Translocation Of PKC-alpha Research Articles

Abstract 5315: Quantitative proteomic assessment of differential ligand responses downstream of protein kinase C activation

Abstract Bryostatin 1 (bryo 1) is an anticancer agent currently in multiple clinical trials with a unique mechanism of action. Whereas bryo 1 binds to and activates protein kinase C (PKC) like the phorbol esters, it paradoxically antagonizes many but not all phorbol ester responses. In LNCaP prostate cancer cells the typical phorbol ester PMA induces TNFalpha secretion and apoptosis while bryo 1 fails to do so and antagonizes the PMA effect when used in combination. The mechanisms responsible for the unique effect of bryo 1 are still unknown. We have shown previously, using transcription factor analysis of microarray data from LNCaP cells, that multiple transcription factor families are involved in the dramatic transcriptional response to PMA and bryo 1 (3131 genes for PMA and 1569 genes for bryo 1). Further, analysis of 45 signaling pathways using reporter assays identified significant changes in 7 pathways induced both by PMA and bryo 1 at 6 hours. To further investigate the altered pathways, the novel Simple Western capillary immunoassay system (ProteinSimple) was used to analyze protein level changes following PKC activation. Simple Western system performs automated western analysis in capillaries using nanograms of lysate and provides digital quantitation with good sensitivity and excellent reproducibility. The system runs at a through-put of 96 sample / target combinations in a single overnight run. We analyzed 35 signals in total lysates (for detection of changes in protein levels and phosphorylation) and in nuclear extracts (for detection of translocation) from LNCaP cells treated for 60 min and 6 hrs. Changes downstream of PKC activation were measured, including PKC/PKD1 phosphorylation, activation of multiple members of the MEK/ERK pathway and levels of transcription factors of the NFKB, AP1, Stat and EGR families. Simple Western results correlated very well with conventional western analysis. The analysis validated the changes described previously using other methods on activation of MEK/ERK pathway and the changes in PKC phosphorylation, translocation and degradation. Bryo1 is different from PMA in failing to induce phosphorylation of PKCdelta at Tyr311 and the translocation of PKCalpha, delta, epsilon, and PKD1 to the nuclear fraction. Bryo1 is similar to PMA in inducing many of the early responses detected at 60 min (pPKCdeltaSer299, pMEK, pERK1/2, pPKD1 in total lysates, decrease in STAT3 levels etc.) but it fails to sustain the induced responses or to induce the late responses detected at 6 hours (pNFκBp65, increase in Fra1 and FosB levels, degradation of cMyc, translocation of cRel and RelB etc.). Using Simple Western system, we developed an approach for precise and accurate measurement of differential PMA and bryo 1 signaling in PKC activation. Further investigation needs to be performed to identify the mechanism/s responsible for the early termination of bryo 1 induced responses. Citation Format: Jin-Qiu Chen, Noemi Kedei, Michelle A. Herrmann, Peter M. Blumberg. Quantitative proteomic assessment of differential ligand responses downstream of protein kinase C activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5315. doi:10.1158/1538-7445.AM2014-5315

Abstract 3195: Transient duration of action is the primary mechanism responsible for the unique biology of bryostatin 1

Abstract Bryostatin 1 (bryo 1) is a natural compound tested in several clinical trials as an anticancer agent and in preclinical studies for treatment of Alzheimer disease. Despite the clinical interest, the mechanisms responsible for its unique behavior are not fully understood. Bryo 1 is a protein kinase C activator that paradoxically antagonizes many but not all phorbol ester responses. To obtain a broader view of the differences in response to bryo 1 and to the phorbol ester PMA, we have compared their effects on global RNA expression as well as on the levels of important regulatory proteins in two cellular systems (LNCaP and U937 cell lines) where bryo 1 has different biology than PMA. In LNCaP cells, where bryo 1 does not induce secretion of TNF alpha or cause apoptosis, bryo 1 fails to induce translocation of PKC alpha, delta, epsilon and pPKD1 to the nuclear enriched fraction or the phosphorylation of PKCdelta at Y311. For many other early responses detected at 60 min (activation of MEK/ERK pathway and of several transcription factors including NFKB, AP1, EGR1) bryo 1 acts similarly to PMA. In contrast, bryo 1 fails to induce the late responses detected at 6 hrs (phosphorylation of p65, translocation of cRel and RelB) and induces early termination of the induced responses both at the protein and RNA levels as detected for a selected panel of genes. The early termination of the induced responses by bryo 1 can be detected as well in U937 cells, where bryo 1 fails to induce differentiation and apoptosis, unlike PMA. Microarray analysis of LNCaP and U937 cells treated for 6 and 8 hrs, respectively, revealed significant, extensive changes in gene expression compared to control but did not identify genes uniquely responsive to bryo 1. Typically, the effect of bryo 1 was smaller than that of PMA. qPCR analysis of about 3 dozen genes spanning the breadth of the microarray response patterns indicated a single mechanism - genes were similarly activated by PMA and bryo 1 early on but the effect of bryo 1 was transient. Further microarray analysis was performed on LNCaP and U937 samples treated for 60 min and 6 hrs to address the following questions: 1) Are there genes that are induced selectively by bryo 1 at 1 hr? 2) Does transient response to bryo 1, which was only shown for selected genes by qPCR but was inferred to explain the extensive reduced expression at 6 hr in response to bryo 1 compared to PMA, actually explain the reduced expression? While some genes are selectively induced by bryo 1 treatment, the altered levels of expression are limited, suggesting that it does not reflect a unique site of action. Looking at genes showing a reduced level of expression at 6 hr in response to bryo 1 compared to PMA, we confirm that transiency of action is the primary mechanism. These insights provide a guide for evaluation of second generation bryostatin analogs and a focus on the biochemical basis for the transiency of bryo 1 effect. Citation Format: Noemi Kedei, Aleksandra M. Michalowski, Peter M. Blumberg. Transient duration of action is the primary mechanism responsible for the unique biology of bryostatin 1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3195. doi:10.1158/1538-7445.AM2014-3195

Protein Kinase C-alpha is Expressed and Activated During the Development of Renal Cell Carcinoma

To evaluate protein kinase C-alpha (PKCalpha) expression in human renal cell carcinoma (RCC) tissues and cell lines, and its clinical significance. Expression of PKCalpha was analyzed by Western blot in 90 clinical specimens. The expression of PKCalpha in the cytoplasm or plasma membrane was correlated to clinical stage and grade to assess for potential relationships. A human renal cell carcinoma (RCC) cell line (786-O/PKCalpha) was generated with stable expression of a fusion protein of green fluorescent protein (GFP) and PKCalpha, to facilitate analysis of in situ compartmentalization of PKCalpha during activation. PKCalpha expression and the ratio of PKCalpha expression in the membrane (M) to that in cytosol (C) were greater in cancerous tissues than in normal tissues (P <.05). With an increase in tumor grade and stage, the level of PKCalpha increased significantly in membrane (P <.01) and decreased in cytosol (P <.01). Consistently, there was an increase of M/C with increasing malignancy of tumor. As expected, the translocation of PKCalpha between plasma membrane and cytosol could be observed in 786-O renal carcinoma cells with the treatment of PKCalpha agonist or inhibitor. We demonstrated differential PKCalpha expression in RCC tissues, indicating that abnormal PKCalpha activation may contribute to the development of RCC.

Abstract 4500: What distinguishes a PKC activator like phorbol ester from a PKC functional antagonist like bryostatin 1? Bryologues permit mechanistic insight

Abstract Bryostatin 1, a promising anticancer agent in clinical trials, is a potent PKC activator in vitro that paradoxically often antagonizes the effects of the typical PKC activator phorbol 12-myristate 13-acetate (PMA) in cellular systems. Understanding the underlying mechanism(s) responsible for this functional PKC antagonism should afford a new generation of drugs for PKC, a validated therapeutic target. We use the synthetic bryostatin look-alike (bryologue) Merle 23 that differs structurally from bryostatin 1 to only a modest degree as a tool to dissect mechanisms responsible for the unique effects of bryostatin 1. We have previously reported that Merle 23 is PMA like, bryostatin-1 like, intermediate or unique in its behavior depending on the system and response examined. In LNCaP prostate cancer cells Merle 23 is bryostatin 1 like in failing to inhibit cell proliferation, to induce apoptosis, or to induce secretion of TNF-alpha, responses induced by PMA. Using siRNAs and PKC inhibitors we show that PKC delta, but not PKC alpha or epsilon, was responsible for induction of apoptosis. Nuclear localized PKC delta was previously shown to be responsible for the apoptotic response, and we found that Merle 23 resembled bryostatin 1 and was unlike PMA in that failed to induce translocation of PKC delta and phospho-PKD1 to the nucleus. In contrast, Merle 23 was PMA-like in inducing nuclear translocation of PKC alpha and PKC epsilon. Bryostatin 1 is unique in down-regulating PKC alpha very efficiently in LNCaP cells and Merle 23 is unique for quick and very efficient down-regulation of PKC delta. If proteosome inhibitors were used to prevent down-regulation of PKC delta, the bryostatin 1-like effects of Merle 23 on proliferation and TNF-alpha secretion were transformed to PMA-like effects: Merle 23 inhibited LNCaP cell proliferation and induced secretion of TNF-alpha when co-applied with lactacystin or MG-132. Similarly, for tyrosine phosphorylation of PKC delta at position 311 and for cFos activation, responses where Merle 23 had effects intermediate between bryostatin 1 and PMA, Merle 23 became more PMA-like when co-applied with proteosome inhibitors. Finally, consistent with stability of PKC signaling elements varying between ligands, we found that phosphorylation of ERK, Mek1 and Mek2, and AKT in response to bryostatin 1, PMA, and Merle 23 was similar at 30 min but returned to the basal level at 150 min for bryostatin 1, but not PMA or Merle 23, as determined using capillary iso-electrofocusing immune-assay. We conclude that differential regulation of PKC isoforms by bryostatin and the bryologues is an important contributor to their differential biological activity relative to PMA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4500.

Reorganization of cytoskeletal proteins by Escherichia coli heat-stable enterotoxin (STa)-mediated signaling cascade

IP(3)-mediated calcium mobilization from intracellular stores activates and translocates PKC-alpha from cytosol to membrane fraction in response to STa in COLO-205 cell line. The present study was undertaken to determine the involvement of cytoskeleton proteins in translocation of PKC-alpha to membrane from cytosol in the Escherichiacoli STa-mediated signaling cascade in a human colonic carcinoma cell line COLO-205. Western blots and consequent densitometric analysis were used to assess time-dependent redistribution of cytoskeletal proteins. This redistribution was further confirmed by using confocal microscopy. Pharmacological reagents were applied to colonic carcinoma cells to disrupt the microfilaments (cytochalasin D) and microtubules (nocodazole). STa treatment in COLO-205 cells showed dynamic redistribution and an increase in actin content in the Triton-insoluble fraction, which corresponds to an increase in polymerization within 1min. Moreover, pharmacological disruption of actin-based cytoskeleton greatly disturbed PKC-alpha translocation to the membrane. These results suggested that the organization of actin cytoskeleton is rapidly rearranged following E. coli STa treatment and the integrity of the actin cytoskeleton played a crucial role in PKC-alpha movement in colonic cells. Depolymerization of tubulin had no effect on the ability of the kinase to be translocated to the membrane. In the present study, we have shown for the first time that in colonic carcinoma cells, STa-mediated rapid changes of actin cytoskeleton arrangement might be involved in the translocation of PKC-alpha to membrane.

20918 血管内皮細胞内Ca^&lt;2+&gt;上昇時におけるPKCalpha局在の変化(OS9 バイオエンジニアリング(4),オーガナイズドセッション)

20918 血管内皮細胞内Ca^&lt;2+&gt;上昇時におけるPKCalpha局在の変化(OS9 バイオエンジニアリング(4),オーガナイズドセッション)

Specific subcellular targeting of PKCα and PKCε in normal and tumoral lactotroph cells by PMA-mitogenic stimulus

The variations of the intracellular localization of the individual protein kinase C (PKC) isoforms are related with their different biological functions. In this study, we have investigated the precise intracellular translocation of endogenous PKCalpha and PKCepsilon in PMA-stimulated normal and tumoral lactotroph cells by using confocal and immunogold electron microscopy, which was correlated with the rate of cell proliferation of both pituitary cell phenotypes. The present results showed that the short phorbol ester incubation stimulated the proliferation of normal and tumoral lactotroph cells, as determined by the measurement of the BrdU-labelling index. The translocation of PKCalpha to plasma and nuclear membranes induced by PMA was more marked than that observed for PKCepsilon in normal and tumoral lactotroph cells. Our results showed that PKCs translocation to the plasma and nuclear membranes varied from isozyme to isozyme emphasizing that PKCalpha could be related with the mitogenic stimulus exerted by phorbol ester. These data support the notion that specific PKC isozymes may exert spatially defined effects by virtue of their directed translocation to distinct intracellular sites.

Roles of phosphatidylinositol 3‐kinase and p38 mitogen‐activated protein kinase in the regulation of protein kinase C‐α activation in interferon‐γ‐stimulated macrophages

Members of the protein kinase C (PKC) family are activated by interferon-gamma (IFN-gamma) and modulate IFN-gamma-induced cellular responses by regulating the activity of transcription factors. We previously reported that PKC-alpha enhances the ability of IFN regulatory factor-1 to transactivate the class II transactivator (CIITA) promoter IV in IFN-gamma-stimulated macrophages. In addition, we showed that IFN-gamma induces the nuclear translocation of PKC-alpha but the mechanisms for this remain to be elucidated. In this study, we sought to identify signalling pathways involved in IFN-gamma-induced activation of PKC-alpha and to characterize their potential roles in modulating IFN-gamma-induced responses in macrophages. IFN-gamma-mediated nuclear translocation of PKC-alpha was a Janus activated kinase 2 (JAK2)-independent process, which required phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK). However, PKC-alpha phosphorylation was independent of PI3K and p38 MAPK, indicating that IFN-gamma-induced phosphorylation and nuclear translocation of PKC-alpha are mediated by distinct mechanisms. In addition, inhibition of PI3K, but not of p38 MAPK, strongly impaired IFN-gamma-induced CIITA and MHC II gene expression. Finally, PKC-alpha associated with signal transducer and activator of transcription 1 (STAT1) and was required for the phosphorylation of STAT1 on serine 727 in IFN-gamma-stimulated macrophages. Taken together, our data indicate that PI3K and p38 MAPK modulate IFN-gamma-stimulated PKC-alpha nuclear translocation independently of JAK2 activity and that both PI3K and PKC-alpha are required for type IV CIITA and MHC II gene expression in IFN-gamma-stimulated macrophages.

Glucosamine induces translocation of protein kinase C isoenzymes in mesangial cells.

Activation of protein kinase C (PKC) has been implicated in the high glucose-induced stimulation of matrix protein production in mesangial cells. Since we have found (Kolm-Litty et al., 1998) that glucosamine, similar to the PKC activator phorbol myristate acetate (PMA), mimicks high glucose-induced TGF-beta1 overexpression and subsequent matrix overproduction, the action of these agents on the translocation of PKC isoenzymes was studied in cultured mesangial cells. Exposure to 12 mM glucosamine resulted in rapid and specific translocation of PKC-isoenzymes in mesangial cells i.e. glucosamine caused an increased and sustained translocation of PKC-alpha, -beta and -epsilon while PKC-zeta was essentially unaffected. Comparison with PMA-induced translocation exhibited distinct differences. Exposure to high glucose concentrations of mesangial cells induced translocation of PKC-beta and down-regulation of PKC-epsilon while PKC-alpha and -zeta were essentially unaltered. Presence of azaserine an inhibitor of glutamine: fructose-6-phosphate amidotransferase, the key enzyme of the hexosamine pathway, attenuated the high glucose-induced effects on the membrane fraction of PKC-beta. Our results indicate that i) glucosamine is a potent stimulator of PKC-translocation exhibiting an isoenzyme specific translocation kinetic which is different from PMA-induced PKC-isoenzyme translocation ii) the hexosamine pathway may be possibly involved in the high glucose-induced activation of PKC.

Acute memory phase of sevoflurane preconditioning is associated with sustained translocation of protein kinase C-α and &amp;epsiv;, but not δ, in isolated guinea pig hearts

Anaesthetic preconditioning (APC) exerts cardioprotective effects by reducing infarct size and improving recovery of contractile function after ischaemia-reperfusion. The interval between brief exposure to volatile anaesthetic and sustained ischaemia, the acute memory phase, is dependent on intracellular signalling mediating this cardioprotection. Intramyocyte translocation of protein kinase C (PKC) is known to be a key mediator in APC. We examined the relationship between the time frame of the acute memory phase of sevoflurane preconditioning and intramyocyte translocation of PKC-alpha, delta and epsilon to the particulate fraction. Isolated perfused guinea pig hearts were subjected to 30 min ischaemia and 120 min reperfusion. APC was elicited with one minimum alveolar concentration sevoflurane for 10 min. Washout times of 10, 30, 60 and 90 min were studied. Contractile recovery was assessed by monitoring left ventricular developed pressures. Infarct size was determined by triphenyltetrazolium chloride staining. Translocation of PKC was examined by western blot analysis. After ischaemia-reperfusion, left ventricular developed pressure recovered to a greater degree with APC compared with control for washout times of 10 and 30 min, but not 60 and 90 min. Similarly, infarct size was reduced for washout times of 10 and 30 min, but not 60 and 90 min. Sustained translocation of PKC-alpha and epsilon, but not delta, was associated with the time frame of the acute memory phase. The acute memory phase of sevoflurane preconditioning is limited to less than 60 min. Sustained translocation of PKC-alpha and epsilon, but not delta, correlates with this acute memory phase of sevoflurane preconditioning.

IL‐1β induces urokinse‐plasminogen activator expression and cell migration through PKCα, JNK1/2, and NF‐κB in A549 cells

Breakdown of the extracellular matrix (ECM) is accomplished by the concerted action of several proteases, including the urokinase plasminogen-activator (uPA) system and matrix metalloproteinases (MMPs), which is crucial for cancer invasion and metastasis. Several reports have shown that the levels of IL-1 beta and MMPs in plasma of the patients with lung cancer are significantly elevated and link to the invasion of tumor cells. Therefore, we investigated whether IL-1 beta-induced expression of uPA participated in lung cancer progression. In this study, IL-1 beta significantly induced uPA expression and activity via PKC alpha-dependent JNK1/2 and NIK cascades, linking to IKK alpha/beta activation, p65 translocation and transcription activity, using pharmacological inhibitors and transfection with dominant negative mutants and siRNAs. IL-1 beta-induced uPA protein and mRNA expression in a time- and concentration-dependent manner, which was inhibited by pretreatment with the inhibitors of JNK1/2 (SP600125), PKC (Ro31-8220, Gö6976), or NF-kappaB (helenalin), and transfection with dominant negative mutants of PKC alpha, NIK, and IKK beta, and siRNAs of JNK1/2 and p65. IL-1 beta stimulated PKC alpha translocation to plasma membrane leading to phosphorylation of JNK1/2, which was attenuated by PKC inhibitors and transfection with shRNAs of JNK1/2, but not by helenalin. In addition, IL-1beta stimulated p65 phosphorylation and translocation into nucleus concomitant with I kappaB alpha phosphorylation and I kappaB alpha degradation, which was mediated via activation of PKC alpha-dependent JNK1/2-NIK/IKK beta cascade. These results demonstrated that in A549 cells, activation of p50/p65 heterodimer through sequential activation of PKC alpha-JNK-NIK-IKK beta-NF-kappaB was required for IL-1 beta-induced uPA expression associated with migration of tumor cells.

A potential mechanism for short time exposure to hypoxia‐induced DNA synthesis in primary cultured chicken hepatocytes: Correlation between Ca2+/PKC/MAPKs and PI3K/Akt/mTOR

Less information is available concerning the molecular mechanisms of cell survival after hypoxia in hepatocytes. Therefore, this study examined the effect of hypoxia on DNA synthesis and its related signal cascades in primary cultured chicken hepatocytes. Hypoxia increased [3H] thymidine incorporation, which was increased significantly after 0-24 h of hypoxic exposure. Indeed, the percentage of cell population in the S phase was increased in hypoxia condition. However, the release of LDH indicating cellular injury was not changed under hypoxic conditions. Hypoxia increased Ca2+ uptake and PKC translocation from the cytosol to the membrane fraction. Among the PKC isoforms, hypoxia stimulated the translocation of PKC alpha and epsilon. Hypoxia also phosphorylated the p38 and p44/42 mitogen-activated protein kinases (MAPKs), which were blocked by the inhibition of PKC. On the other hand, hypoxia increased Akt and mTOR phosphorylation, which was blocked in the absence of intra/extracellular Ca2+. The inhibition of PKC/MAPKs or PI3K/Akt pathway blocked the hypoxia-induced [3H] thymidine incorporation. However, hypoxia-induced Ca2+ uptake and PKC translocation was not influenced by LY 294002 or Akt inhibitor and hypoxia-induced MAPKs phosphorylation was not changed by rapamycin. In addition, LY 294002 or Akt inhibitor has no effect on the phosphorylation of MAPKs. It suggests that there is no direct interaction between the two pathways, which cooperatively mediated cell cycle progression to hypoxia in chicken hepatocytes. Hypoxia also increased the level of the cell cycle regulatory proteins [cyclin D(1), cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4] and p-RB protein but decreased the p21 and p27 expression levels, which were blocked by inhibitors of upstream signal molecules. In conclusion, short time exposure to hypoxia increases DNA synthesis in primary cultured chicken hepatocytes through cooperation of Ca2+/PKC, p38 MAPK, p44/42 MAPKs, and PI3K/Akt pathways.

Inhibition of fibroblast growth factor 2-induced apoptosis involves survivin expression, protein kinase C&amp;alpha; activation and subcellular translocation of Smac in human small cell lung cancer cells

To investigate the mechanism by which fibroblast growth factor 2 (FGF-2) inhibits apoptosis in the human small cell lung cancer cell line H446 subjected to serum starvation, apoptosis was evaluated by flow cytometry, Hoechst 33258 staining, caspase-3 activity, and DNA fragmentation. Survivin expression induced by FGF-2 and protein kinase C alpha (PKC alpha) translocation was detected by subcellular fractionation and Western blot analysis. In addition, FGF-2-induced release of Smac from mitochondria to the cytoplasm was analyzed by Western blotting and immunofluorescence. FGF-2 reduced apoptosis induced by serum starvation and up-regulated survivin expression in H446 cells in a dose-dependent and time-dependent manner, and inhibited caspase-3 activity. FGF-2 also inhibited the release of Smac from mitochondria to the cytoplasm induced by serum starvation and increased PKC alpha translocation from the cytoplasm to the cell membrane. In addition, PKC inhibitor inhibited the expression of survivin. FGF-2 up-regulates the expression of survivin protein in H446 cells and blocks the release of Smac from mitochondria to the cytoplasm. PKC alpha regulated FGF-2-induced survivin expression. Thus, survivin, Smac, and PKC alpha might play important roles in the inhibition of apoptosis by FGF-2 in human small cell lung cancer cells.

Sevoflurane-induced cardioprotection depends on PKC-α activation via production of reactive oxygen species

We previously demonstrated the involvement of the Ca2+-independent protein kinase C-delta (PKC-delta) isoform in sevoflurane-induced cardioprotection against ischaemia and reperfusion (I/R) injury. Since sevoflurane is known to modulate myocardial Ca2+-handling directly, in this study we investigated the role of the Ca2+-dependent PKC-alpha isoform in sevoflurane-induced cardioprotective signalling in relation to reactive oxygen species (ROS), adenosine triphosphate-sensitive mitochondrial K+ (mitoK+(ATP)) channels, and PKC-delta. Preconditioned (15 min 3.8 vol% sevoflurane) isolated rat right ventricular trabeculae were subjected to I/R, consisting of 40 min superfusion with hypoxic, glucose-free buffer, followed by normoxic glucose-containing buffer for 60 min. After reperfusion, contractile recovery was expressed as percentage of force development before I/R. The role of PKC-alpha, ROS, mitoK+(ATP) channels, and PKC-delta was established using the following pharmacological inhibitors: Go6976 (GO; 50 nM), n-(2-mercaptopropionyl)-glycine (MPG; 300 microM), 5-hydroxydecanoic acid sodium (5HD; 100 microM), and rottlerin (ROT; 1 microM). Preconditioning of trabeculae with sevoflurane improved contractile recovery after I/R [65 (3)% (I/R + SEVO) vs 47 (3)% (I/R); n = 8; P < 0.05]. This cardioprotective effect was attenuated in trabeculae treated with GO [42 (4)% (I/R + SEVO + GO); P > 0.05 vs (I/R)]. In sevoflurane-treated trabeculae, PKC-alpha translocated towards mitochondria, as shown by immunofluorescent co-localization analysis. GO and MPG, but not 5HD or ROT, abolished this translocation. Sevoflurane improves post-ischaemic contractile recovery via activation of PKC-alpha. ROS production, but not opening of mitoK+(ATP) channels, precedes PKC-alpha translocation towards mitochondria. This study shows the involvement of Ca2+-dependent PKC-alpha in addition to the well-established role of Ca2+-independent PKC isoforms in sevoflurane-induced cardioprotection.

The Differential Effects of Intravenous Anesthetics on Myofilament Ca2+ Sensitivity in Pulmonary Venous Smooth Muscle

Pulmonary venous contraction can increase pulmonary capillary pressure and pulmonary edema. In the present study, we investigated the direct effects of ketamine, etomidate, thiopental, and midazolam on pulmonary venous contraction and myofilament Ca2+ sensitivity in permeabilized pulmonary venous smooth muscle (PVSM). The effects of these IV anesthetics on acetylcholine contraction were assessed in isolated canine pulmonary vein rings. Tension and [Ca2+]i were measured simultaneously in fura-2 loaded endothelium-denuded PVSM strips after being permeabilized with alpha-toxin. The effects of the IV anesthetics on tension ([Ca2+]i remains constant) in the absence or the presence of muscarinic receptor activation (acetylcholine) were assessed. The immunofluorescence technique and confocal microscopy were used to localize the cellular distribution of protein kinase C (PKC) isoforms in PVSM cells before and after the addition of ketamine. Ketamine, etomidate, and midazolam each attenuated acetylcholine contraction dose-dependently, whereas thiopental had no effect. None of the IV anesthetics alone had an effect on tension in strips at constant [Ca2+]i (i.e., they had no direct effect on myofilament Ca2+ sensitivity). Acetylcholine increased tension by 56% +/- 7% at constant [Ca2+]i. In acetylcholine-stimulated strips, etomidate, midazolam, and thiopental had no additional effect on tension at constant [Ca2+]i, whereas ketamine decreased tension by 33% +/- 3%. Activation with acetylcholine induced translocation of PKC from cytoplasm to membrane, and this effect was blocked by ketamine. Ketamine, etomidate, and midazolam each attenuated acetylcholine-induced pulmonary venous contraction. Ketamine attenuates acetylcholine contraction by inhibiting the acetylcholine-induced increase in myofilament Ca2+ sensitivity and the acetylcholine-induced translocation of PKCalpha.

Ursodeoxycholic Acid Stimulates Cholangiocyte Fluid Secretion in Mice via CFTR-Dependent ATP Secretion

Cholangiopathies are characterized by impaired cholangiocyte secretion. Ursodeoxycholic acid (UDCA) is widely used for cholangiopathy treatment, but its effects on cholangiocyte secretory functions remain unclear and are the subject of this study. Polarized mouse cholangiocytes in tubular (isolated bile-duct units [IBDU]) or monolayer configuration were obtained from wild-type (WT) and B6-129-Cftr(tm1Kth) and Cftr(tm1Unc) mice that are defective in CFTR, an adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl(-) channel expressed in cholangiocytes. Fluid secretion was assessed by video-optical planimetry, Cl(-) and Ca(2+) efflux by microfluorimetry (6-methoxy-N-ethylquinolinium chloride, fura-2, and fluo-4), adenosine triphosphate (ATP) secretion by luciferin-luciferase assay, and protein kinase C (PKC) by Western blot. UDCA stimulated fluid secretion and Cl(-) efflux in WT-IBDU but not in CFTR-KO-IBDU or in WT-IBDU exposed to CFTR inhibitors. UDCA did not affect intracellular cAMP levels but increased [Ca(2+)]i in WT and not in CFTR-KO cholangiocytes. UDCA stimulated apical ATP secretion in WT but not in CFTR-KO cholangiocytes. UDCA-stimulated [Ca(2+)]i increase was inhibited by suramin, a purinergic 2Y-receptor inhibitor. UDCA stimulated the translocation of PKC-alpha and PKC-epsilon to the plasma membrane. UDCA-stimulated secretion was inhibited by 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid and by phospholipase C and PKC inhibitors. UDCA increased ATP output in isolated perfused livers from WT but not from CFTR-KO mice. Our data indicate that UDCA stimulates a CFTR-dependent apical ATP release in cholangiocytes. Secreted ATP activates purinergic 2Y receptors, and, through [Ca(2+)]i increase and PKC activation stimulates Cl(-) efflux and fluid secretion. These data support the concept that CFTR plays a role in modulating purinergic signaling in secretory epithelia and suggest a novel mechanism explaining the choleretic effect of UDCA.

Effect of cigarette smoke extract on proliferation of rat pulmonary artery smooth muscle cells and the relevant roles of protein kinase C

Increased proliferation of pulmonary vascular cells and muscularisation of pulmonary vessels are frequently observed in human smokers and in animals exposed to cigarette smoke. To elucidate the molecular mechanisms leading to these changes, we studied the in vitro effect of cigarette smoke extract (CSE) on proliferation of pulmonary artery smooth muscle cells (PASMCs) and activation of protein kinase C (PKC), an important kinase implicated in cell proliferation. PASMCs cultured from 12 normal Wistar rats were studied in the following conditions: (1) PASMCs were exposed to different concentrations of CSE for 24 hours, then MTT colorimetric assay was used for detection of cell proliferation. Cell viability was assessed by trypan blue exclusion. (2) PASMCs were pre-incubated with phorbol 12-myristate 13-acetate (PMA) for 24 hours or Ro31-8220 for 30 minutes before exposure to 5% CSE for 24 hours. Cell proliferation was examined by MTT colorimetric assay, cell cycle analysis and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. (3) PASMCs were exposed to 5% CSE for 24 hours. Then PKC-alpha mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) and protein expression by Western blotting, while PKC-alpha translocation was observed by immunofluorescence staining and confocal microscopy. (4) PASMCs were transfected with specific antisense oligodeoxynucleotides against PKC-alpha 6 hours before exposure to 5% CSE for 24 hours. PKC-alpha protein expression and cell proliferation were detected by methods described previously. (1) Low concentration of CSE (5%) increased proliferation of PASMCs, whereas high concentrations (20%, 30%) were inhibitory as a result of cytotoxicity. (2) The value of absorbance (Value A), proliferation index (PI), S-phase cell fraction (SPF) and average optical density of PCNA staining in PASMCs from 5% CSE exposure group (0.306 +/- 0.033, 0.339 +/- 0.033, 0.175 +/- 0.021, 0.315 +/- 0.038, respectively) were significantly increased compared with those of control group (0.249 +/- 0.018, 0.177 +/- 0.055, 0.092 +/- 0.023, 0.187 +/- 0.022, respectively) (P < 0.05). PKC down-regulation by PMA pretreatment or PKC inhibition by Ro31-8220 pre-incubation abolished the effect of 5% CSE on PASMCs proliferation. (3) After exposure to 5% CSE for 24 hours, PKC-alpha mRNA and protein expression in PASMCs (1.054 +/- 0.078, 1.185 +/- 0.041, respectively) were much higher than in untreated cells (0.573 +/- 0.054, 0.671 +/- 0.055, respectively) (P < 0.01). Moreover, 5% CSE induced a translocation of PKC-alpha from cytoplasm toward the perinuclear area and into the nucleus. (4) Specific antisense oligodeoxynucleotides against PKC-alpha reduced 5% CSE-induced expression of PKC-alpha protein (0.713 +/- 0.047 vs 1.180 +/- 0.056), also abolished the effect of 5% CSE on PASMCs proliferation significantly. CSE can be cytotoxic at high concentrations. But at low concentrations, it makes a mitogenic effect on cultured PASMCs. PKC, especially its alpha isozyme, seems to play an important role in CSE-induced proliferation of PASMC.

Upregulation of AT1 receptor gene on activation of protein kinase Cβ/nicotinamide adenine dinucleotide diphosphate oxidase/ERK1/2/c-fos signaling cascade mediates long-term pressor effect of angiotensin II in rostral ventrolateral medulla

Angiotensin II induces the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) 1/2 via the activation of nicotinamide adenine dinucleotide diphosphate (NADPH) oxidase on stimulation of the angiotensin subtype 1 receptor (AT1R) in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for the maintenance of vasomotor tone and blood pressure are located. Angiotensin II-activated p38 MAPK in RVLM promotes a short-term pressor effect via augmented glutamatergic neurotransmission. We tested the hypothesis that the NADPH oxidase-dependent phosphorylation of ERK1/2 after the activation of conventional protein kinase C (PKC) mediates the AT1R-dependent long-term pressor effects of angiotensin II via transcriptional induction of the proto-oncogene c-fos gene in RVLM. In Sprague-Dawley rats, a microinjection of angiotensin II bilaterally into the RVLM induced membrane-bound translocation of the conventional PKCalpha, PKCbeta or PKCgamma isoform, phosphorylation of the p47 subunit of NADPH oxidase and ERK1/2, followed by phosphorylation of the transcription factor cyclic adenosine monophosphate response element binding protein (CREB), and c-fos induction. The PKC inhibitor antagonized angiotensin II-induced p47 phosphorylation, and an antisense oligonucleotide (ASON) complementary to PKCbeta messenger RNA suppressed angiotensin II-induced ERK1/2 activation, phosphorylation or DNA binding activity of CREB, and upregulation of c-fos mRNA expression in the ventrolateral medulla. Furthermore, a microinjection of ERK1/2, CREB or c-fos ASON into the RVLM significantly reduced the long-term pressor effect and augmented AT1R expression in the ventrolateral medulla induced by intracerebroventricular infusion of angiotensin II. We concluded that the PKCbeta/NADPH oxidase/ERK1/2/CREB/c-fos cascade represents a novel signaling cascade that mediates the long-term pressor effect induced by angiotensin II in the RVLM.

TGF‐β3 inhibits chondrogenesis of cultured chick leg bud mesenchymal cells via downregulation of connexin 43 and integrin β4

Transforming growth factor beta (TGF-beta) is a multifunctional cytokine that regulates a number of biological responses including chemotaxis, cell cycle progression, differentiation, and apoptosis of cells. Even though temporal and spatial expression of TGF-beta3 suggests its role in chick limb development, it is not well characterized how TGF-beta3 regulates chondrogenic differentiation of limb bud mesenchymal cells. In this study, differential display polymerase chain reaction (DD-PCR) screening and reverse transcription PCR analysis revealed that the mRNA expression of the gap junction protein, connexin 43 (Cx43), was significantly decreased during the first treatment of TGF-beta3 for 24 h in cultured chick leg bud mesenchymal cells. Treatment of these cells with lindane, a general gap junction blocker, or expression of dominant negative Cx43 increased apoptotic cell death and decreased the level of integrin beta4 protein, in a manner similar to that observed when these cells were exposed to TGF-beta3. Similarly, exposure of cultured leg chondroblasts to a functional blocking antibody against integrin-beta4 induced an increase in apoptosis. Treatment of cells with TGF-beta3 decreased the membrane translocation of PKC-alpha, leading to activation of ERK. The increase in apoptotic cell death triggered by TGF-beta3 and dominant negative Cx43 was blocked by inhibition of ERK but increased by inhibition of PKC. Collectively, these data indicate that, in cultured chick leg bud mesenchyme cells, TGF-beta3 treatment downregulates Cx43 and induces apoptotic cell death via downregulation of integrin beta4, activation of ERK and suppression of PKC-alpha activation.

The essentiality of PKCα and PKCβI translocation for CD14+monocyte differentiation towards macrophages and dendritic cells, respectively

Human peripheral CD14(+)monocytes have been known to differentiate into monocyte-derived macrophages (MDMs) or dendritic cells (MoDCs) upon suitable stimulation. However, the key intracellular molecule(s) associated with their differentiation towards specific cell types was(were) not fully understood. This study was designated to determine the association of PKC isoenzymes with the differentiation of CD14(+)monocytes into MDMs or MoDCs. Purified human peripheral CD14(+)monocytes were cultured with GM-CSF, or GM-CSF plus IL-4 for 7 days to induce cell differentiation. The phenotypic changes were analyzed by Flow-Cytometry using various specific antibodies to cell type-specific surface markers. The immunological functions of these differentiated cells were determined by measuring the amounts of TNF-alpha secretion for MDMs, and the capacities of antigen-capturing and bacterial phagocytosis for MoDCs. The translocations of PKC isoenzymes in these cells from cytosol to plasma membrane were examined by Western Blot analysis and Confocal Microscopic observation. The treatment of CD14(+)monocytes with either GM-CSF or PMA elicited PKCalpha translocation and consequently induced their differentiation into MDMs. The inclusion of PKCalpha/beta(I) specific inhibitor, Go6976, greatly inhibited the GM-CSF-induced PKCalpha translocation and dose-dependently reduced the GM-CSF- induced MDM differentiation. On the other hand, the simultaneous pretreatment of CD14(+)monocytes with Go6976 and PKCbeta-specific inhibitor predominantly suppressed the GM-CSF/IL-4-induced generation of MoDCs. Further study demonstrated that GM-CSF/IL-4 selectively induced the translocation of PKCbeta(I), not PKCalpha or PKCbeta(II), in CD14(+)monocytes. In conclusion, the cell fate commitment of CD14(+)monocytes towards MDMs or MoDCs appears to be steered by the selective activation of PKCalpha or PKCbeta(I), respectively.