(Received for publication, January 15, 1997, and in revised form, March 20, 1997)
From the Turku Centre for Biotechnology, University
of Turku, Åbo Akademi University, P. O. Box 123, FIN-20521 Turku,
Finland, ¶ Department of Biology, Åbo Akademi University,
BioCity, FIN-20520 Turku, Finland, and
Department of
Biochemistry, Chandler Medical Center, University of Kentucky,
Lexington, Kentucky 40536-0084
Acquisition of heat shock factor 2 (HSF2) DNA
binding activity is accompanied by induced transcription of heat shock
genes in hemin-treated K562 cells undergoing erythroid differentiation. Previous studies revealed that HSF2 consists of two alternatively spliced isoforms, HSF2- and HSF2-
, whose relative abundance is
developmentally regulated and varies between different tissues. To
investigate whether the molar ratio of HSF2-
and HSF2-
isoforms is crucial for the activation of HSF2 and whether the HSF2 isoforms play functionally distinct roles during the hemin-mediated erythroid differentiation, we generated cell clones expressing different levels
of HSF2-
and HSF2-
. We show that in parental K562 cells, the
HSF2-
isoform is predominantly expressed and HSF2 can be activated
upon hemin treatment. In contrast, when HSF2-
is expressed at levels
exceeding those of endogenous HSF2-
, the hemin-induced DNA binding
activity and transcription of heat shock genes are repressed, whereas
overexpression of HSF2-
results in an enhanced hemin response.
Furthermore, the hemin-induced accumulation of globin, known as a
marker of erythroid differentiation, is decreased in cells
overexpressing HSF2-
. We suggest that HSF2-
acts as a negative
regulator of HSF2 activity during hemin-mediated erythroid differentiation of K562 cells.
Heat shock factors (HSFs)1 function as transcriptional activators of the cellular stress response (1). To date, three mammalian HSF proteins, HSF1, HSF2, and HSF4, have been identified (2-5). HSFs have a common core structure consisting of the well conserved DNA binding and oligomerization domains. The DNA binding domain located in the amino-terminal part of the protein mediates binding to a highly conserved heat shock response element (HSE) found in multiple copies in the promoter of heat shock genes. The oligomerization domain adjacent to the DNA binding domain consists of the hydrophobic heptad repeats A/B (HR-A/B) (also termed the leucine zipper repeats 1-3). This domain is responsible for trimerization, which is essential for the high affinity binding of HSFs to the HSE. The carboxyl-terminal hydrophobic heptad repeat HR-C (leucine zipper repeat 4) is also well conserved and has been implicated in maintaining HSFs in the inactive non-DNA binding form (6, 7). External to DNA binding and oligomerization domains, however, the homology between HSF1 and HSF2 is limited (<40%).
A fundamental difference between HSF1 and HSF2 is that they respond to distinct signals. HSF1 is the functional homologue of the general HSF that is activated by diverse forms of stress, such as elevated temperatures, heavy metals, and amino acid analogs (8-12). Upon activation, HSF1 is rapidly converted from a monomer to a trimer, hyperphosphorylated, and translocated into the nucleus (6, 11-13). Unlike HSF1, HSF2 is not activated by acute stress but is constitutively active in mouse embryonal carcinoma cells, at the blastocyst stage during mouse embryogenesis, and during spermatogenesis (14-16). These findings suggest that HSF2 functions as a regulator of heat shock gene expression during development and differentiation. The functional significance of HSF2 DNA binding activity in these biological processes is, however, unclear. The inducible binding of HSF2 to the hsp70 promoter, which closely correlates with the transcriptional induction of the hsp70 gene during the hemin-mediated erythroid differentiation of K562 cells, provides a well studied example of HSF2 acting as a signal-responsive transcription factor (17, 18). Unlike HSF1, the non-DNA binding form of HSF2 is a dimer, but similar to HSF1, HSF2 trimerizes upon activation. Other significant differences between HSF1 and HSF2 are that HSF2 is activated by slower kinetics, is not constitutively or inducibly phosphorylated, recognizes HSEs slightly differently, and is a less potent activator of heat shock gene expression than HSF1 (12, 17, 19).
In most cell types, HSF2 is present in a latent non-DNA binding form.
However, in cells in which HSF2 is constitutively active such as
embryonal carcinoma cells, mouse blastocysts, and mouse testis, the
expression levels of HSF2 are markedly elevated when compared with
other cell types (14-16). Similarly, the acquisition of HSF2 DNA
binding activity correlates with the increased levels of HSF2 in
hemin-treated K562 cells (18). The existence of two distinct isoforms,
HSF2- and HSF2-
, provides another level of regulation that adds
further complexity to the function of HSF2 (20, 21). The smaller
isoform, HSF2-
, is generated from the nascent transcript by an
additional splice, resulting in the deletion of an 18-amino acid
sequence present in the larger HSF2-
isoform (21). The deletion is
located adjacent to the HR-C and may cause modulation of the
transcriptional activity of HSF2 since the HSF2-
isoform has been
shown to be a more potent transcriptional activator than HSF2-
in
NIH 3T3 cells (21). Interestingly, the ratio of HSF2-
and HSF2-
isoforms varies significantly between different mouse tissues such as
brain, heart, and testis (21). In this study, we show that the
hemin-mediated induction of hsp70 transcription is
negatively regulated in K562 cells overexpressing HSF2-
. Our results
also suggest that the molar ratio of HSF2-
and HSF2-
is likely to
be of central relevance in the regulation of HSF2 activity. Finally, we
propose that HSF2 activation is essential for the hemin-mediated
erythroid differentiation of K562 cells.
Human K562 erythroleukemia
cells were cultured in a humidified 5% CO2 atmosphere at
37 °C in RPMI 1640 supplemented with 10% fetal calf serum. Cells
were heat-shocked at 42 °C or treated with 30 µM hemin
as described earlier (22). To generate permanent cell clones
overexpressing HSF2- and HSF2-
, 10 µg of the expression plasmids
-actin-HSF2-
and
-actin-HSF2-
(12, 21) were
transfected to K562 cells using electroporation. Host cells were washed
twice with phosphate-buffered saline and electroporated (Gene
PulserTM, Bio-Rad) in phosphate-buffered saline with 25 microfarad capacitance and 1.35 kV. Transfected cells were allowed to
recover for 2 days, and neomycin-resistant cells were selected in
medium containing G418 (500 µg/ml, Life Technologies, Inc.) for 2 weeks. Drug-resistant cells were diluted and selected for single
cell clones for another 2 weeks. Cell clones that stably express
HSF2-
and HSF2-
were routinely maintained in medium containing
G418 (500 µg/ml). Mock-transfected cells were used as a control.
Before exposure to heat shock or hemin treatment, cells were plated in
medium without G418.
Whole cell extracts were prepared as described (10). Protein samples (10-20 µg) were separated on SDS-polyacrylamide gels (23) and transferred to a nitrocellulose filter using a semi-dry transfer apparatus (Bio-Rad). Western blotting was performed using immunoserum against mouse HSF1 and HSF2 as described (12). The inducible form of Hsp70 was detected by monoclonal antibody 4g4 (Affinity Bioreagents, Inc.). Hsp90 was detected by monoclonal antibody SPA-835 (StressGen Biotechnologies Corp.) and Hsc70 by monoclonal antibody SPA-815 (StressGen). Horseradish peroxidase-conjugated secondary antibodies were purchased from Promega and Amersham Life Science, Inc. The blots were developed with an enhanced chemiluminescence method (Amersham). Globin content was detected by Coomassie staining. Quantitation was performed using a computerized image analysis (Microcomputer Imaging Device version M4, Imaging Research Inc.).
Gel Mobility Shift AssayA gel mobility shift analysis of protein·DNA complexes was performed as described previously (10). Briefly, whole cell extracts were incubated with a 32P-labeled oligonucleotide representing the proximal HSE of the human hsp70 promoter. Protein·DNA complexes were resolved on a 4% nondenaturing polyacrylamide gel.
Nuclear Run-on AnalysisTranscription run-on analysis was
performed with equal numbers of isolated nuclei in the presence of 100 µCi of [-32P]dUTP as described previously (24).
Radiolabeled RNA was isolated and hybridized to
nitrocellulose-immobilized plasmids specific for hsp70 (25),
hsp90 (26), and
-actin (27). Bluescript (Stratagene) was
used as a vector control. The hybridization and washing conditions were
as described (17). Quantitation was performed using phosphoimaging
(Bio-Rad).
Total RNA was extracted from
hemin-treated cells using a single-step method (28). 10 µg of RNA was
separated on a 1% formaldehyde-agarose gel and transferred to a nylon
membrane (Hybond-N, Amersham). The membranes were hybridized with
[-32P]dCTP-labeled probes specific for
hsp70 (25) and
-actin (27) according to the
manufacturer's instructions.
To examine the molar ratio of the HSF2- and HSF2-
isoforms and the effect of hemin treatment on the expression of HSF2, K562 cells were exposed to hemin for various time periods and analyzed
by Western blotting (Fig. 1A). In untreated
cells, the levels of HSF2-
were approximately 2-fold higher as
compared with those of HSF2-
(Fig. 1C). Hemin treatment
increased the abundance of both HSF2-
and HSF2-
isoforms, and the
increased amounts of HSF2 isoforms were observed within 6 h, in
close correlation with the appearance of HSF2 DNA binding activity
(Fig. 1B).
The amounts of HSF1 as well as Hsp70 and Hsp90 were analyzed in
comparison with HSF2 in the same samples (Fig. 1A). Hemin treatment of K562 cells did not affect the accumulation of HSF1. The
heat shock response was shown to be typical (Fig. 1A), as characterized by the retarded mobility on SDS-PAGE due to the inducibly
phosphorylated state of HSF1 (12, 29) and the kinetics of DNA binding
activation (Fig. 1B). Accumulation of Hsp70 protein in cells
exposed to either heat shock or hemin treatment correlated well with
previous results showing transcriptional induction of hsp70
upon heat shock and hemin treatments (Refs. 17 and 18; see also Fig.
3), whereas the levels of Hsp90 did not markedly change upon exposure
to heat shock or hemin.
Altering the Molar Ratios of HSF2 Isoforms by Overexpression of HSF2-
The result showing that
HSF2- is predominantly expressed in K562 cells (Fig. 1C)
raised the possibility that the relative abundance of HSF2 isoforms
could be crucial for HSF2 activation and that HSF2-
and HSF2-
might play functionally distinct roles in the hemin-mediated regulation
of heat shock gene expression. For this purpose, we generated cell
clones that stably express mouse HSF2-
and HSF2-
under the
control of the human
-actin promoter (30). K562 cells were used as
recipients for the expression plasmids (12, 21). More than 15 single
cell clones for each transfection were isolated, and the expression of
the distinct HSF2 isoforms was determined by Western blotting. Table
I summarizes the characteristics of the cell clones that
were chosen for further analysis. As the mouse HSF2 protein shows
extensive homology with the human HSF2 (>95%), it is likely to
function also in human cells. However, different mobilities of mouse
and human HSF2 on SDS-PAGE (12) allowed us to estimate the relative
expression levels of the exogenous HSF2 isoforms. Among several single
cell clones transfected with
-actin-HSF2-
, the 2
-F4 and
2
-C7 cells expressed the highest levels of HSF2-
and were chosen
for further studies. Quantitation revealed that the ratio of total
HSF2-
to HSF2-
was increased 2-2.5-fold in 2
-F4 cells and
2
-C7 cells, respectively, as compared with the parental K562 cells
and the mock-transfected cells (vector). The representative HSF2-
clones (2
-C8, 2
-D5, 2
-E7) expressed 3-5-fold higher levels of
HSF2-
than the control cells.
|
To examine the effect of the exogenously expressed HSF2- and
HSF2-
on the hemin-mediated increase in HSF2 levels, the
transfectants were treated with hemin and analyzed by Western blotting
(Fig. 2). Similar to the parental K562 cells (Fig. 1),
hemin treatment resulted in increased accumulation of the HSF2 isoforms
in mock-transfected cells (Fig. 2, vector). In 2
-C7 cells
expressing exogenous HSF2-
, both endogenous and exogenous HSF2
levels were elevated. Surprisingly, the hemin-mediated increase in the
endogenous HSF2 was repressed in HSF2-
-overexpressing cells
(2
-D5). This repression was detected in all HSF2-
cell clones.
Analysis of HSF1 revealed that none of the mock-transfected cells or
cells expressing exogenous HSF2 isoforms showed changes in accumulation
or phosphorylation state of the endogenous HSF1 (data not shown).
We also analyzed the accumulation of Hsp70 and Hsc70 during hemin
treatment of the transfected cell clones (Fig. 2). Interestingly, the
accumulation of Hsp70 was markedly reduced in the hemin-treated cells
overexpressing HSF2- as compared with the mock-transfected and
HSF2-
-transfected cells. As expected, the levels of the
constitutively expressed Hsc70 were not affected. Based on these
results, we conclude that overexpression of HSF2-
inhibits the
increase in HSF2 amounts and consequently prevents the hemin-mediated
accumulation of Hsp70.
Since our results presented in Fig. 2 suggest that
HSF2- functions as a repressor of the hemin-induced increase in
Hsp70 accumulation, we wanted to determine at which molecular level the
repression occurs. First, we analyzed whether the reduced accumulation
of Hsp70 was caused by a corresponding decrease in hsp70
transcription. The production of hsp70 and hsp90
transcripts were measured by nuclear run-on assay using nuclei isolated
from the parental K562 cells, the mock-transfected cells (vector), and
the cells transfected with HSF2-
(2
-F4) and HSF2-
(2
-D5, 2
-C8). The results presented in Fig. 3A
and quantitated in Fig. 3B show that the repression of Hsp70
indeed occurs at the transcriptional level in the hemin-treated cells
overexpressing HSF2-
, as only a weak, if any, hemin-mediated
induction of hsp70 transcription could be detected.
Similarly, the hemin-mediated induction of hsp90
transcription was repressed due to overexpression of HSF2-
. In
contrast, all other cell clones showed the characteristic
transcriptional induction of heat shock genes upon hemin treatment. The
transcriptional repression of hsp70 and hsp90
genes in HSF2-
-overexpressing cells was specific for hemin treatment
since exposure to heat shock resulted in an equally prominent induction
of heat shock gene transcription in all cells. The repression of
hsp70 gene expression in HSF2-
-overexpressing cells was
observed also at the steady-state mRNA level as analyzed by
Northern blotting (Fig. 3C).
HSF2- and HSF2-
have identical DNA binding and oligomerization
domains. The only alteration in HSF2-
is the deletion of an 18-amino
acid sequence located immediately adjacent to the carboxyl-terminal
HR-C (21). Thus, HSF2-
would be expected to compete with HSF2-
for binding to HSE. To test this assumption, we next analyzed HSE
binding activity in untreated and hemin-treated cells (Fig.
4). As expected, hemin treatment induced the HSE binding activity in parental K562 cells (Fig. 1B), mock-transfected
cells (vector), and cells overexpressing HSF2-
(2
-C7, 2
-F4). Surprisingly, despite the
presence of a DNA binding domain in the HSF2-
isoform, the formation
of a protein·HSE complex was prevented in the hemin-treated cells
overexpressing HSF2-
. This inhibition of DNA binding activity was
detected in all HSF2-
cell clones (2
-D5,
2
-C8, 2
-E7). Quantitation revealed that
overexpression of HSF2-
in 2
-C7 cells potentiated the binding to
HSE. In accordance with the transcriptional induction of heat shock
genes (Fig. 3A), DNA binding activity was strongly induced
upon heat shock (Table I and data not shown), indicating that the
HSF1-mediated heat shock response is not affected by overexpression of
HSF2-
.
HSF2 Activation Is Involved in the Hemin-mediated Erythroid Differentiation of K562 Cells
Hemin-induced erythroid
differentiation of K562 cells was confirmed by analyzing globin
accumulation using Coomassie staining. In agreement with previous
studies (31, 32), increased accumulation of globin was apparent after 3 days of exposure to hemin (Fig. 5). To elucidate whether
HSF2 activity is involved in the hemin-induced erythroid
differentiation, the globin content was determined in HSF2- and
HSF2-
transfectants. As shown in Fig. 5, overexpression of HSF2-
decreased the hemin-induced globin accumulation by 40-60% (2
-D5, 2
-E7), whereas globin induction was
potentiated in HSF2-
-overexpressing cells (2
-F4,
2
-C7). Taken together, our results indicate that the
HSF2-
isoform functions as a negative regulator of heat shock gene
expression and that HSF2 activity is essential for the hemin-induced erythroid differentiation of K562 cells.
Signal-dependent regulation of transcription factors
plays a critical role in the processes underlying cellular
differentiation and development. HSF2 has been shown to be active
during mouse embryogenesis at blastocyst stage, during mouse
spermatogenesis, during hemin-induced erythroid differentiation of K562
cells, and in mouse embryonal carcinoma cells (14-17). Characteristic of the cells involved in these processes is abundant expression of HSF2
as compared with other cell types. Furthermore, HSF2- is the
predominantly expressed isoform in spermatogenic cell types in testis
(21). Based on these results, it has been suggested that increased
expression of HSF2 as well as the relative abundance of the HSF2
isoforms might contribute to HSF2 activation. Our results provide
evidence that besides increased expression of HSF2, a molar excess of
HSF2-
is required for HSF2 activation in K562 cells. Our results
further suggest that HSF2-
and HSF2-
have distinct functions
during hemin-mediated erythroid differentiation, as HSF2-
acts as a
potential activator and HSF2-
as a suppressor of the hemin-induced
transcription of heat shock genes in K562 cells.
Using cell lines that express altered molar ratios of HSF2- and
HSF2-
, we examined the regulation and the functional significance of
the alternatively spliced HSF2 isoforms during the hemin-mediated erythroid differentiation. We observed that excess of HSF2-
prevents the hemin-mediated HSE binding activity and subsequent induction of
heat shock gene transcription. Furthermore, overexpression of HSF2-
specifically inhibits the hemin-mediated increase in the levels of both
endogenous and exogenous HSF2. These results raise the interesting
possibility that HSF2-
negatively controls the amount of HSF2
protein accumulated in a cell. This type of regulation could be a
relevant mechanism to attenuate the hemin-mediated induction of heat
shock gene expression. Our results further suggest that HSF2-
could
counteract the inhibitory effect of HSF2-
on HSF2 DNA binding and
transcriptional activity only when the molar ratio of HSF2-
to
HSF2-
is favorable, i.e. HSF2-
is predominantly expressed. However, overexpression of HSF2-
alone is not sufficient to activate HSF2 in K562 cells, but another factor, such as hemin, is
required for activation.
Yet, it is not clear whether hemin mediates the action on HSF2 directly
by binding to the molecule or by triggering some post-translational modifications thereby resulting in conformational change in HSF2. Interestingly, as hemin-mediated activation of HSF2 is accompanied by
an increase in the levels of both HSF2 isoforms, the conformations of
HSF2- and HSF2-
might be differentially affected by hemin. An
example of a hemin-regulated transcription factor is HAP1, which in
yeast activates transcription of genes encoding cytochromes in response
to oxygen and hemin (33, 34). HSF2, however, does not contain a
sequence similar to the HAP1 repeat, termed hemin regulatory motif,
that binds hemin directly (35). Furthermore, the kinetics of HSF2
activation upon hemin treatment is relatively slow, varying between 6 and 16 h of hemin exposure (Ref. 17 and this study). Based on
these results and the fact that hemin is known to be an important
regulator of protein synthesis (36), it is tempting to speculate that
the hemin-induced increase in HSF2 levels and acquisition of HSF2 DNA
binding activity could be consequences of changes in protein synthesis
leading to a requirement for elevated levels of molecular
chaperones.
There are multiple examples where changes in RNA splicing result in
transcription factor isoforms with altered activities. For example,
certain AP-2 isoforms function as inhibitors of transactivation due to
a deletion in the oligomerization domain, which is necessary for DNA
binding (37). Splicing can also result in deletion of sequences
required for full transactivation potential as exemplified by the
Fos-related proteins (38-40). Analyses of different family members of
nuclear steroid hormone receptors, c-ErbA and ROR, have revealed
that their distinct DNA binding properties are dictated by the specific
amino-terminal domains (41, 42). In the case of HSF2-
, alternative
splicing eliminates 18 amino acids adjacent to the HR-C, and HR-C has
been implicated in maintaining HSFs in the inactive, non-DNA binding
form (6, 7). According to Goodson et al. (21), the deletion
in HSF2-
resulted in poor transactivation capacity in transiently
transfected NIH 3T3 cells without affecting the DNA binding activity.
However, our results showing that overexpression of HSF2-
in K562
cells interferes with HSF2 DNA binding lead us to conclude that the
hemin-induced HSF2 DNA binding activity is controlled by this specific
sequence adjacent to the HR-C located in the carboxyl-terminal region
of the protein. We propose that the different results obtained earlier and in this study are likely to be due to experimental differences reflecting specific features of the different cell types and distinct expression levels of HSF2-
. Interestingly, HSF4, a novel human HSF
lacking the properties of a transcriptional activator, has recently
been identified (5). In HeLa cells, which do not express endogenous
HSF4, overexpression of HSF4 was shown to lead to constitutive HSE
binding activity and repression of target genes, which could be related
to the fact that HSF4 lacks the HR-C. Thus, despite the similar effects
of overexpression of HSF4 and HSF2-
on gene expression, these
factors seem to be functionally distinct, and the mechanism(s) by which
overexpression of HSF2-
prevents HSF2 DNA binding activity remains
to be established.
The biologically relevant finding that HSF2 activity is essential for
the regulation of hemin-induced erythroid differentiation of K562 cells
is based on our results showing that globin accumulation upon hemin
treatment is differentially affected by overexpression of HSF2
isoforms. The enhancing and inhibiting effects of HSF2- and
HSF2-
, respectively, on the signaling cascades leading to activation
of HSF2 and subsequently to accumulation of Hsp70 and globin suggest
that HSF2 plays a critical role during progression of cells to
erythroid differentiation. We also demonstrate that the HSF1-mediated
heat shock response is not affected by overexpression of HSF2-
and
thus provides support for earlier studies implying that hemin-induced
accumulation of Hsp70 in the differentiated erythroblasts of both K562
and bone marrow reflects a differentiation event rather than a general
response to stress conditions (43). Future studies will be directed
toward elucidating whether activation of HSF2 functions as a trigger in
the initiation phase of erythroid differentiation and determining by
which mechanisms HSF2 activity is regulated in cells undergoing
differentiation.
We thank Minna Lönnström for expert technical assistance and Fiorenzo Peverali for valuable advice on photography. Dirk Bohmann, John E. Eriksson, and Päivi J. Koskinen are acknowledged for discussions and critical review of the manuscript.