Inhibition of angiogenesis by vitamin D-binding protein: Characterization of anti-endothelial activity of DBP-maf

Angiogenesis is a complex process involving coordinated steps of endothelial cell activation, proliferation, migration, tube formation and capillary sprouting with participation of intracellular signaling pathways. Regulation of angiogenesis carries tremendous potential for cancer therapy. Our earlier studies showed that vitamin D-binding protein-macrophage activating factor (DBP-maf) acts as a potent anti-angiogenic factor and inhibits tumor growth in vivo. The goal of this investigation was to understand the effect of DBP-maf on human endothelial cell (HEC) and the mechanism of angiogenesis inhibition. DBP-maf inhibited human endothelial cell (HEC) proliferation by inhibiting DNA synthesis ( μg/ml). DBP-maf significantly induced S- and G0/G1-phase arrest in HEC in 72 h. DBP-maf potently blocked VEGF-induced migration, tube-formation of HEC in a dose dependent manner. In addition, DBP-maf inhibited growth factor-induced microvessel sprouting in rat aortic ring assay. Moreover, DBP-maf inhibited VEGF signaling by decreasing VEGF-mediated phosphorylation of VEGFR-2 and ERK1/2, a downstream target of VEGF signaling cascade. However, Akt activation was not affected. These studies collectively demonstrate that DBP-maf inhibits angiogenesis by blocking critical steps such as HEC proliferation, migration, tube formation and microvessel sprouting. DBP-maf exerts its effect by inhibiting VEGR-2 and ERK1/2 signaling cascades. Understanding the cellular and molecular mechanisms of anti-endothelial activity of DBP-maf will allow us to develop it as an angiogenesis targeting novel drug for tumor therapy.     

Melatonin stimulates calmodulin phosphorylation by protein kinase C

Calmodulin (CaM)-dependent processes can be modulated by the availability of Ca(+2), the subcellular distribution of both CaM and its target proteins, CaM antagonism, and post-translational modifications such as CaM phosphorylation. Melatonin, the pineal secretory product synthesized during the dark phase of the photoperiod is an endogenous CaM antagonist. This indolamine causes CaM subcellular redistribution in epithelial MDCK and MCF-7 cells, and selectively activates protein kinase C alpha (PKC alpha) in neuronal N1E-115 cells. In the present work we have characterized the phosphorylation of CaM mediated by PKC alpha and its stimulation by melatonin in an in vitro reconstituted enzyme system. Additionally, the participation of MAPK and ERKs, downstream kinases of the PKC signaling pathway, was explored utilizing MDCK cell extracts as source of these kinases. Phosphorylation of CaM was characterized in the whole cells by MDCK cell metabolic labeling with [(32)P]-orthoposhospate, and CaM separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, as well as by immunocolocalization of phosphorylated threonine/serine residues and CaM in cultured cells incubated with melatonin. Our results show that melatonin increased CaM phosphorylation by PKC alpha with an EC(50) of 10(-8) m in the presence of the phorbol ester, phorbol-12-myristate-13-acetate (PMA) in the in vitro reconstituted enzyme system. An increase in phosphorylated CaM was also observed in cells cultured with melatonin, or PMA for 2 hr, while, PKC, MAPK, or ERK inhibitors abolished CaM phosphorylation elicited by melatonin in MDCK cell extracts. Our data show that melatonin can stimulate phosphorylation of CaM by PKC alpha in the in vitro reconstituted system and suggest that in MDCK cells this phosphorylation is accomplished by PKC. Modification of CaM by melatonin can be another route to inhibit CaM interaction with its target enzymes.     

Potent activation of multiple signalling pathways by C-peptide in opossum kidney proximal tubular cells

Aims/hypothesis Proinsulin C-peptide is generally believed to be inert without any appreciable biological functions. However, it has been shown to modulate a variety of cellular processes important in the pathophysiology of diabetic complications. We therefore investigated the ability of C-peptide to stimulate intracellular signalling pathways in kidney proximal tubular cells, the altered activation of which may possibly be related to the development of diabetic nephropathy.Methods Extracellular signal-regulated kinase (ERK) and Akt phosphorylation were evaluated by western blotting. ERK activity was measured by in vitro kinase assay. Intracellular Ca²⁺ was evaluated by confocal imaging. The membrane and cytosol-associated fractions of protein kinase C (PKC) isoforms were evaluated by western blotting. Proliferation was assessed by thymidine incorporation assay.Results Using the opossum proximal tubular kidney cell line as a model, we demonstrated that at high picomolar to low nanomolar concentrations, C-peptide stimulates extracellular signal-regulated mitogen-activated kinase (3.3±0.1-fold over basal at 3 minutes) and phosphatidylinositol 3-kinase (4.1±0.05-fold over basal at 5 minutes). ERK activation was attenuated by pre-treatment with a PKC inhibitor and abolished by pertussis toxin. Elevations of intracellular [Ca²⁺] are seen in response to 5 nmol/l C-peptide with consequent activation of PKC-. Pre-treatment with pertussis toxin abolished PKC-. C-peptide is also a functional mitogen in this cell type, stimulating significantly increased cell proliferation. Proliferation was attenuated by wortmannin and pertussis toxin pre-treatments. None of these effects is reproduced by scrambled C-peptide.Conclusions/interpretation This study provides evidence that C-peptide, within physiological concentration ranges, stimulates many signalling pathways in opossum kidney cells.Abbreviations Akt/PKB protein kinase B - DTT dithiothreitol - ERK extracellular signal-regulated kinase - GPCR G-protein coupled receptor - MAPK mitogen-activated protein kinase - OK opossum kidney - PI3-K phosphoinositide 3-kinase - PKC protein kinase C - PMA phorbol myristate acetate - PMSF phenylmethansulfonylfluoride - PTC proximal tubular cells - PTX pertussis toxin     

Insulin induces PKC-dependent proliferation of mesenteric vascular smooth muscle cells from hypertensive patients

Background and objectives Proliferation of human vascular smooth muscle cells (VSMCs) induced by hyperinsulinemia is a very common clinical pathology. Extensive research has focused on PKC (Protein kinase C)-MAPK (mitogen-activated protein kinase) intracellular signal transduction and the phenotypic modulation accompanied by reorganization of intracellular F-actins in VSMCs. Methods DNA synthesis, signaling of ERK1/2 MAPKs, and changes in a-smooth muscle (SM) actin and F-actin were studied in hypertensive and normotensive human arterial VSMCs exposed to insulin and PMA with and without the PKC inhibitor, GF109203X. Results Differences among cell types in MAPK signaling, a-SM actin, and F-actin isoforms in VSMCs harvested from the arteries of patients with essential hypertension (EH) and normotension (NT) were identified in response to insulin treatment. Proliferation and activation of MAPK were more pronounced in EH VSMCs than in NEH VSMCs. Insulin exposure decreased expression of a-SM actin and was accompanied by rearrangement of intracellular F-actins in VSMCs, especially in the EH group. These effects were reversed by treatment with the PKC inhibitor. Conclusions Human mesenteric VSMCs of EH and NT patients differed in proliferation, MAPK signaling, and degree of changes in a-SM actin and F-actin isoforms immediately following insulin exposure in vitro.     

甲状腺激素促进肿瘤细胞增殖及血管新生信号通路的研究

目的 探讨甲状腺激素促进肿瘤细胞增殖及血管新生的信号通路.方法 体外培养人胶质母细胞瘤细胞系（SNB19）,给予甲状腺激素（主要为T4,100 nmol/L）、四碘甲腺乙酸（tetraiodothyroacetic acid,Tetrac,100 nmol/L）、蛋白激酶C（PKC）抑制剂（2.5 μmol/L）作用后,采用Western印迹方法检测磷酸化蛋白激酶D1（PKD1）、磷酸化组蛋白去乙酰化酶（HDAC）5、磷酸化细胞外信号调节激酶（ERK）1/2的表达,ELISA方法检测细胞培养上清血管内皮生长因子（VEGF）的表达量,3-（4,5）-2-唑噻-（2,5）-二苯基溴化四氮唑蓝（MTT）比色法检测细胞增殖.结果 与对照组相比,T4干预组磷酸化PKD1、磷酸化HDAC5、磷酸化ERK1/2水平均增加（P均＜0.05）,Tetrac干预组及PKC抑制剂干预组磷酸化PKD1、磷酸化HDAC5、磷酸化ERK1/2水平均降低（P均＜0.05）.ELISA结果显示,与对照组相比,T4干预组VEGF浓度升高[（56.763±2.611） ng/L vs.（36.597±0.933） ng/L,P＜0.05],Tetrac＋T4干预组VEGF浓度降低[（22.215±1.531） ng/L vs.（36.597±0.933） ng/L,P＜0.05].MTT结果显示,与对照组相比,T4干预组OD值较高[（0.333±0.020）vs.（0.243±0.006）,P＜0.05],Tetrac干预组OD值较低[（0.060±0.016） vs.（0.243±0.006）,P＜0.05].结论 甲状腺激素通过结合整合素αvβ3,激活ERK1/2信号通路促进肿瘤细胞增殖,激活PKC/PKD1/HDAC5信号通路促进血管新生.     

Fibronectin- and protein kinase C-mediated activation of ERK/MAPK are essential for proplateletlike formation

The megakaryoblastic CHRF-288 cell line was used to investigate signal transduction pathways responsible for proplateletlike formation (PPF). The role of fibronectin (FN) and protein kinase C (PKC) activation in PPF were examined. In the presence of serum and phorbol 12-myristate 13-acetate (PMA), a PKC activator, cells exhibited full megakaryocytic differentiation, manifested by adhesion, shape change, increased cell size, polyploidy, PPF, and expression of CD41(+), CD61(+), and CD62P(+). The same morphologic and phenotypic features were observed in serum-free cultures in the presence of FN/PMA. Only partial differentiation occurred when other integrin ligands were substituted for FN. FN alone induced minimal cell adhesion and spreading, while PMA alone induced only polyploidy without adhesion. Signal transduction changes involved the activation of the extracellular signal-regulated protein kinase 1 (ERK1)/ERK2 as well as c-Jun amino-terminal kinase 1 (JNK1)/stress-activated protein kinase (SAPK). Phosphoinositide-3 kinase and p38 were not stimulated under these conditions. Inhibitors were used to identify the causal relationship between signaling pathways and PPF. PD98059 and GF109203X, inhibitors of ERK1/ERK2 pathway and PKC, respectively, blocked PPF, while adhesion, spreading, and polyploidy were normal. These studies show that activation of ERK1/ERK2 mitogen-activated protein kinase pathway plays a critical role in PPF. The elucidation of the signal transduction pathway on megakaryocyte development and PPF is of crucial importance for understanding this unique biological process.     

Protein kinase Cδ oxidation contributes to ERK inactivation in lupus T cells

Objective

CD4+ T cells from patients with active lupus have impaired ERK pathway signaling that decreases DNA methyltransferase expression, resulting in DNA demethylation, overexpression of immune genes, and autoimmunity. The ERK pathway defect is due to impaired phosphorylation of T⁵⁰⁵ in the protein kinase Cδ (PKCδ) activation loop. However, the mechanisms that prevent PKCδ T⁵⁰⁵ phosphorylation in lupus T cells are unknown. Others have reported that oxidative modifications, and nitration in particular, of T cells as well as serum proteins correlate with lupus disease activity. We undertook this study to test our hypothesis that nitration inactivates PKCδ, contributing to impaired ERK pathway signaling in lupus T cells.

Methods

CD4+ T cells were purified from lupus patients and controls and then stimulated with phorbol myristate acetate (PMA). Signaling protein levels, nitration, and phosphorylation were quantitated by immunoprecipitation and immunoblotting of T cell lysates. Transfections were performed by electroporation.

Results

Treating CD4+ T cells with peroxynitrite nitrated PKCδ, preventing PKCδ T⁵⁰⁵ phosphorylation and inhibiting ERK pathway signaling similar to that observed in lupus T cells. Patients with active lupus had higher nitrated T cell PKCδ levels than did controls, which correlated directly with disease activity, and antinitrotyrosine immunoprecipitations demonstrated that nitrated PKCδ, but not unmodified PKCδ, was refractory to PMA‐stimulated T⁵⁰⁵ phosphorylation, similar to PKCδ in peroxynitrite‐treated cells.

Conclusion

Oxidative stress causes PKCδ nitration, which prevents its phosphorylation and contributes to the decreased ERK signaling in lupus T cells. These results identify PKCδ as a link between oxidative stress and the T cell epigenetic modifications in lupus.

     

Induction of c-fos and c-jun messenger ribonucleic acid expression by prostaglandin F2alpha is mediated by a protein kinase C-dependent extracellular signal-regulated kinase mitogen-activated protein kinase pathway in bovine luteal cells

PGF2alpha triggers the demise of the corpus luteum whereby progesterone synthesis is inhibited, the luteal structure regresses, and the estrus cycle resumes. Upon binding to its heterotrimeric G-protein-coupled receptors, PGF2alpha initiates the phospholipase C/diacylglycerol and inositol-1,4,5-trisphosphate/Ca(2+)-protein kinase C (PKC) signaling pathway. More recently, we have demonstrated that PGF2alpha activates extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase signaling through a Raf-dependent mechanism in bovine luteal cells. However, the relationship between PKC and ERK activation in PGF2alpha signaling has not been clearly defined. Moreover, the signaling pathway that PGF2alpha uses to regulate gene expression is unknown. In this report, primary cultures of bovine luteal cells were used to address the role of PKC in ERK activation and the signaling pathway for induction of c-fos and c-jun messenger RNA (mRNA) expression in response to PGF2alpha. By using a PKC inhibitor and a PKC-deficient luteal cell model, we observed that phorbol ester-responsive isoforms of PKC were required for ERK phosphorylation and activation by PGF2alpha (1 microM) or phorbol 12-myristate 13-acetate (PMA) (20 nM). In PGF2alpha- and PMA-treated cells, active ERK MAP kinase was localized in the nucleus. PGF2alpha-induced ERK phosphorylation was dose-dependently inhibited by the MEK1 inhibitor PD098059 (1-50 microM). The expression of c-fos and c-jun mRNA in luteal cells was markedly increased by treatment with PGF2alpha (1 microM) or PMA (20 nM) for 30 min. We also observed that activation of ERK MAP kinase was required for the expression of c-fos and c-jun mRNA in response to PGF2alpha and PMA because it was abrogated by blocking the ERK pathway with PD098059. In addition, PGF2alpha and PMA-induced c-fos and c-jun mRNA expression was abolished in the PKC-deficient cells. Taken together, our data demonstrate that a PKC-dependent ERK MAP kinase pathway mediates the expression of c-fos and c-jun mRNA in PGF2alpha-treated bovine luteal cells.     

Superoxide anion production by neutrophils is associated with prevalent clinical manifestations in systemic lupus erythematosus

To determine the relation between neutrophil function and the clinical characteristics of systemic lupus erythematosus (SLE), the superoxide anion () production by neutrophils, mediated by FcγR and FcγR/CR cooperation, was studied in 64 SLE patients classified according to their prevalent clinical manifestations. Three clinically distinct patterns were designated: (1) manifestations associated with the occurrence of cytotoxic antibodies (SLE-I group); (2) manifestations associated with circulating immune complexes (IC; SLE-II group), and (3) manifestations associated with IC and cytotoxic antibodies (SLE-III group). production was evaluated by a lucigenin-dependent chemiluminescent assay in neutrophils stimulated with IC-IgG opsonized or not with complement. No difference in production was observed when neutrophil responses from healthy controls were compared to the unclassified patients. However, when the SLE patient groups were considered, the following differences were observed: (1) SLE-I neutrophils showed lower production mediated by the IgG receptor (FcγR) with the cooperation of complement receptors (FcγR/CR) than observed in the SLE-II, SLE-III, and healthy groups; (2) neutrophils from the SLE-II group showed a decreased production mediated by FcγR/CR compared to the SLE-III group, (3) SLE-III neutrophils produced more than neutrophils from the SLE-II and control groups, and (4) CR showed inefficiency in mediating the production by neutrophils from the SLE-I group. Comparative experiments on the kinetics of chemiluminescence (CL; T _max and CL_max) disclosed differences only for the SLE-I group. Taken together, these results suggest that differences in oxidative metabolism of neutrophils mediated by FcγR/CR may reflect an acquired characteristic of disease associated with distinct clinical manifestations.     

Effect of phorbol ester and platelet-derived growth factor on protein kinase C in rat hepatic stellate cells.

BACKGROUND/AIMS: Hepatic stellate cells (HSC) play a key role in hepatic fibrogenesis and thus, it is important to understand the intracellular signalling pathways that influence their behaviour. This study investigated the expression and regulation of protein kinase C (PKC) in HSC. RESULTS: Western blot analysis indicates that rat HSC express at least four PKC isoforms, PKC-alpha, PKC-delta, PKC-epsilon and PKC-zeta. PKC-alpha and PKC-zeta were located predominantly in the cytosol and were redistributed to the membrane by the PKC agonist, phorbol 12-myristate 13-acetate (PMA), while PKC-delta and PKC-epsilon were highly membrane-bound and did not undergo translocation by PMA. PKC-alpha, PKC-delta and PKC-zeta were rapidly downregulated by PMA. However, PKC-epsilon was resistant to downregulation. We also examined phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS), a specific substrate of PKC, as another approach to assess activation of PKC. Platelet-derived growth factor (PDGF) and PMA increased the phosphorylation of MARCKS, suggesting that PDGF can induce PKC activation. PDGF-induced stimulation of extracellular signal-regulated kinase, phosphatidylinositol 3-kinase and p70-S6 kinase was not abrogated by downregulation of PKC-alpha, PKC-delta and PKC-zeta. Prolonged PKC inhibition did not inhibit the fibrogenic phenotype. CONCLUSION: Multiple PKC isoforms are expressed in rat HSC and are differentially regulated by PMA. PDGF activates certain mitogenic signalling pathways independent of PKC-alpha, PKC-delta and PKC-zeta. Specific PKC isoforms may modulate different cell functions in HSC.     

Protein kinase D participates in cardiomyocyte hypertrophy by regulating extracellular signal-regulated and myocyte enhancer factor 2D

ObjectiveCardiomyocyte hypertrophy is an important feature of hypertension. However, its molecular underpinnings, especially the signaling cascades, remain unclear. Here we hypothesized that a protein kinase D (PKD)-dependent extracellular signal-regulated kinase 5 (ERK5) pathway was able to regulate downstream myocyte enhancer factor 2D (MEF2D), affecting prohypertrophic responses to angiotensin II (Ang II).MethodsNeonatal rat cardiomyocytes from 2- to 3-day-old Sprague-Dawley rats were prepared and Western blot, real-time quantitative PCR and immunofluorescence staining were used to assess the activation and translocation of pathway signaling molecules. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) expression and [3H]-leucine (Leu) incorporation were measured to determine cell hypertrophy.ResultsElevated levels of phosphorylated PKD (p-PKD) and ERK5 (p-ERK5) were observed in cardiomyocytes stimulated with Ang II, while silencing protein kinase C epsilon (PKC?) resulted in significantly lower levels of p-PKD. Furthermore, Ang II-induced ERK5 activated translocation was mediated by the PKD pathway. Consequently, inhibiting PKC?, PKD and ERK5 by siRNA significantly attenuated Ang II-induced MEF2D activation, ANP and BNP mRNA expression, and [3H]-Leu incorporation.ConclusionsOur studies are the first to show that the PKC?/PKD/ERK5/MEF2D pathway plays an important role in the cardiomyocyte hypertrophy response to Ang II.     

C-peptide stimulates ERK1/2 and JNK MAP kinases via activation of protein kinase C in human renal tubular cells

Aims/hypothesis Accumulating evidence indicates that replacement of C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, but the molecular mechanisms involved are incompletely understood. C-peptide shows specific binding to a G-protein-coupled membrane binding site, resulting in Ca²⁺ influx, activation of mitogen-activated protein kinase signalling pathways, and stimulation of Na⁺, K⁺-ATPase and endothelial nitric oxide synthase. This study examines the intracellular signalling pathways activated by C-peptide in human renal tubular cells.Methods Human renal tubular cells were cultured from the outer cortex of renal tissue obtained from patients undergoing elective nephrectomy. Extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and Akt/protein kinase B (PKB) activation was determined using phospho-specific antibodies. Protein kinase C (PKC) and RhoA activation was determined by measuring their translocation to the cell membrane fraction using isoform-specific antibodies.Results Human C-peptide increases phosphorylation of ERK1/2 and Akt/PKB in a concentration- and time-dependent manner in renal tubular cells. The C-terminal pentapeptide of C-peptide is equipotent with the full-length C-peptide, whereas scrambled C-peptide has no effect. C-peptide stimulation also results in phosphorylation of JNK, but not of p38 mitogen-activated protein kinase. MEK1/2 inhibitor PD98059 blocks the C-peptide effect on ERK1/2 phosphorylation. C-peptide causes specific translocation of PKC isoforms and to the membrane fraction in tubular cells. All stimulatory effects of C-peptide were abolished by pertussis toxin. The isoform-specific PKC- inhibitor rottlerin and the broad-spectrum PKC inhibitor GF109203X both abolish the C-peptide effect on ERK1/2 phosphorylation. C-peptide stimulation also causes translocation of the small GTPase RhoA from the cytosol to the cell membrane. Inhibition of phospholipase C abolished the stimulatory effect of C-peptide on phosphorylation of ERK1/2, JNK and PKC-.Conclusions/interpretation C-peptide signal transduction in human renal tubular cells involves the activation of phospholipase C and PKC- and PKC-, as well as RhoA, followed by phosphorylation of ERK1/2 and JNK, and a parallel activation of Akt.     

Protein kinase C protects preconditioned rabbit hearts by increasing sensitivity of adenosine A2b-dependent signaling during early reperfusion

Although protein kinase C (PKC) plays a key role in ischemic preconditioning (IPC), the actual mechanism of that protection is unknown. We recently found that protection from IPC requires activation of adenosine receptors during early reperfusion. We, therefore, hypothesized that PKC might act to increase the heart's sensitivity to adenosine. IPC limited infarct size in isolated rabbit hearts subjected to 30-min regional ischemia/2-h reperfusion and IPC's protection was blocked by the PKC inhibitor chelerythrine given during early reperfusion revealing involvement of PKC at reperfusion. Similarly chelerythrine infused in the early reperfusion period blocked the increased phosphorylation of the protective kinases Akt and ERK1/2 observed after IPC. Infusing phorbol 12-myristate 13-acetate (PMA), a PKC activator, during early reperfusion mimicked IPC's protection. As expected, the protection triggered by PMA at reperfusion was blocked by chelerythrine, but surprisingly it was also blocked by MRS1754, an adenosine A(2b) receptor-selective antagonist, suggesting that PKC was somehow facilitating signaling from the A(2b) receptors. NECA [5'-(N-ethylcarboxamido) adenosine], a potent but not selective A(2b) receptor agonist, increased phosphorylation of Akt and ERK1/2 in a dose-dependent manner. Pretreating hearts with PMA or brief preconditioning ischemia had no effect on phosphorylation of Akt or ERK1/2 per se but markedly lowered the threshold for NECA to induce their phosphorylation. BAY 60-6583, a highly selective A(2b) agonist, also caused phosphorylation of ERK1/2 and Akt. MRS1754 prevented phosphorylation induced by BAY 60-6583. BAY 60-6583 limited infarct size when given to ischemic hearts at reperfusion. These results suggest that activation of cardiac A(2b) receptors at reperfusion is protective, but because of the very low affinity of the receptors endogenous cardiac adenosine is unable to trigger their signaling. We propose that the key protective event in IPC occurs when PKC increases the heart's sensitivity to adenosine so that endogenous adenosine can activate A(2b)-dependent signaling.     

Enhanced activity of the myocardial Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger contributes to left ventricular hypertrophy in the Goto–Kakizaki rat model of type 2 diabetes: critical role of Akt

Aims/hypothesis Diabetes mellitus is a strong risk factor for the development of heart failure, and left ventricular (LV) hypertrophy has been detected in a significant proportion of diabetic patients. Because several studies have suggested that the Na⁺/H⁺ exchanger (NHE1) plays a part in the molecular mechanisms involved in cardiac hypertrophy, we investigated its activity and its role in LV myocytes from the Goto–Kakizaki (GK) rat model of type 2 diabetes. Materials and methods Fluorometric measurements were used to assess sarcolemmal NHE1 activity in isolated myocytes. NHE1 levels and the possible molecular pathways driving and/or related to NHE1 activity were investigated in relation to the diabetic LV phenotype. Results Enhanced NHE1 activity was associated with LV myocyte hypertrophy. This occurred in the absence of any change in NHE1 protein levels; however, activation of several molecular pathways related to NHE1 activity was demonstrated. Thus, phosphorylation of the extracellular signal-regulated protein kinase (Erk), of the protein kinase Akt (also known as protein kinase B) and of the Ca²⁺/calmodulin-dependent kinase II was increased in GK LV myocytes. Intracellular Ca²⁺ levels were also increased. Chronic treatment (10–12 weeks) with the NHE1 inhibitor cariporide normalised NHE1 activity, decreased levels and reduced LV myocyte hypertrophy. Moreover, among the various activated pathways, cariporide treatment markedly reduced Akt activity only. Conclusions/interpretation These findings indicate that activation of the Akt pathway represents a likely mechanism mediating the hypertrophic effect of increased NHE1 activity in the GK model of type 2 diabetes.     

Activation of conventional PKC isoforms increases expression of the pro-apoptotic protein Bad and TRAIL receptors

Background. Pancreatic cancer is a leading cause of cancer death worldwide; current treatment options have been ineffective in prolonging survival. Agents that target specific signaling pathways (e.g., protein kinase C [PKC]) may regulate apoptotic gene expression rendering resistant cancers sensitive to the effects of other chemotherapeutic drugs. The purpose of our study was to assess the effect of PKC stimulation on apoptotic gene expression in pancreatic cancer cells. Methods. The human pancreatic cancer cell line, PANC-1, was treated with PKC-stimulating agents, phorbol 12-myristate 13-acetate (PMA) or bryostatin-1, and analyzed for expression of apoptosis-related genes. Results. Both PMA and bryostatin-1 induced expression of the pro-apoptotic gene Bad in a dosedependent fashion. The expression of Bad was blocked by the PKC inhibitors GF109203x, G?6983, and Ro-31-8220, suggesting a role for the conventional isoforms of PKC. In addition, treatment with the MEK inhibitors PD98059 or UO126 reduced PMA-mediated induction of Bad gene expression. PMA also increased the expression of TRAIL receptors DR4 and DR5; this expression was inhibited by the PKC inhibitors GF109203x, G?6983, and Ro-31-8220 and the MEK inhibitor UO126, suggesting a role for conventional PKC isoforms and MEK in the regulation of TRAIL receptor expression. Conclusions. PKC stimulation in PANC-1 cells increases expression of the pro-apoptotic gene Bad and the TRAIL receptors, DR4 and DR5, through both conventional PKC- and MEK-dependent pathways. Agents that stimulate PKC may sensitize pancreatic cancer cells to apoptosis and provide a potential adjuvant therapy for the treatment of chemoresistant pancreatic cancers.     

Melatonin induces neuritogenesis at early stages in N1E-115 cells through actin rearrangements via activation of protein kinase C and Rho-associated kinase

Melatonin increases neurite formation in N1E-115 cells through microtubule enlargement elicited by calmodulin antagonism and vimentin intermediate filament reorganization caused by protein kinase C (PKC) activation. Microfilament rearrangement is also a necessary process in growth cone formation during neurite outgrowth. In this work, we studied the effect of melatonin on microfilament rearrangements present at early stages of neurite formation and the possible participation of PKC and the Rho-associated kinase (ROCK), which is a downstream kinase in the PKC signaling pathway. The results showed that 1 nm melatonin increased both the number of cells with filopodia and with long neurites. Similar results were obtained with the PKC activator phorbol 12-myristate 13-acetate (PMA). Both melatonin and PMA increased the quantity of filamentous actin. In contrast, the PKC inhibitor bisindolylmaleimide abolished microfilament organization elicited by either melatonin or PMA, while the Rho inhibitor C3, or the ROCK inhibitor Y27632, abolished the bipolar neurite morphology of N1E-115 cells. Instead, these inhibitors prompted neurite ramification. ROCK activity measured in whole cell extracts and in N1E-115 cells was increased in the presence of melatonin and PMA. The results indicate that melatonin increases the number of cells with immature neurites and suggest that these neurites can be susceptible to differentiation by incoming extracellular signals. Data also indicate that PKC and ROCK are involved at initial stages of neurite formation in the mechanism by which melatonin recruits cells for later differentiation.     

Intracellular signaling pathways involved in cell growth inhibition of human umbilical vein endothelial cells by melatonin

Melatonin, an indolamine mainly produced in the pineal gland, has received a great deal of attention in the last decade because of its oncostatic effects, which are due to its immunomodulatory, antiproliferative, antioxidant and its possible antiangiogenesis properties. Herein, we document its antiproliferative action on human umbilical vein endothelial cells (HUVECs). Moreover, the possible cell signaling pathways when melatonin inhibited HUVEC proliferation were explored in this study. Primary HUVECs were isolated, cultured, purified and identified before the studies were performed. HUVECs were found to possess G-protein-coupled membrane receptors for melatonin (MT1 and MT2) and also nuclear melatonin receptors (RORalpha and RORbeta, especially RORbeta). No obvious expression of RORgamma was found. We investigated the membrane receptors and several intracellular signaling pathways including mitogen-activated protein kinases (MAPK)/extracellular signal-related kinases (ERK), phosphoinositol-3-kinase (PI3K)/Akt and protein kinases C (PKC) involved in antiproliferative action of melatonin on HUVECs. The blockade of these pathways using special inhibitors decreased cell growth. Furthermore, the constitutive activation of nuclear factor kappa B (NF-kappaB) contributed to the proliferation of HUVECs. High concentrations of melatonin inhibited both NF-kappaB expression and its binding ability to DNA, possibly through inactivation of ERK/Akt /PKC pathways. Taken together, high concentrations of melatonin markedly reduced HUVEC proliferation; the antiproliferative action of melatonin was closely correlated with following pathway: melatonin receptors/ERK/PI3K/Akt/PKC/ NF-kappaB.