(Received for publication, October 12, 1994; and in revised form, November 17, 1994)
From the
Because of their differentiating effects in neoplastic cells in vitro, the use of retinoids in the treatment of various malignant and premalignant conditions is under investigation. To date, signal transduction pathways involved in retinoid-induced differentiation remain poorly understood. Differentiation of HL-60 cells by all-trans-retinoic acid (tRA) is directly mediated by down-regulation of the serine protease myeloblastin (mbn). In this report, we investigate the possibility that the 28-kDa heat shock protein (hsp28), previously linked to differentiation of normal and neoplastic cells including HL-60, may be regulated by mbn. Using NB4 promyelocytic leukemic cells as a differentiative model, we show that tRA induces initial suppression and subsequent up-regulation of hsp28 protein, mirroring tRA-induced changes in mbn protein. The progressive reduction in hsp28 mRNA levels in response to tRA suggests that changes in hsp28 protein levels might be posttranscriptionally mediated, raising the possibility that hsp28 may be targeted by mbn. To address this, we developed an assay using purified mbn and recombinant hsp28 and now show that hsp28 is hydrolyzed by mbn but not its homologue, human neutrophil elastase. Moreover, mbn does not indiscriminately hydrolyze other proteins. Identifying hsp28 as a substrate of mbn strongly suggests that hsp28 may be a key component of the tRA signaling pathway involved in regulating cell differentiation.
Vitamin A and its retinoid derivatives exert marked biological
effects in a variety of normal and neoplastic cells. For example, RA ()induces human myeloid leukemic cells to exit the cell
cycle and terminally differentiate into mature granulocytes, both in vitro(1, 2) and in vivo(3, 4, 5) . In this context, a novel
serine protease, myeloblastin, has been shown to be directly involved
in RA-induced differentiation of human HL-60 myeloid leukemic
cells(6) . Constitutively expressed in proliferating HL-60
cells, mbn is rapidly down-regulated in response to
RA(6, 7) . Inhibition of mbn protein expression
appears to correlate with the cessation of cell growth. To directly
link mbn down-regulation to changes in cell growth and differentiation,
mbn protein expression was specifically inhibited in HL-60 cells using
antisense oligonucleotides. This manipulation induced spontaneous
growth arrest and differentiation, directly implicating mbn as a key
component of the signaling pathway involved in transducing the
biological effects of RA (6) . To elucidate on this pathway, we
sought to identify proteolytic targets of mbn.
In addition to their role in the development of thermotolerance, members of the highly conserved heat shock protein family have been linked to cell growth and differentiation(8) . Targeting of the 28-kDa mammalian heat shock protein by mitogens(9, 10) , growth regulatory cytokines(11, 12, 13) , and differentiating agents(14, 15, 16, 17, 18, 19) implicated hsp28 as a potential component of signal transduction pathways in both normal and neoplastic cells. Several lines of evidence made it tempting to speculate that hsp28 might be a substrate of mbn. First, hsp28 mRNA was markedly down-regulated by RA during differentiation of HL-60 cells whereas hsp28 protein levels actually increased coincident with the onset of growth arrest(16) . This finding suggested that changes in hsp28 protein levels might be posttranscriptionally regulated. Second, mbn and hsp28 proteins appear to be reciprocally regulated during granulocytic differentiation of HL-60 cells. Third, amino acid sequence analysis of hsp28 revealed several potential serine protease target sites(20) . In the current study, we establish hsp28 as the first reported biological substrate of mbn. Considering hsp28 is a molecular chaperone (21) and key actin binding protein(22) , its regulation by mbn sheds light on potential pathways involved in transducing the differentiative effects of RA in human myeloid malignancies.
In order to establish a link between hsp28 and mbn during
RA-induced cell differentiation, we examined the regulation of hsp28
protein in the NB4 acute promyelocytic leukemia (APL) cell line. APL
cells remain one of the prototypic models with which to study cellular
and molecular differentiative events since these cells undergo
granulocytic differentiation in response to
RA(1, 2, 3, 4, 5) . As the
only established APL cell line, NB4 cells contain the
t(15;17)(q22;q12-21) cytogenetic marker, which is uniquely
associated with APL and thought to confer RA sensitivity through the
expression of a hybrid retinoic acid receptor-
protein(24, 25, 26, 27, 28) .
Decreased mbn protein levels appear to correlate with RA-induced growth
arrest of NB4 cells(7) . However, in contrast to HL-60 cells
where mbn is rapidly down-regulated, mbn is initially induced in
RA-treated NB4 cells and not inhibited until 96 h, at which time NB4
cells growth arrest(7) . hsp28 protein levels were examined at
various time points prior to and following treatment of NB4 cells with
tRA (1 µM). During the first 72 h, levels of hsp28 protein
progressively decreased, during which time
[
H]thymidine incorporation remained unchanged
from that of untreated cells (Fig. 1). After nadiring at 72 h,
hsp28 protein levels increased at 96 h with continued induction above
base-line levels at 4 and 6 days (Fig. 1). Increased hsp28
protein appeared to correlate with cessation of cell proliferation and
55 and 82% inhibition of [
H]thymidine
incorporation at 4 and 6 days, respectively (Fig. 1). Thus, the
initial down- and subsequent up-regulation of hsp28 mirrors that
previously described for mbn, where mbn is up-regulated during the
initial 24-72 h following tRA and not down-regulated until 96
h(7) .
Figure 1:
Increased
hsp28 marks tRA-induced growth arrest of NB4 cells. Exponentially
growing NB4 cells were cultured in the presence of tRA (1
µM). At the indicated times prior to and following
treatment with tRA, equal numbers of NB4 cells were lysed in buffer
containing 0.5% Nonidet P-40. Protein concentrations in cell lysates
were determined by a modification of the Bradford method(50) .
Equal amounts of protein (50 µg/lane) were separated by 12%
SDS-PAGE and transferred to a nitrocellulose filter. hsp28 was
immunoblotted using an -hsp28 mAb (1:1500 dilution) and visualized
with a goat
-mouse horseradish peroxidase-conjugated secondary
antibody (1:5000 dilution). The blot was developed using the ECL method
of detection. The position of hsp28 is indicated. Corresponding cell
proliferation was determined by pulsing 10
cells/well with
1 µCi of [
H]thymidine (Thym.) for 4
h and then measuring radioactive incorporation into DNA. The percent
control = counts/min (indicated time point)/counts/min (day
0).
Relatively little is known about the mechanism regulating hsp28 protein levels during cell differentiation. To shed light on this process, we examined the expression of hsp28 mRNA during RA-induced differentiation of NB4 cells. Total cellular RNA was isolated at various time points prior to and following treatment with tRA. The predicted 0.9-kilobase size hsp28 transcript was markedly down-regulated between 2 and 6 days following treatment with tRA (Fig. 2A). Intactness and equal loading of RNA were verified by ethidium bromide staining (Fig. 2B) and cross-hybridization to the 28 S ribosomal band (Fig. 2A).
Figure 2:
Down-regulation of hsp28 mRNA during
differentiation of NB4 cells. NB4 cells were either cultured in control
media alone (0 and 48 h) or in the presence of tRA (1 µM)
for the indicated periods of time. Total cellular RNA was isolated from
cells at each time point using a modification of the guanidinium
thiocyanate/CsCl method. Equal amounts of RNA (5 µg/lane) were
separated by 1.3% agarose gel electrophoresis and transferred by
Northern blotting to a nitrocellulose membrane and then hybridized with
a P-labeled cDNA probe cloned from the hsp28 gene. As
shown, the probe hybridizes to a 0.9-kDa size transcript (panelA). Cross-hybridization to the 28 S ribosomal band
suggests comparable loading of intact RNA. A photograph of the ethidium
bromide-stained 28 S ribosomal band is shown as a further control of
intactness and equal loading of RNA (panelB). The
positions of the 28 S and 18 S ribosomal bands are
indicated.
The discordant relationship between hsp28
protein and mRNA suggested that RA might regulate hsp28 protein
posttranscriptionally. Analysis of the known amino acid sequence of
hsp28 revealed several potential serine protease target sites. To
determine whether hsp28 is a substrate of mbn, we isolated mbn from
myeloblasts and purified it to homogeneity using a modification of the
method described by Kao et al.(23) . ()The
purity of the product was verified by mass spectrometry, N-terminal
amino acid sequence determination, and SDS-PAGE (data not shown). mbn
and rhsp28 were incubated at various substrate:enzyme ratios for 12 h
at 37 °C, and then hsp28 protein degradation was determined by
immunoblot analysis using an
-hsp28 mAb as the primary antibody.
When incubated with mbn at an hsp28:mbn ratio of either 1:250 (Fig. 3, lane2) or 1:500 (lane3), the 28-kDa ``parent'' heat shock protein
was almost completely hydrolyzed to two or three lower molecular weight
immunoreactive bands (Fig. 3, arrowheads) corresponding
to previously described hsp28 digestion products(9) . As a
control, hsp28 did not undergo spontaneous degradation when incubated
alone (Fig. 3, lane1). To further
characterize the specificity of mbn proteolysis, we examined whether
mbn also hydrolyzed proteins such as human Ig, bovine serum albumin,
and lysozyme. Representative of these proteins was human IgG, which was
not proteolytically cleaved by mbn (Fig. 4).
Figure 3:
Proteolysis of hsp28 by mbn. Purified mbn
and rhsp28 were incubated together for 12 h at 37 °C in a shaking
water bath and then separated by 12% SDS-PAGE and transferred to a
nitrocellulose filter. The filter was blotted with an -hsp28 mAb
(1:1500 dilution) and visualized using a goat
-mouse AP-conjugated
secondary antibody (1:5000 dilution). Proteolysis was examined at an
hsp28:mbn ratio (mol:mol) of 1:250 (lane2) and 1:500 (lane3). Arrowheads indicate the location
of three new immunoreactive bands. As a control, rhsp28 (5 µg) was
run in lane1. The relative position of molecular
mass standards is indicated.
Figure 4:
mbn does not hydrolyze human IgG. Purified
human IgG was incubated either alone (lane1) or with
mbn at a substrate:enzyme ratio of 1:500 (lane2) for
12 h at 37 °C in a shaking water bath and then separated by 12%
SDS-PAGE and transferred to a nitrocellulose filter. Human IgG was
immunoblotted using goat -human IgG mAb (1:5000 dilution) and
visualized using the ECL detection method. Human IgG was separated by
SDS-PAGE under reducing conditions. The relative position of molecular
mass standards is indicated.
Since mbn is homologous to human neutrophil elastase(6) , another protease down-regulated by RA during HL-60 cell differentiation, we examined the effect of hNE on hsp28 protein expression. rhsp28 and purified hNE were incubated together under conditions identical to that described above and were shown to hydrolyze elastin, a known substrate of hNE (data not shown). However, in contrast to mbn, rhsp28 was not affected by hNE (Fig. 5, lane2). rhsp28 alone was included as a control (lane3).
Figure 5:
hsp28 is not degraded by human neutrophil
elastase. rhsp28 was incubated with either myeloblastin (lane1) or hNE (lane2), in both cases at an
hsp28:enzyme ratio of 1:500. Samples were separated by 12% SDS-PAGE and
immunoblotted using an -hsp28 mAb (1:1500 dilution) and visualized
using a goat
-mouse horseradish peroxidase-conjugated secondary
antibody (1:5000). Blots were developed using the ECL detection method.
The arrowhead indicates the position of a complex of
immunoreactive bands. rhsp28 (0.25 µg) was run as a control (lane3).
The mechanism by which decreased mbn protein signals cessation of growth and induction of cell differentiation remains unknown, in part due to the paucity of information regarding biological targets of this key protease. In this report, we identify hsp28 as the first reported protein substrate of mbn. As we have shown, this relationship is rather specific since hsp28 was not hydrolyzed by a homologous protease (Fig. 5), nor do these observations represent indiscriminate hydrolysis (Fig. 4). These findings are intriguing in light of the integral role played by hsp28 in physiological processes that directly impact on cell growth and differentiation. For example, cytoskeletal proteins are important components of signal transduction pathways. In fact, a number of growth regulatory molecules appear to exert their effects through interactions with cytoskeletal components(29, 30, 31) . In this context, hsp28 directly modifies the composition of actin microfilaments through its actin binding activity(22) . As a capping protein, the avian low molecular weight analogue of hsp28 inhibits actin polymerization(32, 33) . The effects of hsp28 on actin appear to be phosphorylation-dependent(22) , which is of interest in light of the identification of a serine kinase involved in the MAP kinase cascade being responsible for hsp28 phosphorylation(34, 35) . Originally associated with mitogen stimulation, activation of MAP kinases has also been linked to cell differentiation, including HL-60 cells(36, 37, 38, 39, 40) . Thus, RA appears to exert a dual effect on hsp28 protein expression through its down-regulation of mbn and activation of MAP kinases.
In
addition, several members of the heat shock protein family, including
hsp28, regulate peptide folding, intracellular protein trafficking, and
signal transduction as molecular
chaperones(21, 41, 42, 43, 44) .
These interactions have been shown to modify the activity of associated
proteins, which has significant consequences when the chaperoned
protein is itself involved in growth regulation, as in the case of
hsp90 and the oncogenic tyrosine kinase
pp60(45, 46, 47) .
Similarly, following transfection of baby rat kidney cells with either
oncogenic or non-oncogenic adenovirus constructs, the presence of a
22-kDa protein associated with the rat low molecular weight hsp
correlates inversely with oncogenicity of transformed
cells(48) . Regulation of hsp28 by mbn could therefore directly
impact on chaperonin activity.
Although the direct involvement of hsp28 in cell differentiation remains to be demonstrated, its identification as a target of mbn provides evidence that hsp28 is a component of the signal transduction pathway mediating differentiation of human myeloid leukemic cells. The identification of mbn in hematopoietic stem cells (49) suggests that a better understanding of mbn and its substrates may ultimately provide insight into the regulation of normal hematopoiesis.