(Received for publication, May 31, 1994; and in revised form, December 21, 1994)
From the
We have found that C ceramide, a cell-permeable
ceramide analog, partially restored the brefeldin A (BFA) sensitivity
in a BFA-resistant mutant of Vero cells (BER-40) and in the naturally
BFA-resistant Madin-Darby canine kidney (MDCK) cells. Incubation of
BER-40 and MDCK cells with low concentrations of C
ceramide
resulted in (i) a pronounced increase in BFA cytotoxicity as measured
by the inhibition of [
H]thymidine incorporation
and the inhibition of colony formation by BFA, (ii) a significant
protection by BFA against ricin cytotoxicity, and (iii) an inhibition
of bulk protein secretion by BFA in BER-40 and MDCK cells. Related
sphingolipids including sphingosine, sphingomyelin, and
lactosylceramide and other unrelated lipid second messengers such as
arachidonic acid and 1,2-diacylglycerol did not elicit the protection
of BER-40 and MDCK cells against ricin cytotoxicity by BFA. C
ceramide was the most effective among the ceramides with
different acyl chain lengths. Interestingly, dihydro-C
ceramide, which lacks the trans double bond in the
sphingoid base, had no effect. On the other hand, C
ceramide did not enhance BFA sensitivity in BFA-sensitive Vero
cells. The LD
of C
ceramide were similar in
Vero and BER-40 cells. Fluorescence microscopic studies revealed that
C
ceramide induced the redistribution of
-COP from the
Golgi membranes to a more dispersed localization in both BFA-sensitive
and BFA-resistant cell lines, mimicking the effect of BFA. Suboptimal
concentration of C
ceramide also restored the effect of BFA
on the
-COP distribution in BER-40 and MDCK cells. These results
indicate that C
ceramide restores the BFA sensitivity in
BFA-resistant BER-40 and MDCK cells.
Brefeldin A (BFA), a fungal antibiotic, has been
shown to affect the structure and function of the Golgi apparatus,
resulting in an inhibition of protein secretion and the redistribution
of the Golgi proteins into the endoplasmic reticulum (1, 2) . An early event in the action of BFA is the
dissociation of the
-COP (110-kDa protein) from the Golgi
membranes(3, 4, 5, 6, 7) .
Yoshida et al.(8, 9) have found that BFA
inhibits the cytotoxicity of ricin, Pseudomonas toxin, and
modeccin in Vero and other cell lines, and these results suggest an
involvement of the Golgi apparatus in the intoxication process of these
toxins. Increasing evidence suggests that BFA may modulate the
functions of GTP-binding proteins that are involved in intracellular
vesicular transport system (10, 11, 12, 13, 14, 15) .
Recently, it has been reported that a water-soluble, cell-permeable
ceramide analog, C ceramide (C
Cer), inhibits
the viral glycoprotein transport through the Golgi complex and reduces
the number of infectious virions released from infected cells in a
concentration-dependent manner(16) . These results suggest that
ceramide may regulate intracellular membrane traffic. In the present
study, we found that incubation of BFA-resistant cell lines, BER-40 and
MDCK cells, with C
Cer substantially restored the
sensitivities of these cell lines to BFA as measured by the inhibition
of DNA synthesis and the inhibition of colony formation, the protection
against ricin cytotoxicity, the inhibition of bulk protein secretion,
and the redistribution of
-COP by BFA in these BFA-resistant cell
lines. The enhancement of BFA sensitivity in BER-40 and MDCK cells
appear to be specific for ceramide as related sphingolipids have no
effect.
To examine the effect of CCer on BFA
cytotoxicity, Vero, BER-40, and MDCK cells pretreated with or without
C
Cer (10 µM) for 2 h at 37 °C were
incubated with varying concentrations of BFA in the presence or absence
of C
Cer for 17 h. After removal of the medium, the
incorporation of [
H]thymidine into perchloric
acid/phosphotungstic acid-insoluble materials was determined. From the
dose-response curves of BFA in the presence or absence of
C
Cer, ID
value of BFA in each cell line was
estimated. As shown in Table 1, both BER-40 and MDCK cells were
rendered BFA-sensitive by the pretreatment with C
Cer, as
evidenced by a significant reduction in ID
of BFA in
C
Cer-treated BER-40 and MDCK cells as compared with the
respective controls; incubation with 10 µM C
Cer alone for 19 h did not significantly inhibit
[
H]thymidine incorporation (see legend to Table 1). Moreover, the LD
of BFA in BER-40 and MDCK
cells, based on its inhibition of colony formation, was reduced
5-8-fold in the presence of sublethal concentration of
C
Cer (1 µM); the viabilities of Vero, BER-40,
and MDCK cells at 1.25 µM C
Cer were 87, 78,
and 95%, respectively. In contrast, C
Cer did not alter the
ID
or LD
of BFA in Vero cells.
BFA has
been shown to inhibit ricin cytotoxicity in Vero cells in a
dose-dependent manner, and 1 µg/ml of BFA completely abolished
ricin cytotoxicity in Vero cells. Even at 0.1 µg/ml of BFA, the
ID of ricin increased from 4.4 to >1000 ng/ml (Table 2). However, 1 µg/ml of BFA had no significant effect
on ricin cytotoxicity in BFA-resistant BER-40 and MDCK
cells(9, 18, 22, 23, 24, 25) (Table 2).
To further characterize the effect of C
Cer on BFA action,
we examined the effect of C
Cer on the protection of ricin
cytotoxicity by BFA in Vero, BER-40, and MDCK cells at different
concentrations of BFA which by itself did not protect each cell line
from ricin cytotoxicity. C
Cer (35 µM) restored
strong protective effects of BFA on ricin cytotoxicity in BFA-resistant
BER-40 and MDCK cells, whereas C
Cer by itself had a very
small protective effect on ricin cytotoxicity in Vero and BER-40 cells;
C
Cer alone increased ID
of ricin
2-3-fold in Vero and BER-40 cells but not in MDCK cells (Table 2). In the presence of 35 µM C
Cer, BFA caused an 26.3- and 87-fold increase in the
ID
of ricin in BER-40 and MDCK cells, respectively. Such a
synergistic effect between BFA and C
Cer was not observed in
Vero cells (Table 2). Especially in MDCK cells, marked protective
effect of BFA against ricin cytotoxicity was evident in
C
Cer-treated cells, even though BFA or C
Cer
alone had no effect. Furthermore, lower concentration of
C
Cer (15 µM), which did not have any
protective effect on ricin cytotoxicity, restored the protective effect
of BFA against ricin cytotoxicity in BER-40 and MDCK cells but not in
Vero cells (Table 2).
To gain insight into the relationship
between the structure of ceramide and its effect on BFA-induced
protection of ricin cytotoxicity in BFA-resistant cell lines, we
examined various ceramide analogs with different acyl chain lengths.
CCer and C
Cer also restored the protective
effect of BFA on ricin cytotoxicity in BER-40 and MDCK cells, albeit
less effectively than C
Cer (Table 2). However,
dihydro-C
Cer and natural ceramide (C
Cer) were
ineffective. These results suggest that the restoration of BFA
sensitivity by ceramide is dependent on the presence of a double bond
in the sphingoid backbone as well as an optimal acyl chain length.
Sphingomyelinase, which has been shown to increase the intracellular
ceramide level(26, 27) , also had no effect. This may
be related to the lack of an effect of C
Cer on BFA
sensitivity, since C
Cer would be the predominant species
produced by sphingomyelinase treatment. Moreover, other related
sphingolipids, including sphingosine, sphingomyelin, lactosylceramide,
and unrelated lipid second messengers such as 1,2-diacylglycerol and
arachidonic acid, did not show any significant effect on ricin
cytotoxicity, nor on the protective effect of BFA against ricin
cytotoxicity (Table 2). These results suggest that short chain
ceramide is a relatively specific lipid effector on BFA action and
ceramide itself, rather than its metabolites or other related lipid
second messengers, is responsible for restoring BFA sensitivity in
BFA-resistant cell lines. It is interesting to note that the activation
of heterotrimeric protein phosphatase 2A by ceramide is also dependent
on the presence of a double bond in the sphingoid backbone and is
influenced by acyl chain composition(28) . These data suggest
that a double bond in the sphingoid base and short hydrophobic acyl
chain are important structural features for ceramide-mediated
bioactivity.
One of the characteristic biological effects of BFA is
its inhibition of bulk protein
secretion(1, 2, 29) , and such an inhibition
is not elicited by BFA in BFA-resistant cell
lines(18, 21) . As shown in Table 3,
pretreatment of BER-40 and MDCK cells with CCer rendered
these cell lines susceptible to BFA in inhibiting bulk protein
secretion. Such an enhancement of BFA-induced inhibition of protein
secretion by C
Cer was not observed in Vero cells.
To
ascertain whether or not the failure of CCer to alter BFA
sensitivity in Vero cells is due to a difference in sensitivity toward
C
Cer between Vero and BER-40/MDCK cells, we compared the
LD
of C
Cer in these cell lines. The LD
values of C
Cer of Vero, BER-40, and MDCK cells were
found to be 1.97, 1.56, and 1.76 µM, respectively. It is
therefore unlikely that the differential enhancement of BFA effect in
BER-40 and MDCK cells by C
Cer and its lack thereof in Vero
cells are due to an intrinsic resistance of Vero cells to
C
Cer. In fact, bulk protein secretion was as strongly
inhibited by C
Cer in a dose-dependent manner in Vero cells
as in BER-40 and MDCK cells (data not shown).
In contrast to BER-40
and MDCK cells, no significant enhancement of BFA effect by
CCer was observed in PtK
cells, another
naturally BFA-resistant cell line(23) . An intrinsic resistance
of PtK
cells to C
Cer action may be one reason
for the inability of C
Cer to enhance the BFA action; the
LD
of C
Cer for PtK
cells was
7-8-fold higher than those for BER-40 and MDCK cells, and higher
concentrations of C
Cer were required to inhibit bulk
protein secretion in PtK
cells (data not shown).
An
early event in the action of BFA is the dissociation of a 110-kDa
Golgi-associated protein (-COP) from the Golgi membrane to
cytosol, resulting in the dispersed distribution of
-COP in the
cytosol in a dose-dependent manner. The BFA resistance in BER-40 and
MDCK cells is also manifested in the inability of BFA to cause the
redistribution of
-COP. 0.005 µg/ml of BFA induced a dispersed
distribution of
-COP in BFA-sensitive Vero cells (data not shown),
whereas 0.1 µg/ml BFA did not affect the
-COP distribution in
BER-40 and MDCK cells (Fig. 1). To examine the effect of
C
Cer on BFA-induced redistribution of
-COP, we used
suboptimal concentration of BFA in each cell line, i.e. 0.001
µg/ml, 0.1 µg/ml, and 10 µg/ml in Vero, BER-40, and MDCK
cells, respectively. Fluorescence microscopic studies revealed that low
concentration of C
Cer (1 µM) rendered both
BER-40 and MDCK cells susceptible to BFA in causing a dispersed
distribution of
-COP, whereas C
Cer (1 µM)
alone had no effect on the distribution of
-COP in these cell
lines (Fig. 1). However, C
Cer (1 µM)
did not render Vero cells susceptible to 0.001 µg/ml BFA in causing
a redistribution of
-COP (Fig. 1). Higher concentrations of
C
Cer itself induced redistribution of
-COP in Vero,
BER-40, and MDCK cells; treatment of these cell lines with 35
µM C
Cer resulted in a dispersed distribution
of
-COP in all three cell lines (data not shown).
Figure 1:
Dissociation of -COP from the
Golgi apparatus by BFA in C
Cer-treated BFA-resistant cell
lines. Cells were treated with BFA, C
Cer (1
µM), or BFA plus C
Cer (1 µM) for
30 min in
-MEM. The localization of
-COP was examined using
immunofluorescence microscopy as described under ``Experimental
Procedures.'' Concentrations of BFA were 0.001 or 0.1 µg/ml in
Vero cells, 0.1 µg/ml in BER-40, and 10 µg/ml in MDCK
cells.
In the
present study we have shown that CCer partially restores
BFA sensitivity in BER-40 and MDCK cells based on four distinct effects
of BFA; i.e. the cytotoxicity of BFA, the protection of ricin
cytotoxicity by BFA, the inhibition of bulk protein secretion by BFA,
and the BFA-induced morphological alterations of the Golgi apparatus.
It is worth noting that like BFA, C
Cer inhibits the
transport of vesicular stomatitis virus G protein through the Golgi
complex, and electron microscopic studies have revealed the disruption
of the Golgi apparatus in C
Cer-treated cells (16) .
On the other hand, unlike BFA(9) , C
Cer by itself
has a marginal protective effect on the cytotoxicity of ricin in Vero
cells, and BFA-resistant cell lines are not cross-resistant to
C
Cer. Although C
Cer by itself affects the
structure and function of the Golgi apparatus, its mode of action
appears to be distinct from those of BFA and epidermal cell
differentiation inhibitor. The mode of action of epidermal cell
differentiation inhibitor is also distinct from BFA and
C
Cer, since it does not inhibit bulk protein secretion (15) .
It has been postulated that BFA may mimic an
endogenous lipid ligand in regulating a heterotrimeric G
protein-coupled receptor that controls intracellular membrane traffic
and lipid metabolism(30) . It has been demonstrated that an
inhibitor of sphingolipid synthesis affects protein and lipid
trafficking through the secretory pathway, suggesting that vesicular
transport along the secretory pathway is coupled to sphingolipid
metabolism(31) . A linkage between lipid composition and
protein transport is also suggested by the defective protein secretion
in yeast mutants deficient in phospholipid transfer
protein(32, 33) . Related to these findings is the
recent study showing that BFA reduces cellular SM level in HL 60 cells
without affecting PC level(34) . We have examined the effect of
BFA on SM and ceramide level in Vero, BER-40, and MDCK cells. As shown
in Fig. 2, SM level was slightly increased by BFA treatment in
Vero cells without affecting PC level, whereas SM levels in BER-40 and
MDCK cells were not affected by BFA. In addition, BFA decreased
ceramide levels in Vero and BER-40 cell, whereas MDCK cells were
resistant to these effects of BFA. Interestingly, SM levels in BER-40
and MDCK cells were significantly higher than that in Vero cells,
whereas the PC and ceramide levels were similar in these three cell
lines (data not shown), suggesting that an alteration in SM metabolism
may be associated with increased BFA resistance in BER-40 and MDCK
cells. On the other hand, sphingomyelinase at 1 unit/ml decreased SM
levels and concomitantly increased ceramide levels in Vero and BER-40
cells as reported in other cell lines(35) . MDCK cells were
resistant to sphingomyelinase as well as BFA. Since recent studies
suggest that SM is synthesized in the Golgi apparatus from endoplasmic
reticulum-derived ceramide(36, 37, 38) , the
effects of BFA on sphingolipids metabolism in Vero cells may result
from a perturbation of vesicle trafficking of sphingolipids through the
Golgi complex by BFA, and such processes may be BFA-resistant in MDCK
cells. Ceramide is a precursor for sphingolipid synthesis(39) ,
and the Golgi apparatus is the major site of sphingomyelin
metabolism(37) . Exogenously added ceramide, known to
accumulate in the Golgi complex (38, 40, 41, 42) , may alter the
physical properties of the Golgi membranes in BFA-resistant cell lines
to render them susceptible to BFA action. These results suggest that
the Golgi membranes are structurally and/or functionally altered in
these BFA-resistant cell lines and consequently are refractory to BFA.
BFA has shown to form cation channels in planar lipid
bilayers(43) , and a change in lipid composition or
lipid/protein ratio in the Golgi membranes may affect the
susceptibility of the endomembrane systems to structural and functional
perturbation by BFA. Ceramide reverses the BFA resistance in BER-40 and
MDCK cells by restoring the Golgi membranes to the ``normal''
structural or functional state. On the other hand, it has been shown
that ceramide activates okadaic acid-sensitive cytosolic protein
phosphatases (44) as well as a membrane-bound protein kinase
activity(45, 46) . CCer may exert its
effect through the modulation of
phosphorylation/dephosphorylation-related signal transduction that
regulates vesicle trafficking through the Golgi region. Further studies
of the relationship between ceramide and BFA action may provide new
insights to our understanding of the mode of action of BFA and ceramide
as well as the mechanism of BFA resistance in BFA-resistant cell lines.
Figure 2:
Effect of BFA and sphingomyelinase (SMase) on SM, PC, and Cer levels in Vero, BER-40, and MDCK
cells. Cells, labeled with [C]choline or
[
C]serine as described under ``Experimental
Procedures,'' were treated with indicated concentrations of BFA or
sphingomyelinase. After 1 h, lipids were extracted with
chloroform:methanol (1:2, v/v) and analyzed for lipids content by TLC
as described. Results are expressed as percent of control. These data
(mean ± S.E.) represent triplicate determinations from three
experiments. Upper panel, sphingomyelin; middle
panel, phosphatidylcholine; lower panel, ceramide. Open columns, Vero cells; hashed columns, BER-40 cells; shaded
columns, MDCK cells.