Delta Isoform Of Protein Kinase Research Articles

Protein kinase C delta mediates Pasireotide effects in an ACTH-secreting pituitary tumor cell line

PurposeClinical control of corticotroph tumors is difficult to achieve since they usually persist or relapse after surgery. Pasireotide is approved to treat patients with Cushing’s disease for whom surgical therapy is not an option. However, Pasireotide seems to be effective only in a sub-set of patients, highlighting the importance to find a response marker to this approach. Recent studies demonstrated that the delta isoform of protein kinase C (PRKCD) controls viability and cell cycle progression of an in vitro model of ACTH-secreting pituitary tumor, the AtT-20/D16v-F2 cells. This study aims at exploring the possible PRKCD role in mediating Pasireotide effects.MethodsIt was assessed cell viability, POMC expression and ACTH secretion in AtT20/D16v-F2 cells over- or under-expressing PRKCD.ResultsWe found that Pasireotide significantly reduces AtT20/D16v-F2 cell viability, POMC expression and ACTH secretion. In addition, Pasireotide reduces miR-26a expression. PRKCD silencing decreases AtT20/D16v-F2 cell sensitivity to Pasireotide treatment; on the contrary, PRKCD overexpression increases the inhibitory effects of Pasireotide on cell viability and ACTH secretion.ConclusionOur results provide new insights into potential PRKCD contribution in Pasireotide mechanism of action and suggest that PRKCD might be a possible marker of therapeutic response in ACTH-secreting pituitary tumors.

Establishment of transgenic lettuce plants producing potentially antihypertensive ShRNA

Development of RNAi-based therapeutics is a fast growing field of the pharmaceutical industry. Using plants for production of pharmaceutically valuable siRNAs may have significant advantages of costeffectiveness, scalability, and low risk of contamination with human pathogens. If edible plant species are genetically engineered to synthesize siRNAs, the costly stage of target product purification may be omitted. We describe the establishment of transgenic lettuce plants producing shRNA targeting delta isoform of protein kinase C (PKC-delta), an effective target for RNAi-based treatment of arterial hypertension. Transgenic lettuce plants were obtained by Agrobacterium-mediated transformation with genetic constructs harboring antiPKC and scrambled (control) shRNA genes. The presence of transgenes was proven by PCR analysis, and the accumulation of antiPKC shRNA was estimated using the RT-qPCR technique. Six transgenic lettuce lines showed varying levels of antiPKC shRNA expression with the highest value reaching 14 ± 9% of highly abundant endogenous lettuce micro RNA (miR156a), or 12.7 fmol/g dry weight. Plants carrying either antiPKC or scrambled shRNA genes flowered normally but did not produce seeds. The described transgenic lettuce plants accumulating antiPKC siRNA are the subject for animal testing and can be considered as raw material for the development of novel antihypertensive drugs.

MTOR Complexes Repress Hypertrophic Agonist-Stimulated Expression of Connective Tissue Growth Factor in Adult Cardiac Muscle Cells.

Connective tissue growth factor (CTGF) is a fibrogenic cytokine that promotes fibrosis in various organs. In the heart, both cardiomyocytes (CM) and cardiac fibroblasts have been reported as a source of CTGF expression, aiding cardiac fibrosis. Although the mammalian target of rapamycin (mTOR) forms 2 distinct complexes, mTORC1 and mTORC2, and plays a central role in integrating biochemical signals for protein synthesis and cellular homeostasis, we explored its role in CTGF expression in adult feline CM. CM were stimulated with 10 μM phenylephrine (PE), 200 nM angiotensin (Ang), or 100 nM insulin for 24 hours. PE and Ang, but not insulin, caused an increase in CTGF mRNA expression with the highest expression observed with PE. Inhibition of mTOR with torin1 but not rapamycin significantly enhanced PE-stimulated CTGF expression. Furthermore, silencing of raptor and rictor using shRNA adenoviral vectors to suppress mTORC1 and mTORC2, respectively, or blocking phosphatidylinositol 3-kinase (PI3K) signaling with LY294002 (LY) or Akt signaling by dominant-negative Akt expression caused a substantial increase in PE-stimulated CTGF expression as measured by both mRNA and secreted protein levels. However, studies with dominant-negative delta isoform of protein kinase C demonstrate that delta isoform of protein kinase C is required for both agonist-induced CTGF expression and mTORC2/Akt-mediated CTGF suppression. Finally, PE-stimulated CTGF expression was accompanied with a corresponding increase in Smad3 phosphorylation and pretreatment of cells with SIS3, a Smad3 specific inhibitor, partially blocked the PE-stimulated CTGF expression. Therefore, a PI3K/mTOR/Akt axis plays a suppressive role on agonist-stimulated CTGF expression where the loss of this mechanism could be a contributing factor for the onset of cardiac fibrosis in the hypertrophying myocardium.

A Diacylglycerol-Dependent Signaling Pathway Contributes to Regulation of Antibacterial Autophagy

Autophagy mediates the degradation of cytoplasmic contents in the lysosome and plays a significant role in immunity. Lipid second messengers have previously been implicated in the regulation of autophagy. Here, we demonstrate a signaling role for diacylglycerol (DAG) in antibacterial autophagy. DAG production was necessary for efficient autophagy of Salmonella, and its localization to bacteria-containing phagosomes preceded autophagy. The actions of phospholipase D and phosphatidic acid phosphatase were required for DAG generation and autophagy. Furthermore, the DAG-responsive delta isoform of protein kinase C was required, as were its downstream targets JNK and NADPH oxidase. Previous studies have revealed a role for the ubiquitin-binding adaptor molecules p62 and NDP52 in autophagy of S. Typhimurium. We observed bacteria-containing autophagosomes colocalizing individually with either DAG or ubiquitinated proteins, indicating that both signals can act independently to promote antibacterial autophagy. These findings reveal an important role for DAG-mediated PKC function in mammalian antibacterial autophagy.

Neurotrophins Induce Neuregulin Release through Protein Kinase Cδ Activation

Proper, graded communication between different cell types is essential for normal development and function. In the nervous system, heart, and for some cancer cells, part of this communication requires signaling by soluble and membrane-bound factors produced by the NRG1 gene. We have previously shown that glial-derived neurotrophic factors activate a rapid, localized release of soluble neuregulin from neuronal axons that can, in turn promote proper axoglial development (Esper, R. M., and Loeb, J. A. (2004) J. Neurosci. 24, 6218-6227). Here we elucidate the mechanism of this localized, regulated release by implicating the delta isoform of protein kinase C (PKC). Blocking the PKC delta isoform with either rottlerin, a selective antagonist, or small interference RNA blocks the regulated release of neuregulin from both transfected cells and primary neuronal cultures. PKC activation also leads to the rapid phosphorylation of the pro-NRG1 cytoplasmic tail on serine residues adjacent to the membrane-spanning segment, that, when mutated markedly reduce the rate of NRG1 activity release. These findings implicate this specific PKC isoform as an important factor for the cleavage and neurotrophin-regulated release of soluble NRG1 forms that have important effects in nervous system development and disease.

Comparative Neuroprotective Properties of Stilbene and Catechin Analogs: Action Via a Plasma Membrane Receptor Site?

Various studies have reported on the neuroprotective effects of polyphenols, widely present in food, beverages, and natural products. For example, we have shown that resveratrol, a polyphenol enriched in red wine and other foods such as peanuts, protects hippocampal cells against beta-amyloid (Abeta)-induced toxicity, a key protein involved in the neuropathology of Alzheimer disease. This effect involves, at least in part, the capacity of resveratrol to activate the phosphorylation of delta isoform of protein kinase C (PKC-delta). The neuroprotective action of resveratrol is shared by piceatannol, a stilbene derivative, as well as by tea-derived catechin gallate esters. The thioflavin T assay indicated that all these polyphenols inhibited the formation of Abeta fibrils, suggesting that this action likely also contributes to their neuroprotective effects. Binding and autoradiographic studies revealed that the effects of polyphenols might involve specific binding sites that are particularly enriched in the choroid plexus in the rat brain. Interestingly, the choroid plexus secretes transthyretin, a protein that has been shown to modulate Abeta aggregation and that may be critical to the maintenance of normal learning capacities in aging. Taken together, these data suggest that polyphenols target multiple enzymes/proteins, leading to their neuroprotective actions, possibly through action via specific plasma membrane binding sites.

Protein kinase Cδ functions downstream of Ca 2+ mobilization in FcεRI signaling to degranulation in mast cells

Mobilization of Ca 2+ plays an important role in the degranulation of mast cells. Although events upstream of Ca 2+ mobilization in the regulation of degranulation are relatively well characterized, the downstream mediators of Ca 2+ remain largely unknown. We sought to characterize the downstream signaling mechanism by which Ca 2+ mobilization mediates degranulation in antigen-stimulated mast cells. The effect of various inhibitors was examined in the antigen-induced or Ca 2+ ionophore A23187-induced degranulation process, and the effect of inhibitors on histamine release was tested in the mouse model of asthma. The delta isoform of protein kinase C (PKC) functions downstream of Ca 2+ in the signaling pathway from FcepsilonRI to degranulation in RBL-2H3 mast cells. Stimulation of cells with either antigen or the Ca 2+ ionophore A23187 induced a rapid translocation of PKC-delta from the cytosol to the cellular membranes, and either treatment with the PKC-delta-specific inhibitor rottlerin or infection with an adenovirus encoding a dominant negative mutant of PKC-delta markedly inhibited degranulation induced with antigen or A23187. Furthermore, both the translocation of PKC-delta and degranulation induced by A23187 were inhibited by prevention of the accumulation of reactive oxygen species normally elicited by the ionophore. Finally, intraperitoneal injection of rottlerin prevented the increase in the concentration of histamine in bronchoalveolar lavage fluid induced by means of antigen challenge in a mouse model of allergic asthma. conclusion: PKC-delta plays an essential downstream mediatory role in the degranulation elicited by Ca 2+ mobilization, and reactive oxygen species mediate the activation of PKC-delta by Ca 2+ in the regulation of degranulation.

PKCδ requires p53 for suppression of the transformed phenotype in human colon cancer cells

We have previously demonstrated that the delta isoform of Protein Kinase C (PKCdelta) acts as a tumor suppressor in HCT116 human colon cancer cells, and that p21(waf1/cip1) is an essential downstream effector of PKCdelta. Our data suggested that p53 might also be involved in the suppression of the neoplastic phenotype induced by PKCdelta. Here we show that homozygous knockout of p53 renders the HCT116 cell line unresponsive to PKCdelta overexpression. Whereas reconstitution of p53 alone did not modify the morphology and growth properties of HCT116/p53null cells, overexpression of both p53 and PKCdelta induced a number of alterations indicating suppression of the transformed phenotype. Interestingly, PKCdelta was ineffective when overexpressed in HT29 cells, a human colon cancer line characterized by the Arg273His dominant-negative mutation of p53. Thus, our data indicate that wild-type p53 is an essential effector of PKCdelta in human colon cancer cells.

P21(Waf1/Cip1) and p53 are downstream effectors of protein kinase C delta in tumor suppression and differentiation in human colon cancer cells.

We have previously demonstrated that the delta isoform of protein kinase C (PKCdelta) is importantly involved in cell growth inhibition and tumor suppression in colon cancer cells. To investigate further the activity and mechanism of action of PKCdelta, we have retrovirally transduced a PKCdelta cDNA in HCT116 human colon cancer cells. PKCdelta-overexpressing cells (HCT116/PKCdelta) were growth-inhibited, showed marked morphologic changes and underwent multinucleation and phenotypic changes characteristic of mitotic catastrophe. Compared to controls, HCT116/PKCdelta cells showed a highly attenuated tumorigenic profile and poor anchorage-independent growth. In addition, transfected cells established junction-coordinated intercellular communications, expressed cell surface microvilli and overexpressed the colon differentiation marker alkaline phosphatase. HCT116/PKCdelta cells also produced the 89 kDa, carboxy-terminal catalytic domain of PARP. In HCT116/PKCdelta cells, p21(Waf1/Cip1) and p53 were transiently upregulated for 48 hr after PKCdelta transduction. In a p21 null subline of HCT116 cells (HCT116/p21null), overexpression of PKCdelta did not affect tumorigenicity or differentiation, indicating that p21 is essential for the antitumorigenic activity of PKCdelta. Similarly, overexpression of PKCdelta caused no significant phenotypic changes in HCT116/E6 cells, an HCT116 subline in which the p53 protein is downregulated by the human papillomavirus E6 gene product. We conclude that overexpression of PKCdelta in human colon cancer cells induces multiple antineoplastic effects that depend on the activities of p21(Waf1/Cip1) and p53.

Evidence that protein kinase Cdelta is not required for palmitate-induced cytotoxicity in BRIN-BD11 beta-cells.

Chronic exposure of pancreatic beta-cells to saturated fatty acids leads to loss of viability, an effect that has been implicated in the process of beta-cell 'lipotoxicity' associated with the progression of type 2 diabetes. The mechanisms involved are unknown but recent evidence has implicated the delta isoform of protein kinase C (PKCdelta) in mediating fatty acid toxicity. We have investigated this proposition in the clonal insulin-secreting cell line, BRIN-BD11. BRIN-BD11 cells were found to undergo apoptosis when exposed to palmitate and this response was attenuated by the purportedly selective inhibitor of PKCdelta, rottlerin. However, activation of PKCdelta with the phorbol ester, phorbol-12-myristate-13-acetate (PMA), failed to promote cell death and down-regulation of PKCdelta did not prevent the cytotoxic effects of palmitate. Moreover, rottlerin remained effective as a blocker of the palmitate response in cells depleted of PKCdelta. Since rottlerin can inhibit various other kinases in addition to PKCdelta, a range of additional kinase inhibitors was also tested. Of these, only the putative Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) inhibitor, KN-62, was found to inhibit palmitate-induced cell death. However, this effect was not reproduced by a more selective pseudo-substrate inhibitor of CaM kinase II. Therefore, the present results reveal that palmitate induces cell death in BRIN-BD11 cells and suggest that this may involve the activation of a rottlerin (and KN-62)-sensitive kinase. However, it is clear that PKCdelta is not required for this response.

Phenotypic modulation of human articular chondrocytes by bistratene A.

Chondrocytes undergo phenotypic alterations following extended periods in monolayer culture, i.e., they become bipolar and flattened, proliferate, and synthesise type I as opposed to type II collagen. This process has been termed chondrocyte dedifferentiation. Bistratene A is a macrolide polyether that specifically activates the delta isoform of protein kinase C (PKCdelta) in some cell types. Here, we show that dedifferentiated human articular chondrocytes became rounded and underwent cell growth arrest after treatment with bistratene A. In addition, bistratene A-treated chondrocytes became more immunopositive for type II collagen, but less immunopositive for type I collagen. These phenotypic changes were associated with a prior and extensive disruption of actin microfilaments and translocation of PKCdelta to the nuclear membrane. Concurrent treatments of chondrocytes with a specific inhibitor of PKCdelta, rottlerin, partially blocked the morphological effects of bistratene A.

Overexpression of nPKCdelta in BL6 murine melanoma cells enhances TGFbeta1 release into the plasma of metastasized animals.

Transforming growth factor-beta (TGFbeta) contributes to the promotion of invasion, metastasis, angiogenesis and even immunosuppression. Since overexpression of the delta isoform of protein kinase C (nPKCdelta) in BL6 murine melanoma cells (BL6T cells) increases their metastatic capacity, we investigated the possible involvement of TGFbeta1 in this process. Immunohistochemical analysis demonstrated lower levels of TGFbeta1 in BL6T lung metastases compared with BL6 lung metastases. On the other hand, higher levels of this cytokine, in particular in its active form, occur in the plasma of BL6T metastasized animals, suggesting a nPKCdelta-dependent TGFbeta1 release. Therefore, nPKCdelta-dependent TGFbeta1 release and activation may be involved in the greater angiogenic and metastatic capacity of murine melanoma BL6T cells.

Approaches to determine the specific role of the delta isoform of protein kinase C

Two dimensional gel electrophoresis of proteins from HL-60 human leukaemia cells treated with bistratene A, a specific activator of protein kinase C (PKC)δ, was performed in conjunction with sequencing in order to identify components of the signal transduction pathway of this isoform of PKC. Stathmin (oncoprotein 18) was identified in this way and the phosphorylation of this protein after treatment with bistratene A, was confirmed by Western blotting of 2D gels. Since stathmin has phosphorylation sites for mitogen activated protein (MAP) kinases, cyclin dependent kinases and calcium/calmodulin dependent protein kinases, it is assumed that one of these enzymes, acting downstream from PKCδ, is responsible for the phosphorylation. Another approach to determining the role of PKCδ involves the identification of interacting proteins using the yeast two hybrid screen. The sequence of nine out of ten independently isolated clones from a two hybrid screen showed perfect homology to human ribosomal protein L8. This protein has previously been shown to exist in complexes with ribosomal RNA, aminoacyl-tRNA and elongation factor-1α, a known substrate of PKCδ, suggesting a role for PKCδ in protein synthesis regulation.

Presence or absence of TrKA protein distinguishes subsets of small sensory neurons with unique cytochemical characteristics and dorsal horn projections

Investigations into the biological actions of nerve growth factor (NGF) have shown that dorsal root ganglion (DRG) neurons subserving nociception require NGF for survival and maintenance of phenotype. This discovery suggests that the signaling NGF receptor, TrkA, can be used as a marker for nociceptive neurons. In this study, we have used antibodies to TrkA, in conjunction with cell biological markers that show a restricted distribution in the DRG, to further characterize subsets of DRG neurons that are dependent upon NGF. Staining for TrkA labeled small and medium-sized neurons that composed 47% of all neurons in thoracic ganglia. Double-labeling with antibodies to the high molecular weight neurofilament protein (NFH), a marker for neurons with myelinated axons, demonstrated that TrkA staining is found in only a small subset of myelinated neurons. Surprisingly, many DRG neurons were not labeled by either TrkA or NFH. These neurons had small soma areas, contained the intermediate filament protein peripherin, and were labeled by the lectin BSI, identifying them as neurons likely to have unmyelinated axons. In addition, small TrkA-negative neurons were extensively labeled by antibodies to the intermediate filament protein alpha-internexin, the delta isoform of protein kinase C, and by the BSI isolectin BSI-B4. In order to assess the potential functions of TrkA-negative small neurons, we examined their projections to the dorsal horn of the spinal cord. TrkA-immunoreactivity in the spinal cord was restricted to lamina I and the outer region of lamina II (IIo), similar to staining for calcitonin gene-related peptide. In contrast, the central projections of TrkA-negative neurons, as visualized by BSI-B4 staining, were particularly dense in lamina IIi. Our results suggest that TrkA-expressing and non-TrkA-expressing small neurons compose functionally distinct populations of DRG neurons.

Tyrosine phosphorylation of protein kinase C-delta in response to the activation of the high-affinity receptor for immunoglobulin E modifies its substrate recognition.

The delta isoform of protein kinase C is phosphorylated on tyrosine in response to antigen activation of the high-affinity receptor for immunoglobulin E. While protein kinase C-delta associates with and phosphorylates this receptor, immunoprecipitation of the receptor revealed that little, if any, tyrosine-phosphorylated protein kinase C-delta is receptor associated. In vitro kinase assays with immunoprecipitated tyrosine-phosphorylated protein kinase C-delta showed that the modified enzyme had diminished activity toward the receptor gamma-chain peptide as a substrate but not toward histones or myelin basic protein peptide. We propose a model in which the tyrosine phosphorylation of protein kinase C-delta regulates the kinase specificity toward a given substrate. This may represent a general mechanism by which in vivo protein kinase activities are regulated in response to external stimuli.

Chemical cross-linking of IgE-receptor complexes in RBL-2H3 cells.

Aggregation of the high-affinity receptors for IgE (Fc epsilon RI) on mast cells activates nonreceptor kinases and other enzymes, thereby initiating a complex biochemical cascade. We have employed a chemical cross-linker in order to stabilize the association of Fc epsilon RI with other cellular proteins that are involved in the early events. We reacted the water-soluble, membrane-impermeant chemical cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) with permeabilized rat mucosal mast cells of the RBL line and analyzed immunoprecipitates of the receptor in detergent lysates of cells that had biosynthetically incorporated [35S]cysteine. Gel electrophoresis revealed substantial amounts of nonreceptor components only when the cells had been reacted with DTSSP. Receptors isolated from cells whose receptor-bound IgE had been aggregated with antigen prior to cross-linking yielded a similar pattern of 35S-labeled proteins. However, when the cells had also been exposed to [gamma-32P]ATP, the proteins associated with the cross-linked, aggregated receptors revealed enhanced incorporation of 32P compared to those associated with cross-linked, unaggregated receptors. A variety of antibodies were used to try to identify the associated proteins. Of those tested for, two, the src-like kinase p53/56lyn and the delta isoform of protein kinase C, were associated with the cross-linked Fc epsilon RI in amounts much greater than could be accounted for by nonspecific cross-linking.