Sherry C. Morash

Phosphatidylcholine biosynthesis in cultured glioma cells: evidence for channeling of intermediates

The major pathway of choline (Cho) incorporation into phosphatidylcholine (PtdCho) in mammalian cells is sequential conversion of Cho to phosphocholine (PCho), cytidinediphosphate choline (CDP-Cho) and PtdCho. In intact cells, this sequence is usually demonstrated using radiolabeled Cho since PCho and CDP-Cho do not enter the cell intact. We have studied the incorporation of radiolabeled Cho, PCho and CDP-Cho into rat glioma (C6) cells following electropermeabilization. C6 cells were permeable as judged by [U-14C]sucrose and Erythrosin B uptake and more rapid incorporation of [1,2,3-3H]glycerol into cell lipids, and viable as assessed by uptake and incorporation of [methyl-3H]Cho, [1-14C]oleate and [1,2,3-3H]glycerol into complex lipids. Despite rapid incorporation of [methyl-3H]Cho into PtdCho in permeabilized cells, there was no incorporation of [methyl-14C]PCho or CDP-[methyl-14C]Cho into PtdCho. PCho (300 microM) and CDP-Cho (300 microM) failed to significantly reduce incorporation of 28 microM [methyl-3H]Cho into PtdCho. Radioactivity in PtdCho of cells prelabeled with [methyl-3H]Cho prior to permeabilization could be chased with 4 mM Cho but not with 4 mM PCho or 4 mM CDP-Cho. The water-soluble products of Cho metabolism--PCho, CDP-Cho and glycerophosphocholine--were retained at 37 degrees C in permeabilized cells compared with controls while there was uniform leakage from permeabilized cells at 4 degrees C. Hemicholinium-3, an inhibitor of high-affinity Cho transport, decreased [methyl-3H]Cho incorporation into PtdCho in permeabilized cells, as in controls, suggesting that even in permeabilized cells, Cho incorporation into PtdCho is linked to the transport system. We propose that individual steps of the cytidine pathway of PtdCho biosynthesis are functionally linked and that reaction intermediates are not freely diffusible within the cell but are channeled to PtdCho biosynthesis.

Phosphatidylcholine metabolism in cultured cells: catabolism via glycerophosphocholine

The catabolism of phosphatidylcholine (PtdCho) has been studied in cultured murine neuroblastoma (N1E-115), C6 glioma, rat brain primary glia, and human fibroblast cells. Cells were pulse labelled for 96 h with [methyl-3H]choline followed by a chase for up to 24 h in medium containing 4 mM choline. Measurement of the radioactivity and mass of choline-containing compounds in these cells indicated that the major degradative pathway is PtdCho----lysophosphatidylcholine (lysoPtdCho)----glycerophosphocholine (GroPCho)----choline. At all times during the chase, PtdCho, sphingomyelin and lysoPtdCho comprised 72-92% of the cell-associated radioactivity; the remaining 10-30% was water-soluble and was chiefly GroPCho (30-80%) in all cell lines. In fibroblasts, however, phosphocholine (PCho) was also a major labelled water-soluble component (33-54%). The specific activity of GroPCho closely parallelled that of PtdCho in fibroblasts, but decreased faster than PtdCho in C6 and N1E-115 cells. We postulate that this may be due to distinct pools of PtdCho in the cell with differing rates of turnover. The changes in specific activity of PCho suggest that the major portion is formed by synthesis rather than as a degradative product. However, the inability to reduce the specific activity of this fraction to that of the intracellular choline suggests that a portion may be derived from either PtdCho or GroPCho.

Lysophosphatidylcholine as an intermediate in phosphatidylcholine metabolism and glycerophosphocholine synthesis in cultured cells: an evaluation of the roles of 1-acyl- and 2-acyl-lysophosphatidylcholine

Previous studies in our laboratory have shown that the principal pathway of phosphatidylcholine (PtdCho) degradation in cultured mouse N1E-115 neuroblastoma, C6 rat glioma, primary rat brain glia and human fibroblasts is PtdCho----lysophosphatidylcholine (lysoPtdCho)----glycerophosphocholine (GroPCho)----glycerophosphate plus choline (Morash, S.C. et al. (1988) Biochim. Biophys. Acta 961, 194-202). GroPCho is the first quantitatively major degradation product in this pathway, and could be formed by phospholipases A1 or A2, followed by lysophospholipase, or by a co-ordinated attack releasing both fatty acids by phospholipase B. The quality and quantities of lysoPtdCho present in cells reflect the nature of the initial hydrolysis step (A1 or A2), specificities of the lysophospholipases, and activities of acyltransferases that form PtdCho from lysoPtdCho. The present study was undertaken to elucidate the relative importance of these pathways by examining the fate of exogenous 1-acyl and 2-acyl-lysoPtdCho incubated with N1E-115 and C6 cells in culture. By fatty acid composition, endogenous lysoPtdCho was found to be mainly 1-acyl in both cell types based on a predominance of saturated acyl species; this suggested either preferential further deacylation or reacylation of 2-acyl-lysoPtdCho, or that 2-acyl-lysoPtdCho was not formed. Exogenous 1- and 2-acyl-lysoPtdCho specifically radiolabelled with choline and/or fatty acid were incubated either singly or as equimolar mixtures with cells. Cell association was rapid and not reversible by washing and both species were taken up at similar rates. The 2-acyl species was acylated to PtdCho faster than the 1-acyl species in both cell lines. Acylation of both lyso species was higher in C6 compared to N1E-115 cells. Hydrolysis of lysoPtdCho to GroPCho was higher in N1E-115 cells and with 1-acyl-lysoPtdCho. Transacylation between two molecules of lysoPtdCho was a minor pathway. These results document the variety and relative importance of reactions of lysoPtdCho metabolism; under similar conditions, 1- and 2-acyl-lysoPtdCho are handled differently. Both species turn over actively, but only the 1-acyl species accumulates while 2-acyl-lysoPtdCho is likely to be reacylated to form PtdCho.

Lipopolysaccharide stimulates differential expression of myristoylated protein kinase C substrates in murine microglia.

Microglia rapidly respond to lipopolysaccharide (LPS) by transformation from resting to active states and secretion of several neuro‐ and immuno‐regulators including tumour necrosis factor alpha (TNF‐α), interleukin 1β (IL‐1β), and interleukin 6 (IL‐6). With longer LPS treatment, microglia are converted to reactive or phagocytic states with characteristics similar to macrophages in inflammation and injury processes. We have investigated LPS‐mediated changes in two myristoylated substrates of protein kinase C (PKC): MARCKS (myristoylated alanine‐rich C kinase substrate) and MRP (MARCKS‐related protein). Within 6 hours of addition, LPS induced a twofold increase in [3H]myristoylated and immunoreactive MARCKS protein and a sevenfold increase in MRP. The differential effect of LPS on expression of MRP vs. MARCKS was even more dramatic at the level of transcription: S1 nuclease protection assays revealed a 40‐fold increase in MRP mRNA levels (maximum at 4–6 hours), whereas a threefold increase was observed for MARCKS. TNFα and colony‐stimulating factor 1 (CSF‐1), two cytokines which are induced by LPS, did not reproduce the observed effect of LPS on MARCKS and MRP gene transcription. CSF‐1 also induced differential transcription of MRP, but of lower magnitude (threefold) and more sustained than by LPS. Accordingly, these two substrates for PKC are differentially up‐regulated by LPS, apparently independent of TNFα or CSF‐1.

Phorbol ester stimulation of phosphatidylcholine synthesis in four cultured neural cell lines: correlations with expression of protein kinase C isoforms.

Phosphatidylcholine (PtdCho) can provide lipid second messengers involved in signal transduction pathways. As a measure of phospholipid turnover in response to extracellular stimulation, we investigated differential enhancement of [3H]choline incorporation into PtdCho by phorbol esters. In C6 rat glioma and SK-N-SH human neuroblastoma cells, [3H]PtdCho synthesis was 2–4 fold stimulated by β-12-O-tetradecanoylphorbol-13-acetate (β-TPA) when [3H]choline was incubated simultaneously with, or 15 min prior to, β-TPA treatment. By contrast, in N1E-115 mouse and SK-N-MC human neuroblastoma cells, phorbol esters had no appreciable effect on [3H]choline incorporation; however, in all cells, 200 μM oleic acid enhanced PtdCho synthesis, indicating a stimulable process. Alterations by thymeleatoxin (TMT), an activator of conventional PKC isoforms (α, β and γ), were similar to β-TPA. We investigated whether expression of specific PKC isoforms might correlate with these effects of phorbol esters on PtdCho synthesis. All cell lines bound phorbol esters, had PKC activity that was translocated by phorbol esters and differentially expressed isoforms of PKC. Northern and western blot analyses, using specific cDNA and antibodies for PKC-α,-β,-γ,-δ,-ε, and-ζ, revealed that expression of α-isoform predominated in C6 and SK-N-SH cells. In contrast, TPA-responsive β-isoform predominated in SK-N-MC cells. γ-PKC was not detected in any cells and only in C6 cells was PKC-δ present and translocated by β-TPA treatment. PKC-ε was not detected in SK-N-MC cell lines but translocated with TPA treatment in the other three cell lines. PKC-ζ was present in all cells but was unaltered by TPA treatment. Accordingly, stimulation of PtdCho turnover by phorbol esters correlated only with expression of PKC-α; presence of PKC-β alone was insufficient for a TPA response.

Overexpression of MARCKS, but Not Protein Kinase C‐α, Increases Phorbol Ester‐Stimulated Synthesis of Phosphatidylcholine in Human SK‐N‐MC Neuroblastoma Cells

Abstract: To investigate the regulation of phorbol ester‐stimulated synthesis of phosphatidylcholine (PtdCho), myristoylated alanine‐rich protein kinase C substrate (MARCKS) and the α‐isoform of protein kinase C (PKC‐α) were overexpressed in a human neuroblastoma (SK‐N‐MC) cell line that does not increase PtdCho synthesis in response to 4β‐12‐O‐tetradecanoylphorbol 13‐acetate (TPA). In five clones with a less than fivefold increase in MARCKS protein level, the synthesis of PtdCho from [methyl‐3H]choline was stimulated 1.88–2.34‐fold in the presence of 100–200 nM TPA. In clones overexpressing PKC‐α (30–40‐fold increased level of protein) or in mock‐transfected vector controls, TPA had much less of a stimulatory effect (1.04–1.43‐fold) on PtdCho synthesis. TPA caused translocation of PKC‐α and increased phosphorylation of MARCKS, indicating that both overexpressed proteins responded to stimulation. Thus, in SK‐N‐MC cells, MARCKS is required for TPA‐stimulated synthesis of PtdCho, and PKC‐α alone is insufficient for supporting enhanced synthesis.

Activation of phospholipase D by PKC and GTPγS in human neuroblastoma cells overexpressing MARCKS

Regulation of phospholipase D (PLD) activity participating in signal transduction involves complex interactions with small G-proteins (ARF, Rho) and protein kinase C isoforms (PKCalpha). In SK-N-MC human neuroblastoma cells, phorbol ester (TPA) activation of PLD was enhanced by overexpressing myristoylated alanine-rich C kinase substrate (MARCKS). To study MARCKS interactions with PLD, we investigated PLD isoform expression and activation by TPA and GTPgammaS in intact and digitonin-permeabilized clones transfected with MARCKS (M22). PLD2 was in both cytosol and membrane fractions while PLD1 was primarily membrane-associated in both vector control and M22 cells; location or quantities were unaltered by TPA treatment. TPA-stimulated PLD activity was higher in both intact and digitonin-permeabilized M22 cells than in vector controls. In contrast, GTPgammaS-stimulated PLD activity was independent of MARCKS expression but was additive with MARCKS-PKC-dependent activation in permeabilized cells. Combinations of PKC inhibition and down-regulation in intact and permeabilized (with GTPgammaS present) cells indicated that a PKC-mediated phosphorylation event was necessary in intact cells without access to GTPgammaS, stimulation of PLD mediated by GTPgammaS was independent of PKC, and PLD activation by PKC in permeabilized cells was kinase-independent. Western blot analysis showed that MARCKS, PKCalpha, PLD1 and PLD2 were present in a detergent-insoluble fraction (DIF); GTPgammaS increased recovery of PLD2 in DIF. Disruption of cholesterol-rich DIFs with digitonin, cyclodextrin or filipin potentiated activation of PLD by TPA. Our studies suggest that activation of PLD by PKC requires MARCKS and can involve both phosphorylation-independent and -dependent processes. As PLD activation by GTPgammaS is PKC-MARCKS-independent, MARCKS may provide a fine tuning component in conjunction with G-protein-mediated mechanisms for regulation of PLD.

Expression of MARCKS Effector Domain Mutants Alters Phospholipase D Activity and Cytoskeletal Morphology of SK-N-MC Neuroblastoma Cells

Stable overexpression of myristoylated alanine-rich C-kinase substrate (MARCKS) is known to enhance phorbol ester stimulation of phospholipase D (PLD) activity and protein kinase Cα (PKCα) levels in SK–N–MC neuroblastoma cells. In contrast, expression of MARCKS mutants (S152A or S156A) lacking key PKC phosphorylation sites within the central basic effector domain (ED) had no significant effect on PLD activity or PKCα levels relative to vector control cells. Like control cells, those expressing wild type MARCKS were elongated and possessed longitudinally oriented stress fibers, although these cells were more prone to detach from the substratum and undergo cell death upon phorbol ester treatment. However, cells expressing MARCKS ED mutants were irregularly shaped and stress fibers were either shorter or less abundant, and cell adhesion and viability were not affected. These results suggest that intact phosphorylation sites within the MARCKS ED are required for PLD activation and influence both membrane-cytoskeletal organization and cell viability.

Protein kinase C isoforms and growth, differentiation and phosphatidylcholine turnover in human neuroblastoma cells.

Neuroblastoma and glioma cells differentially express isoforms of protein kinase C (PKC) and myristoylated PKC substrates (e.g. MARCKS). Correlation with metabolism of membrane phospholipids suggests that PKC-alpha and MARCKS may be required to mediate phosphatidylcholine turnover stimulated by phorbol ester (beta-TPA). To evaluate relationships to neural cell differentiation, SK-N-SH human neuroblastoma cells were treated with 20 nM beta-TPA. In beta-TPA-treated cells, growth arrest and differentiation occurred (neurite extension; 40-60% decrease in cell number, total protein and RNA). By day 4, mRNA for PKC-alpha and MARCKS increased and, after an initial decrease, PKC-alpha protein also increased. At day 4, phosphatidylcholine synthesis was 3-5 fold greater than in control cells. In contrast, C6 glioma cells treated with beta-TPA showed no growth arrest, decreased PKC-alpha protein (< 20%) and lower phosphatidylcholine synthesis. Thus, induced differentiation of human neuroblastoma cells involved increased expression of PKC-alpha and MARCKS and synthesis of phosphatidylcholine, consistent with involvement of PKC-alpha and MARCKS in regulation of phosphatidylcholine turnover during neurite growth.

Phospholipase D Activities and Phosphatidylcholine Turnover are Differentially Related to Expression of Protein Kinase C Isoforms and Marcks in Control and Transfected Neural Cells

Phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) are major membrane phospholipids constituting 70–80% of the total phospholipids in most ceils (Vance, 1991; Kent, 1995). Accordingly, they have major roles in maintaining the structural integrity of membrane boundaries between cells and within subcellular organelles and compartments. Both PtdCho and PtdEtn are potential contributors to the generation of lipid second messengers following activation of phospholipases A2 (PLA2), C (PLC) and D (PLD) (Kiss et al, 1991; Kiss et al., 1994; Exton, 1990). Following activation of PLA2, release of polyunsaturated fatty acids may result in bioactive oxygenated products including eicosanoids, leukotrienes and other derivatives. There is increasing evidence that fatty acids also may activate kinases or alter gene expression through receptors and nuclear response elements (Ailhaud et al., 1994; Graber et al., 1994). Release of diacylglycerol (DAG) and phosphatidic acid by PLC and PLD, respectively, also is important in signal transduction (Exton, 1990; Nishizuka, 1992). Initial production of DAG is part of a dual release of second messengers (DAG and inositol tris-phosphate) when phosphatidylinositol bisphosphate is hydrolyzed by PLC. Subsequent, sustained production of DAG appears to involve hydrolysis of PtdCho and PtdEtn (Nishizuka, 1992).