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Cell and Molecular Biology Program and the Department of Pharmacological and Physiological Science, St. Louis University School of Medicine, St. Louis, Missouri 63104
Address all correspondence and requests for reprints to: Dr. Nicola C. Partridge, Department of Pharmacological and Physiological Science, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, Missouri 63104. E-mail: partrinc{at}slu.edu
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Introduction |
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The PTH receptor is found only on osteoblasts in bone and is a seven-transmembrane, G protein-coupled receptor. When bound by PTH, the receptor indirectly activates adenylyl cyclase to increase cAMP levels and increases levels of diacylglycerol and intracellular calcium through the activation of phospholipase C. Previous work in our laboratory has determined that the effect of PTH on c-fos expression is mediated predominantly by the actions of cAMP acting through the major cAMP response element (CRE) within the c-fos promoter (1, 14). In addition, we have previously demonstrated that the major CRE is bound by the CRE-binding protein (CREB) and is phosphorylated at serine 133 (pS133) in response to PTH treatment (14). Others have shown previously that this phosphorylation event is required for the ability of CREB to activate transcription (15, 16), and that many enzymes are able to phosphorylate CREB at S133, including protein kinase A (PKA) and protein kinase C (PKC) (16), calcium/calmodulin-dependent kinases (CaMK) II and IV (17, 18), and members of the ribosomal S6 kinase family, p70S6K, p90RSK, and RSK2 (19, 20). Although evidence presented by Clohisy et al. (1) suggests against roles for PKC and calcium in mediating the effects of PTH on c-fos expression, this evidence is limited to the effect of down-regulation by prolonged treatment with phorbol ester and the lack of effect of the calcium ionophore ionomycin. It is still possible, however, that a phorbol ester-insensitive PKC isoform is involved with or without an increase in intracellular calcium levels. As PTH can potentially activate many different kinases that have been shown to phosphorylate CREB at S133, any one of these enzymes may be responsible for this event in response to PTH. In this study we provide strong evidence that PKA is the enzyme responsible for phosphorylating CREB at S133 and that PKA activity is required for the PTH-mediated induction of c-fos transcription.
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Materials and Methods |
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Plasmid construction
The cDNA of human PKI was amplified by PCR from the clone
obtained from American Type Culture Collection (see
Antibodies and reagents) using the following
oligonucleotides as primers: sense, 5'-GAATTCGTGGATATTTGGTAGCAATGAC;
and antisense, 5'-TCTAGATGGTCGAGGGTCAAAGT. It was subcloned into the
eukaryotic expression vector pcDNA3 (Invitrogen, Carlsbad,
CA). The correct sequence of PKI was verified.
Cell culture and lysate preparation
UMR 106–01 (UMR) cells were cultured in 100-mm dishes or
six-well plates (35-mm diameter wells) as previously described (22) in
MEM containing penicillin/streptomycin, nonessential amino acids, and
5% FBS and were used between passages 17–23. Lysates for
immunoblotting were prepared by first washing cells with wash buffer
[50 mM Tris-HCl (pH 7.4), 10 mM EDTA, 10
mM sodium pyrophosphate, and 5 mM EGTA]. Cells
were then scraped into 1 mL wash buffer/100-mm dish and centrifuged at
14,000 x g for 30 sec. The supernates were removed,
and cell pellets were resuspended in 100 µl wash buffer. Resuspended
cells were then boiled for 10 min and centrifuged at 14,000 x
g for 2 min. Supernates were transferred to new tubes, and
60 µl of 3 x SDS sample buffer (1 x SDS sample buffer is
50 mM Tris, 2 mM ß-mercaptoethanol, 20%
glycerol, 6% SDS, and 0.08% bromphenol blue) were added to the
samples to be used for immunoblotting.
Immunoblotting
Equal amounts of protein were separated by 12% SDS-PAGE at 200
V for 45 min and electrotransferred to polyvinylidene difluoride
membrane (Bio-Rad Laboratories, Inc., Hercules, CA) in
transfer buffer (20% methanol, 192 mM glycine, and 56
mM Tris) at 100 V for 1 h. The membranes were blocked
with 5% nonfat dry milk in 1 x Tris-buffered saline (pH 7.4 at
room temperature) containing 0.1% Tween-20 (TBST). Antibodies were
diluted 1:1000 in 1% nonfat dry milk in TBST and incubated on
membranes for 1 h at room temperature. Horseradish
peroxidase-conjugated goat antirabbit IgG was used at a 1:10,000
dilution for 30 min at room temperature to detect the primary
antibodies. Detection was performed using the enhanced
chemiluminescence (ECL) kit and Hyperfilm ECL from Amersham (Arlington
Heights, IL). Immunoblotting was first performed using the anti-pCREB
antibody with lengths of exposure usually from 10–20 min. Membrane was
reused directly (without stripping) for use with anti-CREB antibody
with exposure times of approximately 20 sec to 1 min. At these times,
no reactivity due to anti-pCREB antibody was detectable.
Transfections
Cells were seeded at approximately 105 cells/well of
a six-well plate and allowed to attach overnight at 37 C in 5%
CO2. The next day, cells were washed once with MEM without
serum. Transfections were performed using Lipofectamine reagent
(Life Technologies, Gaithersburg, MD) according to the
manufacturer’s protocol. Each single well transfection contained 500
ng Qiagen-purified plasmid DNA (Qiagen, Chatsworth, CA) and 5 µl
Lipofectamine reagent in 750 µl serum-free MEM.
Northern blotting
Total RNA was isolated using Tri-Reagent (Sigma Chemical Co., St. Louis, MO) according to the manufacturer’s protocol.
Twenty micrograms of total RNA per sample were separated on a 1%
agarose gel containing 8% formaldehyde and transferred to positively
charged nylon membranes (Micron Separations, Inc., Westborough, MA)
using 10 x SSC. Membranes were dried, and RNA was cross-linked to
the membrane using UV light in a Stratalinker (Stratagene,
La Jolla, CA). The membranes were prehybridized using RapidHyb solution
(Amersham, Arlington Heights, IL) at 65 C for 1 h. Hybridization
was performed at 65 C for 2 h with 106 cpm
specifically labeled probe/mL RapidHyb solution. The probes used were
the 1-kb PstI fragment of v-fos and the
full-length chicken ß-actin cDNA and were prepared using the
oligolabeling kit (Pharmacia Biotech, Piscataway, NJ)
according to the manufacturer’s instructions.
CAT assays and luciferase assays
CAT assays were performed as described previously (14) with
slight modification. Briefly, cells were lysed with 125 µl Reporter
Lysis Buffer (Promega Corp., Madison, WI)/well of a
six-well plate, scraped, and transferred into 1.5-mL microcentrifuge
tubes. Cell debris was pelleted by centrifugation at 14,000 x
g. Fifty microliters of the cell supernatant fraction were
mixed with CAT assay buffer [250 mM Tris-HCl (pH 7.4),
n-butyryl coenzyme A, and
[14C]chloramphenicol] and incubated at 37 C for 1
h. Reactions were stopped by adding 200 µl mixed xylenes and
vortexing for 10 sec. Phases were separated by microcentrifugation at
14,000 x g for 5 min, and 180 µl organic phase were
back extracted with 100 µl Tris-HCl, pH 7.4. Samples were centrifuged
at 14,000 x g for 5 min, and 157 µl of the organic
phase were analyzed by scintillation counting.
Statistical analysis
Data represented with bar graphs are representative
experiments performed in triplicate, with points
representing the means and bars indicating the
SEMs. To determine differences among the samples, one-way
ANOVA with a post-hoc analysis of multiple comparison using
the pairwise Student-Newman-Keuls method was performed. Analysis was
performed using SigmaStat 2.0 for Windows (SPSS, Inc.,
Chicago, IL). All other data shown are representative of at least three
separate experiments with similar results.
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Results |
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). At 5 min, the level of
phosphorylation was easily detectable, and phosphorylation was maximal
by 20 min. S133 remained phosphorylated for more than 4 h
(our unpublished observation). The length of time of PTH
treatment that corresponds to maximal pS133 correlates with the time
required for PKA to become maximally activated and translocate to the
nucleus (23, 24). Bands reactive with the anti-pCREB antibody that
migrate faster than CREB are probably CREB family members that have
homologous sequences in the region of phosphorylation (25). The bands
labeled with the arrow were determined to be CREB
by the ability of the anti-CREB antibody to react with the identical
regions on the same membrane, and as recombinant rat CREB
demonstrates the identical mobility on SDS-PAGE.
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). When UMR cells were pretreated with
the phosphodiesterase inhibitor isobutylmethylxanthine for 20 min
before PTH treatment, the minimal dose at which pS133 was detectable
was lowered to 10-12 M (Fig. 2B), whereas
maximal phosphorylation occurred at 10-9 M
PTH, strongly implicating a cyclic nucleotide second messenger cascade
leading to CREB phosphorylation after PTH stimulation.
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). Similarly, only PTH-(1–34) and
-(1–31) can increase c-fos mRNA levels (Fig. 3B). As PTH
peptides containing the first two amino acids, which are required to
activate adenylyl cyclase, are the only peptide fragments that can
cause CREB phosphorylation and c-fos expression, this
strongly implicates the cAMP pathway in this process. Furthermore, as
none of the other PTH peptide fragments could cause CREB to be
phosphorylated at S133 or cause the increase in c-fos mRNA
levels, these activities cannot be mediated by either a rise in
intracellular calcium or activation of PKC. The fact that PTH-(1–31),
which does not activate PKC, is able to cause CREB phosphorylation and
increase c-fos mRNA levels strongly suggests that PKC is not
involved in mediating these events in PTH action on the osteoblast.
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); 20
µM PKCi also had no effect on these activities, whereas
chelerythrine chloride actually increased the phosphorylation of CREB
at S133 and only slightly inhibited PTH-induced c-fos
expression. Only H-89, a specific inhibitor of PKA, inhibited both the
PTH-induced phosphorylation of CREB at S133 and c-fos
expression. To further assess the efficacy of H-89 inhibition of
PTH-mediated CREB phosphorylation and increased c-fos
expression, various doses of H-89 were added to UMR cells before PTH
treatment. Increasing concentrations of H-89 demonstrated increasing
potencies for inhibiting CREB phosphorylation at S133 as well as
c-fos expression (Fig. 5). To
further assess the specific role of PKA on the activation of the
c-fos promoter by PTH, experiments were performed in which
increasing amounts of the heat-stable inhibitor of PKA (PKI) were
cotransfected with a c-fos promoter reporter construct
(-151fosCAT). When 100 ng or more of the PKI expression
plasmid were cotransfected with the reporter plasmid, there was
complete inhibition of the PTH-induced promoter activity (Fig. 6).
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Discussion |
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A previous report of ours demonstrated that comparable c-fos
expression could be mimicked by the cAMP analog 8-bromo-cAMP and was
independent of PKC activity (1). Contrary to the findings of Clohisy
et al. (1), Kano et al. (31) demonstrated a much
reduced expression of c-fos when the cAMP analogs
(Bu)2cAMP and adenosine 3',5'-cyclic monophosphorothioate,
Sp-isomer were employed. In addition, this group showed that the
PKC inhibitor H-7 dramatically reduced the expression of
c-fos, suggesting a role for PKC in the PTH-induced
expression of c-fos in UMR cells. The differences in these
two reports probably arise from the variability of the activities of
8-bromo-cAMP vs. (Bu)2cAMP and adenosine
3',5'-cyclic monophosphorothioate, Sp-isomer on isoforms of PKA
and the possibility that H-7 has effects on PKA as well as PKC. The
results reported by Clohisy et al. (1) demonstrating that
the inability of PKC to mediate the PTH activation of c-fos
expression is limited to the phorbol ester-sensitive PKC isoforms,
leaves open the possibility that PKC
or PKC
, the phorbol
ester-insensitive isoforms, can mediate this response. The evidence put
forward in the present work is in agreement with the previous data
suggesting no role for PKC in mediating PTH effects on c-fos
expression. First, PTH peptide fragments that do not activate adenylyl
cyclase but retain the ability to activate PKC [PTH-(3–34),
-(13–34), and -(28–48)] cannot cause the phosphorylation at S133 or
induce c-fos mRNA levels. In addition, the phorbol
ester-insensitive PKC isoforms (PKC and PKC) are still sensitive
to the PKC inhibitors chelerythrine chloride (34) and PKCi (35, 36),
and these agents do not inhibit the PTH-induced phosphorylation of CREB
at S133 or the increase in c-fos mRNA.
There have been many reports that Ca2+/CaMK can phosphorylate CREB at S133 (17, 18, 37, 38, 39). Several lines of evidence go against a role of calcium/calmodulin-dependent kinases mediating the PTH-induced phosphorylation of CREB at S133 and the increase in c-fos expression. First, the PTH peptide fragment containing amino acids 3–34, which can still increase intracellular calcium levels (28), cannot mediate these events. Also, when EGTA is used to chelate extracellular calcium before PTH treatment, no inhibition of the PTH effect on CREB phosphorylation or c-fos expression is detected (our unpublished observation). Furthermore, the calmodulin antagonist calmidazolium and the CaMK-specific inhibitor KN-93 were each unable to inhibit the PTH-induced responses.
In support of the hypothesis that PKA is mediating the effects of PTH, the doses of PTH that induce CREB S133 phosphorylation correlate with the doses required to activate PKA (23, 24). The times of PTH treatment also correspond to PKA activation. Specifically, the length of time between PTH treatment and detection of maximal levels of CREB pS133 corresponds to the time required for maximal activation of PKA and translocation to the nucleus, and the time required for maximal phosphorylation of CREB immediately preceded the time of maximal c-fos expression (1). In addition, the first two amino acids of PTH that are required for the activation of adenylyl cyclase are absolutely required for the phosphorylation of CREB at S133 as well as the activation of c-fos expression. Lastly, specific inhibitors of PKA can inhibit the ability of PTH to phosphorylate CREB at S133, to increase c-fos expression, and to increase the CRE-dependent increase in c-fos promoter activity in a dose-dependent manner.
Several other enzymes appear to be able to phosphorylate CREB at S133 in various systems (16, 19, 20). Although this study did not specifically examine the possibility that these enzymes are involved in the response to PTH, the data presented here provide strong evidence that PKA is the enzyme activated by PTH that is responsible for phosphorylating CREB at S133. Furthermore, the activity of PKA is required to mediate the PTH-induced increase in c-fos expression in osteoblasts.
The requirement of CREB S133 phosphorylation for its increased transcriptional activity appears to be absolute, however, regardless of whether other phosphorylation events occurring on CREB play a role in regulating its transcriptional activity, which remains to be conclusively proven. We are currently investigating the possibility that other phosphorylation events occur on CREB in response to PTH and that these events are involved in regulating its activity in response to this hormone.
Received April 28, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 integrin gene expression is mediated by
protein kinase C-. J Cell Biol 134:1301–1311 and noninvolvement of diacylglycerol-sensitive PKCs in
insulin-stimulated glucose transport in L6 myotubes. Endocrinology 138:4721–4731 substrate. Connection between cell surface
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