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.

Cell death by apoptosis can be caused by the DNA mutagen UV light whose exposure causes the direct activation of both the caspase 9 regulated cell damage intrinsic pathway and the caspase 8 regulated plasma membrane extrinsic pathway. We determined that increased activity of the plasma membrane phospholipid scramblase, PLSCR1, amplified UV mediated apoptosis primarily through the activation of the intrinsic apoptotic pathway. The caspase 8 inhibitor z-IETD-fmk was not as effective an inhibitor of PLSCR1 augmented UV induced apoptosis compared to treatment with caspase 3, caspase 9, or pan-caspase inhibitors. The inability of the caspase 8 inhibitor to decrease UV induced apoptosis was dependent on PLSCR1, as UV induced apoptosis was decreased by a similar amount in the control cells in the presence of inhibitors of caspase 8, caspase 9, caspase 3, or the pan-caspase inhibitor. PKC-δ directly phosphorylates human PLSCR1 resulting in increased PLSCR1 scramblase activity. PKC-δ can also be activated by caspase mediated cleavage resulting in the release of a constitutively active kinase domain. We observed that replacing the PKC-δ phosphorylation site of PLSCR1 with an alanine did not affect the ability of PLSCR1 to enhance UV induced apoptosis implying that PKC-δ does not directly phosphorylate PLSCR1 to increase plasma membrane scramblase activity during apoptosis. Cells transfected with a PLSCR1 mutant that contained an alanine substitution at its known PKC-δ phosphorylation site underwent UV induced apoptosis at a level similar to those transfected with wild type PLSCR1. The combined results indicate that UV exposure in cells possessing PLSCR1 increases apoptosis primarily by enhancement of the intrinsic apoptotic pathway, and also imply that the increased apoptosis observed upon exposure to UV light is not through direct phosphorylation of PLSCR1 by PKC-δ.

Externalization of PtdSer (phosphatidylserine) is an important event in signalling removal of apoptotic cells. In contrast with previous work [Yu, Byers, Ridgway, McMaster and Cook (2000) Biochim. Biophys. Acta 1487, 296-308] with U937 cells showing that specific stimulation of PtdSer biosynthesis during apoptosis was caspase dependent, PtdSer biosynthesis in CHO (Chinese-hamster ovary)-K1 increased 2.5-fold during UV-induced apoptosis but was not reversed by a caspase inhibitor, Z-VAD-FMK (benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone). Also, in CHO-K1 cells, stimulation of synthesis was less specific for PtdSer as similar levels of stimulation were observed for sphingomyelin biosynthesis. Involvement of PtdSer synthase isoforms was tested in CHO-K1 cells overexpressing PSS I (PtdSer synthase I) and PSS II. Both types of transformed cells showed resistance to UV-induced apoptosis based on the decreased levels of caspase 3 activation and morphology changes; externalization of PtdSer was reduced with UV treatment even though expression of endogenous scramblase increased slightly. Serine-labelling experiments showed that PSS I- or PSS II-expressing cells had higher basal levels of PtdSer biosynthesis compared with vector control cells. When cells were exposed to UV light to induce apoptosis, PtdSer biosynthesis was further stimulated 1.5- and 2-fold in PSS I- and PSS II-expressing cells respectively compared with UV-treated vector cells. Caspase activation was not required, as Z-VAD-FMK did not change PtdSer synthesis. Although enhanced PtdSer synthesis was supposed to facilitate apoptosis, cells overexpressing PSS I and II were actually resistant to UV-induced apoptosis. Whereas enhanced PtdSer synthesis was associated with apoptosis, potential anti-apoptotic effects were observed when excess activity of these synthetic enzymes was present. This suggests a tightly regulated role for PtdSer synthesis and/or an important dependence on compartmentation of PSS enzymes in association with scramblase facilitated enrichment of this phospholipid at the cell surface.

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) are essential proteins that are implicated in coordination of membrane-cytoskeletal signalling events, such as cell adhesion, migration, secretion, and phagocytosis in a variety of cell types. The most prominent structural feature of MARCKS and MRP is a central basic effector domain (ED) that binds F-actin, Ca2+-calmodulin, and acidic phospholipids; phosphorylation of key serine residues within the ED by protein kinase C (PKC) prevents the above interactions. While the precise roles of MARCKS and MRP have not been established, recent attention has focussed on the high affinity of the MARCKS ED for phosphatidylinositol 4,5-bisphosphate (PIP2), and a model has emerged in which calmodulin- or PKC-mediated regulation of these proteins at specific membrane sites could in turn control spatial availability of PIP2. The present review summarizes recent progress in this area and discusses how the above model might explain a role for MARCKS and MRP in activation of phospholipase D and other PIP2-dependent cellular processes.

Members of the phospholipid scramblase (PLSCR) family play active roles in altering lipid asymmetry at the plasma membrane including phosphatidylserine (PtdSer) exposure on the cell surface. To determine whether PtdSer biosynthesis and externalization are altered by PLSCR activities during apoptosis, Chinese hamster ovary K1 cell lines stably overexpressing PLSCR1 and PLSCR2 were established. PLSCR1 was localized on the plasma membrane, whereas PLSCR2 was predominantly in the nucleus. Cells overexpressing PLSCR1 showed suppressed growth, altered cell morphology, and higher basal levels of cell death. Following UV irradiation, these cells showed earlier and enhanced PtdSer exposure, increased caspase-3 activation, apoptotic nuclear changes, and PARP cleavage indicative of apoptosis. UV irradiation in cells overexpressing PLSCR1 led to a 4-fold stimulation of PtdSer synthesis (accompanied by increased movement of newly made PtdSer into microvesicles) relative to untreated PLSCR1 cells, whereas PtdSer formation in UV-irradiated vector control cells increased only by 2-fold. No differences in these responses were observed between PLSCR2-expressing cells and vector controls. PtdSer synthesis and its transbilayer movement stimulated by PLSCR1 overexpression were blocked by a caspase inhibitor along with progression of apoptosis. Thus, our studies showed that overexpression of PLSCR1 in Chinese hamster ovary K1 cells stimulated caspase-dependent PtdSer externalization and synthesis, implying an up-regulation of PtdSer formation in response to enhanced outward movement of this phospholipid to the cell surface during apoptosis. PLSCR1 also appears to influence progression of UV-induced apoptosis and could be a point of regulation or intervention during programmed cell death.

Microglial activation by amyloid β-protein in senile plaques contributes to neurodegeneration in Alzheimer disease. In BV-2 microglial cells, amyloid β-protein 1–40 (Aβ 1–40) elicited a dose-dependent increase (3–4 fold) of Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP), two protein kinase C substrates implicated in membrane-cytoskeletal alterations underlying microglial adhesion, migration, secretion, and phagocytosis. Neither MARCKS nor MRP was induced by the amyloid fragment Aβ 25–35, although both Aβ 1–40 and Aβ 25–35 caused extensive aggregation of BV-2 cells. Interferon-γ synergistically enhanced the induction by Aβ 1–40 of inducible nitric oxide synthase, but not MARCKS or MRP. Our results suggest that MARCKS and MRP may play important roles in microglia activated by amyloid peptides.

This chapter describes the chain elongation of long-chain fatty acids, formation of monounsaturated fatty acids by oxidative desaturation, formation of polyunsaturated fatty acids, unsaturated fatty acids with trans double bonds, abnormal patterns of distribution and metabolism of long-chain saturated and unsaturated fatty acids, and regulation through sensors and receptors. Within membranes, acyl chain length and the number and position of double bonds markedly influence the fluidity, permeability, and stability of biological membranes. De novo synthesis of fatty acids produces mainly 16-carbon palmitic acid, with minor amounts of 18-carbon stearic acid. In liver, brain, and other tissues, two primary systems—one in the endoplasmic reticulum (ER) and the other in mitochondria—provide chain elongation. Monounsaturated fatty acids are formed in mammalian systems by direct oxidative desaturation of a preformed long-chain saturated fatty acid. Although application of cloning, molecular probes, and genetic technologies to desaturation and chain elongation enzymes has substantially progressed recently, however the full power of molecular probes and techniques is yet to be realized in this realm.

Myristoylated alanine‐rich C kinase substrate (MARCKS) and MARCKS‐related protein (MRP) have been implicated in membrane‐cytoskeletal events underlying cell adhesion, migration, secretion, and phagocytosis. In BV‐2 microglial cells, lipopolysaccharide (LPS) elicited a dose‐dependent increase in mRNA of both MRP (sixfold) and MARCKS (threefold) with corresponding increases in [3H]myristoylated and immunoreactive protein levels. LPS also produced significant increases in protein kinase C (PKC)‐β twofold and PKC‐ε (1.5‐fold). Pro‐inflammatory cytokines produced by activated microglia (IL‐1β, IL‐6, TNF‐α) did not mimic LPS effects on MARCKS or MRP expression when added individually or in combination. LPS and IFN‐γ produced a synergistic induction of iNOS but not MARCKS or MRP. Induction of MARCKS and MRP by LPS was completely blocked by inhibitors of NF‐κB (PDTC) and protein tyrosine kinases (herbimycin A), partially blocked by the p38 kinase inhibitor SB203580, and unaffected by the MEK inhibitor PD98059. LPS induction of iNOS was considerably more sensitive to all these inhibitors. The Src kinase inhibitor PP2 had no effect, while the closely related inhibitor PP1 actually increased LPS induction of MARCKS and MRP. Our results suggest that MARCKS and MRP may play an important role in LPS‐activated microglia, but are not part of the neuroinflammatory response produced by cytokines.

Oxysterol binding protein (OSBP) translocation between Golgi and vesicular/cytoplasmic compartments is affected by conditions that alter cholesterol and sphingomyelin homeostasis, indicating a role in lipid and sterol regulation in this organelle. In this study, we show that OSBP dissociation from the Golgi apparatus was inhibited when LDL cholesterol efflux from lysosomes was blocked in Niemann-Pick C (NPC) or U18666A [3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one]-treated fibroblasts. Dissociation of OSBP from the Golgi apparatus in response to LDL was independent of de novo cholesterol biosynthesis. OSBP did not localize with filipin-stained lysosomal cholesterol, and the NPC defect did not alter OSBP expression or phosphorylation. However, OSBP in the Golgi apparatus was progressively dephosphorylated (as assessed by a molecular mass shift on SDS-PAGE) in U18666A-treated fibroblasts or Chinese hamster ovary cells as a result of combined inhibition of LDL cholesterol transport and de novo cholesterol synthesis. In vivo phosphopeptide mapping and mutagenesis of OSBP was used to identify the cholesterol-sensitive phosphorylation sites at serines 381, 384, and 387 that were responsible for the altered mobility on SDS-PAGE. NPC-1 protein-mediated release of LDL-derived cholesterol and de novo biosynthesis regulates OSBP localization and phosphorylation. This indicates that OSBP responds to or senses altered cellular sterol content and transport.

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.

Externalization of phosphatidylserine (PtdSer) is a common feature of programmed cell death and plays an important role in the recognition and removal of apoptotic cells. In this study with U937 cells, PtdSer synthesis from [3H]serine was stimulated and newly synthesized PtdSer was transferred preferentially to cell-free medium vesicles (CFMV) from cells when apoptosis was induced with a topoisomerase I inhibitor, camptothecin (CAM). When CAM-induced apoptosis was blocked by a caspase inhibitor, z-VAD-fmk, stimulation of PtdSer synthesis and movement to CFMV were abolished. In contrast, changes in synthesis and transport of sphingomyelin (SM) or phosphatidylethanolamine (PtdEtn) were minor; total phosphatidylcholine (PtdCho) synthesis was below control levels. All phospholipids appeared in CFMV but PtdSer displayed a 6-fold increase relative to controls compared to 3-fold for SM, 2-fold for PtdCho and 1.8-fold for PtdEtn. Even greater effects on specificity of PtdSer synthesis, movement to CFMV and inhibition by z-VAD-fmk were observed in apoptotic cells induced by UV irradiation or tumor necrosis factor-α/cycloheximide treatment. Thus, PtdSer biosynthesis stimulated during apoptosis in U937 cells was specific for this phospholipid and was correlated with caspase-mediated exposure of PtdSer at the cell surface and preferential movement to vesicles during apoptosis.

The lethal consequences of imbalances in lipid and sterol metabolism in human diseases such as atherosclerosis and lipid storage disorders underscores our need to know how cholesterol, phospholipid and sphingolipid metabolism is integrated. Accumulation and abnormal localization of lipids and sterol affects cellular function not only by perturbing membrane activity but also by increasing production of bioactive lipids derived from cholesterol, phospholipids and sphingolipids. For example in the NPC mouse model, accumulation of intracellular cholesterol and sphingomyelin is accompanied by increased sphingosine [187], a potent regular of protein kinase C and cell proliferation [152]. Oxidized LDL has an important role in the pathology of atherosclerosis by promoting foam cell formation and cytotoxicity [65]. 7-Hydroxycholesterol and 7-ketocholesterol are involved in many aspects of oxidized LDL activity including initiation of apoptosis in a number of cell types [188, 189] and enhancing cholesterol accumulation by inhibiting efflux [190]. Oxysterols formed intracellularly or from oxidized lipoproteins could have an important role in regulating lipid metabolism in the foam cell. Bioactive metabolites of phospholipids, such as diglyceride, phosphatidic acid and lysolipids, could also increase in circumstances of elevated deposition and have profound and varied effects on cell physiology. In addition to elucidating mechanisms for integration of lipid metabolism, we should determine when these responses go awry and assess the influence of bioactive compounds formed under these circumstances on cell viability and growth.

Progesterone inhibits intracellular transport of lysosomal cholesterol in cultured cells, and thus at least in part mimics the biochemical phenotype of Niemann–Pick type C disease (NPC) in human fibroblasts. The goal of this study was to determine whether metabolism of progesterone to other steroids is affected by the NPC mutation or by P-glycoprotein (a known progesterone target). We found that human fibroblasts metabolize progesterone in three steps: rapid conversion to 5α-pregnane-3,20-dione, which is then reduced to 5α-pregnane-3β(α)-ol-20-one with subsequent 6α-hydroxylation. The pattern and rates of progesterone metabolism were not significantly different in a variety of fibroblasts from normal individuals, NPC patients, and obligate heterozygotes. Inhibition of steroid 5α-reductase with finasteride completely blocked metabolism of progesterone but had no effect on inhibition of LDL-stimulated cholesterol esterification (IC50=10 μM). Progesterone also partially inhibited 25-hydroxycholesterol-induced cholesterol esterification, with similar dose-dependence in normal and NPC fibroblasts. P-glycoprotein levels varied significantly among the various fibroblasts tested, but no correlation with NPC phenotype or rate of progesterone metabolism was noted, and P-glycoprotein inhibitors did not affect conversion of progesterone to products. These results indicate that metabolism of progesterone in human fibroblasts is largely independent of its ability to interfere with cholesterol traffic and P-glycoprotein function.