The RacGAP β2-Chimaerin Selectively Mediates Axonal Pruning in the Hippocampus

SummaryAxon pruning and synapse elimination promote neural connectivity and synaptic plasticity. Stereotyped pruning of axons that originate in the hippocampal dentate gyrus (DG) and extend along the infrapyramidal tract (IPT) occurs during postnatal murine development by neurite retraction and resembles axon repulsion. The chemorepellent Sema3F is required for IPT axon pruning, dendritic spine remodeling, and repulsion of DG axons. The signaling events that regulate IPT axon pruning are not known. We find that inhibition of the small G protein Rac1 by the Rac GTPase-activating protein (GAP) β2-Chimaerin (β2Chn) mediates Sema3F-dependent pruning. The Sema3F receptor neuropilin-2 selectively binds β2Chn, and ligand engagement activates this GAP to ultimately restrain Rac1-dependent effects on cytoskeletal reorganization. β2Chn is necessary for axon pruning both in vitro and in vivo, but it is dispensable for axon repulsion and spine remodeling. Therefore, a Npn2/β2Chn/Rac1 signaling axis distinguishes DG axon pruning from the effects of Sema3F on repulsion and dendritic spine remodeling

Differential Regulation of Gene Expression in Lung Cancer Cells by Diacyglycerol-Lactones and a Phorbol Ester Via Selective Activation of Protein Kinase C Isozymes

Despite our extensive knowledge on the biology of protein kinase C (PKC) and its involvement in disease, limited success has been attained in the generation of PKC isozyme-specifc modulators acting via the C1 domain, the binding site for the lipid second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters. Synthetic eforts had recently led to the identifcation of AJH-836, a DAG-lactone with preferential afnity for novel isozymes (nPKCs) relative to classical PKCs (cPKCs). Here, we compared the ability of AJH-836 and a prototypical phorbol ester (phorbol 12-myristate 13-acetate, PMA) to induce changes in gene expression in a lung cancer model. Gene profling analysis using RNA-Seq revealed that PMA caused major changes in gene expression, whereas AJH-836 only induced a small subset of genes, thus providing a strong indication for a major involvement of cPKCs in their control of gene expression. MMP1, MMP9, and MMP10 were among the genes most prominently induced by PMA, an efect impaired by RNAi silencing of PKCα, but not PKCδ or PKCε. Comprehensive gene signature analysis and bioinformatics eforts, including functional enrichment and transcription factor binding site analyses of dysregulated genes, identifed major diferences in pathway activation and transcriptional networks between PMA and DAG-lactones. In addition to providing solid evidence for the diferential involvement of individual PKC isozymes in the control of gene expression, our studies emphasize the importance of generating targeted C1 domain ligands capable of diferentially regulating PKC isozyme-specifc function in cellular models.Centro de Investigaciones Inmunológicas Básicas y Aplicada

Non-Small Cell Lung Carcinoma Cell Motility, Rac Activation and Metastatic Dissemination Are Mediated by Protein Kinase C Epsilon

Background: Protein kinase C (PKC) e, a key signaling transducer implicated in mitogenesis, survival, and cancer progression, is overexpressed in human primary non-small cell lung cancer (NSCLC). The role of PKCe in lung cancer metastasis has not yet been established. Principal Findings: Here we show that RNAi-mediated knockdown of PKCe in H358, H1299, H322, and A549 NSCLC impairs activation of the small GTPase Rac1 in response to phorbol 12-myristate 13-acetate (PMA), serum, or epidermal growth factor (EGF). PKCe depletion markedly impaired the ability of NSCLC cells to form membrane ruffles and migrate. Similar results were observed by pharmacological inhibition of PKCe with eV1-2, a specific PKCe inhibitor. PKCe was also required for invasiveness of NSCLC cells and modulated the secretion of extracellular matrix proteases and protease inhibitors. Finally, we found that PKCe-depleted NSCLC cells fail to disseminate to lungs in a mouse model of metastasis. Conclusions: Our results implicate PKCe as a key mediator of Rac signaling and motility of lung cancer cells, highlighting its potential as a therapeutic target

Diacylglycerol-Stimulated Endocytosis of Transferrin in Trypanosomatids Is Dependent on Tyrosine Kinase Activity

Small molecule regulation of cell function is an understudied area of trypanosomatid biology. In Trypanosoma brucei diacylglycerol (DAG) stimulates endocytosis of transferrin (Tf). However, it is not known whether other trypanosomatidae respond similarly to the lipid. Further, the biochemical pathways involved in DAG signaling to the endocytic system in T. brucei are unknown, as the parasite genome does not encode canonical DAG receptors (e.g. C1-domains). We established that DAG stimulates endocytosis of Tf in Leishmania major, and we evaluated possible effector enzymes in the pathway with multiple approaches. First, a heterologously expressed glycosylphosphatidylinositol phospholipase C (GPI-PLC) activated endocytosis of Tf 300% in L. major. Second, exogenous phorbol ester and DAGs promoted Tf endocytosis in L. major. In search of possible effectors of DAG signaling, we discovered a novel C1-like domain (i.e. C1_5) in trypanosomatids, and we identified protein Tyr kinases (PTKs) linked with C1_5 domains in T. brucei, T. cruzi, and L. major. Consequently, we hypothesized that trypanosome PTKs might be effector enzymes for DAG signaling. General uptake of Tf was reduced by inhibitors of either Ser/Thr or Tyr kinases. However, DAG-stimulated endocytosis of Tf was blocked only by an inhibitor of PTKs, in both T. brucei and L. major. We conclude that (i) DAG activates Tf endocytosis in L. major, and that (ii) PTKs are effectors of DAG-stimulated endocytosis of Tf in trypanosomatids. DAG-stimulated endocytosis of Tf may be a T. brucei adaptation to compete effectively with host cells for vertebrate Tf in blood, since DAG does not enhance endocytosis of Tf in human cells

Further evidence of interaction between vasodilator β\u3csub\u3e2\u3c/sub\u3e- and vasoconstrictor α\u3csub\u3e2\u3c/sub\u3e-adrenoceptor-mediated responses in maintaining vascular tone in anesthetized rats

The importance of the interaction of α- and β-adrenoceptors in maintaining vascular tone in rats was studied. This interaction after clenbuterol (CLEN) treatment indicates an important contribution of the circulating epinephrine (EPI) levels. In urethane-anesthetized rats, the β2-adrenoceptor antagonist (CI 118.551 was more effective in antagonizing isoproterenol-induced hypotension (mainly β2-mediated) than tachycardia (mainly β1-mediated). Intravenous (i.v.) administration of the α2-adrenoceptor agonist clonidine (CLO) induced an initial pressor response followed by a more prolonged hypotension and bradycardia. The initial hypertensive effect was potentiated by previous acute administration of ICI 118.551 as well as by the nonselective β-adrenoceptor antagonist propranolol, but not by metoprolol, a more selective β1-blocker. Fourteen days of administration of the β2-adrenoceptor agonist CLEN [0.3 mg/kg, subcutaneously (s.c.) twice daily], a treatment that induces desensitization of β2-mediated vasodilation, increased the pressor response induced by CLO, an effect that was not observed in pentobarbital-anesthetized rats. In any case, neither β-blockers nor CLEN treatment affects the hypotension and bradycardia induced by CLO. Mean blood pressure (BP) of CLEN-treated rats was increased under urethane anesthesia but not under pentobarbital anesthesia. Catecholamine levels (principally EPI) were higher in urethane-anesthetized rats. These results provide further evidence of a functional interaction between α2- and β2-adrenoceptor-mediated responses in rat vasculature and suggest that vasodilator β2-adrenoceptors might contribute to the determination of peripheral vascular tone when circulating EPI is substantially elevated

Pressor response induced by clenbuterol treatment in immobilized normotensive rats

Short-term treatment with clenbuterol [0.6 mg/kg-1 subcutaneously (s.c.) daily] produces a pressor effect in stressed rats after a period of immobilization (40 min). The stress applied markedly increases the plasma concentrations of norepinephrine (NE) and epinephrine. After bilateral adrenal demedullation, the increased levels of catecholamines and the hypertensive response obtained after clenbuterol treatment in the stressed animals were reduced to the values of the control rats. Clenbuterol treatment produced desensitization of the β2-adrenoceptor-mediated effect and thus reduced the vasodilator response induced by isoproterenol and increased the vasoconstriction produced by epinephrine but not that caused by NE. This desensitization may be responsible for the hypertensive response after clenbuterol treatment in stressed animals which is attenuated after adrenal demedullation. In conclusion, our results provide evidence that clenbuterol treatment induces pressor effect in normotensive animals under stress

Regulation of Transcriptional Networks by PKC Isozymes: Identification of c-Rel as a Key Transcription Factor for PKC-Regulated Genes

<div><p>Background</p><p>Activation of protein kinase C (PKC), a family of serine-threonine kinases widely implicated in cancer progression, has major impact on gene expression. In a recent genome-wide analysis of prostate cancer cells we identified distinctive gene expression profiles controlled by individual PKC isozymes and highlighted a prominent role for PKCδ in transcriptional activation.</p><p>Principal Findings</p><p>Here we carried out a thorough bioinformatics analysis to dissect transcriptional networks controlled by PKCα, PKCδ, and PKCε, the main diacylglycerol/phorbol ester PKCs expressed in prostate cancer cells. Despite the remarkable differences in the patterns of transcriptional responsive elements (REs) regulated by each PKC, we found that c-Rel represents the most frequent RE in promoters regulated by all three PKCs. In addition, promoters of PKCδ-regulated genes were particularly enriched with REs for CREB, NF-E2, RREB, SRF, Oct-1, Evi-1, and NF-κB. Most notably, by using transcription factor-specific RNAi we were able to identify subsets of PKCδ-regulated genes modulated by c-Rel and CREB. Furthermore, PKCδ-regulated genes condensed under the c-Rel transcriptional regulation display significant functional interconnections with biological processes such as angiogenesis, inflammatory response, and cell motility.</p><p>Conclusion/Significance</p><p>Our study identified candidate transcription factors in the promoters of PKC regulated genes, in particular c-Rel was found as a key transcription factor in the control of PKCδ-regulated genes. The deconvolution of PKC-regulated transcriptional networks and their nodes may greatly help in the identification of PKC effectors and have significant therapeutics implications.</p></div