(Received for publication, February 7, 1995; and in revised form, October 19, 1995)
From the
We investigated the intracellular events involved in the
3,3`,5-triiodo-L-thyronine (T)-induced
accumulation in acetylcholinesterase (AChE) activity in neuroblastoma
cells (neuro-2a) that overexpress the human thyroid receptor
1
(hTR
1). Treatment of these cells with T
increased AChE
activity and its mRNAs after a lag period of 24-48 h, and these
levels increased through stabilization of the transcripts by
T
. T
had no effect on the transcriptional rate
or processing of AChE transcripts. The protein kinase inhibitor H7
inhibited T
-induced accumulation in AChE activity and its
mRNAs, whereas okadaic acid (a potent inhibitor of phosphatases 1 and
2A) potentiated the effect of T
. Okadaic acid and H7 have
no effect on the binding of hTR
1 to T
or the
transcriptional rate of the AChE gene. Finally, treatment of cells with
T
stimulated cytosolic serine/threonine, but not tyrosine
kinase, activities. The time course analysis reveals that the increase
in serine/threonine activity precedes the effect of T
on
AChE mRNAs. These results suggest that activation of a serine/threonine
protein kinase pathway might be a link between nuclear thyroid hormone
receptor activation and stabilization of AChE mRNA.
Acetylcholinesterase (AChE; EC 3.1.1.7) ()is
responsible for the hydrolysis of acetylcholine at peripheral and
central cholinergic synapses. This enzyme is encoded by a single gene
in those vertebrate species examined to date, and the structural
diversity in gene products arises from alternative mRNA processing and
post-translational associations of catalytic and structural
subunits(1, 2, 3, 4, 5) .
Several lines of evidence suggest that thyroid hormones are involved
in regulating AChE activity in neural cells: AChE activity is decreased
in the brains of neonate thyroidectomized
rats(6, 7, 8) , and
triiodo-L-thyronine (T) increases AChE activity in
primary neuronal cultures initiated from fetal rat brains(9) .
Much less is known about the molecular basis for hormone control. It is
widely accepted that thyroid hormones exert their actions via nuclear
receptors, which regulate the transcription of thyroid
hormone-responsive genes. Three forms of thyroid receptors have been
identified in neural cells: TR
1, TR
1, and TR
2 (for
review, see (10) ). Recently, we showed that T
increases the activity of AChE and its mRNAs in neuro-2a cells, which
specifically overexpress human thyroid receptor
1 (hTR
1),
indicating that the control of AChE gene expression by T
occurs at a pretranslational step(11) . Furthermore, the
lag time of several hours following hormone administration suggests
that this effect is likely to be indirect, mediated by unknown factors.
Phosphorylation events regulate AChE activity.
1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor
of protein kinase, stimulates AChE activity in neuro-2a
cells(12) . Phosphorylation events also participate in thyroid
hormone action. Continued protein phosphorylation is required for
stimulation of transcription of the lipogenic genes by
T(13) , and transient transfection assays showed
that phosphorylation of TR isoforms increases their transcriptional
activity(14, 15) . Phosphorylation of the TRs could
lead to a change in either hormone binding or DNA binding of the
receptor(16) . Another possibility is that the target for the
phosphorylation events may be one or more of the auxiliary proteins or
coactivators that are important in T
-dependent
transcription(17, 18) . Alternatively, phosphorylation
could mediate the post-transcriptional effects of thyroid hormones.
Thyroid hormones regulate the turnover of numerous
mRNAs(19, 20, 21, 22, 23) .
Most of these effects are typically blocked by inhibitors of protein
synthesis, suggesting the possible involvement of one or more
components that influence the degradative pathway for specific mRNAs.
Phosphorylation events also participate in the control of mRNA
turnover. The turnover of several mRNAs is regulated by agents that
activate the protein kinase C or A pathway either directly (such as
phorbol esters or cAMP) or indirectly (such as via receptor
activation)(24, 25, 26, 27, 28, 29, 30, 31) .
It is therefore possible that activation of a protein kinase pathway might be the link between thyroid receptor activation and mRNA
turnover. However, there is no example of such a mechanism for thyroid
hormones.
In the present study, we investigated the mechanism of
regulation of AChE gene expression by T using neuro-2a
cells that overexpress hTR
1 as a model. Our results suggest that
activation of a serine/threonine protein kinase pathway may be the link
between thyroid receptor activation and the stabilization of AChE mRNA
induced by T
.
Specific oligonucleotides (nucleotides 1779-1824 and
1936-1979 of mouse AChE cDNA) were synthesized, 3` end-labeled,
and used to probe endogenous AChE mRNAs as described(11) . The
recovery of RNAs and poly(A) was determined by
hybridization with either a 3` end-labeled 18 S rRNA-specific
oligonucleotide (35) or a
P-labeled cDNA
cyclophilin probe, an unrelated gene constitutively expressed in
cells(36) , respectively. All values were normalized to
cyclophilin or 18 S rRNA signal. Quantitation of relative RNA levels of
autoradiograms was determined by densitometry (BioImage-Visage 110S
from Millipore Corp., Ann Arbor, MI).
Transcription assays were performed
essentially as described(38) . Nuclei were resuspended in
buffer containing 250 µCi of [P]UTP
(Amersham Corp.) and 1 mM each of ATP, CTP, and GTP and 5
mM dithiothreitol. For experiments with
-amanitin (from
Sigma),
-amanitin (2 µg/ml) was added to medium prior
initiating synthesis. After incubation at 30 °C for 30 min, the
radiolabeled RNAs were isolated by ethanol precipitation after DNase I
and proteinase K treatment. Total incorporation of
[
P]UTP into RNAs was 3-6
10
cpm/10
nuclei.
The radiolabeled RNAs were
hybridized to dot blots containing 5 µg of plasmid DNA. Plasmids
containing the 2.1-kb AChE insert and a control plasmid (pcDNA3) were
linearized with HindIII. In previous experiments, we showed
that the transcription factor NGFI-A (39) (also known as
zif268(40) , Krox-24(41) , Erg-1(42) ,
TIS28(43) , and CEF-5(44) ) is transcriptionally
regulated by T in these cells. (
)Thus, a plasmid
containing the 3.2-kb NGFI-A insert (from ATCC) was used as positive
control. 2
10
cpm were hybridized to the blots in
10 mM Tris-HCl, pH 7.4, 0.2% SDS, 10 mM EDTA, 300
mM NaCl, 1
Denhardt's solution, and 200 mg/ml Escherichia coli tRNA. Blots were incubated for 36 h at 65
°C, washed in 2
SSC and 0.1% SDS at room temperature, and
then washed at high stringency (0.2
SSC and 0.1% SDS at 65
°C) for 1 h.
Figure 1:
Triiodothyronine stimulates AChE
activity and levels of AChE mRNA. Neuro-2a was prepared and incubated
for the specific times with T (30 nM). Upper
panel, cells were homogenized in 10 mM Tris buffer, pH
7.0, containing 1% Triton X-100 and 1 M NaCl, and AChE
activity was determined. Lower panel,
poly(A)
-selected mRNA isolated from parallel culture
plates was electrophoresed, blotted, and hybridized to AChE-specific
oligonucleotides. Blots were exposed to Kodak XAR-5 film and quantified
by densitometry. Cyclophilin cDNA probe was used to standardize for
sample loading. Results represent the mean ± S.E. of four and
two experiments for AChE activity and mRNA,
respectively.
Figure 2:
Effect of triiodothyronine on AChE gene
transcription. [-
P]UTP-labeled transcripts
were synthesized in nuclei isolated from cells treated or not for 24 h
with 30 nM T
. Hybridizations were performed with
plasmids containing cDNA sequences for NGFI-A, c-jun, AChE,
and cyclophilin. A plasmid without insert (pcDNA3) was used as
control. Upper panel: A, a photo of an
autoradiograph; exposure time: 3 days. B, effect of
-amanitin on AChE gene transcription; exposure time: 6 days. Lower panel, quantitation of the signals was performed by
densitometry analysis. Open bars, control; dashed
bars, T
-treated cells. Values were normalized to the
cyclophilin signals and represent the mean ± S.E. of three
independent experiments.
The adenosine analog DRB specifically inhibits RNA polymerase
II(51, 52) , and its capacity to block the
transcription of AChE gene has rendered it useful in determining the
half-life of AChE mRNAs(53) . In the absence of T,
the AChE mRNA level decayed significantly after cells were treated with
DRB (Fig. 3). The calculated AChE mRNA half-life is about
4-5 h, which is in agreement with the AChE mRNA half-life
reported in muscle cells(53) . In cells pretreated with
T
for 72 h, no decrease in AChE mRNA levels was evident
over the next 24 h, suggesting the presence of a very stable mRNA
following a global block of transcription (Fig. 3). Furthermore,
the addition of OA (10 nM) prevented the decrease in AChE mRNA
levels observed 48 h after DRB treatment, suggesting that OA further
stabilizes the mRNAs. The addition of DRB 15 min before treatment with
T
completely inhibited T
's ability to
increase AChE mRNA levels (Fig. 4).
Figure 3:
Quantitation of AChE mRNA after DRB
treatment of neuro-2a cells. Neuro-2a cells were maintained for 3 days
in cultured medium with and with T (30 nM). At day
3, cells were extensively washed and treated for the specified times
with 20 µg/ml DRB. Upper panel, Northern blot analysis of
total RNA isolated from cultures grown in the absence of T
and treated with DRB for the indicated times (0, 1, 3, 6, 8, and
24 h). Lower panel, total RNA was isolated and AChE mRNA
levels were quantified by Northern blot. Filled dot represents
the levels of AChE mRNAs in cultures grown in the absence of T
and treated with DRB for various period of times (0, 1, 2, 3, 6,
8, and 24 h). The open dot represents the levels of AChE mRNAs
in T
-pretreated cells and treated with DRB for various
period of times (0, 2, 3, 12, and 24 h). The signals were quantified by
densitometry and the data standardized to the 18 S rRNA signals. Values
represent mean ± S.E. of three separate
experiments.
Figure 4:
DRB inhibits the T-induced
accumulation of AChE mRNA. Neuro-2a cells were cultured for 3 days in
the absence of T
. At day 3, DRB (20 µg/ml) was added to
the medium 15 min before treatment of cells with 30 nM T
. The cells were harvested after an additional 48 h
and total RNA isolated and analyzed as described in the legend to Fig. 3.
Figure 5:
Effect of H7 and OA on the
T-induced accumulation in AChE activity. Neuro-2a cells
were cultured for 3 days in the absence of T
. On day 3, the
medium was changed to one of the same composition plus or minus T
(30 nM) and (or) either H7 or OA at the indicated
concentration. AChE activity was determined 48 h latter. Upper
panel, effect of okadaic acid on AChE activity; open
bars, control; dashed bars, T
-treated cells. Lower panel, effect of H7 on the T
-induced
accumulation in AChE activity. Results are expressed as nmol of AChE
hydrolyzed per min/mg of protein and represent the mean ± S.E.
of three separate experiments.
To determine whether phosphorylation mediates the effect of T on AChE mRNA, we examined the effect of H7 and OA on the
T
-induced accumulation in AChE mRNA. H7 (20
µM), added coincidentally with T
, caused an
almost 90% inhibition of the T
-induced accumulation of AChE
mRNA after 24 h, whereas treatment of cells with OA for 24 h
potentiated the effect of T
on AChE mRNA levels (not
shown). A transcriptional effect of H7 and OA on AChE gene was excluded
by run-on transcriptional analysis (not shown).
In neuro-2a cells that overexpress hTR1, we previously
showed that the effect of T
on AChE gene expression is
independent of its antiproliferative effect, indicating that both
effects are regulated by distinct mechanisms(11) .
We show
here that the T-induced accumulation in AChE activity and
its mRNA result from stabilization of the mRNAs. No effect of T
was observed on the transcriptional rate or processing of AChE
mRNA. Although we previously reported that the increase in 3.2-kb mRNAs
was much greater than the increase of 2.4-kb mRNAs after T
treatment(11) , this result was not confirmed in the
present study, in which we analyzed a larger number of Northern blots.
It is likely that nuclear thyroid receptors indirectly participate in
regulated AChE mRNA turnover, because they are required for the
transcriptional regulation of one or more components that influence the
degradative pathway of AChE mRNAs. Consistent with this point is the
suppression of the T
-induced accumulation in AChE mRNAs
after a global block of transcription.
The protein kinase inhibitor
H7 blocks the ability of T to increase AChE activity and
its mRNAs. H7 had no effect on AChE activity, AChE mRNA levels, or the
rate of AChE gene transcription in the absence of T
which
suggests that H7 selectively inhibits the increase in AChE mRNA induced
by T
. At 500 µM, however, H7 increases AChE
activity in neuro-2a cells(12) . Isoquinoline sulfonamide
derivatives vary with respect to the affinity with which they bind to
different protein kinases(55) . In our study, half-maximal
inhibition of the activity of AChE enzyme occurs at about 10-15
µM H7. This concentration of H7 is slightly higher than
the K
values of protein kinase C (6
µM). It is likely that at higher concentration, H7 may
inhibit other protein kinases that are also involved in the regulation
of AChE enzyme activity. In contrast, OA potentiated the effect of
T
on AChE activity and its mRNA. The finding that OA had no
effect on the transcription of the AChE gene indicates that OA
selectively potentiates the effect of T
at a
post-transcriptional step. This effect was observed at a concentration
of 5 nM OA, which is in the range of the concentration
necessary to inhibit protein phosphatases 1 and 2A (0.1-10
nM)(54) . At this concentration, OA does not inhibit
protein phosphatase 2B and 2C, protein-tyrosine phosphatases, acid and
alkaline phosphatase(54) , indicating that OA selectively
inhibits serine/threonine phosphatases 1 and 2A. Furthermore, we showed
that T
stimulated cytosolic serine/threonine but not
protein tyrosine kinase activities. The time course analysis revealed
that the effect of T
on serine/threonine protein kinase
activity precedes its effect on AChE mRNAs, supporting the hypothesis
that activation of a serine/threonine protein kinase pathway might be
the intermediate between thyroid receptor activation and stabilization
of the AChE mRNA. Attempts were made to identify the serine/threonine
kinase(s). However, our study suggests that the endogenous
serine/threonine kinase (s) involved in the stabilization of AChE mRNAs
did not have specificities similar to those of cyclic AMP-dependent
protein kinase, protein kinase C, phosphorylase kinase, glycogen
synthetase kinase 3, and casein kinases I and II.
Our working
hypothesis is that T activates a serine/threonine protein
kinase, which in turn causes the stabilization of AChE mRNAs. The
mechanisms by which protein phosphorylation controls the stability of
AChE mRNA are unknown. The binding activity of an inducible stabilizing
factor that recognizes the AU-rich motif in the 3`-untranslated region
of granulocyte-macrophage colony-stimulating factor is dependent on its
phosphorylation state (56) . It is therefore possible that
phosphorylation of cytosolic turnover factor(s) could influence their
binding activity to specific sequence in AChE mRNAs, which in turn
causes the stabilization of AChE transcripts.
In conclusion, the present study provides the first evidence that activation of the protein kinase pathway might be the linkage between nuclear thyroid receptor activation and a post-transcriptional effect of thyroid hormones.