(Received for publication, November 8, 1994)
From the
Members of the low molecular weight heat shock protein (hsp) family show regulated expression in both Drosophila and mice during development and differentiation. Here we have examined whether similar regulation of the single low molecular weight hsp (hsp 28) of humans exhibits differences in either its expression and/or phosphorylation during the course of in vitro differentiation of hematopoietic cells. In the promyelocytic leukemic cell line, HL-60, we show that early after commitment of the cells to a macrophage-like phenotype (via exposure to phorbol ester myristate, PMA) there occurs an accompanying increased phosphorylation of hsp 28. Over time and as the cells become terminally differentiated the levels of hsp 28 increase significantly. In contrast, cells stimulated to adopt a granulocyte-like phenotype (e.g. exposed to either dimethyl sulfoxide or retinoic acid) show no changes in either the phosphorylation or expression of hsp 28. Moreover, once differentiated the granulocyte-like cells no longer appear capable of phosphorylating hsp 28. Human K562 cells, in response to hemin, rapidly increase their expression and phosphorylation of hsp 28 during the course of their differentiation into erythroid-like cells. Addition of PMA to the K562 cells induces differentiation into a megakaryocyte-like phenotype but is not accompanied by changes in hsp 28 phosphorylation/expression. In the case of the HL-60 cells, differentiation toward the macrophage like lineage is accompanied by an increased adherence of the cells to their substratum and an apparent association of hsp 28 with the actin cytoskeleton.
Most members of the heat shock or stress protein family are
constitutively expressed proteins whose synthesis increases
significantly in cells exposed to different environmental insults. Such
an increase in the expression of the stress proteins appears to provide
the traumatized cell with an added degree of protection, thereby
contributing to the cell's overall ability to survive the
particular environmental insult(1) . For at least some of the
stress proteins, in particular the so-called hsp ()70 and
hsp 60 families, recent evidence has implicated their role in
facilitating the early events of protein maturation. Members of these
two families, now also referred to more generally as molecular
chaperones, interact with proteins which are in the course of
synthesis, folding, and/or assembly into higher ordered structures. Via
such an interaction the maturing polypeptide is temporarily provided a
somewhat shielded environment thereby reducing the possibility of
nonproductive intra- or intermolecular interactions which might occur
otherwise during the course of its maturation (reviewed in Refs. 2, 3).
Relative to our new insights regarding the structure-function of
those stress proteins which function as molecular chaperones, less is
known regarding the function of the so-called low molecular weight heat
shock proteins (low molecular weight hsps). Members of this family,
with apparent molecular masses of 20-30 kDa, exist in
vivo as higher ordered structures of
600-900
kDa(4) . At least four related members of this family have been
described for insects like Drosophila(5, 6) .
However. yeast, avian, and animal cells express only a single form of
the protein, referred to by different investigators as hsp 25, hsp 27,
or hsp 28 (the latter designation which will be used
here)(7, 8, 9) . All of the members of this
family exhibit sequence homology with the
-crystallin proteins,
which like hsp 28, exist as higher ordered oligomeric structures in
vivo(10) . While the exact function of both the low
molecular weight hsps and
-crystallins is still unclear, both
groups of proteins have been reported to function as molecular
chaperones in vitro, facilitating both the refolding of
denatured proteins and/or the stabilization of proteins in response to
thermal treatments(11, 12, 13, 14) .
In contrast to the hsp 70 and hsp 60 chaperones, however, such
chaperoning activity provided by the low molecular weight hsps appeared
independent of an added energy source such as ATP. Perhaps our best
insight regarding potential function is a study reporting that the low
molecular weight hsp purified from avian sources can both inhibit the
polymerization of actin filaments as well as sever pre-existing actin
filaments using in vitro assays(15) . Following up on
this observation Landry and colleagues(16, 17) reported that animal cells overexpressing the low
molecular weight hsp, via transfection of the corresponding cDNA,
exhibited changes in their actin microfilament cytoskeleton and
acquired a thermotolerant phenotype.
Expression of the low molecular
weight hsps has been observed to vary as a function of development
and/or differentiation in Drosophila and
mice(18, 19, 20, 21) . In both yeast
and mammalian tissue culture lines, expression of the low molecular
weight hsp increases as the cell progress into their stationary growth
phase, and in the case of yeast, during sporulation (22, 23, 24) . ()In addition to
changes in its expression, changes in the phosphorylation of hsp 28
occur in response to a wide number of stimuli, some of which are
associated with growth and/or differentiation (reviewed in (25) ). For example, hsp 28 is heavily phosphorylated within
minutes after cells have been exposed to different mitogens, tumor
promoters such as phorbol esters, calcium ionophores, or
cytokines(26, 27, 28, 29) . In
addition, the protein is heavily phosphorylated in cells subjected to
heat shock and certain other metabolic
stressors(26, 30) . Recent studies concluded that
phosphorylation results in a reduction in the native size of the
protein(31, 32) .
More than 10 years ago it was shown that an early event accompanying phorbol ester myristate (PMA) induced in vitro differentiation of promyelocytic HL-60 cells was a rapid increased phosphorylation of two proteins with apparent molecular masses of 17 and 27 kDa(33) . Subsequent work by the same investigators concluded that such phosphorylation of these two proteins was transient and likely dependent upon the calcium- and phospholipid-dependent protein kinase C(34) . Subsequently, it was reported that heat shock treatment also appeared to result in the initiation of HL-60 cell differentiation (35) . Because both heat shock and PMA result in a rapid increase in the phosphorylation of hsp 28, we undertook a careful study of the pattern of protein phosphorylation changes which accompany the differentiation of HL-60 cells in vitro. Here, we show that the 27-kDa protein that is rapidly phosphorylated during the early stages of PMA-induced differentiation of HL-60 cells into a macrophage-like phenotype indeed is hsp 28. Prompted by these observations we examined for possible changes in either the expression and/or phosphorylation of hsp 28 in other cell types capable of in vitro differentiation. We show that changes in hsp 28 expression/phosphorylation vary significantly as a function of both the cell type and the differentiation agent employed.
For metabolic labeling, cells were
washed in leucine-free RPMI and then incubated with
[H]leucine (Amersham Corp.; specific activity 75
Ci/mM) in leucine-free RPMI supplemented with 2% fetal calf
serum. Alternatively, cells were washed in phosphate-free RPMI or DMEM,
incubated with [
P] or
[
P]orthophosphate (Amersham; carrier-free, 10
mCi/ml) in phosphate-free RPMI or DMEM for 1 h, and then subjected to
various treatments as described in the figure legends. After labeling,
cells were washed in phosphate-buffered saline (PBS), solubilized in
Laemmli sample buffer (LSB), heated to 100 °C for 5 min, and the
labeled proteins analyzed by one-dimensional SDS-polyacrylamide gel
electrophoresis (PAGE). For two-dimensional SDS-PAGE, cell lysates were
digested with DNase I and RNase A (each 10 mg/ml), quickly frozen, and
then lyophilized. Samples were redissolved in isoelectric focusing
buffer containing 9.9 M urea, 4% Nonidet P-40, 2% ampholytes,
and 100 mM dithiothreitol.
Changes in the phosphorylation of hsp 28 were examined in
HL-60 cells during their early commitment to the macrophage-like
phenotype following exposure to PMA. Cells were incubated with
[P]orthophosphate and then exposed to 100 ng/ml
of PMA for 30 min. As a control, the cells also were subjected to
either a 44 °C heat shock treatment, or exposure to sodium
arsenite, two potent inducers of the stress response and both known to
stimulate the rapid increased phosphorylation of hsp 28 in other cell
types(26, 30) . Following the different treatments,
cell lysates were prepared and analyzed either by high resolution
two-dimensional gel electrophoresis or immunoprecipitation using an
antibody specific for hsp 28. The two-dimensional gel analysis revealed
the previously reported(33, 34) increased
phosphorylation of two proteins of
17 and 27 kDa. The 27-kDa
protein phosphorylated after PMA treatment migrated in the
two-dimensional gels in a position identical with that of hsp 28 (data
not shown). That the 27-kDa protein which exhibited increased
phosphorylation following PMA treatment was in fact hsp 28 was
confirmed by immunoprecipitation from the cell lysates using an
antibody specific for hsp 28. As is shown in Fig. 1, exposure to
PMA, like heat shock or sodium arsenite treatment, resulted in a marked
increase in the phosphorylation of hsp 28.
Figure 1: The 27-kDa protein rapidly phosphorylated in HL-60 cells following PMA addition is hsp 28. HL-60 cells growing at 37 °C were incubated with [32P]-H3PO4 for 1 h in DMEM lacking phosphate. The cells then were: maintained at 37 °C (control); subjected to a 44 °C heat shock; exposed to 100 ng/ml PMA; treated with 200 µM sodium arsenite; or 1 mM dibutyl cAMP. After 30 min the cells were harvested by the addition of Laemmli sample buffer and the lysates immediately heated at 95 °C for 5 min. Immunoprecipitations using a mouse monoclonal anti-hsp 28 antibody were performed and the resultant immunoprecipitates analyzed by SDS-PAGE. Shown is an autoradiograph of the gel. 37 °C control cells (lane 1); heat shock-treated cells (lane 2); PMA-treated cells (lane 3); sodium arsenite-treated cells (lane 4); dibutyl cAMP-treated cells (lane 5).
To rule out that PMA exposure, like heat shock or sodium arsenite treatment, simply was initiating a classical stress response and an accompanying stress-induced increase in hsp 28 phosphorylation, HL-60 cells were examined for possible changes in the expression of the different stress proteins following their exposure to PMA. In contrast to the increased rates of synthesis of hsp 90, hsp 73, hsp 72, and hsp 28 in cells placed under stress via exposure to sodium arsenite, no obvious changes in the overall expression of the stress proteins were observed in the PMA-treated HL-60 cells during their first 12 h of commitment toward the macrophage-like phenotype (Fig. 2).
Figure 2:
PMA stimulation of HL-60 cells does not
result in an induction of the stress response. Undifferentiated HL-60
cells were treated with either sodium arsenite (150 µM) or
PMA (100 ng/ml) for 90 min. The culture medium was then removed and the
cells labeled with [H]leucine for 14 h. As a
control, untreated cells were labeled in a similar manner. The labeled
cells were harvested in LSB, and an equal amount of radiolabeled
material (as determined by trichloroacetic acid precipitation) was
analyzed by two-dimensional gel electrophoresis. Shown are the
fluorographs of the gels. Panel A, control, untreated cells. Panel B, arsenite-treated cells. Panel C, PMA-treated
cells. The arrow near the bottom of the gels indicates the
position of the of hsp 28 (seen only in the arsenite-treated cells, panel B). The positions of hsp 73 (a), hsp 72 (b), hsp 90 (c), and actin (ac) are
indicated.
Complete differentiation of HL-60 cells to the macrophage-like phenotype requires approximately 48-72 h following their initial exposure to the differentiation agent, PMA. Moreover, in the case of PMA, once the cells have become committed to the differentiation process (within a few hours), the PMA can be removed from the cells without affecting the overall differentiation process (reviewed in (42) ). We examined next whether changes in the overall levels of hsp 28 might accompany the transition of the cells into their terminally differentiated phenotype. HL-60 cells were treated with PMA for 4 h, the culture medium containing the drug was removed, and the cells further incubated in complete medium lacking PMA for varying periods of time. At different times during the course of the differentiation process, cells were harvested, and the relative levels of hsp 28 were determined via Western blotting (Fig. 3). For each time point, an equal amount of total cellular protein was applied to the gels. After the first 12 h following administration of PMA, no changes in hsp 28 levels were observed. By 24 h, however, the levels of the protein had increased, and by 36 h the cells exhibited a significant increased accumulation of hsp 28. By 72 h, a time by which greater than 75% of the cells have terminally differentiated into the macrophage-like phenotype, the cells contained relatively high amounts of hsp 28. Again such an increase in the amounts of hsp 28 did not appear to result from the cells having undergone a stress-like response. For example, we did not observe any increases in the expression or accumulation of the other most highly stress-inducible protein, hsp 72, during the course of the PMA-induced differentiation (data not shown but see Fig. 5).
Figure 3: The levels of hsp 28 gradually increase over the course of PMA-induced HL-60 cell differentiation. Undifferentiated HL-60 cells were treated with PMA (100 ng/ml) for 4 h, after which the culture medium was removed, the cells extensively washed with and further incubated in fresh culture medium for various times as indicated below (12-72 h). At the indicated time, the cells were harvested, total protein content determined, and then 20 µg of total protein from each cell lysate was applied to an SDS-gel. Following transfer of the proteins to nitrocellulose, the blots were probed with the mouse monoclonal antibody specific for hsp 28. The primary antibody was visualized by subsequent incubation with horseradish peroxidase-conjugated goat anti-mouse antibody and chemilluminescence. Lane 1, undifferentiated HL-60 cells. Lanes 2-7, hours after PMA treatment began (12 h, 2; 24 h, 3; 36 h, 4; 48 h, 5; 60 h, 6; and 72 h, 7. Shown in lane 8 are HeLa cells grown at 37 °C and in lane 9 HeLa cells subjected to a 43 °C/90 min heat shock treatment and subsequently recovered at 37 °C for 12 h.
Figure 5:
Unlike
PMA, differentiation of HL-60 cells with either retinoic acid or
dimethyl sulfoxide does not result in any changes in the expression of
hsp 28. Duplicate plates of HL-60 cells growing at 37 °C were; left
untreated (control); exposed to 100 ng/ml of PMA for 4 h, the medium
removed, and the cells washed with and further incubated in complete
medium lacking PMA for 56 h; exposed to 140 µM dimethyl
sulfoxide continuously for 60 h; exposed to 1 mM retinoic acid
continuously for 60 h. The cells, treated as described, then were
either maintained at 37 °C for an additional 12 h or were subjected
to a 43 °C/45-min heat shock treatment and then returned to 37
°C for an additional 12 h. Cells were harvested in LSB, 20 µg
of total protein from each cell lysate was applied to an SDS gel, the
proteins separated, and then transferred to nitrocellulose.
Simultaneous Western blot analyses was performed using monoclonal
antibodies specific for hsp 28 and hsp 72. Visualization of the primary
antibodies was done by incubation with horseradish
peroxidase-conjugated goat anti-mouse antibodies and
chemilluminescence. Panel A shows the results using the hsp 72
antibody and panel B the results using the hsp 28 antibody. Lane 1, PMA-differentiated cells incubated at 37 °C; lane 2, MeSO-differentiated cells incubated at 37
°C; lane 3, RA-differentiated incubated at 37 °C; lane 4, undifferentiated cells heat shock-treated; lane
5, PMA-differentiated cells heat shock-treated; lane 6,
Me
SO-differentiated cells heat shock-treated; lane
7, RA-differentiated cells heat
shock-treated.
Having shown that hsp 28 levels increase in the
HL-60 cells following their differentiation into a macrophage-like
phenotype, we examined whether the protein, observed to exhibit rapid
increased phosphorylation immediately following PMA addition (Fig. 1), might still be phosphorylated 72 h after exposure of
the cells to PMA. In addition, we wondered whether the fully
differentiated cells, like the undifferentiated cells, would still be
able to phosphorylate hsp 28 in response to either metabolic stress or
the readdition of PMA. HL-60 differentiation toward the macrophage-like
phenotype was initiated by the addition of PMA. 4 h later the medium
containing the drug was removed, the cells washed with and further
incubated in complete medium (in the absence of PMA) for an additional
68 h to allow for complete differentiation. These now differentiated
HL-60 cells, along with HeLa cells (as a control), were then labeled
with [P]orthophosphate for 1 h and the pattern
of phosphoproteins analyzed by two-dimensional gel electrophoresis. In
addition both the PMA-differentiated HL-60 cells and HeLa cells were
examined for their pattern of hsp 28 phosphorylation following heat
shock treatment, exposure to sodium arsenite, or re-exposure to PMA (Fig. 4). In contrast to the 37 °C HeLa cells (Fig. 4E), the PMA-differentiated HL-60 cells incubated
at 37 °C exhibited basal phosphorylation of hsp 28, as evidenced by
the single phosphorylated isoform (Fig. 4A). Following
heat shock treatment, two phosphorylated isoforms of hsp 28 were
readily apparent in the HeLa cells (Fig. 4F). In the
differentiated HL-60 cells subjected to the heat shock treatment, the
relative intensity of the phosphorylated isoform seen in the 37 °C
cells increased, along with the appearance of a minor amount of a
second isoform (Fig. 4B). Following sodium arsenite
treatment, the three previously described hsp 28-phosphorylated
isoforms (26, 30) were apparent in both cell types (Fig. 4, C and G). Finally, and as shown
previously(26) , PMA treatment resulted in an obvious increased
phosphorylation of hsp 28 in the HeLa cells (Fig. 4H).
Once differentiated by exposure to PMA 68 h earlier, the HL-60 cells
now no longer displayed a significant increase in the phosphorylation
of hsp 28 in response to the readdition of PMA (Fig. 4D).
Figure 4:
Comparison of hsp 28 phosphorylated
isoforms in Hela and PMA-differentiated HL-60 cells subjected to heat
shock, sodium arsenite, or PMA treatment. HeLa cells and
PMA-differentiated HL-60 cells (72 h after exposure to PMA) were
labeled with [P]H
PO
for
1 h, and the cells, while in the presence of the radiolabel were then:
maintained at 37 °C (control); given a 44 °C heat shock
treatment; exposed to 200 uM sodium arsenite; treated with 200
ng/ml PMA. After 30 min, the cells were harvested in LSB and an equal
percentage of the cell lysates were analyzed by two-dimensional gel
electrophoresis. Shown only are those portions of the autoradiograms
revealing the position of the hsp 28 isoforms (acidic end is at the
left of each panel). In panels A-D are shown the
PMA-differentiated HL-60 cells and in panels E-H the HeLa
cells. Panels A and E, untreated, control cells; panels B and F, heat shock-treated cells; panels
C and G, arsenite-treated cells; panels D and H, PMA-treated cells. The arrows indicate the
positions of the various isoforms of hsp
28.
Because PMA induced differentiation of
HL-60 cells toward a macrophage-like phenotype resulted in changes in
both the expression and phosphorylation of hsp 28, we investigated
whether similar changes might accompany the differentiation of the
cells toward a granulocyte-like phenotype. Previous studies have shown
that both dimethyl sulfoxide (MeSO) and retinoic acid
treatment of HL-60 cells results in their differentiation into a
granulocyte-like phenotype (again see (42) for review). In
contrast to PMA treatment, we saw no evidence of any increase in the
levels of hsp 28 accumulating in the HL-60 cells treated with either
Me
SO or retinoic acid for 60 h, a period of time sufficient
for their almost complete differentiation into the granulocyte-like
phenotype (Fig. 5B, lanes 2 and 3,
respectively). However, both the Me
SO and retinoic
acid-differentiated cells were clearly capable of expressing hsp 28 as
well as the stress-inducible hsp 72 after heat shock treatment (Fig. 5, A and B, lanes 6 and 7, respectively). With regard to possible changes in the
phosphorylation state of hsp 28, HL-60 cells continuously exposed to
Me
SO for 72 h to elicit the granulocyte phenotype were
incubated with [
P]orthophosphate. The cells were
maintained at 37 °C or subjected to the different agents/treatments (e.g. heat shock, arsenite, the calcium ionophore A23187,
cAMP, and lysophosphatidic acid) known to result in the rapid increased
phosphorylation of hsp 28 in other cell types (as well as in the
undifferentiated HL-60 cells). Analysis of the cell lysates by SDS-PAGE
revealed changes in the patterns of phosphorylated proteins following
these different treatments (data not shown). However, via
immunoprecipitation analyses we could not detect any phosphorylated hsp
28, regardless of the treatment used to elicit its phosphorylation
(negative data not shown). Thus, differentiation of the HL-60 cells to
the granulocyte-like phenotype is not accompanied by either an increase
in the levels or phosphorylation state of hsp 28. Moreover, once
differentiated toward the granulocyte phenotype the cells no longer are
able to mediate phosphorylation of hsp 28 in response to any of the
different agents/treatments which normally induce high level
phosphorylation of the protein in other cell types or in the
undifferentiated HL-60 cells. Although the granulocyte-like cells still
appear capable of up-regulating hsp 28 expression, for example
following heat shock treatment, they are unable to mediate
phosphorylation of the protein.
Another human-derived cell line capable of undergoing in vitro differentiation events is the erythroleukemia K562 cells (43) . These cells in response to added hemin acquire an erythroid-like phenotype (i.e. red blood cell). Following their exposure to PMA however, the cells adopt a megakarocyte-like phenotype (i.e. platelet producing). Consequently, we were curious to know whether changes in hsp 28 expression/phosphorylation might accompany either of these two differentiation processes. Both hemin-differentiated and PMA-differentiated K562 cells were examined for their relative expression of hsp 28 (Fig. 6B). As a positive control, hsp 72 levels were analyzed owing to previous studies showing its increased expression after hemin induced K562 differentiation (44, 45) (Fig. 6A). Little or no expression of hsp 28 was observed in the K562 cells in their resting, undifferentiated state (at the protein concentrations used to program the gels for comparative Western blotting, Fig. 6B, lane 1) Hsp 72 levels, although relatively low, could be detected in the undifferentiated K562 cells (Fig. 6A, lane 1). Both stress proteins were observed to accumulate following heat shock treatment of the undifferentiated cells (Fig. 6, A and B, lane 2). Following exposure to hemin and differentiation toward the erythroid-like phenotype, the levels of both hsp 72 and hsp 28 were found to be increased relative to that observed for the undifferentiated cells (Fig. 6, A and B, lane 3). Heat shock treatment of the hemin-differentiated cells resulted in even higher levels of the two proteins (Fig. 6, A and B, lane 4). In contrast, no expression of either hsp 72 nor hsp 28 was observed in those cells differentiated toward the megakarocyte lineage following PMA exposure (Fig. 6, A and B, lane 5). After heat shock treatment, however, the PMA-differentiated K562 cells did exhibit a typical stress response, increasing their levels of both stress proteins (Fig. 6, A and B, lane 6).
Figure 6: Levels of hsp 28 and hsp 72 increase in hemin-differentiated but not in PMA-differentiated K562 cells. K562 cells were incubated with 30 µM hemin for 24 h to initiate their differentiation into a erythroid-like phenotype, or were incubated with 100 ng/ml of PMA for 72 h to initiate differentiation into a megakaryocyte-like phenotype. Once fully differentiated the cells were either maintained at 37 °C or subjected to a 43 °C/60-min heat shock treatment and subsequent recovery at 37 °C for 8 h. The cells were harvested, total protein content determined, and then 20 µg of total protein applied to a gel. Following separation by SDS-PAGE, the proteins were transferred to nitrocellulose and the relative amounts of both hsp 72 and hsp 28 determined via Western blotting as described earlier. Shown in panel A are the results using the hsp 72 antibody and in panel B the results using the hsp 28 antibody. Lanes 1 and 2, undifferentiated K562 incubated at 37 °C or subjected to heat shock, respectively; Lanes 3 and 4, hemin-differentiated cells incubated at 37 °C or subjected to heat shock, respectively; Lanes 5 and 6, PMA-differentiated cells incubated at 37 °C or subjected to heat shock, respectively.
The ability of the
undifferentiated and hemin-differentiated K562 cells to carry out
phosphorylation of hsp 28 was examined. The undifferentiated cells and
the hemin-differentiated cells were incubated with
[P]orthophosphate and then stimulated with the
different agents/treatment which, in most cells, results in the rapid
increased phosphorylation of the hsp 28 (Fig. 7). The
undifferentiated K562 cells maintained at 37 °C, and shown earlier
to express little or no hsp 28, contained little if any phosphorylated
hsp 28 (Fig. 7A, lane b). Upon stimulation of
the undifferentiated K562 cells by heat shock treatment, exposure to
sodium arsenite, addition of PMA, or with the protein kinase inhibitor
staurosporine, hsp 28 phosphorylation was observed to increase
significantly (Fig. 7A, lanes c-f,
respectively). In contrast to the undifferentiated K562 cells, the K562
cells exposed to hemin and now which had increased their level of
expression of hsp 28 (Fig. 6) also exhibited relatively high
level phosphorylation of hsp 28 (Fig. 7B, lane
b). Only a slight increase above this basal level of hsp 28
phosphorylation was observed when the hemin-differentiated cells were
subjected to heat shock treatment (Fig. 7B, lane
c). Interestingly, upon treatment of the hemin-differentiated
cells with either sodium arsenite or PMA hsp 28 phosphorylation was
further increased (Fig. 7B, lanes d and e, respectively). Finally, the PMA-differentiated K562 cells,
which showed little or no expression of hsp 28 (Fig. 6), also
exhibited little or no phosphorylation of the protein regardless of the
treatment used (negative data not shown). An interesting difference
revealed by these studies is the fact that while PMA treatment does
stimulate the increased phosphorylation of hsp 28 very early after its
addition to the K562 cells (see Fig. 7A, lane
e), once the cells have fully differentiated into the
megakaryocyte-like phenotype the extent of hsp 28 phosphorylation
declines. Indeed, once fully differentiated to the megakaryocyte like
phenotype (via PMA) the cells now appear refractory to most of the
classical stimuli known to result in increased hsp 28 phosphorylation.
In contrast, the hemin-differentiated K562 erythroid-like cells, like
the PMA-differentiated HL-60 macrophage-like cells, express hsp 28 at
high levels and continue to mediate relatively high phosphorylation of
the protein.
Figure 7:
Hemin-induced differentiation of K562
cells results in the sustained and high level phosphorylation of hsp
28. Undifferentiated K562 cells along with K562 cells induced to
differentiate into an erythroid-like phenotype via a prior 24-h
exposure to 30 µM hemin were incubated with
[P]H
PO
in DMEM lacking
phosphate for 1 h. While still in the presence of the radiolabel the
cells were: left untreated (control); subjected to a 43 °C heat
shock treatment; exposed to 200 µM sodium arsenite;
treated with 200 ng/ml of PMA; or treated with 50 nM of the
protein kinase inhibitor, staurosporine. After 1 h of the different
treatments the cells were harvested in LSB, the lysates heated to 95
°C, and an equal percentage of the cell lysate used for
immunoprecipitation of hsp 28. Shown is an autoradiograph of the gel. Panel A shows the hsp 28 immunoprecipitation results from the
undifferentiated K562 cells while panel B shows the hsp 28
immunoprecipitation results from the hemin-differentiated K562 cells.
Lane designations in the two panels are the same. Lane a,
immunoprecipitation using preimmune serum from the undifferentiated and
hemin-differentiated cells labeled at 37 °C; lanes b-f represent immunoprecipitations using hsp 28 antibody. Lane
b, untreated cells; lane c, heat shock-treated cells; lane d, arsenite-treated cells; lane e, PMA-treated
cells; lane f, staurosporine-treated
cells.
As a final step in our study we examined the relative distribution of both hsp 28 and actin in the PMA-differentiated HL-60 cells. The rationale for such studies was the fact that hsp 28 previously has been suggested to play a role in the regulation of the actin filament cytoskeleton(15, 16, 17) . Moreover, others had reported that accompanying PMA differentiation of HL-60 cells toward the macrophage-like phenotype was an increase in both the substratum adherence properties of the cells and their levels of actin(46) . HL-60 cells, after 72 h of differentiation by PMA exposure, were examined for the simultaneous distribution of both hsp 28 and polymerized F-actin. Hsp 28 distribution was analyzed by indirect immunofluorescence using an antibody specific for the protein while the distribution of F-actin was determined by incubation of the cells with rhodamine-conjugated phalloidin (Fig. 8). Although not shown, the undifferentiated HL-60 cells, when examined by phase-contrast microscopy were small, well rounded cells which adhered very poorly to their substratum. Upon differentiation into the macrophage-like phenotype, the cells now appeared larger, were no longer rounded, appeared to be motile, and were found to adhere rather strongly to their substratum (Fig. 8). In addition the differentiated cells, in most cases, exhibited an apparent co-localization of hsp 28 with a subset of the actin filaments. Such a co-distribution was most obvious in the phase-dense regions near the perimeter of the cells, this likely representing the leading edge of the cells.
Figure 8: A subpopulation of hsp 28 co-localizes with F-actin in PMA-differentiated HL-60 cells. HL-60 cells were induced to differentiate with PMA as described earlier. Cells were differentiated on glass coverslips for 72 h. The cells were fixed in 3.7% formalin, permeabilized in 0.1% Triton X-100 in PBS, and then analyzed for the simultaneous distribution of hsp 28 and F-actin. Hsp 28 locale was determined by incubation with a mouse monoclonal anti-hsp 28 antibody followed by incubation with fluorescein-conjugated goat anti-mouse antibody. F-actin distribution was determined via incubation with rhodamine-conjugated phalloidin. Shown in panel A is the phase-contrast micrograph, in panel B the distribution of hsp 28, and in panel C the distribution of F-actin. The results of two independent experiments (topversusbottom panels) are shown.
The studies presented here demonstrate that one of the
proteins whose phosphorylation increases during the early stages of
HL-60 differentiation toward a macrophage-like phenotype is the low
molecular weight heat shock protein, hsp 28. In addition, over the
course of their commitment to the macrophage lineage is the gradual
increase in the overall levels of hsp 28. Our results are similar to
those of Spector et al.(47) , who similarly reported
increased phosphorylation and accumulation of the low molecular weight
heat shock protein during PMA-induced HL-60 differentiation. On the
basis of these observations, we examined whether similar changes in the
phosphorylation and expression of hsp 28 might represent a common event
accompanying other cell differentiation events in vitro. The
impetus for these studies was to establish different in vitro systems where we might study changes in both the expression and
phosphorylation of hsp 28. Once established we could further
investigate the proposed connection between hsp 28 and components of
the cytoskeleton. To our surprise, however, we observed a tremendous
variation in the expression and phosphorylation of the protein in
several hematopoietic model systems routinely used to study cell
differentiation. To summarize, upon PMA stimulation HL-60 cells adopt a
macrophage-like phenotype. Commitment toward the macrophage-like
phenotype is accompanied by the rapid phosphorylation of hsp 28 and
over time, an accumulation of the protein. In contrast, differentiation
toward the granulocyte-like phenotype, via exposure of the HL-60 cells
to either MeSO or retinoic acid, did not result in any
changes in either the expression or phosphorylation of hsp 28. Rather,
once differentiated to a granulocyte-like phenotype, the cells now
appeared incapable of mediating the phosphorylation of hsp 28 under any
of the conditions known to result in the increased phosphorylation of
the protein in other cell types, or even in the undifferentiated HL-60
cells. Although apparently unable to mediate hsp 28 phosphorylation,
both the Me
SO and retinoic acid-differentiated HL-60 cells
still were able to increase the synthesis of hsp 28 following heat
shock treatment. In the case of the erythroleukemia cell line K562, the
levels of both hsp 72 and hsp 28 were found to increase during the
course of hemin-induced differentiation. Once fully differentiated, the
erythroid-like cells, similar to the PMA-differentiated HL-60 cells
(macrophage-like), expressed appreciable amounts of hsp 28. In
contrast, addition of PMA, sufficient to elicit the transition of the
K562 cells toward the megakaryocyte-like phenotype, did not result in
any increased synthesis or phosphorylation of hsp 28. These admittedly
somewhat complicated results are summarized in Table 1.
Thus while changes in the expression/phosphorylation of hsp 28 do accompany some in vitro cell differentiation events, the phenomenon is by no means a universal one. In some respects our results appear similar to the lack of consensus regarding hsp 28 levels and the growth status of different cells. In particular, we hypothesized that as the cells exited their cell cycle upon terminal differentiation, there would occur an increase in the expression and overall levels of hsp 28. The premise for such a hypothesis was the fact that in both yeast and certain mammalian cell lines, expression of the low molecular weight hsp increase as the cells reach stationary phase, and in the case of yeast, during sporulation(22, 23, 24) . In addition, others have shown that overexpression of hsp 28 in Ehrlich ascites tumor cells, via transfection of hsp 28 cDNA, results in a slowing down or even complete inhibition of cell growth(48) . There are, however, exceptions to this connection between hsp 28 expression and a decreased rate or inhibition of cell proliferation. For example some cell lines, such as the breast tumor-derived cell line MCF-7, express extremely high levels of hsp 28, yet grow at very high rates in vitro (reviewed in (49) ). In addition, in Drosophila some of the related members of the low molecular weight hsp family are found to accumulate in cells that apparently are undergoing multiple rounds of cell division during the developmental process(20) . Interestingly, a number of drugs that act as teratogens in mammalian systems, and which have been found to inhibit muscle and neuron differentiation in Drosophila, have as a common property their ability to induce the high level expression of one or more of the low molecular weight hsps(50) .
Thus, the possible connection between the low molecular weight hsps and growth control does not appear to be a simple black versus white situation. Clouding the issue as well is the fact that the low molecular weight hsps undergo phosphorylation in response to a bewildering number of agents/treatments which have no common phenotypic end point; e.g. some which stimulate growth while others which appear to inhibit or slow down growth. For example, increased phosphorylation of the low molecular weight hsp in various animal cells is observed in response to various mitogens (e.g. fresh serum or defined growth factors like epidermal growth factor), cytokines (e.g. interleukin 1, tumor necrosis factor), so-called tumor promoters (e.g. PMA, calcium ionophores), and finally different types of metabolic stress (e.g. heat shock treatment, sodium arsenite exposure, 26-30). Not surprisingly, there have been numerous reports of different kinases and phosphatases which function alone or together in mediating the phosphorylation on serine residues of the low molecular weight hsps in different systems. Indeed, two very recent studies have concluded that there may exist at least two distinct pathways by which phosphorylation of hsp 28 is mediated, one more associated with mitogenic activation, while the other perhaps activated by different types of physiologic stress(51, 52) . With regard to the significance of hsp 28 phosphorylation, recent work from two different laboratories have concluded that phosphorylation of hsp 28 may result in the dissociation of the homo-oligomeric state of the protein (normally somewhere between 400-800 kDa) into a stable tetrameric or dimeric form(31, 32) .
Exactly how expression of
the low molecular weight hsp is controlled during development and
differentiation, either in vivo or in vitro, remains
unclear. At least in vitro, the differentiation of human K562
cells toward a red cell-like phenotype following hemin exposure has
been shown previously, as well as here, to result in the increased
expression of the highly stress-inducible hsp 72
gene(44, 45) . A significant amount of work has shown
that the regulation of hsp 72 expression in cells undergoing metabolic
stress is controlled by a transcription factor generally referred to as
the heat shock transcription factor (HSF-1). Within the last few years
additional genes encoding proteins with similar DNA-binding domains as
that of HSF have been identified (reviewed in (53) ). Moreover,
one of these genes, termed HSF-2, has been shown to be activated during
hemin-induced K562 differentiation and is responsible for the
aforementioned increase in hsp 72 expression which accompanies the
differentiation process(54) . Thus we suspect that the
increased levels of hsp 28, which also accompany the hemin-induced
differentiation of the K562 cells, is, like hsp 72 expression, mediated
by activation of HSF-2. Our observations that hsp 28 expression
increases only under certain differentiation stimuli in both the HL-60
cells (i.e. in response to PMA but not MeSO and
retinoic acid) and K562 cells (i.e. in response to hemin but
not PMA) provides an ideal system to further study possible differences
in the activation of HSF-2 during in vitro cell
differentiation events.
Our results represent another contribution to study of in vitro cell differentiation and corresponding changes in stress protein expression. A number of studies using different in vitro systems have observed either increased or decreased expression of one or more of the different stress proteins accompanying the differentiation process (reviewed in (55) ). Of potential clinical relevance, Hanash and colleagues (56, 57) have concluded that specific changes in the relative amounts of hsp 28 isoforms (arising from differential phosphorylation) in childhood acute lymphoblastic leukemia carries prognostic significance. One cannot help but wonder whether these latter observations are related to the earlier mentioned studies in Drosophila showing that inappropriate expression and accumulation of one or more of the low molecular weight hsps very often is associated with developmental abnormalities(50) .
A
crucial but still unanswered question concerns the biochemical function
of the low molecular weight hsps. Once determined, perhaps new insights
regarding the reason for the regulated expression/phosphorylation of
the low molecular weight hsps during development and differentiation
might be realized. Like the well characterized hsp 70 and hsp 60
families of stress proteins, both the low molecular weight hsps and
their related counterparts, the -crystallins, have been reported
to function as molecular chaperones, facilitating the refolding of
denatured polypeptides and/or protecting target proteins from thermal
denaturation(11, 12, 13, 14) .
However, unlike hsp 70 and hsp 60, a dependence on ATP of such
chaperoning activities has not been demonstrated. Perhaps most telling
with regards to a possible function were the observations of Miron et al.(15) , reporting that hsp 25 purified from
avian sources was capable of inhibiting the growth of actin filaments in vitro. Subsequently, Landry and colleagues (16, 17) reported both changes in the actin
cytoskeleton and the acquisition of a thermotolerant-like phenotype in
cells overexpressing the mammalian low molecular weight hsp. These
observations, as well as previous studies reporting changes in the
levels of both actin and actin-associated proteins during the
differentiation of myeloid cells in vitro prompted us to
examine the locales of hsp 28 and the actin microfilaments. As was
shown in the HL-60 cells following their differentiation toward the
more motile macrophage-like phenotype, we did observe a portion of hsp
28 to localize in close proximity to the actin filaments, especially
those filaments present within the leading edge of the cell. Whether
hsp 28, through phosphorylation/dephosphorylation events triggered by
various external stimuli, somehow controls changes in the organization
of the actin filament network in the macrophage-like cells is currently
under study.