The signaling axis atypical protein kinase C λ/ι-Satb2 mediates leukemic transformation of B-cell progenitors.

Epigenetically regulated transcriptional plasticity has been proposed as a mechanism of differentiation arrest and resistance to therapy. BCR-ABL leukemias result from leukemic stem cell/progenitor transformation and represent an opportunity to identify epigenetic progress contributing to lineage leukemogenesis. Primary human and murine BCR-ABL+ leukemic progenitors have increased activation of Cdc42 and the downstream atypical protein kinase C (aPKC). While the isoform aPKCζ behaves as a leukemic suppressor, aPKCλ/ι is critically required for oncogenic progenitor proliferation, survival, and B-cell differentiation arrest, but not for normal B-cell lineage differentiation. In vitro and in vivo B-cell transformation by BCR-ABL requires the downregulation of key genes in the B-cell differentiation program through an aPKC λ/ι-Erk dependent Etv5/Satb2 chromatin repressive signaling complex. Genetic or pharmacological targeting of aPKC impairs human oncogenic addicted leukemias. Therefore, the aPKCλ/ι-SATB2 signaling cascade is required for leukemic BCR-ABL+ B-cell progenitor transformation and is amenable to non-tyrosine kinase inhibition.

Pexelizumab and survival in cardiac surgery.

A recent international consensus conference on the reduction in mortality in cardiac anesthesia and intensive care included pexelizumab, a recombinant monoclonal antibody to the component 5 of the complement system, among the ancillary (i.e. non-surgical) drugs/techniques/strategies that might influence survival rates in patients undergoing cardiac surgery. The consensus conferences state that "A subgroup analysis of a meta-analysis of randomized controlled trials suggested that pexelizumab might reduce mortality (longest follow up available, up to 6 months) in patients undergoing coronary artery bypass grafting. Pexelizumab was not included among the most important topics of the consensus conference as it was the only topic that did not receive a sufficient percentage of votes from the audience (32% at the first round and 35% at the second round). Pexelizumab is no longer on the market, however, the concept of reducing the generalized inflammatory process accompanying cardiopulmonary bypass deserves further investigation.

The effects of load on systolic mitral annulus movements by tissue Doppler imaging in cardiac surgery patients.

AIM: The main indexes of ventricular function used in clinical practice are strongly influenced by the ventricular load conditions. Tissue Doppler imaging (TDI) has been reported to be a powerful modality that enables assessment of ventricular wall motion with a high temporal and spatial resolution. The study of the systolic movements of the mitral annulus has been proposed as a valid index of myocardial contractility. The aim of this study was to verify if the systolic movements of the mitral annulus by TDI are load-dependent in patients undergoing coronary artery bypass grafting. We evaluated the effect of load changes on systolic (S(M)) and isovolumic (S(IVC)) velocity waves, and on isovolumic acceleration (S(IVA)). METHODS: Twenty-three patients undergoing coronary artery bypass grafting were evaluate. Load was changed with a rapid infusion of 7 cc/kg of a gelatin solution. The sample volume of TDI was placed at the lateral side of the mitral annulus in the mid-esophageal 4-chamber view. RESULTS: The increase preload resulted in a statistically significant increase of S(M) (8.7+/-1.04 cm/s vs. 9.99+/-0.88 cm/s P=0.01), and a statistically significant increase of S(IVC) (5.65+/-1.95 cm/s vs. 6.75+/-1.53 cm/s P=0.03). S(IVA) does not appear to be changed after the increase of preload (203.45+/-45.69 cm/s(2) vs. 211.41+/-71.28 p=0.81). CONCLUSION: Only S(IVA) seems to be an independent index of cardiac functionality in cardiac surgery patients and appears to be an optimum index in the clinical evaluation of these patients.

Of the atypical PKCs, Par-4 and p62: recent understandings of the biology and pathology of a PB1-dominated complex.

The recent identification of a novel protein-protein interaction module, termed PB1, in critical signaling molecules such as p62 (also known as sequestosome1), the atypical PKCs, and Par-6, has unveiled the existence of a new set of signaling complexes, which can be central to several biological processes from development to cancer. In this review, we will discuss the most recent advances on the role that the different components of these complexes have in vivo and that are relevant to human disease. In particular, we will review what we are learning from new data from knockout mice, and the indications from human mutations on the real role of these proteins in the physiology and biology of human diseases. The role that PKCzeta, PKClambda/iota, and Par-4 have in lung and prostate cancer in vivo and in humans will be extensively covered in this article, as will the multifunctional role of p62 as a novel hub in cell signaling during cancer and inflammation, and the mechanistic details and controversial data published on its potential role in aggregate formation and signaling. All this published information is shedding new light on the proposed pathological implications of these PB1-regulators in disease and shows their important role in cell physiology.

Polarized subcellular localization of Jak/STAT components is required for efficient signaling.

Three protein complexes control polarization of epithelial cells: the apicolateral Crumbs and Par-3 complexes and the basolateral Lethal giant larvae complex. Polarization results in the specific localization of proteins and lipids to different membrane domains. The receptors of the Notch, Hedgehog, and WNT pathways are among the proteins that are polarized, with subcellular receptor localization representing an important aspect of signaling regulation. For example, in the WNT pathway, differential DFz2 receptor localization results in activation of either the canonical or the planar polarity pathway. Despite the large body of research on the vertebrate JAK/STAT pathway, there are no reports indicating polarized signaling. By using the conserved Drosophila JAK/STAT pathway as a system, we find that the receptor and its associated kinase are located in the apical membrane of epithelial cells. Unexpectedly, the transcription factor STAT is enriched in the apicolateral membrane domain of ectoderm epithelial cells in a Par-3-dependent manner. Our results indicate that preassembly of STAT and the Receptor/JAK complex to specific membrane domains is a key aspect for signaling efficiency. Our results also suggest that receptor polarization in the ectoderm cell membrane restricts the cell's response to ligands provided by neighboring cells.

Aortocoronary bypass grafting in high-risk patients over 75 years. Propensity score analysis of on versus off-pump, early and midterm results.

AIM: Bypass surgery in high risk patients over the age of 75 results in increased mortality and morbidity, which may be also related to the cardiopulmonary bypass system. METHODS: Using the propensity score analysis, we have selected two homogeneous groups of high-risk elderly patients undergoing coronary surgery: 41 patients operated with cardiopulmonary bypass- coronary artery bypass graft (CPB-CABG), and 78 patients operated without cardiopulmonary bypass (off-pump coronary artery bypass graft, OPCABG). All preoperative and operative variables were similar and outcomes were compared. RESULTS: Perioperative mortality was higher in the patient group operated with CPB (12.2%) as compared to patients operated without CPB (1.3%, P = 0.01). Perioperative complications were more frequent in the CPB-CABG group. Logistic regression analysis showed that avoiding CPB was an independent protective factor for mortality and morbidity. Midterm survival, freedom from angina, freedom from reintervention, and Canadian Cardiovascular Society (CSS) class were comparable between the 2 groups. CONCLUSION: OPCABG is safe in the high risk elderly population and significantly reduces postoperative mortality and morbidity. There are no differences in midterm results between the 2 groups of patients in our study.

PKCzeta at the crossroad of NF-kappaB and Jak1/Stat6 signaling pathways.

The atypical protein kinase C (PKC) isoforms (aPKC) have been implicated in the regulation of a number of essential signaling events. Early studies using dominant-negative mutants suggested that they are important intermediaries in the activation of the canonical nuclear factor (NF)-kappaB pathway. More recent data using knockout mice genetically demonstrate that in fact the PKCzeta isoform is essential for the adequate activation of this cascade both upstream and downstream the IkappaB kinase complex. In this review, we summarize the mechanistic details whereby the aPKC pathway regulates important cellular functions and how this is achieved by the ability of these kinases to interact with different protein regulators and adapters, as well as to impinge in NF-kappaB-independent signaling cascades such as the Janus kinase-1/signal transducer and activator of transcription 6 system, which plays a critical role in T-cell-mediated hepatitis and asthma.

Mortality and morbidity from intra-aortic balloon pumps. Risk analysis.

BACKGROUND: The purpose of this study, was to assess the incidence of and predictors for mortality and morbidity in patients who required postoperative intra-aortic balloon pump (IABP) support. METHODS: We have retrospectively estimated 116 patients and data were statistically analyzed, and significant variables were evaluated with multivariate analysis. RESULTS: Mortality rate was 57.8% (67 patients). Nineteen patients (16.3%) had major vascular complications: 12 patients (10.3%) limb ischemia, 1 patient (0.9%) aortic dissection, 6 patients (5.2%) mesenteric infarction. Thirty patients (25.8%) had minor vascular complication: 23 patients (19.8%) bleeding from insertion site, 7 (6%) patients infection of insertion site. Limb ischemia was resolved by IABP removal (10 patients), thrombectomy (2 patients). No patient required limb amputation. Sixty patients (51.7%) had renal insufficiency, of which 40 patients needed dialysis. Fifteen patients (10.3%) had neurological complications, 13 patients (11.2%) thrombocytopenia and 5 patients (4.3%) sepsis. CONCLUSIONS: The incidence of IABP insertion in our institution was 1.5%. Mortality rate is similar to mortality of other studies in which the IABP has been inserted in the postoperative period. We have found that timing of IABP insertion, thrombocytopenia, presence of peripheral vascular disease and the redo intervention are independent predictors of mortality. We also found that female sex, diabetes, history of cigarette smoking and preoperative use of antiplatelet drugs are independent predictors of limb ischemia. The following factors are instead independent predictors of renal insufficiency: postoperative ejection fraction lower than 40% and non use of dobutamine in the postoperative period.

Nerve growth factor stimulates multisite tyrosine phosphorylation and activation of the atypical protein kinase C's via a src kinase pathway.

Atypical protein kinase C (PKC) isoforms are required for nerve growth factor (NGF)-initiated differentiation of PC12 cells. In the present study, we report that PKC-iota becomes tyrosine phosphorylated in the membrane coincident with activation posttreatment with nerve growth factor. Tyrosine phosphorylation and activation of PKC-iota were inhibited in a dose-dependent manner by both PP2 and K252a, src and TrkA kinase inhibitors. Purified src was observed to phosphorylate and activate PKC-iota in vitro. In PC12 cells deficient in src kinase activity, both NGF-induced tyrosine phosphorylation and activation of PKC-iota were also diminished. Furthermore, we demonstrate activation of src by NGF along with formation of a signal complex including the TrkA receptor, src, and PKC-iota. Recruitment of PKC-iota into the complex was dependent on the tyrosine phosphorylation state of PKC-iota. The association of src and PKC-iota was constitutive but was enhanced by NGF treatment, with the src homology 3 domain interacting with a PXXP sequence within the regulatory domain of PKC-iota (amino acids 98 to 114). Altogether, these findings support a role for src in regulation of PKC-iota. Tyrosine 256, 271, and 325 were identified as major sites phosphorylated by src in the catalytic domain. Y256F and Y271F mutations did not alter src-induced activation of PKC-iota, whereas the Y325F mutation significantly reduced src-induced activation of PKC-iota. The functional relevance of these mutations was tested by determining the ability of each mutant to support TRAF6 activation of NF-kappaB, with significant impairment by the Y325F PKC-iota mutant. Moreover, when the Y352F mutant was expressed in PC12 cells, NGF's ability to promote survival in serum-free media was reduced. In summary, we have identified a novel mechanism for NGF-induced activation of atypical PKC involving tyrosine phosphorylation by c-Src.

Targeted disruption of the zetaPKC gene results in the impairment of the NF-kappaB pathway.

Here we have addressed the role that zetaPKC plays in NF-kappaB activation using mice in which this kinase was inactivated by homologous recombination. These mice, although grossly normal, showed phenotypic alterations in secondary lymphoid organs reminiscent of those of the TNF receptor-1 and of the lymphotoxin-beta receptor gene-deficient mice. The lack of zetaPKC in embryonic fibroblasts (EFs) severely impairs kappaB-dependent transcriptional activity as well as cytokine-induced phosphorylation of p65. Also, a cytokine-inducible interaction of zetaPKC with p65 was detected which requires the previous degradation of IkappaB. Although in zetaPKC-/- EFs this kinase is not necessary for IKK activation, in lung, which abundantly expresses zetaPKC, IKK activation is inhibited.

Haemolysis due to active venous drainage during cardiopulmonary bypass: comparison of two different techniques.

To facilitate mini-access for cardiac surgery, two different methods of active venous drainage are used: vacuum assisted drainage and centrifugal pump aspiration on the venous line. The aim of this study was to compare the haemolysis produced using these two techniques. From June to December 1999, 50 consecutive patients were operated on using a ministernotomy. All of these patients had valvular surgery for either valve repair or valve replacement (9 MVRepair, 11 MVR, 29 AVR, 1 AVR + MVR). They were randomized into two groups: Group A, 25 patients who underwent surgery where vacuum assisted drainage was used, and Group B, 25 patients where kinetic asssisted venous drainage with centrifugal pump venous aspiration was used. Patient characteristics of both groups were similar for age, gender, body weight, body surface area, height, cardiopulmonary bypass (CPB) time, aortic crossclamp time, priming volume, cardioplegia volume, haemoglobin concentration, haematocrit, serum creatinine, bilirubin, lactate dehydrogenase (LDH), serum glutamic oxaloacetic transaminase (sGOT), serum glutamic pyruvic transaminase (sGPT), aptoglobin, reticulocytes, and platelet count. We checked all these laboratory parameters preoperatively, at the end of CPB, and 2 and 24 h after operation. We also checked haemoglobinuria at these same time points. We assessed blood loss at 6, 12, and 24 h after the operation and calculated total postoperative bleeding. There was a tendency towards a greater increase in LDH, sGOT and sGPT in Group A more than in Group B, but these data did not reach statistical significance. Platelet count was always lower in Group A and aptoglobin increased in Group A more than in Group B. More patients in Group A had haemoglobinuria. These findings indicate that haemolysis is increased more in patients treated with vacuum assisted drainage, when compared to the rise in haemolysis in those treated with centrifugal pump venous drainage. Total bleeding is also greater in Group A.

The atypical protein kinase C-interacting protein p62 is a scaffold for NF-kappaB activation by nerve growth factor.

Nerve growth factor (NGF) binding to both p75 and TrkA neurotrophin receptors activates the transcription factor nuclear factor kappaB (NF-kappaB). Here we show that the atypical protein kinase C-interacting protein, p62, which binds TRAF6, selectively interacts with TrkA but not p75. In contrast, TRAF6 interacts with p75 but not TrkA. We demonstrate the formation of a TRAF6-p62 complex that serves as a bridge linking both p75 and TrkA signaling. Of functional relevance, transfection of antisense p62-enhanced p75-mediated cell death and diminished NGF-induced differentiation occur through a mechanism involving inhibition of IKK activity. These findings reveal a new function for p62 as a common platform for communication of both p75-TRAF6 and TrkA signals. Moreover, we demonstrated that p62 serves as a scaffold for activation of the NF-kappaB pathway, which mediates NGF survival and differentiation responses.

MEK5, a new target of the atypical protein kinase C isoforms in mitogenic signaling.

The MEK5-extracellular signal-regulated kinase (ERK5) tandem is a novel mitogen-activated protein kinase cassette critically involved in mitogenic activation by the epidermal growth factor (EGF). The atypical protein kinase C isoforms (aPKCs) have been shown to be required for cell growth and proliferation and have been reported to interact with the adapter protein p62 through a short stretch of acidic amino acids termed the aPKC interaction domain. This region is also present in MEK5, suggesting that it may be an aPKC-binding partner. Here we demonstrate that the aPKCs interact in an EGF-inducible manner with MEK5 and that this interaction is required and sufficient for the activation of MEK5 in response to EGF. Consistent with the role of the aPKCs in the MEK5-ERK5 pathway, we show that zetaPKC and lambda/iotaPKC activate the Jun promoter through the MEF2C element, a well-established target of ERK5. From all these results, we conclude that MEK5 is a critical target of the aPKCs during mitogenic signaling.

The MKK(3/6)-p38-signaling cascade alters the subcellular distribution of hnRNP A1 and modulates alternative splicing regulation.

Individual members of the serine-arginine (SR) and heterogeneous nuclear ribonucleoprotein (hnRNP) A/B families of proteins have antagonistic effects in regulating alternative splicing. Although hnRNP A1 accumulates predominantly in the nucleus, it shuttles continuously between the nucleus and the cytoplasm. Some but not all SR proteins also undergo nucleo-cytoplasmic shuttling, which is affected by phosphorylation of their serine/arginine (RS)-rich domain. The signaling mechanisms that control the subcellular localization of these proteins are unknown. We show that exposure of NIH-3T3 and SV-40 transformed green monkey kidney (COS) cells to stress stimuli such as osmotic shock or UVC irradiation, but not to mitogenic activators such as PDGF or EGF, results in a marked cytoplasmic accumulation of hnRNP A1, concomitant with an increase in its phosphorylation. These effects are mediated by the MKK(3/6)-p38 pathway, and moreover, p38 activation is necessary and sufficient for the induction of hnRNP A1 cytoplasmic accumulation. The stress-induced increase in the cytoplasmic levels of hnRNP A/B proteins and the concomitant decrease in their nuclear abundance are paralleled by changes in the alternative splicing pattern of an adenovirus E1A pre-mRNA splicing reporter. These results suggest the intriguing possibility that signaling mechanisms regulate pre-mRNA splicing in vivo by influencing the subcellular distribution of splicing factors.

The atypical PKC-interacting protein p62 channels NF-kappaB activation by the IL-1-TRAF6 pathway.

The atypical protein kinase C (aPKC)-interacting protein, p62, has previously been shown to interact with RIP, linking these kinases to NF-kappaB activation by tumor necrosis factor alpha (TNFalpha). The aPKCs have been implicated in the activation of IKKbeta in TNFalpha-stimulated cells and have been shown to be activated in response to interleukin-1 (IL-1). Here we demonstrate that the inhibition of the aPKCs or the down-regulation of p62 severely abrogates NF-kappaB activation by IL-1 and TRAF6, suggesting that both proteins are critical intermediaries in this pathway. Consistent with this we show that p62 selectively interacts with the TRAF domain of TRAF6 but not that of TRAF5 or TRAF2 in co-transfection experiments. The binding of endogenous p62 to TRAF6 is stimulus dependent, reinforcing the notion that this is a physiologically relevant interaction. Furthermore, we demonstrate that the N-terminal domain of TRAF6, which is required for signaling, interacts with zetaPKC in a dimerization-dependent manner. Together, these results indicate that p62 is an important intermediary not only in TNFalpha but also in IL-1 signaling to NF-kappaB through the specific adapters RIP and TRAF6.

The atypical protein kinase Cs. Functional specificity mediated by specific protein adapters.

Since its discovery more than 10 years ago, the atypical PKC (aPKC) subfamily has attracted great interest. A number of reports have shown that the kinases of this subfamily play critical roles in signaling pathways that control cell growth, differentiation and survival. Recently, several investigators have identified a number of aPKC-interacting proteins whose characterization is helping to unravel the mechanisms of action and functions of these kinases. These interactors include p62, Par-6, MEK5 and Par-4. The details of how these adapters serve to link the aPKCs to different receptor signaling pathways and substrates in response to specific stimuli are crucial not only for developing an understanding of the roles and functions of the aPKCs themselves, but also for more generally establishing a view of how specificity in signal transduction is achieved.

The downregulation of the pro-apoptotic protein Par-4 is critical for Ras-induced survival and tumor progression.

Inhibition of apoptosis is an important characteristic of oncogenic transformation. The Par-4 gene product has recently been shown to be upregulated in cells undergoing apoptotic cell death, and its ectopic expression was shown to be critical in apoptosis. We demonstrate that expression of oncogenic Ras promotes a potent reduction of Par-4 protein and mRNA levels through a MEK-dependent pathway. In addition, the expression of permanently active mutants of MEK, Raf-1 or zetaprotein kinase C but not of phosphatidylinositol 3-kinase (PI 3-kinase) is sufficient to decrease Par-4 levels. These effects are independent of p53, p16 and p19, and were detected not only in fibroblast primary cultures but also in NIH 3T3 and HeLa cells, indicating that they are not secondary to Ras actions on cell cycle regulation. Importantly, restoration of Par-4 levels to normal in Ras-transformed cells makes these cells sensitive to the pro-apoptotic actions of tumor necrosis factor-alpha under conditions in which PI 3-kinase is inhibited and also severely impairs colony formation in soft agar and tumor development in nude mice, as well as increases the sensitivity of these tumors to camptothecin. This indicates that the downregulation of Par-4 by oncogenic Ras is a critical event in tumor progression.

Inactivation of the inhibitory kappaB protein kinase/nuclear factor kappaB pathway by Par-4 expression potentiates tumor necrosis factor alpha-induced apoptosis.

Par-4 is a novel protein identified in cells undergoing apoptosis. The ability of Par-4 to promote apoptotic cell death is dependent on the binding and inactivation of the atypical protein kinases C (PKCs). This subfamily of kinases has been reported to control nuclear factor kappaB (NF-kappaB) through the regulation of the IkappaB kinase activity. NF-kappaB activation by tumor necrosis factor alpha (TNFalpha) provides a survival signal that impairs the TNFalpha-induced apoptotic response. We show here that expression of Par-4 inhibits the TNFalpha-induced nuclear translocation of p65 as well as the kappaB-dependent promoter activity. Interestingly, Par-4 expression blocks inhibitory kappaB protein (IkappaB) kinase activity, which leads to the inhibition of IkappaB phosphorylation and degradation, in a manner that is dependent on its ability to inhibit lambda/iotaPKC. Of potential functional relevance, the expression of Par-4 allows TNFalpha to induce apoptosis in NIH-3T3 cells. In addition, the down-regulation of Par-4 levels by oncogenic Ras sensitizes cells to TNFalpha-induced NF-kappaB activation.