From the Department of Neurochemistry, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan
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ABSTRACT |
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1-(5-Isoquinolinesulfonyl)-2-methylpiperazine
(H7) has often been used in combination with protein kinase inhibitor
(N-(2-guanidinoethyl)-5-isoquinolinesulfonamide) (HA1004) to assess the contribution of protein kinase C (PKC) to
cellular processes, including the induction of gene expression. This
use of H7 and HA1004 is based upon the fact that H7 inhibits PKC more
potently than HA1004 in in vitro assays. Thus, although both compounds are broad spectrum protein kinase inhibitors, inhibition by H7, but not by HA1004, has often been interpreted as evidence for
the involvement of PKC in the cellular process under study. Here we
describe experiments that show that this interpretation is not correct
with regard to the induction of two immediate-early genes,
zif268 and c-fos, in PC12D cells. In
these studies we confirmed that H7, but not HA1004, potently blocks the
induction of zif268 and c-fos mRNA
by nerve growth factor, carbachol, phorbol ester, Ca2+
ionophore, or forskolin. Surprisingly, however, H7 has no effect on the
ability of these agents to activate mitogen-activated protein kinase
(MAPK), an upstream activator of zif268 and
c-fos gene expression. H7 also does not inhibit
preactivated MAPK in vitro. Taken together, these results
suggest that H7 blocks gene expression by acting at a site downstream
from MAPK. H7 has previously been shown to block transcription in
vitro by blocking the phosphorylation of the carboxyl-terminal
domain of RNA polymerase II (Yankulov, K., Yamashita, K., Roy, R.,
Egly, J.-M., and Bentley, D. L.(1995) J. Biol.
Chem. 270, 23922-23925). In this study, we show that pretreating
PC12D cells with H7, but not with HA1004, significantly reduces levels
of phosphorylated RNA polymerase II in vivo. These results
suggest that H7 blocks gene expression by inhibiting the phosphorylation of RNA polymerase II, a step required for progression from transcription initiation to mRNA chain elongation.
The immediate-early genes zif268 (also termed
NGFI-A, egr-1, krox24, TIS8; reviewed in Ref. 1) and
c-fos (2) encode transcription factors that have been
proposed to function as "third messengers" in intracellular signal
transduction cascades that convert information conveyed by
extracellular stimuli into genomic responses that underlie growth,
differentiation, and long term changes in the behavior of cells (3, 4).
We have previously shown that
NGF1 (1) and the carbachol
(carbamylcholine) cause the rapid induction of zif268
mRNA in PC12D cells (5). Induction of zif268
mRNA by NGF is mediated by the high affinity NGF receptor, TrkA,
which activates the Ras/MAPK cascade (6). Induction by carbachol is
mediated by the m1 subtype of muscarinic acetylcholine receptor, which
activates phospholipase C to produce the second messengers inositol
1,4,5-trisphosphate and diacylglycerol (5). Increased intracellular levels of inositol 1,4,5-trisphosphate trigger the release of Ca2+ from internal stores, which in turn opens
"capacitative influx" Ca2+ channels in the cell
membrane, resulting in a sustained influx of extracellular
Ca2+.2 Increased
levels of diacylglycerol activate PKC. Both the sustained increase in
intracellular Ca2+ and the activation of PKC contribute to
the induction of zif268 mRNA (5), at least in
part by activating the MAPK cascade (81). Activation of the MAPK
cascade is therefore a common element in the intracellular signaling
events leading to gene expression that are initiated by NGF and
carbachol in PC12D cells.
In the course of investigating the involvement of PKC in the induction
of zif268 mRNA by NGF and carbachol, we compared
the effects of pretreating PC12D cells with the protein kinase
inhibitor H7 (7) with pretreatment of the cells with the related
compound HA1004 (8). Both H7 and HA1004 are broad spectrum protein
kinase inhibitors, but H7 inhibits PKC more potently than HA1004
(Ki values = 6 and 40 µM for H7
and HA1004, respectively) in in vitro assays (7). Based upon
this difference, many investigators have used these inhibitors in
combination to evaluate the role of PKC in various cellular processes,
including the induction of gene expression. In many of these studies,
inhibition by H7 in the absence of inhibition by HA1004 was taken as
evidence for a role for PKC in the process under investigation. The
data presented in this paper, however, shows that inhibition of gene
expression by H7 does not necessarily imply that PKC is involved.
Rather, we found that although H7 potently inhibits the induction of
zif268 and c-fos mRNAs following
activation of PKC with phorbol ester, it fails to prevent activation of
MAPK by phorbol ester. This shows that H7 can block the induction of
gene expression without blocking PKC.
Examination of the literature indicates that H7 blocks the induction of
a broad spectrum of rapidly inducible genes by a variety of stimuli,
including stimuli not previously associated with the activation of PKC.
These observations suggest that H7 may block a site, different from
PKC, that is universally required for the induction of rapidly
inducible genes. A previous report that H7 blocks transcription
in vitro by inhibiting the phosphorylation of RNA polymerase
II by a TFIIH-associated kinase (9), led us to examine the effect of H7
on phosphorylation of RNA polymerase II in vivo. In the
present study we show that pretreatment of PC12D cells with H7
significantly reduces levels of phosphorylated RNA polymerase II
in vivo, suggesting that H7 blocks gene expression by
directly inhibiting transcription.
Materials--
H7 was purchased from Seikagaku Kogyo and
Calbiochem. This H7 is the authentic compound originally described by
Hidaka et al. (7) and not the less potent iso-H7, which has
sometimes been sold under the H7 label (10). HA1004, H8, and H89 were from Seikagaku Kogyo. NGF, carbachol, ATP, chloramphenicol,
Ca2+ ionophore A23187, thapsigargin, forskolin,
dideoxyforskolin, and dimethyl sulfoxide were purchased from Wako
Chemical Industries. K252a and staurosporin were from Kyowa Medex Co.,
Inc. GF109203x, PD098059, and
5,6-dicholoro-1- Cell Culture--
PC12D cells (12), a rapidly differentiating
subline of rat pheochromocytoma-derived PC12 cells (13), were a gift
from Mamoru Sano (Dept. of Biology, Faculty of Medicine, Kyoto
Prefectural University of Medicine). PC12D cells were cultured in
Dulbecco's modified Eagle's medium (DMEM, Nissui) supplemented with
5% fetal bovine serum, 5% horse serum, 0.16% sodium bicarbonate, 3.6 mM glutamine, 10 units/ml penicillin, 45 ng/ml streptomycin
at 37 °C under 5% CO2. Non-differentiated PC12D cells
were used in all of the experiments. Unless noted, drugs were added
directly to the culture medium and were present until the time at which
the cells were harvested. The corresponding vehicle (water,
Me2SO, or ethanol) was added to control cells.
MAPK Assays--
MAPK assays were performed as described by Cook
and McCormick (14) with some modifications. Briefly, PC12D cells grown
to 80-90% confluency in 3.5 cm in uncoated plastic culture dishes (Corning or Iwaki Glass Co.) were stimulated with various agents for 10 min and then lysed by addition of 200 µl of lysis buffer containing
20 mM Tris-Cl (pH 8.0), 137 mM NaCl, 10%
glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 50 mM NaF, 1 mM
Na3VO4, 1 mM phenylmethanesulfonyl
fluoride (PMSF), 20 µM leupeptin, 10 µg/ml aprotinin.
After brief centrifugation to remove cellular debris, 0.1 µg of
anti-Erk-1 and 0.1 µg of anti-Erk-2 antibodies were added to the
supernatant fractions, and these were incubated for 1 h at 4 °C
with rotation to provide gentle mixing. Protein A-agarose (10 µl of
resin suspension, Santa Cruz Biotechnology catalog number sc-2001) was
subsequently added to each sample and the incubation continued with
rotation at 4 °C for 1 h. The resin in each sample was then
collected by centrifugation (2500 rpm) and washed twice with 200 µl
of lysis buffer and once with 200 µl of 2× reaction buffer. 1×
reaction buffer contained 25 mM MOPS (pH 7.2), 25 mM sodium RNA Purification and Northern Blot Analysis--
RNA was
isolated from PC12D cells grown to 80-90% confluency in 6-cm uncoated
plastic dishes, and Northern analysis was carried out as described
previously (5). The amount of RNA in each sample was determined by
optical spectroscopy, and the integrity of the RNA in each sample was
assessed by examining the ethidium bromide-stained RNA in denaturing
gel used for Northern blot analysis. Unless noted, 10 µg of total
cellular RNA was electrophoresed in each lane. After blotting onto Pall
Biodyne type B transfer membranes (0.45-µm pore size), hybridization
was carried out simultaneously using DNA probes prepared from
zif268, c-fos (coding regions), and
cyclophilin DNA fragments isolated from agarose gels and labeled using
the Amersham Pharmacia Biotech Oligolabeling kit and
[ Plasmid Construction--
An expression vector, pGLzif420,
containing a firefly luciferase reporter gene linked to the rat
zif268 promoter was constructed using pGL2 (Promega).
Briefly, the zif268 promoter region (from Transfection of PC12D Cells and Assay of Reporter
Genes--
Transfections were performed using
LipofectAMINETM reagent (Life Technologies, Inc.)
essentially as recommended by the manufacturer. Cells were seeded in
6-cm plastic culture dishes (Corning or Iwaki Glass) at a density of
4 × 106 cells/dish and cultured for 1 day prior to
transfection. 0.92 µg of pEF-CAT DNA, 2.3 µg of luciferase
expression vector DNA, 13.8 µl of LipofectAMINETM reagent
were added to each dish of cells and incubated for 4 h, prior to
adding the medium containing twice the normal concentration of serum.
After incubation overnight, the cells in each 6-cm dish were
resuspended and distributed into 12 × 1.1-cm wells. The following day, the medium was replaced with normal DMEM, and the cells were cultured for 1 more day. Drugs were added directly to the culture medium, and cells were harvested after 4 h. Luciferase expression was carried out using the Promega Luciferase or Packard LucLiteTM, and
luciferase activities were quantified using a Packard Tri-Carb or Top
count scintillation counter as described in the manuals supplied by
Promega and Packard. Background luciferase expression was determined
using cells transfected with pGL2, which lacks a promoter for
luciferase gene expression. Transfection efficiency was determined by
cotransfection with pEF-CAT. CAT activities were measured as described
by Nordeen et al. (17), and these values were used to
calculate normalized luciferase activities for each sample.
Assay of RNA Polymerase II Phosphorylation in Vivo--
PC12D
cells were labeled with 32P in vivo as described
(18). Briefly, PC12D cells grown to 80-90% confluency in 10-cm
culture plates were washed twice with 10 mM Tris (pH
7.0)-buffered saline (TBS) and overlaid with 5 ml of phosphate-free
DMEM (Life Technologies, Inc.) supplemented with 25 mM
HEPES, 5% dialyzed horse serum, and 5% dialyzed fetal bovine serum.
50 µCi of [32P]orthophosphate was added to medium, and
the cells were incubated at 37 °C for 3 h in the absence of
CO2. H7 (Seikagaku Kogyo) or other inhibitors were added 30 min prior stimulation with 5 ng/ml NGF for 15 min. Cells were washed
twice with ice-cold phosphate-buffered saline supplemented with 0.2 mM PMSF, scraped into tubes, and centrifuged at 800 × g for 5 min. Cells were resuspend in 1 ml of buffer
containing 10 mM Tris-Cl (pH 7.9), 1 mM
CaCl2, 1.5 mM MgCl2, 0.25 M sucrose, 0.2 mM PMSF, 0.5% Triton X-100,
homogenized with Dounce homogenizer, and centrifuged at 800 × g for 5 min. Nuclei pellets were washed once with 1 ml of
the above buffer lacking Triton X-100. Pellets were resuspended in 100 µl of buffer containing 25 mM Tris-Cl (pH 8.0), 2.5 mM magnesium acetate, 2 mM CaCl2,
0.05 mM EDTA, 0.1 mM DTT, 0.2 mM
PMSF, and 12.5% glycerol and treated with 10 µg of DNase and RNase
on ice for 30 min. Nuclei were lysed by adding 100 µl of 2% SDS,
boiled for 3 min, and centrifuged at 15,600 × g for 3 min to remove debri. Nuclei extracts (15 µl/lane) were resolved by
SDS-PAGE (5% polyacrylamide stacking gel containing 125 mM
Tris-Cl (pH 6.8), 0.1% SDS; 5% polyacrylamide resolving gel (prepared
from 29.5% acrylamide + 0.5% bisacrylamide stock solution) containing
375 mM Tris-Cl (pH 8.8), 0.1% SDS; running buffer
containing 25 mM Tris, 250 mM glycine, 0.1%
SDS). Electrophoresis was carried out at 25 mA per gel until the
tracking dye entered the resolving gel, after which the current was
increased to 45 mA per gel. Proteins were then electrophoretically
blotted (0.5 A, 45 min) onto polyvinylidene difluoride membranes
(ImmobilonTM transfer membrane, Millipore) using a Nihon
Eido Western blotting apparatus (20 × 20 cm) in buffer containing
100 mM Tris, 192 mM glycine, 10% methanol, and
0.02% SDS. Following transfer, the membranes were blocked by
incubation in phosphate-buffered saline containing 5% skim milk and
0.5% Tween 20 overnight at room temperature. Phosphorylated proteins
were detected by autoradiography using Fuji RX-U x-ray film. The
membranes were then exposed to 0.02 µg/ml antibodies that recognize
the carboxyl-terminal domain (CTD) of RNA polymerase II (C-21, catalog
number sc-900, Santa Cruz Biotechnology) in phosphate-buffered saline
containing 0.5% skim milk and 0.05% Tween 20 for 2 h at room
temperature, washed 3 times (10 min each) with buffer, and incubated in
buffer containing anti-rabbit IgG antibodies cross-linked with
horseradish peroxidase (Jackson ImmunoResearch, catalog number
111-035-003; 5000-fold final dilution) for 2 h at room
temperature. After washing 3 times for 20 min/wash and 3 times for 10 min/wash with buffer, immune complexes were visualized by enhanced
chemiluminescence (ECL kit, Amersham Pharmacia Biotech).
H7 Blocks the Induction of zif268 and c-fos mRNA by NGF
and Carbachol--
As shown in Fig. 1,
levels of both zif268 and c-fos mRNAs
are low or undetectable in unstimulated cells but increase rapidly following exposure to NGF or carbachol, reaching high levels after 45 min. The fact that NGF-stimulated increases in zif268
and c-fos mRNA are blocked by the inhibitor K252a (19,
20) indicates that these inductions require the activation of the
tyrosine kinase of the high affinity NGF receptor, TrkA. Likewise, the
ability of atropine to block the inductions of zif268
and c-fos mRNAs by carbachol indicates the involvement
of muscarinic acetylcholine receptors. Pretreatment of the cells for 30 min with 100 µM H7 completely blocks the induction of
zif268 and c-fos mRNAs by NGF and
carbachol. By contrast, pretreatment with 100 µM HA1004
has essentially no effect on these inductions. Neither H7 nor HA1004 affect background levels of zif268 and
c-fos mRNAs.3 As shown in Fig.
2, H7 blocks the induction of
zif268 and c-fos mRNAs in a
dose-dependent manner, with complete inhibition observed at
concentrations of 50 µM and greater.
The complete inhibition of zif268 and
c-fos mRNA inductions by H7 is surprising for two
reasons. First, although NGF has previously been reported to activate
PKC in PC12 cells (21, 22), the inductions of zif268
and c-fos mRNAs were not expected to require the
activation of PKC, since down-regulation of PKC by prolonged exposure
to phorbol ester has only a small effect on the induction of these
mRNAs by NGF.4 We have
also previously observed (5) that induction of zif268 mRNA by NGF is not affected by the specific PKC inhibitor GF109203x (23, 24). Second, although we have previously shown that PKC contributes to m1 muscarinic acetylcholine
receptor-mediated induction of zif268 mRNA in
PC12D cells, this induction is only partially blocked by pretreatment
with GF109203x (5). Thus, the total block of zif268
and c-fos gene induction by H7 seems to be too large an
effect. To understand better how H7 blocks gene expression, we examined
its effect on the induction of zif268 and
c-fos mRNAs by additional agents.
H7 Also Blocks the Induction of
zif268 and c-fos mRNAs by Phorbol Ester, Ca2+
Ionophore, and Forskolin--
Phorbol ester is expected to stimulate
increases in immediate-early gene mRNA by activating PKC, and
therefore this induction would be expected be inhibited by H7. By
contrast, activation of immediate-early gene expression by elevated
level of intracellular Ca2+ in PC12 cells has not
previously been suggested to require PKC. Rather, activation of
Ca2+/calmodulin kinases (25, 26) and/or activation of MAPK
cascade (27, 28) is thought to be sufficient. Similarly, forskolin, which increases intracellular levels of cAMP by stimulating adenylate cyclase, is thought to activate gene expression via activation of PKA
(29) and/or the MAPK cascade (30-33). The experiment depicted in Fig.
3, however, shows that H7 inhibits the
induction of zif268 and c-fos mRNAs by
each of these agents. These results strongly suggest that H7 blocks
gene expression by acting at a site distinct from PKC.
MAPK Contributes to the Induction of zif268 and c-fos RNAs
by NGF, Carbachol, Phorbol Ester, and Increases in Intracellular
Ca2+ or cAMP--
MAPK is likely to play a central role in
the induction of zif268 and c-fos by NGF and
carbachol in PC12 cells. MAPK functions as part of the Ras/MAPK cascade
(Ras-Raf-MEK-MAPK), which transmits signals from tyrosine kinase- and G
protein-linked membrane receptors to the nucleus (6, 34, 35). Activated
MAPK enters the nucleus where it phosphorylates and activates
"ternary complex" transcription factors, e.g. Elk-1 or
SAP-1, which stimulate gene expression by forming a complex with serum
response factor bound to the serum response element (SRE) (36-38). The
c-fos promoter contains 1 SRE (2) and the
zif268 promoter contains 6 SREs (16, 39), which have
been shown to play a role in the induction of zif268
mRNA by serum and NGF (16, 40, 41). In PC12 cells, MAPK has
previously been shown to be strongly activated by NGF (42-45), phorbol
ester (44), and by elevated levels of intracellular Ca2+
(27, 28, 46) or cAMP (30-33). Together, these observations suggest
that activation of MAPK plays a central role in the induction of
c-fos and zif268 mRNAs by each of
these agents in PC12D cells. Evidence supporting this inference is
shown in the Northern blot depicted in Fig.
4, where induction of c-fos
and zif268 RNAs by each of the agents tested was
inhibited by pretreating PC12D cells with PD098059 (48, 49), a specific
inhibitor of MEK, the immediate upstream activator of MAPK.
Concentrations of H7 Sufficient to Block the Induction of
zif268 and c-fos mRNAs Do Not Block the Activation of
MAPK--
The data in Fig. 5 show that
concentrations of H7 that block the induction of
zif268 and c-fos mRNAs have no effect
on the activation of MAPK. Pretreatment with HA1004 also does not block activation of MAPK. The inability of H7 to block the activation of MAPK
by phorbol ester is particularly surprising, since H7 is expected to
inhibit PKC. To determine if PKC can be effectively blocked in PC12D
cells, we examined the effects of pretreatment with the specific PKC
inhibitor GF109203x. The data in Fig. 6 show that pretreatment with GF109203x is effective in blocking both
phorbol ester-mediated induction of zif268 and
c-fos mRNAs and phorbol ester-mediated activation of
MAPK. That phorbol ester activation of immediate-early gene expression
and MAPK activation is mediated by PKC is supported by the observation
that exposure to the same concentration of a less active phorbol ester,
4- H7 Does Not Inhibit PKA in PC12D Cells--
Although H7 has been
reported to potently block PKA activity in vitro
(Ki = 3 µM) (7), the failure of H7 to
block PKC in PC12D cells suggests that it may also not be effective in
blocking PKA. To demonstrate that PKA in PC12D cells can be pharmacologically blocked, we pretreated the cells with the specific PKA inhibitor H89 (47). The results depicted in Fig.
7 show that pretreatment with 30 µM H89 blocks both forskolin-stimulated induction of
zif268 and c-fos mRNAs and
forskolin-stimulated activation of MAPK. The specificity of the effects
of forskolin is demonstrated by the lack of mRNA induction and MAPK
activation by the related compound dideoxyforskolin, which does not
activate adenylate cyclase. These results suggest that the ability of
H7 to block the induction of c-fos and
zif268 mRNA is unrelated to PKA.
H7 Does Not Inhibit Preactivated MAPK--
The results presented
so far show that although H7 potently inhibits the induction of
zif268 and c-fos mRNAs, this is not caused by inhibition of PKC or PKA or by blocking the activation of
MAPK. To determine if the block of mRNA induction is caused by
inhibition of MAPK itself, we examined the ability of H7 to inhibit
preactivated MAPK in vitro. As shown in Fig.
8, preactivated MAPK is essentially
unaffected by H7, even at high concentrations of the inhibitor. Thus,
it is unlikely that H7 blocks the induction of immediate-early gene
RNAs by blocking MAPK. Rather, these results suggest that H7 acts at a
site downstream from MAPK.
H7 Blocks NGF-mediated Induction of a Luciferase Reporter Gene
Linked to zif268 Promoter--
The data in Fig.
9 show that preincubation with H7, but
not HA1004, blocks the induction of luciferase reporter gene linked to
the zif268 promoter. By contrast, GF109203x and H89
have only a small effect on the induction of the luciferase reporter by NGF. The ability of H7 to block the induction of gene expression mediated by the zif268 promoter in a heterologous
system suggests that the mechanism of inhibition involves the
functioning of the promoter rather than post-transcriptional controls
such as transcriptional pausing, which is mediated by sequences in the
first intron of the c-fos gene (50, 51). The small effects
of GF109203x and H89 on the expression of the reporter gene suggest
that neither PKC nor PKA is essential for activation of the
zif268 promoter by NGF.
H7 Inhibits the Phosphorylation of RNA Polymerase II in
Vivo--
The data presented up to this point show that H7 does not
inhibit zif268 and c-fos gene expression
by blocking the activation of MAPK or by directly inhibiting MAPK.
Rather, the site at which H7 acts seems to be downstream from MAPK. One
possibility we considered was that H7 blocks gene expression, not by
inhibiting a step in the intracellular signaling cascade, but rather by
inhibiting some step essential for transcription. A search of the
literature for kinases known to be inhibited by H7 brought our
attention to the general transcription factor TFIIH, which contains an
associated kinase (cyclin-dependent kinase (cdk)
MO15/Cdk-7) that phosphorylates the CTD of RNA polymerase II (9,
52-54). Phosphorylation of the CTD of RNA polymerase II is required
for efficient RNA chain elongation (9, 54), and blocking CTD
phosphorylation with H7 has been shown to inhibit transcription of
human immunodeficiency virus and c-myc mRNAs by RNA
polymerase II in vitro (9). Based upon these results we
decided to determine if H7 blocks the phosphorylation of RNA polymerase
II in vivo. The experiment depicted in Fig. 10A shows that pretreatment
of PC12D cells with H7 specifically reduces levels of phosphorylated
RNA polymerase II, as evidenced by (i) a reduction in the intensity of
32P-labeled RNA polymerase II detected by autoradiography
(left panel, upper band within the
bracket) and (ii) a reduction in levels of the
"shifted-up" form of RNA polymerase II detected by immunochemical
staining (right panel, upper band within the bracket). The slowly migrating, "shifted-up" form of RNA
polymerase II has been previously shown to correspond to the
phosphorylated form of the enzyme (18). By contrast, pretreatment of
PC12D cells with HA1004 does not reduce levels of phosphorylated RNA polymerase II or change its electrophoretic mobility (Fig. 10, B and C). Inhibition of RNA polymerase II
phosphorylation similar to that obtained with H7 is also observed
following pretreatment of PC12D cells with two additional compounds
known to block phosphorylation of the CTD of RNA polymerase II in
vitro, the protein kinase inhibitor H8 (7, 55) and the classic RNA
polymerase II inhibitor DRB (9, 56) (Fig. 10B). By contrast,
the MEK inhibitor PD098059 and broad spectrum protein kinase inhibitor
staurosporine have no effect on phosphorylation of RNA polymerase II
(Fig. 10B). Taken together, these data suggest that H7
blocks the induction of zif268 and c-fos
mRNAs in vivo by inhibiting transcription
elongation.
In this study we show that H7 blocks the induction of
zif268 and c-fos mRNA in PC12D cells
by NGF, carbachol, phorbol ester, and agents that increase
intracellular Ca2+ or cAMP (Figs 1-3 and 5) but fails to
block the activation of MAPK, an enzyme that contributes to the
induction of these mRNAs (Fig. 4). The inability of H7 to block the
activation of MAPK by phorbol ester is surprising, since H7 is a potent
inhibitor of PKC in vitro and is often used as a PKC
inhibitor in in vivo experiments. Likewise, the inability of
H7 to block the activation of MAPK by forskolin was not expected since
H7 is also a potent inhibitor of PKA in vitro.
The inability of H7 to block PKC and PKA in vivo could be
caused by an inability to efficiently penetrate the cells. Thus, H7 may
not reach sufficient concentrations to block PKC or PKA, even though
concentrations sufficient to block gene expression are attained. By
contrast, the more potent PKC inhibitor GF109203x (in vitro
Ki = 14 nM; Ref. 23) and PKA inhibitor
H89 (in vitro Ki = 48 nM;
Ref. 47) evidently do reach sufficient intracellular concentrations to
block PKC and PKA, respectively (Figs. 6 and 7). The observation that
HA1004, a potent inhibitor of PKA in vitro
(Ki = 2.3 µM; Ref. 7), fails to block the activation of MAPK or induction of zif268 and
c-fos mRNA by forskolin in vivo suggests that
HA1004 may also not efficiently permeate the cells. If it is true that
H7 fails to block intracellular PKC and PKA because only a small amount
enters the cells, then its ability to block mRNA induction may
depend upon high affinity binding of H7. Inhibition by H7 apparently
takes place very rapidly, since H7 was found to be equally effective at
inhibiting the induction of c-fos and
zif268 mRNAs when added 30 min before, at the
same time, or up to 5 min after stimulation with NGF.3
The fact that H7 does not effectively block PKC- and PKA-mediated
activation of MAPK in PC12D cells suggests that it inhibits the
induction of zif268 and c-fos mRNAs by
acting at a site unrelated to PKC or PKA. The observation that H7 also
does not block preactivated MAPK (Fig. 8) suggests that H7 blocks the
induction of zif268 and c-fos mRNAs by
acting at a site downstream from MAPK or at a site unrelated to the
MAPK intracellular signaling cascade. Recent work has shown that MAPK
activates two additional kinases that may be important for the
induction of c-fos mRNA in PC12 cells. The
serine/threonine kinases RSK-1 (p90RSK) and RSK-2 (CREB
kinase) are activated upon phosphorylation by MAPK and then enter the
nucleus and activate the transcription factors serum response factor
(57) and CREB (58, 59), respectively. Studies by Greenberg and
co-workers (58) have shown that, although SRE and CRE function
independently when linked to heterologous minimal promoters, both are
required for activation of c-fos gene expression by the
native c-fos promoter. Thus, it may be that activation of
c-fos and zif268 gene expression requires
the activation of RSK-1 and/or RSK-2 in addition to activation of MAPK.
Significantly, Yin and Yang (60) have shown that H7 inhibits
preactivated RSK-1 in vitro, although the effect of H7 on
RSK-2 is still unknown. Taken together, the above observations suggest
that H7 might inhibit gene expression by inhibiting RSK-1 and/or
RSK-2.
Another possibility is that H7 interferes with gene expression by
blocking the processing of mRNA. H7 has previously been reported to
inhibit the splicing of c-fos mRNA in PC12 cells, as
evidenced by an accumulation of prespliced c-fos mRNA in
cells pretreated with high concentrations of H7 (61). (The "H7"
used in that study was probably actually iso-H7 (10).) By contrast, we
have never observed the accumulation of c-fos or
zif268 precursor mRNAs in PC12D cells pretreated
with authentic H7, and thus we have no evidence to suggest that H7 is
blocking mRNA induction by inhibiting mRNA splicing.
Nevertheless, our data do not rule out the possibility that H7 blocks
gene expression by interfering with the processing of mRNA. The
observation that H7 blocks the activation of the
zif268 promoter linked to a luciferase reporter gene
(Fig. 9), however, suggests that its effects are not restricted to the
native zif268 gene.
A survey of published papers that use H7 to study gene expression
reveals that H7 blocks the induction of a broad spectrum of rapidly
inducible mRNAs by diverse stimuli. For example, H7 blocks the
induction of mRNA by cytokines and lymphokines, including the
induction of tumor necrosis factor mRNA by interleukin-1 In many studies using H7, inhibition of gene expression has been taken
as evidence for the involvement of PKC. Whereas PKC certainly functions
in the induction of some genes, there are many cases where the authors
have noted that the inhibition of mRNA induction by H7 is not
consistent with a role for PKC. For example, Nakajima and Wall (63),
Kahle et al. (69), Ohtsuki and Massague (71), Abboud (72),
and Nose et al. (74) reported that down-regulation of PKC by
long-term exposure to phorbol ester failed to block the gene expression
under study, even though H7 was effective. Authors of other studies
have also concluded that H7 blocks inducible gene expression by
blocking a kinase distinct from PKC (60, 66).
In a few studies, H7 had no effect on gene expression or, instead,
stimulated gene expression. In most of these cases, the lack of
inhibition can in retrospect be accounted for by the use of iso-H7,
rather than authentic H7 (76, 77), or very low concentrations of
authentic H7 (78, 79). In almost every study to date, authentic H7 used
at concentrations between 50 and 100 µM has been found to
block gene induction. Together, these results suggest that H7 may be
inhibiting some step that is generally required for the induction genes.
An attractive hypothesis to explain the breadth of the inhibitory
activity of H7 is that it affects some aspect of the general transcriptional machinery, rather than the kinase cascades that activate specific transcription factors. Consistent with this idea is
the observation that H7 blocks the kinase associated with the general
transcription factor TFIIH, which phosphorylates the carboxyl-terminal
domain of RNA polymerase II (9, 52). TFIIH participates in the late
stages of transcription initiation, and the associated kinase is
required for efficient elongation mRNA by RNA polymerase II (9,
52). Inhibition of TFIIH kinase with H7, in fact, blocks
transcriptional elongation of human immunodeficiency virus and
c-myc RNAs by RNA polymerase II in vitro (9).
Based upon these observations, we decided to determine whether
pretreatment of PC12D cells with H7 affects the phosphorylation of RNA
polymerase II. The results depicted in Fig. 10 show that a 30-min
exposure to H7 is sufficient to reduce significantly levels of
phosphorylated RNA polymerase II in both unstimulated cells and cells
stimulated with NGF. By contrast, pretreatment of the cells with HA1004
has no effect on phosphorylation of RNA polymerase II. To our
knowledge, this is the first demonstration that H7 inhibits the
phosphorylation of RNA polymerase in vivo, although H7 has
previously been shown to block c-myc transcriptional elongation in vivo (80). Together, these results suggest
that H7 blocks the induction of gene expression by inhibiting
elongation of RNA transcripts. A scheme indicating the site at which H7
inhibits gene expression is depicted in Fig.
11. Further work will be required to
prove this model, but the data in this study provide a plausible explanation for the general inhibition of gene expression by H7 and
suggest that caution must be taken when interpreting the effects of H7
on inducible gene expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-ribofuranosylbenzidazole (DRB) were
purchased from Calbiochem. PMA, 4-
-PMA, acetyl-CoA, and S-acetyl-coenzyme A synthetase were from Sigma.
[
-32P]ATP and [
-32P]dCTP were
obtained from Amersham Pharmacia Biotech and [3H]sodium
acetate was from NEN Life Science Products. Anti-Erk-1 and anti-Erk-2
antibodies were obtained from Santa Cruz Biotechnology. Restriction
enzymes and other reagents for modification of DNA were obtained from
Toyobo Corp., Takara Shuzo Co., and New England Biolabs. Murine
zif268 cDNA (ATCC number 63027), murine
c-fos genomic DNA (ATCC number 41041), and the expression
vector pBLCAT2 were obtained from the American Tissue Culture
Collection. Human cyclophilin cDNA was a gift from Toshio Watanabe
(Tohoku University), and pEGF-BOS (11) was a gift from Shigeki Nagata
(Osaka Bioscience Institute).
-glycerophosphate, 15 mM
MgCl2, 1 mM EGTA, 0.1 mM NaF, 4 mM DTT, 1 mM Na3VO4. 22 µl of reaction mix containing 25 µM ATP, 1 µCi of
[
-32P]ATP, 15 µM myelin basic protein in
1× reaction buffer was added to the resin, and the mixture was
incubated at 30 °C for 30 min. Reaction mixes were directly spotted
on Whatman phosphocellulose filters, and filters were washed 6 times in
1% phosphoric acid for 5 min each wash. Radioactivity retained on the
filters was quantified by liquid scintillation counting. During the
course of this study, we determined that, unlike the anti-Erk-1
antibodies (Santa Cruz C-16), the anti-ErK-2 antibodies (Santa Cruz
Biotechnology catalog number C-14) were not very effective in
immunoprecipitating Erk-2 from cell
lysates.3 MAPK activities
reported in this paper therefore reflect primarily Erk-1 activities.
Qualitative assays of MAPK activation determined by measuring the
activation-correlated shift-up in electrophoretic mobilities (15)
showed, however, that Erk-1 and Erk-2 responded in the same manner to
all treatments examined.3
-32P]dCTP. The intensities of bands in Northern blots
were quantified using a Fuji Bioimaging analyzer BAS2000.
420 to 0 base pairs) containing 6 SRE sites and 2 CRE sites was amplified by
polymerase chain reaction using synthetic oligonucleotide primers
(forward primer corresponding to nucleic acid residues 121-149 of the
rat zif268 promoter in the numbering system of Changelion et al. (Ref. 16; GenBankTM accession
number J04154), 5'-AACACCATATAAGGAGCAGGAAGGATCCC-3'; backward primer
containing a synthetic EcoRI site followed by nucleic
acid residues 941-920 (14) of the rat zif268
gene, 5'-(GCGAATTC)TTGCTCAGCAGCATCATCTCCT-3'). This
polymerase chain reaction product was blunt-ended, digested with
NruI, and the fragment containing the
zif268 promoter sequences cloned into the blunt-ended
HindIII site of pGL2. An expression vector containing the
bacterial chloramphenicol acetyltransferase (CAT) gene under the
control of the human elongation factor 1
promoter was constructed as
follows: pBLCAT2 was digested with BamHI and
BglII and then self-ligated. Digestion of the resulting plasmid with SalI and SmaI yielded a 1.5-kilobase
pair fragment containing the CAT gene and a polyadenylation signal
derived from SV40. This fragment was isolated, blunt-ended, and cloned
into the XbaI site (after converting XbaI-cut
ends to blunt ends) of pEF-BOS. The resulting vector, pEF-CAT, was used
as an internal control in transfection experiments using
zif268-luciferase expression vectors.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
H7 blocks the induction of
zif268 and c-fos mRNAs by
NGF and carbachol. Northern blot analysis of
zif268 and c-fos mRNA in PC12D cells
pretreated with water (W), 200 nM K252a
(K) for 15 min, 1 µM atropine (A)
for 2 min, or 100 µM Calbiochem H7 (H7(C)),
100 µM Seikagaku Kogyo H7 (H7(S)), or 100 µM HA1004 (HA) for 30 min prior to stimulation
with water (W), 5 ng/ml NGF, or 500 µM
carbachol for 45 min. Northern blots were carried out as described
under "Experimental Procedures" using 10 µg of total cellular RNA
per lane. These data are representative of experiments performed 2 times with similar results.
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Fig. 2.
H7 inhibits the induction of
zif268 and c-fos gene
expression in a dose-dependent manner. Northern blot
analysis of zif268 and c-fos mRNA in
PC12D cells pretreated with water (W), HA1004, or H7. PC12D
cells were preincubated with the indicated concentrations of HA1004 or
H7 for 30 min prior to stimulation with 5 ng/ml NGF (left)
or 100 µM carbachol (right) for 45 min. These
data are representative of experiments performed 3 times with similar
results.
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Fig. 3.
H7 inhibits the induction of
zif268 and c-fos mRNAs by
phorbol ester, Ca2+ ionophore, and forskolin. Northern
blot analysis of zif268, c-fos, and
cyclophilin mRNAs was carried out as described in the legend to
Fig. 1 (10 µg of total cellular RNA per lane). PC12D cells were
preincubated with water (W), 50 µM H7, or 50 µM HA1004 (HA) for 30 min prior to stimulation
with 1 µM PMA, 5 µM A23187, or 10 µM forskolin for 45 min. These data are representative of
experiments performed 3 times with similar results.
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Fig. 4.
MEK inhibitor PD098059 blocks the induction
of zif268 and c-fos mRNAs
by NGF, carbachol, PMA, thapsigargin, and forskolin. Northern blot
analysis of zif268 and c-fos mRNA in
PC12D cells pretreated with Me2SO (0) or the
indicated concentrations of PD098059 for 30 min prior to stimulating
the cells with water (W), 5 ng/ml NGF, 1 mM
carbachol, 1 µM PMA, 0.2 µM thapsigargin,
or 10 µM forskolin for 45 min. Northern blots were
carried out as described under "Experimental Procedures." These
data are representative of experiments preformed 3 times with similar
results.
-PMA, failed to increase zif268 and
c-fos mRNA levels or activate MAPK. These results are
also consistent with the conclusion that H7 blocks
zif268 and c-fos gene expression by acting
at a site distinct from PKC.
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Fig. 5.
H7 does not inhibit the activation of MAPK by
NGF, carbachol, phorbol ester, Ca2+ ionophore, or
forskolin. PC12D cells were preincubated with water (W,
shaded box), 50 µM H7 (dotted box), or 50 µM HA1004 (open box) prior to stimulation with
water (W), 1 ng/ml NGF, 100 µM carbachol, 1 µM PMA, 5 µM A23187, or 10 µM
forskolin for 10 min. Preparation of cell extracts, immunoprecipitation
of p42 and p44 MAPK isoforms, and in vitro phosphorylation
of the MAPK substrate myelin basic protein in the presence of
[ -32P]ATP were carried out as described under
"Experimental Procedures." The heights of the
bars represent the average of two independent measurements
of incorporation of 32P into myelin basic protein. These
data are representative of two experiments that yielded similar
results.
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Fig. 6.
The PKC inhibitor GF109203x blocks the
induction of zif268 and c-fos
mRNAs and the activation of MAPK by phorbol ester. PC12D
cells were preincubated with dimethyl sulfoxide (D) or 10 µM GF109203x (G) for 10 min prior to
stimulation with 1 µM PMA or 1 µM -PMA.
Cells were harvested for Northern blot analysis (A) 45 min
after the stimulations. Duplicate sets of cells were harvested for MAPK
assays (B) 10 min after stimulation. These data shown are
representative of the results of three experiments. Me2SO,
dark gray box; GF109203x, light gray box.
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Fig. 7.
The PKA inhibitor H89 blocks the induction of
zif268 and c-fos mRNAs and
the activation of MAPK by forskolin. PC12D cells were preincubated
with dimethyl sulfoxide (D) or 30 µM H89
(H) for 10 min prior to stimulation with 10 µM
forskolin or 10 µM dideoxyforskolin. Cells were harvested
for Northern blot analysis (A) 45 min after stimulation.
Duplicate sets of cells were harvested for MAPK assays (B)
10 min after stimulation. These data shown are representative of the
results of three experiments. Me2SO, dark gray
box; GF109203x, light gray box.
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Fig. 8.
H7 does not inhibit preactivated MAPK
in vitro. Activated forms of p42 and p44 MAPK
were isolated by immunoprecipitation from PC12D cells that had been
stimulated with water (W) or 5 ng/ml NGF for 5 min. The
indicated concentrations of H7 or HA1004 were added to the standard
in vitro MAPK assay reaction mix containing buffer,
immunoprecipitated MAPK, [ -32P]ATP, and myelin basic
protein (MBP). Reaction mixtures were incubated at 30 °C
for 30 min, and the incorporation of 32P into myelin basic
protein was determined by scintillation spectroscopy. H7, dark
gray box; HA1004, dotted box.
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Fig. 9.
H7 blocks NGF-mediated induction of a
luciferase reporter gene linked to the zif268
promoter. PC12D cells transfected with pGLzif420, an
expression vector containing a luciferase reporter gene under the
control of the zif268 promoter, were pretreated with
100 µM HA1004 (HA), 1 µM
GF109203x (GF), 30 µM H89, or 100 µM H7 for 30 min prior to stimulation with water
(W) or 5 ng/ml NGF. Cells were harvested 4 h after
stimulation and luciferase assays performed as described under
"Experimental Procedures."
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Fig. 10.
H7 blocks phosphorylation of RNA polymerase
II in vivo. A, PC12D cells were
cultured for 3 h in phosphate-free cellular medium containing 10 µCi/ml [32P]orthophosphate, pretreated with water ( )
or 100 µM H7 (+) for 30 min, and then stimulated with
water (W) or 5 ng/ml NGF for 15 min. Nuclei extracts were
prepared, electrophoresed in a 5% SDS-polyacrylamide gel, and blotted
onto a polyvinylidene difluoride membrane as described under
"Experimental Procedures." Left panel, autoradiogram of
the membrane showing proteins labeled with 32P in
vivo (2-day exposure to x-ray film at
80 °C using an
intensifying screen). Right panel, immunochemically enhanced
chemiluminescence (ECL) detection of RNA polymerase II (pol
II) using the same membrane as that on the left (20-s
exposure at room temperature). Arrows indicate the position
of the 208-kDa molecular mass marker. B, autoradiogram of
nuclear extracts resolved by electrophoresis in 5% SDS-polyacrylamide
gel and blotted onto polyvinylidene difluoride membrane. PC12D cells
were incubated in medium containing [32P]orthophosphate
as described in A and pretreated with water (W),
100 µM H7, 100 µM HA1004 (HA),
200 µM PD098059 (PD), 100 µM H8,
100 µM DRB, or 50 nM staurosporine
(stauro) of 30 min prior to stimulating with water (
) or 5 ng/ml NGF (+) for 15 min. The band corresponding to RNA polymerase II
is indicated. C, immunochemical-ECL detection of RNA
polymerase II. PC12D cells in DMEM were pretreated with water
(0), 50 µM H7, 100 µM H7, or 100 µM HA1004 (HA) for 30 min prior to treatment
with 5 ng/ml NGF or 15 min.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(62)
and the induction of immediate-early genes by interleukin-6 (60,
63-65), interleukin-1
(60, 66),
- and
-interferons (67),
leukemia inhibitory factor (60, 68), and tumor necrosis factor (67). H7
also blocks mRNA induction by trophic/growth factors, including the
induction of c-fos (69) and neuropeptide Y (70) mRNA by
NGF, junB mRNA (71), and platelet-derived growth factor
A and B chain mRNAs (72) by transforming growth factor (TGF)-
,
and c-fos mRNA by growth hormone (73). Additional
examples, include the inhibition by H7 of the induction of
c-fos and c-jun mRNAs by
H2O2 (74) and the induction of metallothionein
mRNA induction by Cd2+ or Zn2+ (75).
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Fig. 11.
Working model, H7 blocks transcription of
c-fos and zif268 mRNAs by
inhibiting phosphorylation of RNA polymerase II. PKC and PKA
function upstream of MAPK, which activate gene expression by
phosphorylating ternary complex factor. Ternary complex factor
stimulates transcription by forming a ternary complex with the
transcription factor serum response factor, which is prebound to the
SRE. There is 1 SRE in the c-fos promoter and 6 SRE in the
zif268 promoter (shown in diagram). H7 blocks
elongation of nascent mRNAs by inhibiting phosphorylation of RNA
polymerase II.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Tatsuya Haga for helpful discussions and critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by Grant-in-Aid for Scientific Research in Priority Areas 07279107, on "Functional Development of Neural Circuits," the Ministry of Education, Science, Sports and Culture of Japan (to D. S.), and by grants from the Japan Society for the Promotion of Science (Research for Future Program), and the Japan Science and Technology Corp. (CREST).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Neurochemistry, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-3-5689-7331; Fax: 81-3-3814-8154; E-mail: kumahara{at}m.u-tokyo.ac.jp.
§ Current address: Laboratory of Molecular Neurobiology, National Institute of Bioscience and Human Technology, 1-1 Higashi Tsukuba-city, Ibaraki 305-8566, Japan.
2 F.-F. Guo, T. Ebihara, and D. Saffen, D., manuscript in preparation.
3 E. Kumahara and D. Saffen, unpublished observations.
4 T. Ebihara and D. Saffen, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
NGF, nerve growth
factor;
PMA, phorbol 12-myristate, 13-acetate;
PKC, protein kinase C;
PKA, protein kinase A;
MAPK, mitogen-activated protein kinase;
SRE, serum response element;
CRE, cAMP response element;
CTD, carboxyl-terminal domain (of RNA polymerase II);
TFIIH, transcription
factor IIH;
H7, [1-(5-isoquinolinesulfonyl)-2-methylpiperazine];
H8, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide;
H89, N-[2-(p-bromocinnamino)ethyl]-5-isoquinolinesulfonamide;
DRB, 5, 6-dicholoro-1--D-ribofuranosylbenzidazole;
MOPS, 4-morpholinepropanesulfonic acid;
HA1004, [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide];
CAT, chloramphenicol acetyltransferase;
DMEM, Dulbecco's modified Eagle's
medium;
MEK, MAP kinase/ERK-activating kinase;
PMSF, phenylmethanesulfonyl fluoride.
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REFERENCES |
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