Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Long-chain (sphingoid) bases may serve as another category of "lipid second messenger" because they inhibit protein kinase C and affect multiple cellular functions. Free sphingosine has been found in rat liver (Merrill, A. H., Jr., Wang, E., Mullins, R. E., Jamison, W. C. L., Nimkar, S., and Liotta, D. C. (1988) Anal. Biochem. 171, 373-381); hence, this study determined if liver plasma membranes contain free long-chain bases and have the ability to form them from endogenous enzymes and substrates. Isolated plasma membranes contained 0.45 nmol of sphingosine/mg of protein which, based on the recovery of the membranes, was equivalent to 3.5 +/- 1.2 nmol/g of liver and at least half of the total free sphingosine in liver. When the membranes were incubated at 37 degrees C, the amount increased at an initial rate of 5-25 pmol/min/mg, resulting in a 2-3-fold increase over an hour. Sphingosine formation required divalent cations, was optimal at neutral to alkaline pH, and was temperature-dependent. Activities with these characteristics were not identified in microsomes or lysosomes (lysosomal activities with acidic pH optima were detected, however); hence, they appear to reflect a separate plasma membrane system. Sphingosine formation was stimulated by ceramides either added exogenously or formed endogenously by treating the membranes with sphingomyelinase (but not endoglycoceramidase). Sphingomyelin hydrolysis to ceramide was also observed during incubation of the plasma membranes alone. Some of the properties of this system resembled the neutral sphingomyelinase and ceramidase activities of liver. While the physiological significance of this endogenous sphingosine is not known, this system has the appropriate subcellular location to provide sphingosine as a participant in signal transduction.
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PMID:Free sphingosine formation from endogenous substrates by a liver plasma membrane system with a divalent cation dependence and a neutral pH optimum. 249 78

Sphingosine is a product of sphingolipid metabolism that has been linked to a protein kinase C-independent mitogenic response. In previously published data, utilizing an in vitro model system for platelet-derived growth factor (PDGF)-induced vascular smooth muscle proliferation, we have demonstrated that sphingosine is increased at the expense of a concomitant decrease in ceramide formation, implicating an altered ceramidase activity. To explore mechanisms of growth factor-stimulated sphingosine formation, we have developed and investigated a cell-free model system assessing ceramidase activity. We now report that an alkaline, membrane-associated, ceramidase activity in the rat glomerular mesangial cell, a smooth muscle-like pericyte, is up-regulated by growth factors, apparently via a tyrosine kinase phosphorylation mechanism. PDGF also stimulated sphingomyelinase activity which generates sufficient substrate to drive the subsequent ceramidase reaction. Inflammatory cytokines, including interleukin-1, and tumor necrosis factor-alpha, stimulated sphingomyelinase but not ceramidase activity, a result consistent with the cellular accumulation of the ceramide, apoptidic, differentiating second messenger. Mitogenic vasoconstrictor peptides such as endothelin-1 stimulated neither sphingomyelinase nor ceramidase activities. An inhibitor of ceramidase activity, N-oleoylethanolamine, reduced PDGF- but not endothelin-1-stimulated proliferation. Thus, we conclude that, in mesangial cells, growth factors but not vasoconstrictor peptides or cytokines induce mitogenesis, in part, through ceramidase-mediated sphingosine formation.
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PMID:Differential regulation of sphingomyelinase and ceramidase activities by growth factors and cytokines. Implications for cellular proliferation and differentiation. 755 85

The present studies were undertaken to characterize the potential role of sphingosine in the regulation of apoptosis in HL-60 promyelocytic leukemia cells. A 6-h exposure of HL-60 cells to sphingosine or its methylated derivative, N,N-dimethylsphingosine, caused internucleosomal DNA fragmentation and stereotypical morphological changes characteristic of apoptosis (i.e., cell shrinkage, nuclear condensation, and the formation of apoptotic bodies), as well as that to pharmacological inhibitors of protein kinase C such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine and staurosporine. Apoptosis by sphingosine and N,N-dimethylsphingosine was measured using a flow cytometric method. The percentages of apoptotic cells in cultures treated with sphingosine (10 microM) and N,N-dimethylsphingosine (10 microM) for 6 h were 55.6 +/- 7.8% and 84.2 +/- 11.6%, respectively. HL-60 cells were induced to differentiate toward macrophages by treatment with 5 nM 4 beta-phorbol 12-myristate 13-acetate (PMA). Internucleosomal DNA fragmentation, which was a hallmark of apoptosis, was first detected after 10-h exposure to PMA and increased with longer treatment. Sphingosine concentrations in the cells increased concomitantly with the increasing proportion of apoptotic cells during cell differentiation. Sphingosine level in HL-60 cells differentiated by treatment with PMA for 48 h was about 3.3-fold greater than that in untreated cells. Differentiated HL-60 cells exhibited a markedly increased conversion of exogenously added [3H]ceramide to [3H]sphingosine, indicating elevation of ceramidase activity. Moreover, exposure to sphingosine resulted in down-regulation of c-myc mRNA. These observations suggest the possible role of sphingosine in induction of apoptotic DNA fragmentation during PMA-induced differentiation in myeloid leukemia cells. Sphingosine may function as an endogenous modulator mediating the apoptotic signal.
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PMID:Induction of apoptosis by sphingosine in human leukemic HL-60 cells: a possible endogenous modulator of apoptotic DNA fragmentation occurring during phorbol ester-induced differentiation. 783 42

Embryonic astrocytes respond readily to serine/threonine kinase regulation in terms of cytoskeleton assembly, mitotic activity, and cell fate. We now present evidence that these responses include apoptosis. Staurosporine induced apoptosis in astrocyte cultures derived from chick embryo cerebral hemispheres, as assayed both by immunocytochemical detection of new 3-hydroxy DNA ends and production of 200-bp DNA fragment laddering. Staurosporine treatment also resulted in the prolonged (>24 h) activation of a 60-kDa serine/threonine protein kinase (PK60), increased ceramide formation (fourfold after 24 h), increased glutamine synthetase activity, and significant apoptosis (40%) after 24 h. PK60 was shown to be cytoskeleton associated and its activity, as measured by phosphorylation of myelin basic protein, was rapid, increased for up to 3 h, and was stable for at least 24 h. Other protein kinase C inhibitors, H8, sphingosine, calphostin C, or the protein kinase A inhibitor KT5720 did not induce either PK60 activation or apoptosis. The dose-dependent increase in [3H]palmitate labeling of ceramide and a specific decrease in labeling of its precursor sphingomyelin were not blocked by the biosynthetic inhibitor fumonisin beta1 but were increased (in a dose-dependent manner) by the coaddition of the ceramidase inhibitor oleoylethanolamine. Exogenous addition of C2-ceramide induced apoptosis but did not activate PK60. These results suggest that apoptosis in embryonic astrocytes involves pathways similar to those described in other cell types and that the activation of PK60 and formation of ceramide are early events in the pathway.
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PMID:Programmed cell death in cortical chick embryo astrocytes is associated with activation of protein kinase PK60 and ceramide formation. 942 55

Exogenous NO is able to trigger apoptosis of renal mesangial cells, and thus may contribute to acute lytic phases as well as to resolution of glomerulonephritis. However, the mechanism involved in these events is still unclear. We report here that chronic exposure of renal mesangial cells for 24 h to compounds releasing NO, including spermine-NO, (Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1, 2-diolate (MAHMA-NO), S-nitrosoglutathione (GS-NO), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) results in a potent and dose-dependent increase in the lipid signaling molecule ceramide. Time courses reveal that significant effects occur after 2-4 h of stimulation with NO donors and reach maximal levels after 24 h of stimulation. No acute (within minutes) ceramide production can be detected. When cells were stimulated with NO donors in the presence of phorbol ester, a direct activator of protein kinase C, both ceramide production and DNA fragmentation are completely abolished. Furthermore, addition of exogenous ceramide partially reversed the inhibitory effect of phorbol ester on apoptosis, thus suggesting a negative regulation of protein kinase C on ceramide formation and apoptosis. In contrast to exogenous NO, tumor necrosis factor (TNF)-alpha stimulates a very rapid and transient increase in ceramide levels within minutes but fails to induce the late-phase ceramide formation. Moreover, TNF fails to induce apoptosis in mesangial cells. Interestingly, NO and TNFalpha cause a chronic activation of acidic and neutral sphingomyelinases, the ceramide-generating enzymes, whereas acidic and neutral ceramidases, the ceramide-metabolizing enzymes, are inhibited by NO, but potently stimulated by TNFalpha. Furthermore, in the presence of an acidic ceramidase inhibitor, N-oleoylethanolamine, TNFalpha leads to a sustained accumulation of ceramide and in parallel induces DNA fragmentation. In summary, our data demonstrate that exogenous NO causes a chronic up-regulation of ceramide levels in mesangial cells by activating sphingomyelinases and concomitantly inhibiting ceramidases, and that particularly the late-phase of ceramide generation may be responsible for the further processing of a proapoptotic signal.
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PMID:Nitric oxide donors induce stress signaling via ceramide formation in rat renal mesangial cells. 1006 79

Experiments were designed to examine the role of sphingosine, PP2A phosphatases, and protein kinase C (PKC) inhibition in mediating the vasodilatory effects of ceramide in rat thoracic aorta. Sphingosine did not cause vasorelaxation, and oleoylethanol-amine, a ceramidase inhibitor, did not affect sphingomyelinase-induced relaxation. Okadaic acid potentiated the relaxation response to ceramide. These observations rule out involvement of sphingosine and PP2A phosphatases in mediating ceramide-induced relaxation. Sphingomyelinase attenuated contractile and single-cell intracellular calcium responses to phorbol ester. Chelerythrine incubation potentiated the relaxation response to ceramide. These observations support a role for PKC inhibition in mediating the vasodilatory effects of ceramide.
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PMID:Ceramide-induced vasorelaxation: An inhibitory action on protein kinase C. 1055 83

Ceramide levels are strongly increased by stimulation of renal mesangial cells with nitric oxide (NO). This effect was shown previously to be due to a dual action of NO, comprising an activation of sphingomyelinases and an inhibition of ceramidase activity. In this study we show that the NO-triggered inhibition of neutral ceramidase activity is paralleled by a down-regulation at the protein level. A complete loss of neutral ceramidase protein is obtained after 24 h of stimulation. Whereas the selective proteasome inhibitor lactacystin blocked NO-evoked ceramidase degradation, several caspase inhibitors were ineffective. Moreover, the NO-induced degradation is reversed by the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), and also by the physiological PKC activators platelet-derived growth factor-BB (PDGF), angiotensin II and ATP, resulting in a normalization of neutral ceramidase protein as well as activity. In vivo phosphorylation studies using (32)P(i)-labeled mesangial cells revealed that TPA, PDGF, angiotensin II, and ATP trigger an increased phosphorylation of the neutral ceramidase, which is blocked by the broad spectrum PKC inhibitor Ro-31 8220 but not by CGP 41251, which has a preferential action on Ca(2+)-dependent isoforms, thus suggesting the involvement of a Ca(2+)-independent PKC isoform. In vitro phosphorylation assays using recombinant PKC isoenzymes and neutral ceramidase immunoprecipitated from unstimulated mesangial cells show that particularly the PKC-delta isoform and to a lesser extent the PKC-alpha isoform are efficient in directly phosphorylating neutral ceramidase. In summary, our data show that NO is able to induce degradation of neutral ceramidase, thereby promoting accumulation of ceramide in the cell. This effect is reversed by PKC activation, most probably by the PKC-delta isoenzyme, which can directly phosphorylate and thereby prevent neutral ceramidase degradation. These novel regulatory interactions will provide therapeutically valuable information to target neutral ceramidase stability and subsequent ceramide accumulation.
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PMID:Nitric oxide induces degradation of the neutral ceramidase in rat renal mesangial cells and is counterregulated by protein kinase C. 1235 35

Exposure of 3T3/A31 cells to serum-free medium, one type of apoptotic stimulus, causes a rapid increase in the sphingosine (Sph) level, which initiates a series of processes: (i) activation of caspase 3 through an enhanced "cascade" of caspases, (ii) release of the C-terminal-half kinase domain of PKCdelta (PKCdelta KD) by caspase 3, and (iii) activation of Sph-dependent kinase 1 (SDK1), which was previously identified as PKCdelta KD. The activation of caspase 3 and release of PKCdelta KD are inhibited strongly by the incubation of cells with the ceramidase inhibitor D-erythro-2-tetradecanoylamino-1-phenyl-1-propanol and, to a much lesser extent, by L-cycloserine, an inhibitor of de novo ceramide synthesis. Exogenous addition of Sph or N,N-dimethyl-Sph to U937 cells causes caspase 3 activation and release of PKCdelta KD (SDK1), leading to apoptosis. The Sph-induced apoptotic process associated with activation of caspase 3 and release of PKCdelta KD (SDK1) may promote the proapoptotic effect of BAD or BAX through an increase of phosphorylated 14-3-3. In addition, Sph induces apoptosis through a separate process: the blocking of "survival signal" through the Akt kinase pathway induced by alpha3beta1-mediated cell adhesion to laminin 10/11 in extracellular matrix. We hereby propose a unified concept of Sph-dependent apoptosis based on these multiple mechanisms operating in concert.
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PMID:Sphingosine-dependent apoptosis: a unified concept based on multiple mechanisms operating in concert. 1546

Human keratinocytes (KC), when cultured under conditions to remain undifferentiated or to terminally differentiate, changed their cellular distribution of CD1d. As studied by confocal microscopy, undifferentiated KC had a pool of cytoplasmic CD1d, whereas after terminal differentiation, this molecule localized in the cell membrane, which recapitulates CD1d expression in vivo. A comparison of undifferentiated and differentiated cultured KC did not reveal any differences in the association with beta(2)-microglobulin, invariant chain of class II MHC, or patterns of glycosylation, suggesting that these biochemical properties are not regulating the cellular distribution of CD1d. Time-course studies of CD1d gene expression indicated that KC slowly increased gene expression with CaCl(2)-induced terminal differentiation. Increased CD1d gene expression was dependent on ceramide synthesis, because fumonisin B1, a ceramide synthetase inhibitor, blocked the increase in CD1d gene expression during terminal differentiation. Similarly, exogenous ceramide or the ceramidase inhibitor, B13, induced CD1d gene expression by undifferentiated, but not terminally differentiated, KC. A protein kinase C-zeta (PKC-zeta) inhibitor (a pseudosubstrate oligopeptide), but not a PKC-alphabeta inhibitor, significantly decreased CD1d gene expression by undifferentiated or ceramide-stimulated cultured, undifferentiated KC. As expected, downstream signaling events of PKC-zeta (JNK phosphorylation and NF-kappaBeta accumulation in the nucleus) were also attenuated. The calcineurin phosphatase inhibitor cyclosporine A, which blocks KC terminal differentiation, also blocked CD1d gene expression by cultured KC. In conclusion, this novel function of cellular ceramides extends the importance of this class of biologically active lipids beyond that of terminal differentiation and barrier function in normal human skin.
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PMID:Ceramide-dependent regulation of human epidermal keratinocyte CD1d expression during terminal differentiation. 1645 21

Ceramides are the main lipid component maintaining the lamellae structure of stratum corneum, as well as lipid second messengers for the regulation of cellular proliferation and/or apoptosis. In our previous study, psoriatic skin lesions showed marked decreased levels of ceramides and signaling molecules, specially protein kinase C-alpha (PKC-alpha) and c-jun N-terminal kinase (JNK) in proportion to the psoriasis area and severity index (PASI) scores, which suggested that the depletion of ceramide is responsible for epidermal hyperproliferation of psoriasis via downregulation of proapoptotic signal cascade such as PKC-alpha and JNK. In this study, we investigated the protein expression of serine palmitoyltransferase (SPT) and ceramidase, two major ceramide metabolizing enzymes, in both psoriatic epidermis and non-lesional epidermis. The expression of SPT, the ceramide generating enzyme in the de novo synthesis in psoriatic epidermis, was significantly less than that of the non-lesional epidermis, which was inversely correlated with PASI score. However, the expression of ceramidase, the degradative enzyme of ceramides, showed no significant difference between the lesional epidermis and the non-lesional epidermis of psoriatic patients. This might suggest that decreased expression of SPT protein is one of the important causative factors for decreased ceramide levels in psoriasis.
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PMID:A study on altered expression of serine palmitoyltransferase and ceramidase in psoriatic skin lesion. 1798 36


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