(Received for publication, August 17, 1995; and in revised form, October 10, 1995)
From the
Free cholesterol-loaded macrophages in atheromata synthesize
excess phosphatidylcholine (PC), which may be an important adaptive
response to the excess free cholesterol (FC) load. We have recently
shown that FC loading of macrophages leads to 2-4-fold increases
in PC mass and biosynthesis and to the post-translational activation of
the membrane-bound form of CTP:phosphocholine cytidylyltransferase
(CT), a key enzyme in PC biosynthesis. Herein, we explore further the
mechanism of CT activation in FC-loaded macrophages. First, enrichment
of membranes from control macrophages with FC in vitro did not
increase CT activity, and PC biosynthesis in vivo is
up-regulated by FC loading even when CT and FC appear to be mostly in
different intracellular sites. These data imply that FC activates
membrane-bound CT by a signaling mechanism. That the proposed signaling
mechanism involves structural changes in the CT protein was suggested
by data showing that two different antibodies against synthetic CT
peptides showed increased recognition of membrane-bound CT from
FC-loaded cells despite no increase in CT protein. Since CT is
phosphorylated, two-dimensional maps of peptides from P-labeled control and FC-loaded macrophages were compared:
six peptide spots from membrane-bound CT, but none from soluble CT,
were dephosphorylated in the FC-loaded cells. Furthermore, incubation
of FC-loaded macrophages with the phosphatase inhibitor, calyculin A,
blocked increases in both PC biosynthesis and antipeptide-antibody
recognition of CT. Last, treatment of membranes from control
macrophages with
phage protein phosphatase in vitro increased both CT activity (2-fold) and antipeptide-antibody
recognition of CT; soluble CT activity and antibody recognition were
not substantially affected by phosphatase treatment. In summary, FC
loading of macrophages leads to the partial dephosphorylation of
membrane-bound CT, and possibly other cellular proteins, which appears
to be important in CT activation. This novel regulatory action of FC
may allow macrophages to adapt to FC loading in atheromata.
Macrophages are a prominent cell type in both early and advanced
atherosclerotic lesions (1, 2, 3) and
undoubtedly play important roles in the clinical progression of these
lesions(4, 5) . Most atheroma macrophages are loaded
with both cholesteryl esters (6) and, particularly in advanced
lesions, free cholesterol
(FC)()(7, 8, 9, 10) . In
addition, lesion macrophages appear to have increased rates of
phospholipid biosynthesis (11, 12, 13) and to
have increased phospholipid mass(14, 15) , in the form
of intracellular membrane whorls(10) . This excess phospholipid
may serve to prevent the decreased fluidity of FC-rich membranes (16) and/or to inhibit cholesterol crystal
formation(15) . Thus, the increased phospholipid biosynthesis
seen in atheroma macrophages may be part of an adaptive response to
prevent FC-mediated cellular toxicity.
To gain insight into the biochemistry of this in vivo phenomenon, we recently explored phospholipid metabolism in FC-loaded macrophages in cell culture(17) . Our work revealed that FC loading of macrophages leads to the accumulation of excess PC mass, in the form of intracellular membrane whorls, via activation of the enzyme CTP:phosphocholine cytidylyltransferase, which is a rate-limiting enzyme in PC biosynthesis(17) . Initial mechanistic studies in that report disclosed two important points. First, FC loading of macrophages led to the activation of membrane-bound CT without a substantial change in the activity of soluble CT(17) . Thus, the activation cannot be explained by soluble-to-membrane CT translocation, which is a process thought to be involved in the activation of CT in certain other systems (18, 19, 20) . Second, there was no increase in CT mRNA in FC-loaded macrophages, and stimulation of PC biosynthesis in the FC-loaded cells did not depend upon new protein synthesis(17) . Therefore, the activation occurs via a post-translational mechanism.
The goal of the present study was to explore further the mechanism of CT activation in FC-loaded macrophages. Our new data indicate that FC loading activates CT in macrophages via a signaling mechanism that appears to involve the dephosphorylation of membrane-bound CT and possibly other cellular proteins. This novel intracellular signaling effect of FC may be part of an important adaptive response of macrophages to the potential toxicity of excess FC accumulation.
For immunoblotting, two-dimensional phosphopeptide mapping, and in vitro phosphatase treatment, the macrophages were fractionated by the digitonin treatment method of Wright et al.(32) . In brief, the cell monolayers were washed three times with ice-cold PBS and then incubated for 8 min at 4 °C with digitonin-release buffer, which contained 10 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 0.8 mg/ml digitonin, 33 mM sodium fluoride, 33 µM sodium vanadate, 3.3 mM EDTA, 3.3 mM EGTA, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin. The release buffer was then collected (soluble fraction), and the cell ghosts, still attached to the culture dish, were carefully washed once with PBS and scraped into digitonin-release buffer (membrane fraction). Homogenate fractions were prepared by scraping the monolayer in digitonin-release buffer and homogenizing the suspension by repeated aspiration and expulsion from a Pasteur pipette.
Figure 1:
Effect of FC loading on PC biosynthesis
in CHO-mSRAII cells. A, monolayers of CHO-mSRAII cells were
incubated for 12 h in Ham's F-12 medium, 10% LPDS alone (Control) or Ham's F-12 medium, 10% LPDS containing 50
µg of acetyl-LDL/ml (AcLDL), 5 µg of 58035/ml (58035), or acetyl-LDL plus 58035 (AcLDL + 58035). The cells were then incubated for 1 h in the same medium
containing 2 µCi of [H]choline/ml, and the
radioactivity in cellular [
H]phosphatidylcholine
was determined. B, monolayers of J774 macrophages were
incubated for 12 h in DMEM, 10% LPDS alone (Control) or medium
containing 50 µg of acetyl-LDL/ml plus 5 µg of 58035/ml (AcLDL + 58035). The cells were then assayed for
phosphatidylcholine biosynthesis as above. C, monolayers of
CHO-mSRAII cells (CHO
) and J774
macrophages (Mø) were incubated for 12 h in medium
containing 50 µg of acetyl-LDL/ml plus 5 µg of 58035/ml. The
cells were then assayed for FC content. The FC contents of the unloaded
CHO-mSRAII and J774 macrophages were 88.8 ± 9.9 and 41.3
± 2.1 nmol/mg of cell protein,
respectively.
Figure 2:
Enrichment of membranes from J774
macrophages with FC in vitro: effect on CT activity. Monolayers of J774 macrophages were incubated for 36 h in DMEM,
10% LPDS alone (Con Mø) or containing 50 µg
acetyl-LDL/ml plus 5 µg 58035/ml (FC-loaded Mø).
Membrane fractions from these cells were preincubated in the absence or
presence of 300 µM cholesterol (FC), 30
µM 25-hydroxycholesterol (25OHC), or 300
µM cholesterol in cholesterol/phosphatidylcholine
liposomes (FC-lip). The cholesterol was added from a 50
stock (15 mM) in acetone, and 25-hydroxycholesterol
was added from a 1000
stock (3 mM) in ethanol;
separate controls contained solvent alone (2% acetone or 0.1% ethanol,
respectively) which had no effect on CT or ACAT activities. The
membranes were then assayed, also in the absence or presence of the
above cholesterol preparations, for CT activity (A) or, as a
control, for ACAT activity (B). The CT assay was done in the
absence of PC-oleic acid liposomes.
Figure 3: Indirect immunofluorescence microscopy of control and FC-loaded macrophages using an anti-CT antibody. Monolayers of J774 macrophages were incubated for 12 h in DMEM, 10% LPDS alone (A) or containing 50 µg acetyl-LDL/ml plus 5 µg of 58035/ml (C-D). The cells were then fixed, permeabilized, incubated with an anti-N-terminal CT peptide antibody and then a rhodamine-labeled anti-IgG antibody, and viewed by standard (A-C) or confocal (D) fluorescence microscopy. In C, the anti-CT antibody incubation included 5 µg of purified recombinant CT/ml to determine specific antibody binding. Bar, 10 µm.
To examine where cholesterol accumulates in FC-loaded macrophages, control and FC-loaded macrophages were fixed, stained with filipin to visualize intracellular accumulations of free cholesterol(47) , and viewed by fluorescence microscopy (Fig. 4, A and B). The filipin signal was much brighter in the FC-loaded cells, as expected, and was located predominantly in perinuclear vesicles. Although this localization, rather than a predominantly plasma membrane localization, was somewhat surprising (cf. (48) ), the data clearly show that the filipin signal was almost entirely absent from the nucleus or nuclear envelope (Fig. 4B). This finding, together with the CT immunofluorescence data in Fig. 3, show that most of the cholesterol in FC-loaded macrophages is in a different intracellular location from most of the CT, suggesting that the presence of the bulk of cholesterol and CT in the same intracellular compartment is not necessary for CT activation in intact FC-loaded macrophages.
Figure 4: Fluorescence microscopy of control and FC-loaded macrophages stained for free cholesterol by filipin. Monolayers of J774 macrophages were incubated for 12 h in DMEM, 10% LPDS alone (A), or containing 50 µg acetyl-LDL/ml plus 5 µg 58035/ml (B) or 50 µg of acetyl-LDL/ml plus 1 µM U18666A (C). The cells were then fixed, stained with filipin, and viewed by fluorescence (A-C) or phase (D-F) microscopy. n, nucleus; Bar, 10 µM. In order to visualize the control cells (A), the brightness of the image was increased 5-fold (compared with the images in B and C) prior to printing.
To further support this conclusion, we took advantage of a steroid, called U18666A, which is known to block the exit of lipoprotein-derived cholesterol from lysosomes(49) . Note that the pattern seen in Fig. 4B, although not yet defined in terms of the identity of the fluorescent vesicles, is reminiscent of that seen in cells loaded with cholesterol in the presence of inhibitors of lysosomal cholesterol export, including U18666A(49, 50) . In fact, when macrophages were incubated with acetyl-LDL plus U18666A, the pattern of fluorescence was very similar to that seen with cells incubated with acetyl-LDL plus the ACAT inhibitor 58035 (compare C and B in Fig. 4). The data in Fig. 5clearly show that, under conditions of similar cellular FC loading (inset), the activation of PC biosynthesis in macrophages incubated with acetyl-LDL plus U18666A was very similar to that in cells incubated with acetyl-LDL plus 58035. In this system, we cannot rule out a small amount of contact between FC and CT in the FC-loaded cells, and the FC-containing vesicles in either the U18666A- or 58035-treated cells have not yet been definitively identified (cf.(50) ). Nonetheless, these data, together with those in Fig. 2Fig. 3Fig. 4, strongly suggest that activation of PC biosynthesis in FC-loaded macrophages involves a signaling mechanism rather than direct interaction of CT with cholesterol.
Figure 5:
Effect of inhibition of FC export from
lysosomes on PC biosynthesis in macrophages incubated with acetyl-LDL.
Monolayers of J774 macrophages were preincubated for 30 min in the
absence or presence of 1 µM U18666A and then incubated for
12 h in DMEM, 10% LPDS containing 50 µg of acetyl-LDL/ml alone,
acetyl-LDL plus 1 µM U18666A, or acetyl-LDL plus 5 µg
of 58035/ml. The cells were then incubated for 1 h in the same medium
containing 2 µCi of [H]choline/ml, and the
radioactivity in cellular [
H]phosphatidylcholine
was determined. The cells were also assayed for their content of FC (inset).
Figure 6:
Anti-CT immunoblots of CT from control and
FC-loaded macrophages. A, monolayers of J774 macrophages were
incubated for the indicated timepoints as follows: lane 1,
DMEM, 10% LPDS alone; lane 2, DMEM, 10% LPDS containing 50
µg of acetyl-LDL/ml; lane 3, DMEM, 10% LPDS containing 5
µg of 58035/ml; lane 4, DMEM, 10% LPDS containing
acetyl-LDL plus 58035 (i.e. FC loading conditions).
Homogenates of the cells were then subjected to reducing SDS-10%
polyacrylamide electrophoresis, blotted to nitrocellulose, and
immunoblotted with an anti-N-terminal-CT synthetic peptide antibody.
The bands shown migrated in a region of the gel corresponding to
42 kDa. B, monolayers of J774 macrophages (Mø) were incubated for 12 h in DMEM, 10% LPDS alone (Con) or DMEM, 10%LPDS containing 50 µg of acetyl-LDL/ml
plus 5 µg of 58035/ml (FC). Membrane fractions were
prepared by the digitonin method and subjected to immunoblot analysis
using the anti-N-terminal-CT synthetic peptide antibody employed above (N-term Ab), an anti-mid-molecule-CT synthetic peptide
antibody (Mid-mol Ab), and an anti-holo-CT antibody (Holo
Ab).
The increased recognition of CT by the antipeptide antibody was seen as early as 4 h after FC loading and continued to increase modestly up to 12 h of FC loading. As demonstrated previously(17) , the induction of PC biosynthesis in macrophages first becomes apparent at 4 h of FC loading and continues to peak up to 12 h of FC loading. Furthermore, as shown in Fig. 6B, an antibody made against another synthetic CT peptide (mid-molecule, amino acids 164-176) (26) also reacted more intensely with CT in membranes from FC-loaded macrophages, although the effect was not as marked as that seen with the N-terminal antibody. In contrast, an antibody made against holorecombinant rat liver CT (24) reacted equally well with CT from control and FC-loaded macrophages, which is consistent with our previous conclusion (17) that synthesis of CT is not increased in activated cells. Furthermore, this finding indicates that the increases in CT activity and antipeptide antibody immunoreactivity seen with FC loading cannot be explained by a decrease in degradation of the CT protein or a masking of CT by another 42-kDa protein on the blot. Note that neither the N-terminal nor mid-molecule antibody showed increased recognition of soluble CT from FC-loaded macrophages (not shown), which is important since only membrane-bound CT is activated by FC loading(17) . Although the molecular basis of these findings has not yet been definitively determined (see ``Discussion''), the data suggest that activation of CT in FC-loaded macrophages is associated with an alteration of the structure of the membrane-bound CT molecule.
Figure 7:
Autoradiography of two-dimensional maps of
trypsin-digested CT phosphopeptides from control and FC-loaded
macrophages. Monolayers of J774 macrophages were labeled for 12 h with P
(7.5 mCi/well), in the absence (A and C) or presence (B and D) of 50
µg of acetyl-LDL/ml and 5 µg of 58035/ml. Soluble (A and B) and membrane fractions (C and D), made by digitonin method, were immunoprecipitated using an
anti-N-terminal-CT synthetic peptide antibody. The immunoprecipitates
were subjected to reducing SDS-10% polyacrylamide electrophoresis,
blotted to PVDF membrane, and exposed to x-ray film. CT bands were then
cut out and digested with modified trypsin, and the resulting
phosphopeptides were resolved by electrophoresis and thin-layer
chromatography. Shown are the autoradiograms of the two-dimensional
peptide maps. The arrows in C (membranes from control
macrophages) point out peptide spots that were diminished in D (membranes from FC-loaded
macrophages).
Figure 8:
Effect of the phosphatase inhibitor
calyculin A on PC biosynthesis and recognition of CT by an anti-CT
synthetic peptide antibodies. A, monolayers of J774
macrophages were preincubated for 1 h in the absence or presence of 5
nM calyculin A (CalA) and then incubated for 8 h in
DMEM, 10% LPDS alone, or medium containing 5 µg of 58035/ml plus 50
µg of acetyl-LDL/ml (FC). The cells were then incubated
for 1 h in the same medium containing 2 µCi of
[H]choline/ml, and the radioactivity in cellular
[
H]phosphatidylcholine was determined. The cells
were also assayed for FC content (inset in A). B, homogenates of the cells from the experiment in A were subjected to immunoblot analysis using antibodies against
N-terminal (
-N-term-CT peptide Ab) and mid-molecule (
-mid-CT peptide Ab) CT synthetic
peptides.
The data in Fig. 8B show the effect of calyculin A treatment on the immunoblot pattern of CT from control and FC-loaded macrophages. As above (Fig. 6), both the N-terminal and mid-molecule antibodies showed an increased signal with CT from FC-loaded cells. This increased signal was blunted in FC-loaded cells treated with calyculin A. Note that the CT signal from unloaded macrophages is not affected by calyculin A treatment. In experiments not displayed here, we also showed that calyculin A treatment of FC-loaded macrophages partially prevented the decreased phosphorylation of peptides derived from membrane-bound CT. Thus, treatment of macrophages with a very low concentration of a potent phosphatase inhibitor blunts the induction of PC biosynthesis, the increased recognition of CT on immunoblots by anti-CT peptide antibodies, and the decreased phosphorylation of membrane-CT peptides.
To further support the idea that dephosphorylation can lead to
macrophage CT activation, soluble and membrane fractions from unloaded
macrophages were treated in vitro with phage protein
phosphatase (cf.(23) and (34) ) and then
assayed for CT activity and immunoblot reactivity using the
anti-N-terminal CT peptide antibody (Fig. 9). Phosphatase
treatment led to only very small increases in soluble CT activity and
in anti-N-terminal antibody reactivity. In contrast, membrane-bound CT
was activated 2-fold by phosphatase treatment, and reactivity with the
anti-N-terminal antibody was also markedly increased. These data,
together with the calyculin A data in Fig. 8, strongly suggest
that up-regulation of CT activity by FC loading of macrophages involves
an intracellular protein dephosphorylation signaling mechanism.
Figure 9:
Effect of in vitro phosphatase
treatment of soluble and membrane fractions from unloaded macrophages
on CT enzymatic activity and antipeptide-antibody immunoreactivity.
Soluble and membrane fractions from unloaded macrophages were incubated
for 30 min at 37 °C with buffer alone (hatched bars) or
buffer containing 400 units of phage protein phosphatase/ml (solid bars). The fractions were then assayed for CT activity,
in the presence (soluble fraction) or absence (membrane fraction) of
PC-oleic acid liposomes and for reactivity on immunoblots with
anti-N-terminal CT peptide antibody (
N-term-CT peptide
Ab). The
42-kDa immunoblot band from each condition is
displayed below the bar for the respective
condition.
The mechanistic studies presented in this report have potential importance to three areas of research, namely, FC-mediated intracellular signaling pathways, mechanisms of CT regulation, and adaptive responses of FC-loaded macrophages in atherosclerotic lesions.
Whether it is the dephosphorylation of CT itself (see Fig. 7) which is important in enzyme activation, or whether some protein or proteins other than or in addition to CT need to be dephosphorylated, has not been addressed by this study. The issue as to whether dephosphorylation of rat liver CT can directly lead to enzyme activation has been addressed in several recent publications. Yang and Jackowski (58) measured in vitro enzyme kinetics of baculovirus-expressed recombinant rat CT with deleted or mutated serine phosphorylation sites and concluded that phosphorylation of these sites decreases the affinity of CT for two lipid activators, namely, oleic acid and diacylglycerol, and induces negative cooperativity. On the other hand, Wang and Kent (59) examined a CHO line expressing recombinant rat liver CT with mutations in all 16 of the CT serine phosphorylation sites and concluded that the major effect of CT dephosphorylation in CHO cells may not be to directly activate the enzyme but rather to stabilize CT in a membrane-bound form, which then leads to enzyme activation (cf. (60) ). Whether the dephosphorylation of CT that occurs in FC-loaded macrophages (Fig. 7) affects CT enzymatic activity remains to be studied.
How do the data for activation of CT in FC-loaded macrophages compare with those for activation of CT in other systems? Mechanisms reported for other systems that clearly do not play an important role in FC-loaded macrophages include soluble-to-membrane CT translocation (18, 19, 20) , activation of soluble CT(61, 62) , and increased synthesis of CT(63, 64) . There are, however, two situations in which CT activation has been shown to be inhibited by treatment of the cells with the protein phosphatase inhibitor, okadaic acid: oleic acid-treated rat hepatocytes (43) and phospholipase C-treated CHO cells(44) . Unlike the situation with FC-loaded macrophages, however, CT translocation to membranes is also prominent in these other systems(40, 65) . Since soluble-to-membrane translocation of CT is not substantial when macrophages are FC-loaded (above), it is possible that the mechanism of CT activation in FC-loaded macrophages is fundamentally different from that in these two other situations, despite the fact that in all three cases CT is dephosphorylated and CT activation is blocked by a protein phosphatase inhibitor. In addition, the effect of the phosphatase inhibitor on PC biosynthesis and CT immunoreactivity in macrophages is only seen in FC-loaded cells (Fig. 8). Thus, the maintenance of basal CT activity appears not to involve dephosphorylation, further distinguishing macrophages from these other cell types.
The
apparent differences in the mechanism of CT activation in FC-loaded
macrophages versus other situations could be related to the
cell type (i.e. macrophages) and/or the inducing agent (i.e. FC loading). In FC-loaded CHO cells, unlike FC-loaded
macrophages, PC biosynthesis is not increased (Fig. 1), and
there is no increased recognition of CT on immunoblots by the
anti-N-terminal CT peptide antibody (data not shown). Furthermore, in
preliminary experiments, we found that incubation of macrophages with
oleic acid up-regulates PC biosynthesis and is associated with
increased recognition of CT by the anti-N-terminal CT peptide
antibody. Thus, pending examination of PC biosynthesis in
other cell types loaded with FC and more detailed studies with oleic
acid and other possible inducers of PC biosynthesis in macrophages, the
mechanistic properties described herein may be part of a
macrophage-specific repertoire for the up-regulation of PC
biosynthesis.
Throughout this report, we have monitored the immunoreactivity of CT in fixed cells and on immunoblots and found that two antisynthetic peptide antibodies, but not an anti-holo-CT antibody, gave a much stronger signal with CT from FC-loaded cells ( Fig. 3and Fig. 6). Furthermore, there was a strong correlation between this increased immunoreactivity of CT and its state of activation (e.g.Fig. 8and Fig. 9). How can these antibody data be interpreted? Since the N-terminal and mid-molecule antibodies were made against unmodified peptides whereas the holo-CT antibody was made against phosphorylated CT(24) , and since CT is dephosphorylated when macrophages are FC-loaded (Fig. 7), the simplest explanation is that the antisynthetic peptide antibodies have greater recognition for dephosphorylated, and thus unmodified, CT. The region of CT that is phosphorylated when rat liver CT is expressed in insect cells or CHO cells, however, is the C terminus (amino acids 315-362)(37) , not the N terminus or mid-molecular region used to generate the antipeptide antibodies. Thus, it is possible that the N terminus of CT, which contains two potential protein kinase C sites (66) , and the mid-molecular region, which contains two serine residues (66) , are phosphorylated in macrophages; if so, this may be at least one reason why activation of CT in this cell type is different from that in CHO cells ( Fig. 1and above). Alternatively, the antibody data might be due to the removal of some other modification of CT in the N terminus and mid-molecular region, which might also be unique to macrophages (cf.(66) ). Finally, since proteins from SDS gels may renature after transfer to nitrocellulose or PVDF membranes (e.g. see (67) ), it is theoretically possible that C-terminal dephosphorylation could lead to an N-terminal or mid-molecular conformational change that could be detected by immunoblotting. Interestingly, CT activation in oleic acid-treated HepG2 cells is also associated with increased recognition by an anti-CT antibody(62) , but in this case, the antibody used was one made against native rat liver CT, not against a synthetic peptide. In future studies, as we further define the structural changes in macrophage CT that occur with FC loading, the explanation of our antibody data should become apparent.