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Endocrinology Vol. 140, No. 3 1255-1261
Copyright © 1999 by The Endocrine Society

ARTICLES

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

Darren R. Tyson, John T. Swarthout and Nicola C. Partridge

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH induces c-fos expression rapidly and transiently in osteoblastic cells and requires the activity of the cAMP response element-binding protein (CREB). Here we provide evidence that protein kinase A (PKA) is the enzyme responsible for phosphorylating CREB at serine 133 (S133) and that this event is required for PTH-induced c-fos expression. PTH increases the level of phosphorylation of CREB at S133 in a time- and dose-dependent manner, correlating with the time and level of activation of PKA in response to PTH. PTH-(1–34) and -(1–31), each known to activate the cAMP pathway, induced the phosphorylation of CREB and increased the levels of c-fos messenger RNA, whereas PTH-(3–34), -(13–34), and -(28–48) could not. Specific inhibitors of calcium/calmodulin-dependent protein kinases and protein kinase C could not inhibit CREB phosphorylation or c-fos expression in response to PTH; however, H-89, a specific inhibitor of PKA, could do so in a dose-dependent manner. In addition, PTH-induced c-fos promoter activity was completely inhibited in a dose-dependent fashion by transfection of the heat-stable inhibitor of PKA. Taken together, these data provide strong evidence that PKA is the enzyme responsible for phosphorylating CREB at S133 in response to PTH and that PKA activity is required for PTH-induced c-fos expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH HAS complex effects in bone. It is primarily considered a bone-resorbing agent; however, at low doses with an intermittent treatment schedule, PTH can increase bone mass through as yet undetermined mechanisms. In rat osteoblastic cells PTH causes a rapid and transient increase in expression of the immediate early gene c-fos (1). The role of c-fos in bone remains elusive even though expression of its viral homolog v-fos by the FBJ and FBR murine sarcoma viruses was linked to osteocarcinogenesis as early as 1966 (2). Curran and Teich first described the viral oncoprotein v-fos in 1982 as a gene that was overexpressed in spontaneously induced mouse osteosarcomas (3). It has been shown that mice infected with the FBJ virus or transgenic mice overexpressing c-fos develop osteosarcomas in the same location that c-fos is normally expressed, specifically cartilage, bone, and teeth (4), suggesting a role for c-fos in mitogenesis and/or differentiation in the skeletal system. This effect is specific to Fos expression, as overexpression of other members of the activation protein-1 family (e.g. Jun and FosB) did not produce a higher incidence of osteosarcomas (5). In addition, c-fos expression preceded osteogenic differentiation of cartilage cells in vitro (6) and is up-regulated in proliferating osteoblasts (7, 8) and bone from patients with fibrous dysplasia (9), further implicating c-fos as a regulator of mitogenesis. Fos, in conjunction with Jun, can inhibit the expression of osteocalcin by binding the activator protein-1 site in the osteocalcin promoter, thereby suppressing the mature osteoblast phenotype (8). Mice lacking a functional copy of the c-fos gene (fos-null mice) are viable but develop osteopetrosis and have deficiencies in bone remodeling as a result of a lack of osteoclasts (10). In addition, a role for c-fos has been implicated in an osteogenic response to mechanical stress in osteocytes (11). The effects of osteoblastic expression of c-fos are not limited to osteoblasts, however, as MC3T3-E1 cells constitutively expressing c-fos can stimulate the maturation of osteoclasts as well as osteoclastic resorption presumably by expressing some soluble factor(s) (12). Recent evidence suggests that induction of c-fos expression is required for the PTH-mediated increase in collagenase expression in rat osteoblastic cells (13), which may be important for the initiation of bone resorption by osteoclasts. Thus, c-fos appears to have many pleiotropic and essential effects in bone.

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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies and reagents
The CREB and phosphoCREB-specific antibodies and recombinant PKA and GSK-3 were purchased from New England Biolabs, Inc. (Beverly, MA). The clone of the human heat-stable inhibitor of PKA (PKI; construct HFBCO78) was obtained from American Type Culture Collection (Manassas, VA). -151fosCAT was a gift from M. Gilman (21) and contains the bases from -151 to +91 (relative to the transcriptional start site) of the mouse c-fos promoter 5' of the bacterial chloramphenicol acetyltransferase (CAT) gene. N-[2-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, HCl (H-89); N-[2-N-4-chlorocinnamyl-N-methylaminomethylphenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide (KN-93); and chelerythrine chloride were purchased from Calbiochem (San Diego, CA). Human PTH-(1–31), bovine PTH-(3–34), human PTH-(13–34), and human PTH-(28–48) were obtained from Bachem California, Inc. (Torrance, CA). Calmidazolium chloride was provided by T. C. Westfall (St. Louis University, St. Louis, MO). The plasmid containing the complementary DNA (cDNA) of rat CREB (pET15b-CREB341) was provided by J. Chrivia (St. Louis University). The plasmids used as template for Northern probe generation have been described previously (1). All other reagents and chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).

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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time required for PTH induction of S133 phosphorylation
As amino acids 1–34 of all PTH molecules contain the predominant amount of the biological activity of the full-length molecule (1–84) on osteoblasts, synthetic rat PTH-(1–34) was employed for the majority of the studies. To determine the rate at which CREB is phosphorylated in response to PTH, CREB pS133 was measured in cell lysates prepared from UMR cells treated with rat PTH-(1–34) for various lengths of time using an antibody directed to the phosphorylated form of CREB (at S133). UMR cells that were not treated with PTH showed no detectable pS133 (Fig. 1Go). 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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  		Figure 1. Effect of time of PTH treatment on CREB phosphorylation at S133. Rat UMR 106–01 cells were treated with 10-8 M PTH for the times indicated, and lysates were prepared and analyzed by immunoblotting as described in Materials and Methods.

 
Concentration of PTH required to induce S133 phosphorylation
As the concentrations of PTH required to raise the levels of c-fos messenger RNA (mRNA) in osteoblasts have previously been determined, we compared the doses required for increasing c-fos levels with those required to induce CREB S133 phosphorylation. CREB phosphorylation at S133 was detectable when UMR cells were treated with PTH at concentrations as low as 10-10 M, and maximal stimulation occurred at 10-8 M PTH (Fig. 2AGo). 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. 2BGo), 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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  		Figure 2. Concentrations of PTH required to induce phosphorylation of CREB at S133. A, Dose response. Increasing concentrations of PTH were used to treat UMR cells for 20 min and were analyzed by immunoblotting as described previously. B, Dose response in the presence of IBMX. UMR cells were pretreated with 10-5 M isobutylmethylxanthine (IBMX) or dimethylsulfoxide (DMSO; as a control) for 20 min before PTH treatments. PTH was administered for 20 additional min after IBMX pretreatment. CREB bands are indicated by an arrowhead.

 
Peptide fragments of PTH
Several regions of the PTH peptide have been associated with activating different signal transduction pathways within target cells. For instance, the first two amino acids are absolutely required to activate adenylyl cyclase (26, 27, 28). In addition, residues between 3–7 appear to be important for the rise in intracellular calcium levels (27, 28), and amino acids 29–32 appear to be required for the activation of PKC (29, 30). For our studies we chose to use several PTH peptide fragments to further examine the specific second messenger pathways required for PTH to stimulate CREB phosphorylation at S133 and the rise in c-fos mRNA levels. Only PTH-(1–34) and PTH-(1–31), which both retain the ability to activate adenylyl cyclase, can induce CREB phosphorylation at S133 while PTH-(3–34), -(13–34), and -(28–48) cannot (Fig. 3AGo). Similarly, only PTH-(1–34) and -(1–31) can increase c-fos mRNA levels (Fig. 3BGo). 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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  		Figure 3. PTH peptide fragments differentially activate CREB phosphorylation and c-fos expression. A, Immunoblot. PTH fragments consisting of the amino acids listed above each lane were each used at 10-8 M, and UMR cells were incubated with each for 20 min. In addition to lysates, controls were included to demonstrate the specificity of the antibodies. Recombinant CREB was expressed in E. coli, and lysate was prepared as described for UMR cells (CREB). This lysate was used as a substrate in a phosphorylation reaction catalyzed by recombinant PKA (pCREB). Bands corresponding to CREB are indicated by an arrowhead. B, Northern blot. UMR cells were treated with the various PTH peptide fragments listed for 30 min, and total RNA was isolated and analyzed as described in Materials and Methods.

 
Kinase inhibitors
The fact that treatment of cells with PTH-(1–31) can lead to CREB phosphorylation suggests no role for PKC in this response; however, it does not rule out a role for calcium-dependent kinases such as calcium/calmodulin-dependent kinases II and IV, which have been demonstrated previously to be able to phosphorylate CREB at S133 (17, 18). To determine the kinase(s) responsible for phosphorylating CREB at S133, we employed several cell-permeable enzyme-specific inhibitors: H-89 for PKA, KN-93 and calmidazolium chloride (CMDZ) for calcium/calmodulin-dependent kinases, and chelerythrine chloride and PKCi (a myristoylated peptide inhibitor) for PKC. Each inhibitor was added to UMR cells for 30 min before PTH treatment (using 10-8 M PTH) at doses up to 200-fold greater than their Ki values. At concentrations of 80 µM for KN-93 (a specific inhibitor of calcium/calmodulin-dependent kinases) and 20 µM for CMDZ (a calmodulin antagonist), no inhibition was detected on the PTH induction of CREB phosphorylation or c-fos expression, although CMDZ repeatedly caused increased c-fos expression in response to PTH (Fig. 4Go); 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. 5Go). 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. 6Go).



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  		Figure 4. Effects of kinase inhibitors on CREB phosphorylation and c-fos expression. A, Immunoblot. UMR cells were pretreated for 30 min with the following: 80 µM KN-93, 20 µM calmidazolium (CMDZ), 20 µM PKCi, 120 µM chelerythrine chloride (CC), and 20 µM H-89. Cells were then treated with 10-8 M PTH for 20 min and analyzed as before. Three percent dimethylsulfoxide (DMSO) alone or in the presence of PTH was included as a control. CREB bands are indicated by an arrowhead. B, Northern blot. Kinase inhibitors and DMSO were used at the same concentrations as those used for immunoblotting. The abilities of these agents to inhibit the PTH-mediated increase in c-fos expression were determined by analyzing total RNA for the presence of c-fos mRNA. The amount of mRNA present in each lane was assessed by comparing the amount of ß-actin present in each sample.

 


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  		Figure 5. Dose-dependent inhibition of PTH responses by the PKA inhibitor H-89. A, Immunoblot. H-89 was added to UMR cells for 30 min before PTH treatment (10-8 M for 20 min) at the concentrations listed. Lysates were isolated and analyzed as previously described. CREB bands are indicated by an arrowhead. B, Northern blot. Expression of c-fos was determined by Northern analysis of total RNA isolated from cells pretreated with H-89 (at the concentrations indicated) and subsequently treated with or without 10-8 M PTH for 20 min. The amount of mRNA present was normalized to ß-actin by stripping blots and reprobing.

 


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  		Figure 6. Effects of PKI on PTH-induced c-fos promoter activity. UMR cells were cotransfected with a reporter plasmid (-151fosCAT) and increasing amounts of an expression plasmid for human PKI. Total amounts of DNA added to each sample were normalized with the expression vector lacking the cDNA for PKI. Promoter activity was determined by analyzing CAT activity in the lysates. Three similar experiments were performed. Each experiment was performed with triplicate samples, and a single experiment is shown. a, Statistically significant difference compared with no PTH treatment (P < 0.001); b, significant difference from no PTH treatment (P < 0.05); c, significant difference compared with PTH-treated cells with no PKI (P < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PTH-mediated expression of c-fos has been demonstrated in vivo in bone as well as in cultured UMR cells (1, 31, 32, 33). In UMR cells, PTH rapidly and dose dependently induces transcription of c-fos (1, 31). Three different regions within the c-fos promoter have been identified as the major targets of stimulatory signals: the sis-inducible element at -345 (relative to the transcriptional start site of the human c-fos gene), the serum response element at -300, and the cAMP response element at -60. This laboratory has recently demonstrated that the major CRE at -60 is required for PTH induction of c-fos in UMR cells and that CREB binds to this site apparently as a homodimer and is phosphorylated in a PTH-inducible fashion at S133 (14). In addition, mutating the major CRE causes a significant decrease in the PTH-induced transcriptional activity of the c-fos promoter (14). These data strongly suggest a role for the phosphorylation of CREB at S133 in the mediation of the PTH-induced increase in c-fos expression.

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{zeta} or PKC{iota}, 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{zeta} and PKC{iota}) 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.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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