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Phospholipase A2 inhibits nuclear nucleoside triphosphatase activity and mRNA export in isolated nuclei from rat liver
Life Sciences 73 (2003) 969 – 980 www.elsevier.com/locate/lifescie
Phospholipase A2 inhibits nuclear nucleoside triphosphatase activity and mRNA export in isolated nuclei from rat liver Ju-Xiang Li a,*, Zai-Quan Li b, Yong-Zheng Pang b, Chao-Shu Tang a,b a
Department of Physiology and Pathophysiology, Health-Science Center, Peking University, Beijing 100083, China b Institute of Cardiovascular Research, First Hospital, Peking University, Beijing 100034, China Received 1 July 2002; accepted 14 January 2003
Abstract The present study is undertaken to investigate whether the phospholipase A2 (PLA2) influences mRNA nucleocytoplasmic transport evaluated by nucleoside triphosphatase (NTPase) activity and mRNA export in isolated hepatic nuclear envelope. Isolated hepatic nuclei from rat liver were exposed to PLA2 (10 5 f 10 2/ml) with or without incorporation of nuclei with phosphatidylcholine (PC) liposome. Messenger RNA exports and NTPase activities of nuclear membrane were assayed using ATP and GTP as substrates. We found that the RNA efflux, evaluated by [3H] uridine, was potently decreased in a concentration-dependent manner, by incubation of hepatic nuclei with PLA2, regardless using ATP or GTP as substrates. The PC content in nuclear membrane was also decreased by PLA2-treatment. The PC was incorporated into the nuclear membrane by addition of phospholipid liposomes into the incubation mixture. PC incorporation into the nuclear membrane did not alter mRNA export. However this resulted in a significant increase in mRNA export rate in PLA2-treated group. Messenger RNA export rate in PLA2 (10 3 unit/mL)- treated nuclear membrane was positively correlated with level of PC incorporation, both using ATP and GTP as substrates. The activity of nucleoside triphosphatase, a nuclear membrane-associated enzyme, showed parallel variations with mRNA transport. It is concluded that nuclear PLA2 plays a regulatory role in RNA transport, which can be antagonized by exogenous PC. These might be pathophysiologically significance, although the mechanisms by which this effect takes place remain to be clarified. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Nuclear nucleoside triphosphatase; mRNA export; Phosphatidylcholine; Hepatocyte; Phospholipase A2
* Corresponding author. Tel.: +86-10-62092183; fax: +86-10-66176255. E-mail address: [email protected] (J.-X. Li). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00357-6
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Introduction The nuclear envelope (NE) consists of an inner and an outer nuclear membrane, which are separated by the nuclear pore complexes (Agutter and Prochnow, 1994; Gorlich and Mattaj, 1996). The outer nuclear membrane is continuous with the endoplasmic reticulum, whereas the inner membrane is linked with the nuclear lamina (Franke, 1974). The nuclear phospholipids are mainly localized on the nuclear membranes (Albi et al., 1997; Tomassoni et al., 1999a,b). Phospholipase A2 (PLA2), a class of enzymes that hydrolyze fatty acid from the sn-2 position of phospholipids, plays critical roles in the synthesis of various lipid mediators, such as arachidonic acid, eicosanoids, platelet activating factor and lysophosphatidylcholine (Farooqui et al., 1997). The presence of a neutral PLA2 at the nuclear level was first demonstrated in rat ascite hepatoma cells by Oishi et al. (1996). In the nuclear membranes of hepatocyte, PLA2 hydrolyzing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) at an equal rate was found recently by Neitcheva and Peeva (1995). PC was the major phospholipid in the nuclear membrane, PLA2, hydrolyse PC, was active in the nuclear membrane, taking into consideration the important role of this enzyme for maintaining the phospholipid composition (Neitcheva and Peeva, 1995) Several investigations have hinted at the fact that nuclear PLA2 could be linked to cell proliferation including protein synthesis (Martelli et al., 1999). Transport of mRNA from the nuclei to the cytoplasm through the nuclear pore is a key process of protein synthesis, which includes two major steps: the recognition of RNA molecules to be transported and their transfer through the nuclear pore (Hanover, 1992; Schroder et al., 1990). There is evidence that the latter step is involved in regulating the quantity of the mRNA export out of the nucleus (Schroder et al., 1990). The nucleocytoplasmic transport of mRNA is an energy-consuming process since the translocation of an average size polyA- mRNA requires the hydrolysis of approximately 103 ATP molecules (Agutter and Prochnow, 1994). The energy requirement is associated with the functioning of a nucleoside triphosphatase (NTPase) that, together with an 110kD poly A binding protein, constitutes the complex major regulatory element of mRNA transport (Agutter and Prochnow, 1994). There was data shown that alteration of the nuclear membrane fluidity and lipid metabolism might lead to the change in nuclear membrane structure, which in turn may alter NTPase activity and mRNA export (Tomassoni et al., 1999a,b; Ramjiawan et al., 1996, 1997). As nuclear PLA2 activity was considered as a major factor, which regulated fatty acid composition of nuclear phospholipids and membrane integrity, the present study was therefore undertaken to determine the effect of PLA2 on the nucleocytoplasmic transport of nuclear mRNA through evaluating mRNA export rate and NE NTPase activity. Our experimental approach was to modify the lipid composition of rat liver nuclear membrane by PLA2, incorporation of PC and to examine how such modification influence the mRNA export and NE NTPase activity.
Methods Animals and materials Male Sprague–Dawley (SD) rats weighting from 270 to 300 g were supplied by The Animal Center, Health-Science Center of Peking University. PLA2 and egg PC were purchased from Sigma Chemical Co St. Louis, MO. [3H] ATP (11 Ci/mmol), [3H] GTP (17 Ci/mmol), [3H]-uridine (50Ci/mmol) and [3H] poly (A)(600 Ci/mmol of nucleoside residue) from New England Nuclear.
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Isolation of hepatic nuclei and nuclear envelopes The nuclei and the nuclear membranes were isolated according to the procedure of Kaufman et al. (1983) with modification. All animals were fasted overnight with free access to water. Livers were removed under chloralose and urethane anesthesia and were minced, homogenized in 4 volumes STM/ PMSF buffer, filtered through 4 layers cheesecloth, sedimented at 800 g for 10 min. The pellet was washed once with STM/PMSF buffer. The nuclei were suspended in DS/PMSF buffer, layered over cushions of this buffer, and sedimented at 70,000 g for 60 min. Isolated nuclei were resuspended in STM/PMSF buffer, again layered over cushions of DS/PMSF buffer, and sedimented at 70,000 g for 30 min. Resuspension of the resulted nuclei in STM/PMSF to Ca. 4 108 nuclei/ml as purified nuclei, and stored at 70 jC. The nuclei were resuspended in STM/PMSF buffer (containing to a final concentration of 5 108/ ml, incubated for 60 min with 250 Ag/ml DNase 1 and 250 Ag/ml boiled RNase A, and sedimented for 10 min at 800 g. The nuclei were resuspended in LS/PMSF buffer. HS/PMSF buffer was immediately added drop wise to a concentration of 1.6 mol/L NaCl, 1% h-mercaptoethanol was added with gentle agitation, then incubated for 15 min and sedimented for 30 min at 1600 g. Extraction with 1.6 mol/L NaCl was performed again except that h-mercaptoethanol was omitted. After 15 min, aliquots were sedimented at 1600 g for 30 min. The NEs were stored at 70 jC in 50 mmol/L Tris-HCl, pH 7.4, 5 mmol/L MgCl2, l mmol/L EGTA, 250 mmol/L sucrose, and l mmol/L PMSF, l Amol/L leupeptin, l mmol/L DTT to obtain a final protein concentration of 1 mg/ml (measured using Lowry et al. method). STM buffer in mmol/L: 250 sucrose. 50 Tris/HCl pH 7.4, 5 MgCl2. DS Buffer: 2100 sucrose, 50 Tris/ HCl pH 7.4, 5 MgCl2. LS buffer in mmol/L: 0.2 MgCl2, 10 Tris/HCl, pH 7.4. HS buffer in mmol/L: 2000 NaCl in LS buffer. The designation /PMSF is meaning in the presence of 1 mmol/L serine esterase inhibitor phenylmethylsulfonyl fluoride. Liposome preparation Liposome preparation was carried out according to detergent-dialysis method on LIPOSOMATE previously described by Zhang et al. (1994). The prepared liposome was small unilamellar vesicles (diameter 32 F 6 nm) containing egg PC. Buffer was STM. In some experiments [14C]-labeled DOPC (dioleoyl PC, final concentration 5 ACi/ml) was added to prepare [14C]-labeled liposome. Protocol for in vitro incubation of isolated nuclei Incorporation of liposome into nuclei was done as descried previously by us (Zhang et al., 1994) with modification. Isolated purified nuclei were incubated for 16 f 18 h at 4 jC in the presence (as liposome-pretreated group) or absence of liposome (0.1, 0.5, 1.0 and 5.0 Amol PC/ml). The liposome/ nuclei mixtures were then centrifuged at 104,000 g for 25 min to separate the nuclei from the liposome vesicles. The nuclei pellet was washed once and then resuspended in STM/PMSF. Then, the nuclear suspension (108 nuclei/0.25 ml) were incubated with different agents (dissolved in 0.25 ml) for 10 min at 30 jC: buffer alone, and PLA2 10 5 f 10 2 units/ml. The reaction was stopped by cold (4 jC) centrifuge on microcentrifuge for 2 min, and the nuclei pellet was washed once and then resuspended in STM/PMSF. Then NE was prepared (above described) to assay NTPase activity and mRNA export.
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Assay of NTPase activity NTPase activity was assayed as descried by Ramjiawan et al. (1996), with modification. Prepared NE (25 Ag protein) in 200 Al buffer containing 250 mmol/L sucrose, 50 mmol/L Tris-HCl (pH 7.4), 5 mmol/L EDTA, l mmol/L MgCl2 were preincubated for 10 min at 30 jC. Addition of 1 mmol/L of ATP or GTP initiated the reaction. Ten minutes after incubation, the reaction was stopped by addition of 10% SDS and placing the test tube on ice bath, and inorganic phosphate was measured according to the method of Raess (Raess and Vincenzi, 1980) to calculate the NTPase activity. Preliminary experiments showed a linear relationship of NTPase activity with incubation time of nucleoside triphosphate within 30 min. Measurement of mRNA transport from nuclear envelope (Tiffany et al., 1995) Preparation of [3H]-labeled poly (A)+ mRNA was performed by inoculating Saccharomyces cerevisiae into PGY medium (1 g peptone, 3 g glucose, and 1 g yeast extract per liter) containing 2.5 mCi/L of [3H]-uridine. Yeast was grown for 4 days at 30 jC and harvested by centrifugation. [3H]labeled poly (A)+ mRNA was isolated from the yeast pellet by acid quanidinum thiocyanate-phenolchloroform extraction using a RNA isolation kit (bulletin 1, TEL-TEST), and by oligo (dT)-cellulose type 7 chromatography. mRNA concentration was determined by absorbance at 260 nm, and the purities were assayed by the 260/280 nm ratio. The [3H]-labeled poly (A)+ mRNA specific activity was 101.7 10 cpm/Ag. Poly (A)+ mRNA was trapped inside the NE during their preparation by adding 5 Ag [3H]-poly (A)+ mRNA to 100 Al of the heparin-containing lysis buffer. After the suspension was gently agitated at room temperature for 5 min, the NEs vesicles were pelleted by means of centrifugation at 5000 g for 10 min. The NE were resealed by washing them twice in 10 mmol/L Tris/HCl (pH 8.0), 100 mmol/L NaCl, 30 mmol/L KCl, 3 mmol/L MgCl2, and 0.5 mmol/L CaCl2, and then resuspended at 2 mg protein/ml. The reaction mixture was prepared on ice by layering in succession 10 Al 60% HCl4, 40 Al silicone oil, and 150 Al of the envelope vesicles. The reaction was initiated at 30 jC by adding ATP 1 mmol/L or GTP 1 mmol/L respectively. Blank tube was prepared simultaneously in the absence of ATP and GTP. After the appropriate time, the reaction was terminated by centrifugation. The lower layer, containing NE vesicles, was discarded, and the upper layer, containing exported mRNA, was aspirated and counted in scintillation fluid. The NTPase-dependent transport of mRNA was presented as difference between assayed tube and blank tube. Lipid extraction and analysis fractions After incubation, nuclear membrane was extracted by the method of Folch et al. with modification (Folch et al., 1957). Frozen membrane preparations were thawed at 25j and suspended in 1 mM potassium bicarbonate buffer. The suspensions were extracted twice with chloroform-methanol (2:1 by vol.) and then once with chloroform alone. The chloroform-rich layers were combined and the solvents were evaporated under a stream of N2 without heat. Lipid residues were redissolved in an accurately measured volume of chloroform- methanol and stored at 40j under N2 in tightly sealed vials. The extracts of the control and PLA2-treated nuclear membrane from the same rat were used as a pair of samples. Phospholipids were chromatographed on Silica gel 60 thin layer plates (Military Medical
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Science Institute, China). Chloroform/methanol/acetic acid/H2O (65:25:4:2 by vol.) were used as developing solvents. Identity of components was verified by comparison with authentic reference compounds: L-a- Lysophosphatidylcholine from bovine liver, L-a-phosphatidylcholine (Sigma Co.). The components were measured quantitatively by densitometric analysis of plates. Measurement of [14C]-Labeled liposome incorporation to nuclei Isolated purified nuclei (0.5 ml) were incubated for 16 f 18 h at 4 jC with [14C]-labeled-liposome (0.1, 0.5, 1.0 and 5.0 Amol PC/ml). The liposome/nuclei mixtures were then centrifuged at 104,000 g for 25 min to separate the nuclei form the liposomal vesicles. The nuclei pellet was washed twice, and NE was prepared as above described. The [14C]-radioactivity of NE was measured on Liquid Scintillation Analyzer (Packard, 1600RT). The amount of [14C]-labeled liposome incorporation into NE was presented as pmol PC/mg protein NE. Other measurements Protein content in NE was assayed by the Lowry method (Lowry et al., 1951). Various marker enzyme activities: Mannose-6-phosphatase, in both microsomes and nuclei, was determined as described by Gilchrist (Gilchrist and Pierce, 1993). NAD pyrophosphorylase activity, a specific marker for nuclei, was determined according to Crane and Low (1976). NADPH cytochrome-c reductase activity, a microsomal marker, was measured by the reduction of cytochrome c as described by Kornberg (1955). Data analysis Six independent experiments with duplication were done. All results are expressed as mean F SD. The statistical analysis of the data was performed using one-way analysis of variance followed by Student–Newman–Keuls tests. P < 0.05 was accepted as statistically significant.
Results Marker enzyme activity Experiments for nuclei and nuclear membrane purity showed that the activity of NADH pyrophosphorylase (as a marker enzyme for NE) in the prepared hepatic nuclei and NE were about 7-fold and 22Table 1 Specific activities of marker enzymes Enzymes (nmol mg
1
protein min
NAD pyrophosphorylase Mannose-6-phosphatase NADPH cytochrome-c reductase
1
)
Homogenate
Intact nuclei
Nuclear envelope
3.68 F 0.27 90.70 F 6.40 10.30 F 0.90
25.77 F 1.26 (56.5 F 2.76) 412.00 F 2.00 (36.60 F 0.20) 2.88 F 0.22 (2.30 F 0.20)
88.46 F 6.44 (15.90 F 1.20) 14.48 F 1.30 (0.10 F 0.00) 97.00 F 0.12 (6.20 F 0.00)
The enzyme activities are indicated in parentheses as percentage of total liver homogenate activity expressed as nmol Pi releasedmin 1 gr.liver 1.
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Fig. 1. PC incorporation into nuclear envelope.
folds higher than that in homogenate of whole cells, respectively (P < 0.01), but the activity of NADPH cytochrome-c reductase activities (marker enzyme for microsome) were only 30% and 7% of that in hepatocytes homogenate (P < 0.01), respectively. Activities of mannose-6-phosphatase existing both in microsomes and nuclei were 4 f 5 times greater in nuclei than and only 14% in NE of that in cell homogenate, respectively. It was demonstrated that the isolated cell nuclear fraction was in high purity and little contaminated by other organelle (Table 1). Phosphatidylcholine incorporation into nuclear envelope Incorporation of PC into NE was increased with the incubated liposome in a concentration dependent manner (Fig. 1). Effect of PLA2 on mRNA transport and NTPase activity of nuclear envelope The nuclei were treated separately with various concentrations of PLA2. PC loss from the membrane preparation was achieved. Incubations with PLA2 (10 3 units/ml) removed about 13.59% of the nuclear membrane PC (from 53.47% in control to 39.88% in PLA2 treatment group). PC underwent considerable degradation (Table 2). Table 2 Effect of PLA2 treatment on the major phospholipid content of rat liver nuclear membrane Phospholipids PC LPC
Ag/mg protein
% of TPL
Control
PLA2 treatment
Control
PLA2 treatment
103.40 F 14.51 11.42 F 1.34
77.22 F 11.03** 24.77 F 4.45*
53.47 F 4.88 5.91 F 0.40
39.88 F 3.44 12.77 F 1.49
Values are mean F SD (n = 6). *P < 0.05; **P < 0.01 as compared with control. TPL—total phospholipids; PC— phosphatidylcholine; LPC—lysophosphatidylcholine.
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Fig. 2. Effect of PLA2 on mRNA transport (Fig. 2a) and NTPase activity (Fig. 2b) of NE. mean F SD, n = 6, mRNA transport: ng RNAmg Pro 1min 1; NTPase activity: nmolmg Pro 1min 1. PLA2 [unit/ml] ATP 1 mmol/L as substrate; GTP 1 mmol/ L as substrate. *P < 0.05, **P < 0.01 compared with control ([0] PLA2). Correlation between mRNA export and NTPase activity. [ATP]: r = 0.99;Y = 29 F 145 [GTP]: r = 0.98; Y = 39 F 165 .
The effect of modification of NE by PLA2 on mRNA transport and NTPase activity are present in Fig. 2. Messenger RNA transport rate was decreased by PLA2 treatment. PLA2, at a concentration as low as 10 5 unit/mL, caused a decrease in mRNA efflux rate by 19% (for ATP as substrate) (P < 0.01). At Table 3 Effects of PC-liposome on mRNA transport and NTPase activity of nuclear envelope PC- [mol/L] 0 10 4 5 10 10 3 5 10
4
3
ATP (1 mmol/L) as substrate
GTP 1 (mmol/L) as substrate
mRNA export
NTPase activity
mRNA export
NTPase activity
0.84 0.90 0.86 0.80 0.92
92 90 96 88 89
1.24 1.32 1.18 1.36 1.27
162 155 168 160 158
F F F F F
0.05 0.07 0.07 0.06 0.09
F F F F F
Mean F SD, n = 6, mRNA transport: ng RNA mg Pro
6 7 4 7 6 1
min
1
F F F F F
0.12 0.12 0.13 0.14 0.11
; NTPase activity: nmol mg Pro
1
min
F F F F F 1
.
12 15 17 14 10
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higher concentrations of PLA2 (10 2 mol/L), the mRNA efflux rates was decreased by 52.4% (for ATP as substrate) and 40.3% (for GTP as substrate) (Fig. 2a). In parallel assays, the NTPase activities were determined. It was found that this enzyme also responded to the PLA2 with a change in specific activity similar to the respective alterations in RNA transport rate (Fig. 2b). At the concentration of 10 2 unit/ml of PLA2, a decrease of NTPase activity to 67.4% (for ATP as substrate) and 51.6% (for GTP as substrate) of the control were found. Effect of PC on PLA2-induced changes in mRNA transport and NTPase activity of nuclear envelope As it was shown in Table 3, incorporated the nuclei with various concentrations of PC, efflux of poly (A) mRNA and NTPase activity on the NE remained unchanged as compared with those of the control
Fig. 3. Effects of PC on PLA2-induced changes in mRNA transport and NTPase activity of NE. Mean F SD, n = 6, mRNA transport: ng RNAmg Pro 1min 1; NTPase activity: nmolmg Pro 1min 1. PLA2 (10 3 units/ml) *P < 0.05, **P < 0.01 compared with [0] PC, i.e. PLA2 alone. #P < 0.05, # #P < 0.01 compared with control. Correlation between mRNA export and NTPase activity [ATP]: r = 0.95, Y = 37 F 157 ; [GTP]: r = 0.96; Y = 32 F 154 .
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group (all P values >0.05). It showed that the increase of PC in nuclear membrane could not alter mRNA transport and NTPase activity. However, PC-liposome incorporation could partially antagonize the effect of PLA2 on mRNA transport and NTPase activity (Fig. 3). Incubation of the PC-incorporated nuclei with PLA2 caused a pronounced increase of RNA transport. RNA transport rates in NE from the control were 0.84 F 0.05 (for ATP as substrate) and 1.24 F 0.12 (for GTP as substrate) ng RNAmg Pro 1min 1. Treated the nuclei with PLA2 (10 3 units/ml), the RNA transport were 0.51 F 0.05 (for ATP as substrate) and 0.80 F 0.10 (for GTP as substrate) ng RNAmg Pro 1min 1, which were greatly higher than those of the control. Treated the PC-incorporated nuclei with PLA2 (10 3 unites/ml), export of mRNA was greater than those of PLA2 (10 3 unit/mL)-treated group. When PC-concentration was at 5 10 3unit/mL, the exports of mRNA were 0.72 F 0.08 (for ATP as substrate) and 1.17 F 0.12 (for GTP as substrate) ng RNAmg Pro 1min 1, which increased by 41.2% (P < 0.01) and 46.3% (P < 0.01), respectively as compared with those of the PLA2-treated groups (Fig. 3a). In parallel assays, it was found that NTPase activity also responded to the PLA2, paralleling the alterations in RNA transport. NTPase activities in NE from untreated cellular nuclei were 92 F 6 and 162 F 12 nmolmg Pro 1min 1. The specific activities of the NTPase in PLA2 (10 3 unit/ml)-treatment group were 44 F 4 (for ATP as substrate) and 90 F 10 (for GTP as substrate) nmolmg Pro 1min 1, respectively. Increasing concentrations of PC liposome incorporation antagonized the effect of PLA2 (10 3 units/ml) on NTPase activity, regardless using ATP or GTP as substrate. PC liposome at a concentration of 5 10 3 mol/L, increased the NTPase activity by 73.3% and 64.4% as compared with that of the PLA2-treated group, respectively (all P values less than 0.01), as shown in Fig. 3b.
Discussion Export of mRNA molecules from nucleus to the cytoplasm is an active process involved in energy consumption. Nuclear NTPase, a nuclear membrane-associated enzyme, provided energy for poly (A)+mRNA export through the nuclear pore. The ability of the nuclear NTPase to hydrolyze various nucleotides had been reported in the rank order of UTP > GTP > ITP > CTP > ATP (Ramjiawan et al., 1996, 1997; Tiffany et al., 1995). This enzyme is located on the inner face of the nuclear envelope near the pore, in which also binding sites for poly (A)-RNA are localized (Kondor-Koch et al., 1982; Prochnow et al., 1990). It is obvious that the membrane structure will be important for maintaining nuclear NTPase activity and the function of mRNA export. It is well recognized that PLA2 action towards organized lipid interfaces (vesicles, multilamellar dispersions and monolayers) is much higher than against isotropically dispersed phospholipids (Martelli et al., 1999). PLA2 selectively cleaved phospholipids (mostly PC) containing arachidonic acid at the sn-2 position. The 1-acyl lysoderivates produced are water-soluble and part of them spontaneously leaves the membrane, thus decreasing the total phospholipid protein ratio. Nuclear PLA2 activity might be related to changes in fatty acid composition of nuclear phospholipids (PC and PE) (Neitcheva and Peeva, 1995). In the present study, PC-liposomes incorporated into nuclei, which increased the content of PC, could not alter RNA transport and NTPase, indicating that excess of PC within the nuclei is insufficient to induce a significant change in nucleocytoplasmic export. We also found that RNA nucleocytoplasmic export and NTPase activity were decreased by PLA2 treatment.
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Nuclear membrane PC was also decreased by PLA2 incubation. These showed that degradation of NE phospholipid might affect the NTPase activity and RNA export. The incorporation of PC before PLA2 treatment, causing a PC-replenishment, inhibited the effect of PLA2 on the RNA transport and NTPase activity, indicating that PC composition in the NE might play an important role in the NTPase activity and RNA transport. Changes in the RNA translocation were also accompanied by concomitant alterations in the NTPase activities according to the previous works (Tomassoni et al., 1999a,b; Tiffany et al., 1995). As excessively activated phospholipase A2 might play a deleterious role in nuclear membrane integrity and the impairment of nuclear membrane could influence RNA nucleocytoplasmic transport (Tabuchi et al., 2000), it suggests that this loss of membrane integrity allowed the nuclear NTPase and RNA nucleocytoplasmic transport to be decreased by phospholipase A2. It can be speculated according to our experiment that PC-liposome, provided PLA2 with substrate, might compensate the nuclear membrane impairment induced by PLA2. Therefore, the incorporation of PC into the NE might antagonize the effect of PLA2, through protecting the membrane integrity. Although PC incorporated have the identical polar head groups but different acyl chains, but they still have the ability to cause the changes in nuclear NTPase and RNA nucleocytoplasmic transport in PLA2-treated nuclear membrane. Hence, the ability of PC antagonizing the effect PLA2 on the RNA transport and NTPase seem to depend on the nature of their polar head group but not on their hydrophobic chain. The results showed that nuclear NTPase activity and mRNA transport were attenuated by PLA2, which might act through changing composition of nuclear phospholipids. It has been shown that the nonpolar components of the membrane lipid bilayer exert a greater effect than the phospholipid head group in modulating the activity of membrane-bound enzymes (Tomassoni et al., 1999a,b). Our investigations demonstrated that PC, when incorporated into non-PLA2 -treated nuclei and maybe inducing alterations in membrane fluidity, have no effect on NTPase and RNA transportation. Therefore, it seems likely that the PC action in the present study is not fluidity controlled. However, whether PLA2 action on the nuclear fluidity involved in the mechanism of the decrease in NTPase and RNA transportation need to be further investigated. Since PLA2-treatment caused a decrease in NTPase activity and mRNA transport, and PC replenishment could reverse this effect, it is quite possible that the phospholipid composition of nuclear membrane facilitates the most optimal state of the NTPase and RNA export. Little attention has been given to the pathophysiological role of PLA2 in nucleocytoplasmic transportation in the present study. A variety of techniques indicated endogenous PLA2 to be mainly located in the nucleus in highly proliferative cells whereas its location was cytoplasmatic in nonproliferative cells (Martelli et al., 1999). When non-confluent cells were incubated with exogenous PLA2, the enzyme was internalized and, in the majority of cell, appeared within the nucleus (Martelli et al., 1999). Cytoplasmic nuclear PLA2 activity might associate with liver regeneration (Tabuchi et al., 2000). These findings suggested that excessively activated PLA2 might modify protein synthesis process through influencing NTPase and RNA transport under physiological and pathophysiological conditions, which would benefit from PC compensation according to our experiment. In conclusion, we may propose that the nucleocytoplasmic efflux of mRNA is influenced by PLA2 activity. PC incorporation into the nuclear membrane exerted a protective effect on PLA2-induced decrease in mRNA export.
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Tomassoni, M.L., Albi, E., Magni, M.V., 1999a. Changes of nuclear membrane fluidity during rat liver regeneration. Biochemistry and Molecular Biology International 47 (6), 1049 – 1059. Tomassoni, M.L., Amori, D., Magni, M.V., 1999b. Changes of nuclear membrane lipid composition affect RNA nucleocytoplasmic transport. Biochemical and Biophysical Research Communications 258 (2), 476 – 481. Zhang, L.Z., Ling, S.C., Zhao, J., Tang, C.S., 1994. Study on the targeting treatment for cardiovascular and cerebrovascular diseases with liposome. Journal of Beijing Medical University 26 (2), 144 – 152.
Phospholipase A2 inhibits nuclear nucleoside triphosphatase activity and mRNA export in isolated nuclei from rat liver Ju-Xiang Li a,*, Zai-Quan Li b, Yong-Zheng Pang b, Chao-Shu Tang a,b a
Department of Physiology and Pathophysiology, Health-Science Center, Peking University, Beijing 100083, China b Institute of Cardiovascular Research, First Hospital, Peking University, Beijing 100034, China Received 1 July 2002; accepted 14 January 2003
Abstract The present study is undertaken to investigate whether the phospholipase A2 (PLA2) influences mRNA nucleocytoplasmic transport evaluated by nucleoside triphosphatase (NTPase) activity and mRNA export in isolated hepatic nuclear envelope. Isolated hepatic nuclei from rat liver were exposed to PLA2 (10 5 f 10 2/ml) with or without incorporation of nuclei with phosphatidylcholine (PC) liposome. Messenger RNA exports and NTPase activities of nuclear membrane were assayed using ATP and GTP as substrates. We found that the RNA efflux, evaluated by [3H] uridine, was potently decreased in a concentration-dependent manner, by incubation of hepatic nuclei with PLA2, regardless using ATP or GTP as substrates. The PC content in nuclear membrane was also decreased by PLA2-treatment. The PC was incorporated into the nuclear membrane by addition of phospholipid liposomes into the incubation mixture. PC incorporation into the nuclear membrane did not alter mRNA export. However this resulted in a significant increase in mRNA export rate in PLA2-treated group. Messenger RNA export rate in PLA2 (10 3 unit/mL)- treated nuclear membrane was positively correlated with level of PC incorporation, both using ATP and GTP as substrates. The activity of nucleoside triphosphatase, a nuclear membrane-associated enzyme, showed parallel variations with mRNA transport. It is concluded that nuclear PLA2 plays a regulatory role in RNA transport, which can be antagonized by exogenous PC. These might be pathophysiologically significance, although the mechanisms by which this effect takes place remain to be clarified. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Nuclear nucleoside triphosphatase; mRNA export; Phosphatidylcholine; Hepatocyte; Phospholipase A2
* Corresponding author. Tel.: +86-10-62092183; fax: +86-10-66176255. E-mail address: [email protected] (J.-X. Li). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00357-6
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Introduction The nuclear envelope (NE) consists of an inner and an outer nuclear membrane, which are separated by the nuclear pore complexes (Agutter and Prochnow, 1994; Gorlich and Mattaj, 1996). The outer nuclear membrane is continuous with the endoplasmic reticulum, whereas the inner membrane is linked with the nuclear lamina (Franke, 1974). The nuclear phospholipids are mainly localized on the nuclear membranes (Albi et al., 1997; Tomassoni et al., 1999a,b). Phospholipase A2 (PLA2), a class of enzymes that hydrolyze fatty acid from the sn-2 position of phospholipids, plays critical roles in the synthesis of various lipid mediators, such as arachidonic acid, eicosanoids, platelet activating factor and lysophosphatidylcholine (Farooqui et al., 1997). The presence of a neutral PLA2 at the nuclear level was first demonstrated in rat ascite hepatoma cells by Oishi et al. (1996). In the nuclear membranes of hepatocyte, PLA2 hydrolyzing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) at an equal rate was found recently by Neitcheva and Peeva (1995). PC was the major phospholipid in the nuclear membrane, PLA2, hydrolyse PC, was active in the nuclear membrane, taking into consideration the important role of this enzyme for maintaining the phospholipid composition (Neitcheva and Peeva, 1995) Several investigations have hinted at the fact that nuclear PLA2 could be linked to cell proliferation including protein synthesis (Martelli et al., 1999). Transport of mRNA from the nuclei to the cytoplasm through the nuclear pore is a key process of protein synthesis, which includes two major steps: the recognition of RNA molecules to be transported and their transfer through the nuclear pore (Hanover, 1992; Schroder et al., 1990). There is evidence that the latter step is involved in regulating the quantity of the mRNA export out of the nucleus (Schroder et al., 1990). The nucleocytoplasmic transport of mRNA is an energy-consuming process since the translocation of an average size polyA- mRNA requires the hydrolysis of approximately 103 ATP molecules (Agutter and Prochnow, 1994). The energy requirement is associated with the functioning of a nucleoside triphosphatase (NTPase) that, together with an 110kD poly A binding protein, constitutes the complex major regulatory element of mRNA transport (Agutter and Prochnow, 1994). There was data shown that alteration of the nuclear membrane fluidity and lipid metabolism might lead to the change in nuclear membrane structure, which in turn may alter NTPase activity and mRNA export (Tomassoni et al., 1999a,b; Ramjiawan et al., 1996, 1997). As nuclear PLA2 activity was considered as a major factor, which regulated fatty acid composition of nuclear phospholipids and membrane integrity, the present study was therefore undertaken to determine the effect of PLA2 on the nucleocytoplasmic transport of nuclear mRNA through evaluating mRNA export rate and NE NTPase activity. Our experimental approach was to modify the lipid composition of rat liver nuclear membrane by PLA2, incorporation of PC and to examine how such modification influence the mRNA export and NE NTPase activity.
Methods Animals and materials Male Sprague–Dawley (SD) rats weighting from 270 to 300 g were supplied by The Animal Center, Health-Science Center of Peking University. PLA2 and egg PC were purchased from Sigma Chemical Co St. Louis, MO. [3H] ATP (11 Ci/mmol), [3H] GTP (17 Ci/mmol), [3H]-uridine (50Ci/mmol) and [3H] poly (A)(600 Ci/mmol of nucleoside residue) from New England Nuclear.
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Isolation of hepatic nuclei and nuclear envelopes The nuclei and the nuclear membranes were isolated according to the procedure of Kaufman et al. (1983) with modification. All animals were fasted overnight with free access to water. Livers were removed under chloralose and urethane anesthesia and were minced, homogenized in 4 volumes STM/ PMSF buffer, filtered through 4 layers cheesecloth, sedimented at 800 g for 10 min. The pellet was washed once with STM/PMSF buffer. The nuclei were suspended in DS/PMSF buffer, layered over cushions of this buffer, and sedimented at 70,000 g for 60 min. Isolated nuclei were resuspended in STM/PMSF buffer, again layered over cushions of DS/PMSF buffer, and sedimented at 70,000 g for 30 min. Resuspension of the resulted nuclei in STM/PMSF to Ca. 4 108 nuclei/ml as purified nuclei, and stored at 70 jC. The nuclei were resuspended in STM/PMSF buffer (containing to a final concentration of 5 108/ ml, incubated for 60 min with 250 Ag/ml DNase 1 and 250 Ag/ml boiled RNase A, and sedimented for 10 min at 800 g. The nuclei were resuspended in LS/PMSF buffer. HS/PMSF buffer was immediately added drop wise to a concentration of 1.6 mol/L NaCl, 1% h-mercaptoethanol was added with gentle agitation, then incubated for 15 min and sedimented for 30 min at 1600 g. Extraction with 1.6 mol/L NaCl was performed again except that h-mercaptoethanol was omitted. After 15 min, aliquots were sedimented at 1600 g for 30 min. The NEs were stored at 70 jC in 50 mmol/L Tris-HCl, pH 7.4, 5 mmol/L MgCl2, l mmol/L EGTA, 250 mmol/L sucrose, and l mmol/L PMSF, l Amol/L leupeptin, l mmol/L DTT to obtain a final protein concentration of 1 mg/ml (measured using Lowry et al. method). STM buffer in mmol/L: 250 sucrose. 50 Tris/HCl pH 7.4, 5 MgCl2. DS Buffer: 2100 sucrose, 50 Tris/ HCl pH 7.4, 5 MgCl2. LS buffer in mmol/L: 0.2 MgCl2, 10 Tris/HCl, pH 7.4. HS buffer in mmol/L: 2000 NaCl in LS buffer. The designation /PMSF is meaning in the presence of 1 mmol/L serine esterase inhibitor phenylmethylsulfonyl fluoride. Liposome preparation Liposome preparation was carried out according to detergent-dialysis method on LIPOSOMATE previously described by Zhang et al. (1994). The prepared liposome was small unilamellar vesicles (diameter 32 F 6 nm) containing egg PC. Buffer was STM. In some experiments [14C]-labeled DOPC (dioleoyl PC, final concentration 5 ACi/ml) was added to prepare [14C]-labeled liposome. Protocol for in vitro incubation of isolated nuclei Incorporation of liposome into nuclei was done as descried previously by us (Zhang et al., 1994) with modification. Isolated purified nuclei were incubated for 16 f 18 h at 4 jC in the presence (as liposome-pretreated group) or absence of liposome (0.1, 0.5, 1.0 and 5.0 Amol PC/ml). The liposome/ nuclei mixtures were then centrifuged at 104,000 g for 25 min to separate the nuclei from the liposome vesicles. The nuclei pellet was washed once and then resuspended in STM/PMSF. Then, the nuclear suspension (108 nuclei/0.25 ml) were incubated with different agents (dissolved in 0.25 ml) for 10 min at 30 jC: buffer alone, and PLA2 10 5 f 10 2 units/ml. The reaction was stopped by cold (4 jC) centrifuge on microcentrifuge for 2 min, and the nuclei pellet was washed once and then resuspended in STM/PMSF. Then NE was prepared (above described) to assay NTPase activity and mRNA export.
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Assay of NTPase activity NTPase activity was assayed as descried by Ramjiawan et al. (1996), with modification. Prepared NE (25 Ag protein) in 200 Al buffer containing 250 mmol/L sucrose, 50 mmol/L Tris-HCl (pH 7.4), 5 mmol/L EDTA, l mmol/L MgCl2 were preincubated for 10 min at 30 jC. Addition of 1 mmol/L of ATP or GTP initiated the reaction. Ten minutes after incubation, the reaction was stopped by addition of 10% SDS and placing the test tube on ice bath, and inorganic phosphate was measured according to the method of Raess (Raess and Vincenzi, 1980) to calculate the NTPase activity. Preliminary experiments showed a linear relationship of NTPase activity with incubation time of nucleoside triphosphate within 30 min. Measurement of mRNA transport from nuclear envelope (Tiffany et al., 1995) Preparation of [3H]-labeled poly (A)+ mRNA was performed by inoculating Saccharomyces cerevisiae into PGY medium (1 g peptone, 3 g glucose, and 1 g yeast extract per liter) containing 2.5 mCi/L of [3H]-uridine. Yeast was grown for 4 days at 30 jC and harvested by centrifugation. [3H]labeled poly (A)+ mRNA was isolated from the yeast pellet by acid quanidinum thiocyanate-phenolchloroform extraction using a RNA isolation kit (bulletin 1, TEL-TEST), and by oligo (dT)-cellulose type 7 chromatography. mRNA concentration was determined by absorbance at 260 nm, and the purities were assayed by the 260/280 nm ratio. The [3H]-labeled poly (A)+ mRNA specific activity was 101.7 10 cpm/Ag. Poly (A)+ mRNA was trapped inside the NE during their preparation by adding 5 Ag [3H]-poly (A)+ mRNA to 100 Al of the heparin-containing lysis buffer. After the suspension was gently agitated at room temperature for 5 min, the NEs vesicles were pelleted by means of centrifugation at 5000 g for 10 min. The NE were resealed by washing them twice in 10 mmol/L Tris/HCl (pH 8.0), 100 mmol/L NaCl, 30 mmol/L KCl, 3 mmol/L MgCl2, and 0.5 mmol/L CaCl2, and then resuspended at 2 mg protein/ml. The reaction mixture was prepared on ice by layering in succession 10 Al 60% HCl4, 40 Al silicone oil, and 150 Al of the envelope vesicles. The reaction was initiated at 30 jC by adding ATP 1 mmol/L or GTP 1 mmol/L respectively. Blank tube was prepared simultaneously in the absence of ATP and GTP. After the appropriate time, the reaction was terminated by centrifugation. The lower layer, containing NE vesicles, was discarded, and the upper layer, containing exported mRNA, was aspirated and counted in scintillation fluid. The NTPase-dependent transport of mRNA was presented as difference between assayed tube and blank tube. Lipid extraction and analysis fractions After incubation, nuclear membrane was extracted by the method of Folch et al. with modification (Folch et al., 1957). Frozen membrane preparations were thawed at 25j and suspended in 1 mM potassium bicarbonate buffer. The suspensions were extracted twice with chloroform-methanol (2:1 by vol.) and then once with chloroform alone. The chloroform-rich layers were combined and the solvents were evaporated under a stream of N2 without heat. Lipid residues were redissolved in an accurately measured volume of chloroform- methanol and stored at 40j under N2 in tightly sealed vials. The extracts of the control and PLA2-treated nuclear membrane from the same rat were used as a pair of samples. Phospholipids were chromatographed on Silica gel 60 thin layer plates (Military Medical
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Science Institute, China). Chloroform/methanol/acetic acid/H2O (65:25:4:2 by vol.) were used as developing solvents. Identity of components was verified by comparison with authentic reference compounds: L-a- Lysophosphatidylcholine from bovine liver, L-a-phosphatidylcholine (Sigma Co.). The components were measured quantitatively by densitometric analysis of plates. Measurement of [14C]-Labeled liposome incorporation to nuclei Isolated purified nuclei (0.5 ml) were incubated for 16 f 18 h at 4 jC with [14C]-labeled-liposome (0.1, 0.5, 1.0 and 5.0 Amol PC/ml). The liposome/nuclei mixtures were then centrifuged at 104,000 g for 25 min to separate the nuclei form the liposomal vesicles. The nuclei pellet was washed twice, and NE was prepared as above described. The [14C]-radioactivity of NE was measured on Liquid Scintillation Analyzer (Packard, 1600RT). The amount of [14C]-labeled liposome incorporation into NE was presented as pmol PC/mg protein NE. Other measurements Protein content in NE was assayed by the Lowry method (Lowry et al., 1951). Various marker enzyme activities: Mannose-6-phosphatase, in both microsomes and nuclei, was determined as described by Gilchrist (Gilchrist and Pierce, 1993). NAD pyrophosphorylase activity, a specific marker for nuclei, was determined according to Crane and Low (1976). NADPH cytochrome-c reductase activity, a microsomal marker, was measured by the reduction of cytochrome c as described by Kornberg (1955). Data analysis Six independent experiments with duplication were done. All results are expressed as mean F SD. The statistical analysis of the data was performed using one-way analysis of variance followed by Student–Newman–Keuls tests. P < 0.05 was accepted as statistically significant.
Results Marker enzyme activity Experiments for nuclei and nuclear membrane purity showed that the activity of NADH pyrophosphorylase (as a marker enzyme for NE) in the prepared hepatic nuclei and NE were about 7-fold and 22Table 1 Specific activities of marker enzymes Enzymes (nmol mg
1
protein min
NAD pyrophosphorylase Mannose-6-phosphatase NADPH cytochrome-c reductase
1
)
Homogenate
Intact nuclei
Nuclear envelope
3.68 F 0.27 90.70 F 6.40 10.30 F 0.90
25.77 F 1.26 (56.5 F 2.76) 412.00 F 2.00 (36.60 F 0.20) 2.88 F 0.22 (2.30 F 0.20)
88.46 F 6.44 (15.90 F 1.20) 14.48 F 1.30 (0.10 F 0.00) 97.00 F 0.12 (6.20 F 0.00)
The enzyme activities are indicated in parentheses as percentage of total liver homogenate activity expressed as nmol Pi releasedmin 1 gr.liver 1.
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Fig. 1. PC incorporation into nuclear envelope.
folds higher than that in homogenate of whole cells, respectively (P < 0.01), but the activity of NADPH cytochrome-c reductase activities (marker enzyme for microsome) were only 30% and 7% of that in hepatocytes homogenate (P < 0.01), respectively. Activities of mannose-6-phosphatase existing both in microsomes and nuclei were 4 f 5 times greater in nuclei than and only 14% in NE of that in cell homogenate, respectively. It was demonstrated that the isolated cell nuclear fraction was in high purity and little contaminated by other organelle (Table 1). Phosphatidylcholine incorporation into nuclear envelope Incorporation of PC into NE was increased with the incubated liposome in a concentration dependent manner (Fig. 1). Effect of PLA2 on mRNA transport and NTPase activity of nuclear envelope The nuclei were treated separately with various concentrations of PLA2. PC loss from the membrane preparation was achieved. Incubations with PLA2 (10 3 units/ml) removed about 13.59% of the nuclear membrane PC (from 53.47% in control to 39.88% in PLA2 treatment group). PC underwent considerable degradation (Table 2). Table 2 Effect of PLA2 treatment on the major phospholipid content of rat liver nuclear membrane Phospholipids PC LPC
Ag/mg protein
% of TPL
Control
PLA2 treatment
Control
PLA2 treatment
103.40 F 14.51 11.42 F 1.34
77.22 F 11.03** 24.77 F 4.45*
53.47 F 4.88 5.91 F 0.40
39.88 F 3.44 12.77 F 1.49
Values are mean F SD (n = 6). *P < 0.05; **P < 0.01 as compared with control. TPL—total phospholipids; PC— phosphatidylcholine; LPC—lysophosphatidylcholine.
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Fig. 2. Effect of PLA2 on mRNA transport (Fig. 2a) and NTPase activity (Fig. 2b) of NE. mean F SD, n = 6, mRNA transport: ng RNAmg Pro 1min 1; NTPase activity: nmolmg Pro 1min 1. PLA2 [unit/ml] ATP 1 mmol/L as substrate; GTP 1 mmol/ L as substrate. *P < 0.05, **P < 0.01 compared with control ([0] PLA2). Correlation between mRNA export and NTPase activity. [ATP]: r = 0.99;Y = 29 F 145 [GTP]: r = 0.98; Y = 39 F 165 .
The effect of modification of NE by PLA2 on mRNA transport and NTPase activity are present in Fig. 2. Messenger RNA transport rate was decreased by PLA2 treatment. PLA2, at a concentration as low as 10 5 unit/mL, caused a decrease in mRNA efflux rate by 19% (for ATP as substrate) (P < 0.01). At Table 3 Effects of PC-liposome on mRNA transport and NTPase activity of nuclear envelope PC- [mol/L] 0 10 4 5 10 10 3 5 10
4
3
ATP (1 mmol/L) as substrate
GTP 1 (mmol/L) as substrate
mRNA export
NTPase activity
mRNA export
NTPase activity
0.84 0.90 0.86 0.80 0.92
92 90 96 88 89
1.24 1.32 1.18 1.36 1.27
162 155 168 160 158
F F F F F
0.05 0.07 0.07 0.06 0.09
F F F F F
Mean F SD, n = 6, mRNA transport: ng RNA mg Pro
6 7 4 7 6 1
min
1
F F F F F
0.12 0.12 0.13 0.14 0.11
; NTPase activity: nmol mg Pro
1
min
F F F F F 1
.
12 15 17 14 10
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higher concentrations of PLA2 (10 2 mol/L), the mRNA efflux rates was decreased by 52.4% (for ATP as substrate) and 40.3% (for GTP as substrate) (Fig. 2a). In parallel assays, the NTPase activities were determined. It was found that this enzyme also responded to the PLA2 with a change in specific activity similar to the respective alterations in RNA transport rate (Fig. 2b). At the concentration of 10 2 unit/ml of PLA2, a decrease of NTPase activity to 67.4% (for ATP as substrate) and 51.6% (for GTP as substrate) of the control were found. Effect of PC on PLA2-induced changes in mRNA transport and NTPase activity of nuclear envelope As it was shown in Table 3, incorporated the nuclei with various concentrations of PC, efflux of poly (A) mRNA and NTPase activity on the NE remained unchanged as compared with those of the control
Fig. 3. Effects of PC on PLA2-induced changes in mRNA transport and NTPase activity of NE. Mean F SD, n = 6, mRNA transport: ng RNAmg Pro 1min 1; NTPase activity: nmolmg Pro 1min 1. PLA2 (10 3 units/ml) *P < 0.05, **P < 0.01 compared with [0] PC, i.e. PLA2 alone. #P < 0.05, # #P < 0.01 compared with control. Correlation between mRNA export and NTPase activity [ATP]: r = 0.95, Y = 37 F 157 ; [GTP]: r = 0.96; Y = 32 F 154 .
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group (all P values >0.05). It showed that the increase of PC in nuclear membrane could not alter mRNA transport and NTPase activity. However, PC-liposome incorporation could partially antagonize the effect of PLA2 on mRNA transport and NTPase activity (Fig. 3). Incubation of the PC-incorporated nuclei with PLA2 caused a pronounced increase of RNA transport. RNA transport rates in NE from the control were 0.84 F 0.05 (for ATP as substrate) and 1.24 F 0.12 (for GTP as substrate) ng RNAmg Pro 1min 1. Treated the nuclei with PLA2 (10 3 units/ml), the RNA transport were 0.51 F 0.05 (for ATP as substrate) and 0.80 F 0.10 (for GTP as substrate) ng RNAmg Pro 1min 1, which were greatly higher than those of the control. Treated the PC-incorporated nuclei with PLA2 (10 3 unites/ml), export of mRNA was greater than those of PLA2 (10 3 unit/mL)-treated group. When PC-concentration was at 5 10 3unit/mL, the exports of mRNA were 0.72 F 0.08 (for ATP as substrate) and 1.17 F 0.12 (for GTP as substrate) ng RNAmg Pro 1min 1, which increased by 41.2% (P < 0.01) and 46.3% (P < 0.01), respectively as compared with those of the PLA2-treated groups (Fig. 3a). In parallel assays, it was found that NTPase activity also responded to the PLA2, paralleling the alterations in RNA transport. NTPase activities in NE from untreated cellular nuclei were 92 F 6 and 162 F 12 nmolmg Pro 1min 1. The specific activities of the NTPase in PLA2 (10 3 unit/ml)-treatment group were 44 F 4 (for ATP as substrate) and 90 F 10 (for GTP as substrate) nmolmg Pro 1min 1, respectively. Increasing concentrations of PC liposome incorporation antagonized the effect of PLA2 (10 3 units/ml) on NTPase activity, regardless using ATP or GTP as substrate. PC liposome at a concentration of 5 10 3 mol/L, increased the NTPase activity by 73.3% and 64.4% as compared with that of the PLA2-treated group, respectively (all P values less than 0.01), as shown in Fig. 3b.
Discussion Export of mRNA molecules from nucleus to the cytoplasm is an active process involved in energy consumption. Nuclear NTPase, a nuclear membrane-associated enzyme, provided energy for poly (A)+mRNA export through the nuclear pore. The ability of the nuclear NTPase to hydrolyze various nucleotides had been reported in the rank order of UTP > GTP > ITP > CTP > ATP (Ramjiawan et al., 1996, 1997; Tiffany et al., 1995). This enzyme is located on the inner face of the nuclear envelope near the pore, in which also binding sites for poly (A)-RNA are localized (Kondor-Koch et al., 1982; Prochnow et al., 1990). It is obvious that the membrane structure will be important for maintaining nuclear NTPase activity and the function of mRNA export. It is well recognized that PLA2 action towards organized lipid interfaces (vesicles, multilamellar dispersions and monolayers) is much higher than against isotropically dispersed phospholipids (Martelli et al., 1999). PLA2 selectively cleaved phospholipids (mostly PC) containing arachidonic acid at the sn-2 position. The 1-acyl lysoderivates produced are water-soluble and part of them spontaneously leaves the membrane, thus decreasing the total phospholipid protein ratio. Nuclear PLA2 activity might be related to changes in fatty acid composition of nuclear phospholipids (PC and PE) (Neitcheva and Peeva, 1995). In the present study, PC-liposomes incorporated into nuclei, which increased the content of PC, could not alter RNA transport and NTPase, indicating that excess of PC within the nuclei is insufficient to induce a significant change in nucleocytoplasmic export. We also found that RNA nucleocytoplasmic export and NTPase activity were decreased by PLA2 treatment.
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Nuclear membrane PC was also decreased by PLA2 incubation. These showed that degradation of NE phospholipid might affect the NTPase activity and RNA export. The incorporation of PC before PLA2 treatment, causing a PC-replenishment, inhibited the effect of PLA2 on the RNA transport and NTPase activity, indicating that PC composition in the NE might play an important role in the NTPase activity and RNA transport. Changes in the RNA translocation were also accompanied by concomitant alterations in the NTPase activities according to the previous works (Tomassoni et al., 1999a,b; Tiffany et al., 1995). As excessively activated phospholipase A2 might play a deleterious role in nuclear membrane integrity and the impairment of nuclear membrane could influence RNA nucleocytoplasmic transport (Tabuchi et al., 2000), it suggests that this loss of membrane integrity allowed the nuclear NTPase and RNA nucleocytoplasmic transport to be decreased by phospholipase A2. It can be speculated according to our experiment that PC-liposome, provided PLA2 with substrate, might compensate the nuclear membrane impairment induced by PLA2. Therefore, the incorporation of PC into the NE might antagonize the effect of PLA2, through protecting the membrane integrity. Although PC incorporated have the identical polar head groups but different acyl chains, but they still have the ability to cause the changes in nuclear NTPase and RNA nucleocytoplasmic transport in PLA2-treated nuclear membrane. Hence, the ability of PC antagonizing the effect PLA2 on the RNA transport and NTPase seem to depend on the nature of their polar head group but not on their hydrophobic chain. The results showed that nuclear NTPase activity and mRNA transport were attenuated by PLA2, which might act through changing composition of nuclear phospholipids. It has been shown that the nonpolar components of the membrane lipid bilayer exert a greater effect than the phospholipid head group in modulating the activity of membrane-bound enzymes (Tomassoni et al., 1999a,b). Our investigations demonstrated that PC, when incorporated into non-PLA2 -treated nuclei and maybe inducing alterations in membrane fluidity, have no effect on NTPase and RNA transportation. Therefore, it seems likely that the PC action in the present study is not fluidity controlled. However, whether PLA2 action on the nuclear fluidity involved in the mechanism of the decrease in NTPase and RNA transportation need to be further investigated. Since PLA2-treatment caused a decrease in NTPase activity and mRNA transport, and PC replenishment could reverse this effect, it is quite possible that the phospholipid composition of nuclear membrane facilitates the most optimal state of the NTPase and RNA export. Little attention has been given to the pathophysiological role of PLA2 in nucleocytoplasmic transportation in the present study. A variety of techniques indicated endogenous PLA2 to be mainly located in the nucleus in highly proliferative cells whereas its location was cytoplasmatic in nonproliferative cells (Martelli et al., 1999). When non-confluent cells were incubated with exogenous PLA2, the enzyme was internalized and, in the majority of cell, appeared within the nucleus (Martelli et al., 1999). Cytoplasmic nuclear PLA2 activity might associate with liver regeneration (Tabuchi et al., 2000). These findings suggested that excessively activated PLA2 might modify protein synthesis process through influencing NTPase and RNA transport under physiological and pathophysiological conditions, which would benefit from PC compensation according to our experiment. In conclusion, we may propose that the nucleocytoplasmic efflux of mRNA is influenced by PLA2 activity. PC incorporation into the nuclear membrane exerted a protective effect on PLA2-induced decrease in mRNA export.
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