates in the lipid core of atherosclerotic plaques.1 The interaction of oxLDL with cells is mediated by several receptors.2 Members of the scavenger receptor family predominate in macrophages,3 whereas endothelial cells mainly take up oxLDL by the lectin-like ox-LDL receptor-1 (LOX-1).4 On binding to LOX-1, oxLDL activates a multitude of signaling cascades involving MAP kinases,5,6 protein kinase C (PKC),7,8 and protein kinase B9 in endothelial cells. The consequence of this cellular activation, is apoptosis and superoxide anion production, the latter originating from the NADPH oxidase and uncoupling of the endothelial NO synthase (eNOS). OxLDL therefore is a strong inducer of endothelial dysfunction, a state in which the endothelium, instead of generating NO, produces superoxide anions. OxLDL also modulates the specific activity of NOS. It increases caveolin I expression and the interaction of NOS with this protein impairs NO production.10 Furthermore, oxLDL modulates PKC activity and expression and this family of kinases has been shown to acutely attenuate eNOS-dependent NO production and to promote eNOS uncoupling during prolonged exposure times.11 Several observations suggest that the supply of eNOS with the substrate l-arginine is impaired in the presence of oxLDL: In hypercholesterolemia, supplementation of l-arginine improves endothelium-dependent relaxation and NO production;12,13 l-arginine also attenuates the development of atherosclerosis,14 and normalizes leukocyte adhesion …

Elucidating the role of protein kinase C in chronic lymphocytic leukaemia

While advances have been made in the clinical treatment of chronic lymphocytic leukaemia (CLL) in recent years, it is still an incurable disease and therefore the identification of novel drug therapies is of paramount importance. Understanding the molecular mechanisms that govern the survival of CLL cells is fundamental in achieving this goal. A number of studies indicate that protein kinase C (PKC)- and phosphatidylinositol-3-kinase (PI3K)- mediated signalling pathways are central to CLL cell survival, and as such PKC has gained renewed interest as a potential drug target in CLL. This may be because it represents a closely-related family of ten protein kinases, which due to the redundancy that exists between isoforms offers an opportunity for the design of isoform specific inhibitors drugs that target leukaemic cells whilst showing reduced toxicity for normal cells. Indeed, PKC signalling pathways have already been considered as targets for specific anticancer drugs [1-3]. Therefore, this short review will focus on the effect of modulating PKC activity in CLL cells and explore whether targeting PKCs could represent a valid therapy for this leukaemia.

The linoleic acid derivative DCP-LA selectively activates PKC-ɛ, possibly binding to the phosphatidylserine binding site

This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM-100 microM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-epsilon. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-epsilon. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-epsilon, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-gamma, a conventional PKC, but to a much lesser extent compared with that for PKC-epsilon, by a mechanism distinct from PKC-epsilon activation. Thus, DCP-LA serves as a selective activator of PKC-epsilon, possibly by binding to the phosphatidylserine binding site on PKC-epsilon. These results may provide fresh insight into lipid signaling in PKC activation.

Cellular and molecular mechanisms of tissue protection by lipophilic calcium channel blockers

Long-acting third-generation dihydropyridine calcium channel blockers (CCBs) improve endothelial dysfunction and prevent cardiovascular events in humans, but their cellular and molecular mechanisms of tissue protection are not elucidated in detail. We assessed organ (renal) protection by the highly lipophilic CCB lercanidipine in a double-transgenic rat (dTGR) model with overexpression of human renin and angiotensinogen genes. We randomly treated dTGR with lercanidipine (2.5 mg/kg/day; n=20) or vehicle (n=20) for 3 wk. Furthermore, we explored the influence of lercanidipine on protein kinase C (PKC) signaling in vivo and in vitro using endothelial and vascular smooth muscle cell cultures. Cumulative mortality was 60% in untreated dTGR, whereas none of the lercanidipine-treated animals died (P<0.001). We found significantly less albuminuria and improved renal function in lercanidipine-treated dTGR (both P<0.05). Lercanidipine treatment also significantly (P<0.05) reduced blood levels of the endogenous NOS inhibitor asymmetric dimethylarginine. On histological examination, we observed significantly less tissue inflammation and fibrosis in lercanidipine-treated animals (both P<0.05). Lercanidipine significantly inhibited angiotensin (ANG) I-mediated PKC-alpha and -delta activation in vivo and in vitro, partly due to reduced intracellular calcium flux. As a result, lercanidipine improved endothelial cell permeability in vitro. Lercanidipine prevents tissue injury and improves survival in a model of progressive organ damage. These effects may result, at least in part, from inhibition of tissue inflammation as well as improved NO bioavailability. Modulation of PKC activity may be an important underlying intracellular mechanism.

Modulating protein kinase C (PKC) to increase the efficacy of chemotherapy: stepping into darkness.

The identification of molecules that promote chemotherapeutic resistance would allow rationally designed approaches to abrogate this resistance, thereby possibly improving clinical outcomes for patients with cancer. In this regard, the PKC family is attractive for targeting, because it is comprised of a family of isoforms that play key roles in multiple cellular processes and can contribute to cellular transformation. Encouraging in vitro data originally showed that approaches to modulate PKC activity through small-molecule inhibitors or genetic manipulation could affect tumor cell survival. Recently, some of these approaches have begun clinical testing. Early-stage clinical trials revealed scattered clinical responses to these agents, but the most recent clinical trials have shown that combining modulators of PKC with standard chemotherapy does not improve outcome over chemotherapy alone. In this review, we will trace the development of these approaches, and discuss possible explanations for the recent negative results. Importantly, we will suggest guidelines for the clinical evaluation of PKC modulators.

Effect of novel modulators of protein kinase C activity upon chemotherapy-induced differentiation and apoptosis in myeloid leukemic cells.

Modulation of protein kinase C (PKC) activity has been demonstrated to either prevent or enhance drug-induced apoptosis in various tissue types. We tested four novel modulators of PKC activity in comparison to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) for the capability to affect differentiation, cell cycle progression and apoptosis in the human myeloid leukemia cell lines U937 and HL-60. Farnesyl thiotriazole (FTT) and N-(n-heptyl)-5-chloro-1-naphthalenesulfonamide (SC-10) are both direct activators of PKC, whereas 6-(2-(4-[(4-fluorophe-nyl)phenylmethylene]-1-piperidinyl)ethyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one (R59022) and [3-[2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2,3-dihydro-2-thioxo-4(1H)-quin-azolinone (R59949) are diacyl glycerol kinase inhibitors that activate PKC by enhancing the levels of the endogenous ligand diacyl glycerol. U937 cells displayed a slight reduction in the number of cells in G(2)/M cell cycle phase after exposure to FTT, SC-10, R59022 and R59949, respectively. In contrast, HL-60 cells demonstrated a largely unaltered cell cycle distribution. Whereas TPA treatment resulted in a strong induction of p21(WAF/CIP1), c-Fos and c-Jun levels, neither one of the novel PKC activators altered expression of these proteins. Consequently, we tested the ability of the activators to cause membrane translocation of PKC. While TPA treatment resulted in translocation of the PKC isoforms alpha, delta and epsilon, SC-10 and FTT failed to induce alterations in the PKC content of the membrane and cytosolic fractions, respectively. Expression of the beta(2)-integrin CD11c that is induced during TPA-mediated differentiation remained unaltered after exposure to SC-10 and was partly reduced after treatment with FTT. To further investigate the effect of these activators upon apoptosis in leukemic cells, HL-60 and U937 cells were treated with 1-beta-D-arabinofuranosylcytosine (Ara-C) or etoposide (VP-16). Whereas TPA strongly reduced apoptosis in Ara-C- or VP-16-treated U937 cells, little if any reduction was observed after pretreatment with either FTT, SC-10, R59022 or R59949, respectively, in these cells. In contrast, TPA enhanced apoptosis in Ara-C- or VP-16-treated HL-60 cells. Interestingly, FTT and SC-10 demonstrated a protective effect in Ara-C-treated HL-60 cells. Taken together, these data suggest that the novel PKC activators FTT, SC-10, R59022 and R59949 exhibit modest biological effects upon leukemic blast cells, and are not capable of enhancing the apoptotic response of these cells to cytotoxic drugs.

Protein kinase C changes in diabetes: Is the concept relevant to neuropathy?

Protein kinase C (PKC) comprises a superfamily of isoenzymes, many of which are activated by 1,2-diacylglycerol (DAG) in the presence of phosphatidylserine. In order to be capable of DAG activation, PKC must first undergo a series of phosphorylation at three conserved sites. PKC isoforms phosphorylate a wide variety of intracellular target proteins and have multiple functions in signal transduction-mediated cellular regulation. An elevation in DAG levels and an increase in composite PKC activity and/or certain isoforms occurs in several nonneural tissues from diabetic animals, including the vasculature. The ability of isoform-specific PKC inhibitors to antagonize diabetes-induced abnormalities has implicated altered PKC beta activity in the onset of several diabetic complications, In contrast to many other tissues, DAG levels fall in diabetic nerve and a consistent pattern of change in PKC activity has not been observed. Treatments that alter PKC activity affect nerve Na+, K+-ATPase activity, but the mechanism involved is not well understood, Inhibition of PKC beta in diabetic rats appears to correct reduced nerve blood flow and decreased nerve conduction velocity. These and other findings indicate that changes in the neurovasculature exert adverse effects during the pathogenesis of diabetic neuropathy. Still unresolved is a clear-cut role for PKC in the development of abnormalities in neural cell metabolism. Further progress will depend on a more complete understanding of the functions of individual PKC isoforms in nerve. Future investigation could focus profitably on biochemical processes in nerve cells that modulate PKC activity and that are altered in diabetes, such as vascular endothelial growth factor levels and production of reactive oxygen species arising from oxidative stress.

Effect of modulators of protein kinase C activity on Ca2+ transport in retinal rod microsomes.

The effect of modulators of protein kinase C (PKC) activity on Ca2+ translocation in retinal rod microsomes was studied. It is shown that PKC activators (phorbol 12-myristate-13-acetate (PMA) and diacylglycerol (DAG)) and inhibitors (chelerythrine chloride, polymyxin B, and phloretin) stimulate and inhibit ATP-dependent Ca2+ uptake in retinal rod microsomes, respectively. This effect is apparently due to an influence of PKC on Ca-ATPase contained in these vesicular structures. It was found that PKC inhibitors (chelerythrine chloride, polymyxin B, and phloretin) and activators (PMA and DAG) potentiate Ca2+ release from Ca2+-loaded retinal rod microsomes. Specific and nonspecific mechanisms of Ca-release stimulation by the modulators of PKC activity are discussed.

Protein kinase C pathway is involved in regulating the secretion of prostatic acid phosphatase in human prostate cancer cells.

The stimulated secretion of prostatic acid phosphatase (PAcP) has been known to be a hallmark of androgen action on human prostate epithelial cells for the last five decades. The molecular mechanism of androgen action on PAcP secretion, however, has remained mostly unknown. We investigated the molecular mechanism that promotes PAcP secretion in LNCaP human prostate carcinoma cells which express PAcP and are androgen-responsive. Treatment with 12-o-tetradecanoyl phorbol-13-acetate (TPA), a protein kinase C (PKC) activator, resulted in an increased secretion of PAcP in a dose- and time-dependent fashion. 4Alpha-phorbol, a biologically inactive isomer of TPA, had no effect. This TPA stimulation of PAcP secretion was observed 2 h after exposure, while TPA did not have a significant effect on the mRNA level even with a 6 h treatment. A23187 calcium ionophore, known to mobilize cellular calcium which is a co-factor of PKC, also activated PAcP secretion. This TPA stimulation of PAcP secretion was more potent than the conventional stimulating agent 5alpha-dihydrotestosterone (DHT) at the same concentration of 50 nM. Furthermore, the action of TPA and DHT on PAcP secretion was blocked by five different PKC inhibitors. Results also showed that DHT, as well as TPA, could rapidly modulate PKC activity. Therefore, PKC can regulate PAcP secretion, and may also be involved in DHT action on PAcP secretion.

Shedding of growth hormone-binding protein is inhibited by hydroxamic acid-based protease inhibitors: proposed mechanism of activation of growth hormone-binding protein secretase.

The present study describes events postulated to be involved in the regulated mechanism of proteolytic shedding of growth hormone (GH)-binding protein (GHBP). Using Chinese hamster ovary (CHO) cell lines stably transfected either with the full-length human GH receptor (hGHR) or with the cytoplasmic domain-truncated hGHR (hGHR(tr)), we show that the phorbol ester, phorbol 12-myristate 13-acetate (PMA), caused a rapid time- and dose-dependent increase in GHBP secretion, which, as expected, was matched by a corresponding decrease in cell-surface GHR. Furthermore, PMA equally enhanced GHBP release from CHO/hGHR(tr) cells, suggesting that the cytoplasmic domain of hGHR is not essential for PMA-induced shedding. PMA is known to specifically activate protein kinase C and, indeed, the stimulatory effects of PMA in both cell lines were completely inhibited by the protein kinase inhibitor, staurosporine (100 nM), suggesting that activation of protein kinase C (PKC) may mediate PMA-induced GHBP shedding. Since proteolytic cleavage of several cell-surface proteins was shown to be stimulated by modulators of PKC activity and inhibited by metalloprotease inhibitors, we studied the effects of two hydroxamic acid-based inhibitors of zinc-dependent metalloproteases, BB-3103 and Ro31-9790, on GHBP proteolysis. Pretreatment of CHO/hGHR cells with both these inhibitors reduced PMA-enhanced shedding of GHBP, in a dose-dependent manner, with IC(50) values of approximately 0.41 microM for BB-3103 and approximately 0.97 microM for Ro31-9790. In addition, these inhibitors dose-dependently reduced the shedding enhanced by the sulfhydryl alkylator, N-ethylmaleimide (NEM), with IC(50) values of approximately 0.32 microM and approximately 0.58 microM for BB-3103 and Ro31-9790 respectively. It was of interest to find out that Ro31-9790 acted not only to modulate PMA- or NEM-induced shedding processes, but also markedly reduced the spontaneous, time-dependent accumulation of GHBP released from CHO/hGHR cells growing in serum-containing medium. Taken together, these results suggest that one or more zinc-dependent metalloprotease(s), acting at the cell surface, may be involved in GHBP secretase activity. A scheme is proposed whereby at least part of the regulated maturation and/or activation of the protease activity may involve a cysteine-switch mechanism and/or PKC-dependent phosphorylation. In the long run, specific inhibitors of these processes could be applied in the regulation of GHBP levels and, thus, of GH availability and/or activity.

Resistance to induced apoptosis in the human neuroblastoma cell line SK‐N‐SH in relation to neuronal differentiation

Much evidence suggests that apoptosis plays a crucial role in cell population homeostasis that depends on the expression of various genes implicated in the control of cell life and death. The sensitivity of human neuroblastoma cells SK-N-SH to undergo apoptosis induced by thapsigargin was examined. SK-N-SH were previously differentiated into neuronal cells by treatments with retinoic acid (RA), 4 beta-phorbol 12-myristate 13-acetate (PMA) which increases protein kinase C (PKC) activity, and staurosporine which decreases PKC activity. Neuronal differentiation was evaluated by gamma-enolase, microtubule associated protein 2 (MAP2) and synaptophysin immunocytochemistry. The sensitivity of the cells to thapsigargin-induced apoptosis was evaluated by cell viability and nuclear fragmentation (Hoechst 33258) and compared with pro-(Bcl-2, Bcl-x(L)) and anti-apoptotic (Bax, Bak) protein expression of the Bcl-2 family. Cells treated with RA and PMA were more resistant to apoptosis than controls. Conversely, the cells treated with staurosporine were more susceptible to apoptosis. In parallel with morphological modifications, the expression of inhibitors and activators of apoptosis was directly dependent upon the differentiating agent used. Bcl-2 expression was strongly increased by PMA and drastically decreased by staurosporine as was Bcl-x(L) expression. Bax and Bak expression were not significantly modified. These results demonstrate that drugs that modulate PKC activity may induce a modification of Bcl-2 expression as well as resistance to the apoptotic process. Furthermore, the expression of Bcl-2 was reduced by toxin B from Clostridium difficile and, to a lesser extent, by wortmannin suggesting a role of small G-protein RhoA and PtdIns3 kinase in the control of Bcl-2 expression. Our data demonstrate a relationship between the continuous activation of PKC, the expression of Bcl-2 protein family and the resistance of differentiated SK-N-SH to apoptosis.

Direct Interaction of All-trans-retinoic Acid with Protein Kinase C (PKC): IMPLICATIONS FOR PKC SIGNALING AND CANCER THERAPY

Protein kinase C (PKC) regulates fundamental cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. All-trans-retinoic acid (atRA) modulates PKC activity, but the mechanism of this regulation is unknown. Amino acid alignments and crystal structure analysis of retinoic acid (RA)-binding proteins revealed a putative atRA-binding motif in PKC, suggesting existence of an atRA binding site on the PKC molecule. This was supported by photolabeling studies showing concentration- and UV-dependent photoincorporation of [(3)H]atRA into PKCalpha, which was effectively protected by 4-OH-atRA, 9-cis-RA, and atRA glucuronide, but not by retinol. Photoaffinity labeling demonstrated strong competition between atRA and phosphatidylserine (PS) for binding to PKCalpha, a slight competition with phorbol-12-myristate-13-acetate, and none with diacylglycerol, fatty acids, or Ca(2+). At pharmacological concentrations (10 micrometer), atRA decreased PKCalpha activity through the competition with PS but not phorbol-12-myristate-13-acetate, diacylglycerol, or Ca(2+). These results let us hypothesize that in vivo, pharmacological concentrations of atRA may hamper binding of PS to PKCalpha and prevent PKCalpha activation. Thus, this study provides the first evidence for direct binding of atRA to PKC isozymes and suggests the existence of a general mechanism for regulation of PKC activity during exposure to retinoids, as in retinoid-based cancer therapy.

1,25(OH)(2)-vitamin D(3) affects the subcellular distribution of protein kinase C isoenzymes in rat duodenum: influence of aging.

We have previously shown that the steroid hormone 1, 25-dihydroxy-vitamin D(3) [1,25(OH)(2)D(3)] stimulates total cell protein kinase C (PKC) activity in rat duodenum, an effect that is severely impaired in old animals. We further examined the role of 1, 25(OH)(2)D(3) on PKC as it relates to aging by measuring hormone-induced changes in subcellular localization of PKC activity and isoenzymes in duodenal mucosae from young (three-month-old) and aged (24-month-old) rats. Short treatment of duodenum with 1, 25(OH)(2)D(3) (0.1 nM, 1 min) increased membrane-associated PKC activity, whereas it decreased the activity in the cytosol of young rats but was without significant effect in aged animals. Furthermore, the ability to translocate was present in young animals after a short treatment with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA; 100 nM) or dioctanoyl-glycerol (50 microM), whereas the ability was absent in aged rats, suggesting that PKC function was impaired with aging independent of agonist stimulation. The expression of specific PKC isoenzymes and changes in their subcellular distribution after short exposure of the duodenum to the hormone were determined. Western blot analysis of total homogenates using antibodies to various PKC isoforms allowed detection of PKC alpha, beta, and delta. The expression of the straight theta and the zeta isoforms was in addition demonstrated by reverse transcription-polymerase chain reaction. The pattern of isoenzymes present in the duodenum was unaffected by aging. In young rats, 1, 25(OH)(2)D(3) translocates PKC alpha, beta, and delta to the membrane and nucleus; however, no translocation of PKC isoforms was observed in 24-month-old animals in response to the hormone. In summary, in rat duodenum, 1,25(OH)(2)D(3) modulation of PKC activity and isoenzyme subcellular distribution are impaired with aging and may explain age-induced alterations in the intestinal processes under the control of the hormone.

Protein kinase Ca is involved in the cell condensation during chondrogenesisin vitro

In order to investigate the role of protein kinase C (PKC) in chondrogenic differentiation, we examined the localization of PKC isoforms in a limb bud micromass culture system. PKCα is specifically localized in the regions which would become cartilage nodules, while PKCλ/ι and ζ display widespread distribution in the whole culture. Distribution of PKCa change along with promotion or inhibition of chondrogenesis by lysophosphatidylcholine or phorbol 12‐myristate 13‐acetate. On the other hand, localization of PKCA/i or ζ was not changed by the modulation of chondrogenesis. Peanut agglutinin binding protein which is associated with cell aggregation during chondrogenesis was present in the cell condensation regions and its expression in those regions was influenced by PKC activity. Expression of fibronectin and N‐cadherin in the cell condensing area were also affected by modulation of PKC activity. These results suggest involvement of PKCα in the cell condensation, possibly through regulating expression of fi...

Conformation of the C1 phorbol-ester-binding domain participates in the activating conformational change of protein kinase C

The fluorescent phorbol ester 12-N-methylanthraniloylphorbol 13-acetate [sapintoxin D (SAPD)] was used as both the activator and the probe for the activating conformational change of the C1 domain of recombinant protein kinase C (PKC)alpha. Fluorescence emission spectra and steady-state anisotropy measurements of SAPD in fully active membrane-associated PKC show that there is a relatively hydrophobic environment and restricted motional freedom characterizing the phorbol-ester-binding site. SAPD also interacts with the membrane lipids so that it was necessary to resort to time-resolved anisotropy measurements to resolve the signals corresponding to PKC-bound SAPD from that associated with buffer and lipid. In the presence of membrane lipids (unilamellar vesicles of phosphatidylcholine and phosphatidylserine, 4:1 molar ratio) and Ca(2+), at a concentration sufficient to activate the enzyme fully, a long correlation time characteristic of highly restricted motion was observed for PKC-associated SAPD. The fraction of SAPD molecules displaying this restricted motion, in comparison with the total SAPD including that in lipids and in buffer, increased with increasing concentrations of Ca(2+) and paralleled the appearance of enzyme activity, whereas the rotational correlation time remained constant. This could be rationalized as an increase in the number of active PKC conformers in the total population of PKC molecules. It therefore seems that there is a distinct conformation of the C1 activator-binding domain associated with the active form of PKC. The addition of SAPD and dioleoyl-sn-glycerol together produced an activity higher than that achievable by either activator alone both at concentrations that alone induced maximal activity for the respective activator; this higher activity was associated with a further restriction in SAPD motion. Increasing the cholesterol concentration, the phosphatidylethanolamine concentration, the sn-2 unsaturation in phosphatidylcholine and the vesicle curvature each also elevated SAPD-induced PKC activity and again increased the PKC-associated SAPD rotational correlation time. In summary, the rotational correlation time of PKC-bound SAPD, extractable from a single time-resolved fluorescence anisotropy measurement, provides a novel probe for the involvement of interactions between the C1 domain and phorbol ester in the modulation of PKC activity.

Arachidonic acid induces a long-lasting facilitation of hippocampal synaptic transmission by modulating PKC activity and nicotinic ACh receptors

The present study was conducted to understand the effect of arachidonic acid on nicotinic acetylcholine (ACh) receptor-mediated synaptic plasticity. Arachidonic acid persistently (≥1 h) potentiated currents through neuronal nicotinic ACh receptors (α7 and α4β2) expressed in Xenopus oocytes, and the effect was blocked by the selective protein kinase C (PKC) inhibitors, such as GF109203X, PKCI, and co-expressed active PKC inhibitor peptide. This free fatty acid markedly increased nicotine-sensitive glutamate release from hippocampal slices and enhanced the rate of nicotine-sensitive miniature excitatory postsynaptic currents without affecting the amplitude in cultured hippocampal CA1 neurons under the influence of PKC. Furthermore, arachidonic acid induced a long-lasting (≥3 h) facilitation of hippocampal CA1 synaptic transmission in slices, and the effect was blocked by nicotinic ACh receptor antagonists, α-bungarotoxin and mecamylamine. The facilitation, whereas independent of N-methyl- d-aspartate (NMDA) receptors, shares a common mechanism with long-term potentiation (LTP) induced by tetanic stimulation. The results of the present study thus suggest that arachidonic acid sustains enhanced activity of nicotinic ACh receptors by interacting with a PKC pathway, thereby increasing glutamate release from presynaptic terminals, and then leading to an `LTP-like' facilitation of hippocampal synaptic transmission.

Glycated albumin stimulation of PKC-beta activity is linked to increased collagen IV in mesangial cells.

Albumin modified by Amadori-glucose adducts induces coordinate increases in the expression of extracellular matrix proteins, transforming growth factor (TGF)-beta1, and the TGF-beta type II receptor in glomerular mesangial cells. Because activation of protein kinase C (PKC) accompanies the increased mesangial cell expression of matrix proteins and TGF-beta1 induced by high ambient glucose, we postulated that glycated albumin (GA) modulates PKC activity and that PKC participates in mediating the GA-induced stimulation of matrix production. To test this hypothesis, we examined the effects of PKC inhibitors on collagen type IV production by mouse or rat mesangial cells incubated with GA, and the influence of GA on PKC activity in these cells. Increased collagen type IV production evoked by GA in 5.5 and 25 mM glucose in mouse mesangial cells was prevented by both general (GF-109203X) and beta-specific (LY-379196) PKC inhibitors. Total PKC activity, measured by phosphorylation of a PKC-specific substrate, increased with time after exposure of rat mesangial cells to GA compared with the nonglycated, glucose-free counterpart. GA caused an increase in PKC-beta1 membrane-bound fraction and in total PKC activity in media containing physiological (5.5 mM) glucose concentrations in rat mesangial cells, confirming that the glucose-modified protein, and not a "hyperglycemic" milieu, was responsible. The findings indicate that Amadori-modified albumin stimulates mesangial cell PKC activity, and that activation of the PKC-beta isoform is linked to the stimulation of collagen type IV production.

Staurosporine‐induced versus spontaneous squamous metaplasia in pre‐ and postmenopausal breast tissue

Breast cancers from pre- vs. postmenopausal women display unique characteristics that may be related to differences in epithelial differentiation between these two populations. In addition to lobular development, lactational changes, and involution, breast epithelium can undergo metaplastic alterations, often in association with carcinoma. Because protein kinase C (PKC) regulates differentiation and proliferation in many cell types, we asked whether modulation of PKC activity could define biochemical differences in breast epithelium from pre- vs. postmenopausal women. Organ cultures of normal human breast were treated with PKC agonists and antagonists. Epithelial differentiation was evaluated based on morphologic criteria and the expression of cell-type specific proteins. Staurosporine, a nonspecific but extremely potent inhibitor of PKC, induced squamous metaplasia in eight of eight cases within 2 weeks of treatment. Other inhibitors of PKC, such as calphostin C and tamoxifen, had no effect on epithelial differentiation. Long-term treatment with phorbol esters also did not induce squamous metaplasia. However, stimulation of cAMP levels by forskolin and isobutyl-methyl-xanthene (IMX) rapidly induced squamous metaplasia, as has been previously reported. Surprisingly, squamous metaplasia occurred in 10 of 12 cultures derived from postmenopausal women in the absence of exogenous agents. Untreated cultures derived from premenopausal women never developed this type of epithelium (0 of 11). Therefore, breast epithelium from pre- and postmenopausal women responded differently to in vitro culture. Forskolin/IMX or staurosporine can reproduce these conditions, acting independent of menopausal status. Because staurosporine's action was unique among PKC inhibitors, staurosporine may induce squamous metaplasia of breast epithelium by a PKC-independent mechanism. J. Cell. Physiol. 176:245–254, 1998. © 1998 Wiley-Liss, Inc.

Breast cancer cell invasiveness: correlation with protein kinase C activity and differential regulation by phorbol ester in estrogen receptor-positive and -negative cells.

Increased protein kinase C (PKC) activity in malignant breast tissue and in most aggressive breast cancer cell lines has suggested a possible role of PKC in breast carcinogenesis and tumor progression. We have investigated here the involvement of PKC in the in vitro invasiveness and motility of several breast cancer cell lines. Modulation of PKC activity by treatment with a phorbol ester (TPA), drastically increased the invasiveness of 2 estrogen receptor-positive (ER+) lines (MCF7 and ZR 75.1), whereas it markedly decreased the invasiveness of 2 ER- cell lines (MDA-MB-231 and MDA-MB-435). A PKC inhibitor (H7) reversed the TPA effects in MCF7 cells, whereas it mimicked TPA action in MDA-MB-231 cells. All of these effects of TPA also were observed to a similar extent for cell chemotaxis, and they were not dependent on protein neo-synthesis. In parallel, short TPA treatment induced cell spreading and microtubule organization in MCF7 cells and inverse morphological changes in MDA-MB-231 cells. In ER+ cells, constitutive PKC activity and PKCalpha expression were very low as compared to ER- cells, and this correlated with the invasive potential of the cells. The opposed effects of TPA in ER+ and ER- cells could be due to the abnormal TPA regulation of PKCalpha observed in ER- cells.

A functional homolog of mammalian protein kinase C participates in the elicitor-induced defense response in potato.

The elicitor-induced activation of the potato pathogenesis-related gene PR-10a is positively controlled by a protein kinase(s) that affects the binding of the nuclear factors PBF-1 (for PR-10a binding factor-1) and PBR-2 to an elicitor response element (ERE). In this study, we have identified a kinase that has properties similar to the conventional isoenzymes of the mammalian protein kinase C (PKC) family. the treatment of potato tuber discs with specific inhibitors of PKC abolished the elicitor-induced binding of the nuclear factor PBF-2 to the ERE. This correlated with a reduction in the accumulation of the PR-10a protein. In contrast, treatment of the tuber discs with 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of PKC, led to an increase in binding of PBF-2 to the ERE and the corresponding increase in the level of the PR-10a protein, mimicking the effect seen with the elicitor arachidonic acid. Biochemical characterization of proteins extracted from the particulate fraction of potato tubers demonstrated that a kinase belonging to the conventional isoforms of PKC is present. This was confirmed by immunoprecipitation with antibodies specific to the conventional isoforms of human PKC and in-gel kinase assays. The ability of the immunoprecipitates to phosphorylate the alpha-peptide (a PKC specific substrate) in the presence of the coactivators calcium, phosphatidylserine, and TPA strongly suggested that the immunoprecipitated kinase is similar to the kinase characterized biochemically. Finally, the similar effects of the various modulators of PKC activity on the elicitor-induced resistance against a compatible race of Phytophthora infestans implicate this kinase in the overall defense response in potato.