1 The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA
2 Department of Explorative Science, Biogen, Cambridge, MA 02142, USA
3 The University of Texas-Houston Health Science Center, Houston, TX 77030,
USA
4 Laboratory of Cell and Developmental Genetics, Department of Medicine, Pav.
Marchand, Université Laval, Ste-Foy, QC, G1K 7P4, Canada
5 Institut für Physiologische Chemie, Universität Mainz, D-55099,
Mainz, Germany
Author for correspondence (e-mail:
maul{at}wistar.upenn.edu)
Accepted 31 October 2002
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Summary |
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Key words: ND10, Nuclear depot, PML, Daxx, Heat shock, Heavy metal, Stress, Transcriptional regulation
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Introduction |
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The nucleus of eukaryotic cells is a complex and dynamic structure with
different domains involved in specific processes. For ND10, physiological
functions remain largely unknown, although a depot function has been suggested
(Maul, 1998;
Negorev and Maul, 2001
). The
first ND10 constituent protein molecularly characterized was Sp100, an
autoantigen in primary biliary cirrhosis, which is thought to be a
transcriptional repressor (Sternsdorf et
al., 1999
; Szostecki et al.,
1990
). Another major component of ND10 is PML, a fusion partner of
the RAR
(retinoic acid receptor-
) in the t(15;17) translocation
from patients with acute promyelocytic leukemia (APL). PML has been suggested
to be a tumor suppressor and transcriptional regulator, and might function in
apoptosis (Zhong et al.,
2000b
). PML and Sp100 are both covalently modified by the
ubiquitin-like protein SUMO-1 (sentrin-1), which is thought to regulate the
localization and/or specific protein interactions of the modified proteins
(Kretz-Remy and Tanguay,
1999
). Other proteins including Daxx, BLM helicase, topoisomerase
3, PAX 3 and CBP also colocalize in ND10 (reviewed by
Negorev and Maul, 2001
),
raising the possibility of repressive or activating transcriptional functions
at this site (for a review, see Zhong et
al., 2000b
). However, no newly synthesized RNA
(Ishov and Maul, 1996
;
Grande et al., 1996
) or basal
transcription factors have been reported in ND10.
PML plays a key role in the integrity of ND10, because all other
ND10-associated proteins are dispersed in the absence of PML
(Ishov et al., 1999;
Lallemand-Breitenbach et al.,
2001
; Zhong et al.,
2000a
). However, the possibility of a proto-ND10 at evolutionarily
older eIF-4E sites has been suggested
(Cohen et al., 2001
). In the
present model of ND10 formation and protein recruitment, PML represents the
main scaffold protein to which other proteins such as Daxx bind in a
SUMO-1-dependent way. Under physiological conditions, this is a dynamic
process that might regulate the freely mobile availability of certain
components in the nucleoplasm through a regulatable recruitment-release
process. Thus, recruitment might be favored on at least two different levels:
by increasing the amount of the scaffold protein PML or by enhancing the
post-translational sumolation of PML. Indeed, both interferon (IFN) treatment
to upregulate PML transcription
(Chelbi-Alix et al., 1995
;
Chelbi-Alix et al., 1998
;
Fabunmi et al., 2001
;
Grotzinger et al., 1996
;
Lavau et al., 1995
;
Stadler et al., 1995
) and
overproduction of SUMO-1 enhance the depot function of ND10 by increasing the
capacity to recruit proteins (Ishov et
al., 1999
). However, the mechanism(s) controlling the release has
not been identified. The stress-induced dispersion of ND10-associated proteins
(Maul et al., 1995
) thus
provides the concept and assay to probe for a regulated release mechanism.
Various forms of stress can have profound effects on the physiology of the
organism or cells. Extracellular environmental insults (hyperthermia,
ultraviolet radiation, chemical shock, inflammatory cytokines, heavy metals)
are transduced from the cell surface to the nucleus through stress-activated
signaling pathways, with those involving the mitogen-activated-protein kinases
(MAPKs) being the most conserved. The three major MAPK subfamilies are
represented by the extracellular-regulated kinase (ERK), c-Jun N-terminal
kinase (JNK) and the stress-activated p38 MAPK (SAPK2), all of which induce
the expression of heat-shock proteins (Hsps). Hsps are involved in protecting
damaged proteins from subsequent and/or higher stress levels
(Morimoto and Santoro, 1998).
Upon activation, the Hsp-specific transcription factor HSF1-1 relocates within
seconds into 6-10 nuclear stress granules
(Jolly et al., 1999
), which do
not localize with ND10 (Cotto et al.,
1997
). Pre-existing proteins can be directly activated through one
of these cascades, as in the case of Hsp27, which is phosphorylated within 20
minutes of heat shock by MAPKAP kinase-2, a substrate of the p38 MAPK
(Adler et al., 1995
;
Landry et al., 1991
). The
availability of specific inhibitors permits analysis of the role of such
pathways in the release of ND10-associated proteins upon stress
(Davies et al., 2000
).
A physiological dispersion of ND10-associated proteins occurs during
mitosis (Ascoli and Maul, 1991;
Everett et al., 1998
),
although PML is retained in a few aggregates throughout the mitotic phases
(Maul and Everett, 1994
). The
cytoplasmic PML aggregates apparent in the G1 phase are remnants of
ND10 that were excluded from the nucleus during karyogenesis. Everett et al.
(Everett et al., 1999) described the appearance of a mitosis-specific,
phosphorylated PML isoform and the disappearance of the SUMO-1-modified
isoforms during mitosis, providing some indication that protein modifications
might regulate the release of ND10-associated proteins.
In the present study, we analyzed the effect of two environmental stress
factors, hyperthermia and Cd2+ exposure, on ND10 protein retention,
and the mechanisms controlling the release of proteins from these nuclear
sites. Hyperthermia and Cd2+ also trigger reactivation of latent
herpes simplex virus type 1 (Fawl and
Roizman, 1993; Sawtell and
Thompson, 1992
). Because herpesviruses use ND10 to begin
transcription and replication during lytic infection
(Maul et al., 1996
;
Maul, 1998
; Ishov et al.,
1996; Ishov et al., 1997) and have evolved proteins that disperse
ND10-associated proteins (Maul, 1993;
Everett and Maul, 1994
),
elucidation of mechanisms underlying ND10 protein release and the
physiological consequences of this specific release is relevant to broad areas
of cell physiology.
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Materials and Methods |
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Western blot analysis
Cells were lysed either directly in 6 M guanidine-HCl or in a 3:1 dilution
of RIPA (150 mM NaCl, 50 mM Tris-HCl (pH 8), 1% NP-40, 0.5% DOC, 0.1% SDS) and
SDS sample buffer (250 mM Tris-HCl (pH 6.8), 50% glycerol, 20% SDS, 15%
ß-mercaptoethanol). Samples were boiled for 5 minutes and proteins were
separated by SDS-PAGE, transferred electrophoretically to a nitrocellulose
membrane and incubated with rabbit anti-PML antibody (1:2000), rabbit
anti-Sp100 antibody (1:2000), monoclonal antibody (mAb) 5.14 anti-human Daxx
(Sotnikov et al., 2001)
(1:10), rabbit anti-Daxx antibody (Ishov
et al., 1999
), rabbit anti-mouse-Hsp25 antibody
(Tanguay et al., 1993
)
(1:2500), rabbit anti-Hsp70 antibody (SPA-812) (1:2000) (StressGen
Biotechnologies Group, Victoria, Canada) and rabbit anti-heat-shock-factor-1
(HSF1) antibody (SPA-901) (1:1000) (StressGen Biotechnologies Group, Victoria,
Canada) in 5% milk in PBS/1% Tween as primary antibody.
Horseradish-peroxidase-conjugated secondary antibodies (Vector Laboratories,
Burlingame, CA) were used to develop the blots. For normalization of loading,
all blots were striped and reprobed with anti-tubulin MAb DM 1A (1:20,000
dilution) from Sigma (St Louis, MI).
Immunohistochemistry
Cells were fixed in 1% paraformaldehyde
(Ascoli and Maul, 1991) for 15
minutes and permeabilized in 0.3% Triton-X. ND10 were visualized using MAb
1150 for Sp100 and MAb 5E10 for PML
(Stuurman et al., 1992
).
Polyclonal rabbit antisera were prepared against a
glutathione-S-transferase (GST)-tagged peptide containing PML
residues 334-480 (Sotnikov et al.,
2001
). Human specific anti-Daxx MAb 5.14 were raised against a
His-tagged human Daxx fragment from residues 423-740; the binding epitope has
been localized to residues 495-535
(Sotnikov et al., 2001
). HP-1
MAb was obtained from F. Rauscher (The Wistar Institute) and rabbit anti-Daxx
antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Human
autoimmune serum 21 for fibrillarin
(Yasuda and Maul, 1990
), human
autoimmune serum 44 for lamin B (Maul et
al., 1987
) and autoimmune serum 822 for coilin were used to test
the effects of stress on nuclear integrity. Hsp25, the inducible Hsp70 and
HSF1 were analyzed with rabbit anti-mouse Hsp25
(Tanguay et al., 1993
), rabbit
anti-Hsp70 (SPA-812) and rabbit anti-heat-shock factor 1 (HSF1) (SPA-901)
obtained from StressGen Biotechnologies Group (Victoria, Canada). Anti-SUMO-1
antibodies came from two sources. The antibodies from M. Matunis
(Matunis et al., 1996
) had a
strong tendency also to stain the nuclear envelope, whereas those produced by
Tanguay's group showed strong staining of ND10 and low dispersed nucleoplasmic
staining but no significant staining of the nuclear envelope. FITC- or
Texas-Red-labeled secondary antibodies (Vector Laboratories) served as
fluorescent markers. Cells were examined using a Leica TCS SPII confocal
laser-scanning system. Two or three channels were recorded simultaneously
and/or sequentially, and controlled for possible breakthrough between the FITC
and Texas Red signals and between the blue and red channels. For quantitation
of ND10 and ND10-associated proteins, immunohistochemical samples were
double-labeled for PML and Daxx; for each sample, the total number of PML dots
and the number of colocalizing Daxx dots were counted in 200 cells and
transformed using Microsoft® Excel® for graphical representation.
Separate Daxx antibody-positive dots, which appear in very few cells and are
usually irregularly shaped centromere-associated Daxx deposits (cloud-like
domains found in a subpopulation of the cells during recovery), were not
included.
Reverse-transcription PCR
Total RNA was extracted from cells using the TRI Reagent (Molecular
Research Center, Cincinnati, OH) according to the manufacturer's protocol. The
quality of extracted RNA was determined by spectrophotometry and the
appearance of characteristic 28S and 18S rRNA fragments on a 1% agarose gel.
DNA synthesis was carried out using 5 µg RNA on You-Prime-First-Strand
beads (Amersham Pharmacia Biotech, Piscataway, NJ), and oligo dT15
(0.5 µg, Promega, Medison, WI), according to the manufacturer's protocols.
PCR was performed using 2 µl of the 35 µl cDNA sample. Initial PCR
reactions were performed simultaneously for several cycles to determine the
range of linearity for each gene. Below, we indicate the genes tested, the
size of the expected PCR product, the number of cycles and the sequences of
the 5' and 3' primers.
All PCR reaction were performed with 1.5 mM MgCl2, denatured at 94°C for 1 minute, annealed at 63°C for 40 seconds and extended at 72°C for 40 seconds. PCR products were resolved on 2% agarose gel and photographed with a Polaroid camera.
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Results |
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|
Both SUMO-1 and Daxx staining disappeared from the larger PML aggregates
with increasing treatment time, and the staining did not reappear in the
smaller ones (shown for 1 hour heat shocks for Daxx in
Fig. 1I-K and for SUMO in
Fig. 1L). With these longer
heat shocks, we did not recognize an obvious change in speckles or in Cajal
bodies, but fibrillarin became dispersed by 30 minutes, similar to previous
observations (Liu et al.,
1996). At this treatment time, we also observed HSF1 aggregation
but did not find Daxx localized at these sites
(Fig. 1J). The
immunohistochemistry-based observations provide evidence that ND10-associated
proteins are released from their site of highest concentration, whereas other
nuclear compartments (such as speckles and Cajal bodies) remain intact. The
release of SUMO-1 and Daxx was relatively rapid (less than 12 minutes in most
cells) and occurred in the presence of cycloheximide applied 1 hour before
heat shock and therefore did not depend on new protein synthesis (Maul, 1995).
These observations suggest that the formation of new PML aggregates is
independent of de novo PML synthesis and of the presence of SUMO-1-modified
PML. The latter observation is consistent with previous reports that
unsumofied PML can bind to nuclear deposition sites
(Ishov et al., 1999
;
Lallemand-Breitenbach et al.,
2001
).
After 1 hour exposure to 42°C, the release of SUMO-1, Daxx and Sp100
from ND10 appeared to be complete in most cells. Sp100 had totally dispersed
in 45% of the cells. In the remaining cells, the signal at ND10 had
diminished and was detected as many small dots (shown for a SUMO-1- and
Sp100-stained cell pair in Fig.
1L). Thus, Sp100 disperses more slowly than SUMO-1 and Daxx. After
the initial redistribution of PML into additional and smaller aggregates, the
localization of PML did not change substantially during extended periods of
elevated temperature exposure. The redistribution of ND10 proteins tested
still occurred and Daxx was released in cells pretreated with MG132, an
inhibitor of the proteasomal degradation pathway (data not shown), indicating
that the disappearance of components out of ND10 was not due to a rapid
degradation of proteins.
Cd2+ exposure can also induce a stress-like response and
dispersion of ND10-associated proteins
(Maul et al., 1995). We
therefore tested for potential changes in various nuclear domains using
protocols similar to those for thermal stress. Unlike the rapid dispersion of
SUMO-1 and Daxx and eventually Sp100 in response to hyperthermic exposure, the
response to 50 µM Cd2+ required
2 hours for reliable
observation of dispersion in many cells. After 4 hours of Cd2+
exposure, no changes in speckles, Cajal bodies, nucleoli or nuclear envelope
were observed (Fig. 2A,B). HSF1
granules did form and were not associated with Daxx
(Fig. 2B). However, Daxx
(Fig. 2A,B) and SUMO-1 (not
shown) were dispersed. Importantly, PML was also dispersed in most, but not
all, cells (shown for Daxx and PML stained cells in
Fig. 2C). The retained PML
aggregates were smaller and nucleoplasmic staining was increased. In many
cells, low numbers of Sp100 aggregates that lacked SUMO-1 or Daxx remained
(shown in Fig. 2D for Sp100 and
SUMO-1). The apparent simultaneous loss of PML, Daxx and SUMO, and the
retention of Sp100 in SUMO-1-negative domains suggest that a mechanism
different from that in hyperthermic stress is operating in
Cd2+-exposed cells. Because PML is the protein that retains Daxx at
ND10 (Ishov et al., 1999
), PML
dispersal might cause the dispersal of other ND10-associated proteins but not
necessarily Sp100.
|
ND10 are restored upon recovery from hyperthermic stress or
Cd2+ exposure
To determine whether the heat-shock-induced dispersion of ND10 is
reversible, cells exposed to 42°C for 12 minutes, 1 hour or 3 hours were
left to recover at 37°C for 45 minutes, 2 hours, 5 hours and overnight. In
all recovery experiments, cells showed reformation of normal ND10 (i.e.
recruitment of SUMO-1, Daxx and Sp100 into ND10). However, there was extensive
heterogeneity among and within cells. SUMO-1 was not present in all
PML-positive sites (shown in the upper left cell of
Fig. 1M for 1 hour heat shock
and 2 hours recovery, and the cell at the left, in which almost no SUMO-1
staining is recognizable in PML aggregates). Daxx might be present together
with some PML aggregates (Fig.
1N, right cell) or not at all
(Fig. 1N, left cell). Daxx
might also be deposited at heterochromatin without PML like in
PML-/- cells (irregular green deposits right cell,
Fig. 1N). After 5 hours of
recovery, colocalization was almost completely restored
(Fig. 1O). Overall, the number
of PML aggregates decreased over time and the frequency distribution of PML
aggregates narrowed (Fig. 3A), suggesting release from alternate binding sites that were initially induced by
heat shock. By contrast, Daxx aggregates were totally absent after heat shock
but increased and colocalized with PML during the study period, although they
did not reach the frequency of PML aggregates in the same time
(Fig. 3B and directly seen in
Fig. 1O for 5 hours recovery,
in which some PML aggregates have no Daxx).
|
In cells exposed to 50 µM CdCl2 for 6 hours and monitored
over 24 hours after medium exchange, ND10 presented their normal shape. After
6-18 hours, the number of mitotic figures was increased. A peak of 17% mitotic
cells was recognized at 8 hours recovery and another of 23% at 16 hours
recovery, similar in timing if not in magnitude to that observed after heat
shock (Maul et al., 1995).
Control cells maintained in medium without Cd2+ had a mitotic index
of 2.1. These findings suggest that CdCl2-exposed cells were
accumulated in a specific phase of the cell cycle and then release during
medium exchange, and that the Cd2+ concentration used is subtoxic.
A higher Cd2+ concentration (80 µM) led to cell death during
exposures exceeding the length of a cell cycle, with many cells floating off
the plate.
Heat shock induces changes in desumolation of PML and Sp100
Sumolation of PML is important for the recruitment of Daxx into ND10
(Ishov et al., 1999).
Therefore, the presence of different SUMO-1-modified PML isoforms was analyzed
by western blotting. In non-heat-shocked cells, PML antibodies reacted with an
isoform migrating at
110 kDa and with three major slower-migrating bands
(arrows in Fig. 4A),
corresponding to three SUMO-1-associated isoforms
(Kamitani et al., 1998
;
Everett et al., 1998
;
Muller et al., 1998
). Upon
heat shock, these SUMO-1-associated isoforms gradually diminished, beginning
as early as 12 minutes post-exposure. Reprobing the same blot with anti-Sp100
antibodies revealed a selective lost of the major sumofied isoform of Sp100
(arrow in Fig. 4C). Finally,
use of anti-Daxx mAbs revealed only one major band
(Fig. 4B), with a slight
decrease in intensity after prolonged heat shock. The slow decay of Daxx is
surprising because this protein contains three PEST domains, which are thought
to imply fast turnover through proteasome-dependent hydrolysis
(Rechsteiner and Rogers,
1996
). Thus, Daxx might be released from ND10 and bound to other
sites, preventing its hydrolysis.
|
ND10-associated proteins are released by different mechanisms during
stress
To determine whether the disruption of ND10 upon heat shock is regulated by
the activation of a stress-activated kinase pathways, cells were pretreated
for 1 hour with SB203580 (Cuenda et al.,
1995), which specifically inhibits the stress-activated p38 MAP
kinase, or with PD98059, which inhibits the mitogen-activated MEK/ERK pathway
(Dudley et al., 1995
). Cells
were then heat shocked for 1 hour at 42°C and analyzed for the release of
PML, Daxx and Sp100. No difference was observed between inhibitor-treated
cells and untreated cells. Thus, the rapid heat-shock-induced ND10
modifications in HEp-2 cells appear to be independent of the activation of the
p38 MAP kinase and the MEK/ERK pathways.
Similar analysis of cells preincubated for 1 hour with SB203580 followed by 4 hours of Cd2+ exposure revealed intact ND10 (shown in Fig. 2E for Sp100/SUMO). Thus, a block in p38 MAPK phosphorylation prevented dispersion of ND10-associated proteins upon Cd2+ exposure but not heat shock. Surprisingly, PD98059 had the same effect (shown for Daxx/PML, Fig. 2F) as SB203580 ND10 were retained over prolonged periods of Cd2+ exposure. The different effect of these inhibitors after HS and Cd2+ exposure indicate that the release of ND10-associated proteins is regulated by different pathways.
Overproduction of a SUMO-1 isopeptidase induces release of
ND10-associated proteins
The heat-shock-induced removal of ND10-associated proteins could not be
linked to either of two induced signaling pathways, but the release was
associated with rapid desumolation of PML. We therefore tested whether a
recently described sentrin/SUMO-1 isopeptidase, SENP-1
(Gong et al., 2000) might
account for the release of ND10-associated proteins. In HEp-2 cells
transfected with a SENP-1-expressing plasmid, SENP-1 was found at
ND10 and PML was retained in aggregates of the same size
(Fig. 5A-C, upper right cell is
transfected). No ND10-like staining for SUMO-1 was detected in the
SENP-1-transfected cells, even at very low concentrations of SENP-1.
SUMO-1 staining was retained at the nuclear envelope, where it presumably
modifies RanGAP, suggesting that the SENP-1 effectively and specifically
cleaves SUMO-1 at ND10. Daxx was not detected in ND10 even at low expression
levels of SENP-1 (Fig. 5E-H).
Sp100 was also reduced in most cells but could be found in a few PML
aggregates (Fig. 5I-L). It is
therefore released much more slowly than Daxx, similar to the finding with
heat shock. These findings are consistent with the release of ND10-associated
proteins through desumolation observed after heat shock, except that, under
these conditions, no new PML sites were formed in the SENP-1-transfected
cells. Thus, SENP-1 activity does not induce redistribution of PML to new
sites and PML can remain aggregated in the absence of SUMO-1 over extended
periods of time. These results point to the role of a SUMO-1-cleaving enzyme,
possibly SENP-1, in the release of ND10-associated proteins upon heat shock
and suggest that heat shock activates this enzyme rapidly, although this point
can not be proved at present.
|
Effect of loss of Daxx or PML on the stress response
The supramolecular changes in the nucleus after heat shock suggest that
Daxx is released from ND10 and that some PML is distributed to different
sites, although much remains in distinct domains. Daxx and PML, as repressors,
should have a downstream effect if their release or redistribution has any
physiological relevance. However, the potential repressive effect of the
heat-shock-induced release of Daxx and PML cannot be directly assayed because
many proteins might be inactivated and various transcriptional and splicing
processes are affected by heat shock. Also, proteins other than PML and Daxx
are released from ND10, complicating the analysis. We therefore decided to
test whether the lack of Daxx or PML might affect the induction of two
heat-shock proteins, Hsp70 and Hsp25, using PML and Daxx knockout mouse cell
lines and their respective controls. The control mouse cell lines respond
essentially the same way as human cells with respect to ND10 dispersion after
stress (data not shown). Comparison of Hsp synthesis in normal and
Daxx-/- and PML-/- cells should provide a measure of
Daxx or PML contribution to the heat-shock response as measured by the
accumulation of heat-shock proteins (in these cells, they cannot be released
from a depot). We also used a cell line derived from ES cells
(Michaelson et al., 1999) that
expresses a Daxx fragment lacking the N-terminal 260 residues, as shown by
western blot in Fig. 6. Because
the C-terminus is intact, this protein can bind to PML and be recognized by
immunofluorescence at ND10 (A.M.I. et al., unpublished), although it lacks the
domain involved in binding to other proteins
(Ishov et al., 2002
). Cells
were collected after 1 hour at 42°C and at hourly intervals of recovery at
37°C. Transferred proteins were probed for Hsp25 and reprobed for Hsp70,
HSF1 and tubulin after successive striping of the membranes. As shown in
Fig. 7, all cell lines showed
no difference in the relative amounts of HSF1 and no variation over the time
of recovery from 1 hour at 42°C. We also noticed no significant Hsp70
differences between the respective wild-type control and the Daxx and PML
knockout cells, although there were minor differences early in the recovery
process between sets of cells, which were derived from different stages in
development. The highest accumulation of either Hsp70 or Hsp25 was seen after
6 hours of recovery. However, unlike Hsp70, Hsp25 differed dramatically
between the normal and knock out cells. In Daxx-/- cells, a
substantial amount of Hsp25 was already present in cells not exposed to higher
temperatures and increased from this high level, whereas, in normal cells,
substantial amounts of Hsps were only seen after 2 hours of recovery. A
similar pattern was observed in PML-/- cells. This pattern was
different in the cells containing an N-terminally truncated Daxx, in which
Hsp25 was barely detectable at 6 hours of recovery and normal cells produced
more Hsp25. The results suggest that, in the absence of Daxx or PML, Hsp25 is
produced in appreciable amounts, whereas, in the presence of a truncated form
of Daxx, the production of Hsp25, but not Hsp70, is inhibited. This suggests
that Daxx, presumably through its release-based availability, might bind to
some other protein to inhibit Hsp25 production, whereas the truncated Daxx
retains its repressive activity, which might even be enhanced.
|
|
The inhibition of Hsp25 production might happen at the transcriptional or translational level. To test whether Daxx or PML have an influence on the transcription of Hsp25, cells exposed for 1 hour to 42°C were analyzed for accumulation of Hsp25 mRNA over a range of recovery times using semi-quantitative RT-PCR. For normal and PML-/- cells, induction of Hsp70 mRNA was the same, but Hsp25 mRNA increased slowly over time in the PML+/+ cell. Hsp25 mRNA was present even in unstimulated PML-/- cells and levels increased after stimulation and recovery (Fig. 8A). Again, in Daxx+/+ cells and Daxx-/- cells, there was no significant difference in the Hsp70 mRNA concentration or accumulation (Fig. 8B) but Hsp25 mRNA accumulation in Daxx+/+ cells increased gradually from an insignificant amount before hypothermic exposure to high concentrations after 4 hours of recovery, whereas Hsp25 mRNA in Daxx-/- cells was at maximum after only1 hour exposure and before any recovery time. Message, although at low levels, was also detectable before any exposure to elevated temperatures. Thus, the presence of Daxx and PML, and potentially their heat-shock-induced distribution, might inhibit the transcription and consequent translation of Hsp25. Importantly, the increased transcription during recovery correlates with the resegregation of Daxx and PML into ND10 during recovery and thus with the unavailability of Daxx and PML.
|
CdCl2 exposure induced the stress response and dispersed PML and, along with it, most of the other ND10-associated proteins in most cells. We therefore tested what effect lack of PML would have on the CdCl2-induced heat-shock response. As shown in Fig. 9, exposing cells to various concentrations of CdCl2 for 6 hours decreased slower-migrating PML isoforms or sumolated species, resulting in a higher concentration of the fastest-migrating species. These should become available for binding in the nucleoplasmic space. The exposure of cells to CdCl2 had no effect on the amount of cyclin D1 and tubulin or of HSF1, which might have influenced transcription of Hsp genes. However, the apparent amount of Hsp70 was higher than that of Hsp25 in PML+/+ cells; that is, the ratio of Hsp70 to Hsp25 produced in CdCl2-treated cells was reversed when compared with the heat-shock induction of these proteins. In CdCl2-exposed PML-/- cells, the same ratio of induction was found as with heat shock (i.e. less Hsp70 than Hsp25). Proteins were separated on the same gel and the transferred proteins probed on the same membrane after successive stripping to ensure that the analysis shows comparable concentrations. These results indicate that hyperthermia and CdCl2 in the absence of PML have different effects on Hsp induction.
|
![]() |
Discussion |
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Environmental insults such as hyperthermia, ultraviolet radiation, chemical shock and heavy-metal exposure are transduced from the cell surface to the nucleus of the cell through stress-activated signaling pathways. However, heat-shock-induced dispersion of ND10 proteins, such as SUMO-1, Daxx and Sp100 was not inhibited by blocking the ERK1/2 and p38 MAPK pathways, making it unlikely that the signaling pathways inducing Hsp synthesis are involved in ND10 protein dispersion after heat shock. Instead, a SUMO-1 isopeptidase, such as SENP-1, appears to be rapidly activated, because no protein synthesis is necessary, and SUMO-1, Daxx and Sp100 but not PML were released. A potential cellular defense response located at ND10 might be faster than or precede that of the Hsp synthesis.
Unlike heat shock, Cd2+ exposure induced the dispersion of PML
but not Sp100, suggesting the involvement of a mechanism to disperse PML. The
block in Cd2+-induced ND10 dispersion by inhibiting either p38 MAPK
or ERK1/2 implicates both stress and mitogen signaling pathways in the
dispersion. The apparent communication between the stress-and mitogen-induced
signaling pathways for the release of proteins from ND10 by Cd2+
exposure might resemble that reported for arsenite, in which ERK activation is
strongly enhanced through p38 MAPK (Ludwig
et al., 1998; Rouse et al.,
1994
). Alternatively, both pathways might require the same
substrate, as shown for Mnk1/2 after TPA
(12-O-tetradecanoylphorbol-13-acetate) treatment
(Waskiewicz et al., 1997
),
which would provide a more complex or targeted signaling and response to
Cd2+ exposure. The most direct route for such a possibility would
involve phosphorylation of PML at the three consensus sequences for MAP
kinases (
X(S/T)P), where
is an aliphatic amino acid
(Alvarez et al., 1991
), which
are conserved between human and mouse PML.
The effect of Cd2+ and heat-shock exposure on the release of
ND10 proteins resembles that induced by viruses during lytic infection. In
fact, both Cd2+ and heat-shock exposure reactivate herpes simplex
virus from latency. However, the two stress inducers appear to differ in the
mechanisms that mediate the release. Activation of a specific SUMO
isopeptidase such as or similar to SENP-1 appears to be an immediate event in
the heat-shock response, whereas downstream effects of the mitogen and stress
signaling pathways induced by Cd2+ occur later. Both effects on
ND10 are apparently independent of the classical heat-shock response, because
neither pretreatment by elevated temperatures nor use of protein synthesis
inhibitors influences their actions (Maul
et al., 1995). Our results suggest that both stressors release
ND10-associated proteins by acting on the structure of ND10 at different
levels. The MEK-dependent dissociation of PML by Cd2+ might cause
the removal of proteins associated with PML. This PML dispersal might occur
through direct phosphorylation of PML. Heat shock, however, does not interfere
with PML as a scaffold protein of ND10 but by changing the sumolation level of
PML and Sp100, thereby reducing the ability of PML to retain other components.
Thus, herpes simplex virus reactivation from latency by the two different
stresses might be a consequence of specific proteins released from ND10 and
not of a single, specific, stress-related mechanism.
The complexity of the stress response makes it difficult to analyze whether a directly observed effect such as release of specific proteins from a nuclear domain has physiologically relevant consequences. More than one protein is released into a potentially available pool when such multiprotein aggregates are dispersed. Adding more proteins, such as Daxx or PML, through transient expression would also increase their individual availability but it also induces further aggregation and titration of a host of other effector proteins as well as the stress of the transfection procedure. The availability of PML-/- and Daxx-/- cells and their direct wild-type counterparts from equivalent gestation stages of the mouse enabled analysis of the effect of lack of PML or Daxx on the induction of the Hsp synthesis. Surprisingly, different effects were found for Hsp25 and Hsp70 after hyperthermic treatment of cells. After heat shock, Hsp70 synthesis appeared to be independent of the presence or absence of PML and Daxx, suggesting no general involvement of the heat-shock factor with PML or Daxx. However, Hsp25 synthesis was suppressed by the presence of PML and Daxx in wild-type cells. This suppression was even more evident in cells expressing a truncated Daxx that can bind to PML. The suppressive effect is presumably exerted at the transcriptional level because accumulation of Hsp25 mRNA was very slow in the presence of PML and Daxx but immediate and near maximal in the absence of PML or Daxx. The removal of Daxx and PML from the freely accessible compartment into the bound and therefore inaccessible compartment of ND10 during the recovery period appears to have the same effect as the absence of these proteins. It correlates with the full expression of HSP25. This is consistent with the idea that release of Daxx induces repression of Hsp25. In cells exposed to CdCl2, we find the same pattern for Hsp25 as for thermal stress. It remains unclear how the release of either PML or Daxx can have the same effect of reducing or slowing transcript accumulation. Daxx might still affect the production of Hsp25 in the absence of PML by being released from its alternate binding sites. However, Hsp70 production was strongly inhibited in the absence of PML during CdCl2 exposure in PML-/- cells. PML might have to be released, suggesting that PML can modulate the activation of Hsp70 production. We suggest that the regulated release of Daxx and PML by various types of stress modifies the cell's heat-shock response differently at the transcriptional level. It is astonishing that the cell has mechanisms to slow down the heat-shock response.
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