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INTRODUCTION |
Although the thyroid hormone (triiodothyronine, T3) is essential
for the normal development of the central nervous system, the specific
mechanisms by which these hormones control neuronal proliferation and
differentiation are currently unknown. T3 actions in cells are
initiated by binding to nuclear receptors encoded by two genes,
and
, which give rise to different receptor isoforms (1). The
neuroblastoma cell line N2a expresses low levels of nuclear receptors
(2). However, a stably transfected cell line (N2a-
cells) that
overexpresses the nuclear receptor
1 isoform has been established
(3). T3 treatment of N2a-
cells blocks proliferation by an arrest of
cells in G0/G1 and induces morphological and
functional differentiation (3, 4). These findings in neuroblastoma
cells correlate with similar effects of T3 in primary neuronal cultures
and in the developing brain (5, 6). It has been suggested that T3 may
act as a timing clock by pushing the neuroblasts out of the mitotic
phase, and a role of nuclear receptor
1 in this effect is supported
by the finding that
1 transcripts are predominantly found in zones
of neuroblast proliferation (7). However, to our knowledge no studies
on the effect of T3 on the expression of proteins involved in the
regulation of the cell cycle in neural cells have been reported.
To gain some insights into the mechanisms by which T3 regulates
neuronal cell growth and differentiation, we have analyzed the effect
of this hormone on the expression and activity of cell cycle-regulating
molecules. Our results show that incubation of N2a-
cells with T3
leads to a rapid down-regulation of the c-myc gene, to a
decrease of cyclin D1 levels, and to a sustained induction of the
cyclin kinase inhibitor
(CKI)1 p27Kip1.
This increase is secondary to the augmented levels of
p27Kip1 transcripts as well as to stabilization of the p27
protein. As a consequence of the increased levels of
p27Kip1, the kinase activity associated with the
cyclin-dependent kinase 2 (CDK2) complexes is inhibited and
retinoblastoma protein (pRb) family members are hypophosphorylated in
T3-treated N2a-
cells. This study shows for the first time that
T3-mediated neuronal growth arrest and differentiation are associated
with an increase in the levels of a CKI.
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EXPERIMENTAL PROCEDURES |
Cell Cultures--
The clonal cell line Neuro-2a stably
transfected with the
1 isoform of the human thyroid hormone receptor
(N2a-
cells) was grown as described previously in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% (v/v)
T3-depleted serum (3).
RNA Extraction and Hybridization--
Total RNA was extracted
from the cell cultures with guanidine thiocyanate. The RNA was run in
1% formaldehyde-agarose gels and transferred to nylon-nitrocellulose
membranes (Nytran) for Northern blot analysis. The RNA was stained with
0.02% methylene blue. The blots were hybridized with labeled mouse
cDNA probes for c-myc, cyclin D1, or
p27Kip1. Quantification of mRNA levels was carried out
by densitometric scan of the autoradiograms. The values obtained were
always corrected by the amount of RNA applied in each lane, which was
determined by densitometry of the stained membranes.
Immunoblotting--
The cells were washed in ice-cold
phosphate-buffered saline, and lysed in Nonidet P-40 lysis buffer
(0.1% SDS, 1% Nonidet P-40, 50 mM Tris-HCl, pH 8, 150 mM NaCl, 5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml
aprotinin). Protein concentrations were determined with Bio-Rad protein
assay reagent. Aliquots containing 50 µg of lysate were boiled in 5×
Laemmli sample buffer, and the proteins were separated by
SDS-polyacrylamide gel electrophoresis. Proteins were transferred onto
nitrocellulose membranes (Schleicher & Schüll), the membranes
were then blocked with 5% nonfat milk in Tris-buffered saline and
0.1% Tween 20. This was followed by incubation with the corresponding
diluted antibody for 1 h at room temperature. The antisera used
were the following: anti-c-myc (N-262, sc-764), anti-cyclin
D1 (9, sc-92), anti-p27Kip1 (M-197, sc-776), anti-p130
(C-20, sc-137) purchased from Santa Cruz Biotechnology, and anti-Rb
(14441A, PharMingen). After washing, the membranes were incubated for
1 h with an appropiate secondary antibody, and the proteins were
visualized by chemiluminescence (ECL, Amersham Pharmacia Biotech).
Protein bands were analyzed by densitometry.
Immunoprecipitation and Kinase Assay--
For
immunoprecipitation, 500 µg of cell lysates were incubated overnight
at 4 °C with 5 µl of anti-CDK2 antibody-agarose conjugated (M2-G,
sc-163-G). The immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis, and revealed with the anti-p27 antibody. To
measure kinase activity, immunoprecipitates were washed twice, resuspended in 40 µl of kinase reaction buffer (50 mM
Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM
dithiothreitol, 0.1 mg/ml bovine serum albumin, and 50 µM
ATP) containing 5 µg of histone H1 and 7 µCi of
[
-32P]ATP, and incubated for 30 min at 30 °C. The
reactions were stopped by adding Laemmli sample buffer. The samples
were boiled, and the proteins were separated by SDS-polyacrylamide gel
electrophoresis. 32P-labeled histone H1 was detected by
autoradiography and quantified by densitometry.
35S-Pulse-labeling Chase Experiments--
After a
48-h incubation period in the presence or absence of 5 nM
T3, the cells were detached from the culture plates and pulse-labeled
for 1 h with a mixture of [35S]methionine and
[35S]cysteine (Amersham Pharmacia Biotech) (1.5 mCi/10
ml) in methionine- and cysteine-deficient DMEM. Labeled cells were
washed and incubated in DMEM supplemented with 150 µg/ml unlabeled
amino acids. After a chase for 0, 3.5, and 7 h, the cells were
washed and lysed. The lysates were immunoprecipitated with the
anti-p27Kip1 antibody, and the immunoprecipitates analyzed
by SDS-polyacrylamide gel electrophoresis. After fluorography, the
amount of 35S incorporated into p27Kip1 was
quantitated in an Instantimager (Packard Instruments Co.).
Proliferation Assays--
N2a-
cells were transfected by
calcium phosphate with 0.5 µg of cytomegalovirus
-galactosidase
plasmid and 5 µg of pLPC (8) or pLPC-p27 (human p27 cDNA
subcloned into the pLPC vector). The DNA precipitates were added to
dishes containing coverslips. Cells were exposed to the DNA
precipitates for 16 h, washed, and incubated in DMEM containing
10% T3-depleted serum for 48 h. Cells undergoing DNA synthesis
were identified by incubation with the thymidine analogue
bromodeoxyuridine (BrdUrd) at a final concentration of 10 µM for the last 4 h, and transfected cells were
identified by staining for
-galactosidase. Cells were fixed with
methanol/acetic acid/water (90:5:5, v/v) for 30 min at room
temperature, stained with a 1:500 dilution of
-galactosidase
antibody (Promega), and subsequently with a BrdUrd monoclonal antibody
(Amersham Pharmacia Biotech) as suggested by the supplier. Fluorescein
isothiocyanate-conjugated and rhodamine-conjugated goat anti-mouse
antibodies were used as secondary antibodies to visualize
-galactosidase and BrdUrd-stained cells, respectively. Cells were
then examined using fluorescence microscopy with the appropiate filters.
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RESULTS |
T3 Inhibits c-Myc and Cyclin D1 Expression in N2a-
Cells--
T3 treatment of N2a-
cells strongly blocks DNA synthesis
and causes an arrest of cells in G0/G1 (Ref. 3,
and data not shown). Although the mechanisms connecting c-Myc function
to cell cycle control are not well understood, different signals that arrest growth and elicit cell differentiation suppress c-Myc
expression. As shown in Fig.
1A, the levels of c-Myc were
markedly decreased in N2a-
cells incubated for 48 h in the
presence of 5 nM T3. The reduction of c-Myc is a
consequence of the decreased c-myc mRNA levels.
Incubation with T3 causes a rapid decrease of c-myc transcripts in N2a-
cells. A maximal reduction (approximately 3-fold) was found only after 2 h of incubation with the hormone, and the levels of c-myc transcripts remained low for at
least 72 h (data not shown). This is one of the most rapid effects
of thyroid hormones on gene expression known. As shown in Fig.
1B, the levels of cyclin D1, another important component of
cell cycle progression, were also strongly reduced after 48 h of
T3 treatment. As in the case of c-Myc, a reduction of cyclin D1
transcripts was found, showing that T3 also alters cyclin D1 gene
expression.

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Fig. 1.
T3 decreases c-Myc and cyclin D1 expression
in N2a- cells. A, the
expression of c-Myc and cyclin D1 was determined by immunoblot of
extracts from duplicate cultures of N2a- cells incubated in the
presence or absence of 5 nM T3 for 48 h. B,
c-myc and cyclin D1 transcripts were analyzed by Northern
blot after the same treatment. The upper panels show the
autoradiograms obtained, and the lower panels show the
staining of the 18 S rRNA with 0.02% methylene blue. The results
illustrated are representative of three independent experiments.
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Induction of p27Kip1 by T3--
Because CKIs are
targeted by different growth-inhibitory and differentiation signals,
the possibility that T3 could induce the expression of CKIs was also
analyzed. In Fig. 2A Northern analysis of RNA from N2a-
cells indicated that p27Kip1
transcripts were induced by T3. Fig. 2B shows that this
induction was already observed after 3 h, but the maximal effect
was found at 24 h. p27Kip1 mRNA levels decreased
thereafter, but were still higher than in the untreated controls after
96 h of incubation with T3. This regulation was specific for
p27Kip1 because the level of p21Waf1 mRNA
was not affected by T3 (data not shown). To analyze whether de
novo protein synthesis was required for T3 induction of
p27Kip1 transcripts, treatment with the hormone was
performed in the presence and absence of 10 µg/ml cycloheximide. As
shown in Fig. 2C treatment with T3 for 4 h caused a
detectable increase of p27Kip1 transcripts. Incubation with
cycloheximide alone was able to induce p27Kip1 mRNA
levels, showing that this induction has characteristics similar to
those found with different early response genes, which have transcripts
with short half-lives in which labile proteins are implied. T3 was not
able to induce further p27Kip1 transcripts in the presence
of cycloheximide, indicating that likely activation of
p27Kip1 represents an indirect effect of the T3 receptor,
which requires previous synthesis of a protein or proteins. The
increase of p27Kip1 transcripts could also be secondary to
mRNA stabilization. To analyze this point, p27Kip1
mRNA levels were determined in control cells and in cells induced with T3 for 24 h and then incubated for varying times with 5 µg/ml of actinomycin D. Fig. 2D shows that
p27Kip1 mRNA disappeared with a similar half-life (2 h)
in both untreated and T3-treated N2a-
cells, showing that mRNA
stabilization is not involved in the induction of p27Kip1
transcripts. To directly address the question of whether the increase
of p27Kip1 transcripts by T3 is caused by an increase of
p27 gene transcription, a p27Kip1 reporter construct
containing a 1.6-kilobase upstream genomic fragment was used to
transiently transfect N2a-
cells (9). Treatment with T3 did not
increase p27Kip1 promoter activity. Similar results were
obtained with plasmids containing shorter promoter fragments (data not
shown). These results suggest that either p27Kip1 is not a
transcriptionally responsive target gene of the T3 receptor, or that
the sequences responsible for the transcriptional effect are outside of
the promoter fragments examined.

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Fig. 2.
T3 induces p27 transcripts.
A, total RNA from N2a- cells treated with and without 5 nM T3 for 24 h was hybridized with a
p27Kip1 cDNA probe. The autoradiogram is shown in the
top lane and the stained rRNA in the bottom lane.
B, quantification of p27Kip1 mRNA levels in
cells treated for various intervals with 5 nM T3. The
autoradiograms were quantitated by densitometry, and the values
obtained were corrected by the amount of RNA applied. Data are
mean ± SD values obtained from three independent samples,
expressed as fold-induction over the values obtained in control
untreated cells. C, Northern blot analysis of RNA from cells
treated for 4 h with T3 in the presence or absence of 10 µg/ml
cycloheximide (CHX). Quantification of the corrected
p27Kip1 mRNA expressed as the percentage obtained in
the untreated cells is shown below the autoradiogram. D,
determination of the half-life of p27Kip1 transcripts in
control and in T3-treated cells. The cells were first incubated in the
presence and in the absence of T3 for 24 h. The cultures were then
washed (time 0) and incubated with medium containing 5 µg/ml of
actinomycin D. At the times indicated, RNA was extracted from duplicate
cultures of control and T3-treated cells. The data are expressed as
percentages of the corresponding level of p27Kip17 mRNA
obtained at time 0.
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Fig. 3A shows that
p27Kip1 protein levels were markedly induced in N2a-
cells incubated with the hormone for different time periods. Quantification of different blots revealed that treatment with 5 nM T3 between 24 and 96 h increased by 8-9-fold the
cellular levels of p27Kip1. It has been described that
different antiproliferative signals regulate p27Kip1
accumulation by posttranslational mechanisms (10). To determine whether
p27Kip1 stabilization could also contribute to the elevated
p27Kip1 levels found in T3-treated cells, the incorporation
of [35S]methionine and [35S]cysteine into
p27Kip1 and the half-life of the labeled protein was
determined. As illustrated in Fig. 3B, the half-life of the
p27Kip1 protein was much shorter in the untreated N2a-
cells than in the cells treated with T3. Whereas in control cells
35S-p27Kip1 disappeared with an apparent
half-life of approximately 5 h, most of the label remained after a
7 h chase in T3-treated cells. Thus, protein stabilization clearly
contributes to the accumulation of p27Kip1 protein observed
in the cells incubated with T3.

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Fig. 3.
T3 induces the cellular levels of
p27Kip1 and increases p27Kip1 half-life.
A, lysates from cells treated with 5 nM T3 for
various periods were analyzed by immunoblotting with anti
p27Kip1 antibody. The top panel shows a
representative blot, and the bottom panel illustrates the
quantification obtained from three independent experiments.
B, control cells and cells incubated for 48 h with T3
were pulse-labeled for 1 h with [35S]methionine and
[35S]cysteine and subsequently chased with an excess of
nonradioactive amino acids for the times indicated. Cells were lysed,
extracts were normalized for equal counts per minute, and
p27Kip1 immunoprecipitated. The autoradiogram of the
immunoprecipitates is shown in the top panel, and
quantification of the radioactivity of each band expressed as the
percentage of the corresponding value found at time 0 is shown in the
bottom panel.
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Overexpression of p27Kip1 Blocks Proliferation of
N2a-
Cells--
To analyze whether an increase in the expression of
p27Kip1 protein is able to block cell cycle progression in
N2a-
cells, DNA synthesis was determined in cells that overexpress
p27Kip1. For this purpose, the cells were transfected with
an expression vector encoding p27Kip1. Cotransfection with
a plasmid expressing a
-galactosidase gene was used to identify the
transfected cells, and cells that entered S-phase were identified by
staining for BrdUrd incorporation. Staining with
-galactosidase
showed that approximately 20-25% of the total cell population was
successfully transfected. As shown in Table
I, expression of p27Kip1
caused a dramatic decrease in the percentage of cells in S-phase. Whereas in N2a-
cells transfected with an empty noncoding vector, a
large fraction (more than 70%) of the
-galactosidase-positive cells
were BrdUrd positive, only 14% of the cells transfected with the
p27Kip1-expressing plasmid entered S-phase.
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Table I
p27kip1 blocks N2a- cells proliferation
N2a- cells were cotransfected with the CMV -galactosidase plasmid
and pLPC-p27kip1 vector, which directs the expression of
p27kip1, or with the pLPC vector as a negative control.
Transfected cells were analyzed by fluorescence microscopy and the
percentage of BrdUrd-positive cells was determined in -galactosidase
positive cells. Data are the average ± S.D. of counting at least
twenty different microscopy fields from two separate experiments.
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T3 Increases Association of p27Kip1 with CDK2 and
Inhibits Kinase Activity--
CDK inhibitors are thought to prevent
cell proliferation by interaction with cyclin-CDK complexes. To
elucidate the significance of the induced p27Kip1 by T3, we
studied the complex formation of p27Kip1 with CDK2. As
shown in Fig. 4, the levels of CDK2 did
not change after incubation of N2a-
cells with T3. However, the
amount of p27Kip1 precipitated with anti-CDK2 antibody
increased significantly in T3-treated cells concomitantly with the
increased content of total p27Kip1 under this condition. It
was expected that association of CDK2 with p27Kip1 could
result in an impaired kinase activity. Thus, we next examined this
activity in the immunoprecipitates using histone H1 as a substrate.
Fig. 5A shows that
CDK2-associated kinase activity was indeed strongly reduced by T3 in
N2a-
cells.

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Fig. 4.
T3 increases the association of
p27Kip1 with CDK2. N2a- cells were incubated with 5 nM T3 for 48 h. The total levels of CDK2 (top
panel) and p27Kip1 (middle panel) were
analyzed by immunoblotting (blot) with the corresponding
antibodies. In the bottom panel the cell lysates were
immunoprecipitated (I.P.) with the anti-CDK2 antibody,
followed by immunoblot analysis of co-precipitated p27Kip1
with the p27Kip1 antibody (blot).
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Fig. 5.
A, effect of T3 on kinase activity
associated with CDK2. Histone H1 kinase activity was measured in
immunoprecipitates of CDK2 obtained from cells treated with 5 nM T3 for the indicated times. A representative
autoradiogram is shown at the bottom. The upper
panel shows the quantification of kinase activities obtained from
two independent experiments performed with duplicate cultures.
B, T3 increases the amount of hypophosphorylated pRb
family proteins. Immunoblot analysis was carried out with extracts from
cells incubated with or without T3 for 48 h. The pRb antibody
specifically detects the underphosphorylated forms of the protein
(pRb unphospho), and the p130 antibody detects both the
hyperphosphorylated (p130-phospho) and underphosphorylated
(p130 unphospho) forms.
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pRb and p130 Are Hypophosphorylated in T3-treated N2a-
Cells--
The effects of T3 on pRb family proteins were investigated
by immunoblotting. Fig. 5B shows the results obtained with
anti-pRb antibody that recognizes specifically the underphosphorylated forms of the protein. These forms were almost undetectable in control
N2a-
cells, and T3 produced a significant accumulation of
unphosphorylated pRb. In addition, the hormone induced a shift from the
slower migrating hyperphosphorylated p130 found in control N2a-
cells to their faster migrating hypophosphorylated forms. These results
are consistent with the requirement of CDK2 activity for
phosphorylation of pRb and p130 (11, 12), and with the finding that
during neuronal diiferentiation pRb hypophosphorylation is concomitant
with the loss of CDK2 activity (13).
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DISCUSSION |
Our results show that T3-mediated growth arrest and
differentiation of neuroblastoma cells is associated with
hypophosphorylation of the retinoblastoma protein pRb and the related
protein p130. The pRb family members appear to be essential for
neurogenesis and are highly expressed in cell types undergoing neuronal
differentiation, whereas loss of pRb affects the ability of neurons to
differentiate properly (14-16). One of the molecular events required
for cell cycle progression is the inactivation by hyperphosphorylation of pRb family proteins (12,17). Therefore, T3 maintains pRb family proteins in their active form. Under these conditions, they associate with E2F/DP factors and repress transcription of target genes required
for progression through the restriction point in the cell cycle
(18).
The hyperphosphorylation of pRb family members is catalyzed by the
CDKs. It has been shown that during neuronal differentiation the loss
of pRb phosphorylation correlates with loss of activity of the pRb
kinases CDK2 (associated with cyclins A or E) and CDK4 (associated with
D-type cyclins) (13). Our results in neuroblastoma cells demonstrate
that T3 causes a strong reduction of CDK2 activity without altering the
cellular levels of CDK2. The activities of the CDKs are regulated by
various mechanisms including dimerization with cyclins,
phosphorylation, and association with a group of inhibitory proteins
called CKIs (19, 20). The Cip/Kip family of CKIs, which are targeted by
different growth-inhibitory and differentiation signals, includes
p21Waf1 (21), p27Kip1 (22, 23), and
p57Kip2 (24) that bind to and inhibit all
G1-cyclin·CDK complexes. It has been described that
during differentiation of neuroblastoma cells with Me2SO,
the activities of CDK2 and CDK4 decline by association with
p27Kip1 (13) and that differentiation by nerve growth
factor and an inhibitor of DNA polymerases is also accompanied by the
expression of p21Waf1, another CKI that inhibits cyclin
E-associated kinase activity and is required for neuroblastoma cell
survival (25). Moreover, neuronal differentiation can be induced by
overexpression of p27Kip1 or pRb, suggesting that
inhibition of CDK activity and pRb phosphorylation are the major
determinants for neuronal differentiation and survival (13, 25). In
agreement with the important role of CKIs on neuronal differentiation,
we find that T3 induces a strong and sustained increase of the levels
of p27Kip1 in N2a-
cells. It is generally accepted that
the mRNA level of p27Kip1 remains constant regardless
of the state of cell growth and that p27Kip1 stabilization
or destabilization is the cause of the changes in p27Kip1
levels (10, 26-29). However, our results show that T3 increases the
level of p27Kip1 transcripts. Recent work has shown an
increase of p27Kip1 mRNA levels after incubation of
gastric carcinoma cells by interferon-
(30), or myelomonocytic cells
with vitamin D3 (31, 32). The increase in
p27Kip1 mRNA levels in N2a-
cells is found as soon
as 3 h after treatment with the hormone, which suggests a direct
effect on p27Kip1 gene transcription. Transcriptional
effects of thyroid hormones are mediated by binding of nuclear
receptors to hormone-response elements located normally in the promoter
region of regulated genes and several potential nuclear receptors
binding sequences are present in the 5'-flanking region of the
p27Kip1 gene. However, we did not identify a functional
response element, because T3 did not increase the activity of the p27
promoter in N2a-
cells. Moreover, T3 was not able to induce
p27Kip1 transcripts above the levels found in the presence
of cycloheximide, suggesting the requirement of de novo
protein synthesis for this induction. This suggested that T3 could
increase p27Kip1 expression primarily by stabilization of
the gene transcripts, but the hormone did not increase the half-life of
p27Kip1 mRNA, which was approximately 2 h,
regardless of the treatment. The short half-life as well as the
implication of labile proteins in the mRNA turnover demonstrated by
the induction in the absence of protein synthesis could explain the
rapid induction by T3. The findings that the induction in
p27Kip1 levels by T3 was more pronounced than that of
p27Kip1 mRNA levels, and that p27 transcripts decline
between 24 and 72 h of incubation with T3, when the protein levels
are maximally induced, suggested that p27Kip1 stabilization
could also contribute to the elevated p27Kip1 levels. This
fact was demonstrated by pulse-chase experiments, which showed that T3
significantly increased p27Kip1 half-life in N2a-
cells.
It has been reported that accumulation of p27Kip1 protein
is not sufficient on its own to arrest the cell cycle or to induce
differentiation in oligodendrocytes (33). In this cell model, the
levels of p27Kip1 progressively increase in the precursor
oligodendrocytes as they proliferate, but despite high levels of the
inhibitor the cells tend to keep dividing and not differentiate in the
absence of hydrophobic signals such as T3. In contrast, our results
clearly show that overexpression of p27Kip1 is sufficient
in itself to trigger an exit from the cell cycle in N2a-
cells. Our
results are in agreement with the finding that expression of
p27Kip1 blocks cell cycle progression in G1 in
different cell lines tested (17).
Although the hypophosphorylation of pRb and p130 caused by T3 is most
likely secondary to the increase in p27Kip1 and the
consequent inhibition of CDK2 activity, cyclin D-dependent kinases also induce pRb hyperphosphorylation (34). Cyclin D is rate
limiting for the G1/S transition, and it has been reported that expression of cyclin D1 advances the G1/S transition
and leads to the immediate appearance of hyperphosphorylated pRb. Induction of cyclin D1 promotes cell cycle progression not only by
activating CDKs but also by sequestering p27Kip1 (35-37).
Because T3 reduces cyclin D1 expression, this decrease as well as the
induction of p27Kip1 might contribute to maintain pRb
family proteins in their hyposphosphorylated state.
p27Kip1 and the c-myc proto-oncogene generally
have opposite roles in cell growth control, and c-Myc antagonizes the
growth arrest induced by p27Kip1 (38). Expression of
c-myc is suppressed by different growth-inhibitory or
differentiation signals (39). Therefore, it was not surprising that T3
reduced c-myc gene expression in N2a-
cells. It has been shown that c-Myc induces an as yet unknown cellular activity leading to
sequestration of p27Kip1, derepression of cyclin/CDK
activity and pRb hyperphosphorylation and thus allowing the escape from
the restriction point control in the cell cycle (38). Suppression of
c-Myc levels by T3 assuredly contributes to growth arrest and
differentiation of neuroblastoma cells.
In summary, our results indicate that T3 coordinately regulates the
expression of several genes that play a key role in cell cycle control
and differentiation of neuronal cells. The induction of a CKI, which
inhibits the activity of cyclin-dependent kinases and does
not allow progression through the restriction point in the cell cycle,
provides a mechanism to explain the crucial effects of thyroid hormones
on neuroblast growth and differentiation in the developing brain.