(Received for publication, July 22, 1996, and in revised form, November 21, 1996)
From the Department of Pharmacology and Physiology, and the Neuroscience Program, University of Rochester Medical Center, Rochester, New York 14642
Membrane depolarization of PC12 cells using 50 mM KCl leads to induction of tyrosine hydroxylase (TH)
mRNA. This induction of TH mRNA is apparently due to increased
TH gene promoter activity mediated by the influx of Ca2+.
In PC12 cells transiently transfected with a chimeric gene expressing chloramphenicol acetyltransferase (CAT) driven by the proximal TH gene
5-flanking region, 50 mM KCl increases TH gene promoter activity 3-4-fold. Promoter analysis utilizing TH-CAT constructs containing mutagenized sequences indicates that this response to the
depolarization-mediated influx of Ca2+ is primarily
dependent on both the TH cAMP-responsive element (CRE) and TH
activating protein-1 (AP1) site. Minimal promoter constructs that
contain a single copy of either the TH CRE or TH AP1 site fused
upstream of the TH gene basal promoter are only modestly responsive or
nonresponsive, respectively, to depolarization. However, both these
constructs are strongly responsive to the calcium ionophore, A23187.
Gel shift assays indicate that TH AP1 complex formation is dramatically
increased after treatment with either 50 mM KCl or A23187.
Using antibodies to transcription factors of the Fos and Jun families,
we show that the nuclear proteins comprising the inducible TH AP1
complex include c-Fos, c-Jun, JunB, and JunD. In cAMP-responsive
element binding protein (CREB)-deficient cell lines that express
antisense RNA complementary to CREB mRNA, the response of the TH
gene promoter to cyclic AMP is dramatically inhibited, but the response
to A23187 remains robust. This result indicates that transcription
factors other than CREB can participate in the
Ca2+-mediated regulation of the TH gene. In summary, our
results support the hypothesis that regulation of the TH gene by
Ca2+ is mediated by mechanisms involving both the TH CRE
and TH AP1 sites and that transcription factors other than or in
addition to CREB participate in this response.
Biosynthesis of the catecholamines is tightly regulated by the activity of the rate-limiting enzyme, tyrosine hydroxylase (TH1; EC 1.14.16.2). Experimental manipulations that lead to long-term stimulation of catecholaminergic cells in sympathetic ganglia and adrenal medulla are associated with increases in TH gene expression (1-8). The mechanisms responsible for regulation of the TH gene are complex and have not been fully elucidated. Cultured rat pheochromocytoma cells have been used extensively to study the underlying mechanisms for TH gene regulation. Using this model system, a number of laboratories have shown that cAMP analogs (9-11), active phorbol esters (12), and glucocorticoids (8-10, 13) induce TH mRNA, stimulate TH gene transcription rate, and/or activate TH gene promoter activity. These results are consistent with the hypothesis that protein kinase A, protein kinase C, and glucocorticoid receptors participate in signaling pathways that regulate the TH gene.
Treatments that lead to increased intracellular Ca2+ concentration ([Ca2+]i) also increase expression of the TH gene. Studies by Sabban and co-workers (14, 15) have shown that membrane depolarization or agonist occupation of nicotinic cholinergic receptors leads to the induction of TH mRNA in PC12 cells. Both of these treatments lead to Ca2+ influx through voltage-sensitive Ca2+ channels. Furthermore, chelation of extracellular Ca2+ with EGTA inhibits the depolarization-mediated increase in TH mRNA (14). These results suggest that the influx of Ca2+ plays an important role in regulating TH gene expression.
A number of studies have been conducted in order to understand the
mechanisms that mediate Ca2+- dependent
regulation of different genes. Ca2+ influx elicited by
either membrane depolarization or Ca2+ ionophore treatment
activates and/or induces transcription factors, such as cAMP-responsive
element binding protein (CREB) and c-Fos, in PC12 cells (16-20). CREB
and c-Fos interact with the promoter elements CRE and AP1,
respectively, to transactivate expression of numerous genes (21-23).
Since the 5-flanking region of the TH gene contains CRE and AP1 sites,
it is reasonable to hypothesize that Ca2+ exerts its
effects on TH gene transcription rate by influencing transcription
factors that bind to these promoter elements. Kilbourne et
al. (24) have shown that a minimal promoter containing the TH CRE
is responsive to Ca2+ influx in PC12 cells. Using deletion
analysis, they have also reported that promoter elements upstream of
the CRE do not apparently participate in the response of the TH gene
promoter to Ca2+. Recently, these results have been
extended using TH gene constructs with site-directed mutations (25).
Results from these studies suggest that the TH AP1 site is not involved
in Ca2+-mediated TH gene regulation. In contrast, deletion
analysis by Stachowiak et al. (26) has shown that the TH CRE
is not required for activation of the TH gene promoter by
Ca2+ influx in bovine adrenal chromaffin cells and that, by
inference, promoter elements upstream of the CRE participate in this
response. However, it is not clear from their study which promoter
elements participate in this regulation.
In the present study, we demonstrate that both the CRE and AP1 sites in
the proximal 5-flanking region of the rat TH gene participate in
regulating TH gene expression by Ca2+ influx in PC12 cells.
The newly identified TH CRE2 site does not apparently play a major role
in this response. To further analyze the involvement of the TH AP1 site
in Ca2+-mediated TH gene regulation, we have examined
nuclear protein interactions at this site. Finally, we have tested the
involvement of CREB in Ca2+-mediated TH gene regulation
using PC12 cell-derived CREB-deficient cell lines. Our results indicate
that CREB is not required for Ca2+- dependent
regulation of the TH gene, supporting the hypothesis that multiple
promoter elements are involved in regulation of the TH gene promoter in
response to Ca2+ influx.
RPMI 1640 medium and penicillin/streptomycin were purchased from Life Technologies, Inc. Fetal bovine and donor horse sera were obtained from Hyclone Laboratories, Inc. (Logan, UT) and JRH Biosciences (Lenexa, KS), respectively. Cell culture flasks and dishes were Falcon or Corning brand. Primary antibodies used in the supershift assays and immunoblots were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) or Oncogene Science (Uniondale, NY). All other chemicals were purchased from Sigma. The calcium ionophore A23187 (stock solution, 10 mM) was dissolved in dimethyl sulfoxide. Liposomes for transient transfections were made by combining 1 mg of dioleoyl-L-phosphatidylethanolamine and 200 µg of dimethyldioctadecyl ammonium bromide in 1 ml of water. The lipids were suspended by sonication using a Branson Sonifier (model W140D) at setting 1.5 for 9 min. [14C]Chloramphenicol was obtained from DuPont NEN.
TH(272/+27)-CAT was constructed as described previously (27). This
construct contains the structural gene encoding chloramphenicol acetyltransferase (CAT) fused downstream of the rat TH gene 5
-flanking region from
272 to +27. TH(
272/+27)-CAT constructs, in which the
CRE (
45/
38; TGACGTCA), AP1 (
205/
199; TGATTCA), or TH CRE2 (
97/
90; AGGCGTGG) core sequences were mutagenized, were isolated using site-directed mutagenesis as described previously (27). The
mutagenized constructs contained the following sequences (capital letters show mutagenized sequences; the normal sequences are given below in lowercase letters; Sequence 1).
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The TH gene minimal promoter constructs, TH(44/+27)-CAT, THCRE-CAT,
and THAP1-CAT, were generously provided by Dr. D. M. Chikaraishi (Duke
University, NC). TH (
44/+27)-CAT (also referred to as
44PL TH CAT
in Ref. 28) contains rat TH gene promoter sequences from
44 to +27
upstream of the CAT gene. THCRE-CAT and THAP1-CAT contain a single copy
of either element fused upstream of the basal TH gene promoter as
described by Fung et al. (28). THCRE2-CAT was constructed as
described in Best et al. (27). RSV-CAT was purchased from
ATCC, and CAT-Basic was purchased from Promega Corp. (Madison, WI).
For gel shift assays, oligonucleotide sequences containing the TH CRE, TH AP1, or mutagenized TH AP1 element were synthesized and purified by denaturing gel electrophoresis. Mixtures of sense and antisense oligonucleotides were heated at 95 °C for 2 min in a buffer containing 67 mM Tris-HCl (pH 7.6), 13 mM MgCl2, 6.7 mM dithiothreitol, 1.3 mM spermidine, and 1.3 mM EDTA. The oligonucleotides were then allowed to anneal by slowly cooling the mixture to room temperature. Double-stranded oligonucleotide containing the somatostatin SP1 element was purchased from Promega (Madison, WI). Sequences of the sense oligonucleotides are shown below (the core TH AP1 sequence is underlined and the mutagenized sequence is capitalized, Sequence 2):
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PC12 cells were cultured in RPMI 1640 medium containing 2.5% fetal bovine serum, 10% heat-inactivated donor horse serum, 50 units/ml penicillin, and 50 µg/ml streptomycin. The cells were maintained in 75-cm2 culture flasks at 37 °C with 95% air and 5% CO2. For experiments, PC12 cells were plated in 100-mm dishes and treated with either control or 50 mM KCl-containing (high K+) medium. The control medium contained 5 mM KCl. The high K+ medium contained reduced NaCl concentration (53 mM) to conserve osmotic strength. Both media were supplemented with 2.5% fetal bovine serum and 10% donor horse serum. In some experiments, PC12 cells were treated with 10 µM calcium ionophore A23187 or 100 µM 8-chlorophenylthio-cAMP (8-CPT-cAMP) in control, serum-containing medium.
Transient Transfection and Treatment of PC12 CellsPC12
cells were transiently transfected by the method of Rose et
al. (29), as modified by Best et al. (27). Briefly,
PC12 cells were cultured in 100-mm dishes at approximately
105 cells/cm2. The cells were incubated in 2 ml
of serum-free RPMI 1640 medium containing 60 µl of liposomes and 10 µg of either wild type or mutagenized TH-CAT construct for 4 h
in an atmosphere of 93% air and 7% CO2. Serum-containing
medium (4 ml) was then added to each dish, and the cells were
maintained in 93% air, 7% CO2. Two to three days after
transfection, the cells were treated for 8 h with either control
medium, high K+ medium, and/or different drugs. The cells
were harvested in ice-cold PBS and stored as cell pellets at
80 °C. Most of the transfection experiment results in the figures,
and tables were derived from at least three experiments using three
different sets of TH-CAT plasmid preparations.
Separate dishes of cells (in triplicate) were transfected with 10 µg/dish RSV-CAT to determine transfection efficiency. Transfection efficiency varied dramatically between experiments but was very reproducible between dishes within each experiment. For example, RSV-CAT expression varied from 86,000 ± 6,000 to 377,000 ± 9,800 cpm/mg protein (n = 3) in different experiments. Separate dishes of cells were also transfected with CAT-Basic (a plasmid containing the CAT gene without a promoter to drive its expression). CAT activity in these cells was used as a blank value. In most experiments blank values were less than 20% of the values obtained from cells transfected with TH-CAT or RSV-CAT. Depolarization did not affect these blank values.
CAT AssayThe cell pellets were resuspended in 60-100 µl of 250 mM Tris-HCl (pH 7.8). The cell suspension was frozen and thawed three times and centrifuged at 12,000 × g for 5 min. The supernatant was heated for 10 min at 65 °C, clarified by centrifugation at 12,000 × g for 5 min, and assayed for CAT activity. Each assay contained 0.8 to 1.0 mg of protein, 480 mM Tris-HCl (pH 7.8), 13.5 µM [14C]chloramphenicol, and 670 µM acetyl-CoA in a total volume of 130 µl. The acetylation reaction was carried out at 37 °C for 12-16 h. Under these conditions, the assay was linear with respect to both protein concentration and time. Acetylated and non-acetylated forms of chloramphenicol were extracted and separated by thin layer chromatography (TLC) as described by Gorman et al. (30). The amount of acetylated chloramphenicol was quantified by scraping the TLC plate and counting the radioactivity using a liquid scintillation counter. TH-CAT activity (expressed as pmol of product formed per h) was divided by the mg of protein present in the original extract (prior to heating) and then normalized to RSV-CAT activity (expressed as pmol of product formed per h/mg of protein) measured in companion dishes in each experiment.
Gel Shift AssaysCells were treated with either control medium, medium containing 50 mM KCl, or control medium containing 10 µM A23187 for 1 h. Nuclear proteins were isolated as described by Best et al. (27). Gel shift assays were performed as described previously (27). Briefly, approximately 0.2 pmol (50,000 cpm) of 32P-radiolabeled TH AP1 oligonucleotide probe was incubated with 10 µg of nuclear protein extract on ice for 20 min in a reaction buffer containing 25 mM HEPES (pH 7.5), 6% glycerol, 0.1 M NaCl, 0.1% Triton X-100, 2 mM EDTA, 0.6 mM spermidine, 0.2 mM spermine, 0.1 mM phenylmethylsulfonyl fluoride, 0.25 µg/ml leupeptin, 0.25 µg/ml pepstatin, 10 µg/ml poly(dI-dC), and 0.4 mM dithiothreitol. The reaction mixture was then incubated at room temperature for 5 min. For competition assays, a 20-fold molar excess amount of unlabeled competitor oligonucleotide was added to the reaction mixture 5 min prior to the addition of the radiolabeled probe. Protein-bound DNA was then electrophoretically separated from the free DNA on a 5% polyacrylamide gel. The gel was dried onto Whatman paper, and the radiolabeled DNA was visualized by autoradiography.
In the supershift assays, 10 µg of PC12 cell nuclear proteins were first incubated on ice for 1 h with 1 µg of a rabbit IgG antibody against either c-Fos, c-Jun, Jun-B, or Jun-D. A separate sample of nuclear proteins was incubated with 1 µg of control rabbit IgG antibody against rat IgG or with buffer in the absence of antibody (0.1% sodium azide, 2.5% gelatin in phosphate buffer (pH 7.5)) as controls. The entire antibody/protein mixture was then incubated with radiolabeled TH AP1 probe and processed for the gel shift assay as described above.
Construction of CREB-deficient Cell LinesAntisense CREB
RNA expression vectors, CREB-AS.6 and CREB-AS.3, were constructed in
pBK-RSV (Stratagene, La Jolla, CA). Expression of the antisense CREB
RNAs was under control of the RSV promoter and contained base pairs
1-620 and 1-320, respectively, of rat CREB cDNA in the antisense
orientation. The NIH BLAST program was used to compare CREB cDNA
sequences with other transcription factor sequences. The sequences in
CREB-AS.3 possess weak homology (~40%) to CREM and no significant
homology to ATF1 or any other transcription factor listed in GenBank.
Sequences 321-620 are 68% homologous to CREM but not significantly
homologous to ATF1 or any other listed factor. PC12 cells were
transfected with either CREB-AS.6 or CREB-AS.3, and stably transfected
clonal cell lines (ASCR.6-2 and ASCR.3-6, respectively) were isolated
using G418 selection followed by limiting dilution. Control cell lines
were constructed by stably transfecting PC12 cells with the null
vector, pBK-RSV. These clonal cell lines were then transiently
transfected with TH(272/+27)-CAT and treated with appropriate drugs
as described above.
Nuclear proteins (30 µg) were subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose as described previously (8). The presence of CREB or phospho-CREB protein was detected using a modified protocol of that provided by Upstate Biotechnology Inc., which was the commercial source of the antibodies. The nitrocellulose was first treated with 5% dry milk in PBS for 1 h with gentle shaking at room temperature. The membrane was then incubated with a 1:10,000 or 1:5000 dilution of primary antibody in 3% dry milk in PBS for 1 h at room temperature and rinsed with PBS once for 15 min and twice for 5 min with fresh changes of PBS. The dilution of CREB antibody was adjusted for two lots of antibody used in these experiments. The horseradish peroxidase-linked secondary antibody (purchased from Bio-Rad) was diluted 1:3000 in 3% dry milk in PBS. The blot was incubated with the secondary antibody for 30 min at room temperature, washed one time for 15 min, and four times for 5 min with PBS. Immunolabeled proteins were visualized using a chemiluminescent detection protocol provided with the enhanced chemiluminescence detection kit from Amersham Corp. Densitometric analysis of the autoradiograms obtained from these immunoblots was performed using a Hewlett-Packard ScanJet 4C scanner with a transparency adaptor and NIH Image software.
Statistical AnalysesResults were analyzed by one-way analysis of variance followed by Student-Newman-Keuls or Dunnett multiple comparisons tests, using the computer program INSTAT. Data are presented as means ± standard error (S.E.). A p value less than 0.05 was considered statistically significant.
TH gene promoter activity was measured using a
construct containing TH gene sequences from 272 to +27 fused upstream
of the CAT reporter gene (TH-CAT construct). This region of the TH gene contains promoter elements including CRE, AP1, and TH CRE2. In order to
evaluate the involvement of each of these promoter elements in response
to Ca2+ influx elicited by depolarization, PC12 cells were
transiently transfected with TH-CAT constructs containing either the
wild type TH gene promoter or the same TH gene promoter region with a
mutagenized CRE, AP1, or TH CRE2 site. High K+ treatment
increased the expression of wild-type TH-CAT by approximately 4-fold
(Table I). Mutagenesis of 6 out of the 8 bases that
comprise the core TH CRE lowered basal TH-CAT expression by ~70% and
inhibited the depolarization-mediated increase in TH-CAT expression by
~80%. However, high K+ treatment still elicited a
modest, but significant, increase in TH-CAT expression when the CRE was
mutagenized. This residual induction of TH-CAT suggested that other
elements might participate in the response to Ca2+ influx.
The TH AP1 and TH CRE2 are two other sites within the TH gene that are
responsive to different stimuli (27, 31). Mutagenesis of 2 out of the 7 bases of the core TH AP1 site lowered basal TH-CAT expression (by
~60%) and inhibited the high K+-evoked activation of the
TH gene promoter by ~80%. Mutagenesis of the TH CRE2 site was also
associated with a modest but significant inhibition of the
depolarization-mediated stimulation of the TH gene promoter (by
~30%). In contrast, mutagenesis of the THE2 site (see Ref. 27),
which is downstream and adjacent to the TH CRE2 site in the TH gene
promoter, did not affect the induction of TH-CAT expression in cells
treated with 50 mM K+. These results suggest
that the CRE and AP1 sites within the TH gene promoter participate
significantly in the response of the gene to membrane depolarization
and the consequent influx of Ca2+. The TH CRE2 site plays
only a minor role, if any, in this response.
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In order to test whether each individual promoter
element is independently responsive to Ca2+, we examined
the response of each element in minimal promoter constructs (Table
II). PC12 cells transfected with the minimal TH gene
basal promoter construct, TH(44/+27)-CAT, did not show depolarization-mediated induction of TH-CAT expression, indicating that
the minimal promoter by itself was insufficient to enhance transcription in response to high K+. When cells were
transfected with THCRE-CAT, high K+ treatment significantly
increased CAT expression by ~3-fold. Interestingly, high
K+ treatment did not increase CAT expression in cells
transfected with either THAP1-CAT or THCRE2-CAT. These results,
particularly those using the THAP1-CAT construct, apparently contradict
the mutagenesis data presented in Table I. However, it is possible that
the high K+-mediated increase in
[Ca2+]i was not a strong enough signal to enhance
transcription dramatically from these minimal promoter constructs. In
order to test this hypothesis, Ca2+ influx was amplified by
treatment of cells with 10 µM A23187. This calcium
ionophore increased [Ca2+]i to more than 3 µM when measured using the FURA-2 Ca2+
indicator (data not shown). Treatment with A23187 did not significantly increase CAT expression from the basal promoter. However, A23187 treatment dramatically induced TH-CAT expression driven by a single copy of either the TH CRE or TH AP1 site (Table II). In contrast, THCRE2-CAT did not respond significantly to Ca2+ influx
elicited by A23187. For comparison purposes, 8-CPT-cAMP (0.1 mM) induced THCRE-CAT expression 15-fold and THAP1-CAT
expression 24-fold in PC12 cells (data not shown). THCRE2-CAT
expression was induced approximately 3-fold by 8-CPT-cAMP in PC12 cells
(27). These results suggest that both TH CRE and TH AP1 sites are
independently responsive to Ca2+ influx but that the TH CRE
requires less Ca2+ influx to respond independently than the
TH AP1 site. The TH CRE2 is not capable of independently responding to
Ca2+ influx.
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The involvement of the CRE in Ca2+-mediated TH
gene regulation has been previously demonstrated (24, 25). However, the
present study is the first to demonstrate directly the involvement of the TH AP1 site in response to Ca2+ influx. To confirm
further the role of the TH AP1 in this regulation, nuclear protein
interactions with the TH AP1 element were examined using gel shift
assays. Nuclear protein extracts from control PC12 cells contained
proteins that interacted with the TH AP1 probe to form a number of
protein-DNA complexes (Fig. 1). When the cells were
treated with depolarizing medium for 1 h, the formation of the
slowest migrating complex dramatically increased (see arrow in Fig. 1). Treatment of the cells with A23187 also dramatically increased the formation of this TH AP1 complex (Fig. 2).
The proteins comprising this inducible complex were effectively
competed away with a 20-fold molar excess of unlabeled TH AP1 probe but
not by 20-fold molar excess of unlabeled TH mAP1 probe or SP1 probe (Fig. 1). Similar competition displacement results were obtained when
nuclear proteins from A23187-treated cells were used (data not shown).
These results suggest that Ca2+ influx into PC12 cells
augments specific nuclear protein interaction at the TH AP1 site, which
is consistent with the TH AP1 site participating in the
Ca2+-mediated regulation of the gene. Furthermore, the
competition assay results indicate that the mutagenized AP1 sequence
within the TH(mAP1)-CAT construct did not bind to proteins that
comprise the Ca2+-inducible TH AP1 complex.
Identification of Nuclear Proteins That Comprise the Ca2+-inducible TH AP1 Complex
Nuclear proteins that increase their interaction with the TH AP1 site upon Ca2+ influx were identified using specific antibodies against the AP1 family of transcription factors. These transcription factors included c-Fos, c-Jun, JunB, and JunD. The rabbit IgG antibodies raised against these factors recognized their non-DNA binding domains (Santa Cruz Biotechnology, Inc). In this set of experiments, nuclear protein extracts from A23187-treated PC12 cells were incubated with one of these antibodies prior to the gel shift assay. In some experiments a slight decrease in the intensity of the Ca2+-inducible complex band was observed when nonspecific rabbit IgG was used as a control, indicating a nonspecific interaction of rabbit IgG and the nuclear proteins within the complex (experiment in Fig. 2A but not in Fig. 2B). Incubation of nuclear proteins with each specific antibody decreased the intensity and supershifted the band corresponding to the inducible TH AP1 complex (Fig. 2, A and B). No supershifted bands were observed when nuclear proteins were incubated with the control rabbit IgG antibody. As a further check on the specificity of the antibodies, the analogous supershift assays using the same nuclear protein extracts were performed using the TH CRE oligonucleotide as a probe (Fig. 2C). As expected, A23187 treatment did not increase binding to the TH CRE. The control antibody decreased TH CRE binding slightly; however, none of the antibodies targeting the Fos or Jun proteins diminished TH CRE binding to a greater extent than that observed with the nonspecific control antibody nor were any supershifted bands observed when the TH CRE was used as the probe. Similar results were observed when nuclear proteins from cells treated with 50 mM KCl were used. These results demonstrate that proteins comprising the Ca2+-inducible TH AP1 complex include c-Fos, c-Jun, JunB, and JunD and that these factors do not bind to the TH CRE.
Ca2+-mediated TH Gene Promoter Activation in a CREB-deficient Cell LineThe role of the CRE in
Ca2+-mediated TH gene promoter activation was further
evaluated by examining the participation of the CRE binding protein,
CREB, in this response. The ability of Ca2+ to increase TH
gene promoter activity was examined in CREB-deficient cell lines
derived from PC12 cells. These CREB-deficient cell lines were generated
by stably transfecting PC12 cells with one of two different
antisense-CREB RNA expression vectors to diminish the expression of
CREB in these cells. Both antisense CREB RNA constructs targeted the 5
region of CREB mRNA and did not include sequences encoding the
basic region-leucine zipper domain. A control cell line, CN9, was
generated by stably transfecting PC12 cells with the pBK-RSV plasmid
without the antisense CREB RNA insert. The level of CREB protein in the
CN9 cells was similar to that found in wild type PC12 cells (data not
shown). Both cell lines that were stably transfected with antisense
CREB constructs showed marked decreases in a 43-kDa band which
presumably represents CREB protein (Fig. 3). This band
was the only one recognized by the lot of CREB antiserum used in Fig.
3B. However, one lot of CREB antiserum yielded a number of
nonspecific bands (seen in Fig. 3A). One of these bands
migrated as a 43-kDa protein, which is the molecular size of CREB. To
verify that this band represented CREB protein, we measured
phospho-CREB using a different antibody in wild type PC12 cells and CN9
cells (the data from the wild type PC12 cells is shown in Fig.
3A). The phospho-CREB antibody also recognized a 43-kDa
band, and the intensity of this band increased when using nuclear
extracts isolated from forskolin-treated PC12 cells (Fig.
3A, lanes 3 and 4). This result
indicated that phosphorylation of CREB increased after forskolin
treatment, as would be expected. The phospho-CREB antiserum also
recognized two other bands that we did not identify; however, the
intensities of these bands did not increase with forskolin treatment.
Hence, the evidence suggests that the 43-kDa band is CREB and that CREB is dramatically diminished in the ASCR.6-2 cells (Fig. 3A),
as well as in the ASCR.3-6 cells (Fig. 3B). Densitometric
analysis of these Western blots indicated that CREB protein was
diminished by 80-90% in ASCR.6-2 cells and 70-75% in ASCR.3-6
cells.
Even though we have not yet performed exhaustive analysis of the changes that occur in other transcription factors in these CREB-deficient cell lines, we have compared a number of pertinent parameters in the ASCREB and CN9 cell lines. 1) The basal activity of protein kinase A is essentially identical in ASCREB and control cell lines, and protein kinase A is activated by forskolin in both control and ASCREB cell lines. 2) In both ASCREB cell lines, ATF1 and CREM levels are similar to those observed in control or wild type PC12 cells. 3) c-Fos is induced by Ca2+ influx in both ASCREB and control cell lines. This latter result is not surprising, since Ca2+ induces c-Fos via phosphorylation of the serum response factor, as well as via phosphorylation of CREB (34).
Using these CREB-deficient PC12 cell lines, TH-CAT expression was examined in response to Ca2+ to determine whether the presence of CREB was required for Ca2+-mediated TH gene promoter regulation. The CREB-deficient cells were transiently transfected with wild type TH-CAT and treated with either 0.1 mM 8-CPT-cAMP or 10 µM A23187 (Table III). As in the wild type PC12 cells, TH-CAT expression was increased by 8-CPT-cAMP (~10-fold) or A23187 (~5-fold) in the control CN9 cell line. The induction of TH-CAT expression by 8-CPT-cAMP was markedly attenuated in both CREB-deficient cell lines; in ASCR.6-2 cells, the cAMP-mediated induction of TH-CAT was completely inhibited. Most significantly, the decrease in CREB levels did not affect the ability of A23187 to increase TH-CAT expression, suggesting that CREB is not required for Ca2+-mediated TH gene promoter activation. This result strongly suggests that transcription factors other than CREB can participate in the induction of TH gene promoter activity in response to Ca2+ influx.
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Membrane depolarization of excitable cells and the consequent influx of Ca2+ leads to modulation of a number of intracellular events, including changes in transcription rates of numerous genes. Mechanisms responsible for this gene regulation have been extensively studied, particularly with respect to the c-fos gene (19, 20, 32-35). These studies have identified a Ca2+-responsive element (CaRE) within the c-fos gene, which is strikingly similar to the consensus CRE. In addition, the increase in intracellular Ca2+ leads to activation of CREB by phosphorylation of its Ser-133 site (20, 36). Therefore, it has been postulated that Ca2+ influx evoked by membrane depolarization increases c-fos gene expression via the CRE/CaRE following the phosphorylation of CREB. However, the critical importance of the CRE/CaRE and CREB activation in c-fos gene regulation is unclear, because 1) CREB phosphorylation by itself is not sufficient to increase c-fos gene expression in response to Ca2+ influx in protein kinase A-deficient cell lines (35), and 2) c-fos gene sequences upstream of the CRE/CaRE also participate in the response to Ca2+ (34, 37).
Depolarizing stimuli also induce TH mRNA and stimulate TH gene
promoter activity in PC12 cells (14, 24) and in cultured bovine adrenal
medullary cells (26). Kilbourne et al. (24) have used
deletion mutation analysis to show that the region from 60 to +27,
which contains the consensus CRE core sequence, is as responsive to
veratridine as the region from
773 to +27 in PC12 cells. The
construct containing TH gene sequences from
41 to +27 was
unresponsive. These authors have interpreted these data to support the
participation of the TH CRE (at position
45 to
38) in this
veratridine-mediated response. In addition, a construct, in which
sequences from
209 to
193 (a region referred to as the FSE) are
deleted from TH(
272/+27)-CAT, does not lose responsiveness to
veratridine in PC12 cells. Since the TH AP1 site is found within this
FSE region, it was concluded that this site does not participate in the
response. However, other elements, such as AP2 (
220 to
213),
E2A/MyoD (
194 to
189), and POU/Oct (
175 to
168), that may
participate positively or negatively in regulating the TH gene are
located adjacent to this region. The deletion of this FSE domain alters
the normal spacing between these elements. In contrast, Stachowiak
et al. (26) have used deletion analysis of the bovine TH
gene promoter to show that regions of the promoter upstream of the CRE
are required for the response to veratridine in bovine adrenal
chromaffin cells. These regions contain numerous candidate elements,
including POU/Oct, SP1, and AP1 sites. From these previous deletion
analyses, it is difficult to identify specific promoter elements
involved in the response to depolarization, due to the deletion of
large segments of the TH gene promoter in the TH-CAT constructs.
Furthermore, these deletions alter the normal spacing of elements
within the TH gene promoter and eliminate potential interactions
between proximal and distal elements.
In the present study, we have used a rat TH gene promoter construct similar to that used by Kilbourne et al. (24); however, we have employed site-directed mutagenesis to knock out the function of specific sites within the promoter without altering the length of the promoter region. Using this approach, our results suggest that at least two promoter elements participate in the response to Ca2+ influx; these two elements are the CRE and AP1. This conclusion is strongly supported by the 70-80% decrease in response to depolarization, when either the CRE or AP1 site is mutagenized within the context of the intact TH gene promoter.
The participation of the TH CRE is also supported by the results of the minimal promoter studies. Treatment of PC12 cells with 50 mM KCl leads to a significant increase in the expression of the minimal promoter construct, THCRE-CAT. This response is modest (about 3-fold) compared with that observed when the cells are treated with a cyclic AMP analog (approximately 15-fold). However, this modest response is perhaps due to the limiting amount of Ca2+ influx that occurs during depolarization with 50 mM KCl, because treatment with 10 µM A23187 (which raises [Ca2+]i to levels more than 3 µM) is associated with more than a 10-fold increase in THCRE-CAT expression.
Recently, Nankova et al. (25) have reported a similar study
investigating Ca2+-responsive elements within the TH gene
promoter using TH-CAT constructs with a mutagenized CRE or AP1 site.
Their results with the mutagenized CRE construct agree with our data,
indicating that the CRE site plays a significant role in
Ca2+-mediated regulation of the TH gene. In contrast to our
findings, these investigators do not observe a significant decrease in
Ca2+-induced TH-CAT expression when the TH AP1 site is
mutagenized. One explanation for this discrepancy may be differences in
the number of mutagenized nucleotides within the element. In the
mutagenized AP1 construct used in our study, only the first two base
pairs of the core AP1 element (TGATTCA) are modified (to GAATTCA). This modification was chosen to minimize its influence on factors binding to
the dyad symmetry element that overlaps the 3 region of the core TH
AP1 site (38) or other elements found within this region, such as the
FSE (39) or HIE (40). The competitive displacement gel shift assays
indicate that this 2-bp mutation is sufficient to inhibit the binding
of Ca2+-inducible AP1 factors to the mutagenized site (Fig.
1). Furthermore, this 2-bp mutation produces a dramatic decrease in
basal promoter activity, which is in agreement with previous studies
indicating that the AP1 site or adjacent, overlapping sites participate
in controlling basal expression of the gene in rat pheochromocytoma cells. In contrast, the entire core TH AP1 sequence is mutagenized in
the construct used by Nankova et al. (25) (mutant 2 in Ref. 38). Since a number of regulatory sequences have been mapped to this
region of the gene, it is possible that these different mutations alter
the response to Ca2+ due to changes in the binding of
activating or repressing proteins that interact with these adjacent
sites.
An alternative explanation is that the discrepancy is due to differences in the route of Ca2+ influx in the two experiments. Membrane depolarization using high K+ permits Ca2+ influx into PC12 cells via L-type voltage-sensitive calcium channels.2 Nankova et al. (25) used ionomycin to promote Ca2+ influx. Hence, the route of Ca2+ entry differs between these two studies. It is possible that CRE and AP1 transcription factors are modulated differently depending upon the route of Ca2+ influx. Support for this hypothesis has been demonstrated for the c-fos gene (41).
In order to confirm that the TH AP1 site participates in the response of the promoter to Ca2+ influx, we have performed a number of subsequent studies. Even though the minimal promoter construct, THAP1-CAT, does not respond significantly to 50 mM KCl, a slight, but statistically insignificant, response was observed in each experiment. This lack of significant response is apparently due to either the limited influx of Ca2+ elicited by high K+ treatment or to differences in the route of Ca2+ entry, because treatment of the cells with A23187 elicited a 7-8-fold increase in THAP1-CAT expression. This ability of a single copy of TH AP1 to increase CAT expression upon Ca2+ influx provides evidence that the TH AP1 functions as a Ca2+-responsive element at least under certain stimulus conditions. However, the results using A23187 need to be interpreted with caution, because this ionophore may be producing effects that are not specific to Ca2+ influx.
The gel shift assays demonstrate that Ca2+ influx elicited by either depolarization or treatment with A23187 increases the formation of a TH AP1 complex. These results also strongly support the participation of the TH AP1 site in the response to Ca2+ influx. However, it is also possible that unidentified adjacent sites bind to PC12 cell nuclear proteins to form the observed complex. Hence, we used antibodies targeting specific transcription factors to identify the Ca2+-inducible nuclear proteins that bind to the TH AP1 site. These studies have identified c-Fos, c-Jun, JunB, and JunD as members of the inducible TH AP1 complex. Hence, the increased formation of this complex upon Ca2+ influx is due to the increased binding of Fos and Jun family transcription factors to this site, supporting the role of the TH AP1 site in this response.
The TH CRE2 is a newly discovered site within the TH gene promoter that is homologous to the proenkephalin gene ENKCRE1 and that is responsive to cyclic AMP in PC12 cells (27). The present results suggest that this site does not play a significant role in the response to Ca2+. This conclusion is based on the very modest decrease in response to high K+, when the TH CRE2 is mutagenized within the normal context of the TH gene promoter and the lack of response of the THCRE2 in the minimal promoter studies to either 50 mM KCl or A23187.
A current prevailing hypothesis is that Ca2+ influx stimulates genes by activating a calcium/calmodulin- dependent protein kinase, which then phosphorylates and activates CREB, leading to the transactivation of genes downstream of CRE sites. However, our results using the CREB-deficient cell lines suggest that CREB is not necessary for Ca2+-mediated regulation of the TH gene promoter. These results do not rule out CREB as a participant in the response to Ca2+ influx in wild type cells but suggest that other transcription factors can compensate when CREB levels are diminished. In this regard Greenberg and co-workers (42) have shown that the c-fos gene CRE is responsive to Ca2+ via phosphorylation of ATF1. It is also possible that transactivating forms of the CREM gene family may participate in this response (see Ref. 43 for review). Both of these factors are still present in the ASCREB cell lines.2 Furthermore, when CREB levels are lowered, regulation via the TH AP1 site may predominate. Further work is needed to elucidate these compensatory mechanisms in the CREB-deficient cell lines.
We thank Dr. Dona Chikaraishi for the kind
gift of the minimal promoter constructs, TH(44/+27)-CAT, THCRE-CAT,
and THAP1-CAT, and we acknowledge the excellent technical assistance of
Carol R. Sterling.