(Received for publication, March 15, 1995; and in revised form, June 21, 1995)
From the
Protein B23 is a nucleolar and nuclear matrix-associated phosphoprotein that is involved in ribosome synthesis. Its expression and phosphorylation in rat ventral prostate, an androgen target organ, are profoundly influenced by androgens. Induction of programmed cell death (apoptosis) in the prostatic epithelium by androgen deprivation in the animal induces an early decline in protein B23 in the absence of a corresponding loss of protein B23 mRNA. We have now demonstrated that prostatic nuclei retain the ability to transcribe the B23 mRNA and that a significant amount of this mRNA persists even after 7 days of androgen deprivation when >80% of the prostatic epithelial cells have undergone apoptosis. The B23 mRNA from these nuclei is also translatable in vitro. However, the majority of the B23 mRNA is associated with free and short-stretch polysomes, which may account for the castration-induced decline in synthesis of protein B23 in vivo. In addition, the mechanism of down-regulation of protein B23 in apoptotic prostatic cells appears to relate to two coordinate signals, which include loss of phosphorylation of the protein as well as the expression of a protease active toward dephosphorylated protein B23, under these conditions.
Programmed cell death or apoptosis has been described in diverse
biological systems mediated by a variety of
signals(1, 2, 3) , and the response of the
prostatic glandular epithelium to androgen withdrawal is one of the
frequently studied models(3, 4) . Androgen withdrawal
via orchiectomy in the rat induces an energy-dependent cascade of
biochemical and morphological changes that lead to the death of 80% of
the secretory epithelium between days 2 and 6(4) .
Morphologically, the nucleolus dissolves(5) , and the chromatin
is condensed and fragmented to form the membrane-bound apoptotic bodies
that appear in appreciable numbers after 2 days of androgen
deprivation(6) . Biochemically, there is an increase in
intracellular calcium and enhancement of calcium/magnesium-dependent
endonuclease activity that reaches its maximum 4-5 days after
androgen withdrawal (7) . Apoptosis in the prostate is
associated with modulation of gene expression so that the expression of
some genes is repressed and that of others is enhanced. Among the
latter are c-fos, c-myc, heat shock
proteins(8) , glutathione S-transferase(9) ,
and TRPM-2/sulfated glycoproteins(10) . On the other hand, the
synthesis of ribosomes, especially their assembly into polysomes,
markedly declines after androgen deprivation (11, 12) . Also, prostatic ribosomes from animals
treated with 5-dihydrotestosterone support a significantly higher
incorporation of radiolabeled amino acids into proteins than do
ribosomes isolated from castrated animal controls(11) .
We
have been interested in the mechanisms underlying the decline in
prostatic rRNA synthesis and assembly after androgen deprivation.
Protein B23, a conserved phosphoprotein that is localized to the
granular and fibrillar regions of the nucleolus where rRNA synthesis
and assembly take place(13, 14) , seems to have
different functions at different stages of the cell cycle. It is
capable of binding nucleic acids and exhibits both helix-destabilizing (15) and ribonuclease activities that implicate the protein in
preribosomal RNA processing and transport(16, 17) .
Various observations suggest that protein B23 plays a role in DNA
synthesis (18, 19) and might also have a structural
role as one of the components of the perichromosomal layer that is
involved in chromosome organization in mitosis (20) and as one
of the nuclear matrix-associated proteins(21) . Protein B23 is
phosphorylated by protein kinase CK2 in interphase (22) and by
p34 kinase during mitosis(23) . The
expression and phosphorylation of the protein are enhanced in different
cell types in response to mitogens, growth factors, and hormones,
including androgen in the prostate, suggesting that protein B23
constitutes a common signal required for cell
proliferation(23, 24, 25, 26, 27, 28) .
In a previous report, we documented that expression and phosphorylation of protein B23 started to decline at a modest rate in the first 24 h after androgen withdrawal (26) and that by 48 h post-castration, B23 was undetectable despite the presence of B23 mRNA up to 7 days of androgen withdrawal(26) . These changes in expression coincided with the decline in ribosome synthesis and the morphological alterations associated with apoptosis, suggesting that protein B23 might be involved in these processes(26) . In the present work, we have explored the mechanisms by which the prostatic glandular epithelium controls the expression of protein B23 during apoptosis. Our data indicate that regulation of protein B23 expression after androgen withdrawal is not at the transcriptional level. There appears to be no new cis-acting element that would hinder B23 mRNA translation in vitro. This mRNA shows a differential pattern of association with prostatic polysomes in response to androgen deprivation, but this effect alone might not explain the specific and abrupt decline in protein B23 expression observed after 48 h of androgen deprivation. It appears that the primary means of regulation of protein B23 expression after androgen withdrawal is its proteolytic degradation and that the decline in protein B23 phosphorylation as well as the expression (or release of inhibition) of a specific protease(s) after androgen withdrawal may be the prerequisites for protein B23 degradation.
Figure 1:
In vitro transcription of B23
mRNA in prostatic nuclei. The nuclear run-on assay was carried out on
prostatic nuclei isolated from normal control and castrated rats as
described under ``Methods.'' Lane a, normal control
rats; lane b, 4-day castrated rats; lane c, 7-day
castrated rats. Relative densitometric values for B23 were 1.0 (lane a), 0.8 (lane b), and 0.5 (lane c),
and those for -actin were 1.0 (lane a), 0.7 (lane
b), and 0.4 (lane c).
Figure 2: Effect of androgen deprivation on steady-state level of B23 mRNA using Northern blot analysis. Total prostatic RNA was electrophoresed, transferred to nylon membrane, and probed with randomly labeled B23 cDNA as described under ``Methods.'' Lanesa-c correspond to prostatic total RNA from intact normal and 4- and 7-day castrated rats, respectively. Relative densitometric values were 1, 0.8, and 0.4, respectively.
Figure 3:
Effect of androgen deprivation on B23 mRNA
association with prostatic polysomes. Prostatic cytosol was
fractionated on a sucrose gradient, and RNA from different fractions
was extracted as described under ``Methods.'' Each fraction
was examined for the presence of B23 mRNA, -actin mRNA, and 28 S
RNA-containing ribosomal particles by using the corresponding randomly
radiolabeled probes as described under ``Methods.'' The
amounts of total RNA loaded in each lane are indicated. The polysome
fractions from top to bottom of the slot blot represent the fractions
collected from top to bottom of the sucrose gradient, respectively. A, detection of B23 mRNA in different prostatic polysome
fractions isolated from intact rats (normal control) or from rats after
5 days (5d) of androgen deprivation. To ensure that the
observed signals were due to binding of the radiolabeled probes to RNA
in the polysome fractions, samples from different polysome fractions
were treated with RNase A prior to the hybridization step. B,
effect of androgen on the distribution of B23 mRNA in prostatic
polysomes. The relative density of different bands in A was
plotted against the sucrose concentration to facilitate analysis of the
autoradiogram in A. C, effect of androgen on the
distribution of
-actin mRNA in prostatic polysomes. D,
effect of androgen on the distribution of 28 S ribosomal particles in
prostatic polysomes.
Figure 4: Effect of androgen deprivation on translation of protein B23 in vitro. Prostatic poly(A) mRNA was isolated from animals at the indicated times (days (d)) after androgen withdrawal and translated in vitro, followed by immunoprecipitation of protein B23 using the specific antibody as described under ``Methods.''
Figure 5: Effect of androgen on proteolytic degradation of purified protein B23 added to prostatic homogenate in vitro. Purified radioiodinated protein B23 was incubated, in a final reaction volume of 100 µl, with 20 µl of prostatic cellular homogenate (20%, w/v) from intact or from 4-day castrated rats. After incubation for the indicated periods of time, the reaction was stopped by the addition of gel electrophoresis sample buffer. The material was subjected to SDS-PAGE as described under ``Methods.'' The dried gel was exposed to Kodak X-Omat film overnight at -70 °C. The toppanel shows protein B23 incubated with prostatic homogenate obtained from intact rats. The bottompanel depicts protein B23 incubated with prostatic homogenate prepared from 4-day castrated rats. The control lane in both panels included the addition of protease inhibitors as described under ``Methods.''
Figure 6:
Effects of protein B23 phosphorylation
status on its susceptibility to proteolytic degradation. A,
effect of dephosphorylation of protein B23 on its proteolytic
degradation. Purified radioiodinated protein B23 was treated with
potato acid phosphatase followed by alkaline phosphatase prior to
incubation with prostatic cellular homogenate prepared from intact or
4-day castrated rats as described for Fig. 5. In all panels,
heparin and poly(Glu,Tyr) (poly GT) were added as inhibitors
of protein kinase CK2. After incubation for the indicated periods of
time, the reaction was stopped by the addition of gel electrophoresis
sample buffer. The sample was subjected to SDS-PAGE, and the dried gel
was exposed to Kodak X-Omat film overnight at -70 °C. The toppanel represents dephosphorylated protein B23
incubated with prostatic homogenate obtained from intact rats. The middlepanel shows phosphorylated protein B23
incubated with prostatic homogenate prepared from 4-day castrated rats.
The bottompanel depicts dephosphorylated protein B23
incubated with prostatic homogenate prepared from 4-day castrated rats.
The control lane in all panels included the addition of the protease
inhibitors listed under ``Methods.'' B, effects of
various conditions on proteolytic degradation of protein B23 and actin
by prostatic homogenate from castrated rats. Lane a, I-labeled protein B23 (recombinant, nonphosphorylated; 5
µg) was incubated for 60 min at room temperature in medium
consisting of 30 mM Tris-HCl, pH 7.45, 5 mM MgCl
, 1 mM dithiothreitol, 0.1 mM ATP, 40 mM
-glycerophosphate, 2 µM Microcystin-LR, and 350 ng of CK2 in a final volume of 50 µl
(phosphorylation medium). Subsequently, 50 µl of control buffer
(0.32 M sucrose, 3 mM MgCl
, 5 mM
-mercaptoethanol) were added, and incubation was continued for 4 h
at room temperature (control). Lane b, the conditions were the
same as described for lane a, except that phosphatase
inhibitors (ATP,
-glycerophosphate, and Microcystin-LR) were
omitted, and 0.1 mM Na
HPO
was included
(to provide nonphosphorylation conditions). Instead of the control
buffer, 50 µl of 50% prostatic homogenate (from 4-day castrated
rats) prepared in the control buffer were added, along with inhibitors
of CK2 (50 µg/ml heparin, 1 mg/ml poly(Glu,Tyr)), to prevent any
phosphorylation of protein B23 catalyzed by CK2 during incubation with
the homogenate. Lane c, the conditions were the same as
described for lane a, except that prostatic homogenate was
added as described for lane b, but without inhibitors of CK2
to promote phosphorylation conditions for CK2. Lane d, 4.5
µg of
I-labeled actin were incubated under
nonphosphorylating conditions as described above for 60 min in medium
consisting of 50 mM imidazole HCl, pH 6.3, 2 mM MgCl
, 2.5 units of alkaline phosphatase, and 2.5 units
of acid phosphatase, followed by the addition of control buffer plus
CK2 inhibitors and 2.5 mM CaCl
, and incubation was
continued for 4 h as described above. Lane e, the conditions
were the same as described for lane d, except that the second
incubation (4 h) was carried out following the addition of prostatic
homogenate as described above. C, effect of treatment with CK2
on degradation of protein B23 at 30 min by prostatic homogenate from
4-day castrated rats. Lane a, 5 µg of
I-labeled B23 were incubated under nonphosphorylating
conditions as described for B (lane b), except that
the second phase of incubation was reduced from 4 h to 30 min. Lane
b, the conditions were the same as described for B (lane c), except that the second incubation was for 30
min only as described above. In all panels, arrowheads indicate the position of protein B23. In addition, the position of
a 24-kDa marker is shown in B and C.
The nature of
the role of phosphorylation of protein B23 in its proteolytic
degradation in prostate tissue from castrated rats was further
examined. The result in Fig. 6B (lane b),
employing recombinant protein B23.1, shows that dephosphorylated
protein B23 under conditions that favor dephosphorylation (i.e. presence of excess P, CK2 inhibitors, phosphatases)
was almost completely degraded after 4 h of incubation with prostatic
homogenate from 4-day castrated rats. On the other hand, prior
incubation of protein B23 with CK2 under conditions that favor
phosphorylation (presence of excess ATP, phosphatase inhibitors)
protected a 22-kDa segment of protein B23 from being degraded under the
same conditions (Fig. 6B, lane c). Since these
results suggested that phosphorylation may influence the rate or
pattern of proteolytic degradation of protein B23, further experiments
were undertaken in which proteolytic degradation of protein B23 was
examined at 30 min of reaction time. The results in Fig. 6C (lane b) show that when protein B23 was treated with CK2 (i.e. phosphorylation conditions), some undegraded protein B23
was apparent in addition to the resistant 22-kDa segment that was also
observed after 4 h of incubation (e.g.Fig. 6B, lane c). It is noteworthy that
intact protein B23 was not detected when nonphosphorylated protein B23
was subjected to the same treatment (Fig. 6C, lane
a). Also, it appears that the 22-kDa band has undergone further
degradation under these conditions. However, it is possible that the
rate and pattern of degradation of protein B23 in vivo may
also be influenced by stoichiometry of phosphorylation and/or the
presence of other intrinsic kinases. The present observations on the
effects of the phosphorylation status of protein B23 on its proteolytic
degradation appear to be relatively specific since pretreatment of
actin with protein phosphatases under conditions that favor
dephosphorylation did not demonstrate a corresponding degradation of
actin (or any of the contaminating proteins in the sample) (Fig. 6B, lanes d and e). Thus, the
observed degradative changes in protein B23 are not due to the
treatment conditions, and furthermore, it appears that protein B23
degradation may be catalyzed by specific protease(s) that may act on a
relatively specific set of proteins.
Protein B23 plays an integral role in ribosome synthesis.
Changes in rRNA synthesis and assembly are among the earliest responses
to androgen action in the prostate. Employing this paradigm, we
previously examined the changes in prostatic protein B23
phosphorylation and level in response to androgen withdrawal (i.e. during induction of programmed cell death) and administration (i.e. during prostatic epithelial regeneration)(26) .
These studies established that although protein B23 expression declines
to undetectable levels by 48 h after androgen deprivation, the
steady-state level of its mRNA does not change as rapidly, being
60% of normal even after 7 days of castration (26) when
>80% of the prostatic cells have undergone apoptosis(4) .
The present results demonstrate that persistence of the B23 mRNA after
androgen deprivation is the result of ongoing transcription rather than
enhanced stability. The decline in the rate of in vitro transcription of protein B23 mRNA was comparable to that of
-actin, which suggests that these changes are general effects of
androgen withdrawal and cannot explain the observed specific rapid
decline in protein B23 expression.
B23 mRNA in the prostate gland of
the intact animal exists mainly associated with long-stretch polysomes,
where it is probably being translated. We have now demonstrated that
after androgen withdrawal, B23 mRNA is mainly associated with either
free ribosomes or short-stretch polysomes. However, this effect is also
observed for -actin mRNA. This similar association might be
attributed to the general reduction in rRNA synthesis, as evidenced by
the decline in 28 S RNA-containing ribosomal particles, and accords
with the decline in rRNA synthesis and assembly which is one of the
most dramatic effects of androgen withdrawal in the prostate
gland(11, 12) . The observed pattern of association of
B23 mRNA or actin mRNA with ribosomes after androgen withdrawal might
contribute to the decline in protein expression in both cases. However,
it cannot explain the aforementioned specific abrupt decline in protein
B23 expression (26) in that actin protein is detected despite
the fact that the majority of its mRNA is associated with free or
short-stretch polysomes. Furthermore, it is likely that this
differential pattern of association with polysomes might still be
compatible with some protein expression that is translated from the
mRNA associated with long-stretch polysomes. It is noteworthy that the
expression of many androgen-repressed genes is enhanced after androgen
withdrawal, which again suggests that protein expression can occur
despite the decline in the formation of long-stretch
polysomes(8, 9, 10) . Competition between
different mRNAs to associate with polysomes has been suggested as a
mechanism for translational control(41) . In the case of
protein B23 mRNA, there are no cis-acting elements that would
arise after androgen deprivation and hinder its translation, at least in vitro.
The possibility of proteolytic degradation of prostatic protein B23 as a result of androgen deprivation was examined. The initial incubation of radioiodinated protein B23 with prostatic homogenate obtained after androgen withdrawal did not yield significant degradation products. However, prior incubation of protein B23 with phosphatases led to the appearance of degradation products when incubated with prostatic homogenate obtained from castrated animals, but not when incubated with homogenates from intact animals. This finding strongly indicates that dephosphorylation of protein B23 and the expression (or release of inhibition) of protease(s) after androgen withdrawal are both necessary for the observed decline in protein B23. This mechanism might be restricted to a specific group of proteins that includes B23 since, on androgen withdrawal, the expression of certain proteins is enhanced and actin protein persists. This accords with our observation that actin is not degraded under these conditions. Our data also suggest that phosphorylation of protein B23 enhances its stability and resistance to proteolytic degradation.
The level of phosphorylation of B23 correlates with cellular proliferative activities and is enhanced at mitosis(17, 18, 19, 20, 21, 22) . Protein B23 is phosphorylated by protein kinase CK2(22) , and CK2 is the rate-limiting factor for B23 phosphorylation in the prostate gland after androgen deprivation(26) . Furthermore, both protein B23 (21) and protein kinase CK2 (42) are associated with the nuclear matrix, and phosphorylation of nuclear matrix-associated protein B23 is directly affected by changes in nuclear matrix-associated CK2 activity(43) . Upon androgen deprivation, nuclear matrix-associated CK2 declines rapidly, which affects the rate of phosphorylation of proteins intrinsic to that fraction. On the other hand, after androgen administration to castrated rats, nuclear matrix-associated CK2 is increased within 1 h(43) . The physiological significance of these dynamic changes in the association of CK2 with the nuclear matrix is apparent in regard to protein B23, where the decline in phosphorylation would enhance its susceptibility to proteolytic degradation during programmed cell death. On the other hand, the early availability of nuclear matrix-associated CK2 activity after androgen administration might play a role in the phosphorylation and stability of the newly translated protein B23, which starts to appear within 4 h after androgen administration in the cascade of events leading to epithelial regeneration(26) .
The role of certain proteases in the induction of apoptosis has been
documented in several systems(44, 45) . Apoptosis
induced in thymocytes by staurosporine (a kinase inhibitor) could be
inhibited by a protease inhibitor, which suggests a role for both
kinases and proteases in this process(46) . Nuclear
matrix-associated proteins seem to be a preferential target for
proteolysis during apoptosis. Lamin A, lamin B, poly(ADP-ribose)
polymerase, and topoisomerases are all targets of proteolytic
degradation during apoptosis in many
systems(39, 47, 48) . As is the case for
protein B23, nucleolin is another nuclear matrix protein that is
localized to the nucleolus and involved in rRNA synthesis and has been
shown to be a preferential target of serine proteases during programmed
death induced in target cells by cytotoxic lymphocytes(49) .
Although the nature of the proteases responsible for degradation of
these nuclear matrix proteins, including protein B23, is still unknown,
a Ca-dependent protease exists in the nuclear lamina (39) and may be involved in this process(40) . It is
noteworthy that the addition of Ca
to the incubation
medium markedly enhances the degradation of protein B23. The relation
between the decline in protein B23 or its phosphorylation may have
certain implications for programmed cell death. An obvious effect of a
decline in protein B23 would be on ribosome assembly. Also, protein B23
has been suggested to have a structural role; the decline in protein
B23 and its charge might affect the overall tensional integrity of the
tissue matrix(50) , leading to altered gene expression
associated with programmed cell death.
In summary, we have documented that cells undergoing apoptosis retain the ability to transcribe B23 mRNA. The existing mRNA for this protein declines very slowly on induction of apoptosis. It appears that the long-lived mRNA is available for translation when the cells are stimulated to grow. Disappearance of protein B23 from the cell appears to relate to its proteolytic degradation, and phosphorylation of the protein (by protein kinase CK2) plays a role in preventing its degradation. To our knowledge, this is the first report to document the kinetics and mechanism of protein B23 disposition in cells undergoing programmed cell death.