Termination of immediate-early gene expression after stimulation by parathyroid hormone or isoproterenol

Xin Chen1, Jia-Chun Dai1, and Edward M. Greenfield1,2,3

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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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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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 beta 2-adrenergic receptors (beta 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. 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 Phi 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 beta -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).

PKA activity was measured by using the Protein Kinase A assay system from GIBCO BRL. Briefly, 10 µl of either whole cell lysates or nuclear extracts were incubated in duplicate for 5 min at 30°C with [gamma -32P]ATP (Amersham), 50 µM kemptide, 100 µM ATP, 41 mM MgCl2, 0.25 mg/ml BSA, and 50 mM Tris · HCl, pH 7.5, in the presence or absence of 1 µM PKI(6-22) amide and/or 10 µM cAMP in a total volume of 40 µl. Reactions were terminated by spotting 20 µl onto phosphocellulose discs and washing twice with 1% phosphoric acid and then twice with distilled water. Peptide-incorporated 32P was determined by scintillation counting. PKA activation was calculated as a percentage of the total cAMP-induced PKA activity after subtraction from both values of the activity that was not inhibited by protein kinase inhibitor (PKI).

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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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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Fig. 1.   Rapid and transient stimulation by bPTH(1-34) of interleukin-6 (IL-6) and c-fos mRNA expression (A), cAMP production (B), total cellular PKA activation (C), nuclear PKA activation (D), and CREB/ATF-1 phosphorylation (E) in ROS 17/2.8 cells. ROS 17/2.8 cells were treated with 100 nM bPTH(1-34) or vehicle control (1 uM acetic acid) for the indicated periods of time. A and E: representative results from n = 3 experiments. B: means of n = 6 experiments. C and D: means of n = 3 experiments. In B, all parathyroid hormone (PTH)-treated groups are significantly (P < 0.0002) greater than the control groups at the same time points, and all PTH-treated groups >= 10 min are significantly (P < 0.002) less than the 1 min group. In C, PTH-treated groups from 5 to 60 min are significantly (P < 0.002) greater than the control groups at the same time points, and all PTH-treated groups >= 60 min are significantly (P < 0.002) less than the 5 min group. In D, PTH-treated groups from 15 to 120 min are significantly (P < 0.008) greater than the control groups at the same time points, and all PTH-treated groups are significantly (P < 0.003) less than the 60 min group.

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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Fig. 2.   G protein-coupled receptor kinase 2 (GRK2) antisense phosphorothioate oligonucleotides reduce GRK2 protein levels (A) and inhibit PTH receptor desensitization (B) but do not block the termination of IL-6 and c-fos mRNA expression that occurs after induction by bPTH(1-34) (C). Fresh medium containing 5 µM GRK2 sense oligonucleotides (S-oligo), 5 µM GRK2 antisense oligonucleotides (AS-oligo), or vehicle control (2.5% distilled H2O) were administered to ROS 17/2.8 cells every 8 h for 2 days before GRK2 Western blot analysis (A), PTH receptor desensitization assays (B), or stimulation with 100 nM bPTH(1-34) for the indicated periods of time (C). A and C: representative results from n = 3 experiments. B: means of n = 3 experiments, asterisks indicate P < 0.0001, and NS indicates P > 0.05.

To confirm the results obtained with antisense oligonucleotides, we constructed stable ROS Tet-Off cell lines, as well as plasmids encoding either sense GRK2 (pTRE-GRK2-S), antisense GRK2 (pTRE-GRK2-AS), or control luciferase (pTRE-Luc). The stable ROS pTet-Off cells were cotransfected with pTRE-GRK2-S, pTRE-GRK2-AS, or pTRE-Luc to derive double-stable transfectant clones. In these clones, the pTet-Off plasmids produce a transactivator that activates plasmids containing the gene of interest cloned downstream of a promotor with seven repeats of the TC operator (18). Withdrawal of TC from the culture medium therefore induces the expression of sense or antisense GRK2 mRNA from the transfectants. Of the three kinds of double-stable clones with or without TC examined in these experiments, the pTRE-GRK2-AS cell clone without TC shows dramatically reduced GRK2 protein levels (Fig. 3A). In this clone, TC withdrawal also reduces PTH receptor desensitization (Fig. 3B). Thus PTH pretreatment reduces elevation of cAMP by bPTH(1-34) by 35-52% in the control groups, but only by 9% in the pTRE-GRK2-AS clone without TC (seventh and eighth bars in Fig. 3B). However, none of the clones showed substantial changes in the pattern of transient response of IL-6 and c-fos mRNA expression induced by bPTH(1-34) (Fig. 3C). Both the antisense oligonucleotide and transfectant strategies suggest that mechanisms downstream of PTH receptor desensitization are primarily responsible for the termination of immediate-early gene expression.


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Fig. 3.   GRK2 antisense plasmid transfection reduces GRK2 protein levels (A) and inhibits PTH receptor desensitization (B) but does not block the termination of IL-6 and c-fos mRNA expression that occurs after induction by bPTH(1-34) (C). A stable ROS 17/2.8 pTet-Off clone was transfected with GRK2 sense (pTRE-GRK2-S), GRK2 antisense (pTRE-GRK2-AS), or control (pTRE-Luc) plasmids to obtain double-stable transfectants. Expression of the transfected plasmids was induced by withdrawal of tetracycline (TC) for 1 day before GRK2 Western blot analysis (A), PTH receptor desensitization assays (B), or stimulation with 100 nM bPTH(1-34) for the indicated periods of time (C). Control cultures were maintained in the continuous presence of 0.2 ug/ml TC. A and C: representative results from n = 3 experiments. B: means of n = 3 experiments, asterisks indicate P < 0.0001, and NS indicates P > 0.1.

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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Fig. 4.   Forskolin (FSK) induces sustained elevation of cAMP (A) but transient nuclear PKA activation (B), CREB/ATF-1 phosphorylation (C), and IL-6 and c-fos mRNA expression (D). ROS 17/2.8 cells were treated with 10 µM FSK or 100 nM bPTH(1-34) for the indicated periods of time. All cultures also received a mixture of vehicle controls, such that all contained 1 µM acetic acid and 0.1% dimethyl sulphoxide. C and D: representative results from n = 3 experiments. A and B: means of n = 3 experiments. In A, all FSK-treated groups >= 2 min are significantly (P < 0.008) greater than the control groups at the same time points. In B, all FSK-treated groups from 15 min to 2 h are significantly (P < 0.04) greater than the control groups at the same time points.

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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Fig. 5.   3-Isobutyl-1-methylxanthine (IBMX) blocks cAMP degradation (A) but does not block the termination of nuclear PKA activation (B), CREB/ATF-1 phosphorylation (C), or IL-6 and c-fos mRNA expression (D) that occurs after induction by bPTH(1-34). ROS 17/2.8 cells were treated with 100 nM bPTH(1-34) with or without 100 µM IBMX for the indicated periods of time. All cultures also received a mixture of vehicle controls, such that all contained 1 µM acetic acid and 0.1 mM NaOH. C and D show representative results from n = 3 experiments. A and B show means of n = 3 experiments. In A, all PTH + IBMX-treated groups >= 5 min are significantly (P < 0.03) greater than the PTH-treated groups at the same time points. In B, all PTH + IBMX-treated groups from 1 to 4 h are significantly (P < 0.003) greater than the PTH-treated groups at the same time points.

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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Fig. 6.   In MC3T3-E1 osteoblastic cells (A and B) and NIH3T3 fibroblastic cells (C and D), sustained elevation of cAMP (A and C) induces transient expression of IL-6, c-fos, and leukemia inhibitory factor (LIF) mRNAs (B and D). MC3T3-E1 cells were treated with 100 nM bPTH(1-34), with 100 nM bPTH(1-34) plus 100 µM IBMX, or with 10 µM FSK, while NIH3T3 cells were treated with 10 µM Iso, with 10 µM Iso plus 100 µM IBMX, or with 10 µM FSK for the indicated periods of time. All cultures also received a mixture of vehicle controls, such that all contained 1 µM acetic acid, 0.2% dimethyl sulphoxide, and 0.1 mM NaOH. B and D: representative results from n = 3 experiments. A and C: means of n = 3 experiments. In A and C, the 1 min PTH- or Iso-treated groups and all of the IBMX- or FSK-treated groups are significantly (P < 0.0001) greater than the control groups at the same time points.


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ABSTRACT
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MATERIALS AND METHODS
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DISCUSSION
REFERENCES

Phosphorylation by GRKs is an important mechanism responsible for rapid inactivation of GPCRs (43). The best-studied example of this process is phosphorylation of beta 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 beta -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 beta -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, PKIalpha , PKIbeta , and PKIgamma , inhibit nuclear PKA activity (10, 35, 55, 58) and induce export of PKA from the nucleus (10, 14, 58). Moreover, both PKIalpha and PKIbeta 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.


    ACKNOWLEDGEMENTS

We thank J. Nalepka for the endotoxin measurements and C. Carlin, D. Holderbaum, S. Orellana, and J. Yoo for many helpful discussions.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ahlstrom, M, and Lamberg-Allardt C. Rapid protein kinase A-mediated activation of cyclic AMP-phophodiesterase by parathyroid hormone in UMR-106 osteoblast-like cells. J Bone Miner Res 12: 172-178, 1997[ISI][Medline].

2.   Ahlstrom, M, and Lamberg-Allardt C. Regulation of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in UMR-106 osteoblast-like cells: role of cAMP-phosphodiesterase and cAMP efflux. Biochem Pharmacol 58: 1335-1340, 1999[ISI][Medline].

3.   Alberts, AS, Montminy M, Shenolikar S, and Feramisco JR. Expression of a peptide inhibitor of protein phosphatase 1 increases phosphorylation and activity of CREB in NIH 3T3 fibroblasts. Mol Cell Biol 14: 4398-4407, 1994[Abstract].

4.   Arriza, JL, Dawson TM, Simerly RB, Martin LJ, Caron MG, Snyder SH, and Lefkowitz RJ. The G-protein-coupled receptor kinases beta ARK1 and beta ARK2 are widely distributed at synapses in rat brain. J Neurosci 12: 4045-4055, 1992[Abstract].

5.   Bibb, JA, Snyder GL, Nishi A, Yan Z, Meijer L, Fienberg AA, Tsai LH, Kwon YT, Girault JA, Czernik AJ, Huganir RL, Hemmings HC, Nairn AC, and Greengard P. Phosphorylation of DARPP-32 by Cdk5 modulates dopamine signaling in neurons. Nature 402: 669-671, 1999[ISI][Medline].

6.   Blind, E, Bambino T, Huang Z, Bliziotes M, and Nissenson RA. Phosphorylation of the cytoplasmic tail of the PTH/PTHrP receptor. J Bone Miner Res 11: 578-586, 1996[ISI][Medline].

7.   Blind, E, Bambino T, and Nissenson RA. Agonist-stimulated phosphorylation of the G protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein. Endocrinology 136: 4271-4277, 1995[Abstract].

8.   Chen, X, Nakata H, and Baba H. Effect of beta -adrenergic receptor kinase 1 on the very rapid phase homologous desensitization of PTH/PTHrP receptors in rat osteoblastic osteosarcoma cells. J Kobe Univ Sch Med 62: 1-10, 2001.

9.   Clohisy, JC, Scott DK, Brakenhoff KD, Quinn CO, and Partridge NC. Parathyroid hormone induces c-fos and c-jun messenger RNA in rat osteoblastic cells. Mol Endocrinol 6: 1834-1842, 1992[Abstract].

10.   Collins, SP, and Uhler MD. Characterization of PKIgamma , a novel isoform of the protein kinase inhibitor of cAMP-dependent protein kinase. J Biol Chem 272: 18169-18178, 1997[Abstract/Free Full Text].

11.   Constantinescu, A, Diamond I, and Gordon AS. Ethanol-induced translocation of cAMP-dependent protein kinase to the nucleus. Mechanism and functional consequences. J Biol Chem 274: 26985-26991, 1999[Abstract/Free Full Text].

12.   Dai, JC, and Greenfield EM. PTH and TNFalpha induce differential patterns of IL-6 mRNA and protein production in osteoblasts (Abstract). J Bone Miner Res 16: S486, 2001.

13.   Dicker, F, Quitterer U, Winstel R, Honold K, and Lohse M. Phosphorylation-independent inhibition of parathyroid hormone receptor signaling by G protein-coupled receptor kinases. Proc Natl Acad Sci USA 96: 5476-5481, 1999[Abstract/Free Full Text].

14.   Fantozzi, DA, Harootunian AT, Wen W, Taylor SS, Feramisco JR, Tsien RY, and Meinkoth JL. Thermostable inhibitor of cAMP-dependent protein kinase enhances the rate of export of the kinase catalytic subunit from the nucleus. J Biol Chem 269: 2676-2686, 1994[Abstract/Free Full Text].

15.   Feliciello, A, Giuliano P, Porcellini A, Garbi C, Obici S, Mele E, Angotti E, Grieco D, Amabile G, Cassano S, Li Y, Musti AM, Rubin CS, Gottesman ME, and Avvedimento EV. The v-Ki-Ras oncogene alters cAMP nuclear signaling by regulating the location and the expression of cAMP-dependent protein kinase II beta. J Biol Chem 271: 25350-25359, 1996[Abstract/Free Full Text].

16.   Ferrari, SL, Behar V, Chorev M, Rosenblatt M, and Bisello A. Endocytosis of ligand-human parathyroid hormone receptor 1 complexes is protein kinase C-dependent and involves beta-arrestin 2. Real-time monitoring by fluorescence microscopy. J Biol Chem 274: 29968-29975, 1999[Abstract/Free Full Text].

17.   Fukayama, S, Kong G, Benovic JL, Meurer E, and Tashjian AH. Beta-adrenergic receptor kinase-1 acutely regulates PTH/PTHrP receptor signaling in human osteoblastlike cells. Cell Signal 9: 469-474, 1997[ISI][Medline].

18.   Gossen, M, and Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89: 5547-5551, 1992[Abstract].

19.   Greenfield, EM, and Einhorn TA. Calcium homeostasis. In: Orthopaedics, edited by Fitzgerald RH, Kaufer H, and Malkani A.. Philadelphia, PA: Mosby, 2002, p. 195-200.

20.   Greenfield, EM, Gornik SA, Horowitz MC, Donahue HJ, and Shaw SM. Regulation of cytokine expression in osteoblasts by parathyroid hormone: rapid stimulation of interleukin-6 and leukemia inhibitory factor mRNA. J Bone Miner Res 8: 1163-1171, 1993[ISI][Medline].

21.   Greenfield, EM, Horowitz MC, and Lavish SA. Stimulation by parathyroid hormone of interleukin-6 and leukemia inhibitory factor expression in osteoblasts is an immediate-early gene response induced by cAMP signal transduction. J Biol Chem 271: 10984-10989, 1996[Abstract/Free Full Text].

22.   Greenfield, EM, Shaw SM, Gornik SA, and Banks MA. Adenyl cyclase and interleukin 6 are downstream effectors of parathyroid hormone resulting in stimulation of bone resorption. J Clin Invest 96: 1238-1244, 1995[ISI][Medline].

23.   Greengard, P, Allen PB, and Nairn AC. Beyond the dopamine receptor: the DARPP-32/protein phosphatase-1 cascade. Neuron 23: 435-447, 1999[ISI][Medline].

24.   Grey, A, Mitnick MA, Masiukiewicz U, Sun BH, Rudikoff S, Jilka RL, Manolagas SC, and Insogna K. A role for interleukin-6 in parathyroid hormone-induced bone resorption in vivo. Endocrinology 140: 4683-4690, 1999[Abstract/Free Full Text].

25.   Hagiwara, M, Alberts A, Brindle P, Meinkoth J, Feramisco J, Deng T, Karin M, Shenolikar S, and Montminy M. Transcriptional attenuation following cAMP induction requires PP-1-mediated dephosphorylation of CREB. Cell 70: 105-113, 1992[ISI][Medline].

26.   Hagiwara, M, Brindle P, Harootunian A, Armstrong R, Rivier J, Vale W, Tsien R, and Montminy M. Coupling of hormonal stimulation and transcription via the cyclic AMP-responsive factor CREB is rate limited by nuclear entry of protein kinase A. Mol Cell Biol 13: 4852-4859, 1993[Abstract].

27.   Hassig, CA, Tong JK, Fleischer TC, Owa T, Grable PG, Ayer DE, and Schreiber SL. A role for histone deacetylase activity in HDAC1-mediated transcriptional repression. Proc Natl Acad Sci USA 95: 3519-3524, 1998[Abstract/Free Full Text].

28.   Herschman, HR. Primary response genes induced by growth factors and tumor promoters. Annu Rev Biochem 60: 281-319, 1991[ISI][Medline].

29.   Huang, YF, Harrison JR, and Kream BE. CRE and C/EBP sites in the human interleukin-6 promoter are important for cAMP induction of promoter expression in osteoblastic MC-3T3-E1 cells (Abstract). Bone 23: 448, 1998.

30.   Huang, YF, Harrison JR, Lorenzo JA, and Kream BE. Parathyroid hormone induces interleukin-6 heterogeneous nuclear and messenger RNA expression in murine calvarial organ cultures. Bone 23: 327-332, 1998[ISI][Medline].

31.   Majeska, RJ, Rodan SB, and Rodan GA. Parathyroid hormone-responsive clonal cell lines from rat osteosarcoma. Endocrinology 107: 1494-10503, 1980[Abstract].

32.   Malecz, N, Bambino T, Bencsik M, and Nissenson RA. Identification of phosphorylation sites in the G protein-coupled receptor for parathyroid hormone. Receptor phosphorylation is not required for agonist-induced internalization. Mol Endocrinol 12: 1846-1856, 1998[Abstract/Free Full Text].

33.   Michael, LF, Asahara H, Shulman AI, Kraus WL, and Montminy M. The phosphorylation status of a cyclic AMP-responsive activator is modulated via a chromatin-dependent mechanism. Mol Cell Biol 20: 1596-1603, 2000[Abstract/Free Full Text].

34.   Molina, CA, Foulkes NS, Lalli E, and Sassone-Corsi P. Inducibility and negative autoregulation of CREM: an alternative promoter directs the expression of ICER, an early response repressor. Cell 75: 875-886, 1993[ISI][Medline].

35.   Olsen, SR, and Uhler MD. Inhibition of protein kinase A by overexpression of the cloned human protein kinase inhibitor. Mol Endocrinol 5: 1246-1256, 1991[Abstract].

36.   Onyia, JE, Bidwell J, Herring J, Hulman J, and Hock JM. In vivo, human parathyroid hormone fragment (hPTH 1-34) transiently stimulates immediate early response gene expression, but not proliferation, in trabecular bone cells of young rats. Bone 17: 479-484, 1995[ISI][Medline].

37.   Onyia, JE, Libermann TA, Bidwell J, Arnold D, Tu Y, McClelland P, and Hock JM. Parathyroid hormone (1-34)-mediated interleukin-6 induction. J Cell Biochem 67: 265-274, 1997[ISI][Medline].

38.   Partridge, NC, Kemp BE, Veroni MC, and Martin TJ. Activation of adenosine 3',5'-monophosphate-dependent protein kinase in normal and malignant bone cells by parathyroid hormone, prostaglandin E2, and prostacyclin. Endocrinology 108: 220-225, 1981[Abstract].

39.   Pearman, AT, Chou WY, Bergman KD, Pulumati MR, and Partridge NC. Parathyroid hormone induces c-fos promoter activity in osteoblastic cells through phosphorylated cAMP response element (CRE)-binding protein binding to the major CRE. J Biol Chem 271: 25715-25721, 1996[Abstract/Free Full Text].

40.   Penn, RB, Panettieri RA, Jr, and Benovic JL. Mechanisms of acute desensitization of the beta 2-AR-adenylyl cyclase pathway in human airway smooth muscle. Am J Respir Cell Mol Biol 19: 338-348, 1998[Abstract/Free Full Text].

41.   Penn, RB, and Benovic JL. Regulation of G protein-coupled receptors. In: Handbook of Physiology. The Endocrine System. Cellular Endocrinology. Bethesda, MD: Am. Physiol. Soc, 1998, sect. 7, vol. 1, chapt. 7, p. 125-164.

42.   Pierce, KL, and Lefkowitz RJ. Classical and new roles of beta-arrestins in the regulation of G-protein-coupled receptors. Nat Rev Neurosci 2: 727-733, 2001[ISI][Medline].

43.   Pitcher, JA, Freedman NJ, and Lefkowitz RJ. G protein-coupled receptor kinases. Annu Rev Biochem 67: 653-692, 1998[ISI][Medline].

44.   Pollock, JH, Blaha MJ, Gornik SA, Stevenson S, and Greenfield EM. In vivo demonstration that parathyroid hormone and parathyroid hormone-related protein stimulate expression by osteoblasts of interleukin-6 and leukemia inhibitory factor. J Bone Miner Res 11: 754-759, 1996[ISI][Medline].

45.   Ragab, AA, Van De Motter RR, Lavish SA, Goldberg VM, Ninomiya JT, Carlin CR, and Greenfield EM. Measurement and removal of adherent endotoxin from titanium particles and implant surfaces. J Orthop Res 17: 803-809, 1999[ISI][Medline].

46.   Segre, GV. Receptors for parathyroid hormone and parathyroid hormone-related protein. In: Principles of Bone Biology, edited by Bilezikian JP, Raisz LG, and Rodan GA.. San Diego, CA: Academic, 1996, p. 377-403.

47.   Suda, T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, and Martin TJ. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 20: 345-357, 1999[Abstract/Free Full Text].

48.   Sudo, H, Kodama HA, Amagai Y, Yamamoto S, and Kasai S. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 96: 191-198, 1983[Abstract].

49.   Tash, JS, Dedman JR, and Means AR. Protein kinase inhibitor in sertoli cell-enriched rat testis. Specific regulation by follicle-stimulating hormone. J Biol Chem 254: 1241-1247, 1979[ISI][Medline].

50.   Tawfeek, H, Qian F, and Abou-Samra A. Phosphorylation of the receptor for PTH and PTHrP is required for internalization and regulates receptor signaling. Mol Endocrinol 16: 1-13, 2002[Abstract/Free Full Text].

51.   Tetradis, S, Nervina JM, Nemoto K, and Kream BE. Parathyroid hormone induces expression of the inducible cAMP early repressor in osteoblastic MC3T3-E1 cells and mouse calvariae. J Bone Miner Res 13: 1846-1851, 1998[ISI][Medline].

52.   Tyson, DR, Swarthout JT, Jefcoat SC, and Partridge NC. PTH induction of transcriptional activity of the cAMP response element-binding protein requires the serine 129 site and glycogen synthase kinase-3 activity, but not casein kinase II sites. Endocrinology 143: 674-682, 2002[Abstract/Free Full Text].

53.   Tyson, DR, Swarthout JT, and Partridge NC. Increased osteoblastic c-fos expression by parathyroid hormone requires protein kinase A phosphorylation of the cyclic adenosine 3',5'-monophosphate response element-binding protein at serine 133. Endocrinology 140: 1255-1261, 1999[Abstract/Free Full Text].

54.   Van Patten, SM, Donaldson LF, McGuinness MP, Kumar P, Alizadeh A, Griswold MD, and Walsh DA. Specific testicular cellular localization and hormonal regulation of the PKIalpha and PKIbeta isoforms of the inhibitor protein of the cAMP-dependent protein kinase. J Biol Chem 272: 20021-20029, 1997[Abstract/Free Full Text].

55.   Van Patten, SM, Ng DC, Th'ng JP, Angelos KL, Smith AJ, and Walsh DA. Molecular cloning of a rat testis form of the inhibitor protein of cAMP-dependent protein kinase. Proc Natl Acad Sci USA 88: 5383-5387, 1991[Abstract].

56.   Wadzinski, BE, Wheat WH, Jaspers S, Peruski LF, Lickteig RL, Johnson GL, and Klemm DJ. Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell Biol 13: 2822-2834, 1993[Abstract].

57.   Wheat, WH, Roesler WJ, and Klemm DJ. Simian virus 40 small tumor antigen inhibits dephosphorylation of protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell Biol 14: 5881-5890, 1994[Abstract].

58.   Wiley, JC, Wailes LA, Idzerda RL, and McKnight GS. Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A. J Biol Chem 274: 6381-6387, 1999[Abstract/Free Full Text].


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