PKCβ Activation Research Articles

PKC activation affects, via ELAVL1/HuR protein, VEGF expression in pericytic/endothelial coculture

AbstractPurpose To explore whether, following direct contact, there is a mutual influence between pericytes (PC) and endothelial cells (EC), and to establish whether PKC activation, a condition associated with hyperglycaemia in diabetic retinopathy (DR), can affect, via the mRNA‐binding ELAVL1/HuR protein, VEGF expression.Methods Retinal PC and EC were cultured separately (MONO) or in direct contact (1:1 ratio; coculture; COCU), and exposed or not to PKC activator phorbol esthers (100 nM PMA, 15 min.). PKCβI/βII, HuR, VEGF proteins were analysed by Western blotting; VEGF secretion in cell culture medium was evaluated by ELISA. VEGF mRNA analysis was performed by real‐time qPCR. Immunocytochemistry was performed to evaluate PMA‐induced HuR translocation.Results In MONO, VEGF mRNA/protein basal levels are higher in PC than EC. In COCU, VEGF protein amount is up‐regulated in EC and down‐regulated in PC, respectively. VEGF release is favoured by physical contact between PC and EC and further increased by PMA exposure. PKCβ activation induces HuR translocation from nucleus to cytoplasm, and it increases the protein levels of the kinase itself, HuR and VEGF in PC and EC in both culture conditions.Conclusion VEGF expression varies in function of the cell culture conditions. In all cultures, PKC activation increases PKCβI/βII, HuR and VEGF protein levels, changes which may occur also at very early stages of DR. The COCU model may be useful to study PC/EC interactions in both physiological and pathological conditions.

Hyperglycemia-Induced Protein Kinase C β2 Activation Induces Diastolic Cardiac Dysfunction in Diabetic Rats by Impairing Caveolin-3 Expression and Akt/eNOS Signaling

Protein kinase C (PKC)β2 is preferably overexpressed in the diabetic myocardium, which induces cardiomyocyte hypertrophy and contributes to diabetic cardiomyopathy, but the underlying mechanisms are incompletely understood. Caveolae are critical in signal transduction of PKC isoforms in cardiomyocytes. Caveolin (Cav)-3, the cardiomyocyte-specific caveolar structural protein isoform, is decreased in the diabetic heart. The current study determined whether PKCβ2 activation affects caveolae and Cav-3 expression. Immunoprecipitation and immunofluorescence analysis revealed that high glucose (HG) increased the association and colocalization of PKCβ2 and Cav-3 in isolated cardiomyocytes. Disruption of caveolae by methyl-β-cyclodextrin or Cav-3 small interfering (si)RNA transfection prevented HG-induced PKCβ2 phosphorylation. Inhibition of PKCβ2 activation by compound CGP53353 or knockdown of PKCβ2 expression via siRNA attenuated the reductions of Cav-3 expression and Akt/endothelial nitric oxide synthase (eNOS) phosphorylation in cardiomyocytes exposed to HG. LY333531 treatment (for a duration of 4 weeks) prevented excessive PKCβ2 activation and attenuated cardiac diastolic dysfunction in rats with streptozotocin-induced diabetes. LY333531 suppressed the decreased expression of myocardial NO, Cav-3, phosphorylated (p)-Akt, and p-eNOS and also mitigated the augmentation of O2−, nitrotyrosine, Cav-1, and iNOS expression. In conclusion, hyperglycemia-induced PKCβ2 activation requires caveolae and is associated with reduced Cav-3 expression in the diabetic heart. Prevention of excessive PKCβ2 activation attenuated cardiac diastolic dysfunction by restoring Cav-3 expression and subsequently rescuing Akt/eNOS/NO signaling.

PKCβ Promotes Vascular Inflammation and Acceleration of Atherosclerosis in Diabetic ApoE Null Mice

Subjects with diabetes mellitus are at high risk for developing atherosclerosis through a variety of mechanisms. Because the metabolism of glucose results in production of activators of protein kinase C (PKC)β, it was logical to investigate the role of PKCβ in modulation of atherosclerosis in diabetes mellitus. ApoE(-/-) and PKCβ(-/-)/ApoE(-/-) mice were rendered diabetic with streptozotocin. Quantification of atherosclerosis, gene expression profiling, or analysis of signaling molecules was performed on aortic sinus or aortas from diabetic mice. Diabetes mellitus-accelerated atherosclerosis increased the level of phosphorylated extracellular signal-regulated kinase 1/2 and Jun-N-terminus kinase mitogen-activated protein kinases and augmented vascular expression of inflammatory mediators, as well as increased monocyte/macrophage infiltration and CD11c(+) cells accumulation in diabetic ApoE(-/-) mice, processes that were diminished in diabetic PKCβ(-/-)/ApoE(-/-) mice. In addition, pharmacological inhibition of PKCβ reduced atherosclerotic lesion size in diabetic ApoE(-/-) mice. In vitro, the inhibitors of PKCβ and extracellular signal-regulated kinase 1/2, as well as small interfering RNA to Egr-1, significantly decreased high-glucose-induced expression of CD11c (integrin, alpha X 9 complement component 3 receptor 4 subunit]), chemokine (C-C motif) ligand 2, and interleukin-1β in U937 macrophages. These data link enhanced activation of PKCβ to accelerated diabetic atherosclerosis via a mechanism that includes modulation of gene transcription and signal transduction in the vascular wall, processes that contribute to acceleration of vascular inflammation and atherosclerosis in diabetes mellitus. Our results uncover a novel role for PKCβ in modulating CD11c expression and inflammatory response of macrophages in the development of diabetic atherosclerosis. These findings support PKCβ activation as a potential therapeutic target for prevention and treatment of diabetic atherosclerosis.

Peroxisome Proliferator-Activated Receptor γ Agonists Attenuate Hyperglycaemia-Induced Hyaluronan Secretion in Vascular Smooth Muscle Cells by Inhibiting PKCβ2

Changes in the composition and assembly of extracellular matrix (ECM) are the most prominent structure abnormalities of the vascular system encountered in early diabetes. Hyaluronan (HA) is a key biologically active element of ECM that plays a crucial role in vascular remodelling in atherosclerosis and restenosis following percutaneous coronary intervention. Hyperglycaemia led to significant increase in HA secretion by vascular smooth muscle cells. Hyperglycaemia also strongly induced HA synthase mRNA levels, notably HAS1-HAS3 mRNA. Remarkably, peroxisome proliferator-activated receptor (PPAR-γ) agonists pioglitazone (Pio) and rosiglitazone (Rosi), a class of anti-diabetic drugs, attenuated hyperglycaemia-induced HA secretion and reduced HAS2 mRNA expression. In vitro experiment with siRNA specific to PPAR-γ demonstrated that the attenuation of hyperglycaemia-induced HA secretion by Pio and Rosi was independent of PPAR-γ activity. Furthermore, hyperglycaemia-induced increase in HA secretion and HAS2 mRNA expression involved protein kinase Cβ2 (PKCβ2) activation, while Pio and Rosi exerted their attenuating effect on HA secretion by inhibiting PKCβ2.

MiR-150 regulates high glucose-induced cardiomyocyte hypertrophy by targeting the transcriptional co-activator p300

p300, a transcriptional co-activator with histone acetyl transferase (HAT) activity, plays an essential role in the pathogenesis of cardiomyocyte hypertrophy in response to multiple pro-hypertrophic stimuli including hyperglycemia. However, the precise mechanisms by which p300 expression is regulated remain unclear. The purpose of this study was to investigate the role of miR-150, a potential p300-targeting microRNA (miRNA), in the post-transcriptional control of p300 expression and cardiomyocyte hypertrophy induced by high glucose. We observed that the expression of miR-150 was significantly reduced, whereas the expression of p300 was strongly elevated, concomitant with cardiomyocyte hypertrophy, in the hearts of diabetic rats compared with normal controls. Similar alterations were observed in neonatal rat cardiomyocytes that had been exposed to high levels of glucose. miR-150 mimics inhibited p300 3′-UTR luciferase reporter activity, as well as endogenous p300 expression. In addition, miR-150 mimics prevented glucose-induced cardiomyocyte hypertrophy. Co-transfection with a p300 expression vector and miR-150 mimics reversed the protective effect of miR-150 on cardiomyocyte hypertrophy. We further showed that the high glucose-mediated activation of PKCβ2 in turn mediated the down-regulation of miR-150 expression. These data demonstrated a novel upstream role for miR-150 in p300-mediated cardiomyocyte hypertrophy and revealed a previously uncharacterized miRNAs and HATs cross-talk mechanism for the hypertrophic phenotype induced by high glucose.

Protective Effects of GLP-1 on Glomerular Endothelium and Its Inhibition by PKCβ Activation in Diabetes

To characterize glucagon-like peptide (GLP)-1 signaling and its effect on renal endothelial dysfunction and glomerulopathy. We studied the expression and signaling of GLP-1 receptor (GLP-1R) on glomerular endothelial cells and the novel finding of protein kinase A–dependent phosphorylation of c-Raf at Ser259 and its inhibition of angiotensin II (Ang II) phospho–c-Raf(Ser338) and Erk1/2 phosphorylation. Mice overexpressing protein kinase C (PKC)β2 in endothelial cells (EC-PKCβ2Tg) were established. Ang II and GLP-1 actions in glomerular endothelial cells were analyzed with small interfering RNA of GLP-1R. PKCβ isoform activation induced by diabetes decreased GLP-1R expression and protective action on the renal endothelium by increasing its degradation via ubiquitination and enhancing phospho–c-Raf(Ser338) and Ang II activation of phospho-Erk1/2. EC-PKCβ2Tg mice exhibited decreased GLP-1R expression and increased phospho–c-Raf(Ser338), leading to enhanced effects of Ang II. Diabetic EC-PKCβ2Tg mice exhibited greater loss of endothelial GLP-1R expression and exendin-4–protective actions and exhibited more albuminuria and mesangial expansion than diabetic controls. These results showed that the renal protective effects of GLP-1 were mediated via the inhibition of Ang II actions on cRaf(Ser259) and diminished by diabetes because of PKCβ activation and the increased degradation of GLP-1R in the glomerular endothelial cells.

Diabetes-induced increased oxidative stress in cardiomyocytes is sustained by a positive feedback loop involving Rho kinase and PKCβ2

We previously reported that acute inhibition of the RhoA/Rho kinase (ROCK) pathway normalized contractile function of diabetic rat hearts, but the underlying mechanism is unclear. Protein kinase C (PKC) β(2) has been proposed to play a major role in diabetic cardiomyopathy at least in part by increasing oxidative stress. Further evidence suggests that PKC positively regulates RhoA expression through induction of inducible nitric oxide synthase (iNOS) in diabetes. However, in preliminary studies, we found that inhibition of ROCK itself reduced RhoA expression in diabetic hearts. We hypothesized that there is an interaction between RhoA/ROCK and PKCβ(2) in the form of a positive feedback loop that sustains their activation and the production of reactive oxygen species (ROS). This was investigated in cardiomyocytes isolated from diabetic and control rat hearts, incubated with or without cytochalasin D or inhibitors of ROCK, RhoA, PKCβ(2), or iNOS. Inhibition of RhoA and ROCK markedly attenuated the diabetes-induced increases in PKCβ(2) activity and iNOS and RhoA expression in diabetic cardiomyocytes, while having no effect in control cells. Inhibition of PKCβ(2) and iNOS also normalized RhoA expression and ROCK overactivation, whereas iNOS inhibition reversed the increase in PKCβ(2) activity. Each of these treatments also normalized the diabetes-induced increase in production of ROS. Actin cytoskeleton disruption attenuated the increased expression and/or activity of all of these targets in diabetic cardiomyocytes. These data suggest that, in the diabetic heart, the RhoA/ROCK pathway contributes to contractile dysfunction at least in part by sustaining PKCβ(2) activation and ROS production via a positive feedback loop that requires an intact cytoskeleton.

Protein kinase cβ phosphorylates occludin regulating tight junction trafficking in vascular endothelial growth factor-induced permeability in vivo.

Vascular endothelial growth factor (VEGF)–induced breakdown of the blood-retinal barrier requires protein kinase C (PKC)β activation. However, the molecular mechanisms related to this process remain poorly understood. In this study, the role of occludin phosphorylation and ubiquitination downstream of PKCβ activation in tight junction (TJ) trafficking and endothelial permeability was investigated. Treatment of bovine retinal endothelial cells and intravitreal injection of PKCβ inhibitors as well as expression of dominant-negative kinase was used to determine the contribution of PKCβ to endothelial permeability and occludin phosphorylation at Ser490 detected with a site-specific antibody. In vitro kinase assay was used to demonstrate direct occludin phosphorylation by PKCβ. Ubiquitination was measured by immunoblotting after occludin immunoprecipitation. Confocal microscopy revealed organization of TJ proteins. The results reveal that inhibition of VEGF-induced PKCβ activation blocks occludin Ser490 phosphorylation, ubiquitination, and TJ trafficking in retinal vascular endothelial cells both in vitro and in vivo and prevents VEGF-stimulated vascular permeability. Occludin Ser490 is a direct target of PKCβ, and mutating Ser490 to Ala (S490A) blocks permeability downstream of PKCβ. Therefore, PKCβ activation phosphorylates occludin on Ser490, leading to ubiquitination required for VEGF-induced permeability. These data demonstrate a novel mechanism for PKCβ targeted inhibitors in regulating vascular permeability.

Gender differences in aortic endothelial function of streptozotocin‐induced diabetic rats: possible involvement of protein kinase C beta and nitric oxide production

Little is known of the interaction between diabetes and gender in the vasculature. The objective of this study was to investigate whether there are gender differences in rat aortic endothelial function in streptozotocin (STZ, 60mg/kg, iv)‐induced diabetes and the roles of protein kinase C beta (PKCβ) and nitric oxide (NO). Endothelium‐dependent vasodilatation (EDV) to acetylcholine (ACh; 10−8 to 10−5M) was measured in aortic rings precontracted with phenylephrine (PE; 2μM) before and after pretreatment with LY341684 (1μM), a selective PKCβ inhibitor. Constrictor response curves to PE (10−8 to 10−5M) were also generated before and after incubation with L‐NAME (200μM), an endothelial nitric oxide synthase inhibitor. STZ‐induced diabetes impaired aortic EDV to ACh only in females. Inhibition of PKCβ increased the sensitivity to ACh in both control and diabetic male rats. However, PKCβ inhibition enhanced the sensitivity to ACh only in aorta taken from diabetic female rats. Addition of L‐NAME resulted in a significant potentiation of the contractile responses to PE in all groups. However, aorta from control females had a greater maximal potentiation of the PE responses than others. These data suggest that the predisposition of female rat aorta to vascular injury in diabetes is possibly due to differences in enhanced PKCβ activation and decreased basal NO production (supported by NIH/NIDCR).

Protein kinase C inhibitor prevents renal apoptotic and fibrotic changes in response to partial ureteric obstruction

What's known on the subject? and What does the study add? Protein kinase C inhibitor (PKCI) can decrease glomerular and tubular cell apoptosis and mitosis and attenuate collagen accumulation and fibronectin expression in a PUUO rat model. Although the role of PKC has been well studied in diabetic nephropathy, there is no report on its role in obstructive nephropathy. This investigation evaluated the processes that were associated with the activation of PKCα and PKCβ pathways and showed that PKCI played an important role in the protection of renal function during ureteric obstruction. • To investigate the expression of the protein kinase C (PKC) pathway after partial unilateral ureteric obstruction (PUUO). • To evaluate the therapeutic potential of a PKC inhibitor (PKCI) in obstructive nephropathy. • Thirty-six rats were divided into three groups. One sham-operated group served as the control. The other two groups received PUUO surgery, after which one group received no treatment and the other group was treated with PKCI, chelerythrine. • The severity of hydronephrosis and renal morphology were assessed: tubular and glomerularcell apoptosis, mitosis and interstitial fibrosis were examined using immunohistochemistry. • Western immunoblots were performed to determine fibronectin, transforming growth factor-β (TGF-β), and PKC isoform levels. • Two weeks after PUUO surgery, hydronephrosis progressively developed. Tubular-interstitial fibrosis, collagen deposition and fibronectin expression were increased. • PUUO also activated the expression of PKCα and PKCβ and the translocation of PKCs from cell cytosol to cell membranes. • Treatment with PKCI significantly decreased PKCα and PKCβ expression and translocation in the renal cortex. • Treatment with PKCI also reduced the severity of hydronephrosis, decreased both glomerular and tubular cell apoptosis and mitosis, and attenuated the collagen and fibronectin accumulation in renal interstitium. • Renal tubular apoptosis and interstitial fibrosis after obstructive nephropathy are associated with PKCα and PKCβ activation. • The PKCI, chelerythrine, is capable of decreasing PKC expression and translocation in the renal cortex, suggesting that this inhibitor may have therapeutic potential in the protection of renal function in the first few weeks after PUUO surgery.

Phospholipase C and Protein Kinase C-β 2 Mediate Insulin-Like Growth Factor II-Dependent Sphingosine Kinase 1 Activation

We recently reported that IGF-II binding to the IGF-II/mannose-6-phosphate (M6P) receptor activates the ERK1/2 cascade by triggering sphingosine kinase 1 (SK1)-dependent transactivation of G protein-coupled sphingosine 1-phosphate (S1P) receptors. Here, we investigated the mechanism of IGF-II/M6P receptor-dependent sphingosine kinase 1 (SK1) activation in human embryonic kidney 293 cells. Pretreating cells with protein kinase C (PKC) inhibitor, bisindolylmaleimide-I, abolished IGF-II-stimulated translocation of green fluorescent protein (GFP)-tagged SK1 to the plasma membrane and activation of endogenous SK1, implicating PKC as an upstream regulator of SK1. Using confocal microscopy to examine membrane translocation of GFP-tagged PKCα, β1, β2, δ, and ζ, we found that IGF-II induced rapid, transient, and isoform-specific translocation of GFP-PKCβ2 to the plasma membrane. Immunoblotting of endogenous PKC phosphorylation confirmed PKCβ2 activation in response to IGF-II. Similarly, IGF-II stimulation caused persistent membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, which does not dissociate from the membrane after translocation. IGF-II stimulation increased diacylglycerol (DAG) levels, the established activator of classical PKC. Interestingly, the polyunsaturated fraction of DAG was increased, indicating involvement of phosphatidyl inositol/phospholipase C (PLC). Pretreating cells with the PLC inhibitor, U73122, attenuated IGF-II-dependent DAG production and PKCβ2 phosphorylation, blocked membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, and reduced sphingosine 1-phosphate production, suggesting that PLC/PKCβ2 are upstream regulators of SK1 in the pathway. Taken together, these data provide evidence that activation of PLC and PKCβ2 by the IGF-II/M6P receptor are required for the activation of SK1.

PKCβ inhibition with ruboxistaurin reduces oxidative stress and attenuates left ventricular hypertrophy and dysfuntion in rats with streptozotocin-induced diabetes

Oxidative stress plays critical roles in the development of diabetic cardiovascular complications, including myocardial hypertrophy. The β isoform of PKC (protein kinase C) is preferentially overexpressed in the myocardium of diabetic subjects accompanied with increased activation of the pro-oxidant enzyme NADPH oxidase, which may exacerbate oxidative stress. We hypothesized that myocardial PKCβ is a major upstream mediator of oxidative stress in diabetes and that PKCβ inhibition can attenuate myocardial hypertrophy and dysfunction. Control or streptozotocin-induced diabetic rats were treated with the selective PKCβ inhibitor RBX (ruboxistaurin; 1mg/kg of body weight per day) or the antioxidant NAC (N-acetylcysteine) for 4weeks. LV (left ventricular) dimensions and functions were detected by echocardiography. 15-F2t-isoprostane (a specific index of oxidative stress) and myocardial activities of superoxide dismutase as well as protein levels of NADPH oxidase were assessed by immunoassay or Western blotting. Echocardiography revealed that the LV mass/body weight ratio was significantly increased in diabetic rats (P<0.01 compared with the control group) in parallel with the impaired LV relaxation. A significant increase in cardiomyocyte cross-sectional area was observed in diabetic rats accompanied by an increased production of O2- (superoxide anion) and 15-F2t-isoprostane (all P<0.05 compared with the control group). RBX normalized these changes with concomitant inhibition of PKCβ2 activation and prevention of NADPH oxidase subunit p67phox membrane translocation and p22phox overexpression. The effects of RBX were comparable with that of NAC, except that NAC was inferior to RBX in attenuating cardiac dysfunction. It is concluded that RBX can ameliorate myocardial hypertrophy and dysfunction in diabetes, which may represent a novel therapy in the prevention of diabetic cardiovascular complications.

PKC-β Activation and Pharmacologically Induced Weight gain During Antipsychotic Treatment

Atypical antipsychotics (APDs) are now the most widely used drugs in the treatment of several mental disorders such as schizophrenia, bipolar disorder and other severe behavioral disturbances [1]. Thanks to great patient benefits such as the reduction of serious side effects, for example extrapyramidal symptoms, including tardive dyskinesia and heart arrhythmias [2], APDs are replacing the traditional antipsychotics, butyrophenones and phenothiazines. In recent years, however, APDs have been found to exert a very strong impact on body metabolism, leading to the development of severe obesity, dys lipidemia and changes in insulin sensitivity, which are major risk factors associated with the development of cardiovascular complications [3]. The WHO considers this as a problem of global importance, not only because metabolic syndrome is a major cause of death in the general population, but also because it affects patients treated with APDs; up to 60% of deaths in this group are caused by this disease (with a mortality ratio of 1:57, compared with the general population) [4,5]. There is still uncertainty regarding the mechanism by which APDs can lead to severe weight gain and all of its effects, most notably the development of diabetes. In this article, we present evidence highlighting the involvement of the b isoform of the protein kinase C family (PKC-b) in this unfortunate side effect of APDs related to weight gain [6]. First, we have to keep in mind that high glucose levels resultant of PKC-b activation, drive mesenchymal stem cells from both adipose tissue (adipose derived stem cells [ADSc]) and muscle (muscle derived stem cells [MDSCs]) towards an adipogenic potential in a reactive oxygen species (ROS)-dependent mechanism [7].

Protein Kinase CβII-Mediated Phosphorylation of Endothelial Nitric Oxide Synthase Threonine 495 Mediates the Endothelial Dysfunction Induced by FK506 (Tacrolimus)

FK506 [tacrolimus; hexadecahydro-5,19-dihydroxy-3-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylethenyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-8-(2-propenyl)-15,19-epoxy-3H-pyrido[2,1-c][1,4]oxa-azacyclotricosine-1,7,20,21(4H,23H)-tetrone] is used clinically to reduce the incidence of allograft rejection; however, chronic administration leads to endothelial dysfunction and hypertension. We have previously shown that FK506 activates Ca(2+)/diacylglycerol-dependent conventional protein kinase C (cPKC), which phosphorylates endothelial nitric oxide synthase (eNOS) at one of its inhibitory sites, Thr495. However, which cPKC isoform is responsible for phosphorylating eNOS Thr495 is unknown. The aim of the current study was to determine the cPKC isoform that is activated by FK506, leading to decreased endothelial function. FK506 reduced endothelium-dependent relaxation responses, yet had no effect on endothelium-independent relaxation responses in aortas from control mice. Of the various cPKC isoforms, only the administration of a PKCβ(II) isoform-specific peptide inhibitor restored aortic relaxation responses to that of controls. In aortic endothelial cells, FK506 significantly increased PKCβ(II) activation compared with vehicle-treated controls, and this was prevented by a PKCβ(II) isoform-specific peptide inhibitor. In addition, a PKCβ(II) isoform-specific peptide inhibitor prevented the increase in eNOS Thr495 phosphorylation induced by FK506. Taken together, our results indicate that β(II) is the cPKC isoform responsible for phosphorylating eNOS at the inhibitory site Thr495 in response to FK506. PKCβ(II) inhibition could prove beneficial in ameliorating the endothelial dysfunction and hypertension in patients treated with FK506.

Amphetamine-induced ERM Proteins Phosphorylation Is through PKCβ Activation in PC12 Cells

Amphetamine, a synthetic psychostimulant, is transported by the dopamine transporter (DAT) to the cytosol and increases the exchange of extracellular amphetamine by intracellular dopamine. Recently, we reported that the phosphorylation levels of ezrin-radixin-moesin (ERM) proteins are regulated by psychostimulant drugs in the nucleus accumbens, a brain area important for drug addiction. However, the significance of ERM proteins phosphorylation in response to drugs of abuse has not been fully investigated. In this study, using PC12 cells as an in vitro cell model, we showed that amphetamine increases ERM proteins phosphorylation and protein kinase C (PKC) β inhibitor, but not extracellular signal-regulated kinase (ERK) or phosphatidylinositol 3-kinases (PI3K) inhibitors, abolished this effect. Further, we observed that DAT inhibitor suppressed amphetamine-induced ERM proteins phosphorylation in PC12 cells. These results suggest that PKCβ-induced DAT regulation may be involved in amphetmaine-induced ERM proteins phosphorylation.

Selective activation of specific PKC isoforms dictating the fate of CD14+ monocytes towards differentiation or apoptosis

In this study, phorbol-12-myristate-13-acetate (PMA) at low concentrations (<10 nM; L-PMA) induces the differentiation of CD14(+) monocytes into monocyte-derived macrophages (MDMs) while PMA at high concentrations (>100 nM; H-PMA) causes the apoptosis of these cells. The pre-treatment with Go6976 (a PKC-α/β(1) selective inhibitor), not anilinemonoindolylmaleimide [a PKC-β inhibitor (PKC-β inh.)], significantly (P < 0.05) reduces the L-PMA-induced generation of MDMs in the cultured CD14(+) monocytes. On the other hand, either of the above two PKC inhibitors is capable of suppressing the H-PMA-induced apoptosis of CD14(+) monocytes. However, only the inclusion of PKC-β inh., not Go6976, prevents the cells from serum deprivation-induced cell apoptosis. Although the membrane translocation of conventional PKC-α, β(1), and β(2) isoforms was observed in the H-PMA-treated CD14(+) monocytes, only PKC-β(2) exhibits a mitochondrial translocation activity among those PKCs responsive to H-PMA treatment. Moreover, the activation of DEVD-dependent caspases (DEVDase) was also detected in the H-PMA-treated CD14(+) monocytes, indicating the involvement of a caspase-dependent signaling pathway in the H-PMA-induced cell apoptosis of CD14(+) monocytes. Together with our previous findings that the selective activation of PKC-α or PKC-β(1) induces the differentiation of CD14(+) monocytes into MDMs or dendritic cells (MoDCs), respectively, the results in this study further demonstrate that PKC-β(2) activation is responsible for relaying the apoptotic signal to intrinsic mitochondria-dependent caspase signaling cascades in the CD14(+) monocytes. It is likely that the selective activation of specific PKC isoforms provides a new strategy to manipulate the differential cell fate commitment of multipotent CD14(+) monocytes towards apoptosis or differentiation into MDMs, MoDCs, and other cell types.

The PKCβ/HuR/VEGF pathway in diabetic retinopathy

We investigated whether the diabetes-related PKCβ activation affects VEGF expression through the mRNA-stabilizing human embryonic lethal abnormal vision (ELAV) protein, HuR, in the retina of streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in rats by STZ injection. Retinal tissues were processed to detect PKCβI, PKCβII, VEGF and HuR contents, as well as HuR phosphorylation. Immunoprecipitation coupled to RT-PCR was employed to evaluate HuR binding to VEGF mRNA in RiboNucleoProteic (RNP) complexes. Statistical analysis was performed by ANOVA followed by an appropriate post hoc comparison test. Following experimental diabetes PKCβI and PKCβII levels were increased compared to sham; there was also a PKC-mediated phosphorylation/activation of HuR. These effects were blunted by the in vivo co-administration of a selective PKCβ inhibitor. A specific binding between the HuR protein and the VEGF mRNA was also detected. The PKCβ/HuR activation was accompanied by enhanced VEGF protein expression that was, again, blunted by the PKCβ inhibitor. These findings first demonstrate the activation, in the retina, of the PKCβ/HuR/VEGF pathway following experimental diabetes and disclose a new potential pharmacological target to counteract pathologies implicating VEGF deregulation, such as diabetic retinopathy.

Protein Kinase C-β Gene Variants, Pathway Activation, and Enzastaurin Activity in Lung Cancer

BackgroundProtein kinase C-β2 (PKCβ2) is a splice-variant of the PRKCB1 gene and belongs to a family of serine/threonine-specific kinases that are predominantly activated by diacylglycerol, calcium, and phorbol ester. Cellular functions associated with PKCβ2 activation include transformation, proliferation, and inhibition of apoptosis. Enzastaurin (LY317615) is an oral, selective, potent inhibitor of the PKCβ2 kinase. Preclinical activity for this agent was predominantly reported in lymphoma, glioblastoma, and colorectal cancer. In patients with advanced non–small-cell lung cancer (NSCLC) whose previous therapy had failed, 13% of patients had disease control for 6 months with single-agent therapy. Patients and MethodsWe investigated whether biologically relevant variants of PRKCB1 exist in lung cancer cell lines in the context of enzastaurin-induced proliferation and kinase inhibition, using exon sequencing, immunoblotting, and cytotoxicity assays in NSCLC and small-cell lung cancer (SCLC) cell lines. ResultsWe discovered a total of 6 single-nucleotide variants, but only 1 resulted in an amino acid substitution (T40I). This substitution was not located in the kinase domain of PKCβ2 and did not affect enzastaurin's antiproliferative or phosphorylation-inhibitory activity. We found enzastaurin to be equally active in NSCLC and SCLC cell lines, with values of the 50% inhibitory concentration in a range of 0.05-0.2 μM. ConclusionThe inhibition of phosphorylation of PKCβ2 and the downstream molecules glycogen synthase kinase-3β, S6RP, Akt, and forkhead transcription factor was evident in the same concentration range, which suggests the premise that the determination of phosphorylation levels of these molecules in human tissue specimens may be a useful pharmacodynamic parameter for in vivo target inhibition by enzastaurin.

Ca2+ influx and protein scaffolding via TRPC3 sustain PKCβ and ERK activation in B cells

Ca(2+) signaling mediated by phospholipase C that produces inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] and diacylglycerol (DAG) controls lymphocyte activation. In contrast to store-operated Ca(2+) entry activated by Ins(1,4,5)P(3)-induced Ca(2+) release from endoplasmic reticulum, the importance of DAG-activated Ca(2+) entry remains elusive. Here, we describe the physiological role of DAG-activated Ca(2+) entry channels in B-cell receptor (BCR) signaling. In avian DT40 B cells, deficiency of transient receptor potential TRPC3 at the plasma membrane (PM) impaired DAG-activated cation currents and, upon BCR stimulation, the sustained translocation to the PM of protein kinase Cbeta (PKCbeta) that activated extracellular signal-regulated kinase (ERK). Notably, TRPC3 showed direct association with PKCbeta that maintained localization of PKCbeta at the PM. Thus, TRPC3 functions as both a Ca(2+)-permeable channel and a protein scaffold at the PM for downstream PKCbeta activation in B cells.

Weight gain related to treatment with atypical antipsychotics is due to activation of PKC-β

Atypical antipsychotics (APDs) are currently used in clinical practice for a variety of mental disorders such as schizophrenia, bipolar disorder and severe behavioral disturbances. A well-known disadvantage of using these compounds is a propensity for weight gain, resulting frequently in obesity. The mechanisms underlying pharmacologically induced weight gain are still controversial. The objective of this study was to evaluate in vitro the effects of different APDs on adipogenic events in cultured human pre-adipocytes and in rat muscle-derived stem cells (MDSCs), aiming to identify a common intracellular event contributable to these drugs. Culture behavior was evaluated in terms of cell proliferation, lipid accumulation, gene expression and morphological features. Results indicate that APDs influence adipogenic events through changes in the differentiation and proliferation of pre-adipocytes and MDSCs that are brought on by protein kinase C-β (PKC-β) activation. These data identify a signaling route that could be a potential target of pharmacological approaches for preventing the weight gain associated with APD treatment.