Departments of 1 Orthopaedics, 2 Pathology, and 3 Physiology and Biophysics, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 44106-5000
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ABSTRACT |
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cAMP/PKA signaling transiently stimulates mRNA expression of immediate-early genes, including IL-6 and c-fos. We confirmed that these mRNAs are transiently stimulated by parathyroid hormone (PTH) in ROS 17/2.8 osteoblastic cells. Consistent with the role for cAMP/PKA signaling in this response, PTH induces transient cAMP elevation, PKA activation, and cAMP-responsive element-binding protein (CREB) phosphorylation. Our goal was to determine whether termination of immediate-early gene expression is due to receptor desensitization or cAMP degradation. The approaches used were 1) inhibition of PTH receptor desensitization with G protein-coupled receptor kinase 2 (GRK2) antisense oligonucleotides or antisense plasmids, 2) sustained activation of adenyl cyclase with forskolin, and 3) inhibition of cAMP degradation with 3-isobutyl-1-methylxanthine. These experiments show that mechanisms downstream of receptor desensitization and cAMP degradation are primarily responsible for termination of PKA activity, CREB phosphorylation, and immediate-early gene expression. Similar conclusions were also obtained in response to PTH in a second osteoblastic cell line (MC3T3-E1) and in response to isoproterenol in NIH3T3 fibroblasts. This conclusion may therefore reflect a general mechanism for termination of immediate-early gene expression after induction by cAMP/PKA.
receptor desensitization; cAMP degradation; IL-6; c-fos; osteoblast
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INTRODUCTION |
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PARATHYROID HORMONE (PTH) is one of the major hormonal regulators of calcium homeostasis, and stimulation of bone resorption is one of the primary means by which PTH increases extracellular calcium levels (19). Interestingly, PTH does not directly stimulate osteoclasts, the cells that resorb bone (47). Instead, PTH stimulates bone resorption indirectly by binding to receptors on osteoblasts, which, in turn, upregulate production of secreted and cell-surface cytokines that increase osteoclast differentiation and activity (47). Interleukin-6 (IL-6) is one of the important cytokines secreted by osteoblasts in response to PTH. Neutralizing antibody studies have shown that IL-6 secretion mediates the ability of PTH to stimulate osteoclast activity in vitro (22), as well as bone resorption in vivo (24). Moreover, gene knockout studies have confirmed the in vivo role of IL-6 in stimulation of bone resorption by PTH (24).
Stimulation of IL-6 protein secretion by PTH is due to increased transcription of IL-6 mRNA (12). Thus it is associated with increases in IL-6 mRNA levels and activity of promoter constructs in osteoblasts both in vitro (20, 21, 30, 37) and in vivo (36, 44). Stimulation of IL-6 mRNA by PTH is a classic immediate-early gene response because it is rapid and transient (20, 21, 30, 36, 44), dependent on transcription (21, 30, 37), and superinduced by inhibition of translation (21). It is likely that transient stimulation of IL-6 mRNA is physiologically important because it limits the amount of IL-6 protein that is secreted in response to PTH (12). PTH also induces other immediate-early genes, including leukemia inhibitory factor (LIF) (21), c-fos (9, 36), c-jun (36), and c-myc (36). Because PTH-mediated induction of IL-6, LIF, and c-fos mRNAs is primarily dependent on cAMP signaling (21, 30, 37, 53) and the resultant phosphorylation of transcription factors such as cAMP-responsive element-binding protein (CREB) and CCAAT/enhancer-binding protein (C/EBP) (29, 39, 52, 53), the molecular mechanisms that are responsible for their transient expression may also be similar.
Immediate-early gene expression requires that the mRNA be rapidly
degraded and that the stimulation of mRNA transcription be rapidly
terminated (28). Because stimulation of cAMP signaling by
PTH is transient (38), this study was designed to
determine whether termination of immediate-early gene transcription is
due to termination of cAMP signaling. In support of this concept, a
number of other G protein-coupled receptors (GPCRs) are known to be
rapidly phosphorylated and desensitized (43). The
best-studied example of this is phosphorylation and desensitization of
2-adrenergic receptors (
2-ARs) by G
protein-coupled receptor kinase 2 (GRK2) (43). PTH
receptors are also phosphorylated in response to ligand occupancy
(6, 7). It is controversial whether binding of GRK2 is
sufficient to induce desensitization of the PTH receptor or whether the
subsequent phosphorylation of the receptor is also necessary (13,
32, 50). Nevertheless, both antisense (8) and
dominant-negative (17) experiments have shown that PTH
receptor desensitization is primarily due to GRK2. Moreover, the rapid decrease in cAMP levels after stimulation by PTH also depends on
degradation of cAMP by phosphodiesterases (PDEs) (2),
which are rapidly activated by PTH (1). However, we found
that inhibition of either GRK2 or PDE activity does not block the
termination of immediate-early gene expression induced by PTH.
Similarly, forskolin (FSK) induces an extended period of cAMP elevation
but transient expression of the immediate-early genes. Taken together, these results strongly support the conclusion that the transient nature
of the expression of immediate-early genes induced by PTH is due to
mechanisms that act downstream of receptor desensitization and cAMP degradation.
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MATERIALS AND METHODS |
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Materials. Cell culture media including F-12 nutrient mixture (Ham's F-12), minimum essential medium (MEM), and Dulbecco's modified Eagle's medium (DMEM) were obtained from GIBCO BRL (Grand Island, NY). Fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT). Nonessential amino acids, L-glutamine, and penicillin-streptomycin were purchased from Mediatech (Herndon, VA). bPTH(1-34) and bPTH(3-34) were obtained from Bachem (Torrance, CA). FSK was purchased from Calbiochem (La Jolla, CA). Tetracycline (TC), 3-isobutyl-1-methylxanthine (IBMX), and isoproterenol (Iso) were from Sigma (St. Louis, MO). All of these reagents were screened for endotoxin contamination by using the high-sensitivity version of the colorimetric limulus amoebocyte lysate assay (QCL-1000; Whittaker Bioproducts, Walkersville, MD) as we have previously described (45). Endotoxin levels were <0.002 EU/ml for the concentration of each reagent used in the experiments.
Cell culture. The rat osteoblastic osteosarcoma cell line ROS 17/2.8 (31) was maintained in Ham's F-12 with 10% FBS, 100 IU/ml penicillin, and 100 mg/ml streptomycin. Murine MC3T3-E1 osteoblastic cells (48) were maintained in MEM with 10% FBS, 1% nonessential amino acids, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin. Mouse NIH3T3 fibroblastic cells (ATCC CRL 1658) were maintained in DMEM with 4.5 g/l glucose, 10% FBS, 1% nonessential amino acids, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin. All cell lines were cultured at 37°C in a humidified atmosphere containing 95% air and 5% CO2 and routinely passaged every 3 or 4 days. For experiments, cells were seeded at a density of 2.5 × 104 cells/cm2 and incubated for 3 days until they reached confluence. At confluence, the culture medium was changed. The cells were incubated 24 h later with 100 nM bPTH(1-34), 100 nM bPTH(3-34), 10 uM FSK, or 10 uM Iso in medium containing 0.1% BSA rather than FBS for the indicated periods of time.
Treatment of ROS 17/2.8 cells with phosphorothioate oligonucleotides. 20-Mer sense and antisense GRK2 phosphorothioate oligonucleotides corresponding to positions 144-163 of rat GRK2 cDNA (4) were obtained from Integrated DNA Technologies (Coralville, IA). Fresh medium containing 5 uM sense or antisense GRK2 phosphorothioate oligonucleotides was administered to newly confluent ROS 17/2.8 cells every 8 h for 2 days before stimulation with 100 nM bPTH(1-34).
Plasmid preparation and stable transfection. Polymerase chain reaction (PCR) products of nucleotides 82-1103 from rat GRK2 cDNA (4) were subcloned into pTRE expression plasmids (Clontech, Palo Alto, CA). Plasmid clones containing either sense GRK2 mRNA (pTRE-GRK2-S) or antisense GRK2 mRNA (pTRE-GRK2-AS) were obtained and purified. ROS 17/2.8 cells in six-well plates were transfected with 1 µg/well of pTet-Off (Clontech) mixed with 6 µg/well of DOSPER Liposomal Transfection Reagent (Boehringer, Germany). A stable transfectant clone (ROS pTet-Off) was selected with 100 µg/ml of G418 (Sigma). ROS pTet-Off cells were then transfected with pTRE-GRK2-S, pTRE-GRK2-AS, or control pTRE-Luc (Clontech) and selected with 20 µg/ml hygromycin B (Sigma) to obtain double-stable transfectant clones. In this system (18), withdrawal of TC from the medium induces the expression of sense or antisense GRK2 mRNA.
RT-PCR.
IL-6, c-fos, and LIF mRNA levels were assessed by reverse
transcriptase-PCR (RT-PCR) as described (20, 21). RNA was
isolated by using the ToTALLY RNA kit (Ambion, Austin, TX), and 4 µg
of each sample were reverse transcribed to cDNA with RNAse H-free reverse transcriptase (GIBCO BRL, Gaithersburg, MD). The IL-6, LIF, and
actin primers and PCR conditions were previously described (20,
21), while c-fos was amplified using primers from and PCR conditions recommended by Stratagene (La Jolla, CA). Controls without cDNA were used in all PCR reactions. The PCR products were
electrophoresed with 2% agarose gels containing ethidium bromide using
HaeIII restriction fragments of X174 DNA as molecular weight markers.
cAMP production analysis. For cAMP assays, cells in 24-well plates were incubated with 100 nM bPTH(1-34), 10 µM FSK, or 10 µM Iso in 500 µl of HEPES-buffered Krebs-Ringer's bicarbonate buffer (pH 7.4) containing 1 mg/ml bovine serum albumin (BSA) and 2.5 mM glucose (KRBG) at 37°C for the indicated periods of time. The reactions were terminated by adding 500 µl of 12% trichloroacetic acid. After three freeze-thaw cycles, the cells were scraped off the plates and centrifuged at 3,000 g for 15 min. Supernatant (500 µl) was extracted with 2 ml of water-saturated diethylether three times. The concentration of cAMP in the aqueous phase was determined in duplicate by radioimmunoassay (RPA509; Amersham, Buckinghamshire, UK).
Desensitization assays. PTH receptor desensitization assays were performed as previously described (8). Briefly, cells in 24-well plates were washed 3 times with PBS, incubated with 100 nM bPTH(1-34) or vehicle (1 µM acetic acid) in 500 µl of KRBG buffer for 5 min at 37°C, and incubated with 3 changes of PBS at 37°C (2 min per incubation). All groups were then incubated with 100 nM bPTH(1-34) plus 100 µM IBMX in 500 µl of KRBG buffer at 37°C for 2 min, and the reactions were terminated by adding 500 µl of 12% trichloroacetic acid. cAMP concentrations were determined as described above.
Western blots. For GRK2 Western blot assays, cells in 60-mm dishes were washed with ice-cold PBS twice and lysed in 250 µl of RIPA buffer and aliquots containing 60 µg of protein electrophoresed on 12% SDS-PAGE gels (BioWhittaker Molecular Applications, Rockland, ME). For transcription factor Western blot assays, cells in 60-mm dishes were washed with ice-cold PBS twice and lysed in 100 µl of SDS sample buffer (62.5 mM Tris · HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, and 0.1% bromphenol blue), and 15-µl aliquots were electrophoresed. Proteins were electrotransferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 and probed with polyclonal rabbit antibodies specific for GRK2 (Santa Cruz Biotechnology, Santa Cruz, CA), CREB (Cell Signaling Technology, Beverly, MA), or phosphoCREB/phosphoATF-1 (Cell Signaling Technology). Bound antibody was detected with horseradish peroxidase-conjugated goat anti-rabbit IgG and enhanced chemiluminescence (Amersham).
PKA activity assays.
To prepare whole cell lysates, cells in 60-mm dishes were rinsed with
PBS twice and lysed in 50 mM Tris · HCl, pH 7.5, and 5 mM EDTA.
The lysates were sonicated on ice for 10 s and centrifuged at
12,000 g for 2 min at 4°C. For nuclear extraction, cells
were lysed in AT buffer (60 mM KCl, 15 mM NaCl, 14 mM
-mercaptoethanol, 2 mM EDTA, 15 mM HEPES, pH 7.9, 0.3 M sucrose, 5 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml pepstatin, and 0.5 mM phenylmethylsulfonyl fluoride) containing 0.1% Triton X-100 and
incubated on ice for 5 min. The lysates were layered on 1 volume of a
sucrose cushion (AT buffer with 1 M sucrose) and centrifuged at 10,000 g for 5 min (15). Nuclear extracts were
prepared by incubating the pellets for 2 h at 4°C in 10 mM
Tris · HCl, pH 7.4, 3 mM MgCl2, 2 mM
dithiothreitol, 0.5 M NaCl, 0.1 mM EDTA, protease inhibitors (0.1 mM
phenylmethylsulfonyl fluoride, 100 µg/ml aprotinin, and 100 µg/ml
leupeptin), and phosphatase inhibitors (20 mM NaF and 1 mM sodium
orthovanadate) and centrifuging at 67,000 g for 20 min
(11).
Statistical analysis. All presented RT-PCR and Western blot results are representative of at least three independent experiments. All quantitative data are presented as the mean ± SE of all available experiments. Symbols without error bars represent standard errors smaller than the symbols. The number of the experiments of each type are indicated in the figure legends. All PKA activity data are normalized by setting the PTH-treated time point with maximal activity at 100%. Statistical analyses were by ANOVA with Fisher's protected least significant difference post hoc tests.
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RESULTS |
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bPTH(1-34) rapidly and transiently stimulates
IL-6 and c-fos mRNA expression in ROS 17/2.8 cells.
Our previous studies showed that stimulation of IL-6 mRNA expression by
bPTH(1-34) is rapid and transient in MC3T3-E1 cells, UMR106-01 cells, primary cultures of rat osteoblasts, and in vivo (20, 21, 44). Figure
1A shows that ROS 17/2.8
osteoblast-like osteosarcoma cells exhibit a similar response and
extends these observations to another immediate-early gene,
c-fos. Both IL-6 and c-fos mRNA levels are
maximal 1 h after bPTH(1-34) addition and return
to baseline within 4 h, a pattern that is characteristic of
immediate-early gene expression (21). We and others have previously shown that induction of IL-6 and c-fos gene
expression by PTH depends on activation of the PKA signal transduction
pathway (21, 30, 37, 53). Consistent with this,
bPTH(1-34) also induces rapid and transient elevation
of cAMP levels (Fig. 1B), activation of PKA, both total
cellular and nuclear (Fig. 1, C and D), and
phosphorylation of both CREB and the related transcription factor,
ATF-1 (Fig. 1E). Maximal stimulation of cAMP elevation, total cellular PKA activation, nuclear PKA activation, and CREB/ATF-1 phosphorylation occur at 1-5 min (Fig. 1B), 5-30
min (Fig. 1C), 30-60 min (Fig. 1D), and
20-45 min (Fig. 1E), respectively, after bPTH(1-34) addition. The delay between activation of
total cellular PKA and the activation of nuclear PKA reflects the
relatively slow translocation of PKA to the nucleus (26).
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Blocking GRK2-mediated PTH receptor desensitization does not block
the rapid decline of IL-6 and c-fos mRNA expression that occurs after
induction by bPTH(1-34).
The rapid termination of immediate-early gene expression that occurs
after stimulation by PTH is likely to be physiologically important
because it limits the amounts of the proteins that are produced
(12). The best-studied mechanism for terminating PKA signal transduction is receptor desensitization caused by
phosphorylation. We have previously used antisense approaches to show
that PTH receptor desensitization is primarily due to GRK2
(8). To determine whether PTH receptor desensitization is
responsible for termination of immediate-early gene expression, ROS
17/2.8 cells were pretreated with GRK2 antisense phosphorothioate
oligonucleotides (AS-oligo), GRK2 sense phosphorothioate
oligonucleotides (S-oligo), or vehicle for 2 days with medium changes
containing fresh oligonucleotides performed every 8 h. Figure
2A shows that the antisense
oligonucleotide effectively reduces GRK2 protein levels. In addition,
the antisense oligonucleotide substantially inhibits PTH receptor
desensitization (Fig. 2B). Thus PTH pretreatment for 5 min
reduces elevation of cAMP by bPTH(1-34) by
39-47% in the control and sense oligonucleotide-treated groups,
which is the amount of desensitization expected for short-term treatment with PTH (46) or many other GPCR agonists
(40, 41). In contrast, PTH pretreatment only induced a
13% decrease in cAMP elevation in the antisense
oligonucleotide-treated group (fifth and sixth bars in Fig.
2B). However, none of the oligonucleotides has a substantial
effect on the time course of IL-6 and c-fos mRNA expression
induced by bPTH(1-34) (Fig. 2C).
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FSK induces sustained elevation of cAMP but transient induction of
IL-6 and c-fos mRNA expression.
Because FSK directly stimulates adenyl cyclase, we compared the effects
of FSK and bPTH(1-34) on ROS 17/2.8 cells. Whereas bPTH(1-34) rapidly and transiently elevates cAMP, FSK
acts in a slower but sustained fashion with maximal cAMP levels
observed beginning at 30 min and lasting for the 24 h duration of
the experiment (Fig. 4A).
Despite this sustained elevation of cAMP, FSK transiently induces
nuclear PKA activation (Fig. 4B), CREB/ATF-1 phosphorylation (Fig. 4C), and IL-6 and c-fos mRNA expression
(Fig. 4D) with time courses that are indistinguishable from
those induced by bPTH(1-34). These results suggest
that mechanisms downstream of adenyl cyclase are primarily responsible
for the termination of immediate-early gene expression, as well as
termination of both PKA activation and transcription factor
phosphorylation. These results also suggest that the cAMP signal
transduction pathway is sufficient not only for the stimulation of
immediate-early gene expression by bPTH(1-34) (21, 30, 37, 53) but also for the transient nature of that
stimulation.
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IBMX blocks cAMP degradation but does not block the termination of
IL-6 and c-fos mRNA expression that occurs after induction by
bPTH(1-34).
Rapid cAMP degradation by PDE after PTH stimulation (1, 2)
is another mechanism that might be responsible for the rapid decline in
IL-6 and c-fos mRNA levels. To test this possibility, we
used IBMX, a PDE inhibitor, to stabilize the cAMP levels. Compared with
its ability to maintain a sustained high level of cAMP for at least
10 h (Fig. 5A), IBMX has
a much shorter, 1- to 2-h effect on nuclear PKA activation (Fig.
5B), CREB/ATF-1 phosphorylation (Fig. 5C), and
IL-6 and c-fos mRNA expression (Fig. 5D) that
occurs after stimulation with bPTH(1-34). Thus,
although the downstream responses are extended for a short period by
IBMX (Fig. 5, B-D), they are still rapidly
terminated compared with the long-term elevation of cAMP levels (Fig.
5A). These results suggest that mechanisms downstream of
cAMP degradation are primarily responsible for the termination of
immediate-early gene expression, as well as termination of both PKA
activation and transcription factor phosphorylation.
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Sustained cAMP elevation but transient immediate-early gene
expression in MC3T3-E1 osteoblastic cells and NIH3T3 fibroblastic
cells.
To determine whether the above results are a unique feature of the ROS
17/2.8 cell line, we performed similar experiments with MC3T3-E1
osteoblastic cells and NIH3T3 fibroblastic cells. Use of the MC3T3-E1
and NIH3T3 cells also allowed examination of LIF, an additional
immediate-early gene (21) which is not detectably
expressed in ROS 17/2.8 cells. bPTH(1-34) induces
transient elevation of cAMP and transient gene expression in MC3T3-E1
cells (Fig. 6, A and
B), whereas Iso induces
similar effects in NIH3T3 cells (Fig. 6, C and
D). In both cell lines, IBMX prevents the decline in cAMP
levels seen subsequent to hormone stimulation (Fig. 6, A and
C) but does not block the rapid decline in gene expression
(Fig. 6, B and D). Similar results are seen after
stimulation with FSK (Fig. 6, A-D). Taken
together, these results suggest that mechanisms downstream of cAMP
degradation are primarily responsible for the termination of
immediate-early gene expression, not only in ROS 17/2.8 cells but also
in a second osteoblastic cell line, as well as in NIH3T3 fibroblasts.
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DISCUSSION |
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Phosphorylation by GRKs is an important mechanism responsible for
rapid inactivation of GPCRs (43). The best-studied example of this process is phosphorylation of 2-ARs by GRK2
(43). Similarly, the PTH receptor is rapidly
phosphorylated in response to ligand occupancy (6, 7), and
both antisense (8) and dominant-negative (17)
experiments have shown that GRK2 is involved in PTH receptor desensitization. Our experiments confirmed the role of GRK2 in termination of cAMP elevation by PTH in osteoblastic cells. However, we
also found that termination of cAMP elevation is not primarily responsible for termination of downstream responses, including activation of nuclear PKA, phosphorylation of the prototypic
transcription factors CREB and ATF-1, and expression of mRNAs encoding
the immediate-early genes IL-6, c-fos, and LIF. This
conclusion is based on evidence that extended cAMP elevation due to
either GRK2 antisense oligonucleotides, GRK2 antisense transfection, or
inhibition of PDEs by IBMX does not block termination of the downstream
responses that are induced by PTH in ROS 17/2.8 osteoblastic cells.
Similar results were also observed after stimulation of ROS 17/2.8
cells with FSK, as well as in osteoblastic MC3T3-E1 cells treated
either with FSK or with IBMX plus bPTH(1-34) and in
fibroblastic NIH3T3 cells treated either with FSK or with IBMX plus
Iso. Taken together, these results strongly support the conclusion that
mechanisms downstream of receptor desensitization and cAMP degradation
are primarily responsible for the termination of immediate-early gene expression, as well as termination of both nuclear PKA activation and
transcription factor phosphorylation. Thus these results indicate that
PTH receptor internalization, which is mediated by
-arrestin (16, 42), is also not the primary mechanism responsible
for these effects because receptor internalization would act upstream of cAMP degradation. Receptor internalization, receptor
desensitization, and cAMP degradation, mediated by
-arrestin, GRKs,
and PDEs, respectively, may, nonetheless, be important regulators of
other physiological processes that are induced by PTH and other GPCR agonists.
Our results demonstrate that termination of PTH-induced immediate-early gene expression is primarily due to mechanisms that act downstream of cAMP degradation. Possibilities include activation either of inhibitory transcription factors, such as inducible cAMP early repressor (ICER) (34), or of histone deacetylases (HDACs) (27) that would negatively regulate transcription of the immediate-early genes. In this regard, PTH increases ICER mRNA levels in osteoblasts (51), and HDACs have been proposed to contribute to transient stimulation of somatostatin mRNA by FSK (33). However, because both inhibitory transcription factors and HDACs would act downstream of PKA and CREB/ATF-1 phosphorylation, they would not account for the termination of PKA activation and transcription factor phosphorylation observed in our study. Thus it is also less likely that these mechanisms account for the termination of immediate-early gene expression by PTH and other cAMP-dependent stimulators. Similarly, CREB dephosphorylation would not account for the termination of PKA activation. Nonetheless, Montminy and colleagues (3, 25) reported that inhibition of protein phosphatase-1 (PP1) with phosphatase inhibitor-1 prolongs CREB phosphorylation and augments the transcriptional response induced by cAMP, whereas inhibition of PP2A by SV40 small t antigen has no effect. In contrast, Klemm and colleagues (56, 57) reported the opposite finding that SV40 small t antigen prolongs CREB phosphorylation and augments the transcriptional response induced by cAMP, whereas inhibition of PP1 by phosphatase inhibitor-2 has no effect.
In contrast to the mechanisms described in the previous paragraph,
there are at least two possibilities that might account for termination
of PKA activation, CREB/ATF-1 phosphorylation and immediate-early gene
expression. For example, the three known members of the PKI family,
PKI, PKI
, and PKI
, inhibit nuclear PKA activity (10, 35,
55, 58) and induce export of PKA from the nucleus (10, 14,
58). Moreover, both PKI
and PKI
mRNAs can be induced by
cAMP (49, 54). Thus PKI can efficiently terminate
PKA-induced gene expression (10, 14, 35, 49, 55, 58). More
recently, dopamine and cAMP-regulated phosphoprotein-32 (DARPP-32) have
been shown to act as a molecular switch in the nervous system that
potentiates cAMP signaling by inhibiting PP-1 when phosphorylated on
threonine-34 (23) but that both inhibits PKA and activates
PP-1 when phosphorylated on threonine-75 (5). Thus, if
DARPP-32 or a related molecule is phosphorylated on threonine-75 in
osteoblasts or fibroblasts, it might terminate PKA activation, CREB/ATF-1 phosphorylation, and immediate-early gene expression. Additional studies are needed to determine the relative roles of PKI
and DARPP-32 in our system.
In summary, PTH-induced cAMP signaling in osteoblasts induces transient activation of nuclear PKA, transient phosphorylation of the prototypic transcription factors CREB and ATF-1, and transient induction of immediate-early genes, including IL-6, c-fos, and LIF. In this study, we found that termination of these responses is not blocked by any of the following approaches: inhibition of receptor desensitization by GRK2 antisense oligonucleotides, inhibition of receptor desensitization by GRK2 antisense transfection, sustained activation of adenyl cyclase by FSK, or inhibition of cAMP degradation by IBMX. Similar results were also obtained in fibroblasts stimulated with Iso, FSK, or Iso plus IBMX. Thus mechanisms that act downstream of receptor desensitization and cAMP degradation are primarily responsible for the termination of immediate-early gene expression, as well as termination of both nuclear PKA activation and transcription factor phosphorylation.
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ACKNOWLEDGEMENTS |
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We thank J. Nalepka for the endotoxin measurements and C. Carlin, D. Holderbaum, S. Orellana, and J. Yoo for many helpful discussions.
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FOOTNOTES |
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This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Research Grant AR-41674 (to E. M. Greenfield).
Address for reprint requests and other correspondence: E. M. Greenfield, Dept. of Orthopaedics, Case Western Reserve Univ., 11100 Euclid Ave., Cleveland, OH 44106-5000 (E-mail: emg3{at}po.cwru.edu).
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.
July 3, 2002;10.1152/ajpcell.00221.2002
Received 16 May 2002; accepted in final form 1 July 2002.
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