The purpose of this study was to investigate the potential roles of protein kinase C beta (PRKCB) in the pathogenesis of Alzheimer's disease (AD). We identified 2,254 differentially expressed genes from 19,245 background genes in AD versus control as well as PRKCB-low versus high group. Five co-expression modules were constructed by weight gene correlation network analysis. Among them, the 1,222 genes of the turquoise module had the strongest relation to AD and those with low PRKCB expression, which were enriched in apoptosis, axon guidance, gap junction, Fc gamma receptor (FcγR)-mediated phagocytosis, mitogen-activated protein kinase (MAPK) and vascular endothelial growth factor (VEGF) signaling pathways. The intersection pathways of PRKCB in AD were determined, including gap junction, FcγR-mediated phagocytosis, MAPK and VEGF signaling pathways. Based on the performance evaluation of the area under the curve of 75.3%, PRKCB could accurately predict the onset of AD. Therefore, low expressions of PRKCB was a potential causative factor of AD, which might be involved in gap junction, FcγR-mediated phagocytosis, MAPK and VEGF signaling pathways.

Protein kinase C beta (PKCβ) expression in breast cancer is associated with a more aggressive tumor phenotype, yet the mechanism for how PKCβ is pro-tumorigenic in this disease is still unclear. Interestingly, while it is known that PKCβ mediates angiogenesis, immunity, fibroblast function and adipogenesis, all components of the mammary tumor microenvironment (TME), no study to date has investigated whether stromal PKCβ is functionally relevant in breast cancer. Herein, we evaluate mouse mammary tumor virus-polyoma middle T-antigen (MMTV-PyMT) induced mammary tumorigenesis in the presence and absence of PKCβ. We utilize two model systems: one where PKCβ is deleted in both the epithelial and stromal compartments to test the global requirement for PKCβ on tumor formation, and second, where PKCβ is deleted only in the stromal compartment to test its role in the TME. MMTV-PyMT mice globally lacking PKCβ live longer and develop smaller tumors with decreased proliferation and decreased macrophage infiltration. Similarly, when PKCβ is null exclusively in the stroma, PyMT-driven B6 cells form smaller tumors with diminished collagen deposition. These experiments reveal for the first time a tumor promoting role for stromal PKCβ in MMTV-PyMT tumorigenesis. In corroboration with these results, PKCβ mRNA (Prkcb) is increased in fibroblasts isolated from MMTV-PyMT tumors. These data were confirmed in a breast cancer patient cohort. Combined these data suggest the continued investigation of PKCβ in the mammary TME is necessary to elucidate how to effectively target this signaling pathway in breast cancer.

Accumulating evidence supports the early involvement of monocyte/macrophage recruitment to activated endothelial cells by leukocyte adhesion molecules during atherogenesis. CD40 and its ligand CD40L are highly expressed in vascular endothelial cells, but its impact on monocyte adhesion and the related molecular mechanisms are not fully understood. The present study was designed to evaluate the direct effect of CD40L on monocytic cell adhesion and gain mechanistic insight into the signaling coupling CD40L function to the proinflammatory response. Exposure of cultured human aortic endothelial cells (HAECs) to clinically relevant concentrations of CD40L (20 to 80 ng/mL) dose-dependently increased human monocytic THP-1 cells to adhere to them under static condition. CD40L treatment induced the expression of vascular cell adhesion molecule-1 (VCAM-1) mRNA and protein expression in HAECs. Furthermore, exposure of HAECs to CD40L robustly increased the activation of protein kinase C beta (PKCβ) in ECs. A selective inhibitor of PKCβ prevented the rise in VCAM-1 and THP-1 cell adhesion to ECs. Moreover, stimulation of ECs to CD40L induced nuclear factor-κB (NF-κB) activation. PKCβ inhibition abolished CD40L-induced NF-κB activation, and NF-κB inhibition reduced expression of VCAM-1, each resulting in reduced THP-1 cell adhesion. Our findings provide the evidence that CD40L increases VCAM-1 expression in ECs by activating PKCβ and NF-κB, suggesting a novel mechanism for EC activation. Finally, administration of CD40L resulted in PKCβ activation, increased VCAM-1 expression and activated monocytes adhesiveness to HAECs, processes attenuated by PKCβ inhibitor. Therefore, CD40L may contribute directly to atherogenesis by activating ECs and recruiting monocytes to them.

Protein kinase C (PKC) influences myocardial contractility and susceptibility to long-term cardiac dysfunction after ischemia-reperfusion injury. In diabetes, PKC inhibition has a protective effect in terms of microvascular dysfunction. SK-channel dysfunction also influences endothelial dysfunction in cardioplegic hypoxia-reoxygenation (CP-H/R). Here, we examine whether acute inhibition of PKC beta protects against CP-H/R-induced coronary endothelial and SK channel dysfunction.

The transmembrane protein amyloid-beta precursor protein (APP) and the vesicle-associated protein c-Jun NH(2)-terminal kinase-interacting protein-1 (JIP-1) are transported into axons by kinesin-1. Both proteins may bind to kinesin-1 directly and can be transported separately. Because JIP-1 and APP can interact, kinesin-1 may recruit them as a complex, enabling their cotransport. In this study, we tested whether APP and JIP-1 are transported together or separately on different vesicles. We found that, within the cellular context, JIP-1 preferentially interacts with Thr(668)-phosphorylated APP (pAPP), compared with nonphosphorylated APP. In neurons, JIP-1 colocalizes with vesicles containing pAPP and is excluded from those containing nonphosphorylated APP. The accumulation of JIP-1 and pAPP in neurites requires kinesin-1, and the expression of a phosphomimetic APP mutant increases JIP-1 transport. Down-regulation of JIP-1 by small interfering RNA specifically impairs transport of pAPP, with no effect on the trafficking of nonphosphorylated APP. These results indicate that the phosphorylation of APP regulates the formation of a pAPP-JIP-1 complex that accumulates in neurites independent of nonphosphorylated APP.

Endoxifen, a secondary tamoxifen metabolite, is a potent antiestrogen exhibiting estrogen receptor alpha (ERα) binding at nanomolar concentrations. Phase I/II clinical trials identified clinical activity of Z-endoxifen (ENDX), in endocrine-refractory metastatic breast cancer as well as ERα+ solid tumors, raising the possibility that ENDX may have a second, ERα-independent, mechanism of action. An unbiased mass spectrometry approach revealed that ENDX concentrations achieved clinically with direct ENDX administration (5 µM), but not low concentrations observed during tamoxifen treatment (<0.1 µM), profoundly altered the phosphoproteome of the aromatase expressing MCF7AC1 cells with limited impact on the total proteome. Computational analysis revealed protein kinase C beta (PKCβ) and protein kinase B alpha or AKT1 as potential kinases responsible for mediating ENDX effects on protein phosphorylation. ENDX more potently inhibited PKCβ1 kinase activity compared to other PKC isoforms, and ENDX binding to PKCβ1 was confirmed using Surface Plasma Resonance. Under conditions that activated PKC/AKT signaling, ENDX induced PKCβ1 degradation, attenuated PKCβ1-activated AKTSer473 phosphorylation, diminished AKT substrate phosphorylation, and induced apoptosis. ENDX's effects on AKT were phenocopied by siRNA-mediated PKCβ1 knockdown or treatment with the pan-AKT inhibitor, MK-2206, while overexpression of constitutively active AKT diminished ENDX-induced apoptosis. These findings, which identify PKCβ1 as an ENDX target, indicate that PKCβ1/ENDX interactions suppress AKT signaling and induce apoptosis in breast cancer.

Despite extensive efforts, cancer therapies directed at the Protein Kinase C (PKC) family of serine/threonine kinases have failed in clinical trials. These therapies have been directed at inhibiting PKC and have, in some cases, worsened disease outcome. Here we examine colon cancer patients and show not only that PKC Beta II is a tumour suppressor, but patients with low levels of this isozyme have significantly decreased disease free survival. Specifically, analysis of gene expression levels of all PKC genes in matched normal and cancer tissue samples from colon cancer patients revealed a striking down-regulation of the gene coding PKC Beta in the cancer tissue (n = 21). Tissue microarray analysis revealed a dramatic down-regulation of PKC Beta II protein levels in both the epithelial and stromal diseased tissue (n = 166). Of clinical significance, low levels of the protein in the normal tissue of patients is associated with a low (10%) 10 year survival compared with a much higher (60%) survival in patients with relatively high levels of the protein. Consistent with PKC Beta II levels protecting against colon cancer, overexpression of PKC Beta II in colon cancer cell lines reveals that PKC Beta II reverses transformation in cell based assays. Further to this, activation of PKC Beta II results in a dramatic downregulation of IGF-I-induced AKT, indicating a role for PKCs in regulating IGF-1 mediated cell survival. Thus, PKC Beta II is a tumour suppressor in colon cancer and low levels serve as a predictor for poor survival outcome.

Activation of the nuclear factor (NF)-kappaB transcription complex by signals derived from the surface expressed B cell antigen receptor controls B cell development, survival, and antigenic responses. Activation of NF-kappaB is critically dependent on serine phosphorylation of the IkappaB protein by the multi-component IkappaB kinase (IKK) containing two catalytic subunits (IKKalpha and IKKbeta) and one regulatory subunit (IKKgamma). Using mice deficient for protein kinase C beta (PKCbeta) we show an essential role of PKCbeta in the phosphorylation of IKKalpha and the subsequent activation of NF-kappaB in B cells. Defective IKKalpha phosphorylation correlates with impaired B cell antigen receptor-mediated induction of the pro-survival protein Bcl-xL. Lack of IKKalpha phosphorylation and defective NF-kappaB induction in the absence of PKCbeta explains the similarity in immunodeficiencies caused by PKCbeta or IKKalpha ablation in B cells. Furthermore, the well established functional cooperation between the protein tyrosine kinase Bruton's tyrosine kinase (Btk), which regulates the activity of NF-kappaB and PKCbeta, suggests PKCbeta as a likely serine/threonine kinase component of the Btk-dependent NF-kappaB activating signal transduction chain downstream of the BCR.

Previous in vitro studies demonstrated that aldosterone rapidly activates sodium-hydrogen exchangers 1 and 3 (NHE 1 and 3). In vitro investigations revealed that protein kinase C (PKC) regulates NHE properties. We previously demonstrated that aldosterone rapidly enhances PKCα protein abundance in the rat kidney. There are no reports of renal PKCβ (I and II) protein levels related to the regulation by aldosterone. There are also no in vivo data regarding the rapid effects of aldosterone on renal protein levels of NHE (1 and 3) and PKCβ (I and II), simultaneously. In the current study, rats received normal saline solution or aldosterone (150 μg/kg BW, i.p.). After 30 minutes, abundance and immunoreactivity of these proteins were determined by Western blot analysis and immunohistochemistry, respectively. Aldosterone increased NHE1 and NHE3 protein abundance to 152% and 134%, respectively (P < 0.05). PKCβI protein level was enhanced by 30%, whereas PKCβII declined slightly. Aldosterone increased NHE protein expression mostly in the medulla. PKCβI immunostaining intensity was increased in the glomeruli, renal vasculature, and thin limb of the loop of Henle, while PKCβII was reduced. This is the first in vivo study to simultaneously demonstrate that aldosterone rapidly elevates PKCβI and NHE (1 and 3) protein abundance in the rat kidney. Aldosterone-induced NHE (1 and 3) protein levels may be related to PKCβI activation.

Activation of protein kinase C (PKC) is closely linked with endothelial dysfunction. However, the effect of PKCβII on endothelial dysfunction has not been characterized in cultured endothelial cells. Here, using adenoviral PKCβII gene transfer and pharmacological inhibitors, the role of PKCβII on endothelial dysfucntion was investigated in cultured endothelial cells. Phorbol 12-myristate 13-acetate (PMA) increased reactive oxygen species (ROS), p66shc phosphorylation, intracellular adhesion molecule-1, and monocyte adhesion, which were inhibited by PKCβi (10 nM), a selective inhibitor of PKCβII. PMA increased the phosphorylation of CREB and manganese superoxide dismutase (MnSOD), which were also inhibited by PKCβi. Gene silencing of CREB inhibited PMA-induced MnSOD expression, suggesting that CREB plays a key role in MnSOD expression. Gene silencing of PKCβII inhibited PMA-induced mitochondrial ROS, MnSOD, and ICAM-1 expression. In contrast, overexpression of PKCβII using adenoviral PKCβII increased mitochondrial ROS, MnSOD, ICAM-1, and p66shc phosphorylation in cultured endothelial cells. Finally, PKCβII-induced ICAM-1 expression was inhibited by Mito-TEMPO, a mitochondrial ROS scavenger, suggesting the involvement of mitochondrial ROS in PKC-induced vascular inflammation. Taken together, the results suggest that PKCβII plays an important role in PMA-induced endothelial dysfunction, and that the inhibition of PKCβII-dependent p66shc signaling acts as a therapeutic target for vascular inflammatory diseases.

1. Resveratrol, an active ingredient of red wine extracts, has been shown to exhibit neuroprotective effects in several experimental models. 2. The present study evaluated the neuroprotective effects of resveratrol against amyloid beta(Abeta)-induced toxicity in cultured rat hippocampal cells and examined the role of the protein kinase C (PKC) pathway in this effect. 3. Pre-, co- and post-treatment with resveratrol significantly attenuated Abeta-induced cell death in a concentration-dependent manner, with a concentration of 25 microm being maximally effective. 4. Pretreatment (1 h) of hippocampal cells with phorbol-12-myristate-13-acetate, a PKC activator, at increasing concentrations (1-100 ng x ml(-1)), resulted in a dose-dependent reduction in Abeta-induced toxicity, whereas the inactive 4alpha-phorbol had no effect. 5. Pretreatment (30 min) of hippocampal cells with GF 109203X (1 microm), a general PKC inhibitor, significantly attenuated the neuroprotective effect of resveratrol against Abeta-induced cell death. 6. Treatment of hippocampal cells with resveratrol (20 microm) also induced the phosphorylation of various isoforms of PKC leading to activation. 7. Taken together, the present results indicate that PKC is involved in the neuroprotective action of resveratrol against Abeta-induced toxicity.

Both mechanistic features and recent correlative findings suggest a potential role for protein kinase C-beta (PKC-beta) in tumor pathogenesis, particularly in B-cell malignancies. To evaluate the role of this gene in lymphoid malignancies, we analyzed global gene expression data to quantify PKC-beta expression across diagnostic groups and, when possible, determined correlations between PKC-beta expression and survival.

Genomic integrity is preserved by the action of protein complexes that control DNA homeostasis. These include the sliding clamps, trimeric protein rings that are arranged around DNA by clamp loaders. Replication factor C (RFC) is the clamp loader for proliferating cell nuclear antigen, which acts on DNA replication. Other processes that require mobile contact of proteins with DNA use alternative RFC complexes that exchange RFC1 for CTF18 or RAD17. Phosphoinositide 3-kinases (PI3K) are lipid kinases that generate 3-poly-phosphorylated-phosphoinositides at the plasma membrane following receptor stimulation. The two ubiquitous isoforms, PI3Kalpha and PI3Kbeta, have been extensively studied due to their involvement in cancer and nuclear PI3Kbeta has been found to regulate DNA replication and repair, processes controlled by molecular clamps. We studied here whether PI3Kbeta directly controls the process of molecular clamps loading. We show that PI3Kbeta associated with RFC1 and RFC1-like subunits. Only when in complex with PI3Kbeta, RFC1 bound to Ran GTPase and localized to the nucleus, suggesting that PI3Kbeta regulates RFC1 nuclear import. PI3Kbeta controlled not only RFC1- and RFC-RAD17 complexes, but also RFC-CTF18, in turn affecting CTF18-mediated chromatid cohesion. PI3Kbeta thus has a general function in genomic stability by controlling the localization and function of RFC complexes.

The choroid plexus (CP), constituting the blood-cerebrospinal fluid barrier, has the capacity to remove beta-amyloid (Abeta) from the cerebrospinal fluid. Our previous work indicates that exposure to lead (Pb) results in Abeta accumulation in the CP by decreasing the expression of low density lipoprotein receptor protein-1 (LRP1), a protein involved in the transport and clearance of Abeta. The current study was designed to explore the relationship between Abeta accumulation, protein kinase C (PKC) activity, and LRP1 status in the CP following Pb exposure. Confocal microscopy revealed that LRP1 was primarily localized in the cytosol of the CP in control rats and migrated distinctly towards the apical surface and the microvilli following acute Pb exposure (27 mg Pb/kg, i.p., 24h). Co-immunostaining revealed a co-localization of both PKC-delta and LRP1 in the cytosol of control rats, with a distinct relocalization of both towards the apical membrane following Pb exposure. Preincubation of the tissues with PKC-delta inhibitor rottlerin (2 microM) prior to Pb exposure in vitro, resulted in abolishing the Pb-induced relocalization of LRP1 to the apical surface. Importantly, a significant elevation in intracellular Abeta levels (p<0.01) was observed in the cytosol of the CP following Pb exposure, which was abolished following preincubation with rottlerin. In addition, rottlerin caused a relocalization of Abeta from the cytosol to the nucleus in both Pb-treated and control CP tissues. Finally, co-immunoprecipitation studies revealed a strong protein-protein interaction between LRP1 and PKC-delta in the CP. These studies suggest that Pb exposure disrupts Abeta homeostasis at the CP, owing partly to a Pb-induced relocalization of LRP1 via PKC-delta.

Excessive activation of glutamate receptors in spinal dorsal horn neurons is a key mechanism leading to abnormal neuronal activation in pathological pain conditions. Previous studies have shown that activation of glutamate receptors in the spinal dorsal horn is enhanced by impaired glial glutamate transporter functions and proinflammatory cytokines including interleukin-1 beta (IL-1β). In this study, we for the first time revealed that spinal glial glutamate transporter activities in the neuropathic animals are attenuated by endogenous IL-1β. Specifically, we demonstrated that nerve injury results in an increased expression of IL-1β and activation of PKC in the spinal dorsal horn as well as suppression of glial glutamate uptake activities. We provided evidence that the nerve-injury induced suppression of glial glutamate uptake is at least in part ascribed to endogenous IL-1β and activation of PKC in the spinal dorsal horn. IL-1β reduces glial glutamate transporter activities through enhancing the endocytosis of both GLT-1 and GLAST glial glutamate transporters. The IL-1β induced trafficking of glial glutamate transporters is through the calcium/PKC signaling pathway, and the dynamin-dependent endocytosis, which is dependent on the integrity of actin filaments. The signaling pathway regulating glial glutamate transporters revealed in this study provides novel targets to attenuate aberrant activation of glutamate receptors in the spinal dorsal horn, which could ultimately help the development of analgesics.

Human immunodeficiency virus (HIV)-1 Tat is a multifunctional protein involved in viral replication, inflammation and apoptosis. Tat activates phospholipase C-beta (PLC-beta), presumably via a pertussis toxin (PTX) sensitive G(i) protein, which is critical for neuronal apoptosis. In this study, we show that Tat-mediated intracellular Ca(2+) release in rat pheochromocytoma (PC-12) cells and rat primary cortical neuronal cultures was abrogated by pretreatment with either pertussis toxin and/or its B-oligomer subunit (PTX-B), devoid of ADP ribosyltransferase activity. PTX-B pretreatment also inhibited intracellular Ca(2+) release by bradykinin and 2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl) benzenesulfonamide (m-3M3FBS), a director activator of phospholipase C. Activation of protein kinase C (PKC) by phorbol 12,13-dibutyrate (PdBu) mimicked the PTX-B-mediated inhibition of m-3M3FBS-stimulated intracellular Ca(2+) increase, while inhibition of PKC by bisindolylmaleimide I hydrochloride (BIM) reversed the inhibitory action of PTX-B. Functionally, PTX-B reduced Tat-induced Bax and caspase-3 proteins and reduced cell apoptosis. We conclude that PTX inhibition of Tat-mediated intracellular Ca(2+) release is independent of ADP ribosylation of the G(i) protein via the A protomer, but mediated by the B-oligomer. Furthermore, PTX-B suppresses HIV-1 Tat-mediated apoptosis by reducing its activation of PLC-beta through a PKC activation pathway.

Medulloblastoma (MB) is the most common and aggressive pediatric intracranial tumor. Estrogen receptor β (ERβ) expression correlates with MB development and its phosphorylation modifies its transcriptional activity in a ligand-dependent or independent manner. Using in silico tools, we have identified several residues in ERβ protein as potential targets of protein kinases C (PKCs) α and δ. Using Daoy cells, we observed that PKCα and PKCδ associate with ERβ and induce its phosphorylation. The activation of ERβ promotes MB cells proliferation and invasion, and PKCs downregulation dysregulates these steroid receptor mediated processes. Our data suggest that these kinases may play a crucial role in the regulation of the ERβ transcriptional activity. Overexpression of both PKCα and PKCδ in MB biopsies samples supports their relevance in MB progression.

Alzheimer's disease (AD) is a neurodegenerative disorder with prominent individual morbidity and mortality among elderly people. Germacrone (Germ) has been reported to exert dominant protective roles in multiple human diseases, and neurological diseases are also included. The intention of this paper is to determine the impacts of Germ on okadaic acid (OA)-treated PC12 cells and confirm the hidden regulatory mechanism. First, PC12 cells were induced by OA in the absence or presence of Germ. Cell counting kit-8 assay was to monitor cell proliferation. Western blot was to test the protein levels of cholinergic muscarinic M1 receptor (CHRM1), Galphaq (Gq), phospholipase C beta (PLCβ) and protein kinase C (PKC). The levels of reactive oxygen species (ROS) and other oxidative stress markers were evaluated using corresponding kits. ELISA was used to estimate the levels of AD markers. RT-qPCR was used to examine the mRNA levels of beta-site amyloid-precursor-protein-cleaving enzyme 1 (BACE-1) and apolipoprotein E (APOE). The results uncovered that Germ enhanced the proliferation of OA-insulted PC12 cells, elevated the protein level of CHRM1 and activated the Gq/PLCβ/PKC signaling. Moreover, after OA-induced PC12 cells were administered with Germ, insufficiency of CHRM1 impeded cell proliferation, enhanced oxidative stress and neuron injury and inactivated the Gq/PLCβ/PKC signaling. Furthermore, the addition of Gq inhibitor UBO-QIC, PLCβ inhibitor U73122 or PKC inhibitor Go6983 reversed the enhanced proliferation, the reduced oxidative stress and neuron injury in OA-treated PC12 cells caused by Germ. Collectively, Germ modulated M1 muscarinic receptor-mediated Gq/PLCβ/PKC signaling, thereby alleviating OA-induced PC12 cell injury.

Impaired cardiovascular function in diabetes is partially attributed to pathological overexpression of inducible nitric oxide synthase (iNOS) in cardiovascular tissues. We examined whether the hyperglycemia-induced increased expression of iNOS is protein kinase C-beta(2) (PKCbeta(2)) dependent and whether selective inhibition of PKCbeta reduces iNOS expression and corrects abnormal hemodynamic function in streptozotocin (STZ)-induced diabetic rats.

Induction of protein kinase C (PKC) pathway in the vascular tissues by hyperglycemia has been associated with many of the cellular changes observed in the complications of diabetes. Recently, we have reported that the use of a novel, orally effective specific inhibitor of PKC beta isoform (LY333531) normalized many of the early retinal and renal hemodynamics in rat models of diabetes. In the present study, we have characterized a spectrum of biochemical and molecular abnormalities associated with chronic changes induced by glucose or diabetes in the cultured mesangial cells and renal glomeruli that can be prevented by LY333531. Hyperglycemia increased diacylglycerol (DAG) level in cultured mesangial cells exposed to high concentrations of glucose and activated PKC alpha and beta1 isoforms in the renal glomeruli of diabetic rats. The addition of PKC beta selective inhibitor (LY333531) to cultured mesangial cells inhibited activated PKC activities by high glucose without lowering DAG levels and LY333531 given orally in diabetic rats specifically inhibited the activation of PKC beta1 isoform without decreasing PKC alpha isoform activation. Glucose-induced increases in arachidonic acid release, prostaglandin E2 production, and inhibition of Na+-K+ ATPase activities in the cultured mesangial cells were completely prevented by the addition of LY333531. Oral feeding of LY333531 prevented the increased mRNA expression of TGF-beta1 and extracellular matrix components such as fibronectin and alpha1(IV) collagen in the glomeruli of diabetic rats in parallel with inhibition of glomerular PKC activity. These results suggest that the activation of PKC, predominately the beta isoform by hyperglycemia in the mesangial cells and glomeruli can partly contribute to early renal dysfunctions by alteration of prostaglandin production and Na+-K+ ATPase activity as well as the chronic pathological changes by the overexpression of TGF-beta1 and extracellular matrix components genes.