Activation Of Protein Kinase C Beta Research Articles

Mechanistic understanding of dendritic cell activation in skin sensitization: additional evidences to support potency classification

Allergic contact dermatitis (ACD) is an important occupational and environmental disease caused by topical exposure to chemical allergens. In the EU, it has been calculated that 4 % of animals are used in toxicity test for the assessment of skin sensitization (Peiser et al., 2012). To come a complete replacement of animals, evaluation of relative skin sensitization potency is necessary. The identification of mechanisms influencing allergen potency requires a better understanding of molecular events that trigger cell activation. Therefore, (i) the effects of selected allergens on surface markers expression and cytokines release in contact allergen-induced cell activation were assessed, and (ii) the role of Protein Kinase C (PKC) beta activation in contact allergen-induced cell activation was investigated. The human pro-myelocytic cell line THP-1 was used as experimental model surrogate of dendritic cells. Cells were exposed to select contact allergens of different potency and cell surface marker expression (CD80, CD86, HLA-DR) was determined by flow cytometry analysis. Cytokines production (IL-6, IL-8, IL-10, IL-12p40, IL-18) was evaluated with specific sandwich ELISA. The effective contribution of PKC beta in chemical allergen-induced cell activation was assessed by Western Blot analysis (PKC beta activation) and using a specific PKC beta inhibitor (PKC beta pseudosubstrate). In addition, to investigate if contact allergens are able to induce indeed dendritic cells (DCs) maturation, THP-1 cells were differentiated to immature DC and then exposed to contact allergen of different potency. Overall, our finding provides insights into the process of sensitization and strength of cell activation associated with allergens of different potency. Results obtained suggest that contact allergens of different potency are able to induce a different degree of activation of dendritic cells maturation involved in the process of ACD.

Abstract 1888: Protein kinase C beta II suppresses colorectal cancer by regulating IGF1 mediated cell survival

Abstract 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 in an IGF-1 dependent manor showed a downregulation of 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. Citation Format: Catriona M. Dowling, James Phelan, Julia A. Callender, Mary Clare Cathcart, Brian Mehigan, Paul McCormick, Tara Dalton, John C. Coffey, Alexandra C. Newton, Jacintha O’Sullivan, Patrick A. Kiely. Protein kinase C beta II suppresses colorectal cancer by regulating IGF1 mediated cell survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1888.

Protein kinase C beta II suppresses colorectal cancer by regulating IGF-1 mediated cell survival.

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.

P47phox contributes to albuminuria and kidney fibrosis in mice

Reactive oxygen species (ROS) play an important pathogenic role in the development of many diseases, including kidney disease. Major ROS generators in the glomerulus of the kidney are the p47phox-containing NAPDH oxidases NOX1 and NOX2. The cytosolic p47phox subunit is a key regulator of the assembly and function of NOX1 and NOX2 and its expression and phosphorylation are up-regulated in the course of renal injury, and have been shown to exacerbate diabetic nephropathy. However, its role in non-diabetic-mediated glomerular injury is unclear. To address this, we subjected p47phox-null mice to either adriamycin-mediated or partial renal ablation-mediated glomerular injury. Deletion of p47phox protected the mice from albuminuria and glomerulosclerosis in both injury models. Integrin α1-null mice develop more severe glomerulosclerosis than wild type mice in response to glomerular injury mainly due to increased production of ROS. Interestingly, the protective effects of p47phox knockout were more profound in p47phox/integrin α1 double knockout mice. In vitro analysis of primary mesangial cells showed that deletion of p47phox led to reduced basal levels of superoxide and collagen IV production. Thus, p47phox-dependent NADPH oxidases are a major glomerular source of ROS, contribute to kidney injury, and are potential targets for antioxidant therapy in fibrotic disease.

BMS-214662 Eliminates Quiescent and Proliferating Acute Myeloid Leukemia Cells through Activation of Protein Kinase Cβ and Enhances the Efficacy of Cytosine Arabinoside

AbstractAbstract 2167Despite major advances in our understanding of the pathophysiology of acute myeloid leukemia (AML), the prognosis remains poor in most patients. While the majority of patients (∼80%) who tolerate induction therapy enter remission, both early and late relapses are a significant problem. It is postulated that, in some cases, these relapses are due to a population of primitive AML stem cells which are not effectively targeted by conventional chemotherapy agents such as cytosine arabinoside (Ara-C) or standard chemotherapy combinations (e.g. DA, ADE or FLAG-IDA). BMS-214662 (Bristol-Myers Squibb), a cytotoxic farnesyltransferase inhibitor (FTI), preferentially kills non-dividing malignant cells (Lee et al, Proc AACR 2001;42:260S) and in a clinical trial as a single agent had anti-leukemia activity in AML (Cortes et al, J Clin Oncology 2005;23:2805). Previous studies have shown that, in chronic myeloid leukemia (CML), BMS-214662 induces mitochondrial apoptosis of primitive progenitor cells via activation of protein kinase Cbeta (PKCβ; Copland et al, Blood 2008;111:2843; Pellicano et al, Blood 2009;114:4186). We therefore assessed the efficacy of BMS-214662, alone and in combination with Ara-C, in patient-derived CD34+ and CD34- AML cells in vitro using a CFSE-based flow cytometry method to track cell division and caspase-3 activity, PARP cleavage, mitochondrial membrane potential (MMP) and measurement of reactive oxygen species (ROS) to assess apoptosis. PKCβ was assessed by Western blotting ± BMS-214662, ± bryostatin-1, a PKCβ modulator. Primary AML cells were cultured for 72 hours in serum free medium supplemented with 5 growth factors (IL-3, IL-6, Flt-3 ligand, G-CSF and SCF). Conditions studied were: (1) no drug control (NDC), (2) BMS-214662 (250nM; IC50 dose), (3) Ara-C 500nM, and (4) BMS-214662 250nm + Ara-C 500nM (BMS-214662+Ara-C). Total viable cells were reduced in all treatment arms compared to NDC. The combination of BMS-214662+Ara-C produced the greatest cytotoxic effect with total viable cells reduced to <20% of NDC after 72 hours (P=0.05). While Ara-C alone did not reduce the number of undivided CFSEmax AML cells compared to NDC, both BMS-214662 alone and BMS-214662+Ara-C resulted in reductions of 47 and 75%, respectively. Furthermore, BMS-214662+Ara-C significantly reduced undivided CFSEmax AML cells compared to either BMS-214662 or Ara-C alone, (P<0.04 and P<0.03, respectively), indicating at least additive activity of this combination. In primary AML cells, BMS-214662 triggered apoptosis via the intrinsic pathway with loss of MMP, generation of ROS, cleavage of PARP and caspase-3 activation. Further experiments demonstrated that PKCβ was up-regulated in a dose-dependent manner in the presence of BMS-214662. Importantly, BMS-214662 at the concentrations effective against AML CD34+ cells did not initiate apoptosis of normal CD34+ hematopoietic cells. Finally, the addition of the PKCβ modulator, bryostatin-1, abrogated the effects of BMS-214662, resulting in reduced activation of caspase-3. These results are interesting as they identify a common mechanism for induction of apoptosis in quiescent CD34+ cells from different myeloid leukemias (all phases of CML and different AML subtypes) which may be unrelated to the FTI activity of this compound. In conclusion, these results indicate that BMS-214662 induces apoptosis of both quiescent and proliferating AML cells via up-regulation of PKCβ and activation of the intrinsic apoptotic pathway. The combination of BMS-214662 with conventional chemotherapy may represent a novel strategy for remission induction therapy in AML, reducing the risk of relapse. Disclosures:Holyoake: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Copland: Novartis Pharma: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Insulin Sensitivity is Related to Fat Oxidation and Protein Kinase C Activity in Children With Acute Burn Injury

Impaired fatty acid oxidation occurs with type 2 diabetes and is associated with accumulations of intracellular lipids, which may increase diacylglycerol (DAG), stimulate protein kinase C activity, and inactivate insulin signaling. Glucose and fat metabolism are altered in burn patients, but have never been related to intracellular lipids or insulin signaling. Thirty children sustaining >40% total body surface area burns were studied acutely with glucose and palmitate tracer infusions and a hyper-insulinemic euglycemic clamp. Muscle triglyceride, DAG, fatty acyl CoA, and insulin signaling were measured. Liver and muscle triglyceride levels were measured with magnetic resonance spectroscopy. Muscle samples from healthy children were controls for DAG concentrations. Insulin sensitivity was reduced and correlated with whole body palmitate beta-oxidation (P = .004). Muscle insulin signaling was not stimulated by hyper-insulinemia. Tissue triglyceride concentrations and activated protein kinase C-beta were elevated, whereas the concentration of DAG was similar to the controls. Free fatty acid profiles of muscle triglyceride did not match DAG. Insulin resistance following burn injury is accompanied by decreased insulin signaling and increased protein kinase C-beta activation. The best metabolic predictor of insulin resistance in burned patients was palmitate oxidation.

Etiology and effect on outcomes of hyperglycemia in hospitalized patients

The prevalence of diabetes in U.S. hospitals, etiology and pathophysiology of hyperglycemia in hospitalized patients, impact of hyperglycemia on patient outcomes, and benefits of hyperglycemia correction are described. Diabetes mellitus is a common and costly condition with an increasing prevalence in the U.S. Hyperglycemia is common among hospital patients with and without diabetes. Possible causes of hyperglycemia include illness-related metabolic stress, parenteral nutrition, and pharmacotherapy. The deposition of advanced glycosylation end products, capillary basement membrane thickening, impaired immune function, oxidative stress, impaired lipid metabolism, prothrombotic changes, activation of protein kinase C-beta, vascular leakage, capillary non perfusion, and induction of vascular endothelial growth factor are among the harmful effects of hyperglycemia. In various types of patients and hospital settings, hyperglycemia increases the mortality rate, risk of postoperative nosocomial infection, need for intensive care unit admission, length of hospital stay, and hospital charges. The benefits of using intensive therapy to correct hyperglycemia in hospitalized patients in reducing mortality and the risk of infections and other adverse outcomes are well documented. Efforts are needed to manage hyperglycemia in hospitalized patients with and without diabetes to minimize the morbidity, mortality, and costs associated with hyperglycemia.

RBCK1, a protein kinase C βI binding partner and its splice variant RBCK2: potential role in cardiac hypertrophy?

The protein kinase C (PKC) family participates in signal transduction in a large number of mammalian cell types. Several PKC isozymes are expressed in rat cardiac myocytes. Many studies have shown that different PKC isozymes are involved in the modulation of diverse functions in the heart, including the developmental and pathological hypertrophic growth of cardiomyocytes.We are focused on understanding the individual role of each PKC isozyme and how they regulate cardiac functions. Our laboratory and others have demonstrated that activation of PKC beta plays a critical role in the development of cardiac hypertrophy. Recently, the PKC-binding partner designated RBCK1 for RBCC protein interacting with PKC 1, has been shown to interact specifically with PKCβI when coexpressed in cultured COS-7 cells. In addition, two different studies have established a correlation between the variation of RBCK1 mRNA levels and cardiac hypertrophy. However, whether the product protein mediate hypertrophy or whether the changes in its levels are the result of hypertrophy cannot be determined from such an analysis. Based on these results, the goal of our present study is therefore to determine if RBCK1 participates actively in the processes leading to cardiac hypertrophy and characterize the molecular mechanisms involved. Does RBCK1 bind PKCβI in cardiac myocytes and is their interaction altered during the induction of hypertrophy? Is the level of expression of PKCβI regulated by the level of expression of RBCK1? A splice variant of RBCK1 named RBCK2 has been cloned and characterized. Does RBCK2 also participate in the development of cardiac hypertrophy? To investigate these questions, adenoviralmediated gene transfer of wild-type RBCK1 and RBCK2 fused to the green fluorescent protein (GFP) was carried out in cultured neonatal rat ventricular cardiac myocytes. By identifying regulators and binding proteins of PKC, we may identify new means to modulate the progression of pathological hypertrophy of the heart. In this study, we showed the preferential interaction of RBCK1 with inactive PKCβI in vitro and the perinuclear localization of endogenous PKCβI and overexpressed RBCK1 in unstimulated cells. This result suggests that RBCK1 could be a binding partner for inactive PKCβI in rat neonatal cardiac myocytes. We also found an increase in the level of expression of endogenous PKCβI and RBCK1 proportional to the time in culture and the induction of hypertrophy with phenylephrine (PE). The level of expression of PKCβI is directly correlated to the level of expression of RBCK1. An isoform specific motility shift of PKCβI was also seen upon the overexpression of RBCK1-GFP, which could reflect a dephosphorylation state. Finally, there was an Nesiritide Improves Urine Output in Severely Ill Pediatric Patients Awaiting Heart Transplantation Without Severe Hypotension Ruchir Sehra, Karen R. Underwood2—Pediatrics/Pediatric Cardiology, Loma Linda University, Loma Linda, CA; Critical Care Medicine, Chlidren’s Hospital of Los Angeles, Los Angeles, CA

Diabetic retinopathy and diabetic macular edema: pathophysiology, screening, and novel therapies.

Diabetic retinopathy (DR) and diabetic macular edema (DME) are leading causes of blindness in the working-age population of most developed countries. The increasing number of individuals with diabetes worldwide suggests that DR and DME will continue to be major contributors to vision loss and associated functional impairment for years to come. Early detection of retinopathy in individuals with diabetes is critical in preventing visual loss, but current methods of screening fail to identify a sizable number of high-risk patients. The control of diabetes-associated metabolic abnormalities (i.e., hyperglycemia, hyperlipidemia, and hypertension) is also important in preserving visual function because these conditions have been identified as risk factors for both the development and progression of DR/DME. The currently available interventions for DR/DME, laser photocoagulation and vitrectomy, only target advanced stages of disease. Several biochemical mechanisms, including protein kinase C-beta activation, increased vascular endothelial growth factor production, oxidative stress, and accumulation of intracellular sorbitol and advanced glycosylation end products, may contribute to the vascular disruptions that characterize DR/DME. The inhibition of these pathways holds the promise of intervention for DR at earlier non-sight-threatening stages. To implement new therapies effectively, more individuals will need to be screened for DR/DME at earlier stages-a process requiring both improved technology and interdisciplinary cooperation among physicians caring for patients with diabetes.

Signaling through HLA-DR induces PKC beta-dependent B cell death outside rafts.

Signals through HLA-DR molecules contribute to optimal activation of antigen-presenting cells (APC) during T cell/APC interactions participating in the generation of productive interactions, and to the induction of APC death, which has been postulated to play a role in the termination of the immune response. To understand how these molecules accommodate both cellular responses, we studied the not yet well-defined signaling events and the biochemical requirements for HLA-DR-mediated death. We demonstrate that in B cells the HLA-DR-activated protein kinase C (PKC) beta is required for HLA-DR-mediated death whereas the HLA-DR-activated Src family of PTK is redundant. In contrast to HLA-DR-mediated activation of Src kinase Lyn, the aggregation of HLA-DR molecules in lipid rafts is not required for HLA-DR-mediated PKC beta activation nor for the induction of cell death. Indeed, the bulk of HLA-DR-activated PKC beta reside outside rafts. This is the first report showing that HLA-DR-induced PKC beta activation is essential for the induction of B cell death via HLA-DR, and that these HLA-DR-mediated events do not require the integrity of rafts.

Obligatory role of protein kinase Cbeta and MARCKS in vesicular trafficking in living neurons.

Neurotransmitter release from neurons involves both vesicular trafficking and subsequent fusion of synaptic vesicles with the plasma membrane. The mechanisms involving the formation and fusion of vesicles that allow the exocytotic release of transmitters are understood well. Little is known, however, about the signaling mechanism involved in the trafficking of vesicles along the neurites. In this study, we used real-time confocal microscopy to search for evidence that vesicular trafficking in neurons requires the activation of protein kinase Cbeta (PKCbeta) and the myristoylated alanine-rich C kinase substrate (MARCKS) signaling pathway. Dopamine-beta-hydroxylase fused to green fluorescent protein has been used to trace vesicular movement. Angiotensin II, an established neuromodulatory hormone, stimulates translocation of green fluorescent protein-dopamine-beta-hydroxylase vesicles from the cell body to neurites. This translocation was blocked by an antisense oligonucleotide to PKCbeta and MARCKS. Stimulation of PKC by other means, such as phorbol-12-myristate-13-acetate or carbachol, also resulted in the redistribution of fluorescence in a manner similar to that observed for angiotensin II. These observations demonstrate that PKCbeta-MARCKS signaling may be a general mechanism for the stimulation of vesicular trafficking in brain neurons.

Role of protein kinase C isozymes in cellular functions and pathological conditions

Protein kinase C (PKC) is a superfamily of lipid-dependent protein Ser/Thr kinases consisting of at least 10 isozymes. The present article summarizes the papers presented at the congress symposium of the 74th Annual Meeting of the Japanese Pharmacological Society, in which six special topics regarding PKC isozyme-dependent cellular functions and pathological disorders were discussed. Using a GFP-tagged PKC expression technique, each PKC subtype was suggested to vary its targeting-site in each cell in response to each stimulus and that the targeting to the specific compartment is necessary for the specific cellular responses (NS). A cardioprotective agent, JTV519, was shown to attenuate post-ischemic myocardial injury by mimicking ischemic preconditioning through specific activation of PKC delta (YK). Using an antisense technique, PKC alpha and delta/epsilon were shown to be necessary for gene expression of inducible NO synthase by interleukin-1, one of the proinflammatory cytokines, by a stimulated transactivation of NF-kappa B (TH). In canine cerebral artery, PKC delta and PKC alpha play important roles in the development and the maintenance of vasospasm induced by subarachnoid hemorrhage, respectively (SN); and stretch-induced MLC20 phosphorylation involves MLCK and PKC alpha but not PKC delta activities facilitated by inactivation of myosin phosphatase through Rho activity (KO & KN). To clarify the role of PKC isozymes in insulin resistance, the effects of insulin on glucose uptake, PKC isozyme activation and PI3K activation in rat adipocytes were shown and then platelet PKC beta activation in diabetic patients with various diabetic complications, including diabetic retinopathy, was reported (TI). These studies will promisingly open the way to a new era for the development of novel drugs controlling an isozyme-specific activity of the protein kinase C superfamily and improvement in the knowledge about the role of the protein kinase in health and disease.

Implication of acyl chain of diacylglycerols in activation of different isoforms of protein kinase C.

We synthesized diacylglycerols (DAGs) containing omega-6 or omega-3 polyunsaturated fatty acids [i.e., 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG), 1-stearoyl-2-docosahexaenoyl-sn-glycerol (SDG), and 1-stearoyl-2-eicosapentaenoyl-sn-glycerol (SEG)] and assessed their efficiency on activation of conventional (alpha, beta I, gamma) and novel (epsilon, delta) protein kinase C (PKC). SAG exerted significantly higher stimulatory effects than SDG and SEG on activation of PKC alpha and PKC delta. Activation of PKC beta I by SEG and SDG was higher than that by SAG. Activation of PKC gamma did not differ significantly among DAG molecular species. Addition of SAG to assays containing SEG and SDG exerted additive effects on activation of alpha and epsilon, but not on beta I and gamma, isoforms of PKC. SDG- and SEG-induced activation of PKC delta was significantly curtailed by the addition of SAG. Three DAG species significantly curtailed the PMA-induced activation of beta Iota, gamma, and delta, but not of alpha and epsilon, isoforms of PKC. Our study demonstrates for the first time that in vitro activation of different PKC isoenzymes vary in response to different DAG species, and one can envisage that this differential regulation may be responsible for their in vivo effects on target organs.

The Protein-tyrosine-phosphatase SHP2 is phosphorylated on serine residues 576 and 591 by protein kinase C isoforms alpha, beta 1, beta 2, and eta.

To study whether protein kinase C (PKC) isoforms can interact with protein-tyrosine-phosphatases (PTPs) which are connected to the insulin signaling pathway, we co-overexpressed PKC isoforms together with insulin receptor, docking proteins, and the PTPs SHP1 and SHP2 in human embryonic kidney (HEK) 293 cells. After phorbol ester induced activation of PKC isoforms alpha, beta 1, beta 2, and eta, we could show a defined gel mobility shift of SHP2, indicating phosphorylation on serine/threonine residues. This phosphorylation was not dependent on insulin receptor or insulin receptor substrate-1 (IRS-1) overexpression and did not occur for the closely related phosphatase SHP1. Furthermore, PKC phosphorylation of SHP2 was completely blocked by the PKC inhibitor bisindolylmaleimide and was not detectable when SHP2 was co-overexpressed with kinase negative mutants of PKC beta 1 and -beta 2. The phosphorylation also occurred on endogenous SHP2 in Chinese hamster ovary (CHO) cells stably overexpressing PKC beta 2. Using point mutants of SHP2, we identified serine residues 576 and 591 as phosphorylation sites for PKC. However, no change of phosphatase activity by TPA treatment was detected in an in vitro assay. In summary, SHP2 is phosphorylated on serine residues 576 and 591 by PKC isoforms alpha, beta 1, beta 2, and eta.

A new look at the heart in diabetes mellitus: from ailing to failing.

This review discusses some of the mechanisms inherent in diabetes that predispose patients to increased cardiac morbidity and mortality. Single photon emission computerized tomography or photon emission tomography with radioactive labeled analogues of norepinephrine have shown that cardiac sympathetic dysfunction and incompetence are early and also late abnormalities in patients with Type I (insulin-dependent) and Type II (non-insulin-dependent) diabetes mellitus. Furthermore, myocardial blood flow assessment with photon emission tomography has shown that in patients without myocardial perfusion deficits, endothelial-dependent vasodilatation is severely reduced in relation to cardiac sympathetic dysfunction. In addition, signs of endothelial activation have also been found early in patients with Type I and Type II diabetes in whom vascular disease has not been clinically detected. This activation in conjunction with glycaemic control is important in determining macrovascular mortality. Cardiac sympathetic dysfunction is partially restored to normal with near normalisation of glycaemia. Recently unrecognized "subtle" changes predispose the heart to failure, after ischaemia-induced remodelling, and arteriosclerotic plaques to instability and rupture. These changes act in conjunction with effects, driven by hyperglycaemia and diabetes, on the endothelium of large blood vessels, e.g. on nitric oxide release or on protein kinase-C beta activation. Meticulous glucose control early on and rapid recompensation of hyperglycaemia in patients with acute coronary syndrome are part of a successful intensive multifactorial approach to prevent the heart in diabetes converting from ailing to failing.

Inhibition of ras p21 membrane localization and modulation of protein kinase C isozyme expression during regression of chemical carcinogen-induced murine skin tumors by lovastatin.

We investigated the ras p21 membrane localization and the expression and activation of protein kinase C (PKC) isozymes in activated ras oncogene-containing tumors and assessed whether these events were related to tumor growth. We used 7,12-dimethylbenz[a]anthracene-initiated and 12-O-tetradecanoylphorbol-13-acetate-promoted SENCAR mouse skin tumors, which were shown to contain Ha-ras oncogene activated by point mutation at codon 61, as an in vivo model for these studies. Compared with levels in epidermis, highly elevated levels of membrane-bound Ha-ras p21 were observed in growing tumors, which also showed strong expression and membrane translocation of PKC zeta and beta II and weak expression of PCK alpha. However, when ras p21 membrane localization was blocked in vivo in growing tumors by lovastatin, opposite results were evident. Compared with saline-treated animals, in which tumor growth continued, lovastatin-treated animals had significantly inhibited tumor growth, which led to tumor regression with concomitant inhibition of Ha-ras p21 membrane localization. These regressing tumors from lovastatin-treated animals also showed a decrease in the expression and membrane translocation of PKC zeta and beta II but increased expression of PKC alpha. Taken together, our results indicate that ras p21 membrane localization and the expression and activation of PKC zeta, beta II, and alpha may be the critical events in the regulation of the growth of tumors that contain activated ras oncogenes.

Activation of beta-isozyme of protein kinase C (PKC beta) is necessary and sufficient for phorbol ester-induced differentiation of HL-60 promyelocytes. Studies with PKC beta-defective PET mutant.

12-O-Tetradecanoylphorbol-13-acetate (TPA) induces growth arrest and differentiation of a number of leukemia cell lines including HL-60 human promyelocytic leukemia. We investigated the involvement of protein kinase C (PKC) isotypes in phorbol ester-induced differentiation using the phorbol ester-tolerant PET mutant of HL-60 cells, which (in contrast to the parent phorbol ester-sensitive (wild-type) S variant of HL-60 cells) does not growth-arrest, become adherent, or undergo apoptosis when exposed to TPA (Macfarlane, D. E., Gailani, D., and Vann, K. (1988) Br. J. Haematol. 68, 291-302). In comparison to S cells, we found that proliferating PET cells markedly underexpress mRNA for PKC beta, but do express PKC alpha and PKC delta. The PKC beta-selective activator 12-deoxyphorbol 13-phenylacetate 20-acetate induces growth arrest, adherence, surface expression of CD11a, and apoptosis in S cells, but not in PET cells. Expression of PKC beta in PET cells can be restored by exposing them to dihydroxyvitamin D3, and this treatment restores the ability of subsequently added 12-deoxyphorbol 13-phenylacetate 20-acetate or TPA to induce immediate cell adherence and growth arrest of PET cells. These data led us to conclude that activation of PKC beta is both necessary and sufficient for phorbol ester-induced growth arrest and adherence in these myeloid cells.

Human endothelial cells are targets for platelet-activating factor (PAF). Activation of alpha and beta protein kinase C isozymes in endothelial cells stimulated by PAF.

We evaluated the role of the protein kinase C (PKC) and its isozymes in the activation of human endothelial cells (EC) stimulated by platelet-activating factor (PAF). Exposure of confluent EC to PAF resulted in a rapid and concentration-dependent redistribution of PKC from cytosol to plasma-membrane, rearrangement of cytoskeleton (i.e. decrease in F-actin content and redistribution of vinculin), and finally increase in the transendothelial flux of 125I-albumin. Stimulation of EC with oleylacetylglycerol or phorbol 12-myristate 13-acetate induced the modification of the cytoskeletal structures and the increase of 125I-albumin clearance. Inhibitors of PKC prevented the effects induced by PAF on the cytoskeleton and on the barrier function of the EC monolayer. Confluent EC expressed only alpha, beta, and epsilon PKC isoforms. Biochemical and immunochemical analysis showed that the time course of the PKC isozymes translocation from cytosol to the membrane fraction of EC stimulated by PAF was different: beta isoform was redistributed more quickly than alpha isoform. PAF did not induce translocation of PKC epsilon. These results suggest that activation of PKC alpha and beta is an important signal transduction pathway by which PAF activates endothelial monolayer and modify its function of barrier to macromolecules.