Antimycin A increases bronchopulmonary C-fiber excitability via protein kinase C alpha.

Inflammation can increase the excitability of bronchopulmonary C-fibers leading to excessive sensations and reflexes (e.g. wheeze and cough). We have previously shown modulation of peripheral nerve terminal mitochondria by antimycin A causes hyperexcitability in TRPV1-expressing bronchopulmonary C-fibers through the activation of protein kinase C (PKC). Here, we have investigated the PKC isoform responsible for this signaling. We found PKCß1, PKCδ and PKCε were expressed by many vagal neurons, with PKCα and PKCß2 expressed by subsets of vagal neurons. In dissociated vagal neurons, antimycin A caused translocation of PKCα but not the other isoforms, and only in TRPV1-lineage neurons. In bronchopulmonary C-fiber recordings, antimycin A increased the number of action potentials evoked by α,ß-methylene ATP. Selective inhibition of PKCα, PKCß1 and PKCß2 with 50 nM bisindolylmaleimide I prevented the antimycin-induced bronchopulmonary C-fiber hyperexcitability, whereas selective inhibition of only PKCß1 and PKCß2 with 50 nM LY333531 had no effect. We therefore conclude that PKCα is required for antimycin-induced increases in bronchopulmonary C-fiber excitability.

miR-216a promotes breast cancer cell apoptosis by targeting PKCα.

Breast cancer (BC) is the most common cause of death in women throughout the world. MicroRNAs (miRNAs, miR) have been identified as key regulators in carcinogenesis of several cancers, including BC. MicroRNA-216a (miR-216a) is downregulated in several cancers. Here, we evaluated the effects of miRNA-216a on breast cancer cells and the underlying mechanisms. miR-216a level was quantified by real-time RT-PCR. Cell viability was analyzed by MTT assay. Wound-healing assay was performed for detection of cell migration. Apoptosis was detected by TUNEL and caspase-3 activity assay. Moreover, the level of protein expression was determined by Western blot. We found that miR-216a expression was remarkably decreased in both human BC tissues and MCF-7 cells. miR-216a overexpression dramatically suppressed the migration and promoted the apoptosis in cultured MCF-7 cells. We validated PKCα (protein kinase C alpha, PRKCA) as a direct target of miR-216a. Knockdown of PKCα induced apoptosis and inhibited migration in cultured MCF-7 cells which were reversed by miR-216a inhibitor. Moreover, the level of miR-216a is negatively correlated with PKCα in cell lines. Our results collectively suggest that miR-216a suppressed migration and promoted apoptosis in breast cancer cells by targeting PKCα. These findings indicate that manipulation of miR-216a expression may represent a novel therapeutic strategy in the treatment of breast cancer.

Effect of melatonin on attenuating the isoflurane-induced oxidative damage is related to PKCα/Nrf2 signaling pathway in developing rats.

Isoflurane, an inhalational anesthesia, has frequently been used in pediatric anesthesia. However, research indicates that isoflurane can induce oxidative stress and affect neural and cognitive development. Melatonin, an endogenous hormone that exhibits antioxidant functions, can play a neuroprotective role by activating the PKCα/Nrf2 signaling pathway in response to oxidative stress. This study aims to determine whether the effect of melatonin on isoflurane-induced oxidative stress is related to activation of the PKCα/Nrf2 signaling pathway. Rat pups at postnatal day 7 were treated with control or 1.5% isoflurane for 4 h after pretreatment for 15 min with either melatonin (10 mg/kg i.p.) or 1% ethanol. The hematoxylin and eosin staining and transmission electron microscopic examination were used for observation of histopathology. The oxidative stress-related indicators were detected by using assay kits. The western blotting, immunohistochemistry and immunofluorescence were used to detect the activation of PKCα/Nrf2 signaling pathway. Results showed that isoflurane induced nerve damage in the hippocampus, and melatonin could reduce this injury. Oxidative stress-related indicators suggested that isoflurane can significantly increase reactive oxygen species and malondialdehyde levels, and decrease superoxide dismutase and glutathione activity compared with the control group, whereas melatonin ameliorated these indices. Expression of proteins associated with the PKCα/Nrf2 signaling pathway indicated that the neuroprotective effect of melatonin is related to activation of the PKCα/Nrf2 signaling pathway. These results suggest that the attenuating effect of melatonin on isoflurane-induced oxidative stress is related to activation of the PKCα/Nrf2 signaling pathway. These findings promote further research into underlying mechanisms and effective treatments to attenuate anesthesia neurotoxicity.

Inhibition of A-Type K+ Channels by Urotensin-II Induces Sensory Neuronal Hyperexcitability Through the PKCα-ERK Pathway.

Previous studies have implicated urotensin-II in the nociception of sensory neurons. However, to date the relevant mechanisms remain unknown. In the current study we determined the role of urotensin-II in the regulation of transient outward A-type potassium currents (IA) and neuronal excitability in trigeminal ganglion (TG) neurons. We found that application of urotensin-II to small-diameter TG neurons decreased IA in a dose-dependent manner, whereas the delayed rectifier potassium current was unaffected. The IA decrease induced by urotensin-II depended on the urotensin-II receptor (UT-R) and was associated with a hyperpolarizing shift in the steady-state inactivation curve. Exposure of TG cells to urotensin-II markedly increased protein kinase C (PKC) activity, and PKC inhibition eliminated the UT-R-mediated IA decrease. Antagonism of PKCα, either pharmacologically or genetically, but not of PKCß prevented the decrease in IA induced by urotensin-II. Analysis of phospho-extracellular signal-regulated kinase (p-ERK) revealed that urotensin-II significantly increased the expression level of p-ERK, whereas p-p38 and p-c-Jun N-terminal kinase remained unchanged. Inhibition of mitogen-activated protein kinase/ERK signaling by the kinase antagonist U0126 and PD98059 completely abolished the UT-R-mediated IA decrease. Moreover, urotensin-II significantly increased the action potential firing rate of small TG neurons; pretreatment with 4-aminopyridine prevented this effect. In summary, our findings suggest that urotensin-II selectively attenuated IA through stimulation of the PKCα-dependent ERK1/2 signaling pathway. This UT-R-dependent mechanism might contribute to neuronal hyperexcitability in TG neurons.

Synthesis and biological activity of pyrazole analogues of the staurosporine aglycon K252c.

A derivative of the staurosporine aglycon (K252c), in which the lactam ring was replaced by a pyrazole moiety, was synthesized. The resulting indolopyrazolocarbazole (3) inhibited Pim isoforms 1-3 whereas it did not impair the activity of two known targets of K252c, protein kinase C isoforms α and γ. Compound 3 exhibited moderate cytotoxic activity toward human leukemia and colon carcinoma cell lines (K562 and HCT116), strongly suggesting that this new scaffold deserves further investigations for treatment of malignancies associated with Pim activity.

Mechanistic analysis of taxol-induced multidrug resistance in an ovarian cancer cell line.

OBJECTIVES: To establish a taxol-resistant cell line of human ovarian carcinoma (A2780/Taxol) and investigate its biological features. METHODS: The drug-resistant cell line (A2780/Taxol) was established by continuous stepwise selection with increasing concentrations of Taxol. Cell morphology was assessed by microscopy and growth curves were generated with in vitro and in vivo tumor xenograft models. With rhodamine123 (Rh123) assays, cell cycle distribution and the apoptotic rate were analyzed by flow cytometry (FCM). Drug resistance-related and signal associated proteins, including P-gp, MRPs, caveolin-1, PKC-α, Akt, ERK1/2, were detected by Western blotting. RESULTS: A2780/Taxol cells were established with stable resistance to taxol. The drug resistance index (RI) was 430.7. Cross-resistance to other drugs was also shown, but there was no significant change to radioresistance. Compared with parental cells, A2780/Taxol cells were significantly heteromorphous, with a significant delay in population doubling time and reduced uptake of Rh123 (p < 0.01). In vivo, tumor take by A2780 cells was 80%, and tumor volume increased gradually. In contrast, with A2780/Taxol cells in xenograft models there was no tumor development. FCM analysis revealed that A2780/Taxol cells had a higher percentage of G0/G1 and lower S phase, but no changes of G2 phase and the apoptosis rate. Expression of P-gp, MRP1, MRP2, BCRP, LRP, caveolin-1, PKC-α, Phospho-ERK1/2 and Phospho-JNK protein was significantly up-regulated, while Akt and p38 MARK protein expression was not changed in A2780/Taxol cells. CONCLUSION: The A2780/Taxol cell line is an ideal model to investigate the mechanism of muti-drug resistance related to overexpression of drug-resistance associated proteins and activation of the PKC-α/ERK (JNK) signaling pathway.

Cyclosporine attenuates arginine transport, in human endothelial cells, through modulation of cationic amino acid transporter-1.

BACKGROUND: The spectrum of cardiovascular toxicity by cyclosporine (CsA) includes hypertension, accelerated atherosclerosis, and thrombotic microangiopathy, all of which are the result of endothelial cell dysfunction. Endothelial cell dysfunction is characterized by decreased endothelial nitric oxide synthase (eNOS) activity. Cationic amino acid transporter-1 (CAT-1) is the specific arginine transporter for eNOS. CsA has been shown to attenuate nitric oxide (NO) generation. However, the mechanism remains elusive. We hypothesize that CsA inhibits eNOS activity through modulation of its selective arginine supplier CAT-1. METHODS: We studied the effect of CsA on arginine uptake, NO2/NO3 generation, and CAT-1, protein kinase Cα (PKCα), and phosphorylated PKCα protein expression in human umbilical vein endothelial cell cultures (HUVEC) in the absence and presence of L-arginine. RESULTS: CsA (0.5-2 µg/ml) significantly attenuated arginine transport in a dose- and time-dependent manner, a phenomenon which was prevented by co-incubation with L-arginine (1 mM). The aforementioned findings were accompanied by increased protein nitration, a measure for peroxynitrite accumulation. In contrast, no changes were observed in NO2/NO3 generation. CsA significantly decreased the abundance of CAT-1 protein, an effect that was attenuated by L-arginine. PKCα and phosphorylated PKCα (CAT-1 inhibitors) protein contents were not affected by CsA. CONCLUSION: CsA inhibits arginine transport and induces protein nitration in HUVEC through modulation of CAT-1.

Interaction between nitric oxide and superoxide in the macula densa in aldosterone-induced alterations of tubuloglomerular feedback.

Tubuloglomerular feedback (TGF)-mediated constriction of the afferent arteriole is modulated by a balance between release of superoxide (O(2)(-)) and nitric oxide (NO) in macula densa (MD) cells. Aldosterone activates mineralocorticoid receptors that are expressed in the MD and induces both NO and O(2)(-) generation. We hypothesize that aldosterone enhances O(2)(-) production in the MD mediated by protein kinase C (PKC), which buffers the effect of NO in control of TGF response. Studies were performed in microdissected and perfused MD and in a MD cell line, MMDD1 cells. Aldosterone significantly enhanced O(2)(-) generation both in perfused MD and in MMDD1 cells. When aldosterone (10(-7) mol/l) was added in the tubular perfusate, TGF response was reduced from 2.4 ± 0.3 µm to 1.4 ± 0.2 µm in isolated perfused MD. In the presence of tempol, a O(2)(-) scavenger, TGF response was 1.5 ± 0.2 µm. In the presence of both tempol and aldosterone in the tubular perfusate, TGF response was further reduced to 0.4 ± 0.2 µm. To determine if PKC is involved in aldosterone-induced O(2)(-) production, we exposed the O(2)(-) cells to a nonselective PKC inhibitor chelerythrine chloride, a specific PKCα inhibitor Go6976, or a PKCα siRNA, and the aldosterone-induced increase in O(2)(-) production was blocked. These data indicate that aldosterone-stimulated O(2)(-) production in the MD buffers the effect of NO in control of TGF response, an effect that was mediated by PKCα.

NADPH oxidase and PKC contribute to increased Na transport by the thick ascending limb during type 1 diabetes.

Type 1 diabetes triggers protein kinase C (PKC)-dependent NADPH oxidase activation in the renal medullary thick ascending limb (mTAL), resulting in accelerated superoxide production. As acute exposure to superoxide stimulates NaCl transport by the mTAL, we hypothesized that diabetes increases mTAL Na(+) transport through PKC-dependent and NADPH oxidase-dependent mechanisms. An O(2)-sensitive fluoroprobe was used to measure O(2) consumption by mTALs from rats with streptozotocin-induced diabetes and sham rats. In sham mTALs, total O(2) consumption was evident as a 0.34±0.03 U change in normalized relative fluorescence (ΔNRF)/min per mg protein. Ouabain (2 mmol/L) reduced O(2) consumption by 69±4% and 500 µmol/L furosemide reduced O(2) consumption by 58±8%. Total O(2) consumption was accelerated in mTAL from diabetic rats (0.74±0.07 ΔNRF/min/mg protein; P<0.05 versus sham), reflecting increases in ouabain- and furosemide-sensitive O(2) consumption. NADPH oxidase inhibition (100 µmol/L apocynin) reduced furosemide-sensitive O(2) consumption by mTAL from diabetic rats to values not different from sham. The PKC inhibitor calphostin C (1 µmol/L) or the PKCα/ß inhibitor Gö6976 (1 µmol/L) decreased furosemide-sensitive O(2) consumption in both groups, achieving values that did not differ between sham and diabetic. PKCß inhibition had no effect in either group. Similar inhibitory patterns were evident with regard to ouabain-sensitive O(2) consumption. We conclude that NADPH oxidase and PKC (primarily PKCα) contribute to an increase in O(2) consumption by the mTAL during type 1 diabetes through effects on the ouabain-sensitive Na(+)-K(+)-ATPase and furosemide-sensitive Na(+)-K(+)-2Cl(-) cotransporter that are primarily responsible for active transport Na(+) reabsorption by this nephron segment.

PKCα take part in CCR7/NF-κB autocrine signaling loop in CCR7-positive squamous cell carcinoma of head and neck.

Metastatic squamous cell carcinoma of head and neck (SCCHN) has been shown to express chemokine receptor 7 (CCR7). The role of nuclear factor-κB (NF-κB) in propagating an autocrine signaling loop in CCR7-positive SCCHN cells may provide a clinically useful biomarker of disease status and response to therapy. In this article, we hypothesized that PKCα might be involved in the CCR7/NF-κB autocrine signaling loop. Results showed that CCL19 induced the activation of PKCα, and the increased activity of PKCα was abolished by CCR7 mAb. PKCα inhibition with Gö6976 led to significant reduction in the activation and nuclear translocation of NF-κB induced by CCL19. Immunohistochemical assay also showed that CCR7 and PKCα were highly expressed in SCCHN and correlated with each other, which was significantly related to lymph node metastasis and clinical stage. Taken together, PKCα is involved in the CCR7/NF-κB autocrine signaling loop.

Modulation of Ca(2+) release through ryanodine receptors in vascular smooth muscle by protein kinase Calpha.

The role of protein kinase C (PKC) in Ca(2+) release through ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (SMCs) is not well understood. Caffeine was used to activate RyRs and the intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured in both freshly isolated and cultured mouse aortic SMCs (ASMCs). Pre-activation of PKC with 1,2-dioctanoyl-sn-glycerol (DOG) prevented caffeine-induced [Ca(2+)](i) transients. Application of the PKC inhibitor calphostin C caused [Ca(2+)](i) transients which were not blocked by nifedipine or by removing extracellular Ca(2+) but were abolished after inhibition of the SR Ca(2+)-ATPase with thapsigargin or after inhibition of RyRs with ryanodine. In addition, chelerythrine and GF109203X also elevated resting [Ca(2+)](i) but no further [Ca(2+)](i) increase was seen with subsequent application of caffeine. Selective inhibition of PKCalpha with safingol blocked caffeine-induced [Ca(2+)](i) transients, but the PKCepsilon inhibitory peptide V1-2 did not. In cells expressing a EGFP-tagged PKCalpha, caffeine-induced [Ca(2+)](i) transients were associated with a rapid focal translocation near the cell periphery, while application of ionomycin and DOG caused translocation to the plasma membrane. Western blot showed that caffeine increased the relative amount of PKCalpha in the particulate fraction in a time-dependent manner. Co-immunoprecipitation of RyRs and PKCalpha indicated that they interact. In conclusion, our studies suggest that PKC activation can inhibit the gating activity of RyRs in the SR of ASMCs, and this regulation is most likely mediated by the Ca(2+)-dependent PKCalpha isoform.

Platelet-activating factor-induced synaptic facilitation is associated with increased calcium/calmodulin-dependent protein kinase II, protein kinase C and extracellular signal-regulated kinase activities in the rat hippocampal CA1 region.

Platelet-activating factor (PAF) is an important inflammatory lipid mediator affecting neural plasticity. In the present study, we demonstrated how PAF affects synaptic efficacy through activation of protein kinases in the rat hippocampal CA1 region. In cultured hippocampal neurons, 10 to 1000 nM PAF stimulated autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) and phosphorylation of synapsin I and myristoylated alanine-rich protein kinase C substrate (MARCKS). In hippocampal CA1 slices, field excitatory postsynaptic potentials (fEPSPs) induced by stimulation of the Schaffer collateral/commissural pathways were significantly increased 10-50 min after exposure to 100 to 1000 nM PAF. Immunoblotting analysis showed that 100 nM PAF treatment for 10 or 50 min significantly and persistently increased CaMKII autophosphorylation in the hippocampal CA1 region. Increased protein kinase Calpha (PKCalpha) autophosphorylation was also seen at the same time point after PAF exposure. By contrast, extracellular signal-regulated kinase (ERK) phosphorylation was slightly but significantly increased at 10 min after PAF exposure. Consistent with increased CaMKII autophosphorylation, AMPA-type glutamate receptor subunit 1 (GluR1) (Ser-831) phosphorylation as a CaMKII postsynaptic substrate significantly increased after 10 or 50 min of treatment, whereas synapsin I (Ser-603) phosphorylation as a presynaptic substrate increased at 10 min in the hippocampal CA1 region. Phosphorylation of MARCKS (Ser-152/156) and NMDA receptor subunit 1 (NR1) (Ser-896) as PKCalpha substrates also significantly increased after 10 min but had not further increased by 50 min in the CA1 region. Increased of fEPSPs induced by PAF treatment completely and/or partly inhibited by KN93 and/or U0126 treatment. These results suggest that PAF induces synaptic facilitation through activation of CaMKII, PKC and ERK in the hippocampal CA1 region.

Enhancement of glucagon secretion in mouse and human pancreatic alpha cells by protein kinase C (PKC) involves intracellular trafficking of PKCalpha and PKCdelta.

AIMS/HYPOTHESIS: Protein kinase C (PKC) regulates exocytosis in various secretory cells. Here we studied intracellular translocation of the PKC isoenzymes PKCalpha and PKCdelta, and investigated how activation of PKC influences glucagon secretion in mouse and human pancreatic alpha cells. METHODS: Glucagon release from intact islets was measured in static incubations, and the amounts released were determined by RIA. Exocytosis was monitored as increases in membrane capacitance using the patch-clamp technique. The expression of genes encoding PKC isoforms was analysed by real-time PCR. Intracellular PKC distribution was assessed by confocal microscopy. RESULTS: The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated glucagon secretion from mouse and human islets about fivefold (p < 0.01). This stimulation was abolished by the PKC inhibitor bisindolylmaleimide (BIM). Whereas PMA potentiated exocytosis more than threefold (p < 0.001), BIM inhibited alpha cell exocytosis by 60% (p < 0.05). In mouse islets, the PKC isoenzymes, PKCalpha and PKCbeta1, were highly abundant, while in human islets PKCeta, PKCepsilon and PKCzeta were the dominant variants. PMA stimulation of human alpha cells correlated with the translocation of PKCalpha and PKCdelta from the cytosol to the cell periphery. In the mouse alpha cells, PKCdelta was similarly affected by PMA, whereas PKCalpha was already present at the cell membrane in the absence of PMA. This association of PKCalpha in alpha cells was principally dependent on Ca(2+) influx through the L-type Ca(2+) channel. CONCLUSIONS/INTERPRETATION: PKC activation augments glucagon secretion in mouse and human alpha cells. This effect involves translocation of PKCalpha and PKCdelta to the plasma membrane, culminating in increased Ca(2+)-dependent exocytosis. In addition, we demonstrated that PKCalpha translocation and exocytosis exhibit differential Ca(2+) channel dependence.

Protein kinase Calpha: disease regulator and therapeutic target.

Protein kinase Calpha (PKCalpha) is a member of the AGC (which includes PKD, PKG and PKC) family of serine/threonine protein kinases that is widely expressed in mammalian tissues. It is closely related in structure, function and regulation to other members of the protein kinase C family, but has specific functions within the tissues in which it is expressed. There is substantial recent evidence, from gene knockout studies in particular, that PKCalpha activity regulates cardiac contractility, atherogenesis, cancer and arterial thrombosis. Selective targeting of PKCalpha therefore has potential therapeutic value in a wide variety of disease states, although will be technically complicated by the ubiquitous expression and multiple functions of the molecule.

H3 histamine receptor-mediated activation of protein kinase Calpha inhibits the growth of cholangiocarcinoma in vitro and in vivo.

Histamine regulates functions via four receptors (HRH1, HRH2, HRH3, and HRH4). The d-myo-inositol 1,4,5-trisphosphate (IP(3))/Ca(2+)/protein kinase C (PKC)/mitogen-activated protein kinase pathway regulates cholangiocarcinoma growth. We evaluated the role of HRH3 in the regulation of cholangiocarcinoma growth. Expression of HRH3 in intrahepatic and extrahepatic cell lines, normal cholangiocytes, and human tissue arrays was measured. In Mz-ChA-1 cells stimulated with (R)-(alpha)-(-)-methylhistamine dihydrobromide (RAMH), we measured (a) cell growth, (b) IP(3) and cyclic AMP levels, and (c) phosphorylation of PKC and mitogen-activated protein kinase isoforms. Localization of PKCalpha was visualized by immunofluorescence in cell smears and immunoblotting for PKCalpha in cytosol and membrane fractions. Following knockdown of PKCalpha, Mz-ChA-1 cells were stimulated with RAMH before evaluating cell growth and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation. In vivo experiments were done in BALB/c nude mice. Mice were treated with saline or RAMH for 44 days and tumor volume was measured. Tumors were excised and evaluated for proliferation, apoptosis, and expression of PKCalpha, vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF receptor 2, and VEGF receptor 3. HRH3 expression was found in all cells. RAMH inhibited the growth of cholangiocarcinoma cells. RAMH increased IP(3) levels and PKCalpha phosphorylation and decreased ERK1/2 phosphorylation. RAMH induced a shift in the localization of PKCalpha expression from the cytosolic domain into the membrane region of Mz-ChA-1 cells. Silencing of PKCalpha prevented RAMH inhibition of Mz-ChA-1 cell growth and ablated RAMH effects on ERK1/2 phosphorylation. In vivo, RAMH decreased tumor growth and expression of VEGF and its receptors; PKCalpha expression was increased. RAMH inhibits cholangiocarcinoma growth by PKCalpha-dependent ERK1/2 dephosphorylation. Modulation of PKCalpha by histamine receptors may be important in regulating cholangiocarcinoma growth.

Antiapoptotic effects of vasopressin in the neuronal cell line H32 involve protein kinase Calpha and beta.

Activation of V1 vasopressin (VP) receptors prevents serum deprivation-induced apoptosis in neuronal H32 cells, partially through mitogen-activated protein kinase (MAPK) mediated Bad phosphorylation. In this study, we investigated the role of protein kinases C (PKC) and B (PKB) mediating VP-induced antiapoptosis in H32 cells. Serum deprivation increased PKCdelta but not PKCalpha or PKCbeta activity, while VP increased PKCalpha and PKCbeta without affecting PKCdelta activity. Inhibition of PKCdelta prevented caspase 3 activation, indicating that PKCdelta mediates the pro-apoptotic actions of serum deprivation. Simultaneous inhibition of PKCalpha and beta and MAPK abolished VP-induced Bad phosphorylation, but it only partially prevented caspase 3 inhibition. Complete abolition of the protective effect of VP on serum deprivation-induced caspase 3 activity required additional blockade of phosphoinositide 3 kinase (PI3K)/protein kinase B. The data demonstrate that VP exerts antiapoptosis through multiple pathways; while PKCalpha and beta together with extracellular signal-regulated kinases/MAPK activation mediates Bad phosphorylation (inactivation), the full protective action of VP requires additional activation of PKB (PI3K/protein kinase B) pathway.

Epithelial-to-mesenchymal transition and resistance to ingenol 3-angelate, a novel protein kinase C modulator, in colon cancer cells.

Acquired resistance to protein kinase C (PKC) modulators may explain the failure of clinical trials in patients with cancer. Herein, we established a human colon cancer cell line resistant to PEP005, a drug that inhibits PKCalpha and activates PKCdelta. Colo205-R cells, selected by stepwise exposure to PEP005, were >300-fold more resistant to PEP005 than parental Colo205-S cells and were cross-resistant to phorbol 12-myristate 13-acetate, bryostatin, bistratene A, and staurosporine. No PKCalpha or PKCdelta mutation was detected in Colo205-S and Colo205-R cells. Changes in Colo205-R cells were reminiscent of the epithelial-to-mesenchymal transition (EMT) phenotype. Accordingly, Colo205-R cells were more invasive than Colo205-S in Matrigel assays and in mouse xenografts. We also found an increased mRNA expression of several EMT genes, such as those encoding for transforming growth factor-beta and vimentin, along with a decreased mRNA expression of genes involved in epithelial differentiation, such as CDH1 (E-cadherin), CLDN4 (claudin 4), S100A4, and MUC1, in Colo205-R compared with Colo205-S cells in vitro and in vivo. Interestingly, high expression of ET-1 was shown in Colo205-R cells and correlated with low sensitivity to PEP005 and staurosporine in a panel of 10 human cancer cell lines. Inhibition of the ET-1 receptor ETR-A with bosentan restored the antiproliferative effects of PEP005 in Colo205-R cells and decreased the invasive properties of this cell line. Exogenous exposure to ET-1 and silencing ET-1 expression using small interfering RNA modulated cell signaling in Colo205-S and Colo205-R. In summary, acquired resistance to PEP005 was associated with expression of EMT markers and activates the ET-1/ETR-A cell signaling.

Activation of protein kinase Calpha 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 alpha isoform of PKC in CL3 cells. Pb also elicited Raf-1 activation as measured by the induction of phospho-Raf-1S338 and the dissociation from the Raf-1 kinase inhibitor protein. Inhibition of cPKC activity using Gö6976 or depletion of PKCalpha by introducing specific small interfering RNA blocked the induction of phospho-Raf-1S338, phospho-MKK1/2 and phospho-ERK1/2 in cells exposed to Pb. Intriguingly, declining PKCalpha enhanced the Pb cytotoxicity and revealed the Pb mutagenicity at the hprt gene. The results suggest that PKCalpha 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.

Rosiglitazone improves aortic arginine transport, through inhibition of PKCalpha, in uremic rats.

Peroxisome proliferator-activated receptor (PPAR) agonists were shown to inhibit atherosclerosis through augmentation of endothelial nitric oxide synthase (eNOS) activity. In addition, rosiglitazone exerts a beneficial effect in chronic renal failure (CRF). Since l-arginine transport by CAT-1 (the specific arginine transporter for eNOS) is inhibited in uremia, we aimed to explore the effect of rosiglitazone on arginine transport in CRF. Arginine uptake by aortic rings was studied in control animals, rats, 6 wk following 5/6 nephrectomy (CRF) and rats with CRF treated with rosiglitazone. The decrease of arginine transport in CRF was prevented by rosiglitazone. Immunobloting revealed that CAT-1 protein was decreased in CRF but remained unchanged following rosiglitazone administration. Protein content of the membrane fraction of PKCalpha and phosphorylated CAT-1 increased significantly in CRF, effects that were prevented by rosiglitazone. PKCalpha phosphorylation was unchanged but significantly attenuated by rosiglitazone in CRF. Ex vivo administration of phorbol-12-myristate-13-acetate to rosiglitazone-treated CRF rats significantly attenuated the effect of rosiglitazone on arginine uptake. The decrease in cGMP response to carbamyl-choline (eNOS agonist) was significantly attenuated by rosiglitazone in CRF. Western blotting and immunohistochemistry analysis revealed that protein nitration was intensified in the endothelium of CRF rats and this was attenuated by rosiglitazone. In conclusion, rosiglitazone prevents the decrease in arginine uptake in CRF through both depletion and inactivation of PKCalpha. These findings are associated with restoration of eNO generation and attenuation of protein nitration and therefore may serve as a novel mechanism to explain the beneficial effects of rosiglitazone on endothelial function in uremia.

TUDCA prevents cholestasis and canalicular damage induced by ischemia-reperfusion injury in the rat, modulating PKCalpha-ezrin pathway.

Cholestasis, induced by liver ischemia-reperfusion injury (IRI), is characterized by dilatation of bile canaliculi and loss of microvilli. Tauroursodeoxycholic acid (TUDCA) is an anti-cholestatic agent, modulating protein kinase C (PKC) alpha pathway. PKC reduces ischemic damage in several organs, its isoform alpha modulates ezrin, a key protein in the maintenance of cell lamellipoidal extensions. We evaluated the effects of TUDCA on cholestasis, canalicular changes and PKCalpha-ezrin expression in a rat model of liver IRI. Livers flushed and stored with Belzer solution or Belzer + 10 mm TUDCA (4 degrees C for 6 h) were reperfused (37 degrees C with O(2)) with Krebs-Ringer bicarbonate + 2.5 micromol/min of Taurocholate or TUDCA. Bile was harvested for bile flow assessment. Liver tissue was employed for Electron Microscopy (EM) and for PKCalpha and ezrin immunoblot and immunofluorescence. The same experiments were conducted with the PKCalpha inhibitor Go-6976. TUDCA-treated livers showed increased bile flow (0.25+/-0.17 vs. 0.042+/-0.02 microl/min/g liver, P<0.05) and better preservation of microvilli and bile canalicular area at EM. These effects were associated with increased PKCalpha and ezrin expression (P=0.03 and P=0.04 vs. control respectively), as also confirmed by immunofluorescence data. PKCalpha inhibition abolished these TUDCA effects. TUDCA administration during IRI reduces cholestasis and canalicular damage in the liver modulating PKCalpha-ezrin pathway.