Regulation of MARCKS and MARCKS-related protein expression in BV-2 microglial cells in response to lipopolysaccharide.

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)-beta twofold and PKC-epsilon (1.5-fold). Pro-inflammatory cytokines produced by activated microglia (IL-1beta, IL-6, TNF-alpha) did not mimic LPS effects on MARCKS or MRP expression when added individually or in combination. LPS and IFN-gamma 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-kappaB (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.

Golgi localization and phosphorylation of oxysterol binding protein in Niemann-Pick C and U18666A-treated cells.

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.

Activation of phospholipase D by PKC and GTPgammaS 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.

Preferential externalization of newly synthesized phosphatidylserine in apoptotic U937 cells is dependent on caspase-mediated pathways.

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 [(3)H]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-alpha/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.

Myristoylated alanine-rich C-kinase substrate is phosphorylated and translocated by a phorbol ester-insensitive and calcium-independent protein kinase C isoform in C6 glioma cell membranes.

Myristoylated alanine-rich C-kinase substrate (MARCKS), a prominent substrate for conventional and novel protein kinase C (PKC) isoforms, is involved in the regulation of membrane-cytoskeletal interactions. Addition of [gamma-32P]ATP to the membrane fraction of digitonin-permeabilized C6 glioma cells resulted in phosphorylation and release of MARCKS, indicating involvement of an active membrane-bound kinase. Pretreatment of cells with 2 microM 4 beta-12-O-tetradecanoyl-phorbol-13-acetate (beta-TPA) for 18 h downregulated conventional (PKC alpha) and novel (PKC delta) isoforms of PKC by > 90% in both membrane and soluble fractions, but did not inhibit the rate of ATP-dependent phosphorylation or release of MARCKS, or decrease levels of membrane-bound PKC zeta or PKC mu. MARCKS phosphorylation was inhibited by staurosporine, bis-indolylmaleimide (a PKC-specific inhibitor), Gö6983 (inhibits all isoforms except PKC mu), and a peptide from the calmodulin-binding domain of MARCKS, but was unaffected by EGTA or Gö6976 (inhibits cPKCs and PKC mu). Peptide mapping indicated similar in vivo and in vitro phosphorylation at serine residue(s) known to be phosphorylated by PKC. These findings support a novel mechanism by which MARCKS may be regulated by an atypical PKC isoform in phorbol ester-downregulated cells.

U18666A inhibits intracellular cholesterol transport and neurotransmitter release in human neuroblastoma cells.

To determine if neurochemical function might be impaired in cell models with altered cholesterol balance, we studied the effects of U18666A (3-beta-[(2-diethyl-amino)ethoxy]androst-5-en-17-one) on intracellular cholesterol metabolism in three human neuroblastoma cell lines (SK-N-SH, SK-N-MC, and SH-SY5Y). U18666A (< or =0.2 microg/ml) completely inhibited low density lipoprotein (LDL)-stimulated cholesterol esterification in SK-N-SH cells, while cholesterol esterification stimulated by 25-hydroxycholesterol or bacterial sphingomyelinase was unaffected or partially inhibited, respectively. U18666A also blocked LDL-stimulated downregulation of LDL receptor and caused lysosomal accumulation of cholesterol as measured by filipin staining. U18666A treatment for 18 h resulted in 70% inhibition of K+-evoked norepinephrine release in phorbol ester-differentiated SH-SY5Y cells, while release stimulated by the calcium ionophore A23187 was only slightly affected. These results suggest that U 18666A may preferentially block a voltage-regulated Ca2+ channel involved in norepinephrine release and that alterations in neurotransmitter secretion might be a feature of disorders such as Niemann-Pick Type C, in which intracellular cholesterol transport and distribution are impaired.

Chinese hamster ovary cells overexpressing the oxysterol binding protein (OSBP) display enhanced synthesis of sphingomyelin in response to 25-hydroxycholesterol.

25-Hydroxycholesterol negatively regulates cholesterol synthesis and activates cholesterol esterification in a variety of cultured cells. Concurrent with these effects, 25-hydroxycholesterol also stimulates the synthesis of sphingomyelin in Chinese hamster ovary (CHO)-K1 cells. The role of oxysterol binding protein (OSBP), a high affinity receptor for 25-hydroxycholesterol, in activation of SM synthesis was assessed by overexpression in CHO-K1 cells. When compared to mock transfected controls, three CHO-K1 clones overexpressing OSBP by 10- to 15-fold displayed a 2- to 3-fold enhancement of [3H]serine incorporation into sphingomyelin when treated with 25-hydroxycholesterol. Closer examination of one of these clones (CHO-OSBP cells) revealed a >8.5-fold stimulation of sphingomyelin synthesis after a 6-h treatment with 25-hydroxycholesterol compared to 3.5-fold in controls, slightly higher basal levels of sphingomyelin synthesis, and a more rapid response to 25-hydroxycholesterol. [3H]serine incorporation into phosphatidylserine, phosphatidylethanolamine, ceramide, or glucosylceramide was affected by <15%. Synthesis of sphingomyelin from exogenous [3H]sphinganine-labeled ceramide was enhanced in overexpressing cells treated with 25-hydroxycholesterol. However, in vitro activities of sphinganine N-acyltransferase, sphingomyelin synthase, and serine palmitoyltransferase were not affected by OSBP overexpression or 25-hydroxycholesterol. Overexpression of OSBP or 25-hydroxycholesterol did not significantly affect the ceramide content of Golgi-enriched fractions from control or overexpressing cells. However, diglyceride mass was reduced in Golgi-enriched fractions from overexpressing cells and by treatment with 25-hydroxycholesterol. Results from overexpressing cells show that OSBP potentiates the stimulatory effects of 25-hydroxycholesterol on sphingomyelin synthesis. 25-Hydroxycholesterol promotes translocation of OSBP to the Golgi apparatus where it appears to stimulate conversion of ceramide to sphingomyelin.

Integration of phospholipid and sterol metabolism in mammalian cells.

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 metabolism in human fibroblasts is independent of P-glycoprotein levels and Niemann-Pick type C disease.

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 5alpha-pregnane-3,20-dione, which is then reduced to 5alpha-pregnane-3beta(alpha)-ol-20-one with subsequent 6alpha-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 5alpha-reductase with finasteride completely blocked metabolism of progesterone but had no effect on inhibition of LDL-stimulated cholesterol esterification (IC50 = 10 microM). 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.

Differential effects of sphingomyelin hydrolysis and cholesterol transport on oxysterol-binding protein phosphorylation and Golgi localization.

The deposition of de novo synthesized and lipoprotein-derived cholesterol at the plasma membrane and transport to the endoplasmic reticulum is dependent on sphingomyelin (SM) content. Here we show that hydrolysis of plasma membrane SM in Chinese hamster ovary cells by exogenous bacterial sphingomyelinase resulted in enhanced cholesterol esterification at the endoplasmic reticulum and rapid dephosphorylation of the oxysterol-binding protein (OSBP), a cytosolic/Golgi receptor for oxysterols such as 25-hydroxycholesterol. After sphingomyelinase treatment, restoration of OSBP phosphorylation closely paralleled resynthesis of SM and down-regulation of cholesterol ester synthesis. SM hydrolysis activated an okadaic acid-sensitive phosphatase that was not stimulated in Chinese hamster ovary cells by short chain ceramides. Agents that specifically blocked sphingomyelinase-mediated delivery of cholesterol to acyl-CoA:cholesterol acyltransferase (U18666A) or promoted cholesterol efflux to the medium (cyclodextrin) did not inhibit OSBP dephosphorylation. SM hydrolysis also promoted OSBP translocation from a vesicular compartment to the Golgi apparatus. Cyclodextrin and U18666A also caused OSBP translocation to the Golgi apparatus, suggesting that OSBP movement is coupled to changes in the cholesterol content of the plasma membrane or Golgi apparatus. These results identify OSBP as a potential target of SM turnover and cholesterol mobilization at the plasma membrane and/or Golgi apparatus.

Cholesterol regulates oxysterol binding protein (OSBP) phosphorylation and Golgi localization in Chinese hamster ovary cells: correlation with stimulation of sphingomyelin synthesis by 25-hydroxycholesterol.

Sphingomyelin (SM) and cholesterol content is positively correlated in cellular membranes, and in several pathological and experimental conditions there is evidence for coregulation. The potential role of oxysterols and oxysterol binding protein (OSBP) in mediating the coregulation of cholesterol and SM was examined using Chinese hamster ovary (CHO) and cholesterol auxotrophic, sterol regulatory defective (SRD) 6 cells. SRD 6 cells grown in the presence or absence of cholesterol for 24 h displayed a 30-50% reduction in SM synthesis compared with control CHO 7 cells. SM synthesis in CHO 7 and cholesterol-supplemented SRD 6 cells was stimulated 2-fold by 25-hydroxycholesterol, but cholesterol-starved SRD 6 cells were unresponsive. Basal and 25-hydroxycholesterol-stimulated SM synthesis was also inhibited in lovastatin-treated wild-type CHO-K1 cells. Lack of 25-hydroxycholesterol activation of SM synthesis in cholesterol-starved SRD 6 and lovastatin-treated CHO-K1 cells was correlated with dephosphorylation of OSBP. In SRD 6 cells, this was evident after 12 h of cholesterol depletion, it occurred equally at all phosphorylation sites and was exacerbated by 25-hydroxycholesterol. Unlike CHO 7 cells, where OSBP was observed in small vesicles and the cytoplasm, OSBP in cholesterol-starved SRD 6 cells was constitutively localized in the Golgi apparatus. Supplementation with non-lipoprotein cholesterol promoted redistribution to vesicles and the cytoplasm. Similarly, OSBP in CHO-K1 cells grown in delipidated serum was predominantly in the Golgi apparatus. Low-density lipoprotein (LDL) supplementation of CHO-K1 cells caused the redistribution of OSBP to the cytoplasm and small vesicles, and this effect was blocked by pharmacological agents ¿3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one and progesterone¿, which inhibited LDL cholesterol efflux from lysosomes. The results showed that localization of OSBP between the Golgi apparatus and a cytoplasmic/vesicular compartment was responsive to changes in cholesterol content and trafficking. In cholesterol depleted SRD 6 cells, this was accompanied by dephosphorylation of OSBP and attenuation of 25-hydroxycholesterol activation of SM synthesis.

Lipopolysaccharide induction of MARCKS-related protein and cytokine secretion are differentially impaired in microglia from LPS-nonresponsive (C3H/HeJ) mice.

Many events involved in activation of microglia and leukocytes by lipopolysaccharide (LPS) are mediated by protein kinase C (PKC), and we have recently demonstrated that a major PKC substrate, MARCKS-related protein (MRP), is selectively induced by LPS in murine microglia. In microglia from LPS-nonresponsive (C3H/HeJ) mice, induction of MRP and secretion of CSF-1 required much higher LPS concentrations (> or = 100 ng/ml) than in normal (C3H/OuJ) microglia (< or = 10 ng/ml). By contrast, TNF alpha production was not significantly increased in C3H/HeJ microglia even at 1 microgram LPS/ml. Microglia expressed PKC isoforms alpha, beta, delta, and zeta (but not gamma and epsilon); PKC isoform levels were similar in both normal and C3H/HeJ microglia and no significant change in response to LPS was noted. Our results indicate that LPS alters PKC substrate (rather than kinase) expression, and that the Lpsd mutation in C3H/HeJ mice differentially affects regulation of several gene products implicated in microglial function.

Overexpression of myristoylated alanine-rich C-kinase substrate enhances activation of phospholipase D by protein kinase C in SK-N-MC human neuroblastoma cells.

Signal transduction can involve the activation of protein kinase C (PKC) and the subsequent phosphorylation of protein substrates, including myristoylated alanine-rich C kinase substrate (MARCKS). Previously we showed that stimulation of phosphatidylcholine (PtdCho) synthesis by PMA in SK-N-MC human neuroblastoma cells required overexpression of MARCKS, whereas PKCalpha alone was insufficient. We have now investigated the role of MARCKS in PMA-stimulated PtdCho hydrolysis by phospholipase D (PLD). Overexpression of MARCKS enhanced PLD activity 1.3-2.5-fold compared with vector controls in unstimulated cells, and 3-4-fold in cells stimulated with 100 nM PMA. PMA-stimulated PLD activity was blocked by the PKC inhibitor bisindolylmaleimide. Activation of PLD by PMA was linear with time to 60 min, whereas stimulation of PtdCho synthesis by PMA in clones overexpressing MARCKS was observed after a 15 min time lag, suggesting that the hydrolysis of PtdCho by PLD preceded synthesis. The formation of phosphatidylbutanol by PLD was greatest when PtdCho was the predominantly labelled phospholipid, indicating that PtdCho was the preferred, but not the only, phospholipid substrate for PLD. Cells overexpressing MARCKS had 2-fold higher levels of PKCalpha than in vector control cells analysed by Western blot analysis; levels of PKCbeta and PLD were similar in all clones. The loss of both MARCKS and PKCalpha expression at higher subcultures of the clones was paralleled by the loss of stimulation of PLD activity and PtdCho synthesis by PMA. Our results show that MARCKS is an essential link in the PKC-mediated activation of PtdCho-specific PLD in these cells and that the stimulation of PtdCho synthesis by PMA is a secondary response.

Phospholipase D activity is altered in X-linked adrenoleukodystrophy heterozygous carriers, but not in hemizygous patients.

Abnormalities in levels of choline and its metabolites have been reported in the lesions of brains of X-linked adrenoleukodystrophy (X-ALD) patients. We have examined the turnover of the major choline-containing phospholipid, phosphatidylcholine (PtdCho), in fibroblasts from hemizygous X-ALD, heterozygous X-ALD, Zellweger syndrome (ZW), and male and female control individuals to assess possible alterations in PtdCho metabolism mediated by activation of protein kinase C (PKC). Hydrolysis of PtdCho by phospholipase D (PLD) and resynthesis of PtdCho from labeled choline were stimulated 2- to 4-fold by PKC activation with the phorbol ester, 4beta-12-O-tetradecanoylphorbol-13-acetate (beta-TPA), in all cells except those from heterozygous X-ALD individuals. No differences in quantity or intracellular distribution of PKC activity, PKC isoforms by Western blot analysis, or of the PKC substrate, myristoylated alanine-rich C kinase substrate (MARCKS), were apparent in any of the cells. Thus, altered PtdCho metabolism was not directly linked to either of these inherited defects that result in abnormal peroxisomal functions. Further, altered responsiveness of PLD in X-ALD heterozygotes was independent of changes in PKC and MARCKS.

Diacylglycerol molecular species in plasma membrane and microsomes change transiently with endothelin-1 treatment of glioma cells.

Agonist-induced intracellular signal transduction often involves activation of protein kinase C by diacylglycerol (DAG) released from membrane phospholipids by phospholipases. Using either DAG kinase or HPLC assays to quantitatively determine DAG mass, we observed a time-dependent increase in DAG accumulation upon incubation of rat C6 glioma cells with 200 nM endothelin-1 (ET-1). Total cell DAG rapidly increased by 25-35% from a basal level of 4.5 +/- 0.3 nmol/mg protein during one min of ET-1 treatment and remained constant or slightly decreased between 1 and 2 min. Thereafter, DAG increased to a maximum (1.6-fold above basal) by 5-10 min. and remained elevated to 30 min. Resolution of DAG molecular species by HPLC after incubation of cells with ET-1 revealed that accumulation of DAG species differed in total cell lysate and subcellular compartments. In plasma membrane, major DAG species increased at 1 min. followed by a decrease at 10 min. whereas in microsomes DAG species did not change at 1 min. and decreased at 10 min. Although phospholipid sources of DAG species were not identified specifically, there was preferential hydrolysis of molecular species of phospholipid for DAG production. We propose that molecular species of DAG produced at the plasma membrane may be transferred to the endoplasmic reticulum so that phospholipid resynthesis can replenish molecular species initially utilized in signal transduction.

Inhibition of phosphorylation of the oxysterol binding protein by brefeldin A.

Oxysterol binding protein (OSBP), a high affinity receptor for 25-hydroxycholesterol that localizes to a Golgi/vesicular compartment, migrated on SDS-PAGE as a doublet of 96 and 101 kDa. The reduced mobility of the upper band of this doublet is the result of phosphorylation on multiple serine residues. Phosphorylation of rabbit OSBP stably overexpressed in CHO-K1 cells was altered by staurosporine and okadaic acid, while other protein kinase activators and inhibitors such as TPA, sphingosine and bis-indolylmaleimide were without affect. Treatment of overexpressing and control cells with brefeldin A (BFA) caused dephosphorylation of OSBP that coincided with disruption of the Golgi apparatus. [32P]Phosphate pulse-chase and immunoprecipitation experiments showed that BFA inhibited phosphorylation of OSBP, but not its rate of dephosphorylation. Phosphopeptide maps of OSBP from overexpressing and control CHO-K1 cells were similar, and BFA promoted dephosphorylation of all five peptides. Compared to overexpressing cells, one tryptic phosphopeptide was more abundant in control CHO-K1 cells and was preferentially dephosphorylated by BFA treatment. OSBP was phosphorylated in vitro by the Golgi enriched fraction of CHO-K1 cells or rat liver by a staurosporine- and BFA-insensitive kinase. The phosphorylation status of OSBP was not affected by 25-hydroxycholesterol and did not alter in vitro 25-[3H]hydroxycholesterol binding. Furthermore, dephosphorylation of OSBP by staurosporine did not affect 25-hydroxycholesterol-mediated localization to the Golgi apparatus. Rapid phosphorylation/dephosphorylation of OSBP requires interaction with the Golgi apparatus and an associated kinase. (c) 1998 Elsevier Science B.V.

Involvement of phospholipase D and protein kinase C in phorbol ester and fatty acid stimulated turnover of phosphatidylcholine and phosphatidylethanolamine in neural cells.

Hydrolysis of phosphatidylcholine (PtdCho) can provide lipid second messengers involved in sustained signal transduction. Four neural-derived cell lines (C6 rat glioma; N1E-115 mouse and SK-N-MC and SK-N-SH human neuroblastoma) express different protein kinase C (PKC) isoforms and differentially respond to 4beta-12-O-tetradecanoylphorbol-13-acetate (beta-TPA)-stimulation of PtdCho synthesis. We examined involvement of PLD and PKC in the hydrolysis and resynthesis of PtdCho and phosphatidylethanolamine stimulated by beta-TPA, bryostatin (a non-phorbol PKC activator) and oleic acid (18:1n-9) in the four cell lines. beta-TPA or bryostatin produced similar enhancement of [3H]Cho incorporation, loss of stimulated synthesis after down regulation of PKC, and activation of PLD. In C6 cells, staurosporine (STS) and bis-indolylmaleimide (BIM) only partially inhibited basal and beta-TPA-stimulated PLD activity measured as choline or ethanolamine release; phosphatidylbutanol formation after prelabeling with [9,10-3H]18:1n-9, [9,10-3H]myristic acid (14:0), [1-14C]eicosapentaenoic acid (20:5n-3) or 1-O-[alkyl-1', 2-3H]-sn-glyceryl-3-phosphorylcholine gave similar results. STS at >200 nM activated PLD in the presence or absence of beta-TPA. In SK-N-SH cells where PtdCho synthesis was stimulated by beta-TPA or bryostatin, no effect of these agents on PLD was observed. 18:1n-9 stimulated PtdCho synthesis and, to a lesser extent, hydrolysis by PLD both with and without beta-TPA present. Fatty acids had no effect on PKC activities and down regulation of PKC with beta-TPA enhanced fatty acid stimulation of PtdCho synthesis. Thus, activation of PLD hydrolysis preceding resynthesis is involved in the stimulatory effects of beta-TPA on PtdCho synthesis in some but not all of these neural derived cells. Further, PLD hydrolysis of PtdCho and PtdEtn appear to have differing aspects of regulation. Fatty acid regulation of PtdCho synthesis occurs independent of PKC activation. Accordingly, regulation of membrane phospholipid degradation and resynthesis in association with lipid second messenger generation can involve a complex interplay of PLD, PKC, and fatty acids. (c) 1998 Elsevier Science B.V.

trans Fatty acids: infant and fetal development.

This review evaluates scientific data associated with the possibility that trans fatty acids compromise fetal and infant early development. Concerns have been triggered by research that has heightened our awareness of the importance of n-3 and n-6 fatty acids; shown that trans fatty acids inhibit delta6 desaturation of linoleic acid; identified trans fatty acid isomers in fetal, infant, and maternal tissues; and reported an inverse association between the trans fatty acid content of tissue lipids and measures of growth and development. Animal studies provide little evidence that trans fatty acids influence growth, reproduction, or gross aspects of fetal development. However, these models may not have been appropriate for addressing all the subtle effects that influence development of human infant retinal, neural, or brain function. Human studies are hampered by the complexity of the interrelations among nutritional, genetic, and environmental factors and by ethical considerations that constrain the research design. Existing data have not established a causal relation between trans fatty acid intake and early development. Conclusions cannot be drawn from the possible association found between trans fatty acid exposure and lower n-3 and n-6 long-chain polyunsaturated fatty acids and growth because of confounding factors. Few studies addressed the question of whether trans fatty acids adversely affect human fetal growth. One study reported a correlation between the trans fatty acid content of plasma and birth weight of preterm infants and one study reported a relation between preterm births and the trans fatty acid content of maternal plasma. Limited associative data have addressed whether trans fatty acids adversely affect fetal and infant neurodevelopment and growth. The interpretation of existing research and development of recommendations should be done cautiously. Suggestions for research to clarify these issues are made.

Altered regulation of cholesterol and cholesteryl ester synthesis in Chinese-hamster ovary cells overexpressing the oxysterol-binding protein is dependent on the pleckstrin homology domain.

Oxysterol-binding protein (OSBP) is a high-affinity receptor for a variety of oxysterols, such as 25-hydroxycholesterol, that down-regulate cholesterol synthesis and stimulate cholesterol esterification. To examine a potential role for OSBP in regulating cholesterol metabolism, we stably overexpressed this protein in Chinese-hamster ovary (CHO)-K1 cells. Compared with mock-transfected controls, several cell lines overexpressing wild-type OSBP (CHO-OSBP) displayed a 50% decrease in cholesteryl ester synthesis when cultured in medium with delipidated serum, 25-hydroxycholesterol or low-density lipoprotein (LDL). CHO-OSBP cells showed a 40-60% decrease in acyl-CoA:cholesterol acyltransferase activity and mRNA, a 50% elevation in mRNA for three sterol-regulated genes [LDL receptor, 3-hydroxy-3-methylgluraryl (HMG)-CoA reductase and HMG-CoA synthase], and an 80% increase in [14C]acetate incorporation into cholesterol. CHO-K1 cells overexpressing two OSBP mutants with a complete or N-terminal deletion of the pleckstrin homology (PH) domain had cholesterol esterification and synthesis rates that were similar to those shown by mock-transfected controls. Unlike wild-type OSBP, both PH domain mutants displayed diffuse cytoplasmic immunofluorescence staining and did not translocate to the Golgi apparatus in the presence of 25-hydroxycholesterol. CHO-K1 cells overexpressing OSBP have pronounced alterations in cholesterol esterification and synthesis, indicating a potential role for this receptor in cholesterol homoeostasis. The phenotype observed in cells overexpressing OSBP is dependent on the PH domain, which appears to be necessary for ligand-dependent localization of OSBP to the Golgi apparatus.

Decreased phosphatidylcholine biosynthesis and abnormal distribution of CTP:phosphocholine cytidylyltransferase in cholesterol auxotrophic Chinese hamster ovary cells.

Phosphatidylcholine (PtdCho) biosynthesis was examined in the sterol regulatory defective (SRD) Chinese hamster ovary (CHO) cell line SRD 6. SRD 6 cells do not display transcriptional activation of sterol-regulated genes and are cholesterol auxotrophs. Compared to CHO 7 cells (parental line from which the SRD cells were derived), incorporation of [3H]choline during a 2-h pulse into PtdCho and sphingomyelin was reduced 3- and 4.5-fold, respectively, in SRD 6 cells grown with or without cholesterol. SRD 6 cells grown in cholesterol-free medium for 24 h had 8% less phosphatidylcholine (PtdCho) mass compared to CHO 7 cells. Consistent with impaired CTP:phosphocholine cytidylyltransferase activity, [3H]choline-labeled SRD 6 cells had elevated [3H]phosphocholine and delayed conversion to [3H]PtdCho during a 2-h chase period. Compared to CHO 7 cells, cytosolic cytidylyltransferase activity was elevated 2- to 3-fold in SRD 6 cells grown in the absence of cholesterol, but activity in the total membrane fraction was normal. Immunoblot analysis confirmed that cytidylyltransferase mass was increased 2-fold in SRD 6 total cell extracts and cytosol, but not membranes. The amount of [32P]phosphate-labeled cytidylyltransferase in cytosol and membranes of SRD 6 cells were similar to controls. Likewise, cytidylyltransferase mRNA levels were not significantly different between SRD 6 and CHO 7. The defect in PtdCho synthesis in SRD 6 cells could be overcome by treatment with 150 microns oleate, such that after 5 h [3H] choline incorporation into PtdCho and phosphocholine in SRD 6 and CHO 7 cells was similar. Cholesterol auxotrophic SRD 6 cells display reduced PtdCho mass and synthesis and elevated levels of cytosolic cytidylyltransferase, defects that were only partially corrected by growth in exogenous cholesterol. These results indicate a requirement for normal cholesterol regulation and synthesis in the maintenance PtdCho levels and activity of cytidylyltransferase.