(Received for publication, August 21, 1996, and in revised form, December 4, 1996)
From the Department of Pharmacology and Physiological
Science, St. Louis University School of Medicine,
St. Louis, Missouri 63104 and the § Department of
Physiology, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205
In this paper we demonstrate for the first time a
mitogen-induced activation of a nuclear acting
phosphatidylcholine-phospholipase D (PLD) which is mediated, at least
in part, by the translocation of RhoA to the nucleus. Addition of
-thrombin to quiescent IIC9 cells results in an increase in PLD
activity in IIC9 nuclei. This is indicated by an increase in the
-thrombin-induced production of nuclear phosphatidylethanol in
quiescent cells incubated in the presence of ethanol as well as an
increase in PLD activity in isolated nuclei. Consistent with our
previous report (Wright, T. M., Willenberger, S., and Raben, D. M.
(1992) Biochem. J. 285, 395-400), the presence of ethanol
decreases the
-thrombin-induced production of phosphatidic acid
without affecting the induced increase in nuclear diglyceride,
indicating that the increase in nuclear PLD activity is responsible for
the effect on phosphatidic acid, but not that on diglyceride. Our data
further demonstrate that RhoA mediates the activation of nuclear PLD.
RhoA translocates to the nucleus in response to
-thrombin.
Additionally, PLD activity in nuclei isolated from
-thrombin-treated
cells is reduced in a concentration-dependent fashion by
incubation with RhoGDI and restored by the addition of prenylated RhoA
in the presence of guanosine 5
-3-O-(thio)triphosphate.
Western blot analysis indicates that this RhoGDI treatment results in
the extraction of RhoA from the nuclear envelope. These data support a
role for a RhoA-mediated activation of PLD in our recently described
hypothesis, which proposes that a signal transduction cascade exists in
the nuclear envelope and represents a novel signal transduction cascade
that we have termed NEST (
uclear
nvelope
ignal
ransduction).
It is now clear that a PLD1 is activated as a component of a number of signal transduction cascades (2-7). Cleavage of PC by a PLD results in the production of a free, water-soluble choline head group, and PA. Although in some systems this PA is the source of increased DG levels generated via PA phosphohydrolase, it is now becoming clear that PA itself plays important signaling roles (3, 7-14). There is evidence, for example, implicating the PLD-mediated production of PA as an important component of the mitogenic cascade (3, 9, 10).
We recently advanced the hypothesis that a novel nuclear lipid
metabolism is a component of unique nuclear signaling cascades that we
defined as uclear
nvelope
ignal
ransduction (NEST) (15, 16). The canonical model of
lipid-mediated signal transduction assumes that all induced lipid
metabolism occurs at the plasma membrane and that the nuclear envelope
is a passive participant in the transduction cascade. In the NEST
hypothesis, just as the plasma membrane serves as the communication
link between the extracellular environment and the cytoplasm, the
nuclear envelope mediates the transmission of cytosolic signals to the
nucleoplasm. Recently, our laboratory and others have presented
compelling data supporting this hypothesis (15-21).
Previous work from our laboratory demonstrated that PC metabolism is a component of NEST (15, 18). One of the PC-hydrolyzing enzymes, PLD, has been identified in the nucleus of Madin-Darby canine kidney cells (19-21), and further studies indicated that this activity may be modulated by RhoA (21). These data suggest that a nuclear PLD is present in these cells, and its activity can be modulated by known signal transduction components.
Clearly, a central tenet of the NEST hypothesis is that the enzymatic
activities involved in this cascade are induced in an agonist-dependent manner. Such an agonist-induced nuclear
activity has not been demonstrated. The data in this report are the
first to demonstrate an agonist-induced increase in a nuclear PLD
activity. This activity contributes to the production of nuclear PA but does not affect the level of nuclear DG generated in response to
-thrombin. RhoA translocates to the nucleus in response to
-thrombin, and removal of this GTP-binding protein with a RhoGDI results in a dose-dependent decrease in nuclear PLD
activity. Taken together, the data demonstrate that the addition of
-thrombin to quiescent fibroblasts leads to the translocation of
RhoA to the nucleus, which mediates the activation of a nuclear
PLD.
Cell culture media were from Life Technologies,
Inc. Tissue culture dishes were from Falcon. Bovine serum albumin,
highly purified human -thrombin, butylated hydroxytoluene, EGTA,
EDTA, quinacrine, 2-nitro-4-carboxyphenyl
N,N-diphenylcarbamate, tetraphenylboron, and
Trizma base (Tris) were obtained from Sigma. Human transferrin was from
Calbiochem. Phospholipase C (Bacillus cereus), aprotinin, and leupeptin were from Boehringer Mannheim. Silica Gel G TLC plates
were from Analtech. DG standards were generated by PC-PLC (B. cereus)-mediated hydrolysis of commercial PC, PA, or PE (22, 23),
which were purchased from Avanti Polar Lipids. Acetonitrile (high
performance liquid chromatography grade) was from J. T. Baker.
Isopropyl ether was from Aldrich. Diethyl ether (high purity) and
chloroform, methanol, acetone, and hexane (all GC2) were
from Burdick and Jackson. All organic solvents contained 50 µg/ml
butylated hydroxytoluene. RhoGDI synthesized as a GST fusion protein
(plasmid a generous gift from Dr. Gary Bokoch (Scripps Research
Institute, La Jolla, CA) in a Escherichia coli expression system was isolated using a glutathione column (24). Palmitoylated RhoA
containing a histidine tag (plasmid a generous gift from Dr. Alan Hall,
MRC Laboratory of Cellular and Molecular Biology, University College
London, London WC1E 6BT, U. K.) was expressed in Sf9 cells and
purified using a nickel affinity column (25). GTP
S was from
Boehringer Mannheim. Anti-RhoA antibodies were purchased from Santa
Cruz (SC-179G). Radiolabels were purchased from Amersham Corp.
IIC9 cells, a subclone of CHEF18,
were grown and serum deprived as described previously (1, 15, 17, 18,
22, 23, 26-29). Briefly, IIC9 cells were grown in 150-mm dishes for 3 days in -MEM/Ham's F-12 containing 5% fetal calf serum. The medium was removed and replaced with serum-free Dulbecco's modified Eagle's medium containing 1 mg/ml grade bovine serum albumin and supplemented with 5 µg/ml human transferrin (serum-free medium). The cells were
serum deprived for 2 days and then washed twice in serum-free medium.
They were incubated at 37 °C in the fresh serum-free medium for at
least 30 min before beginning the experiment. For each experiment,
cells were then incubated at 37 °C in serum-free medium either alone
or containing 1 NIH unit/ml
-thrombin in the presence or absence of
ethanol as indicated in the figure legends.
Nuclei were isolated essentially as described previously (15, 18). Briefly, incubations were terminated by removal of medium, transferring the dishes immediately to an ice bath and adding 4 ml of ice-cold fractionation buffer (buffer B: 10 mM Tris, 10 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 mM phenylmethanesulfonyl fluoride, 10 µM leupeptin, 10 µg/ml aprotinin, 20 µM quinacrine, and 200 µM 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate, pH 7.5 at 4 °C). The cells were scraped from the dishes and subjected to 15 passes in a Potter type Teflon on glass homogenizer. Homogenates from four dishes were used for quantification of DG levels. Homogenation and subsequent steps were carried out at 4 °C.
Nuclei were isolated by centrifugation of the homogenate at 2,000 rpm (700 × g) for 7 min in an RT6000B centrifuge with a swinging bucket rotor. The pellet was dispersed in 5 ml of fractionation buffer and homogenized using a Dounce-type homogenizer with a tight fitting (type B) pestle for 20 passes and layered over a 5-ml cushion of 45% sucrose in fractionation buffer, followed by centrifugation at 2,800 rpm (1,660 × g) for 30 min. The pellet was resuspended in 0.8 ml of buffer B, and a small aliquot was assessed quickly for gross contamination by whole cells and other large debris by light microscopy.
For nuclear lipid analysis, isolated nuclei (typically 50 µg of nuclear protein) were suspended in 0.8 ml of water and transferred into 1 ml of chloroform. The centrifuge tube was washed twice with 1 ml of methanol, and the wash was added to the water and chloroform. Nuclear lipids were extracted (30) and dried under a stream of dry nitrogen.
All other assays, including in vivo assay for PLD, analysis of PA levels, analysis of DG levels, in vitro assay for PLD, treatment of nuclei with RhoGDI, Western blot analysis, were performed as described in the figure legends. Protein was quantified as described by Bradford (33).
As shown in
Fig. 1, PEt in nuclei from cells exposed to -thrombin
in the presence of ethanol was approximately a 2-fold higher than it
was in nuclei isolated from cells exposed to either alone. These data
are consistent with the notion that
-thrombin induced the activation
of a PLD, which catalyzes the hydrolysis of nuclear PC.
The above data demonstrating an activation of PLD acting on the nucleus
implies that -thrombin-induced increase in nuclear PA should be
blunted in the presence of ethanol. Indeed,
-thrombin induced an
increase in nuclear PA. Radiolabeled PA as a percentage of total
labeled nuclear lipid was 0.233 ± 0.072 in quiescent cells and
0.427 ± 0.034, n = 4, after incubation of cells
for 15 min with
-thrombin (1 NIH unit/ml). In the presence of 1% ethanol, the increase in PA induced by
-thrombin was only 49%, significantly less than the 82% increase induced by
-thrombin without ethanol. These data are consistent with the data presented in
Fig. 1 demonstrating the activation of a PLD, which acts on the nuclear
membrane, and they indicate that this PLD is responsible for most of
the PA generated in the nucleus in response to
-thrombin.
In previous reports, we demonstrated in whole cells that
although -thrombin induced the activation of a PLD resulting in the
formation of PA, a PC-PLC was responsible for the generation of
PC-derived DGs (1). We also demonstrated that
-thrombin induced an
increase in nuclear DGs in IIC9 cells and that these DGs are derived
from PC (1, 15, 17, 18). Because the presence of ethanol inhibited the
formation of PA but not DGs even though approximately 50% of the
whole-cell DGs induced by
-thrombin reside in the nucleus (15), it
is unlikely that the nuclear DGs are derived by a
PLD/PA-phosphohydrolase pathway.
To test this directly, we evaluated the effect of ethanol on the
generation of nuclear DGs in response to -thrombin. As shown in Fig.
2, the presence of ethanol does not significantly affect the production of
-thrombin-induced nuclear DGs generated in radiolabeled cells. Similar results have been obtained when the nuclear
DG mass is quantified using the DG kinase assay (29 and data not
shown). These data demonstrate that the induced nuclear PLD is not
involved in the generation of nuclear DGs. In view of previously
published data demonstrating that these DGs are derived from PC (15,
18), the present data implicate a PC-PLC in the production of these
lipids.
In view of the above, it was important to determine if
the PLD activated in response to -thrombin was a membrane-associated enzyme. We examined, therefore, the PLD activity in nuclei isolated from quiescent and
-thrombin-induced cells. As shown in Fig. 3, PLD activity was increased maximally in the nuclei
isolated from
-thrombin-stimulated cells after a 15- and 20-min
exposure to
-thrombin. The data are consistent with the notion that
this enzyme is not involved in the production of nuclear DGs as the PLD
activity was elevated well after the major increase in nuclear DGs
occurred (15, 18). These data demonstrate that a PLD activated in
response to
-thrombin is associated with the nucleus.
There is now strong evidence to suggest that
small molecular weight GTP-binding proteins, RhoA in particular, are
involved in activating PLD (21). RhoA-mediated PLD activity requires that RhoA be constitutively present in nuclei or translocate to the
nucleus in an agonist-induced manner. Western blot analysis of proteins
in nuclei isolated from quiescent cells and -thrombin-induced cultures demonstrated that RhoA translocates to the nucleus in response
to
-thrombin (Fig. 4).
Extraction of RhoA from Nuclei Decreases Induced Nuclear PLD Activity
To investigate further the role of RhoA, nuclei isolated
from -thrombin-induced cultures were treated with RhoGDI, and the level of PLD activity was quantified. As shown in Fig.
5, treatment of these nuclei with RhoGDI resulted in a
concentration-dependent decrease in nuclear PLD activity.
Because this GDI can interact with several members of the Rho family,
released protein was examined by Western blot analysis. Only RhoA was
found to be released (data not shown). Addition of recombinant,
prenylated RhoA, in the presence of GTP
S, restored the activity in
the RhoGDI-treated membranes (Fig. 6). Interestingly,
this RhoA also activates a PLD activity in nuclei isolated from
quiescent cells (Fig. 6), suggesting that the enzyme resides in the
nucleus. These data provide strong evidence indicating a role for RhoA
in the
-thrombin-induced activation of a nuclear PLD.
The canonical model of signal transduction cascades involves the initiation of signals at the plasma membrane which stimulate specific cascades leading to the stimulation of activities in target organelles such as the nucleus. For some time it has been assumed that the nuclear envelope played a passive role in these signal transduction cascades. It is becoming increasingly clear, however, that the nuclear envelope is an active participant in signaling cascades, a process we have termed NEST, and that a major component of these cascades is the induction of specific nuclear lipid metabolism (15-21).
In this report we present the first evidence for the involvement of a
PC-PLD in NEST and identify one of the components involved in coupling
the canonical plasma membrane signaling cascades to this novel pathway
in the nuclear envelope. Our data demonstrate that the addition of a
potent mitogen, -thrombin, to quiescent IIC9 cells results in
increased nuclear PLD activity. This is evidenced by the
-thrombin-induced increase in PEt (Fig. 1) and the increased nuclear
PLD activity in nuclei isolated from
-thrombin-induced cultures
(Fig. 3). We further demonstrate that RhoA is at least one of the
factors involved in this activation. RhoA translocates to the membrane
in response to
-thrombin, and treating nuclei isolated from
-thrombin-induced cultures with RhoGDI results in a
dose-dependent decrease in PLD activity (Figs. 4 and 5). Taken together, these data suggest that the addition of
-thrombin to
quiescent IIC9 cells induces the translocation of RhoA to the nucleus
resulting in the stimulation of nuclear PLD.
Because the GST-tagged RhoGDI used in these studies is too large (50
kDa) to diffuse through a nuclear pore, the above data suggest that the
-thrombin-activated nuclear PLD is located on the outer nuclear
membrane. In these studies, however, we cannot distinguish between a
PLD resident in the outer nuclear membrane which is activated in
response to
-thrombin and a cytosolic PLD that is translocated to
the nucleus during the activation cascade. The activated PLD may
translocate to the nucleus, or agonist-induced changes in the nuclear
envelope may promote the association of the enzyme with the envelope
where it is then activated. Further experiments are in progress to
discriminate between these possibilities.
These and other data lend further support to the notion that mitogens
activate a PC cycle in the nuclear envelope as a component of NEST. Our
data indicate that a PLD is activated by -thrombin, which hydrolyzes
PC, resulting in the production of PA in the nuclear envelope. Our data
also support the notion that a PC-PLC is involved in the generation of
nuclear DGs (15). If a PC cycle were operating in the nucleus, enzymes
involved in the biosynthesis would be expected to be present in the
nucleus. Indeed, one of the enzymes involved in PC biosynthesis,
CTP:phosphocholine cytidylyltransferase, has also been localized in the
nucleus (40-42). This enzyme is particularly interesting as it often
serves as the regulatory enzyme in PC biosynthesis, and its activity is
regulated by diacylglycerol (43, 44). Taken together, these data
provide strong support for the hypothesis that mitogens activate a PC
cycle in the nuclear envelope as a component of NEST.
We thank Dr. Carolyn Machamer for helpful discussions and critically reading this manuscript.