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* Turku Centre for Biotechnology,
Department of Biology,
Department of Anatomy, and
§ Department of Biochemistry and Pharmacy, Åbo Akademi University, University of Turku, FIN-20521 Turku, Finland
1Correspondence: Turku Centre for Biotechnology, BioCity, 5th Floor, Tykistökatu 6, FIN-20521 Turku, Finland. E-mail: lea.sistonen{at}btk.utu.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: HSF2 • K562 cells • erythroid differentiation • megakaryocytic differentiation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
). Differentiation
lineage-restricted transcription factors are thus good candidates for
mediating the various signals in immature cells to establish or
maintain differentiation lineage-specific gene expression. Therefore,
it is essential to study the expression, complex formation, and
chromatin accessibility of these specific transcription factors to
understand the mechanisms underlying the complex processes of cell
differentiation.
The human K562 erythroleukemia cell line is a multipotent
hematopoietic precursor cell line derived from a patient with chronic
myeloid leukemia in blast crisis (3)
. K562 cells can be induced to
differentiate along several lineages, thus providing a model system to
study gene expression during hematopoiesis. The K562 cell line has been
used extensively in studies of hemin-induced erythroid differentiation
(4
5
6)
. Treatment of K562 cells with hemin, the synthetic form of heme,
induces the synthesis of red cell-specific proteins, such as globin
polypeptides, as a result of transcriptional activation of the
embryonic 
- and ß-like globin genes, 
and 
, respectively, as
well as the fetal 
-globin and adult -globin genes (6)
. Hemin
treatment does not, however, lead to terminal differentiation of K562
cells, thereby dissociating increases in intracellular hemoglobin
content from other events considered central to erythroid
differentiation (5)
. On the other hand, treatment of K562 cells with
the tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate
(TPA)2
shifts these cells
toward megakaryocytic pathway of differentiation, leading to loss of
their erythroid properties and to acquisition of several
megakaryoblastoid characteristics, including synthesis and secretion of
platelet-derived growth factor (PDGF) polypeptides as well as synthesis
and surface expression of glycoprotein IIIa (for review, see ref 7
).
TPA is known to exert its effects on various cellular processes, such
as growth and differentiation, through activation of protein kinase C
(8)
. For example, TPA-induced megakaryocytic differentiation of the
human HEL erythroleukemia cells has been shown to be mediated by
protein kinase C (9)
.
Transcriptional activation of heat shock genes, which ultimately
leads to increased synthesis of heat shock proteins (Hsp's), is
regulated by a family of transcription factors called heat shock
factors (HSFs) that respond to external stimuli, such as elevated
temperatures and diverse physiological and environmental stressors (for
review, see refs 10,
11
). In yeast and Drosophila, only one
HSF-encoding gene has been identified (12,
13)
, whereas in vertebrates
several members of the HSF family (HSF1–4) have been cloned (14
15
16
17
18)
.
In mammalian cells, HSF1 mediates the ubiquitous response to stress
stimuli, whereas HSF2 is regulated by distinct signaling mechanisms.
HSF2 is abundantly expressed and constitutively active in mouse
embryonal carcinoma cells, at the blastocyst stage during mouse
embryogenesis, and during spermatogenesis, suggesting a role for HSF2
as a developmental regulator (19
20
21
22
23)
. In addition, HSF2 binds to a
specific DNA binding sequence (heat shock element, HSE) in the hsp70
gene promoter, leading to abundant expression of Hsp70 protein during
hemin-mediated erythroid differentiation of K562 cells (24
25
26
27)
. During
embryogenesis, however, the pattern of HSF2 DNA binding activity does
not coincide with the expression profile of any of the known Hsp's
(22)
. The expression of thioredoxin (TRX) is induced in K562 cells in
response to hemin in an HSF2-dependent manner (28)
, providing evidence
that HSF2 might regulate genes other than the known heat shock genes.
Recently, the roles of distinct HSFs have been proposed to overlap
depending on stimulatory signals. For example, HSF2 activation and
consequent transcriptional induction of heat shock genes have been
indicated in cells where the ubiquitin-proteasome pathway is inhibited
(29)
. Moreover, mutated yeast cells carrying a lethal HSF deletion can
be rescued by human HSF2, but not by HSF1 (30)
.
More complexity to the regulatory functions of HSF2 is added by the
finding that HSF2 exists as two alternatively spliced isoforms,
HSF2- and HSF2-ß (31,
32)
. According to our recent results,
HSF2- is the predominantly expressed isoform in K562 cells (27)
. A
molar excess of HSF2- is required for the hemin-mediated activation
of HSF2 since overexpression of the HSF2-ß isoform inhibits HSF2
activity, hemin-induced transcription of heat shock genes, and
erythroid differentiation of K562 cells (27)
. In this study, we have
analyzed the expression of human HSF2 in K562 cells induced to
differentiate by hemin or TPA along the erythroid or the megakaryocytic
lineage, respectively. Our results reveal that the expression of HSF2
is strictly and specifically regulated in a lineage-restricted manner,
i.e., hemin enhances and TPA down-regulates HSF2 expression in K562
cells, suggesting that HSF2 might be an important transcriptional
regulator involved in erythroid differentiation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and
HSF2-ß isoforms (2-C7 and 2ß-D5, respectively; ref 27
) were
maintained in RPMI 1640 medium containing G418 (500 µg/ml; Life
Technologies, Inc., Paisley, U.K.). For experimental treatments, cells
were seeded at 5 x 106 cells per 10
cm-diameter plate (HSF2-- and HSF2-ß-overexpressing cells were
plated in RPMI 1640 medium without G418). Hemin (Aldrich, Milwaukee,
Wis.) was added to a final concentration of 30 µM, TPA (Sigma, St.
Louis, Mo.) to 10 nM, and actinomycin D (Sigma) to 6.4 µg/ml; cells
were incubated at 37°C for the time periods indicated. Heat shock was
performed at 42°C in a waterbath.
Gel mobility shift analysis
Whole-cell extracts were prepared from experimentally treated
cells, as described previously (33)
, and incubated (12 µg protein)
with a 32P-labeled oligonucleotide representing
the proximal HSE of the human hsp70 promoter. The protein–DNA
complexes were analyzed on a native 4% polyacrylamide gel as described
previously (33)
. The signal intensities of the protein–DNA complexes
were quantitated using a phosphorimaging scanner (Bio-Rad, Hercules,
Calif.). For antibody supershift experiments to analyze HSF1 and HSF2
composition in the HSE binding complex in K562 cells treated with 30
µM hemin for 18 h or 10 nM TPA for 24 h, dilutions (1:10,
1:50, and 1:100) of antisera against mouse HSF1 and mouse HSF2
(mHSF1 and mHSF2, respectively; a kind gift from Dr. Richard
Morimoto; ref 34
) were added to whole-cell extracts and incubated at
25°C for 15 min prior to gel mobility shift analysis. For the
competition experiment, the binding reaction mixture contained 0.1 ng
of the labeled HSE oligonucleotide and a 50-, 100-, or 200-fold molar
excess of the unlabeled HSE oligonucleotide or a 100-fold molar excess
of an unspecific oligonucleotide.
SDS-PAGE and Western blot analysis
Whole-cell extracts (12 µg protein) were subjected to 8%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred to nitrocellulose filter (Protran Nitrocellulose;
Schleicher & Schuell, Keene, N.H.) using a Bio-Rad semidry transfer
apparatus. HSF2 was detected by a polyclonal antibody specific to mouse
HSF2 (34)
, the inducible form of Hsp70 by 4g4 (Affinity Bioreagents,
Inc., Neshanic Station, N.J.), the constitutively expressed Hsc70 by
SPA-815 (StressGen, Victoria, B.C., Canada), and fetal -globin by
PBF-R (Isolab, Akron, Ohio). Horseradish peroxidase-conjugated
secondary antibodies were purchased from Promega (Madison, Wis.) and
Amersham (Little Chalfont, U.K.). The blots were developed with an
enhanced chemiluminescence method (Amersham).
Northern blot analysis
Poly(A) mRNA was isolated from the treated cells using a poly(A)
mRNA purification kit (Pharmacia, Piscataway, N.J.). RNA was separated
on a 1% agarose-formaldehyde gel, transferred to nylon filter
(Hybond-N; Amersham), and hybridized at 65°C with a
[-32P]dCTP (50 µCi, 3000 Ci/mmol; ICN,
Irvine, Calif.) -labeled 931 bp HindIII/PstI cDNA
insert coding for human HSF2 (hHSF2 cDNA was a kind gift from Dr.
Robert Kingston; ref 16
), a 500 bp cDNA insert coding for human TRX
(ref 28
), a 1510 bp cDNA insert coding for human PDGF-B (a kind gift
from Dr. Kari Alitalo), and
[-32P]dCTP-labeled plasmids for the
following genes: human hsp70 (pH2.3; ref 35
), rat GAPDH (pGAPDH; ref 36
), and human ß-actin (pHFßA-1; ref 37
). After hybridization,
filters were washed with high stringency conditions (0.1X SSC-0.1% SDS
at 65°C; 1X SSC is 0.15 M sodium chloride and 0.015 M sodium
citrate), and visualized by autoradiography. The intensities of
radioactive signals were quantitated using a computerized image
analysis (Microcomputer Imaging Device version M4, Imaging Research,
Inc.) or a phosphorimaging scanner (Bio-Rad).
Nuclear run-on analysis
Nuclear run-on transcription reactions were performed with
nuclei isolated from hemin- or heat shock-treated cells in the presence
of 100 µCi of [-32P]dUTP (3000 Ci/mmol;
Amersham) as described previously (38)
. Radiolabeled RNA was hybridized
to nitrocellulose immobilized 931 bp HindIII/PstI
cDNA insert coding for human HSF2 (16)
and plasmids for the following
genes: human hsp70 (pH2.3; ref 35
), human hsp90/89 (pUCHS801; ref 39
), human ß-actin (pHFßA-1; ref 37
), and a Bluescript vector
(Stratagene, San Diego, Calif.). The hybridizations were carried out in
50% formamide-6X SSC-10X Denhardt's-0.2% SDS at 42°C for 72 h. Filters were washed with high stringency conditions (0.2X SSC-0.2%
SDS at 65°C) and visualized by autoradiography. The intensities of
radioactive signals were quantitated using a phosphorimaging scanner
(Bio-Rad).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The
differentiation lineage specificity of HSF2 expression has, however,
remained unknown. Therefore, we examined the activation and expression
of HSF2 during TPA-induced differentiation of K562 cells toward the
megakaryocytic pathway. First, we confirmed the ability of K562 cells
to differentiate along the erythroid and megakaryocytic lineages by
hemin and TPA by analyzing the expression of specific markers, i.e.,
the fetal -globin and PDGF polypeptide, respectively. In accordance
with earlier studies (5,
40)
, accumulation of the fetal -globin
polypeptide was markedly increased after 48 h of hemin treatment
as compared with untreated cells, whereas during the corresponding
treatment with TPA, the -globin polypeptide could not be detected
(Fig. 1
A, left panel). Abundant expression of PDGF-B mRNA was
observed by 24–48 h of TPA incubation, but not on hemin treatment
(Fig. 1A
, right panel).
|
As shown in the left panel of Fig. 1B
, HSF–HSE complex
formation was induced by 16 h treatment with hemin, but not by
TPA. Consistent with our earlier results, HSF2 was primarily activated
in hemin-treated K562 cells, as shown by antibody supershift assay
using specific antisera against mouse HSF1 and HSF2 (Fig. 1B
, middle panel; ref 26
). TPA did not abolish the
hemin-induced HSF2 DNA binding activity (he+TPA; Fig. 1B
).
Likewise, HSF2 DNA binding could not be activated by hemin after a TPA
pretreatment (TPA+he; Fig. 1B
, left panel), suggesting that
commitment of K562 cells to an erythroid differentiation pathway
requires activation of HSF2. Furthermore, HSF2 activation appears to be
irreversible, as the already activated HSF2 could not be inactivated by
TPA. After several hours of exposure to TPA, a faster migrating,
smaller molecular weight DNA binding complex of unknown origin was
observed (asterisks in Fig. 1B
). It appears, however, that
this DNA binding complex did not contain HSF2, since neither the
specific antisera against mouse HSF1 or HSF2 nor an excess of
nonradiolabeled HSE oligonucleotide displaced the corresponding band
(Fig. 1B
, right panel).
To analyze the effects of hemin and TPA on HSF2 expression, the levels
of HSF2 protein were analyzed upon exposure to hemin or TPA or to
combined hemin and TPA treatment. In contrast to the increased
accumulation of HSF2 protein in K562 cells exposed to hemin for 16 h, treatment with TPA resulted in a dramatic reduction of HSF2 protein
by 4 h (41)
and a complete loss by 16–24 h (Fig. 1C
).
However, a similar decrease in the levels of HSF2 protein was not
detected in cells exposed to TPA for 1–6 h after a 16 h
pretreatment with hemin (he+TPA; Fig. 1C
). When K562 cells
were preincubated with TPA for 1 to 4 h prior to addition of hemin
for 16 h, the TPA-induced loss of HSF2 protein could not be
reversed with hemin (TPA+he; Fig. 1C
). Together with
previous studies (26,
27)
, this provides evidence that the HSE binding
activity induced by hemin (Fig. 1B
) requires presence of
HSF2. The amounts of Hsc70 remained constant on both hemin and TPA
treatment.
To examine whether the hemin-induced increase and the TPA-induced
decrease in HSF2 protein were due to changes in HSF2 mRNA expression,
K562 cells were exposed to hemin or TPA for various time periods and
poly(A) mRNA samples were analyzed by Northern blotting. In untreated
cells, a basal HSF2 mRNA expression was detected, and the levels of
HSF2 mRNA gradually increased up to sixfold by 24 h of hemin
treatment (Fig. 2
A). The amounts of hsp70, TRX, and ß-actin mRNAs were
analyzed in comparison with HSF2 mRNA in the same samples. Consistent
with our previous results (28)
, hsp70 mRNA was also induced by hemin
treatment, but, in contrast to HSF2 mRNA, hsp70 mRNA expression reached
the maximum level already at 16 h and started to decrease
thereafter (Fig. 2A
). mRNA expression of human TRX, another
gene that has been shown to be regulated in concert with HSF2
activation (28)
, was increased by hemin with kinetics similar to HSF2
mRNA (Fig. 2A
). ß-Actin was used to confirm the equal
loading of mRNA samples. In contrast to the results obtained with
hemin, upon exposure to TPA, HSF2 mRNA expression was gradually
down-regulated after 6 h of TPA treatment to the extent that the
HSF2 mRNA levels were decreased to 40% from the levels detected in
untreated cells, as normalized against GAPDH mRNA levels; HSF2 mRNA
levels were barely detectable at 24–48 h of TPA treatment (Fig. 2B
).
|
HSF2 gene expression is regulated both at the transcriptional level
and by mRNA stabilization in hemin-treated K562 cells
Next we wanted to establish whether the induction of HSF2
expression on hemin treatment was regulated on the transcriptional
level; nuclear run-on assay was performed with nuclei isolated from
untreated, heat-shocked, and hemin-treated K562 cells. Transcription
analysis revealed a modest but consistent 1.5- to 2-fold increase in
HSF2 gene transcription upon exposure to hemin for 16 h
(Fig. 3
A), as normalized against ß-actin transcription. As
expected, in contrast to transcriptional induction of the classical
heat shock genes hsp70 and hsp90 in response to hemin and heat shock,
transcription of the HSF2 gene was not induced by heat shock (Fig. 3A
). This is consistent with the finding that HSF2 DNA
binding is activated in hemin-treated but not in heat-shocked K562
cells (26)
. It is worth noting that the transcriptional induction of
HSF2 gene was not detected in the earlier study (42)
. This discrepancy
may be due to the differences in experimental conditions, because a
whole plasmid containing the mouse HSF2 cDNA was used earlier as a
probe instead of the more specific human HSF2 cDNA insert used in
the present study (for details, see Materials and Methods).
|
Because the prominently elevated HSF2 steady-state mRNA levels on
hemin treatment (Fig. 2A
) were unlikely due to the modest,
at most twofold transcriptional induction (Fig. 3A
), we
wanted to determine whether the half-life of HSF2 mRNA was affected by
hemin. To prevent de novo gene transcription, actinomycin D
was added to control, hemin-treated, or heat-shocked cells for
different time periods, and the HSF2 mRNA levels were monitored over
the ensuing 4 h period. TRX mRNA was used as a normalization
control in the quantitative analysis, since the half-life of TRX mRNA
was not affected by hemin (Fig. 3B, C
). Exposure to hemin
led to a marked stabilization of HSF2 mRNA. After 4 h of
actinomycin D treatment, the relative level of HSF2 mRNA in
hemin-treated cells was clearly higher than in control cells when the
values were normalized against TRX mRNA (Fig. 3B, C
). The
half-life of HSF2 mRNA was less than 1 h in heat-shocked cells,
whereas it was ~2 h in control cells. We also analyzed the half-life
of hsp70 mRNA in control, hemin-treated, and heat-shocked cells. As
shown in Fig. 3B
, the half-life of hsp70 mRNA was ~2 h in
control cells and 4 h in hemin-treated and heat-shocked cells,
which is in agreement with earlier studies (43,
44)
. To examine whether
the stabilization of HSF2 mRNA required novel protein synthesis, K562
cells were incubated in the presence of cycloheximide, an inhibitor of
protein synthesis, either alone or in combination with hemin, and
poly(A) mRNA levels were analyzed. However, no marked changes in the
HSF2 mRNA levels were observed during cycloheximide treatment (data not
shown), suggesting that the stabilization of HSF2 mRNA is not dependent
on de novo protein synthesis. However, the amounts of HSF2
protein gradually decreased on cycloheximide treatment (data not
shown), which is consistent with the study recently reported by Mathew
and co-workers (29)
.
Down-regulation of HSF2 mRNA by TPA requires the presence of HSF2
promoter
The decrease in HSF2 mRNA expression by TPA (Fig. 2B
)
prompted us to investigate whether this down-regulation occurred at the
promoter level. For this purpose, we made use of stably transfected
K562 cell clones overexpressing either mouse HSF2- (2-C7) or
HSF2-ß (2ß-D5) isoforms under the control of human ß-actin
promoter (27)
. Consistent with earlier results, hemin-induced
accumulation of both the endogenous and exogenous HSF2 protein was
observed in 2-C7 cells, but not in 2ß-D5 cells (Fig. 4
; ref 27
). As expected, in K562 cells as well as in the transfected cell
clones, the endogenous human HSF2 protein decreased during TPA
treatment (Fig. 4)
. In contrast, the exogenous mouse HSF2 isoforms
expressed under the control of human ß-actin promoter were not
affected by TPA treatment (Fig. 4)
, indicating that the inhibiting
effect of TPA on HSF2 expression is likely to be mediated through the
endogenous HSF2 promoter.
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The differentiation lineage-dependent expression of HSF2 upon
treatment with hemin or TPA is specific for K562 cells
Finally, we wanted to determine the specificity of the
differentiation pathway-dependent expression of HSF2. To this end, in
addition to K562 cells, various human cell lines, such as Raji
(Burkitt's lymphoma), Molt-4 (T-lymphoblastic leukemia), and HeLa
(cervical carcinoma) cells, were treated with either hemin or TPA for
24 h and whole-cell extracts were analyzed by Western blotting. As
shown in Fig. 5
, in all cell lines tested, HSF2 protein was readily detectable also in
samples isolated from untreated cells. Yet the amounts of HSF2 varied
considerably between different cell lines, Raji cells containing the
highest and HeLa cells the lowest levels. A prominent hemin-induced
increase in HSF2 protein was observed only in K562 cells. Similarly,
the TPA-mediated loss of HSF2 protein was specific for K562 cells
induced to differentiate along the megakaryocytic lineage, since in the
other cell lines there was essentially no change in the amounts of HSF2
protein (Fig. 5)
. Hsc70 protein levels are shown as a control for equal
loading of samples.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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42)
. In this study, we have
analyzed the molecular levels at which the regulation of HSF2
expression occurs in K562 cells induced to differentiate along the
erythroid or megakaryocytic lineage. Our results reveal that in
hemin-treated K562 cells, HSF2 expression is up-regulated both at the
transcriptional level and by mRNA stabilization. The enhanced
expression of HSF2 is strictly specific for the erythroid properties of
K562 cells; during TPA-induced megakaryocytic differentiation, HSF2
expression is efficiently and rapidly down-regulated, leading to a
complete loss of HSF2 protein.
The K562 erythroleukemia cell line provides a unique human cell model
with which to study gene expression during hematopoiesis. In
vivo, the proliferation and maturation of megakaryocyte and
erythroid precursors are regulated by two structurally related growth
factors, thrombopoietin and erythropoietin, respectively (for review,
see ref 45
). K562 cells possess several erythroid properties that are
enhanced upon treatment with hemin (3,
46
47
48)
, whereas treatment with
TPA leads to loss of their erythroid characteristics, directing these
cells toward megakaryocytic lineage (49)
. We show here that the
expression of HSF2 is differentially regulated in K562 cells undergoing
either erythroid or megakaryocytic differentiation. HSF2 seems to be
needed for maintaining and enhancing the erythroid properties of K562
cells, as characterized by measuring globin expression (27)
, whereas
HSF2 is rapidly and efficiently down-regulated during TPA-mediated
differentiation along the megakaryocytic lineage. Identification and
characterization of specific markers for the distinct hematopoietic
lineages are important for understanding the molecular mechanisms that
underlie the differentiation processes and malignant transformation.
Recently, differential expression of the Kell blood group and CD10
antigens, two related membrane metallopeptidases, was reported in K562
cells undergoing megakaryocytic and erythroid differentiation (50)
.
Expression of Kell and CD10 antigens represent relatively late
differentiation markers, whereas expression of HSF2 is up-regulated and
down-regulated within a few hours in the presence of hemin and TPA,
respectively. Furthermore, the differential regulation of HSF2 seems to
be specific for the progenitor cell-like ability of K562 cells to
differentiate along several lineages, since in Raji, Molt-4, and HeLa
cells the levels of HSF2 protein did not essentially change when
treated with hemin or TPA. Therefore, HSF2 expression could provide an
early hallmark for lineage-specific differentiation pathways of K562
cells.
Although HSF2 is present in various cell types and tissues (22,
31)
, it is to our knowledge the first transcription factor described
whose expression is strictly regulated in K562 cells differentiating
along the erythroid and megakaryocytic lineages. The importance of HSF2
for maintaining and promoting erythroid properties is further
emphasized by the absence of this factor in K562 cells undergoing
megakaryocytic differentiation. It is well established that certain
hematopoietic-restricted transcription factors, such as GATA-1 and
NF-E2, are coexpressed within the erythroid and megakaryocytic
differentiation lineages, raising the possibility of common programs or
mechanisms of gene activation in the various differentiation pathways
(51
52
53)
. GATA-1 was originally identified through its interaction
within the ß-globin locus control region (54,
55)
. Subsequently, a
consensus DNA binding motif for GATA-1 has been found in the
cis-regulatory elements of virtually all known
erythroid-expressed gene promoters (for review, see ref 56
). In K562
cells, the hemin-induced in vivo binding of HSF2 to the HSEs
within the human hsp70 promoter results in transcriptional induction of
the hsp70 gene (26,
42)
. However, it is evident that HSF2 might have
other target genes apart from the known heat shock genes (22,
28)
. We
speculate that during erythroid differentiation of K562 cells, in
addition to or instead of activating the classical heat shock genes,
HSF2 might be a potential candidate either alone or in combination with
certain erythroid-specific transcription factor(s) to regulate the
expression of erythroid-specific genes. In contrast, during
differentiation along the megakaryocytic lineage, HSF2 seems to be
dispensable, and its expression is down-regulated.
The inducible expression of HSF2 indicates a different regulatory
mechanism as compared with the stress-responsive transcription factor
HSF1. Upon activation, expression of the HSF1 gene appears to remain
unaltered but the HSF1 protein undergoes posttranslational
modifications such as oligomerization (i.e., trimerization) and
hyperphosphorylation (34,
57)
. Although HSF2 is known to be converted
from an inert dimeric state to an active trimer, the activation process
is accompanied by enhanced accumulation of HSF2 protein (42)
. In this
study, the transcriptional induction of HSF2 in hemin-treated K562
cells was found to be 1.5- to 2-fold, and the stability of HSF2 mRNA
was markedly increased in the presence of hemin, in contrast to the
rapid decay of HSF2 mRNA in untreated and heat-shocked cells. In
general, although the mechanisms underlying mRNA stabilization are not
yet well understood, at least certain conserved features affecting the
mRNA half-lives, such as sequence determinants and
trans-acting regulatory factors, have been characterized
(for review, see ref 58
). By using cycloheximide to inhibit protein
synthesis, we show that the stabilization of HSF2 mRNA by hemin does
not involve novel synthesis of an HSF2 mRNA binding protein. Whether
the half-life of HSF2 mRNA is regulated by stable RNA-interacting
protein(s) remains to be elucidated. However, it was found that in the
presence of cycloheximide, the levels of HSF2 protein rapidly
decreased, suggesting that the hemin-mediated increase in HSF2 protein
during erythroid differentiation could be due to a stabilizing effect
of hemin on some yet unknown HSF2-interacting protein(s). The short
half-life of HSF2 protein might serve an important regulatory function
considering the need for rapid down-regulation of HSF2 during the
megakaryocytic differentiation.
In conclusion, HSF2 provides an example of transcription factors, the expression of which is strictly regulated at multiple levels in a differentiation lineage-specific manner. In light of our study, regulation of HSF2 expression could be one of the key determinants in the commitment of K562 cells to either erythroid or megakaryocytic pathway of differentiation. The processes actually governing cell differentiation in vivo are certainly more complex, and only spatially and temporarily organized combinations of various components can ensure normal hematopoietic development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication October 14, 1998.
Revision received January 18, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-, ß-, and -actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed. Mol. Cell. Biol. 3,787-795This article has been cited by other articles:
|
|
 |
|
  P. Ostling, J. K. Bjork, P. Roos-Mattjus, V. Mezger, and L. Sistonen Heat Shock Factor 2 (HSF2) Contributes to Inducible Expression of hsp Genes through Interplay with HSF1 J. Biol. Chem., March 9, 2007; 282(10): 7077 - 7086. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Haussecker and N. J. Proudfoot Dicer-Dependent Turnover of Intergenic Transcripts from the Human {beta}-Globin Gene Cluster Mol. Cell. Biol., November 1, 2005; 25(21): 9724 - 9733. [Abstract] [Full Text] [PDF]
|
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 |
|
 T.-P. Alastalo, M. Hellesuo, A. Sandqvist, V. Hietakangas, M. Kallio, and L. Sistonen Formation of nuclear stress granules involves HSF2 and coincides with the nucleolar localization of Hsp70 J. Cell Sci., September 1, 2003; 116(17): 3557 - 3570. [Abstract] [Full Text] [PDF]
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A. Mathew, S. K. Mathur, C. Jolly, S. G. Fox, S. Kim, and R. I. Morimoto Stress-Specific Activation and Repression of Heat Shock Factors 1 and 2 Mol. Cell. Biol., November 1, 2001; 21(21): 7163 - 7171. [Abstract] [Full Text] [PDF]
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 L. PIRKKALA, P. NYKANEN, and L. SISTONEN Roles of the heat shock transcription factors in regulation of the heat shock response and beyond FASEB J, May 1, 2001; 15(7): 1118 - 1131. [Abstract] [Full Text] [PDF]
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L. Pirkkala, T.-P. Alastalo, X. Zuo, I. J. Benjamin, and L. Sistonen Disruption of Heat Shock Factor 1 Reveals an Essential Role in the Ubiquitin Proteolytic Pathway Mol. Cell. Biol., April 15, 2000; 20(8): 2670 - 2675. [Abstract] [Full Text]
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 C. Rabergh, S Airaksinen, A Soitamo, H. Bjorklund, T Johansson, M Nikinmaa, and L Sistonen Tissue-specific expression of zebrafish (Danio rerio) heat shock factor 1 mRNAs in response to heat stress J. Exp. Biol., January 6, 2000; 203(12): 1817 - 1824. [Abstract] [PDF]
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