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Endocrinology Vol. 138, No. 9 3594-3600
Copyright © 1997 by The Endocrine Society

ARTICLES

Parathyroid Hormone Increases Prostaglandin G/H Synthase-2 Transcription by a Cyclic Adenosine 3',5'-Monophosphate-Mediated Pathway in Murine Osteoblastic MC3T3-E1 Cells1

Sotirios Tetradis2, Carol C. Pilbeam, Yong Liu, Harvey R. Herschman and Barbara E. Kream

Departments of Medicine and Oral Diagnosis (S.T.), University of Connecticut Health Center, Farmington, Connecticut 06030; and the Departments of Biological Chemistry and Pharmacology, Molecular Biology Institute (H.R.H.), and Laboratory of Structural Biology and Molecular Medicine (H.R.H.), University of California School of Medicine, Los Angeles, California 90095

Address all correspondence and requests for reprints to: Barbara E. Kream, Ph.D., Department of Medicine, MC-1850, University of Connecticut Health Center, Farmington, Connecticut 06030. E-mail: kream{at}nso1.uchc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH increased PG synthase-2 transcription in osteoblastic MC3T3-E1 cells at 30 min, as assessed by nuclear run-on assays. To determine the signaling pathways used by PTH, the activity of a construct containing the PG synthase-2 gene between nucleotides -963 and +70 linked to a luciferase reporter was analyzed in stably transfected MC3T3-E1 cells. Agents that activate the cAMP-protein kinase A or protein kinase C pathways increased PG synthase-2 promoter activity. In contrast, the calcium ionophore ionomycin was ineffective. The protein kinase A inhibitor H89 blocked PTH stimulation of PG synthase-2 promoter activity, whereas an overnight preincubation with phorbol ester to down-regulate protein kinase C did not. PTH-(3–34), a peptide that has greatly reduced ability to activate the cAMP-protein kinase A pathway, did not increase PG synthase-2 transcription or promoter activity. PTH could induce PG synthase-2 messenger RNA accumulation and PG synthase-2 transcription in the presence of cycloheximide. In addition, PTH-stimulated PG synthase-2 transcription was maintained at a high level at 2 h in the presence of cycloheximide. We conclude that PTH rapidly increases PG synthase-2 transcription in MC3T3-E1 cells, mainly through a cAMP-protein kinase A-mediated pathway without the need for protein synthesis. In contrast, the attenuation of increased PG synthase-2 transcription by PTH requires de novo protein synthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH IS A potent activator of osteoclastic bone resorption that elicits a wide variety of biological responses in osteoblastic cells. PTH mediates its effects on bone metabolism by binding to the G protein-linked PTH/PTH-related peptide receptor that is expressed in cells of osteoblast lineage (1). PTH binding to this receptor stimulates adenylate cyclase and phospholipase C, thereby activating the cAMP-protein kinase A (PKA), protein kinase C (PKC), and calcium signaling pathways (2). Activation of these pathways leads to changes in osteoblastic gene expression by transcriptional and/or posttranscriptional mechanisms. PTH is thought to mediate most of its biological effects in osteoblastic cells, primarily via cAMP-PKA signaling, although the involvement of other pathways has been suggested (3).

PTH stimulates PG synthesis by rodent calvariae (4, 5) and primary osteoblastic cells derived from calvariae (6). PGs activate bone resorption and can either stimulate or inhibit bone formation in vitro depending on their concentration and the presence of other interacting hormones such as cortisol (7) and insulin-like growth factor I (8). In vivo administration of PGs stimulates bone formation in experimental animals and humans (9). Therefore, it is likely that PGs act as local mediators for some PTH responses in bone.

PG G/H synthases (PGHS) are required for the conversion of arachidonic acid to prostanoids and are encoded by two genes, the constitutively expressed PGHS-1 and the inducible PGHS-2. PGHS-2 was first described as a phorbol ester-inducible primary response gene in murine NIH-3T3 cells (10) and a v-src-inducible gene in chicken fibroblasts (11). Growth factors, cytokines, hormones, and mitogens induce PGHS-2 in many cell lines and tissues, including bone (12). PTH induces PGHS-2 messenger RNA (mRNA) accumulation and PG production in organ cultures of mouse and osteoblastic MC3T3-E1 cells (13, 14). We recently showed that PTH induction of PGHS-2 mRNA is mediated primarily by the cAMP-PKA signaling pathway in MC3T3-E1 cells (15). Here we present evidence that PTH induction of PGHS-2 mRNA is due to a rapid increase in PGHS-2 transcription that is also mediated by the cAMP-PKA pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Synthetic bovine PTH-(1–34), bovine PTH-(1–34) amide, bovine PTH-(3–34) amide, forskolin (FSK), phorbol myristate acetate (PMA), 8-bromo-cAMP (8Br-cAMP), and ionomycin were purchased from Sigma Chemical Co. (St. Louis, MO). PTH was prepared as a stock solution containing 1 mg/ml BSA containing 0.001 N HCl and diluted in culture medium at least 1000-fold. FSK, PMA, and ionomycin were prepared as stock solutions in 100% ethanol and diluted in culture medium at least 1000-fold. 8Br-cAMP was dissolved directly in the culture medium to a final concentration of 1 mM. Murine PGHS-2 complementary DNA (cDNA) purchased from Oxford Biomedical Research (Oxford, MI) was used for Northern blots. Murine PGHS-2 cDNA isolated by Kujubu and co-workers was used for the nuclear run-on assays (10). A construct containing the PGHS-2 gene between nucleotides -963 and +70 and a luciferase reporter (PGHS-Luc963) was used for the stable transfection of MC3T3-E1 cells (16). Murine PGHS-1 cDNA was provided by Drs. William Smith and David Dewitt (Michigan State University, East Lansing, MI). Chick ß-actin cDNA was provided by Dr. Donald Cleveland (Johns Hopkins University, Baltimore, MD). Lipofectamine reagent was purchased from Life Technologies (Gaithersburg, MD).

Cell culture
MC3T3-E1 cells were plated at 5000 cells/cm2 in 35- or 100-mm tissue culture wells, cultured in a humidified atmosphere of 5% CO2 in air at 37 C; fed every 3 days with DMEM supplemented with 10% FCS, 100 U/ml penicillin, and 50 µg/ml streptomycin; and passaged on day 7. For experiments, cells were grown for 6 days (confluence) and treated with effectors in serum-free DMEM containing 1 mg/ml BSA.

Total RNA isolation and Northern blot analysis
MC3T3-E1 cells from three 35-mm wells or one 100-mm plate were washed twice with cold PBS. RNA was extracted by the method of Chomczynski and Sacchi (17). Cells were lysed with a solution containing 4 M guanidinium thiocyanate. RNA was extracted with H2O-saturated phenol/chloroform-isoamyl (24:1); precipitated with an equal volume of isopropanol and 0.2 M sodium acetate, pH 4.0; and centrifuged at 10,000 x g. The pelleted RNA was dissolved in guanidinium thiocyanate solution and precipitated with isopropanol. The final RNA pellet was washed twice with 80% ethanol, lyophilized, dissolved in diethylpyrocarbonate-treated water, and quantitated by absorbance at 260 nm. Twenty micrograms of total RNA were fractionated by electrophoresis on a 1% agarose-6% formaldehyde gel and transferred to a GeneScreen Plus hybridization membrane. Filters were prehybridized for 3–6 h, hybridized at 42 C for 12–16 h with [32P]cDNA, washed, and exposed to x-ray film at -70 C.

Stable transfection of MC3T3-E1 cells
DNA constructs were purified by double banding in a cesium chloride gradient. MC3T3-E1 cells were plated at 40,000/cm2 in a 35-mm tissue culture well and incubated with a solution containing 20 µg PGHS-Luc963, 1 µg pSV2-neo, and 8 µl lipofectamine reagent (2 mg/ml)/ml serum-free medium. Five hours later, 1 ml DMEM with 20% FCS was added to each well, and the incubation was continued for 19 h. Then, fresh medium containing 10% FCS was added to the cells. After 48 h, cells were passaged (1:10) and placed under selection with 400 µg/ml G418 for 2 weeks. Surviving colonies (>200/dish) were pooled to randomize effects of integration site on promoter expression and expanded in medium containing 10% FCS and 200 µg/ml G418.

Luciferase assay
Cells were grown to confluence in 35-mm wells as described above, washed twice with cold PBS, and assayed for luciferase activity using a commercially available kit (Promega, Madison, WI). The cells were lysed in 200 µl of a buffer containing 25 mM Tris-phosphate (pH 7.8), 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N,N,N',N-tetraacetic acid, 10% glycerol, and 15% Triton X-100. The cell lysates were transferred to Eppendorf tubes and centrifuged for 30 sec. Ten microliters of each supernatant were assayed for luciferase activity, which was quantitated on a Berthold LB 9501/16 luminometer (Wallac, Gaithersburg, MD). Luciferase activity was normalized to protein content, which was measured using the indicator bicinchoninic acid (18).

Nuclear run-on transcription assays
Nuclear run-on assays were performed as previously described (19). Untransfected (native) MC3T3-E1 cells from one confluent 100-mm dish were rinsed and scraped into cold PBS and harvested by centrifugation at 1500 rpm for 2 min. Cells were resuspended in cold hypotonic buffer [10 mM KCl, 10 mM Tris-HCl (pH 7.5), 1.5 mM MgCl2, 0.3 M sucrose, 0.25% Nonidet P-40, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonylfluoride] and disrupted by homogenization in a Dounce homogenizer (Kontes Co., Vineland, NJ). Nuclei were collected by centrifugation at 1500 rpm for 5 min, resuspended in 75 µl cold buffer [50 mM Tris-HCl (pH 8.3), 40% glycerol, 5 mM MgCl2, and 0.1 mM EDTA] and stored in liquid nitrogen until use. Transcription was carried out for 20 min at 25 C in a 200-µl mixture containing nuclei; 50 mM Tris-HCl (pH 7.5); 100 mM ammonium sulfate; 1.8 mM dithiothreitol; 1.8 mM MnCl2; 2 µl RNasin; 300 µM each of ATP, CTP, and GTP; and 100 µCi [32P]UTP. Transcription was terminated with 2 µl ribonuclease-free deoxyribonuclease and 2 µl transfer RNA (tRNA; 10 mg/ml). After 15 min at 25 C, 20 µl 10% SDS and 2 µl 100 µg/ml proteinase K were added, and the solution was incubated at 37 C for 45 min. RNA was extracted as described above. One microgram of PGHS-2 cDNA and an equimolar amount of actin cDNA were slot-blotted onto nitrocellulose filter strips. For prehybridization and hybridization, filters were placed individually in rotating 10-ml glass scintillation vials and prehybridized for 2 h at 52 C in 1 ml of a solution containing 49.3% formamide, 4.93 x SSC (standard saline citrate), 0.1% SDS, 1 mM EDTA (pH 7.5), 10 mM Tris (pH 7.5), 4 x Denhardt’s, 0.34 mg/ml yeast tRNA, and 0.34 mg/ml sheared salmon sperm DNA. An equal amount of radioactive RNA was added to each vial, and the filters were hybridized at 52 C in 1 ml of a solution containing 58% formamide, 5.8 x SSC, 0.12% SDS, 1.2 mM EDTA (pH 7.5), 12 mM Tris (pH 7.5), 4.7 x Denhardt’s solution, 0.4 mg/ml yeast tRNA, and 0.4 mg/ml sheared salmon sperm DNA for 48–72 h. Filters were washed and exposed to x-ray film at -70 C. The intensity of the bands was assessed using a Molecular Dynamics PhosphorImager (Sunnyvale, CA).

Statistics
Data were analyzed using one-way ANOVA, and statistical differences between groups were determined using the Student-Newman-Keuls post-hoc test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To determine whether PTH induces PGHS-2 by a transcriptional mechanism, we examined the effect of PTH on PGHS-2 transcription using nuclear run-off assays. PTH (10 nM) increased PGHS-2 transcription relative to actin transcription by 3- to 4-fold. PGHS-2 transcription peaked at 30 min and returned almost to control levels by 2 h (Fig. 1Go, A and B). As another means of assessing transcription, MC3T3-E1 cells were stably transfected with PGHS-Luc963, a pXP-2 vector construct containing the murine PGHS-2 gene between nucleotides -963 and +70 linked to a luciferase reporter (16). PTH rapidly increased PGHS-Luc963 activity (Fig. 2AGo). The PTH concentrations that produced half-maximal and maximal stimulation of PGHS-Luc963 were about 0.03 and 1 nM, respectively (Fig. 2BGo).



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  		Figure 1. PTH increases PGHS-2 transcription in MC3T3-E1 cells. Confluent cells were treated for various times with 10 nM PTH-(1–34) in serum-free medium. A, Transcription of PGHS-2 and actin was determined using nuclear run-on assays. Data are representative of two experiments. B, Quantitation of transcription rates for time-course experiments. PGHS-2 transcription was expressed relative to actin transcription for PTH-treated and control cells. The PGHS-2/actin ratio in PTH-treated cells was expressed relative to that in control cells. Each value is the mean ± SEM of two experiments.

 


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  		Figure 2. PTH induces PGHS-Luc963 expression in stably transfected MC3T3-E1 cells. A, Confluent cells were treated with 10 nM PTH-(1–34) in serum-free medium for various time intervals. Luciferase activity in soluble cell extracts was normalized to protein content and expressed relative to that in the untreated control group at each time point. Each value is the mean ± SEM of six measurements. *, Significant effect of PTH (P < 0.01). B, Confluent cells were treated for 2 h in serum-free medium with various concentrations of PTH. Luciferase activity in soluble cell extracts was normalized to protein content and expressed relative to that in the untreated control group. Each value is the mean ± SEM of nine measurements. *, Significant effect of PTH (P < 0.05).

 
To investigate the signaling pathway(s) that increase PGHS-2 transcription, MC3T3-E1 cells were treated with FSK and 8Br-cAMP (which activate the cAMP-PKA pathway), PMA (which activates the PKC pathway), and the ionophore ionomycin (which increases the level of intracellular calcium). FSK (10 µM), 8Br-cAMP (1 mM), and PMA (100 nM) increased PGHS-Luc963 activity, whereas ionomycin was ineffective (Fig. 3Go). These data show that activation of both the PKA and PKC pathways can increase PGHS-2 promoter activity.



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  		Figure 3. Phorbol ester and cAMP activators induce PGHS-Luc963 expression in stably transfected MC3T3-E1 cells. Confluent cells were treated for 2 h in serum-free medium with 10 nM PTH-(1–34), 10 µM FSK, 1 mM 8Br-cAMP, 0.1 µM PMA, or 1 µM ionomycin (Iono). Luciferase activity in soluble cell extracts was normalized to protein content and expressed relative to that in the untreated control group. Each value is the mean ± SEM of 6–12 measurements. *, Significantly different from the control (P < 0.05).

 
To determine whether the PKC pathway plays a role in PTH stimulation of PGHS-2 promoter activity, MC3T3-E1 cells were treated with 100 nM PMA overnight to down-regulate the PKC pathway (20). An overnight pretreatment of cells with PMA did not reduce the stimulation of PGHS-Luc963 activity by PTH and FSK, but completely abolished the increase produced by PMA (Fig. 4AGo). To block the PKA pathway, MC3T3-E1 cells were treated with H89, a protein kinase inhibitor that is 100-fold more specific for PKA than for PKC in vitro (21). Treatment of the cells with H89 (30 µM) reduced the stimulatory effect of PTH (10 nM) and FSK (10 µM) on PGHS-Luc963 activity by 65% and 75%, respectively (Fig. 4BGo). Treatment of the cells with H89 also decreased the stimulatory effect of PMA on PGHS-Luc963 activity by 45% (Fig. 4BGo). These data suggest that there was cross-talk between the cAMP-PKA and PKC pathways, or that H89 partially inhibited the PKC pathway.



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  		Figure 4. Down-regulation of PKC by PMA does not block FSK or PTH stimulation of PGHS-Luc963 expression in stably transfected MC3T3-E1 cells. A, Confluent cells were pretreated (pretx) for 16 h with vehicle or 0.1 µM PMA and then treated with 10 nM PTH-(1–34), 10 µM FSK, or 0.1 µM PMA for 2 h. Luciferase activity in soluble cell extracts was normalized to protein content and expressed relative to that in the appropriate control group with or without PMA pretreatment. Each value is the mean ± SEM of six measurements. *, Significant effect of PMA pretreatment (P < 0.05). B, Confluent cells were pretreated for 1 h with vehicle or 30 µM H89 and then treated with 10 nM PTH-(1–34), 10 µM FSK, or 0.1 µM PMA for 2 h in the presence of H89. Luciferase activity in soluble cell extracts was normalized to protein content and expressed relative to that in the appropriate control group with or without H89 pretreatment. Each value is the mean ± SEM of 9–12 measurements. *, Significant effect of H89 treatment (P < 0.05).

 
Deletion of the first two N-terminal amino acids of PTH results in a peptide with greatly diminished ability to signal through the cAMP-PKA pathway. However, this peptide can still activate the PKC and calcium pathways (22). PTH-(3–34) at 10 and 100 nM did not affect PGHS-2 transcription as measured by nuclear run-on assays, in contrast to 10 nM PTH-(1–34) (Fig. 5AGo). Moreover, PTH-(3–34) at 0.1–100 nM did not significantly increase PGHS-Luc963 activity, in contrast to 10 nM PTH-(1–34) (Fig. 5BGo).



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  		Figure 5. PTH-(3–34) is unable to induce PGHS-2 gene expression in MC3T3-E1 cells. A, Confluent cells were treated for 30 min with 0.1–100 nM PTH-(3–34) or 10 nM PTH-(1–34). Transcription of PGHS-2 and actin was determined using nuclear run-on assays. Data are representative of two experiments. B, Confluent MC3T3-E1 cells stably transfected with PGHS-Luc963 were treated for 2 h with 0.1–100 nM PTH-(3–34) or 10 nM PTH-(1–34). Luciferase activity in soluble cell extracts was normalized to protein content and expressed relative to that in the untreated control group. Each value is the mean ± SEM of six to nine measurements. *, Significantly different from the control (P < 0.05).

 
PGHS-2 is a primary response gene in other cells (23). To determine whether this was the case in PTH-treated MC3T3-E1 cells, induction of PGHS-2 mRNA by PTH was assessed in the presence of the protein synthesis inhibitor cycloheximide (CHX; 3 µg/ml). We previously showed that PTH rapidly and transiently induced PGHS-2 mRNA levels in MC3T3-E1 cells in the absence of CHX; PGHS-2 mRNA peaked at 1 h and declined almost to control levels by 6 h (15). CHX caused superinduction of PGHS-2 mRNA in response to PTH. PGHS-2 mRNA levels were increased at 30 min and continued to rise even after 4 h of culture (Fig. 6Go). Therefore, PTH induction of the PGHS-2 gene is a primary response not requiring any new protein synthesis.



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  		Figure 6. PTH induction of PGHS-2 in MC3T3-E1 cells is a primary response. Confluent cells were pretreated for 1 h with 3 µg/ml CHX (0 h sample). PTH-(1–34) was then added to the cells in the presence of CHX for the times shown. PGHS-2 mRNA levels were determined by Northern blot analysis. The ethidium bromide-stained 18S and 28S ribosomal RNA bands are shown. Data are representative of three experiments.

 
To determine whether the sustained high level of PGHS-2 mRNA was due in part to increased PGHS-2 transcription, MC3T3-E1 cells were treated with PTH for 30 min or 2 h in the presence or absence of CHX (3 µg/ml), and nuclear run-on assays were performed. PTH increased PGHS-2 transcription in the presence or absence of cycloheximide at 30 min (Fig. 7Go). In the absence of CHX, PGHS-2 transcription in PTH-treated cells returned to control levels by 2 h, in agreement with the data shown in Fig. 1Go. However, in the presence of CHX, the increased level of PGHS-2 transcription in PTH-treated cells was maintained at 2 h (Fig. 7Go). We conclude that the induction of the PGHS-2 gene transcription by PTH does not require de novo protein synthesis. However, the attenuation of the increased level of PGHS-2 transcription in response to PTH does require de novo protein synthesis.



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  		Figure 7. Inhibition of protein synthesis prevents the attenuation of PTH-induced PGHS-2 transcription in MC3T3-E1 cells. Confluent cells were pretreated for 1 h with 3 µg/ml CHX or vehicle and then treated with 10 nM PTH-(1–34) for 30 min or 2 h. Transcription of PGHS-2 and actin was determined using nuclear run-on assays. Data are representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The objectives of this study were 1) to determine whether PTH regulates PGHS-2 expression by a transcriptional mechanism, and 2) if so, to identify the signaling pathway(s) that mediates this effect. PGHS-2 transcription was evaluated by nuclear run-on assays and by measuring the activity of a stably integrated PGHS-2 promoter-luciferase construct in osteoblastic MC3T3-E1 cells. We showed that PTH increased PGHS-2 transcription, which peaked at 30 min and returned almost to control levels by 2 h. PTH also stimulated PGHS-Luc963 activity. The difference in the time course between the increase in nuclear run-on transcription and luciferase activity presumably results from the time required to translate luciferase mRNA to functional protein. PTH regulates {alpha}1(I) collagen (24), vitamin D receptor (25), collagenase (26), tissue plasminogen activator (27), c-fos and c-jun (28), osteopontin (29), interleukin-6 (IL-6), and leukemia inhibitory factor (30) genes by transcriptional mechanisms in osteoblastic cells. The PTH concentration range required for stimulated PGHS-2 promoter expression was similar to that necessary for PTH induction of IL-6 (30), leukemia inhibitory factor (30), c-fos (28), and c-jun (28) mRNAs. PTH caused half-maximal induction of PGHS-Luc963 expression (this report), c-fos mRNA (28), and c-jun mRNA (28) at 0.03, 0.025, and 0.0025 nM, respectively. PTH also regulates vitamin D receptor (25), {alpha}1(I) collagen (31), and osteocalcin expression (32) by additional posttranscriptional mechanisms that may include changes in mRNA half-life and translational or posttranslational modifications. We have not investigated the effect of PTH on PGHS-2 mRNA stability or PGHS-2 protein modifications.

Murine MC3T3-E1 cells were used as the model in this study. MC3T3-E1 cells are osteoblastic cells that express increased levels of osteogenic markers when cultured with differentiation medium containing ascorbic acid and ß-glycerolphosphate (33, 34). It is important to note that MC3T3-E1 cells were not differentiated with ascorbic acid before treatment in the present study and do not express osteocalcin mRNA. Therefore, they probably represent immature osteoblastic cells under the conditions of our experiments. Previously, we showed that PTH increases PGHS-2 mRNA levels in normal neonatal mouse calvariae (13), a response that probably represents an effect on differentiated osteoblasts. Moreover, forskolin continued to induce PGHS-2 mRNA levels in confluent MC3T3-E1 cells that were cultured for an additional 2 weeks in the presence of ascorbic acid to induce osteoblastic differentiation (Pilbeam, C. C., unpublished data). Taken together, it is likely that PTH induces PGHS-2 expression in both immature and mature osteoblastic cells.

PGE2 can also induce PGHS-2 expression, causing an autoamplification loop (35). Therefore, it is possible that PTH induction of PGHS-2 expression leads to autoamplification due to increased production of PGE2 (35). The experiments reported in the manuscript were performed using serum-deprived MC3T3-E1 cells. Under these conditions, the cells appear to be substrate limited and produce little if any PGE2 in response to agonists, including PTH. However, the cells will produce PGE2 if supplemented with serum or arachidonic acid as substrate (35).

Because PTH can activate multiple signaling pathways, we used pharmacological activators and inhibitors of several signaling pathways as an initial approach to determining the signaling pathway by which PTH regulates PGHS-2 transcription. PGHS-2 transcription was increased by agents that activate both the cAMP-PKA and PKC pathways. Blocking the PKC pathway by an overnight incubation with PMA did not affect PTH stimulation of PGHS-Luc963. However, the PKA inhibitor H89 significantly reduced the PTH-induced increase in PGHS-Luc963 activity.

PTH-(3–34), an analog that lacks the two N-terminal amino acids of PTH and signals through the PKC pathway and intracellular calcium (22) but does not activate the cAMP-PKA pathway (2, 36), was unable to induce PGHS-2 gene transcription and PGHS-Luc963 activity, in contrast to PTH-(1–34). It is important to note that PTH-(3–34) can have weak agonist activity in vivo, representing less than 1% of the agonist activity of PTH-(1–34) (37, 38). Collectively, our data suggest that PTH regulates PGHS-2 transcription in MC3T3-E1 cells by the cAMP-PKA pathway. PTH regulates collagenase (26), c-fos (28), {alpha}1(I) collagen (24), insulin-like growth factor I (39), PTH/PTH-related peptide receptor (40), and IL-6 (41) gene expression at least in part via cAMP-PKA signaling. Utilization of other pathways has been suggested for PTH-induced osteoclast-like cell formation (42), bone resorption (36), and PTH-mediated changes in osteoblastic cell proliferation (43, 44).

The data presented here show that the region of the PGHS-2 promoter from nucleotides -963 to +70 is sufficient to confer PTH inducibility in MC3T3-E1 cells. We are currently analyzing constructs carrying progressive 5'-deletions of the PGHS-2 promoter to map the cis-element(s) mediating transcriptional regulation by PTH and cAMP. Preliminary data indicate that the region downstream (i.e. 3') of nucleotide -150 contains the cis-element(s) important for the PTH and FSK induction. This region contains a cAMP response element (CRE) site (CGTCA) located between nucleotides -56 and -52 that binds transcription factors of the CRE-binding protein (CREB) transcription factor family (45). The CRE site mediates phorbol ester induction of the human PGHS-2 promoter in monocytes (46) and synergizes with an upstream nuclear factor-IL-6 (NF-IL6) site located between nucleotides -132 and -124 to confer lipopolysaccharide and phorbol ester induction of the human PGHS-2 promoter in endothelial cells (47). This CRE site also mediates the induction of the murine PGHS-2 promoter by v-src (45), serum (48), and platelet-derived growth factor (48). For serum, v-src, and platelet-derived growth factor, the signal transduction pathway activating PGHS-2 expression involves activation of c-jun N-terminal kinase, phosphorylation of c-Jun, and binding of c-Jun to the CRE site (48, 49).

The CREB transcription factor, acting at a CRE, has been suggested as a mediator of PTH-induced c-fos expression in osteoblastic cells (50). CREB binding to an activating protein-1 site of the collagenase promoter has also been implicated in the PTH induction of collagenase expression in UMR-106–01 cells. CREB can bind to the CRE of the murine PGHS-2 promoter (51). Based on these findings, it is possible that the CRE site located between nucleotides -56 and -52 or the activating protein-1 sites between nucleotides -67 and -61 and +33 to +41 could mediate PTH induction of PGHS-2 by binding of activated CREB. However, it is clear that CREB is not the transcription factor that mediates v-src, serum, and platelet-derived growth factor induction of murine PGHS-2 at the CRE in NIH-3T3 cells (48, 49). In fact, overexpression of CREB acts as a dominant negative, binding to this cis-acting element and preventing transcriptional activation by the endogenous activating transcription factor. A variety of experiments have unequivocally identified the c-Jun protein as the transcription factor that mediates v-src-, serum-, and platelet-derived growth factor activation of the PGHS-2 gene via the CRE (48, 49). It will be of great interest to perform similar experiments to determine which cis-acting element(s) and transcription factor(s) mediate PGHS-2 induction by PTH in MC3T3-E1 cells. The C/EBPß (NF-IL6) sites between nucleotides -139 and -130 and/or between -92 and -84 may also play a role in the PTH response. PKA phosphorylates NF-IL6 (52) and facilitates its translocation from the cytoplasm to the nucleus (53). Interestingly, CREB and NF-IL6 are both leucine zipper transcription factors that are capable of recognizing the same element in the IL-1ß promoter (54).

The PTH-induced increase in PGHS-2 mRNA levels and transcription was not inhibited by CHX and, therefore, did not require de novo protein synthesis. However, de novo protein synthesis was required for attenuation of the increased level of PGHS-2 transcription by PTH. Therefore, we suggest that PTH also induces the synthesis of a transcriptional inhibitor. We have recently shown that PTH induces expression of the inducible cAMP early repressor (ICER) in MC3T3-E1 cells (55). ICER is an isoform of the CRE modulator (CREM) transcription factor that is transcribed from an intronic CREM promoter and strongly induced by cAMP (56). ICER contains the DNA-binding domain of CREM, but lacks transcriptional activation domains and, therefore, acts as a transcriptional repressor (57). It is possible that induction of ICER protein by PTH is responsible for attenuating PGHS-2 transcription in MC3T3-E1 cells by binding to the CRE of the PGHS-2 promoter. Experiments are now underway to map the regions of the PGHS-2 gene that mediate PTH transcriptional activation of PGHS-2 and to determine the role of ICER in modulating PGHS-2 transcription.

The induction of PGHS-2 expression by PTH suggests that PG production may mediate some biological effects of PTH in bone (14). In support of this idea, indomethacin, a specific PG synthesis inhibitor, blocks the ability of PTH to increase the formation of multinucleated cells expressing tartrate-resistant acid phosphatase (58), the formation of osteoclastic cells in bone marrow cultures (59), and the production of inositol 1,4,5-triphosphate in mouse osteoblast cultures (60). Indomethacin partially blocks PTH-stimulated insulin-like growth factor I release from neonatal mouse calvariae (61) and the ability of PTH to sensitize long bones to the bone-resorptive effects of 1,25-dihydroxyvitamin D3 (62). Future experimentation will be required to fully define the role of PG production in mediating PTH responses in bone.


   Footnotes
 
1 This work was supported by Grants AR-29850 (to B.E.K.), DK-48361 (to C.C.P.), and GM-24797 (to H.R.H.) from the NIH. Back

2 Present address: NEB Bone and Joint Institute, Room 237, Beth Israel/Deaconess Medical Center, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, Massachusetts 02115. Back

Received March 18, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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